Download Characterization of Head and Neck Squamous Cell Carcinoma

Department of Otorhinolaryngology
University of Lübeck
Head: Prof. Dr. med. Barbara Wollenberg
Characterization of Head and Neck Squamous Cell
Carcinoma under NF-κB Inhibiting Conditions
and
Identication of EPS 7630 as a Novel NF-κB
Inhibitor
Thesis
for the medical doctorate Dr. med.
issued by the University of Lübeck
Faculty of Medicine submitted by
Christian Meyer
from Münster
Lübeck 2010
1. Reviewer:
Prof. Dr. med. Barbara Wollenberg
2. Reviewer:
Priv.-Doz. Dr. med. Andreas Lubienski
Scientic Supervisor:
Dr. rer. nat. Ralph Pries
Date of Doctoral Defense: 13.09.2011
Approved for Publishment, Lübeck 13.09.2011
Prof. Dr. med. Werner Solbach
-Dean of Medical Faculty-
Adavit
The thesis was written single-handed and without the aid of unfair or unauthorized
resources. Indications of sources are given whenever content was taken directly or
indirectly from other sources.
Lübeck, 13.09.2011
for my parents
"...The greatest story ever drawn.
14 liters of Indian ink, 30 brushes,
62 soft pencils, 1 hard pencil,
27 erasers, 1984 sheets of paper,
16 typewriter ribbons, 2 typewriters,
366 pints of beer went into its creation!"
from "Asterix and Cleopatra", Goscinny/Uderzo, 1965
Abstract
Head and neck cancers make up about 5 % of all cancers with more than 100 000
new cases in Europe each year, the majority being of squamous cell origin (head and
neck squamous cell carcinoma, HNSCC). HNSCC almost exclusively occurs among
middle-aged tobacco and alcohol abusers. 5-year survival rates have remained poor
and unchanged for the last 30 years. HNSCC use a variety of immunosuppressive and
modulatory strategies to escape from ecient anti-tumor immune responses. Cytokine alterations supposedly play a critical role in tumor aggressiveness, its response
to chemo- and radiation therapies and the development of an immunosuppressive
HNSCC micromilieu. Transcriptional activator NF-κB as key mediator in these alterations is well accepted and has been observed in a number of human cancers.
In this context we evaluated the impact of Acetylsalicylic Acid (ASA), Celecoxib,
Dexamethasone, Curcumin and EPs 7630 on proliferation and protein expression
of HNSCC. We additionally measured cytokine levels under these conditions to
assess changes in the tumor micromilieu. Secondly, we analyzed the inuence of
Mycoplasma on HNSCC cell cultures.
Our data demonstrate decreased proliferation in response to incubation with aforementioned agents. TLR3 was down-regulated in response to NF-κB inhibition by
every agent via the IKK-complex. Despite suppression, localization of TLR3 expression was not altered signicantly under NF-κB inhibition. Modulation of TLR3
and NF-κB expression was accompanied by altered proles of prominent HNSCC cytokines. IL-6 and IL-8 were signicantly suppressed whereas no signicant change of
the immune modulatory cytokines IL-4 and IL-10 was observed. Under Mycoplasma
inuence HNSCC showed signicantly increased NF-κB expression and high TLR1
and TLR3 expression in three out of seven cell lines.
For the rst time we were able to show that NF-κB inhibition downregulates TLR3
expression under the light of modulated HNSCC cytokine expression. EPs 7630 was
for the rst time described as a natural NF-κB inhibitor. Mycoplasma had signicant eect on the expression of important proteins in HNSCC. Our results contribute
to our understanding of processes at the interface of TLR/NF-κB signaling and pave
the way for a discriminate immune therapy in the future.
Contents
List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
iv
List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vi
List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
1 Introduction
1.1
1.2
1.3
1.4
1
Head and Neck Squamous Cell Carcinoma . . . . . . . . . . . . . . .
1
1.1.1
Epidemiology and Risk Factors . . . . . . . . . . . . . . . . .
1
1.1.2
Classication . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
1.1.3
Limitations of Therapy . . . . . . . . . . . . . . . . . . . . . .
3
1.1.4
Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
1.1.5
Molecular Mechanisms . . . . . . . . . . . . . . . . . . . . . .
4
Genetic Alterations . . . . . . . . . . . . . . . . . . . .
4
Tumor-associated Antigens . . . . . . . . . . . . . . . .
6
Immune Escape Mechanisms . . . . . . . . . . . . . . .
7
Toll-like Receptors (TLRs) . . . . . . . . . . . . . . . . . . . . . . . .
8
1.2.1
Structure and Function . . . . . . . . . . . . . . . . . . . . . .
8
1.2.2
TLRs and Cancer . . . . . . . . . . . . . . . . . . . . . . . . .
9
Transcription Factor NF-κB . . . . . . . . . . . . . . . . . . . . . . . 10
1.3.1
Role and Regulation . . . . . . . . . . . . . . . . . . . . . . . 10
1.3.2
NF-κB in Malignancy
1.3.3
NF-κB Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . 13
. . . . . . . . . . . . . . . . . . . . . . 12
Aim of Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2 Materials and Methods
20
2.1
Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2
Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2.1
Cell Growth and Subcultivation . . . . . . . . . . . . . . . . . 21
2.2.2
Cell Concentrations . . . . . . . . . . . . . . . . . . . . . . . . 21
2.2.3
Long-term Storage and Thawing . . . . . . . . . . . . . . . . . 21
2.2.4
Cell Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.2.5
Cell Harvesting . . . . . . . . . . . . . . . . . . . . . . . . . . 22
i
Contents
2.3
Western Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.3.1
Protein Isolation . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.3.2
Evaluation of Protein Concentration by Photometry . . . . . . 23
2.3.3
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) . . . . 23
2.3.4
Semi-Dry Blotting . . . . . . . . . . . . . . . . . . . . . . . . 24
2.3.5
Blocking and Antibody Detection . . . . . . . . . . . . . . . . 25
2.4
MTT-Cytotoxicity Assay . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.5
Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.5.1
2.6
2.7
2.8
Preparation and Procedure . . . . . . . . . . . . . . . . . . . . 28
Cytokine Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
TM
2.6.1
Principle of the Bio-Plex
Cytokine Assay . . . . . . . . . . 28
2.6.2
Preparation and Procedure . . . . . . . . . . . . . . . . . . . . 29
ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.7.1
Preparation of Nuclear Extracts . . . . . . . . . . . . . . . . . 30
2.7.2
NF-κB Transcription Factor Assay . . . . . . . . . . . . . . . 31
Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.8.1
Cytospin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.8.2
Immunoxation . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3 Results
33
3.1
Dose Dependent Growth Inhibition of HNSCC Cell Lines . . . . . . . 33
3.2
FACS
TM
3.2.1
Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
NF-κB Inhibition Does Not Change Expression of KI-67 and
HLA-A,B,C . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.2.2
CCR7 Shows Higher Expression in the Cytoplasm of HNSCC
Cell Lines than on the Surface . . . . . . . . . . . . . . . . . . 43
3.2.3
3.3
Localization of TLR3 Expression . . . . . . . . . . . . . . . . 43
NF-κB Regulators are Strongly Expressed in HNSCC and Downregulated by Stimulants . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.4
TLR3 is Downregulated in HNSCC Incubated with Stimulants . . . . 46
3.5
NF-κB Inhibition by Stimulants . . . . . . . . . . . . . . . . . . . . . 50
3.6
Cytokine Proles in HNSCC with or without NF-κB Inhibitory Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.6.1
Non-prominent Cytokines in HNSCC . . . . . . . . . . . . . . 52
IL-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
IL-4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
IL-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Granulocyte Macrophage Colony-stimulating Factor (GM-CSF) 53
ii
Contents
Interferon-gamma (IFN-γ ) . . . . . . . . . . . . . . . . . . . . 54
Tumor Necrosis Factor-alpha (TNF-α) . . . . . . . . . . . . . 54
3.7
3.6.2
IL-6 Expression is Decreased in NF-κB Inhibited HNSCC . . . 56
3.6.3
IL-8 Expression is Decreased in NF-κB Inhibited HNSCC . . . 56
Mycoplasma Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.7.1
TLR1, TLR3 and TLR4 but not TLR2 and TLR6 are Expressed in HNSCC under Mycoplasma Inuence . . . . . . . . 59
3.7.2
NF-κB and c-Myc are Upregulated and EpCAM is Downregulated under Mycoplasma Inuence . . . . . . . . . . . . . . . 61
4 Discussion
4.1
4.2
64
NF-κB as Ambiguous Key Player in Inammation-associated Cancer
64
4.1.1
NF-κB Mediates Proliferation in HNSCC . . . . . . . . . . . . 65
4.1.2
IKK-β as Main Regulator of NF-κB . . . . . . . . . . . . . . . 66
Innate Immunity and Immune Escape: Role of TLR3 and the Microenvironment in HNSCC . . . . . . . . . . . . . . . . . . . . . . . . 67
4.3
Identication of a Novel NF-κB Inhibitor: EPs 7630 . . . . . . . . . . 69
4.4
Inuence of Mycoplasma spp. on HNSCC . . . . . . . . . . . . . . . . 69
4.5
Outlook: Hopes and Pitfalls in NF-κB Inhibition and Immunotherapy 71
References
77
Appendices
German Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Curriculum Vitae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
iii
List of Figures
1.1
Clinical, Histologic and Molecular Progression of HNSCC . . . . . . .
1.2
Signaltransduction Pathways of TLRs . . . . . . . . . . . . . . . . . . 11
1.3
Model of NF-κB Activation and Eects . . . . . . . . . . . . . . . . . 12
1.4
Structural Formula of Acetylsalicylic Acid . . . . . . . . . . . . . . . 15
1.5
Structural Formula of Celecoxib . . . . . . . . . . . . . . . . . . . . . 16
1.6
Structural Formula of Dexamethasone . . . . . . . . . . . . . . . . . . 16
1.7
Structural Formula of Curcumin . . . . . . . . . . . . . . . . . . . . . 17
1.8
Structural Formulas of Constituents of EPs 7630 . . . . . . . . . . . . 18
2.1
Principle of AP-Detection . . . . . . . . . . . . . . . . . . . . . . . . 26
2.2
Principle of MTT-Assay . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.3
Principle of the Bio-Plex
2.4
Schematic of the Transcription Factor Assay . . . . . . . . . . . . . . 32
3.1
Proliferation Assay ASA . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2
Proliferation Assay Curcumin . . . . . . . . . . . . . . . . . . . . . . 34
3.3
Proliferation Assay Celecoxib . . . . . . . . . . . . . . . . . . . . . . 35
3.4
Proliferation Assay Dexamethasone . . . . . . . . . . . . . . . . . . . 35
3.5
Proliferation Assay EPs 7630
3.6
Survey of BHY Growth Curves . . . . . . . . . . . . . . . . . . . . . 37
3.7
Survey of PCI-1 Growth Curves . . . . . . . . . . . . . . . . . . . . . 38
3.8
FACS
TM
Cytokine Assay
5
. . . . . . . . . . . . . . 29
. . . . . . . . . . . . . . . . . . . . . . 36
TM
Histogram PCI-1 I . . . . . . . . . . . . . . . . . . . . . . . 39
TM
3.9 FACS Histogram PCI-1 II . . . . . . . . . . . . . . . . . . . . . . . 40
TM
3.10 FACS Histogram BHY I . . . . . . . . . . . . . . . . . . . . . . . . 41
TM
3.11 FACS
Histogram BHY II . . . . . . . . . . . . . . . . . . . . . . . 42
3.12 Western Blot Analysis of IKK-β and Iκ-Bα in BHY . . . . . . . . . . 44
3.13 Western Blot Analysis of IKK-β and Iκ-Bα in PCI-1 . . . . . . . . . 45
3.14 Western Blot Analysis of Cyclin D1 and c-Myc in BHY . . . . . . . . 46
3.15 Western Blot Analysis of Cyclin D1 and c-Myc in PCI-1 . . . . . . . 47
3.16 Western Blot Analysis of TLR3 in BHY . . . . . . . . . . . . . . . . 47
3.17 Western Blot Analysis of TLR3 in PCI-1 . . . . . . . . . . . . . . . . 48
iv
List of Figures
3.18 Immunohistochemistry of TLR3 labeled HNSCC . . . . . . . . . . . . 49
3.19 NF-κB ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.20 Non-prominent HNSCC Cytokines under NF-κB Inhibition . . . . . . 55
3.21 IL-6 Expression in HNSCC under NF-κB Inhibition . . . . . . . . . . 57
3.22 IL-8 Expression in HNSCC under NF-κB Inhibition . . . . . . . . . . 57
3.23 Inuence of Mycoplasma on TLR1 and TLR3 Expression . . . . . . . 60
3.24 Overview of Protein Expressions +/- Mycoplasma inuence . . . . . . 63
4.1
Model of Immune Escape in HNSCC . . . . . . . . . . . . . . . . . . 73
v
List of Tables
1.1
TNM-Staging of HNSCC . . . . . . . . . . . . . . . . . . . . . . . . .
2.1
Composition of SDS-PAGE Gels . . . . . . . . . . . . . . . . . . . . . 24
2.2
Primary Western Blot Antibodies in 1 x PBS
2.3
Secondary Western Blot Antibodies in 1 % Gelatine Buer . . . . . . 26
2.4
FACS
4.1
Overview of Immunotherapies in HNSCC . . . . . . . . . . . . . . . . 72
TM
3
. . . . . . . . . . . . . 25
Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
vi
List of Abbreviations
A
Ampere
AP
Alkaline Phosphatase
APS
Ammonium Persulfate
ASA
Acetylsalicylic Acid
Bcl
Proto-oncogene B cell lymphoma-Protein
bFGF
basic Fibroblast Growth Factor
BSA
Bovine Serum Albumin
CCR
Chemokine (C-C motif) Receptor
c-Myc
Proto-oncogene chromosomal-Myc
COX
Cyclooxygenase
DMEM
"Dulbecco's Modied Eagle Medium"
DMSO
Dimethyl Sulfoxide
(ds)DNA
(double stranded) Deoxyribonucleic Acid
DSMZ
German Collection of Microorganisms and Cell Cultures
EGF-R
Epidermal Growth Factor Receptor
ELISA
Enzyme-linked Immunoabsorbent Assay
et al.
et alii (and colleagues)
TM
FACS
Fluorescence Activated Cell Sorting
FAS
Apoptosis Stimulating Fragment
FCS
Fetal Calf Serum
Fig.
Figure(s)
FITC
Fluorescein Isothiocyanate
FSC
Forward Scatter
g
Gravitational Force or Metrical Gramms
GM-CSF
Granulocyte Macrophage-Colony Stimulating Factor
GTP
Guanosine Triphosphate
h
hour(s)
vii
List of Abbreviations
HLA
Human Leucocyte Antigen
HNSCC
Head and Neck Squamous Cell Carcinoma
HPV
Human Papilloma Virus
IFN
Interferon
IκBα
nuclear factor of kappa light polypeptide
gene enhancer in B-cells inhibitor alpha
IKK
IκB Kinase
IL
Interleukin
IL-1R
Interleukin 1 Receptor
IRAK
Interleukin-1 Receptor-associated Kinase
JNK
c-Jun N-terminal Kinase
LOH
Loss of Heterozygosity
LPS
Lipopolysaccharide
LRR
Leucine-rich Repeats
M
mili
M
Molarity
µ
MAPK
MHC
MTT
MyD
n
NF-κB
NK cells
ODx
(SDS)-PAGE
p
p53
PAMP
PBS
PE
PDGF
PGE
PIP
micro
Mitogen Activated Protein Kinase
Major Histocompatibility Complex
3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide
Myeloid Dierentiation Primary Response Protein
nano or sample size
Nuclear Factor-kappaB
Natural Killer cells
Optical Density at Wavelength x
(Sodium Dodecylsulfate)-Polyacrylamide Gel Electrophoresis
short arm of chromosome or pico
Tumor suppressor protein 53
Pattern Associated Molecular Pattern
Phosphate Buered Saline
Phycoerythrin
Platelet Derived Growth Factor
Prostaglandine
Phosphatidylinositol Phosphate
viii
List of Abbreviations
PKC
Protein Kinase C
PMSF
Phenylmethylsulfonyl Fluoride
PRR
Pattern Recognition Receptor
pH
potentia Hydrogenii (lat.)
q
long arm of chromosome
RIPA buer
Radioimmunoprecipitation Assay buer
RNA
Ribonucleic Acid
ROS
Reactive Oxygen Species
rpm
rotations per minute
siRNA
small interfering RNA
SSC
Sideward Scatter
TAA
Tumor Associated Antigens
TAB
Transforming Growth Factor-activated Kinase binding Protein
TAK
Transforming Growth Factor-activated Kinase
TEMED
N,N,N',N'-Tetramethylethylendiamin
TGF
Tumor Growth Factor
TIR
Toll/IL-1 Receptor
TNF
Tumor Necrosis Factor
TNM
Tumor, Nodes, Metastases
TLR
Toll like Receptor
TRAF
Tumor Necrosis Factor-receptor-associated Factor
Tris
Tris-(hydroxymethyl)-aminomethane
UICC
Union International Contre Cancer (fr.)
V
Volts
VEGF
Vascular Endothelial Growth Factor
WHO
World Health Organization
ix
1 Introduction
1.1 Head and Neck Squamous Cell Carcinoma
1.1.1 Epidemiology and Risk Factors
Head and neck cancers are a related group of cancers that involve the oral cavity,
pharynx and larynx. At least 95 % of cancers of the head and neck are squamous
cell carcinomas (HNSCC). HNSCC is an aggressive epithelial malignancy that is the
sixth most common neoplasm in the world today with an incidence of approximately
35 000 cases in the United States alone [1]. 500 000 worldwide cases of cancer of
the oral cavity and pharynx are newly diagnosed each year, with 160 000 cancers
of the larynx, resulting in circa 300 000 deaths [2]. Regions with high incidences
include much of Southern Asia as well as parts of Central and Southern Europe [3].
In Germany the incidence of head and neck carcinomas approximate 13 500 new
cases per year [4].
Commonly acknowledged risk factors for HNSCC are tobacco and alcohol use. Tobacco smoking is the single most important risk factor for these cancers and follows
a clear exposure to risk ratio. Heavy smokers, long-term smokers and smokers of
black tobacco or high-tar cigarettes have the highest risk of developing these cancers. Cigar and pipe smoking also pose a risk, whereas stopping smoking decreases
the risk [5]. Even in non-smokers there is an elevated risk to develop HNSCC when
being exposed to a smoking environment [6]. Consumption of alcoholic beverages
also increases the risk of head and neck cancer. Relative to abstainers and very light
drinkers, the risk in heavy drinkers is up to tenfold. This increased risk is unlikely
to be caused by alcohol consumption per se, but might instead be the result of the
inuence of acetaldehyde, an intermediate metabolite of ethanol, which is a known
animal carcinogen [7]. The combined eect of both tobacco smoking and alcohol
consumption account for up to 70 % of all head and neck cancers that occur globally [8]. Having long been considered a disease of middle-aged men who have been
chronic abusers of tobacco and alcohol, the incidence of HNSCC in other populations
is on the rise. Not unexpectedly and concurrent with increased cigarette usage the
incidence of HNSCC in women is increasing. Due to the postulated exposure to risk
1
1 Introduction
ratio, the proportion of those cancers caused by alcohol and tobacco was reduced
with decreasing age, being just 32 % for cancers diagnosed prior to age 45.
Other risk factors for these cancers are therefore important. Several occupational
substances or circumstances such as isopropanol manufacturing, inorganic acid mists
containing sulfuric acid and mustard gas are suspected risk factors for laryngeal cancer [9]. In addition it is now known that at least 50 % of oropharyngeal cancers harbor oncogenic variants of Human Papilloma Virus (HPV) [10, 11]. Especially HPV
16 has a high prevalence in HNSCC and seems to play a role in the oncogenesis of
HNSCC [12, 13]. There is increasing epidemiologic evidence that a family history
of head and neck cancer is a risk factor for the disease, and it is postulated that
inherited genomic instability may make individuals more susceptible to developing
cancer [14]. Interestingly, the incidence of HNSCC in individuals younger than 40
without known risk factors has been on the rise for the past several years by mechanisms which have failed to be elucidated so far [1517].
In up to 20 % chronic inammation is thought to play a pivotal role in the evolvement of malignancies [18]. This is particularly true for the association of HNSCC
to tobacco, alcohol and microorganisms like HPV.
1.1.2 Classication
HNSCC can be found in the oral and nasal cavities, the sinuses, the pharynx with
its oro-, hypo- and nasopharyngeal entities and the larynx. The normal epithelium
undergoes dysplastic patterns like leucoplakia and carcinoma in situ which eventually lead to invasive carcinomas with metastases [19]. Chronic exposure to the
factors mentioned above facilitate this process, but this development may also occur
without them. Depending on their histologic dierentiation carcinomas are graded
according to their malignancy from G1 (well dierentiated) to G4 (undierentiated).
Tumor staging follows the international TNM-system as established by the UICC
(Union International Contre Cancer). Primary tumors are staged by increasing size
and tissue inltration from T1 up to T4 and vary slightly with dierent locations.
Lymph node involvement is classied from N0-N3, the higher number depicting
greater number, size and farther located spread of metastasis. Finally, M0 and M1
stand for non-existent or existent spread to other organ systems, respectively. In
order to reect the severity and progression of the disease the TNM-criteria have
been set up to stages (see Table 1.1 on the following page).
2
1 Introduction
Stage
Tumor
Lymph Nodes
Metastasis
0
Tis
N0
M0
I
T1
N0
M0
II
T2
N0
M0
III
T1
N1
M0
T2
N1
M0
T3
N0, N1
M0
T4
N0, N1
M0
Tx
N2
M0
IV B
Tx
N3
M0
IV C
Tx
Nx
M1
IV A
Table 1.1: TNM-Staging of HNSCC. Stages I and II are considered as early disease, Stages
III and IV as advanced
1.1.3 Limitations of Therapy
After a diagnosis of HNSCC has been made by assessment of the primary tumor site
and its potential ways of metastatic dissemination, appreciated therapeutic modalities include surgery, radiation, chemotherapy and combinations thereof.
Primary treatment varies with the anatomic subsite and stage of disease. For most
early cancers, surgical resection is the cornerstone of treatment [20]. Size and local
invasion of the primary tumor put restraints on surgical interventions which span
from partial resections to the complete removal of the tumor and adjacent tissues,
eventually leading to devastating impairment and loss of organ function for the
patient. If lymphatic dissemination is apparent, surgery is extended by neck dissection, by which lymph nodes and, if needed, the inltrated surrounding tissue are
removed. The aim of surgery is always the complete resection in sano. However, for
certain anatomic sites such as the tonsils, the base of the tongue, and the oor of the
mouth, as well as for all locally advanced cancers, radiotherapy is used, either alone
or combined with surgery. Occasionally, chemotherapy may be used in addition to
radiotherapy. The complete surgical resection is a prerequisite for these subsequent
adjuvant therapies.
These established treatments have meanwhile met their ends for patients with advanced stages of HNSCC. Chance of cure decreases signicantly with increasing size
of the primary tumor and its dissemination to regional lymph nodes [21, 22]. That
3
1 Introduction
is why new approaches like immunostimulative methods are highly sought after and
remain a eld of active research and evaluation [2325].
1.1.4 Prognosis
Despite numerous advances in treatment utilizing the most recent protocols for
surgery, radiation, and chemotherapy, the long-term survival has remained at less
than 50 % for the past 50 years [20, 26]. This dismal outlook is due to a number of
factors. For example, oral cancer is often diagnosed when the disease has already
reached an advanced stage. The 5-year survival rate of early-stage oral cancer is
approximately 80 %, while survival drops to 19 % for late-stage disease [27]. In
addition, the frequent development of multiple primary tumors markedly decreases
survival. The rate of second primary tumors in these patients has been reported
to be 3 % to 7% per year, which is higher than for any other malignancy [2830].
This observation has led to the concept of "eld cancerization". It is postulated that
multiple individual primary tumors develop independently in the upper aerodigestive
tract as a result of years of chronic exposure of the mucosa to carcinogens [31, 32].
Because of such eld cancerization, an individual who is fortunate to live 5 years
after the initial primary tumor has up to a 35 % chance of developing at least one new
primary tumor within that period of time. The occurrence of new primary tumors
can be particularly devastating for individuals whose initial lesions are small. The
5-year survival rate for the rst primary tumor is considerably better than 50 %, but
in such individuals, second primary tumors are the most common cause of death [33].
Moreover, the prognosis is generally better for women and for malignancies of the
oral cavity than for those arising in the hypopharynx. In Europe, 5-year relative
survival rates remained virtually identical from 1983 to 1994, suggesting that no
major progress has been made [34]. Therefore, the early detection of all premalignant
lesions is critical for the long-term survival of these patients.
1.1.5 Molecular Mechanisms
Genetic Alterations Like all epithelial neoplasms, the development of squamous
cell carcinoma is thought to be a multi-step process involving the sequential activation of oncogenes and inactivation of tumor suppressor genes in a clonal population
of cells. A number of genetic alterations, some denitively identied and some inferred from tumor-specic chromosomal alterations, have been found in HNSCC and
correlated to their histological and clinical presentation. While not all of the specic
mutations required for progression have been delineated, a working molecular model
4
1 Introduction
Figure 1.1: Clinical, histologic, and molecular progression of oral cancer. A, The
typical clinical progression of oral cancer. B, The histologic progression of squamous
epithelium from normal, to hyperkeratosis, to mild/moderate dysplasia, to severe
dysplasia, to cancer. C, The sites of the most common genetic alterations identied
as important for cancer development (from Robbins and Cotrane, Pathologic Basis
of Disease, 7th Edt., p.784, Elsevier, New York, 2004.)
has been established (see Fig. 1.1 on page 5). The rst reproducible change is the loss
of chromosomal regions of 3p and 9p21 [35]. Loss of heterozygosity (LOH) in conjunction with promoter hypermethylation at this locus results in the inactivation of
the p16 gene, an inhibitor of cyclin-dependent kinases. This alteration is associated
with the transition from normal to hyperplasia/hyperkeratosis and occurs prior to
the development of histologic atypia, thus underscoring the histologic limitations for
early diagnosis. Subsequent LOH at 17p with mutation of the p53 tumor suppressor
gene is associated with progression to dysplasia [36]. It has been demonstrated that
gross genomic alterations as well as deletions on 4q, 6p, 8p, 11q, 13q, and 14q may
act as predictors of progression to frank malignancy [37]. Ultimately, amplication
and overexpression of the Cyclin D1 gene (located on chromosome 11q13), which
constitutively activates cell cycle progression, is a common late event [38, 39].
However, while this model is a good working draft of the molecular changes involved
in development of HNSCC, it is incomplete. While some of the gross genomic
alterations correlate with genes known to be important in HNSCC (such as p16,
p53, and CyclinD1), many of the specic genes are still unknown. It becomes
increasingly clear that HNSCC is a heterogeneous disease in terms of etiology and
therefore its molecular mechanisms of development.
5
1 Introduction
Tumor-associated Antigens Coming from the level of genetics, Tumor-associated
Antigenes (TAA) are the obvious result of theses changes. They represent a variegated array of proteins involved in triggering and maintaining carcinogenesis. TAA
are secreted by the tumor cells themselves or by their immediate inuence on other
cells. They are detectable in the blood, other body uids or in the tumor itself.
Typically each tumor bears its own TAA ngerprint rendering to its individual malignant transformation. Some of these antigens have found clinical importance as
tumor markers in certain tumor entities where they are commonly measured as controls after therapy. Although specic markers have yet to be found for HNSCC there
are several main players of TAA to be considered.
In order to provide for expanded growth tumors have developed the ability to sprout
capillaries from existing vessels. This process is called angiogenesis and caters to
the need for nutrients and growth factors. Angiogenesis is stimulated by special
factors like VEGF (Vascular Endothelial Growth Factor) and bFGF (basic Fibroblast Growth Factor. VEGF caught special interest when its receptors VEGFR 1-3
were not only detected on the cells of the endothelial lining as expected but also on
HNSCC cells [40]. In HNSCC there is particularly dense angiogenesis compared to
normal mucosa and this has been shown to be accompanied by a strong secretion of
facilitating factors such as VEGF, PDGFα/β (Platelet Derived Growth Factor) and
GM-CSF (Granulocyte Macrophage Colony Stimulating Factor) and IL-8 [41, 42].
Oncogenes are another important type of TAA in HNSCC. Their function lies in the
regulation of cellular signal transduction pathways. Oncogenes are not carcinogenic
per se, but once mutated their coded oncoproteins malfunction, resulting in either
an overexpression of the gene products ("gain of function") or "loss of function".
Predominant and known oncogenes in HNSCC include growth factors (e.g. hast-1,
int-2, EGFR/erB), mediators of signal transduction (ras, raf, stat-3), transcription
factors (c-Myc, fos, jun), cell cycle regulators (cyclin D1) and their respective receptors. One example of such "gain of function" mechanism is the epidermal growth
factor receptor (EGFR), which is overexpressed in a high percentage of HNSCC
and has been successfully targeted in the treatment of this disease [4347]. "Loss
of function" is commonly seen in the mutation of tumor suppressor genes like p53
and p16. They are commonly referred to as "guardian of the genome", depicting
their central role in cancer development [48]. If they are subject to mutation this
natural behavior is halted and results in uncontrolled cell growth. Indeed, p53 is
the most common genetic alteration encountered in cancers. In HNSCC, p53 mutations are correlated to high tobacco consumption with subsequent high expression
of anti-apoptotic protein Bcl-2 and suppression of pro-apoptotic protein Bax [19].
6
1 Introduction
The worsened prognosis implicated by the expression of the TAAs has paved the way
for more detailed research in this eld. Nevertheless, so far no "bench to bedside"
transfer has been achieved. Thus, apart from the identication of these markers,
their global regulation of expression is a eld of expanding interest. The global transcription factor NF-κB might be an interesting transducer of such TAA regulation.
Immune Escape Mechanisms The concept that a tumor is not entirely self and
may be recognized by the immune system was conceived by Paul Ehrlich who proposed that immune reception of autologous tumor cells may be a possible mechanism
of eliminating tumors [49]. The fact that tumors can occur in immunocompetent
individuals as well has led to a more recent concept that not only encompasses the
protective role of the immune system in tumor development, but also the eect of
the immune system in selecting for tumor variants [50]. Several ways by which tumors evade immune recognition have since been proposed and identied:
1. Selective outgrowth of antigen-negative variants: During tumor progression,
strongly immunogenic subclones may be eliminated [51].
2. Loss or reduced expression of MHC molecules: Tumor cells may fail to express
normal levels of HLA (Human Leucocyte Antigen) class I molecules, thereby
escaping attack by cytotoxic T cells. Moreover these cytotoxic T cells may
undergo apoptosis by expressing Fas ligand. It has been postulated that these
tumors kill Fas-expressing T lymphocytes that come in contact with them,
thus eliminating tumor-specic T cells [52]. There is evidence that in HNSCC
up to 24 % of tumor cells lack HLA I presentation [53].
3. Lack of costimulation: Sensitization of T cells requires two signals, one by
foreign peptides presented by MHC molecules and the other by costimulatory
molecules such as cytokines; although tumor cells may express peptide antigens
with class I molecules, they often do not express these costimulatory molecules.
This not only prevents sensitization, but also may render T cells anergic or,
worse, cause them to undergo apoptosis. Patients with HNSCC exhibit a
low TH 1/TH 2 cytokine prole ratio which directly correlates with the tumor
progression [54].
4. Immunosuppression: Many oncogenic agents (e.g., chemicals and ionizing radiation) suppress host immune responses. Tumors or tumor products may also be
immunosuppressive. For example, VEGF (Vascular Endothelial Growth Factor), PGE2 (Prostaglandine E2), Interleukin-10 and TGF-β (Tumor Growth
7
1 Introduction
Factor-β ) secreted by many tumors act as potent immunosuppressants by
downregulating the antigen presentation of dendritic cells, thus interfering
with the normal T cell response [55].
5. Antigen masking: The cell-surface antigens of tumors may be hidden, or
masked, from the immune system by glycocalyx molecules, such as sialic acidcontaining mucopolysaccharides. This may be a consequence of the fact that
tumor cells often express more of these glycocalyx molecules than normal cells
do.
1.2 Toll-like Receptors (TLRs)
1.2.1 Structure and Function
The innate immune response is the rst line in the recognition of self and non-self.
The principal challenge for the host is to detect the pathogen and mount a rapid
defensive response. A group of proteins that comprise the Toll-like family of receptors perform this role in vertebrate and invertebrate organisms [56, 57]. This
reects a remarkable conservation of function which runs through all families of the
animal kingdom [58]. However, the discovery of the TLR family began with the
identication of the Toll protein as an essential factor in the dorso-ventral embryonal development of Drosophila melanogaster [59]. In subsequent studies it emerged
that TLRs play a substantial role in innate and adaptive immunity as well [60].
11 subtypes of human TLRs have been identied so far [61]. Toll-like receptors are
part of the Interleukin-1 receptor family. They share a common cytoplasmatic TIR
(Toll/IL-1R)-homologue domain with a binding site for ligands. This binding site
consists of approximately 160 amino acids and is divided into three regions of homology, Box 1-3, which are crucial for signaling. In contrast, the extracellular domain
of TLRs shares no homology with the Interleukin-1 receptors. It is characterized by
19-25 tandem copies of leucine-rich repeats (LRRs) which facilitate specic ligand
binding [62,63]. Despite this conservation among LRR domains, dierent TLRs can
identify several structurally unrelated ligands [6466]. Toll-like receptors are pattern
recognition receptors (PRRs), which are capable of recognizing archaic pathogenassociated molecular patterns (PAMPs) like LPS, dsDNA or CpG-oligonucleotides
among others. PRRs are expressed intra- and extracellularly or secreted into the
lymphatic system or the blood stream [64]. For example, TLR1, TLR2 and TLR4
are located on the cell membrane whereas TLR3, TLR7 and TLR9 seem to be
8
1 Introduction
more present on endosomal membranes. This corresponds to some extent with the
molecular patterns of their ligands. Whereas TLR1, TLR2 and TLR4 respond to
bacterial pathogenic products, the intracellularly located TLR3, TLR7 and TLR9
answer primarily to virus nucleic acids [67].
So far, four dierent subsystems of TLR signaling have been described [68]. Upon
activation, TLRs dimerize and undergo a conformational change required for the
recruitment of downstream signaling molecules [58]. These include the adaptor
molecule myeloid dierentiation primary-response protein 88 (MyD88), IL-1R-associated kinases (IRAKs), TGF-β -activated kinase (TAK1), TAK1-binding protein 1,
TAB2 and TNF-receptor-associated factor 6 (TRAF6) [69, 70]. This is the main
pathway which almost all TLRs utilize so only a handful of proteins are assembled
to various distinct signals from pathogens. It culminates in the activation of nuclear
factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK), leading to
the induction of target genes such as cytokines that are essential for the innate immune response and the maturation and proliferation of the cell [71, 72]. The second
and third systems seem to be subsystems with a small GTPase module and phosphatidylinositol phosphate (PIP) signaling module, respectively. They seem to be
distinct modules and cannot be merged into a central MyD88 module. Both modules receive inputs directly from receptors and transmit them to various molecules
downstream of MyD88 as well as outside of downstream of MyD88. For example,
the small GTPase module receives inputs from IL-1R, TLR9, TLR4, and TLR2, and
the PIP signaling module receives inputs from IL-1R, small GTPase module, TLR2,
TLR3, and MyD88, whereas components within the MyD88 module such as IRAK4,
IRAK1, IRAK2, and TRAF6 are only activated through MyD88 activation [7375].
The remaining fourth pathway appears to be exclusively stimulated by TLR3 and
TLR4. This MyD88-independent pathway is entertained through the TLR adaptor
molecule (TICAM) 1/2 [76]. It ultimately activates NF-κB in the late phase and can
be reckoned a detour for its ability to induce IL-1 and thereby the MyD88-dependent
pathway as well, leading to a whole TLR/NF-κB-system activation [77].
1.2.2 TLRs and Cancer
Constituting the previously discussed ancient, evolutionary conserved pattern recognition system, TLRs are ubiquitously expressed throughout the human body [78].
Increasing evidence of multiple functionality and diversity suggests TLRs play critical roles in noninfective medical conditions such as cardiovascular, gastrointestinal,
neurologic, musculoskeletal, obstetric, renal, liver, and dermatologic diseases, allergy, autoimmunity, and tissue regeneration [79]. Their function no longer seems
9
1 Introduction
to be limited to the regulation of innate and adaptive immunity. Tumor cells are
able to alter and augment uncontrolled proliferation, resistance to apoptosis, metastasis and to escape from the surveillance by the immune system. Recent evidence
emphasizes that TLRs are also expressed in company with HLA-DR on a variety
of tumors suggesting their active and complementary role in tumor biology [80, 81].
As we have learned activation of TLRs leads to production of proinammatory factors and immunosuppressive molecules, thus forfeiting lymphocytic attack by the
immune system. This way TLR signaling may be usurped to advance cancer progression [82]. However, TLR signaling in cancers does not appear to be a one way
street. It was shown that dsDNA, the direct ligand of TLR3 was able to initiate
apoptosis of human breast cancer lines in a TLR3-dependent manner with the help
of NF-κB downstream of TLR3 and the activation of extrinsic caspases [83, 84].
Concurrently, apoptosis was also established to be TLR3-dependent in prostate carcinoma, however, in an interferon-independent pathway involving protein kinase C
(PKC)-alpha in certain cell lines [85].
TLR analyses in HNSCC showed a strong TLR3 expression [86]. Since no expression of TLR3 in the mucosa of healthy controls has been detected, TLR3 expression in HNSCC makes it a major culprit in carcinogenesis. This correlates with a
strong NF-κB expression in tumors and the mucosa of heavy smokers. TLR3 expression corresponded with localization. TLR3 was expressed in the tumor but not
the surrounding tissue, whereas NF-κB was found in every tissue. Further studies
revealed reduced proliferation in TLR3-inhibited HNSCC cell lines. Since the expression of the oncogene c-Myc, a regulator of cell growth and dierentiation, was
also oppressed by TLR3 silencing, a c-Myc-dependent regulation in HNSCC seems
feasible [87]. TLR3 in HNSCC could stipulate a putative stimulus for further NF-κB
activation as it is thought to be maintained in general carcinogenesis [88, 89].
1.3 Transcription Factor NF-κB
1.3.1 Role and Regulation
Nuclear factor (NF)-κB, rst discovered in 1986 in the nucleus of B cells as an
immunoglobulin-kappa light chain enhancer, is today known as a transcription factor that is ubiquitously present in all cell types. It is conserved from drosophila to
man, binding to the promoter of more than 400 dierent genes [90, 91]. These genes
play important roles in the regulation of immune responses, embryo and cell lineage
development, cell apoptosis, cell-cycle progression, inammation and oncogenesis.
10
1 Introduction
Figure 1.2: Signal transduction pathways of Toll-like receptors. TLRs are located both
intra- and extracellularly. Individual TLRs have various pathways of signal transduction, either MyD88-dependent or MyD88-independent (TLR3 only and partly
TLR1/TLR2). Binding of ligand by TLR results in the activation of cascade of kinases, leading to the entry of transcription factors, such as NF-κB, AP1 and IRF to
the cell nucleus (adapted from [88])
NF-κB comprises a family of distinct subunits which share a common homology
domain of approximately 300 amino acids, the REL-homology domain. Any two
subunits may combine to build hetero- and homodimers. So far, ve subunits have
been discovered: p65 (REL A), REL-B, cytoplasmatic (c)-REL, NF-κB1 (p50) and
NF-κB2 (p52) [71]. In most cell types and at resting state, NF-κB is retained in
the cytoplasm through its tight association with inhibitory proteins called IκBs,
most notably IκBα. Key to NF-κB activation is the phosphorylation of IκBα by
the so-called IκB kinase (IKK) complex, which targets the inhibitory protein for
proteasomal degradation and allows the freed NF-κB to enter the nucleus where
it can transactivate its target genes [92, 93]. Expression of IκBα itself is regulated
11
1 Introduction
by NF-κB, which provides auto-regulation of this signaling pathway by a negative
feedback mechanism [94].
Figure 1.3: Simplied Schematic of NF-κB Activation and Eects on Target Genes
Many inducers of NF-κB have been described, among them bacteria, viruses, cytokines, external stressors, chemicals, drugs and others. In turn, NF-κB itself activates and regulates the expression of a myriad of target genes including cytokines
(e.g. interleukins), immunoreceptors (MHCs), stress response and acute phase proteins (C-reactive protein, COX-2), growth factors and regulators of apoptosis to
name a few [95]. Drawing the big picture, NF-κB has established a reputation as
central mediator and main switch in human disease processes which involve but are
not limited to the innate and adaptive immune response, apoptosis, inammation
and oncogenesis [96101].
1.3.2 NF-κB in Malignancy
NF-κB has been implicated in the development of cancers because of its roles in
cell survival, cell adhesion, inammation, dierentiation and cell growth. For example, IL-6 has been shown to be a growth factor for both multiple myeloma and
12
1 Introduction
HNSCC [102, 103]. Cell cycle regulators like cyclin D1, which is responsible for the
transition from G1 to S phase, are also under the replicative control of NF-κB [104].
In some cells, PGE2 (prostaglandin E2) deriving from COX-2 (a target gene of
NF-κB), induced tumor proliferation [105]. Genes required for negative regulation
of apoptosis like Bcl-2 are controlled by activation of NFκB [106]. Angiogenetic
factors like IL-8 and VEGF as a prerequisite for tumor invasion and metastasis
are linked to NF-κB regulation as well [107]. Carcinogenesis and growth of tumor
progression might be supported by constitutive NF-κB activation, referring to its
persistent location in the nucleus, thus withstanding its usual retention in the cytoplasm as discussed previously. While many stimulants of NF-κB activity have
been identied, the mechanisms underlying constitutive activation remain not fully
understood [108]. In HNSCC, constitutive NF-κB activation has been seen in association with autocrine expression of tumor necrosis factor (TNF), TNF receptors, and
receptor-activators of NF-κB and its ligand but not with autocrine expression of IL1β . Furthermore, treatment of HNSCC cells with anti-TNF antibody downregulated
the expression of constitutively active NF-κB and was associated with inhibition of
IL-6 expression and cell proliferation [109]. Despite the dogmatic role of NF-κB in
tumor development and growth, hints for a more suppressive nature warrant caution
with respect to blockage of NF-κB as a broad strategy in treating cancers. First
seen in epidermal cells and squamous cell carcinoma of the skin, the impression of
tumor-suppressive eects of NF-κB seemed to be organ specic [110, 111]. In the
meantime, however, several other cells and tissues like broblast, endothelial cells
and hepatocellular carcinoma have shown to exhibit a suppressive function of NF-
κB. Main interactions seem to exist in this context between NF-κB and a versatile
kinase JNK, which is able to phosphorylate a number of proteins contributing to
both apoptotic and antiapoptotic responses. A sustained activation of JNK gives
rise to a cellular growth advantage, resulting in progressive and uncontrolled proliferation, a general feature of tumor cells. NF-κB also seems to curtail the generation
of reactive oxygen species (ROS), which in turn is able to activate JNK [112]. Whatever the precise mechanisms are, normal levels of NF-κB activity are important to
impede aberrant JNK activation and ROS generation [113].
1.3.3 NF-κB Inhibitors
Cancer is a hyperproliferative disorder involving transformation, initiation, promotion, angiogenesis, invasion, and metastasis. The diversity of its features mirrors the
participation of NF-κB in almost each of those steps, making it an ideal target for
anticancer research and drug development. Over 785 inhibitors of NF-κB signaling
13
1 Introduction
have been identied so far [114]. Among the many classes of inhibitors are
Proteasom Inhibitors Proteasome inhibitors block the 26S proteasome necessary
to degrade the IκBα inhibitory subunit after its phosphorylation and ubiquitination in the cytoplasm and thus its release from the NF-κB complex [115].
IKK Inhibitors IκBα phosphorylation is a critical step in NF-κB activation, and
compounds that block this phosphorylation prevent NF-κB ubiquitination and
further degradation [116].
Acetylation Inhibitors Histone acetylation modulates gene expression, cellular differentiation and survival and is regulated by the opposing activities of histone
acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylation of
RelA is not only required for transactivation but also prevents nucleocytoplasmic tracking of NF-κB [117].
Antisense RNA and siRNA Antisense agents that inhibit the expression of a target gene in a sequence-specic manner may be used therapeutically against
NF-κB. Three anti-mRNA strategies can be distinguished. The rst involves
the use of single-stranded antisense oligonucleotides to inhibit RNA expression; the second involves the use of catalytically active oligonucleotides (ribozymes) to trigger RNA cleavage; the third involves the application of siRNA
molecules [118].
Peptides Another approach to inhibiting NF-κB activation is to use peptides that
cross the cell membrane and block the nuclear localization of the NF-κB complex [119].
Chemopreventive Agents Because of its critical role in tumor proliferation, invasion, angiogenesis, and metastasis, there has been great interest in agents
that can modulate the NF-κB signaling pathway. Several agents, which have
been described as natural chemopreventive agents, have also been found to be
potent inhibitors of constitutive NF-κB activation [120].
Anti-inammatory Agents Several anti-inammatory agents capable of suppressing NF-κB activation have been identied. Examples are aspirin, ibuprofen,
indomethacin, tamoxifen, dexamethasone, and sulindac. However, their exact
mechanism of action in this regard is not fully understood. It was demonstrated, though, that the anti-inammatory drugs sodium salicylate and aspirin inhibited the activation of NF-κB by preventing the degradation of the
NF-κB inhibitor, IκBα, so that NF-κB was retained in the cytosol [121].
14
1 Introduction
The latter two, chemopreventive and anti-inammatory agents have earned a reputation in cancer treatment and research. Though precise mechanisms of the inammatory link in cancers have yet to be elucidated, they exhibit practical implications.
For instance, expression of the enzyme cyclooxygenase-2 (COX-2), which converts
arachidonic acid into prostaglandins and is a downstream target of NF-κB, is induced
by inammatory stimuli and is increased in HNSCC and other tumors [122, 123].
The development of COX-2 inhibitors for cancer treatment is an active and promising area of research [124]. We can distinguish between selective and non-selective
COX-2 inhibitors. Acetylsalicylic acid (ASA) is a non-selective COX-2 inhibitor and
has been shown to abrogate nuclear translocation of NF-κB in gastric and colon cancer [122, 125, 126].
Figure 1.4: Structural Formula of Acetylsalicylic Acid (ASA)
A more potent suppressor of COX-2 is the selective COX-2 inhibitor Celecoxib. NF-
κB is inhibited by Celecoxib in non-small cell lung cancer through the suppression of
IκBα kinase. It also decreases the activity of the COX-2 promotor, a target gene of
NF-κB [127, 128]. In HNSCC, Celecoxib enhanced tumor cell response to radiation
in vivo and in vitro through inhibition of DNA repair mechanisms [129]. However,
there are also reports contradicting its anti-inammatory eects at high doses. In
this instance, Celecoxib was even able to activate NF-κB [130].
Dexamethasone (Fig. 1.6), an anti-inammatory steroid, is a ligand of the glucocorticoid receptor molecule. A variety of studies has shown that some activated nuclear
receptors (NRs), especially the glucorticoid receptor can inhibit the activity of NF-
κB for example [131]. This is to a large extent cell-type dependent. For example,
Dexamethasone can repress IL-6 expression and p65-dependent transactivation in
15
1 Introduction
Figure 1.5: Structural Formula of Celecoxib
murine endothelial broblasts without changing IκB protein levels or NF-κB DNAbinding activity [132]. On the contrary, Dexamethasone induces the synthesis of
IκBα mRNA in glucocorticoid receptorï¾ 12 expressing Jurkat cells and in monocytic
cells, increasing the level of IκBα and resulting in the cytoplasmic retention of NF-
κB [133, 134].
Figure 1.6: Structural Formula of Dexamethasone
Along with the quest for new and state-of-the-art treatments of cancer came the
resurrection of what we now call chemopreventive agents. They comprise a group of
ancient substances from natural sources like plants and fruits [135]. Deriving from
the rizome curcuma longa and being one of the primary ingredients in turmeric
and curry powders of the Middle-East and the Indian subcontinent, Curcumin
has quickly become the main focus of research on traditional substances. Poten-
16
1 Introduction
tial therapeutic eects have been shown against neurodegenerative, cardiovascular,
pulmonary, metabolic autoimmune and neoplastic diseases [136]. This reects the
potency of Curcumin to interact with a wide variety of proteins, including inammatory cytokines and enzymes, transcription factors, and gene products linked with
cell survival, proliferation and angiogenesis [137]. It has proven to down-regulate expression of cell proliferation and antiapoptotic and metastatic gene products through
suppression of IκBα kinase and thus NF-κB [138]. In HNSCC, Curcumin inhibits
growth and survival via NF-κB in a dose-dependent fashion and by downregulation
of target genes like cyclin D1, IL-6 and COX-2 [139141].
Figure 1.7: Structural Formula of Curcumin
A yet to be evaluated agent concerning its chemopreventive potential is EPs 7630,
an extract from Pelargonium sidoides, a South African cranesbill species. Commercially available as an over-the-counter product, it is approved for use in acute
bronchitis. Despite constant contradiction, meta analyses have shown encouraging evidence for its ecacy compared to placebo for patients with acute bronchitis [142]. Six main groups of constituents, namely unsubstituted and substituted oligomeric prodelphinidins, monomeric and oligomeric carbohydrates, minerals, peptides, purine derivatives and highly substituted benzopyranones seem to
constitute its pharmacologic eect [143]. Although exerting an antimicrobial and
anti-inammatory eect in acute bronchitis, no evaluation about its potency of NF-
κB inhibition or anti-proliferative action has been investigated so far.
17
1 Introduction
Figure 1.8: Structural Formulas of Constituents of EPs 7630
18
1 Introduction
1.4 Aim of Studies
In 1863 Virchow proposed that cancer develops at sites of chronic inammation.
Potential relationships between cancer and inammation have been studied since
then. The precise mechanisms that link inammation and cancer development have
not yet been established.
Inammation is the body's normal reaction to internal and external harmful inuences. Innate and adaptive immunity are tightly linked in the regulation and
interplay of the mediators of inammation. NF-κB is a key player in inammation
and cancer for its wide variety of target genes involved in inammation and carcinogenesis. It may be activated through TLRs and become a self-sustained system
through recurrent activation of its own target genes. We have seen that NF-κB activation is a common theme in carcinogenesis but hints for an ambivalent role exist,
leaving the true nature of NF-κB obscured.
HNSCC is clearly linked to the use of tobacco and alcohol and their inammatory
implications. We have seen, that HNSCC express TLR3 and a high activity of NF-
κB. The cytokine prole of the HNSCC milieu is dominated by tumor promoting
cytokines.
In this study we characterized the eects of ASA, Curcumin, Celecoxib, Dexamethasone and EPs 7630 on HNSCC cell lines. In a separate experiment we investigated
the inuence of Mycoplasma as an external pathogen on various cancer cell lines.
Thus, we investigated whether the mentioned substances inhibit HNSCC proliferation in a cell culture model and if they aect NF-κB activity. With respect to the
ndings of TLR3 expression in HNSCC we wanted to know if there was an inuence
of the stimulants upon cell markers and TLR3 expression. With regard to selfsustainability and feedback regulation of NF-κB and its target genes, we analyzed
the micromilieu of the tumor with regard to cytokine expression. Additionally we
wanted to clarify the impact of Mycoplasma on the expression of TLRs and downstream proteins of dierent cancer cell lines with the aim of drawing a better picture
of the inuence of inammation in HNSCC.
19
2 Materials and Methods
All chemicals and ingredients for solutions, buers and medium were obtained from
Sigma-Aldrich,
Otto Fischar (Saarbrücken, GER), Fluka
(Buchs, CH), and Carl Roth GmBH (Karlsruhe, GER) unless stated otherwise.
(Seelze, GER),
2.1 Cell Lines
For the stimulation protocols with ASS, Curcumin, Celecoxib, Dexamethasone and
EPs 7630 two established HNSCC cell lines were used. BHY (DSMZ # ACC 404)
was derived from a 52-year old Japanese man with highly dierentiated squamous
cell carcinoma of the lower alveolus which was highly invasive to the mandibular bone
and the muscle layer of the oral oor. It is described as anchorage-independent and
to be tumorigenic in nude mice [144]. The other, PCI-1, is an adherent human HNSCC from the hypopharynx and was obtained from the Pittsburgh Cancer Institute
(USA).
The inuence of Mycoplasma was studied on the following cell lines: ANT-1 and
GHD are HNSCC cell lines and were established by the research laboratory of the
Department of Otolaryngology (Ludwig-Maximilians-Universität, Munich, GER).
HaCat is a cell line of in vitro spontaneously transformed keratinocytes from histologically normal skin as rst established by Boukamp et al. [145]. Like PCI-1,
PCI-13 is a human HNSCC cell line from the hypopharynx as established by the
Pittsburgh Cancer Institute (USA). The long standing upper aerodigestive tract
cancer lines Hlac78 and Hlac79 complete the array of cell types studied.
2.2 Cell Culture
All culture work was carried out on an airow workbench under sterile conditions.
The bench was prepared with UV light 20 minutes prior to cultivation and thor-
Minerva Bi-
R(
oughly cleaned with appropriate disinfectant, e.g. Mycoplasma-O
olabs, Berlin, GER). All autoclavable consumption goods like pipette tips were
sterilized at 121◦ C for 15 minutes. Disinfection with 70 % ethanol was applied to
20
2 Materials and Methods
all other items.
Cell lines were regularly checked for contamination with Mycoplasma using the Mycoplasma Detection Kit (
Minerva Biolabs).
This was also used to conrm con-
tamination in the cell lines used for the Mycoplasma studies in this work.
2.2.1 Cell Growth and Subcultivation
Sarstedt, Nüm-
The adherent HNSCC-cell lines were incubated in culture asks (
brecht, GER) containing DMEM (Dulbecco's Modication of Eagle Medium +
Gibco, New York, USA)) at 37 ◦C and 90 % humidity with
a CO2 of 5 %. DMEM was always laced with 10 % fetal calf serum (FCS; Paa,
Pasching, AUS), 1 mM sodium pyruvate (Paa) and non-essential amino acids (Gibco). Medium was exchanged regularly according to individual cell line growth and
4,5 mg/ml glucose (
conuency.
For subcultivation, adherent cells were washed twice with ice-cold 1 x Phosphate
Buered Saline (PBS; (140 mM NaCl, 10 mM Na-Phosphat)) and dispensed in
trypsin-ethylen-diamin-tetraacetic acid (trypsin-EDTA;
Paa) for up to 10 minutes.
Trypsin-EDTA breaks down proteins in the membrane of the cells and thus disintegrates them from the ask surface. It also has the capability to destroy other
cell proteins so the progression of this process was continuously monitored by light
microscopy. To stop proteolysis cells were then collected in DMEM and centrifuged
at 200 g for 8 minutes at 30 ◦ C. The pellet was resuspended in fresh DMEM and
diluted accordingly.
2.2.2 Cell Concentrations
Brandt,
Cell counting was determined using the Neubauer-counting chamber (
Wertheim, GER). Trypan Blue 0.1 % (
Merck, Darmstadt, GER) was added in
an equal ratio to the cell suspension. The polyanionic Trypan Blue crosses the
membrane of dead cells and dyes them. Living cells remain undyed under the microscope. Cells in the four corner squares of the chamber were counted and averaged.
Cell concentrations were determined according to the formula:
average cell count x 2 (dilution factor) x 105 = cell concentration/ml
2.2.3 Long-term Storage and Thawing
For long-term storage, cells were washed and trypsinated as above (section 2.2.1)
and brought into a special culture medium containing 70 % DMEM, 20 % FCS and
21
2 Materials and Methods
10 % dimethylsulfoxide (DMSO;
(
PAA). They were then transferred to cryotubes
Sarstedt) and subsequently put into a slow-freezing container at -80 ◦C.
For revitalization the cryotubes were quickly thawed in a heated water bath at 37 ◦ C
and suspended in the culture medium in order to wash out the DMSO. This step
was repeated and after discarding the supernatant the pellet was nally transferred
to culture asks. Growth examination and change of medium was carried out after
24 hours.
2.2.4 Cell Stimulation
In order to study cell line growth under various NF-κB inhibiting conditions, the
cell lines BHY and PCI-1 were grown and passaged as above. Approximately 1.5 x
10
6
cells in 3 ml DMEM were incubated for 2 hours before stimulation. Adherence
assured 2 ml of the relative stimulant was added in 2.5 x concentration to yield the
desired cumulative molarity (e.g. 2 ml of 2.5 mM ASS would have been added to
yield 5 ml of 1 mM cell/stimulant suspension).
Acetylsalicylic acid (ASA) and Dexamethasone were obtained from
Sigma-Al-
drich, Curcumin from Fluka, Celecoxib from LC Laboratories (Woburn, USA)
and EPs 7630 was a generous donation from Dr. Willmar Schwabe GmbH &
Co. KG, Karlsruhe, GER.
2.2.5 Cell Harvesting
Stimulated cells were harvested at 0, 24, 48 and 72 hours with an average conuency of 80-100 %. Supernatants were preserved in 15 ml tubes and centrifuged at
300 g at 4 ◦ C to exclude any cell remnants. They were equally aliquoted into 1.5 ml
cryotubes and stored at -80 ◦ C.
Cells were washed twice with ice-cold 1 x PBS. 200 µl RIPA-buer (1 x PBS, 1 %
Igepal CA-630, 0,5 % sodium-deoxycholate, 0,1 % sodium dodecylsulfate (SDS))
with protease and phosphatase inhibitors (30 % aprotinin, 10 % phenyl-methylsulfonyl-uoride (PMSF),10 % sodium-orthovanodate, 20 % sodium-uoride, 10 %
phosphatase inhibitor cocktail) were added and the culture asks gently canted to
coat all cells. Cells were detached with scrapers (
Eppendorf).
1.5 ml tubes (
Sarstedt) and transferred to
To alleviate membrane destruction, cells were addi-
tionally toggled through a 21 gauge syringe and subsequently stored at -80 ◦ C until
further workup.
22
2 Materials and Methods
2.3 Western Blotting
2.3.1 Protein Isolation
Deep frozen cells were slowly thawed on ice and incubated for 30 minutes. Tubes
were then centrifuged at 4 ◦ C and 10000 g for 10 minutes. The supernatant was
then transferred to new tubes. After equivalent addition of 2 x SDS loading dye
(0.25 M Tris-HCl, pH 6.8; 15 % ÿ-Mercaptoethanol, 30 % Glycerine, 7 % SDS, 0.3 %
Bromophenol blue) the proteins were denaturated for 5 minutes at 100 ◦ C and then
stored at -20 ◦ C. SDS is an anionic detergent which denatures secondary and nondisulde-linked tertiary structures and applies a negative charge to each protein in
proportion to its mass. It binds to the protein and unfolds it, rendering a uniform
negative charge along the length of the polypeptide. ÿ-Mercaptoethanol supports
this denaturation process by reduction of disulde-bonds.
2.3.2 Evaluation of Protein Concentration by Photometry
Protein concentrations were evaluated using the Bradford Protein Assay (
Biorad)
[146]. The ionic dye Coomassie Brilliant Blue G-250 binds to alkaline amino acids in
proteins and hereby changes the maximum absorption of the dye from 465 nm with-
out protein to 595 nm with protein. This change is recognized by the photometer
and correlates to the protein concentration in solution. A standard curve was generated with Bovine Serum Albumine (BSA;
Sigma) solutions of 10 µl/ml, 25 µl/ml,
50 µl/ml, 75 µl/ml, 100 µl/ml and 150 µl/ml. Bradford reagent was added in a
ratio of 1:5 to every dilution, mixed, incubated at room temperature for 5 minutes
Eppendorf).
and gauged at 595 nm in the photometer (
Before the addition of the loading dye (section 2.3.1), 4 µl of each cell suspension
was added to 80 µl of Bradford reagent and 316 µl water. The mix was also incubated for 5 minutes at room temperature and the samples were then read against
the established standard curve. In case of concentrations beyond the curve further
dilution was carried out and taken into account when calculating the nal protein
concentration.
2.3.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
SDS-PAGE is a widely used technique to separate proteins according to their electrophoretic mobility which is determined by the length of the polypeptide chain or
molecular weight as well as higher order protein folding, posttranslational modications and other factors, leading to fractionation by size [147].
23
2 Materials and Methods
Running Gel
Stacking Gel
% of acrylamide
7.5 %
10 %
12.5 %
4%
1 M Tris-HCL pH 8.8
2 ml
2 ml
2 ml
1 M Tris-HCl pH 6.8
0.3 ml
1 % SDS
0.5 ml
0.5 ml
0.5 ml
0.25 ml
Acrylamide
1.25 ml
1.65 ml
2.1 ml
0.35 ml
dist. H2 O
1.25 ml
0.85 ml
0.4 ml
1.6 ml
APS
50 µl
50 µl
50 µl
25µl
TEMED
5 µl
5 µl
5 µl
2.5 µl
Table 2.1: Composition of SDS-PAGE Gels
Gels were prepared according to Table 2.1 and cast in the
Biorad Mini-PROTEAN
III electrophoresis cell. First, the running gel (usually a 10 % PAGE) was lled either between a 0.75 mm or 1.5 mm plate with spacer and prevented from drying out
by a small layer of distilled water. This also helped to smooth and compress the gel.
After polymerization the water was discarded and the stacking gel was applied. An
appropriate gel comb was placed to form the stacking wells. 10-60 µg of protein and
a molecular weight marker (
Biorad) were subsequently loaded onto the gel sub-
merged in 1 x electrophoresis buer. An electric current of 220 V and 120 mA was
applied, causing the negatively charged proteins to migrate across the gel towards
the anode. Depending on their size, each protein moves dierently through the matrix: short proteins will easily t through the pores with the larger ones having more
diculty due to higher resistance.
2.3.4 Semi-Dry Blotting
Following the SDS-PAGE proteins were blotted to membranes as rst described by
Towbin et al. [148]. SDS-PAGE gels were removed from the gel cassettes and the
stacking gel was gently removed. In order to make the proteins accessible to antibody
detection they were moved from gel to nitrocellulose membranes (
Biorad).
was facilitated using the Semi-Dry Blotter (
Biorad).
This
The gel was placed on the
membrane and any air bubbles between the membrane and the gel were removed.
The two were then placed between two stacks of blot papers that had been presoaked in either anode buer (Roti-Blot A) or cathode buer (Roti-Blot K). With
24
2 Materials and Methods
Antibody
Anti NFκB p65
Anti TLR3
Anti TLR4
Anti Iκ-Bα
Anti IKKβ
Anti c-myc
β -Actin loading control
Isotype
monoclonal
(rabbit)
monoclonal
(mouse)
monoclonal
(mouse)
monoclonal
(mouse)
monoclonal
(mouse)
monoclonal
(mouse)
monoclonal
(mouse)
Concentration Reference
10µl/10 ml
Biomol
(Hamburg, GER)
20 µl/10 ml
Imgenex
(San Diego, USA)
20 µl/10 ml
Imgenex
(San Diego, USA)
20 µl/10 ml
Biosource
(Camarillo, USA)
10 µl/20 ml
BD Biosciences
(San Jose, USA)
14.3 µl/20 ml
Biomol
(Hamburg, GER)
10 µl/10 ml
Abcam
(Cambridge, UK)
Table 2.2: Primary Western Blot Antibodies in 1 x PBS
the cathode buer on top the sandwich was put between the two electrodes and was
blotted for 90 minutes at 120 mA. It is a necessity that the membrane is placed
between the gel and the cathode as the current and sample are running in that
direction because the protein will have a negative charge due to its treatment with
SDS (section 2.3.1).
2.3.5 Blocking and Antibody Detection
After blotting the membranes were cautiously cleared of any adherent gel remnants
with pure water. The uniformity and overall eectiveness of protein transfer from gel
to membrane were checked by staining with Ponceaus S (2 % in 3 % Trichloroacetic
acid) for 1 minute. Trichloroacetic acid supports binding of the proteins on the
membrane. Afterwards the dye was removed and the membrane was washed with
pure water. Since the membrane has the ability to bind proteins, with antibodies and
target samples both being proteins, the membrane was blocked with fresh blocking
buer (1 x PBS with 3 % skim milk powder) for 1 hour. The blocking buer was
removed and the membrane then washed with 1 x PBS for 10 minutes. Subsequently
neoLab) with
the membrane was incubated at 4 ◦ C overnight on a tilting table (
the desired primary antibody (Table 2.2).
Unbound antibodies were removed by washing the membrane three times with 1 x
25
2 Materials and Methods
Antibody
Goat-anti-mouse
detection kit
Goat-anti-rabbit
detection kit
Concentration Reference
BioRad
3.5 µl/10 ml
(München,GER)
BioRad
3.5 µl/10 ml
(München, GER)
Table 2.3: Secondary Western Blot Antibodies in 1 % Gelatine Buer
PBS for 5 minutes. The membrane was then incubated for another 2 hours with
the corresponding secondary antibody (Table 2.3). The secondary antibody is conjugated with Alkaline Phosphatase (AP) and diluted in 1 % Gelatine-PBS buer.
After repeated washing steps with 1 x PBS, binding of the secondary antibodies was
BioRad). The substrates detect
visualized with the AP Conjugate Substrate Kit (
the secondary antibodies and are converted to visible complexes (Figure 2.1). As a
protein loading control, the expression of ÿ-Actin as housekeeping gene was always
checked.
BioRad
Figure 2.1: Principle of AP-Detection (from
AP-Kit manual); 1. Nonfat dry
milk blocks unoccupied sites on the membrane, 2. Primary antibody to a specic
antigen is incubated with the membrane, 3. Biotinylated antibody is added to bind
to the primary antibody, 4. Streptavidin-biotinylated AP complex is incubated with
the membrane, 5. Color development reagent is then added to the blot; amplied
signal at the site of the complex is observed by the formation of a colored precipitate
2.4 MTT-Cytotoxicity Assay
The MTT-test is a colorimetric assay to determine cell proliferation and viability.
In this work it was used to show the inuence of the dierent inhibitors on the cell
26
2 Materials and Methods
growth. MTT stands for 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide.
MTT is a yellow tetrazolium salt which is cleaved to purple formazan by the
succinate-tetrazolium reductase system which belongs to the respiratory chain of the
mitochondria, and is only active in metabolically intact cells (Figure 2.2). Thus the
colorimetric change is directly proportional to the amount of viable cells [149, 150].
Figure 2.2: Example of tetrazolium salt cleavage in presence of electron-coupling
reagent (from
)
Roche Applied Sciences
Approximately 5000 cells in 60 µl DMEM per well were sown in a 96-well plate
and incubated for 3 hours. After adherence 40 µl of the respective stimulant (ASS,
Curcumin, Celecoxib, Dexamethasone, EPs 7630) was added in 2.5 x concentration
to reach the desired overall stimulant concentration (e.g. 40 µl of 2.5 mM ASS would
have been added to the 60 µl cell suspension to yield a 1 mM ASS stimulation
per well). Cells were then incubated for 0-72 hours. Measurements were taken
every 24 hours. The enzymatic reaction was triggered by adding 10 µl of MTTsolution to each well. After one hour the reaction was stopped by 110 µl (1:1 ratio)
MTT-solubilization solution. Plates were then incubated in the dark overnight.
Readings were taken with an absorption of 560 nm against the reference wavelength
Dynatech Laboratories). All data represent
of 650 nm on a MRX plate reader (
cumulative results of several readings (n= 2-4).
2.5 Flow Cytometry
Flow cytometry is a technique of counting, examining and sorting microscopic particles suspended in a stream of uid. It allows for simultaneous multiparametric
27
2 Materials and Methods
analysis of physical and chemical characteristics of single cells owing through a
detection apparatus.
Flow cytometry, more exactly uorescent-activated cell sorting (FACS
TM
), was used
TM
to analyze surface and intracellular proteins of stimulated HNSCC-cells. FACS
registered trademark of
is a
Becton Dickinson (Heidelberg, GER) and depicts a spe-
cialized type of ow cytometry. Analyses were performed using the FACSCanto
TM
and the FACS Diva
Software Version 4.1.2 (
TM
BD Biosciences).
2.5.1 Preparation and Procedure
BHY and PCI-1 HNSCC-cell lines were grown in 12 well plates and stimulated as
shown in section 2.2.4. Supernatant was discarded and cells were trypsinated with
500 µl trypsin-EDTA. 1 ml DMEM was added to stop trypsination and 750 µl containing approximately 3 x 106 cells were transferred to tubes and centrifuged with
2000 g at 4 ◦ C for 5 minutes. Supernatant was discarded and the pellet resuspended
in either 50 µl PBS or saponin. The bioactivity of saponin constitutes their complexation with cholesterol to form pores in cell membrane bilayers. In addition their
amphipathic nature gives them activity as surfactants that can be used to enhance
penetration of proteins, e.g. antibodies, through cell membranes. This approach
allows for estimation of surface and intracellular protein expression. After careful
resuspension 1 µl of each antibody (Table 2.4) was added and the cells were incubated on ice and in the dark. After 20 minutes 450 µl of PBS was added to
each tube, again centrifuged with 2000 g at 4 ◦ C for 5 minutes, the supernatant
discarded and the pellet again resuspended with 500 µl PBS. Cells were then fed
into the FACS
TM
system and unlabeled native cells set as control. For every sample
5000-10000 events were recorded and analyzed.
2.6 Cytokine Analysis
TM
2.6.1 Principle of the Bio-Plex
For analysis of cytokines, Bio-Plex
TM
Cytokine Assay
cytokine assays were implemented. These
assays are multiplexable bead assays, i.e. assays that simultaneously quantitate
human cytokines in cell culture supernatants. The principle of the assay is similar
to a capture sandwich immunoassay an antibody to the target protein is covalently
coupled to internally dyed beads. When the beads are incubated with sample, the
protein of interest is captured and then a biotinylated antibody for a dierent epitope
is added to the reaction, which is then detected with streptavidin-phycoerythrin (Fig.
28
2 Materials and Methods
Conjugated Antibody Concentration Reference
PE Anti-human
TLR3
FITC Anti-human
Ki-67
PeCy7 Anti-human
CCR7
PerCP Anti-human
HLA-DR
APC Anti-human
HLA-ABC
1 µl
BD Biosciences
(San Diego, USA)
1 µl
BD Biosciences
(San Diego, USA)
1 µl
BD Biosciences
(San Diego, USA)
1 µl
BD Biosciences
(San Diego, USA)
1 µl
BD Biosciences
(San Diego, USA)
Table 2.4: FACS
TM
Antibodies
2.3). Using a dual-laser ow-based reader, beads are analyzed for the detection
antibody and the internal bead signature, identifying not only the protein analyzed
but also the level of binding to the bead. Cytokine concentrations are automatically
TM
calculated by Bio-Plex Manager
software using a standard curve derived from a
recombinant cytokine standard.
BioPlexTM
Figure 2.3: Antibody binding and cytokine detection (
array manual).
Beads bind specically to cytokines from the supernatant. Secondary antibodies with
PE-conjugation are detected by the optical unit.
2.6.2 Preparation and Procedure
Supernatants from the NF-κB studies were collected as described in section 2.2.5.
Samples were tested simultaneously for cytokines IL-2, IL-4, IL-6, IL-8, IL-10, GMCSF, IFN-γ and TNF-α with the Bio-Plex
(
Biorad).
TM
Human Cytokine 8-Plex A Panel
The assay was run according to the recommended procedure. In brief,
the premixed standards were reconstituted in 0.5 ml of culture medium, generating
29
2 Materials and Methods
a stock concentration of 50,000 pg/ml for each cytokine. The standard stock was
serially diluted in the same culture medium to generate 8 points for the standard
curve. The assay was performed in a 96-well ltration plate supplied with the assay
kit. Premixed beads (50 µl) coated with target capture antibodies were transferred
to each well of the lter plate (5,000 beads per well per cytokine) and washed twice
TM
with Bio-Plex
wash buer. Premixed standards or samples (50 µl) were added
to each well containing the washed beads. The samples were used directly without
further dilution. The plate was shaken for 30 seconds and then incubated at room
temperature for 30 minutes with low-speed shaking (300 rpm). After incubation and
washing, premixed detection antibodies (50 µl, nal concentration of 2 µg/ml) were
added to each well. The incubation was terminated after shaking for 10 minutes at
room temperature. After 3 washes, the beads were resuspended in 125 µl of BioPlex
TM
TM
assay buer. Beads were read on the Bio-Plex
suspension array system,
and the data were analyzed using Bio-Plex ManagerTM software.
2.7 ELISA
ELISA is an acronym for Enzyme-linked Immunosorbent Assay which already outlines the principle of an antigen-antibody reaction with an enzyme as marker [151,
152]. The antibody is covalently bound to a microtiter plate and links specically
with the epitope of the antigen in the sample. Unspecic bindings are prevented
by several washing steps. Another antibody with an enzyme-linked marker binds to
another distinct epitope of the antigen and reacts with an added substrate resulting
in a color change that can be measured with a photometer.
2.7.1 Preparation of Nuclear Extracts
The rst step in studying transcription factor activity is the preparation of nuclear
extracts. This resembles in part the preparation of proteins for the western blots
discussed in section 2.3.1 with some extra twists. A nuclear extraction kit (
Cayman
Chemicals; Ann Arbor, USA) was used according to the manufacture's protocol.
All the steps were carried out on ice. In short cells were grown and stimulated as
outlined in section 2.2.4. After harvest cells were centrifuged at 300 g and 4 ◦ C and
washed with ice-cold PBS/Phosphatase inhibitor solution twice. Supernatant was
discarded and the pellet was resuspended in ice-cold Hypotonic Buer and transferred to microcentrifuge tubes. Cells were incubated on ice for 15 minutes allowing
them to swell. Nonidet P-40 a nonionic surfactant was added for solubilization
and isolation of membrane complexes. After a 14000 g, 30 second centrifuge run,
30
2 Materials and Methods
the supernatant containing the cytosolic fraction was transferred to new tubes and
stored away. The pellet was resuspended in 50 µl extraction buer with protease
and phosphatase inhibitors, shortly vortexed and put on a shaking platform for 15
minutes. Again the sample was centrifuged at 14000 g for 10 minutes at 4 ◦ C. The
remaining supernatant contains the nuclear fraction and was ash frozen at -80 ◦ C.
2.7.2 NF-κB Transcription Factor Assay
Activity of the p65 subunit of NF-κB was elucidated with the help of the NF-
Rockland
κB Transcription Factor Assay Kit (
, Gilbertsville, USA). A specic
double stranded DNA (dsDNA) sequence containing the NF-κB response element
is immobilized onto the bottom of wells of a 96 well plate. NF-κB contained in a
nuclear extract specically binds to the NF-κB response element. NF-κB (p65) is
detected by addition of a specic primary antibody directed against NF-κB (p65). A
secondary antibody conjugated to HRP (Horseradish-Peroxidase) is added to provide
a sensitive colorimetric readout at 450 nm. Buers and solutions were prepared
as recommended. Concisely, the appropriate amount of Complete Transcription
Factor Binding Buer (CTFB) was applied to designated wells of the ready-touse 96-well plate. Positive controls, competitive dsDNA and samples were added
accordingly. The plate was then incubated overnight at 4 ◦ C without agitation. For
the application of the primary and secondary antibodies the wells were thoroughly
washed, the antibody added and an incubation period of 1 hour followed. Developing
solution was added to each well and the change of color was closely monitored,
eventually stopped and absorbance read at 450 nm within 5 minutes of addition of
the stop solution.
2.8 Immunohistochemistry
2.8.1 Cytospin
Cytospin is a technique to immobilize cell suspensions on slides. Cytospin material
was provided by
Shandon Inc., Pittsburgh, USA. Approximately 6 x 104 cells in
100 µl DMEM +/- stimulant were loaded to each cuvette and spun at 800 rpm for 4
minutes. Excess medium was soaked by lter paper. Cuvette and lter paper were
then carefully detached from the fresh cytospin and the area around the cells was
marked with permanent marker. Cells were then either immediately xed or dried
and stored at room temperature for a maximum of 2 days.
31
2 Materials and Methods
Figure 2.4: Schematic of the Transcription Factor Assay (
Rockland Kit Manual)
2.8.2 Immunoxation
Cytospins were xed with 4 % Paraformaldehyde in PBS for 10 minutes. After a
washing step with PBS membrane proteins were detached from the cell membrane
with the help of 0.4 % Triton X-100 and 1 % BSA in PBS. Unspecic binding was
prevented by blocking with 2 % BSA in PBS for 10 minutes. Slides were incubated
overnight with 4 µl/ml of anti-TLR3 antibody (
Imgenex,
San Diego, USA) in
1 % BSA in a humid dark chamber. Slides were then washed three times with
DAKO A/S, DK) was diluted
PBS and secondary antibody anti-mouse uorescein (
1:1000 and incubated for 1 hour in the dark. Slides were again washed and sealed
Vector Labs, Burlington, CAN) and nail polish. Slides were
R (
with Vectashield
analyzed and photographed with the uorescence microscope Axiovert 200-M and
the attached digital camera AxioCam MRm Firewire ( both
Carl Zeiss, Jena,
GER) and edited using the Axio Vision Imaging Solutions Software.
32
3 Results
In this chapter the inuence of the aforementioned stimulants on HNSCC cell lines
is analyzed. Special attention was paid to NF-κB behavior under these conditions.
We were able to further characterize HNSCC cell lines with respect to downstream
targets of NF-κB signaling. In addition we compared the response of various cell
lines in their protein expression proles to Mycoplasma as a chronic irritant.
3.1 Dose Dependent Growth Inhibition of HNSCC
Cell Lines
To assess to what extent the growth behavior of HNSCC cells was changed, we conducted proliferation assays of the HNSCC cell lines BHY and PCI-1. All agents were
able to alter proliferation of these cell lines. The cell lines responded uniquely to
the stimulants unless stated otherwise, though PCI-1 showed a higher proliferation
rate throughout without changing the extent of alteration. Controls were incubated
with DMEM.
Exposition to ASA suppressed proliferation in a dose-dependent manner. After 24 h
concentrations of 0.1 mM and 1 mM ASA oated around the measurements of the
controls in BHY. 10 mM ASA caused a denite suppression of growth after 24 h
with further deterioration at 48 h and even reaching baseline at 72 h. Interestingly a slight increase in molarity of 0.1 mM compared to controls showed a slightly
increased proliferation over the whole 72 h time period. This was not the case
with PCI-1, where alterations were not as signicant as in BHY. Here, no increased
growth in the 0.1 mM group was noted. Cells treated with 1 mM and 10 mM acted
similarly to BHY with 10 mM representing a clear lethal dose (see Fig. 3.1).
Both cell lines, BHY and PCI-1, displayed the same growth behavior upon incubation with Curcumin. There was a clear correlation of growth inhibition with
increasing doses of Curcumin. Concentrations of 10 µM, 15 µM and 25 µM suppressed BHY by approximately 50 %, 60 % and 70 % (OD 0.17, 0.14 and 0.0945
33
3 Results
Figure 3.1: Proliferation Assay ASA. Incubation of cell lines BHY and PCI-1 with ASA
in concentrations of 0.1 mM and 1 mM showed dose-dependent decrease of growth,
eventually leading to total abrogation of cell viability with 10 mM concentration in
BHY. Interestingly incubation with 0.1 mM ASA led to slightly increased proliferation
in BHY but not PCI-1.
compared to 0.33 of control) respectively (see Fig. 3.2). PCI-1 was inhibited by up
to 80 % by the highest dosage of 25 µM Curcumin (OD 0.035 compared to 0.2065).
Incubation with 10 µM and 15 µM Curcumin showed growth inhibtion of 30 %
(∆OD 0.1475/0.2065) and 60 % (∆OD 0.087/0.2065).
Figure 3.2: Proliferation Assay Curcumin. Incubation with Curcumin decreased cell growth
by up to 70 % in BHY. Incubation with PCI-1 returned less suppression with 10 µM
but followed the same dose-dependent pattern.
In series undertaken with Celecoxib, a concentration of 75 µM was able to exert the
strongest inhibitory eect on both cell lines. Here we observed a change in optical
density of 0.770 for BHY and 0.420 for PCI-1, resulting in a decrease of circa 60 %
and 70 % after 72 h, respectively. Lower concentrations of 25 µM and 50 µM yielded
decreases of 60 % and 50 % for BHY and 60 % and 30 % for PCI-1.
Presenting an incoherent proliferation pattern during preliminary series we conducted several assays using Dexamethasone dosages ranging from 10 nM to 25 µM.
Dexamethasone dosages from 10 nM to 1 µM had a distinctly dierent impact
34
3 Results
Figure 3.3: Proliferation Assay Celecoxib Dose-dependent growth inhibition of BHY and
PCI-1 under the inuence of COX2 inibitor Celecoxib.
on the cell lines than the higher concentrated dosages of 10 µM and 25 µM. Concentrations of the former group undulated around the controls for both cell lines.
Only 10 µM and 25 µM concentrations signicantly altered HNSCC proliferation.
Dierences among the rst group itself were not as clear, either, without any dosedependent pattern. For example, cells incubated with 1 µM Dexamethasone reduced
cell growth in PCI-1 less than 500 nM or 10 nM. Moreover, in PCI-1, the dose response was even ipped around in the order of 1 µM > 500 nM > 1 nM. This was
not the case with BHY. The latter group with concentrations of 10 µM and 25 µM
has to be set apart from this for displaying a regular dose-dependent pattern of inhibition. 10 µM of Dexamethasone reduced growth by approximately 60 % in both,
BHY and PCI-1, and 25 µM set it back by 85 % for both cell lines with the curve
indicating a lethal dose.
Figure 3.4: Proliferation Assay Dexamethasone. No dierences in growth inhibition between 10 nM to 1 µM and upside-down behavior comparing BHY and PCI-1.
Since there are several constituents of EPs 7630, a clear molarity cannot be established. For the purpose of the studies, concentrations were given as x µg/ml.
Without appropriate knowledge about its inuence on cell culture experiments, incubation with increasing dosages of EPs 7630 produced a clear dose-dependent growth
35
3 Results
inhibtion of both cell lines. A concentration of 75 µg/ml represented a negatively
sloped curve after 72 h of incubation. Concentrations of 25 µg/ml and 1 µg/ml
followed suit in a dose-dependent manner, with 25 µg/ml exerting an inhibition of
40 % in BHY and 58 % in PCI-1.
Figure 3.5: Proliferation Assay EPs 7630. Growth curves show clear dose-dependent abrogation of proliferation.
With the exception of ASA and Dexamethasone, all stimulants were able to inhibit
HNSCC cell lines in a dose-dependent manner. To study and characterize the two
HNSCC cell lines further, we focused on the most balanced concentrations for each
stimulant with regard to lethality and normal proliferation. We chose concentrations for both cell lines as follows: ASA 2 mM, Curcumin 15 µM, Celecoxib 25 µM,
Dexamethasone 10 µM and EPs 7630 25 µg/ml (see Fig. 3.6 on the following page
and Fig. 3.7 on page 38). Comparing those specic stimulant concentrations without
regard to their own individual dose-dependent action among another, ASA turned
out to be the least potent suppressor of cell growth in both cell lines. Looking at
BHY, ASA limited proliferation to 65 % after 72 h, whereas EPs 7630 and Celecoxib
limited growth of BHY to 35 % of the control. Curcumin and Dexamethasone were
most potent by cutting cell thriving to 26 % with respect to controls. In PCI-1 the
order of suppression was the same with a atter distribution of inhibition among the
stimulants. ASA, again, was least successful in growth inhibition, with a reduction
∆OD of 0.2 with respect to controls. EPs 7630, Celecoxib and Curcumin represent
mid-elders in the suppression of PCI-1 with OD reductions of about 50 %. Most
potent suppressor of PCI-1 is Dexamethsone, cutting growth to 16 % of the controls.
In conclusion, both HNSCC cell lines were indeed growth inhibited by the chosen
stimulants. Each stimulant itself was able to exercise a dose-dependent inuence
on each cell line. In comparison, the extent of individual growth inhibition was signicantly dierent. ASA and Dexamethasone acted dierently from the remaining
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stimulants in the way that ASA showed a slight increase in proliferation at very low
doses of 0.1 mM. Dexamethasone showed almost no dierences in the concentration
range of 10 nM to 1 µM and even bared an upside-down behavior in the aforementioned range of concentrations in PCI-1, with 1 µM revealing the least potent eect.
BHY
0,6
Control
0,5
0,4
OD560
ASA 2mM
0,3
EPs 7630 25 µg/ml
0,2
Celecoxib 25 µM
Curcumin 15 µM
Dexamethasone 10 µM
0,1
0
0h
Curcumin 15 µM
24 h
Celecoxib 25 µM
48 h
EPs 7630 25 µg/ml
Dexamethasone 10 µM
72 h
ASA 2mM
Control
Figure 3.6: Survey of BHY Growth Curves. Inuence of stimulants with chosen concentration on BHY.
3.2 FACSTM Analysis
To elucidate the inuence of dierent agents on the expression of distinct molecules,
FACS
TM
investigations using antibodies against HLA-A,B,C, TLR3, CCR7 and KI-
67 were carried out. KI-67 is a classic proliferation marker and is present during all
active phases of the cell cycle (G1, S, G2, and mitosis), but absent from resting cells
(G0). HLA-A,B,C depicts human major histocompatibility complex (MHC) class I.
MHC class I antigens are expressed by all human nucleated cells and are central
in cell-mediated immune response and tumor surveillance. CCR7 is a chemokine
receptor which, with its ligands, links innate and adaptive immunity through their
eects on interactions between T cells and dendritic cells. CCR7-positive cancer cell
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PCI-1
0,7
0,6
Control
0,5
0,4
OD560
ASA 2 mM
EPs 7630 25 µg/ml
0,3
Celecoxib 25 µM
Curcumin 15 µM
0,2
0,1
Dexamethasone 10 µM
0
0h
Control
24 h
Curcumin 15 µM
Celecoxib 25 µM
48 h
EPs 7630 25 µg/ml
72 h
Dexamethasone 10 µM
ASA 2 mM
Figure 3.7: Survey of PCI-1 Growth Curves. Inuence of stimulants with chosen concentration on PCI-1.
expression has been associated to lymph node metastasis as well.
Cells were incubated for up to 72 h with the respective stimulants and prepared
for FACS
TM
analysis as per protocol above (see 2.5 on page 27). Cells were either
treated with or without saponin, a detergent used to quantify and compare intracellular and extracellular intensity signals. Readings were taken every 24 h for both
cell lines, BHY and PCI-1.
3.2.1 NF-κB Inhibition Does Not Change Expression of KI-67
and HLA-A,B,C
Looking at the expression of KI-67 and HLA-A,B,C over the course of 72 h incubation with ASA, Curcumin, Celecoxib, Dexamethasone and EPs 7630 no signicant change in expression is noted (Fig. 3.8 on the next page - 3.11 on page 42).
As expected for a molecule mainly presenting antigens on the surface of the cells,
HLA-A,B,C expression is higher on the cells without saponin treatment. Treatment
with neither of the stimulants resulted in a signicant peak shift or skewing of the
histogram curve. Slight alteration in KI-67 occurs only in cells incubated with Cur-
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HLA-A,B,C
KI-67
TLR3
CCR7
Control
ASA
Curcumin
Celecoxib
Dexa
EPs 7630
Figure 3.8: Surface Expression of HLA-A,B,C, KI-67, CCR7 and TLR3 in PCI-1
after 72 h. ((1) A "-" in histograms depicts cells not treated with saponin, exempliTM
fying FACS intensity registration on the cell surface; (2) Dexa = Dexamethasone).
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HLA-A,B,C
KI-67
TLR3
CCR7
Control
ASA
Curcumin
Celecoxib
Dexa
EPs 7630
Figure 3.9: Cytoplasmical Expression of HLA-A,B,C, KI-67, CCR7 and TLR3 in
PCI-1 after 72 h. ((1) A "+" in histograms depicts that cells were treated with
TM
saponin, exemplifying FACS intensity registration of cytoplasmical signals; (2)
Dexa = Dexamethasone).
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HLA-A,B,C
KI-67
TLR3
CCR7
Control
ASA
Curcumin
Celecoxib
Dexa
EPs 7630
Figure 3.10: Surface Expression of HLA-A,B,C, KI-67, CCR7 and TLR3 in BHY after 72 h. ((1) A "-" in histograms depicts cells not treated with saponin, exemplifyTM
ing FACS intensity registration on the cell surface; (2) Dexa = Dexamethasone).
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HLA-A,B,C
KI-67
TLR3
CCR7
Control
ASA
Curcumin
Celecoxib
Dexa
EPs 7630
Figure 3.11: Cytoplasmical Expression of HLA-A,B,C, KI-67, CCR7 and TLR3 in
BHY after 72 h. ((1) A "+" in histograms depicts that cells were treated with
TM
saponin, exemplifying FACS intensity registration of cytoplasmical signals; (2)
Dexa = Dexamethasone).
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cumin. KI-67 is more highly present on surface structures than in the cytoplasm.
Among all stimulants, Curcumin allows for the highest expression of KI-67, surpassing the expression of controls.
3.2.2 CCR7 Shows Higher Expression in the Cytoplasm of
HNSCC Cell Lines than on the Surface
CCR7 is a member of the G-protein-coupled receptor family and normally membrane-
TM
bound. One of its downstream oncogenes is NF-κB. In FACS
analysis of the
HNSCC cell lines BHY and PCI-1 we found CCR7 to have a higher expression in
the cytoplasm of cells treated with saponin as compared to those untreated. This
occurred throughout all groups with specic stimulant incubation. So, NF-κB inhibition does not have an eect on the expression of CCR7. HNSCC cells seem to
have a higher frequency of CCR7 in the cytoplasm contrary to the common notion
of its normal distribution.
3.2.3 Localization of TLR3 Expression
TLR3 is strongly expressed on HNSCC cell lines as previously reported [86]. These
results also indicated that TLR3 is indeed a foremost cytoplasmical protein. To
determine where TLR3 is expressed in HNSCC we measured TLR3 expression under NF-κB inhibiting conditions. Measurements show that TLR3 does not change
its site of expression under stimulation. It remains a predominantly cytoplasmical
protein, but the dierences occur to be dampened by the stimulants with respect to
previous reports [86].
Overall, incubation with the stimulants did not lead to a grossly changed pattern
of expression of HLA-A,B,C, KI-67, CCR7 and TLR3 in either HNSCC cell line.
However, it was observed that the dierence between TLR3 expression in the cytoplasm and on the surface of HNSCC did not occur at the same rate under stimulant
incubation. In contrast to its normal distribution the expression of CCR7 takes
place in the cytoplasm of HNSCC.
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3.3 NF-κB Regulators are Strongly Expressed in
HNSCC and Downregulated by Stimulants
NF-κB as a key mediator in cell function is closely regulated. In its inactive state NF-
κB is located in the cytoplasm, closely tied to its direct inhibitor Iκ-Bα. Upstream
activation is coordinated by IKK-β as part of the IKK-complex, which consists of
IKK-α and -β , the active units, and a regulatory subunit, IKK-γ . Upon activation IKK phosphorylates Iκ-Bα with subsequent release of NF-κB from its ties.
NF-κB then translocates to the nucleus commencing its transcriptional activities.
To ascertain the inuence of the stimulants on upstream processes and the feedback
inhibition of IKK-β and Iκ-Bα by NF-κB, we undertook western hybridization analysis of IKK-β and Iκ-Bα.
Iκ-Bα
IKK- β
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
β -Actin
Figure 3.12: Western Blot Analysis of IKK-β and Iκ-Bα. NF-κB regulators IKK-β
and Iκ-Bα are strongly expressed in BHY and suppressed to dierent extents by
respective stimulants (β -Actin shown as loading control). Selection of samples from
three independent experiments.
HNSCC cell lines PCI-1 and BHY were incubated with the respective stimulants
for 72 h and protein extracts prepared as previously described. BHY had a greater
protein expression overall, i.e. seemed to be less aected by NF-κB inhibition than
PCI-1. Unstimulated HNSCC as control shows a strong expression of IKK-β and
Iκ-Bα. Though no stimulant was able to completely abrogate IKK-β expression,
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ASA and EPs 7630 were among the two most potent, followed by Celecoxib and
Dexamethasone. Curcumin shows the weakest IKK-β suppression in western blot
analysis in BHY (Fig. 3.13 on page 45). In PCI-1, Celecoxib, Dexamethasone and
EPs 7360 most potently diminished IKK-β and Iκ-Bα expression.
The high Iκ-Bα expression of the unstimulated control in BHY is only matched
by Celecoxib. All other stimulants lead to a decreased level of expression, Dexamethasone and ASA almost completely abrogating Iκ-Bα expression. Again, the
expression level of treated PCI-1 cells is signicantly lower than in BHY with all
stimulants almost completely exterminating protein levels.
Iκ-Bα
IKK- β
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
β -Actin
Figure 3.13: Western Blot Analysis of IKK-β and Iκ-Bα in PCI-1. NF-κB regulators
IKK-β and Iκ-Bα are strongly expressed in PCI-1 and suppressed to dierent extents
by respective stimulants (β -Actin shown as loading control). Selection of samples
from three independent experiments.
Downstream targets and collaborators of NF-κB play important roles in the mechanisms of cell growth and dierentiation. Two of the most important molecules in
this context are cyclin D1 and c-Myc. Both proto-oncogenes have proved to be ambivalent in exertion of their tasks. C-Myc is an oncogene that functions both in the
stimulation of cell proliferation and in apoptosis. It may also enhance or reduce the
sensitivity of cancer cells to chemotherapy, but how this dual function is controlled
is largely unclear. Cyclin D1 drives cell cycle progression; it acts as a growth factor
sensor to integrate extracellular signals with the cell cycle machinery, though it may
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also promote apoptosis [153]. In this study we looked at the level of expression of
cyclin D1 and c-Myc in HNSCC under the inuence of the aforementioned stimulants.
Cyclin D1
c-Myc
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
β -Actin
Figure 3.14: Western Blot Analysis of Cyclin D1 and c-Myc in BHY. Expression of
proto-oncogene c-Myc does not change signicantly under stimulant inuence except
for ASA, whereas cell cycle regulator cyclin D1 is downregulated by every stimulant
(β -Actin shown as loading control). Selection of samples from three independent
experiments.
HNSCC cell lines were incubated with the respective stimulants for 72 h and protein
extracts prepared as previously described. Unstimulated control HNSCC cells share
a naturally high expression of both cyclin D1 and c-Myc. In BHY, c-Myc is generally
downregulated by all agents and particularly by ASA. Cyclin D1 is downregulated
in all stimulated cells. Strongest suppression of cyclin D1 is found in ASA, Dexamethasone and EPs 7630. PCI-1 cells share most of these patterns. In comparison to
BHY, ASA and Dexamethasone seem to have a weaker impact on protein expression
in PCI-1.
3.4 TLR3 is Downregulated in HNSCC Incubated
with Stimulants
As we know, TLR3 has previously been shown to contribute to the activation of
NF-κB, a transcription factor which promotes several types of human cancers. Ad-
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Cyclin D1
c-Myc
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
β -Actin
Figure 3.15: Western Blot Analysis of Cyclin D1 and c-Myc in PCI-1. In contrast to
BHY expression of c-Myc is inhibited except for ASA and Dexamethasone, whereas
cell cycle regulator cyclin D1 is foremostly downregulated by Celecoxib and EPs
7630 (β -Actin shown as loading control). Selection of samples from three independent experiments.
TLR3
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
β -Actin
Figure 3.16: Western Blot Analysis of TLR3 in BHY. BHY cells have high expression
of TLR3. All stimulants suppress TLR3 levels (β -Actin shown as loading control).
Selection of samples from three independent experiments.
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ditionally our group has shown before that TLR3 is overexpressed in HNSCC and
that inhibition of TLR3 expression in permanent HNSCC cell lines resulted in decreased expression of the oncoprotein c-Myc, thus resulting in decreased cell proliferation [86]. We therefore tried to evaluate this aspect from the opposite direction
by looking at the expression of TLR3 in HNSCC under NF-κB inhibiting conditions.
TLR3
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
β -Actin
Figure 3.17: Western Blot Analysis of TLR3 in PCI-1. PCI-1 shows high expression of
TLR3. All stimulants suppress TLR3 levels with EPs 7630 almost completely abrogating TLR3 expression (β -Actin shown as loading control). Selection of samples
from three independent experiments.
HNSCC cell lines were incubated with the respective stimulants for 72 h and protein extracts prepared as previously described. As gure 3.16 on the preceding page
and gure 3.17 on page 48 shows, TLR3 is strongly expressed in the unstimulated
control. A signicantly lower level of expression of TLR3 among all cells that were
incubated with ASA, Curcumin, Celecoxib, Dexamethasone and EPs 7630. TLR3
seems to be most suppressed by Celecoxib and Curcumin, whereas ASA, Dexamethasone and EPs 7630 show stronger signals. Thus all stimulants are able to curtail
TLR3 expression in HNSCC.
Constricted by its semiquantitative qualities western hybridization qualies only to
hint for tendencies in protein expression. However, a couple of deductions can be
drawn from our results. First of note, TLR3 is expressed in HNSCC and is downregulated by all stimulants. Secondly, all stimulants alter expression levels of the cyclin
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ASA
Curcumin
Control
Celecoxib
Dexa
EPs 7630
Figure 3.18: Immunohistochemistry of TLR3 labeled HNSCC: Though not a quantiable measure the immunohistochemical stains vividly depict the decreased expression of TLR3 in HNSCC under NF-κB inhibition.
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D1, an important regulator of proliferation. C-Myc does not appear to be entirely
involved in the changes brought about by the stimulants. However, all stimulants
act on the IKK-complex to exert their anti-proliferative action.
3.5 NF-κB Inhibition by Stimulants
As pointed out earlier, NF-κB is considered a key player in cell function and immunity. Much light has been shed on its ubiquitous role since its rst description in
1986. Particularly, its function in the promotion of carcinogenesis and inammation
has opened the door for ongoing research and permanent discoveries are made on
this behalf. To evaluate the degree of activity of NF-κB in HNSCC, we cultivated
cells with or without further treatment. Nuclear extracts were then manufactured
according to protocol and underwent a specic NF-κB ELISA as indicated above.
Figure 3.19 on the following page depicts the degree of NF-κB inhibition in detail.
Results are shown as means of two independent experiments with a total of four
measurements for any given stimulant. As pointed out before, overall NF-κB activity is lower in BHY than in PCI-1. Setting the baseline OD of controls to 100 %,
ASA inhibits NF-κB action by almost 47 % in BHY (OD 0.359/0.672) and by only
12 % in PCI-1 (OD 0.245/0.367). Curcumin cuts NF-κB activity to 53 % compared
to unstimulated controls in BHY, a reduction of 47 % (OD 0.357/0.672); in PCI-1
Curcumin reaches an activity level of 75 % of controls, thus an inhibitory potential
of 25 % (OD 0.278/0.367). Celecoxib shows strong delimiting properties in BHY
with 53 % (OD 0.317/0.672) compared to controls and 32 % (OD 0.245/0.367) in
PCI-1. Dexamethasone shows comparable results in BHY and PCI-1 with an activity reduction of 20 % for both cell lines (OD 0.541/0.672 for BHY and 0.297/0.367
for PCI-1). EPs 7630 limits NF-κB activity to 70 % (OD 0.464/0.672) in BHY and
to 80 % in PCI-1 (OD 0.295/0.672).
Thus, all treatments suced to inhibit NF-κB activity. The degree of inhibition
varied not only between the dierent arms of treatment but also between the distinct cell lines BHY and PCI-1.
50
1,2
1,2
1
1
0,8
0,8
OD 450
OD 450
3 Results
0,6
0,6
0,4
0,4
0,2
0,2
0
0
BHY
PCI-1
BHY
(b) Curcumin
1,2
1,2
1
1
0,8
0,8
OD 450
OD 450
(a) ASA
PCI-1
0,6
0,6
0,4
0,4
0,2
0,2
0
0
BHY
PCI-1
BHY
(c) Celecoxib
PCI-1
(d) Dexamethasone
1,2
1
OD 450
0,8
0,6
0,4
0,2
0
BHY
PCI-1
(e) EPs 7630
Figure 3.19: NF-κB ELISA. Comparison of NF-κB inhibition per stimulant and cell line.
Black boxes indicate unstimulated cells (DMEM), white dotted boxes the respective
stimulants. Results are shown as means of four measurements in two independent
experiments with the respective standard deviation.
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3.6 Cytokine Proles in HNSCC with or without
NF-κB Inhibitory Treatment
Cytokines are an essential part of the tumor milieu and carry out important tasks
in immune response mechanisms and their modulation. It has been shown that
HNSCC are densely inltrated by lymphocytes, so regulation of cytokine expression
is key to the proliferation of the tumor and its possible escape from the immune
surveillance. In HNSCC it has been explicably shown that IL (Interleukine)-6 and
IL-8 play a major role in carcinogenesis for their involvement in various oncogenic
processes like metastasis and angiogenesis [41, 42]. It has also been reported that
patients with HNSCC are biased toward the T-helper cell type 2 (TH 2) phenotype
as they have increased levels of the TH 2 cytokines IL-4, IL-6 and IL-10 and diminished levels of the T-helper cell type 1 (TH 1) cytokine IFN-γ . However, this bias is
incomplete since levels of the TH 1-cytokines IL-2 and GM-CSF are increased.
In this context we assessed supernatants of HNSCC under NF-κB inhibiting conditions with regard to their cytokine milieu as described in section 2.6 on page 28.
Featured cytokines included IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, INF-γ and TNF-
α. Originally looking at each analyte every 24 h for 72 h to parallel growth curves
in section 3.1 on page 33, no signicant changes or patterns were noted, so results
represent means of data over 72 h. PCI-1 yielded nonrepresentative low bead counts
throughout all analytes except for IL-6 and IL-8. BHY had a generally higher level
of cytokine expression in the supernatants and was able to generate signals of every
analyte. In the following sections, cytokines are discussed regarding their generally accepted importance in HNSCC, starting with the low prole or non-prominent
cytokines in HNSCC followed by IL-6 and IL-8 which share high importance in HNSCC.
3.6.1 Non-prominent Cytokines in HNSCC
IL-2
IL-2 is the main cytokine to promote T-helper cell response and maturation. Upon
recognizing an MHC class II molecule, a T-helper cell, normally a TH 1, produces
IL-2, which subsequently acts in an autocrine fashion to promote maturation and
clonal production. IL-2 is an immune stimulatory cytokine which is mandatory for
TH 2-cytokine production of IL-4 and IL-5.
Although IL-2 could be detected in BHY, levels are generally low. However, it can
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be argued that unstimulated controls produce more IL-2 than the stimulated HNSCC. Every stimulant stays under the level of the controls. Curcumin is least able
to suppress IL-2 production.
IL-4
Anti-inammatory IL-4 is a marker cytokine for TH 2-cells. There has been evidence
for its increased expression in HNSCC and an immune suppressive action in this
context. IL-4 is known to antagonize the eects of IFN-γ and thus to inhibit cellmediated immunity [154].
Out of its in vivo environment, as in our experimental setting, we can see only a low
prole IL-4 expression. Again, highest expression is seen in unstimulated controls.
IL-4 is slightly suppressed in all treated cells, the most suppressed in Dexamethasone, the least in EPs 7630.
IL-10
IL-10 shares some common aspects with IL-4. Mainly built by TH 2-cells, it also
works as an anti-inammatory cytokine, suppressing the immune reaction. More
specically it inhibits macrophages in carrying out their tasks like the production
of IL-12 [154].
In contrast to the other cytokines analyzed, both cell lines showed signicant expression of IL-10. PCI-1 has a higher overall level of IL-10 than BHY. Moreover,
each stimulant causes a dierent eect in each cell line. Whereas no signicant difference is noted among the cells incubated with or without stimulants in BHY, with
all groups lingering around the levels of the controls, IL-10 levels even surpassed
the ones of controls in PCI-1 incubated with ASA, Curcumin and Dexamethasone.
Celecoxib and EPs 7630 are the only NF-κB inhibitors to remain under the IL-10
level of the controls.
Granulocyte Macrophage Colony-stimulating Factor (GM-CSF)
GM-CSF is mainly produced by TH 1- and TH 2-helper cells. It is necessary for the
growth and maturation of granulocytes, macrophages and dendritic cells. For HNSCC, GM-CSF was shown to trigger the mobilization of CD34 natural suppressor
cells in the bone marrow and then translocate to the tumor tissue. Here they restrict
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proper functioning of HNSCC inltrating T-cells [155].
Looking at the data generated in the context of this study, primary cultures of controls reached levels of almost 300 pg/ml for GM-CSF. Incubation with respective
stimulants led to signicantly lower levels of GM-CSF throughout. In ASA, Curcumin, Celecoxib, Dexamethasone expression of GM-CSF was almost cut in half,
with only EPs 7630 displaying weaker suppression, yet with still signicant dierence.
Interferon-gamma (IFN-γ )
IFN-γ , produced predominantly by Natural Killer (NK) cells and other cell types,
plays a critical role in killing pathogen-infected cells and in defending against tumor cells. However, overproduction of IFN-γ is also dangerous to the body and can
cause autoimmune diseases. INF-γ belongs to the group of TH 1-cytokines, decreased
levels of which have been observed in patients with HNSCC [156]. Downregulation
of TH 1-cytokines such as IFN-γ , IL-2 and IL-12 represents a signicant parameter
of HNSCC immune escape mechanisms [157]. Traditionally a pro-inammatory cytokine, some evidence emerged toward a role in anti-inammatory actions of IFNγ
as well [158].
One would thus expect to have high INF-γ secretions in cells incubated under NF-
κB inhibition. However, INF-γ levels are not exceedingly higher than in controls.
They linger around the levels of controls with the exception of Dexamethasone which
signicantly decreases IFN-γ levels.
Tumor Necrosis Factor-alpha (TNF-α)
TNF-α is a cytokine involved in systemic inammation and is a member of a group
of cytokines that stimulate the acute phase reaction. The primary role of TNF-α
is in the regulation of immune cells. It is also able to induce apoptotic cell death,
to induce inammation, and to inhibit tumorigenesis and viral replication. Via its
two receptors, TNF-R1 and TNF-R2, TNF-α is able to activate NF-κB, MAPKpathways and the induction of death signalling, which, however is considered weak
in comparison to other members of this subgroup, like FAS, and, in addition might
be masked by the antiapoptotic eects of NF-κB activation. The myriad and oftenconicting eects mediated by the above pathways indicate the existence of extensive
cross-talk. Such complicated signaling ensures that, whenever TNF is released, various cells with vastly diverse functions and conditions can all respond appropriately
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IL-2
IL-4
70
80
70
60
60
50
pg/ml
pg/ml
50
40
40
30
30
20
20
10
10
0
0
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
Celecoxib
Dexamethasone
EPs 7630
IL-10
80
70
60
pg/ml
50
40
30
20
10
0
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
INF-γ
140
300
120
250
100
200
80
pg/ml
pg/ml
GM-CSF
350
150
60
100
40
50
20
0
0
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
Control
ASA
Curcumin
TNF-α
300
250
pg/ml
200
150
100
50
0
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
Figure 3.20: Level of Cytokine Expression in HNSCC. Cytokines IL-2, IL-4, IL-10, GMCSF, IFN-γ and TNF-α as expressed by BHY and PCI-1 where indicated.
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to inammation.
TNF-α is expressed in HNSCC in this study. Though in a low range of 250 pg/ml,
primary HNSCC cultures show the highest expression with stimulated cells trailing
behind. Only Curcumin almost matches this level, not showing a signicant decrease
in TNF-α secretion. ASA, Celecoxib as well as Dexamethsone and EPs 7630 show
signicantly lower levels of TNF-α.
3.6.2 IL-6 Expression is Decreased in NF-κB Inhibited
HNSCC
Traditionally, IL-6 is produced by various cells such as T-lymphocytes, macrophages
and monocytes but also cells of endothelial origin, and is involved in distinct cellular
processes like cell dierentiation, the production of acute phase proteins in the liver,
the proliferation of B-lymphocytes, and the production of neutrophils [159]. IL-6
was shown to have pro- as well as anti-inammatory properties and thus deregulation of IL-6 cytokine signaling often contributes to various kinds of cancer playing
a central role as a dierentiation and growth factor of tumor cells [159, 160].
IL-6 is downregulated in all cells inhibited by respective stimulants (see gure 3.21
on the next page). Curcumin, again, is least able to suppress IL-6 secretion. EPs
7630 and Dexamethasone share a common IL-6 suppression potency, the strongest
among all stimulants.
3.6.3 IL-8 Expression is Decreased in NF-κB Inhibited
HNSCC
IL-8 is associated with coordination of leukocyte migration from the blood to the site
of inammation. IL-8 can be secreted by any cells with toll-like receptors which are
involved in the innate immune response. In HNSCC, IL-8 has reached signicance
in its role in angiogenesis. Along with IL-6, IL-8 is a prominent cytokine found in
HNSCC.
All NF-κB inhibitors were able to cut down IL-8 signicantly (see gure 3.22 on the
following page). Controls scratch the 10.000 pg/ml mark for IL-8. Cells incubated
with ASA, Curcumin and Dexamethasone run around 9000 pg/ml and the remaining two, Celecoxib and EPs 7630 around 8500 pg/ml.
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IL-6
10000
9000
8000
7000
pg/ml
6000
5000
4000
3000
2000
1000
0
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
Figure 3.21: Levels of IL-6 under NF-κB Inhibiting Conditions
IL-8
12000
10000
pg/ml
8000
6000
4000
2000
0
Control
ASA
Curcumin
Celecoxib
Dexamethasone
EPs 7630
Figure 3.22: Levels of IL-8 under NF-κB Inhibiting Conditions
57
3 Results
In summary, non-prominent HNSCC cytokines are only scarcely found in supernatants of pure HNSCC cell cultures. In culture with NF-κB inhibiting agents, they
are regulated according to their natural behavior, i.e. benevolent cytokines like IL-4
or IL-10 either almost match levels of the controls or even exceed them, whereas
unfavorable cytokines like GM-CSF, IL-6 and IL-8 are signicantly downregulated.
58
3 Results
3.7 Mycoplasma Studies
Mycoplasma contamination is a serious and frequent problem in cell culture laboratories. It has led to the development of a vast array of detection methods to prevent
contamination. We scanned established cell lines for the presence of Mycoplasma.
Seven out of these proved to be contaminated by Mycoplasma. To determine if and
to what extent Mycoplasma stimulate the expression of TLR we divided these cell
lines into two groups. One group was purposely left untreated whereas the other
group was cleaned using a commercially available Mycoplasma detection kit. After
preparation protein expression of contaminated and uncontaminated cell lines was
measured. In this context we wanted to assess the eect of a chronic irritant like
Mycoplasma on cancer cells and if the exposure to such irritant led to aberrant protein expression.
3.7.1 TLR1, TLR3 and TLR4 but not TLR2 and TLR6 are
Expressed in HNSCC under
Inuence
Mycoplasma
TLRs are outposts of the innate immune system and react to a vast array of ligands.
Generally, TLR1, TLR2 and TLR6 are known to recognize Mycoplasma epitopes.
TLR2 acts as a partner in heterodimers with either TLR1 or TLR6. Mycoplasma
is a gram-negative bacterium and is thus also expected to stimulate TLR4, which
is sensitive towards lipopolysaccharides (LPS) found in the cell membrane of gramnegative bacteria. These aspects hold especially true in cells of the immune system.
So far, only scarce information about TLR expression in cancer cells and especially
in HNSCC is known.
An extensive scan of expression of TLRs 1-9 resulted in signals of TLR 1, 3 and
4 in certain cell lines. As gure 3.23 on the next page shows, TLR1, typically
sensitive to peptidoglycans from bacterial cell walls and usually active as a heterodimer with TLR2, was expressed in Hlac78, Hlac79 and HaCat cells. Stronger
signals were noted in cell cultures with manifest Mycoplasma contamination. No
other cell line expressed TLR1. Results for TLR3 were obtained in cell lines Ant-1,
GHD and Hlac79. Low signals in Ant-1 mirror higher signals in Hlac79 and GHD.
As opposed to TLR1, TLR3 does not seem to follow suit regarding patterns in
contaminated and non-contaminated cells. Whereas Hlac79 exhibits greater TLR3
abundance in contaminated cells, it is surprising that in HNSCC cell line GHD the
pendulum clearly swings in favor of cells without proven Mycoplasma contamination. β -Actin as housekeeping gene underlines consistent and stable protein loading
59
3 Results
Hlac78
-
Hlac79
+
-
HaCat
+
-
+
TLR1
β-Actin
Ant-1
-
Hlac79
+
-
GHD
+
-
+
TLR3
β-Actin
Figure 3.23: Inuence of Mycoplasma on TLR1 and TLR3 Expression on Dierent
Cancer Cell Lines. Three cell lines showed expression of TLR1 and TLR3.
Signicantly higher expression of TLR1 was observed in contaminated samples of
Hlac79. Hlac79 showed high expression of TLR3 in both subgroups. Interestingly,
GHD showed signicantly higher expression of TLR3 in the decontaminated than
in the contaminated cells.
60
3 Results
in wells throughout.
TLR4 was found in every analyzed cell line. Levels of expression varied, though.
General levels were high in Ant-1, GHD and Hlac79. Among those, contaminated
cells showed higher expressions of TLR4 than non-contaminated cells.
3.7.2 NF-κB and c-Myc are Upregulated and EpCAM is
Downregulated under
Inuence
Mycoplasma
Apart from TLR expression attention was paid to a couple of important proteins
which play critical parts in all cancers. C-Myc and NF-κB have been extensively discussed in previous chapters and sections. EpCAM is transmembrane protein that
was amongst the rst tumor-associated antigens to be discovered. First only described in colon cancer, it has also been found in cervical, ovarian and breast cancer
and HNSCC. In short, EpCAM can be considered one of the most frequently overexpressed tumor-associated antigens in a great variety of cancers, which is highly
immunogenic, a prognostic factor, and moreover a valuable target for immunotherapy [161].
As we can see from gure 3.24 on page 63 , EpCAM is expressed in every cell line
but highest in HaCat, Hlac78 and PCI-13. In these it has a higher expression in uncontaminated cells than in the ones with Mycoplasma. In Hlac78 this aspect might
be blurred by the relatively weak signal in the housekeeping gene β -Actin in Hlac/+,
indicating a low protein load to this well. In Ant-1, GHD and Hlac79, EpCAM is
not as signicantly expressed as in the previously discussed cell lines. Interestingly,
also Hlac79, which was high prole in protein levels throughout, does not exhibit a
strong signal in the case of EpCAM.
NF-κB is found throughout each cell line, with or without Mycoplasma contamination. With Mycoplasma, however, NF-κB activity is upregulated in almost every
cell line. The only exception is Hlac78, but as said before, this might be biased
by low protein load in Hlac78/+. Despite the low load in Hlac78/+, its expression
of c-Myc is signicantly higher than in Hlac78/-, underlining a prospective bias toward a simultaneously high expression of NF-κB as well. In HaCat and Hlac78 and
Hlac79 cells, c-Myc is better expressed in contaminated cells than in the negative
ones. Furthermore, there is better c-Myc expression in PCI-13/+, although general
levels are deemed low. Similar levels of c-Myc appear in either sample of Ant-1 and
GHD.
It comes to no surprise that Mycoplasma contamination, which in most cases goes
61
3 Results
unnoticed, has indeed an eect on the expression of key structures in the metabolism
and gene expression of HNSCC cell lines. Not all cell lines act as expected with
regard to TLR expression, though. With TLR1, TLR2 and TLR6 being the main
targets of Mycoplasma PAMPs, not every cell line showed expression of those TLRs.
Furthermore, as seen earlier, HNSCC are suspected to express TLR3. This holds
true only for three out of six cell lines of this study. Moreover, it is surprising that
in the case of GHD a high TLR3 expression in clean cultures is so markedly downregulated in contaminated cultures. Furthermore, this happens under simultaneous
high expression of NF-κB, slightly lower levels of c-Myc and slightly higher levels
of TLR4 in GHD/+. Finally, EpCAM is strongly expressed in clean cultures and
downregulated in contaminated cultures in three out of six analyzed cell cultures,
leaving space for speculation of a possible interference of Mycoplasma with this cell
adhesion molecule and tumor antigen.
62
3 Results
TLR4
c-Myc
NF-κB
EpCAM
β-Actin
Ant-1
+
-
GHD
+
HaCat
+
Hlac78
+
Hlac79
+
PCI-13
+
Figure 3.24: Expression of TLR4, c-Myc, NF-κB and EpCam in Dierent Cancer Cells +/- Mycoplasma inuence
63
4 Discussion
Chronic inammation is the major culprit in the onset of HNSCC, caused by irritants such as tobacco and alcohol. Our group previously found TLR3 to be expressed
on HNSCC and a concordantly high constitutive expression of transcription factor
NF-κB. TLR3 inhibition leads to a downregulation of c-Myc in HNSCC [86]. We
also found increased cytokine secretion in HNSCC upon activation of p38-MAP kinase activation [162]. We claried that targets of TLR3 activation are deregulated
in HNSCC and contribute to the proliferation of HNSCC. In this work we characterized HNSCC behavior under NF-κB inhibiting conditions. We investigated the
actions of ASA, Curcumin, Celecoxib, Dexamethasone and EPs 7630 on NF-κB and
looked at the eects of this treatment on TLR3 and cytokine levels. We found that
all agents reduced active NF-κB levels and that this inhibition also down-regulated
TLR3 levels in HNSCC under the inuence of a signicantly altered HNSCC microenvironment. Furthermore, we looked at the expression of key proteins of HNSCC
when exposed to Mycoplasma, a common contaminant in cell cultures. Mycoplasma
seems to have a substantial eect on the expression of distinct tumor molecules,
postulating not only a signicant eect of Mycoplasma on HNSCC as an example of
chronic inammation but also emphasizes the need for regular monitoring of possible contamination with this irritant.
4.1 NF-κB as Ambiguous Key Player in
Inammation-associated Cancer
A key player at the crossroads of many important cell signalling ways, NF-κB has
been intensively studied. Although the role of NF-κB in carcinogenesis has been well
described, it is still ambiguous. This is partly due to an incomplete understanding
of the linkage of inammation and cancer. In immunology NF-κB has long been recognized as being involved in inammatory and innate immune responses. Over time
more evidence emerged emphasizing the role of NF-κB in tumor development and
progression [163]. NF-κB has since been proclaimed a regulator of programmed cell
64
4 Discussion
death, either as inductor and inhibitor of apoptosis [164] or necrosis. Improper regulation of programmed cell death by NF-κB can have severe pathologic consequences,
ranging from neurodegeneration to cancer, where its activity often precludes eective therapy. Although NF-κB generally protects cells by inducing the expression
of genes encoding anti-apoptotic and anti-oxidizing proteins, its role in apoptosis
and necrosis can vary markedly in dierent cell contexts, and NF-κB can sensitize
cells to death-inducing stimuli in some instances [165]. Hallmarks of cancer development include self-suciency in growth signals, insensitivity to growth-inhibitors,
evasion of apoptosis, limitless replicative potential, tissue invasion and metastasis,
and sustained angiogenesis. NF-κB signaling has been implicated in each of these
hallmarks [166]. While executing this central role in regulating cell processes, a vast
regulation machinery has to maintain proper functioning. IKK-β has been identied
as one of the most important regulators of NF-κB signaling. In summary, activation
of NF-κB is a crucial mediator of inammation-induced tumor growth and progression, but also an important modulator of tumor surveillance and rejection [167]. To
this end, the role of NF-κB seems to be strongly cell-type and context specic.
4.1.1 NF-κB Mediates Proliferation in HNSCC
One group of NF-κB inhibitors are anti-inammatories like NSAIDs (e.g. Aspirin
and Celecoxib) or steroids like Dexamethasone. Although it was demonstrated that
Aspirin inhibited the activation of NF-κB by preventing the degradation of Iκ-Bα
so that NF-κB was retained in the cytosol, the precise mechanisms remain nebulous
[121]. ASA is known to reduce the risk of colon cancer on the basis of inammatory
bowel disease [168170]. Normally thought to exert its action by inhibiting the
cyclooxygenase enzyme (COX), ASA is also able to curtail proliferation in COXdecient colorectal cell lines, suggesting a dierent, COX-independent pathway of
inhibition [171]. So far, no study has evaluated the impact of ASA on NF-κB in
HNSCC. It is known, however, that ASA was ecient in reducing levels of PGE2 in
the surrounding mucosa of HNSCC [172]. Adding to the duality of function towards
NF-κB inhibition, studies in intestinal neoplasia showed apoptosis induction by ASA
through activation of NF-κB [173,174]. We demonstrate that ASA is indeed able to
stop proliferation of HNSCC via inhibition of NF-κB. However, in our studies ASA
was also able to slightly increase growth of HNSCC at very low concentrations.
This might be an indicator of the dual function of NF-κB in dierent cell contexts.
For Celecoxib there is general evidence for a dose-dependent switch in function.
In high doses Celecoxib was able to activate NF-κB in mouse models [130], thus
losing its anti-inammatory ecacy. Furthermore, both Celecoxib and ASA have
65
4 Discussion
demonstrated NF-κB activation in non-small cell lung cancer lines, thus unable to
induce apoptosis [175]. The majority of studies, however, suggests that Celecoxib
strongly inhibits NF-κB in a variety of cancers, ranging from the above mentioned
non-small cell lung carcinoma, pancreatic, breast and urological cancers [128, 176
178]. The results of our Celecoxib growth studies are in agreement with other studies
that report a signicant growth inhibition by Celecoxib via the NF-κB pathway in
HNSCC [179, 180]. Celecoxib curtails proliferation of both HNSCC cell lines and
shows signicant inhibition of NF-κB. Thus, Celecoxib seems to play a dierent part
in HNSCC when compared to lung cancer cell lines, as mentioned above, underlining
the importance of the context in use. This goes hand in hand with reports of other
tumors like pancreatic cancer, breast cancer which all demonstrate a signicant
inhibition of proliferation and NF-κB activity by Celecoxib. The anti-inammatory
steroid Dexamethasone has to be distinguished from its non-steroidal siblings. In
HNSCC Dexamethasone showed potential to inhibit cell proliferation together with
Bortezomib, increasing its cell toxicity [181]. Our results concur with ndings in
renal cell carcinoma, where Dexamethasone alone showed signicant inhibition of cell
proliferation by inhibition of NF-κB [182]. Curcumin as one of the chemopreventive
compounds studied is widely accepted for its growth suppression in HNSCC [139].
Our results agree with the before mentioned dose-dependent inhibition of HNSCC
cell lines, although individual concentrations varied. This might be due to the highly
individual characteristics of the dierent cell lines used.
4.1.2 IKK-β as Main Regulator of NF-κB
While executing this central role in important cell processes, NF-κB has to be tightly
regulated to provide for proper functioning. In most cell types and at resting state,
NF-κB is retained in the cytoplasm through its tight association with inhibitory
proteins called IκBs, most notably Iκ-Bα. Key to NF-κB activation is the phosphorylation of Iκ-Bα by the so-called IκB kinase (IKK) complex, which targets the
inhibitory protein for proteasomal degradation and allows the freed NF-κB to enter the nucleus where it can transactivate its target genes [92, 93]. It consists of
two molecules, IKK-α and IKK-β , which play critical roles in linking inammation
and cancer. In mouse models of cancer, evidence was obtained for a critical role of
IKK-β in tumor promotion and IKK-α in metastatogenesis. Whereas the major protumorigenic function of IKK-β is mediated via NF-κB, the pro-metastatic function
of IKK-α is NF-κB-independent [183]. Looking at the NF-κB-dependent molecule
IKK-β , we found Iκ-Bα and IKK-β to be downregulated by all compounds, although
Celecoxib showed only minor inhibition and similar levels for Iκ-Bα and IKK-β . Sev-
66
4 Discussion
eral mechanisms of NF-kB inhibition have been reported for Celecoxib. There has
been evidence for TNF-induced IKK inhibition by Celecoxib [128]. However, recent studies have also suggested that Celecoxib potently inhibits TNF-α-induced
transcriptional activity and DNA binding activity of NF-κB downstream of IKK
activation and IκBs degradation without an eect on IKK expression itself [184].
With EPs 7630, we present evidence for NF-κB inhibition by EPs 7630 via the IKK
pathway for the rst time. Although our results show decreased levels of Iκ-Bα along
with IKK-β inhibition for all compounds except Celecoxib, this is not paradoxical.
Literature gives an incoherent picture of NF-κB regulation by Iκ-Bα and IKK and
points toward a strong cell type and context dependent expression of the two regulators of NF-κB activity. For example, Dexamethasone has been shown to have
little eect on pre-existing NF-κB in human myeloid leukaemia cells but exerted
its action on the mRNA level [185]. In contrast, other studies depict the induction
of Iκ-Bα as inhibiting process on NF-κB [134]. To add complexity, our HNSCC
cell lines show high expression of Iκ-Bα and IKK-β naturally. Both molecules are
feedback controlled by NF-κB so that methods looking for phosphorylation of Iκ-Bα
and a change in mRNA expression of the two regulators would be better suited for
an in-depth analysis. Although the precise sites of action have yet to be established
we showed that all agents inhibited NF-κB by means of IKK-β down-regulation in
HNSCC. NF-κB is sure to play a critical part in various systems of cell metabolism
and certainly carcinogenesis. However, one has to be cautious to generalize its function as solely unidirectional, for literary evidence is exhaustive in proving its variety
of function in dierent cell contexts.
4.2 Innate Immunity and Immune Escape: Role of
TLR3 and the Microenvironment in HNSCC
In correlation with the inhibition of NF-κB, decreased levels of cyclin D1 were noted
with all compounds. Thus, the growth inhibitory eects on HNSCC are also mediated through the inhibition of NF-κB. It comes as no surprise that novel NF-κB
inhibitor EPs 7630 also reduces the expression of cyclin D1. C-Myc was also notably
down-regulated by all agents and concurs with the majority of the reports about its
decreased expression upon NF-κB inhibition. We showed earlier, that TLR3 is an
important inductor of c-Myc dependent cell proliferation in HNSCC and that TLR3
inhibition led to a decreased proliferation in HNSCC [86]. Here we present for the
rst time that NF-κB inhibition leads to decreased TLR3 expression in NF-κB and
contributes to diminished HNSCC proliferation without altering its cytoplasmatic
67
4 Discussion
localization.
Along with the expression of NF-κB and TLR3, cytokines constitute another important hallmark in the promotion of HNSCC. We display a possible relationship
between TLR expression and cytokines in HNSCC under NF-κB inhibiting conditions. We could clearly show, that suppressed NF-κB led to a decreased level of
prominent cytokines IL-6 and IL-8, which play central roles as dierentiation and
growth factors of tumor cells [159, 160] and in angiogenesis [186], respectively. Although potency of suppression varied among the dierent inhibitors, every agent
was signicantly able to reduce IL-6 and IL-8 levels. These ndings are supported
by another recent study showing that application of Curcumin down-regulates IL-6
and IL-8 via inhibition of IKK in HNSCC [187]. The striking importance of these
ndings is emphasized by another recent study which found the same principle of
IL-6 and IL-8 down-regulation in renal cell carcinoma [182]. It was encouraging that
the eect was not only found in vitro but in vivo as well. In HNSCC it has been
shown that IL-6 directly inuences cell proliferation and the invasion potential as
the rst step of tumor metastasis [188]. IL-6 is so central to HNSCC promotion
that it has been proposed as a valuable biomarker for predicting recurrence and
overall survival among HNSCC patients thus allowing for earlier identication and
treatment of disease relapse [189].
Less prominent HNSCC cytokines like GM-CSF are also known to act in an immunosuppressive fashion, thereby promoting tumor progression and growth of HNSCC [190]. The decreased levels under NF-κB inhibition in our study support this
notion. It is important to realize that HNSCC in general tend to be skewed toward
a TH 2 cytokine prole, with high levels of IL-4, IL-6 and IL-10 [54]. But this shift
is incomplete due to a parallel increase in TH 1 cytokines IL-2 and GM-CSF and
decreased IFN-γ [191]. Indeed, ASA and Curcumin were able to increase levels of
IFN-γ in our studies, suggesting a possible mechanism of HNSCC growth limitation.
However, solid tumors and cell lines expressed only baseline levels of IFN-γ . In addition Dexamethasone, EPs 7630 and Celecoxib suppressed IFN-γ even further. A TH 2
shift in HNSCC might thus merely show the presence but not the extent of disease.
In addition, it is of note that apart from solid IL-6 and IL-8 expression, HNSCC cell
line PCI-1 featured IL-10 levels that rose under incubation with ASA, Curcumin
and Dexamethasone (data not shown). This is in contrast to BHY, where IL-10
levels solely remained the same under NF-κB inhibition. Once again, this might be
due to the individual tumor microenvironment where an anti-inammatory and theoretically protective cytokine like IL-10 might be hijacked by its TH 2 counterparts
with subsequent inability to maintain its beneciary mechanisms.
68
4 Discussion
4.3 Identication of a Novel NF-κB Inhibitor: EPs
7630
For the rst time we were able to show that EPs 7630, a natural chemopreventive
compound from Pelargonium sidoides was able to inhibit HNSCC proliferation in
vivo. EPs 7630 is a major ingredient of a commonly available over-the-counter medication that is normally used for treatment in acute bronchitis. It is appreciated for
its clinical anti-inammatory and natural antibiotic eects. Little is known about
the way EPs 7630 unfolds these properties. For the rst time we present that at
least part of its action is driven by the inhibition of NF-κB and thus EPs 7630
complements the list of more than 785 known NF-κB inhibitors [114]. It seems
to be certain that at least part of the inhibitory eect stems from the inhibition
of IKK-β and Iκ-Bα. Consisting of six main groups of constituents, namely unsubstituted and substituted oligomeric prodelphinidins, monomeric and oligomeric
carbohydrates, minerals, peptides, purine derivatives and highly substituted benzopyranones [143], this does not preclude for other possible mechanisms of action.
Further studies should thus aim to evaluate which particular constituent of EPs
7630 is major contributor to NF-κB inhibition.
Our ndings might not only pave the way for a better understanding of its actions
but could alter the way chemopreventive agents like Curcumin and EPs 7630 are
viewed as beneciary additions to established therapy regimens.
4.4 Inuence of
Mycoplasma spp.
on HNSCC
Mycoplasmas, the smallest free-living, self-replicating bacteria with diameters of 200
to 800 nm, have long been known to be associated with human diseases, for example Mycoplasma pneumoniae as typical pathogenetic factor in community acquired
pneumonia. Apart from this, Mycoplasma is a frequent contaminant of cell cultures
in research laboratories [192]. The need for detection of contamination remains
highly underestimated and inadequately addressed [193, 194]. For the rst time, we
were able to show the inuence of Mycoplasma contamination on cell cultures of
HNSCC and other cancer cell lines. We were able to exemplify that contamination
does indeed change the natural behavior of cell culture responses. Although we were
only able to show altered protein expressions it is far from speculation that contamination might bias results from cell culture experiments in general. There is clear
indication for the potential of these microbes to blur results of scientic studies and
thus calls for cautious judgement in this regard.
69
4 Discussion
In our specic setting cell cultures contaminated with Mycoplasma served as a natural surrogate for models of chronic inammation. It is well known that the My-
coplasma lipoprotein, whose N-terminal structure is an important factor in inducing
immunity and distinguishing Toll-like receptors (TLRs) [195] is thus able to modulate the host immune system. Our results clarify that Mycoplasmas are able to
signicantly alter the natural protein expression of HNSCC cell lines. A recent
report illustrates that persistent exposure to Mycoplasma spp. initiates the transformation of human prostate cell lines from prostatitis to malignancy, providing
further evidence for the linkage of inammation-driven carcinogenesis [196].
It is generally accepted that TLR1, 2 and 6 are directly involved in recognition of
PAMPs of Mycoplasmas [197]. Not all cell lines in our experiments responded this
way, however. This might be explained by the uncertainty about the duration of
exposure to Mycoplasma before protein preparation and the unknown dose of exposure. In cell lines which responded with increases in TLR1, 2 and 6 one might
expect a concurrent rise in secretion IL-8 and other inammatory cytokines as has
been seen in prostate cell lines and epithelial cells from the genital area that had
been exposed to Mycoplasma hominis et genitalium and subsequently showed an
increase in NF-κB activity. Further studies should evaluate if the microenvironment
of HNSCC reacts in the same way as the prostate cells, something we have not
examined in this study.
Although only a selection of our cell lines had a TLR 1, 2 or 6 reaction, all strongly
responded with an increase in TLR4 upon Mycoplasma contact. TLR4 has also been
reported to be triggered by lipopolysaccharides (LPS) of cell walls of gram-negative
bacteria and has been reported in HNSCC before [81]. TLR4 seems to have the same
eect on HNSCC as TLR3. Just recently a small study established TLR4 as being
consistently expressed on 39 tumors from patients with solid tumors of HNSCC,
HNSCC cell lines, and normal mucosa. The TLR4 expression intensity correlated
with tumor grade. LPS binding to TLR4 on tumor cells enhanced proliferation, induced nuclear NF-κB translocation, and increased production of IL-6, IL-8, VEGF
and GM-CSF thus protecting the tumor from immune attack by inammatory stimulation [198].
In this context we can only propose that the trigger of Mycoplasma exposure to our
cell lines might lead to an auxiliary and general defense switch to either immune
surveillance by a change in downstream cytokine production. Further studies should
not only clarify this hypothesis but should also shed a light on the circumstances of
the general TLR4 upregulation in response to Mycoplasma.
70
4 Discussion
4.5 Outlook: Hopes and Pitfalls in NF-κB
Inhibition and Immunotherapy
As pointed out, current therapy regimens for HNSCC are insucient and thwarted
by undesired adverse eects. This calls for alternative methods and strategies of
treatment. For their supercial growth HNSCC are easily accessible for care and
control and thus represent a suitable entity for immunotherapeutic approaches. HNSCC are richly inltrated by immune cells, which are severely impaired in function
by the altered milieu of the tumor. Several dierent receptors (e.g. TLR3), mediators (e.g. MAP-kinases) and subsequent transcription factors like NF-κB are
involved in the genesis and proliferation of HNSCC and the synthesis of its tumor
microenvironment. This not only holds true for the solid tumor itself but also for
the immune cells in the vicinity of the tumor.
The latest eorts of immunotherapy in HNSCC have, among others (see Table 4.1 on
the next page), primarily targeted the Epidermal Growth Factor-Receptor (EGF-R),
which is expressed on almost all HNSCC with high incidence. Its role in carcinogenesis is well established and its expression correlates with a bad prognosis and relative
therapy resistance [217, 218]. Unfortunately, the use of the specic antibody Cetuximab alone has not reached substantial benecial clinical outcomes. In combination
with additional chemotherapy, however, it increased the rate of response in previous
non-responders. In contrast, some patients benetted tremendously and had unexpectedly long survival times [219]. We propose that EGF-R non-responders could
possibly use the TLR3 pathway to escape the immune system for its high resemblance in signal transduction with EGF-R, so that a concomitant block of EGF-R
and TLR3 could improve the response rates of previous non-responders [86]. In addition, our group has already conducted intensive research on the inuence of HNSCC
on immune cells like plasmacytoid and myeloid dendritic cells, B-lymphocytes, natural killer cells and regulatory T-lymphocytes. We demonstrated that solid tumors
massively undermine the expression of TLRs in HNSCC and modulate essential
functions of these cells [87, 220222]. In accordance with the knowledge about the
EGF-R signal cascades, our eorts resulted in the proposition of a working model
as shown in gure 4.1 on page 73.
It postulates recognition of immune cells by the tumor cells via dierent surface
receptors like TLRs and EGF-R. This is followed by secretion of TH 1 cytokines by
inltrating immune cells and leads to defense and evasion processes by the tumor.
These stimuli assemble an army of transcriptional and biosynthetic cascades with
71
4 Discussion
Active unspecic immunotherapy
Bacille Calmette Guérin
Levamisole
(articial imidazothiazol derivative)
Picinabil (OK-432)
(attenuated vaccine of group A streptococci)
DNA-hsp65
(mycobacterial heat shock protein)
Donaldson [199]
Taylor [200]
Wanebo [201]
Kitahara [202]
Michaluart [203]
Active specic immunotherapy
Vaccine from dendritic cells and
apoptotic tumor cells
Whiteside [204]
Tumor-DNA based vaccines
Whiteside [204]
Autologous tumor cells
and IL-12 secreting broblasts
Tahara [205]
Active unspecic immunotherapy
IL-2 (locally)
IL-2 expressing plasmids
IFN-γ (systemic)
IFN-α (systemic)
Cytokine-Mix IRX2 (locally)
Cortesina
Vlock
Wollenberg
Ikic
Vlock
[206]
[207]
[208]
[209]
[210]
Park [209]
Barrera [211]
Passive specic immunotherapy
Anti-EGFR
Anti-CD44v6
Tumor-associated antigenspecic T-cells
Radio-immunotherapy
Bier [212]
Soulieres [213]
Riechelmann [214]
To [215]
Stroomer [216]
Table 4.1: Overview of Immunotherapies in HNSCC
72
4 Discussion
Fcy-R / TLRs
Tumorpromoting Cytokines
(e.g. IL-1)
?
TH1 Cytokines
?
Immune
cell
SOCS
Fcy-R
Fcy-R
TLRs
TLRs
IL-1
TH1 Cytokines
?
?
Cellular
Interaction
?
Immunmodulating/
TH2 Cytokines
?
RNA,
Necrosis
?
TLR3
EGF-R
TRIF
?
p38
?
NF-kB
TLR3
?
?
SOCS
Akt
GM-CSF
IL-4
IL-6
IL-8
IL-10
HNSCC
Immunomodulating
Cytokines
Figure 4.1: Working Model of Immune Escape Mechanisms in HNSCC. Behavior of
immune cells inltrating the tumor is altered by the self-sustained micro-environment
of the tumor and rearrangement of receptors by central mediator NF-κB, preventing
ecient anti-tumor activity.
the induction of the central relay switch, NF-κB. NF-κB activation makes way to
increased secretion of immune-modulating cytokines, thus contributing to the HNSCC micro-environment and hereby altering the behavior of the inltrating immune
73
4 Discussion
cells. The resulting network is self-sustaining and undermines ecient anti-tumor
responses, contributing to tumor proliferation and spreading.
At the very heart of this model stands NF-κB. It is here that information from
receptors is ltered and intracellular signal cascades accumulate. Thus, inhibition
of this central element remains a promising eld of cancer research. But in spite of
all eorts, NF-κB inhibition as actual treatment of oncologic diseases in general and
HNSCC in particular has not yet made the step from bench to bedside.
The ubiquitous presence of transcription factor NF-κB in both pathologic and physiologic processes puts a hold on any simplistic approach. NF-κB cannot be deemed
a solely malignant factor in tumor progression. Its vast signaling network has to be
acknowledged in the specic cell context it works in. For example, it was found that
ablation of IKK-β in enterocytes prevented the systemic inammatory response,
which culminates in multiple organ dysfunction syndrome (MODS) that is normally
triggered by gut ischemia-reperfusion. IKK-β removal from enterocytes, however,
also resulted in severe apoptotic damage to the reperfused intestinal mucosa. These
results not only show the dual function of the NF-κB system, which is responsible
for both tissue protection and systemic inammation, but also underscore the caution that should be exerted in using NF-κB and IKK inhibitors [223]. Moreover, it
is uncertain whether the constitutive NF-κB activation is due to chronic stimulation
of the IKK pathway or a defect in the gene encoding IκBα.
Although each of our reagent has been used in humans in vivo, neither one has proven
eective as lone treatment in HNSCC patients. But whereas single use might not
do the trick, there is hope that some agents may one day serve as adjuvants to
established therapeutic regimens.
For example, Dexamethasone has been cited as an eective combinator in regimens
for multiple myeloma. It has also been successfully used as a sensitizer in HNSCC
cell models which showed better response to treatment when incubated with Bortezomib and Dexamethasone as opposed to Bortezomib alone [181].
In murine models Celecoxib as adjuvant therapy signicantly reduced tumor wasting
in mouse HNSCC and colon cancer models without any eect on tumor growth itself [224]. It, however, strongly enhanced the sensitivity of HNSCC cells to radiation
via the inhibition of NF-κB [225]. In a phase I clinical study incorporating several
solid human malignancies treated with a combination of Mytomycin C and Irenotecan, Celecoxib however failed to improve the clinical outcome [226]. Compared to
many other traditional NSAIDs that are often contraindicated in chemotherapy regimens for their potential to support bleeding from gastrointestinal and hematopoi-
74
4 Discussion
etic toxicities associated with inhibition of the housekeeping cyclooxygenase enzyme
COX-1, selective COX-2 inhibitor Celecoxib was well tolerated in combination therapy with Bortezomib [227].
ASA has been proposed to have good ecacy against human colon cancer [168]. Recently, a new group of promising new anticancer agents has been evaluated, which
are normal NSAIDs coupled to nitrogen oxide (NO). Compared with their parent
compounds, NO-NSAIDs are up to several hundred times more potent in inhibiting
the growth of cancer cell lines and prevent colon and pancreatic cancer in animal
models. Yet again, ASA and its derivatives could not meet the expectations put
forward by the above mentioned studies in HNSCC. They are awed by extensive
adverse reactions which prevent making them an option in prospective therapies.
Curcumin is one of the single most studied chemopreventive agents in the past
decade. Several phase I clinical trials are under way suggesting a potential role
of Curcumin in a myriad of inammatory conditions and cancerous entities [228].
As one of the most promising agents for single therapy Curcumin will remain the
spearhead of alternative therapeutic approaches.
Diculties in the orchestration of eective and specic anti-NF-κB treatments lie
in the disparity of achievable in vitro and in vivo concentrations. For example the
question whether the concentrations of the agents used in our specic study are
clinically relevant and achievable is very important. While other studies with similar agents tend to use shorter periods of time, our cells were exposed for a period
of up to three days. Moreover, in vitro studies strongly skew away from real life
conditions. In vivo, agents are constantly exposed to metabolical breakdown and
might bind to plasmatic proteins which neutralize their activity. As true and relevant tissue concentrations are unclear and hard to assess, it is dicult to correlate
the concentrations in vivo and in vitro. It must be taken into account, however, that
the mechanisms of our stimulants in vivo might be dierent from those observed in
vitro. This emphasizes the need to carry on with phase I clinical trials evaluating the
benets of each of our agents either as single therapy or as adjuvant in combination
regimens.
In conclusion, our studies emphasize the importance of NF-κB as crucial mediator of
inammation-induced tumor growth and progression. At the same time we showed
that NF-κB is important in tumor surveillance and rejection as well. Our ndings
contribute to the understanding of NF-κB dependent signaling at the crossroads of
inammation and carcinogenesis in HNSCC. We clarify that TLR3 is involved in
75
4 Discussion
HNSCC growth and that its actions are opposed by known and newly discovered
NF-κB inhibitors. We were able to further dene the HNSCC microenvironment as
a major inuence on tumor progression. As commonly accepted, the role of NF-κB
and the tumor microenvironment are cell type specic and show altered behavior
in dierent context. Attempts to genuinely understand the many and ambiguous
roles of NF-κB have led and will lead to the discovery and development of new
therapeutic options like EPs 7630. Future immune therapies will have to incorporate
all aspects of carcinogenesis and tumor promotion to signicantly benet the still
dismal prognoses in HNSCC patients.
76
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Appendices
98
German Summary
Einführung
Das Plattenepithelkarzinom des Kopf-Hals-Bereiches (HNSCC) ist der sechsthäugste Tumortyp weltweit mit einer jährlichen Inzidenz von ca. 13.500 allein in Deutschland. HNSCC zählen zu den Tumoren mit einer äuÿerst schlechten Prognose. Als
Hauptrisikofaktoren für die Entstehung von HNSCC werden Tabak- und Alkoholkonsum angesehen, die zusammen mit bakteriellen sowie viralen Infektionen im Sinne
chronischer Entzündungen möglicher Ausgangspunkt für die Entstehung malignen
Tumorwachstums sind. Ein Schlüsselrolle am Relais von Entzündung und Tumorentstehung hat der Transkriptionsfaktor NF-κB inne. Die Bedeutung von NF-κB
für die Entstehung, Proliferation und Angiogenese von Tumoren ist mittlerweile unbestritten. NF-κB ist in der Lage, Apoptose zu inhibieren und aktivierend auf die
Expression von Proto-Onkogenen wie beispielsweise c-Myc und cyclin D1 zu wirken. Die Funktionen von NF-κB sind jedoch sehr vielfältig und zellspezisch. So
kann die Inhibierung von NF-κB sowohl Apoptose bewirken als auch zur spontanen Tumorenstehung führen. Dysregulationen von Transkriptionsfaktoren wie NF-
κB sind maÿgeblich in verschiedenen Aspekten der Onkogenese involviert. Häuger
Ausgangspunkt dieser Signalkaskaden sind membranständige Rezeptorproteine wie
die Familie der Toll-like Rezeptoren (TLRs), die als Teil sogenannter 'pattern recognition receptors' (PRRs) des menschlichen Immunsystems eine natürliche Barriere
gegen Pathogene wie Viren, Bakterien u.a. bilden. Im Menschen sind 10 verschiedene TLR Proteine bekannt, welche von verschiedenen Zellen des Immunsystems
wie B-Zellen, T-Zellen und NK-Zellen exprimiert werden. Die Expression der unterschiedlichen TLRs wird durch Zytokine reguliert und stellt eine wichtige Verbindung
zwischen angeborenem und erworbenem Immunsystem dar. Transkriptionsfaktoren
wie NF-κB werden durch die Stimulierung von Toll-like Rezeptoren aktiviert, was
schlieÿlich zur Aktivierung einer Vielzahl unterschiedlichster Zielgene führt. Unsere
bisherigen Untersuchungen zeigen, dass TLR3 von HNSCC exprimiert wird und dass
die Expressionslevel von TLR3 und NF-κB in diesen Zellen deutlich korrelieren. Die
Inhibierung der Expression von TLR3 in HNSCC vermindert die Expression von
99
German Summary
c-Myc. Da dieses auch in HNSCC maÿgeblich an Prozessen der Zellproliferation beteiligt ist, weisen diese Ergebnisse eindeutig auf eine tumorrelevante Funktion von
TLR3 in HNSCC hin. Gegenstand dieser Arbeit ist, ob und unter welchen Umständen eine Inhibierung von NF-κB mit den COX-2 Hemmern Aspirin (ASA), Celecoxib, dem Steroid Dexamethason und den chemopräventiven Stoen Curcumin und
EPs 7630 Auswirkungen auf die Proliferation von HNSCC und die Expression von
TLR3 und anderen wichtigen Zielgenen hat. Weiterhin untersuchen wir den Einuss
von Mycoplasmen auf die Proteinexpression in HNSCC.
Material und Methoden
Zunächst wurden die HNSCC Zelllinien BHY und PCI-1 über 72 h mit genannten Stimulanzien inkubiert und die Proliferationsrate mittels MTT-Test festgestellt.
Parallel wurden Extrakte zur Messung der Proteinexpression in der Western Hybridisierung und im ELISA hergestellt. Zellkulturüberstände wurden asserviert und
im Bio-Plex Cytokine Assay auf die Expression von prominenten HNSCC Zytokinen untersucht. Rezeptormoleküle von HNSCC wurden durchusszytometrisch und
immunhistochemisch bestimmt. Der Vergleich von mit Mycoplasmen verunreinigten
und nicht kontaminierten HNSCC Zelllinien wurde mittels Western Hybridisierung
durchgeführt.
Ergebnisse
Alle Stimulanzien sind in der Lage, die Proliferation von HNSCC in einer klaren
Dosis-Wirkungsbeziehung zu hemmen (s. Fig. 3.6 bis 3.7 auf den Seiten 3738).
Im NF-κB ELISA zeigte sich hierbei eine deutliche Inhibierung von NF-κB durch
alle behandelten Reagenzien (s. Fig. 3.19 auf Seite 51). Wie man aus der Analyse der Westernblot Ergebnisse ersehen kann (s. Fig. 3.13 auf Seite 45), wurde die
Inhibierung NF-κBs vor allem durch den IKK-Komplex, den natürlichen Regulator
NF-κBs, gesteuert. Ergebnisse der Kontrollen zeigen eine starke IKK-β und Iκ-Bα
Expression. ASA und EPs 7630 schränkten die Expression von IKK-β am meisten
ein, obwohl die allgemeine Supprimierung allgemein gering war. Dahingegen wird die
Expression von Iκ-Bα durch ASA oder Dexamethason fast komplett aufgehoben. Die
Analyse der NF-κB-Zielgene ist uneinheitlicher. Native HNSCC Kulturen zeigen natürlich hohe Expressionraten der Protoonkogenen c-Myc und cyclin D1. Nur ASA
scheint in unseren Experimenten die Expression von c-Myc unterdrücken zu können.
Dahingegen ist cyclin D1 unabhängig von den Reagenzien gleichmäÿig supprimiert.
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German Summary
Im Hinblick auf die vorherigen Untersuchungen unserer Arbeitsgruppe wurde auch
das Expressionsverhalten von TLR3 unter NF-κB Inhibierung untersucht. Wie aus
den Figuren 3.16 auf Seite 47 und 3.17 auf Seite 48 hervorgeht, konnte eine TLR3Expression durch alle Stoe reduziert werden. Vor allem aber Celecoxib und Curcumin sind in dieser Hinsicht wirksam. FACS Analysen zeigten, dass sich in der Expression von Proliferationsmarker KI-67 und dem Human-Leucocyte-Antigen-A,B,C
keinerlei signikante Unterschiede zeigten. Auallend ist, dass sich in in der Durchusszytometrie der ansonsten transmembranär lokalisierte Zytokinrezeptor CCR7 in
HNSCC vorwiegend intrazellulär ausgeprägt ist. Zytokine haben eine charakteristische Ausprägung in HNSCC und spielen eine spezielle Rolle in der Modulation und
Regulation der Immunantwort. In unseren Untersuchungen zeigten sich repräsentative und Zytokinlevel für IL-6 und IL-8. Es zeigte sich, dass das anti-apoptotische
Zytokin IL-6 von allen Reagenzien herunter geregelt wird. Die suppressive Wirkung
von EPs 7630 und Dexamethason war hierbei am stärksten, wohingegen Curcumin die niedrigste inhibierende Potenz zeigte. Das pro-angiogenetische Zytokin IL-8
wurde von allem Inhibitoren fast gleich stark inhibiert. Von den übrigen untersuchten Zytokinen waren IL-10 und GM-CSF in BHY signikant exprimiert. Hierbei
überstiegen die Level von PCI-1 sogar die Level der sonst stärker exprimierenden
Zellllinie BHY. IL-10 mit seiner vornehmlich anti-inammatorischen Wirkung wurde
entweder in Inkubation mit den Stimulanzien grenzwertig hoch geregelt oder entsprach den Werten der unstimulierten Kontrollen. Der immunsuppressive GM-CSF
wurde von allen Stimulanzien inhibiert. Zusammenfassend werden die potentiell tumorfördernden Zytokine wie IL-6, IL-8 und GM-CSF erfolgreich herunter reguliert,
wohingegen die Expression antitumoraler Zytokine entweder nur minimal inhibiert
oder sogar gesteigert wird. Zur Untersuchung des Einusses von Mycoplasmen auf
HNSCC wurden Expressionsprole mittels Western-Hybridisierung erstellt. TLR1,
normalerweise vorliegend im Heterodimer mit TLR2 wurde in Hlac78, Hlac79 and
HaCat Zellen nachgewiesen. Hierbei waren die Signale in den Zellen mit nachgewiesener Kontamination stärker. TLR3 konnte in den Zelllinien ANT-1, GHD und
Hlac79 nachgewiesen werden, hierbei am stärksten in Hlac79 und GHD. Dabei zeigt
sich in Hlac79 eine höhere Expression in kontaminierten Zellen, wohingegen das Gegenteil in GHD der Fall ist. TLR4 wurde in allen analysierten Linien gefunden. Es
zeigten sich hohe Level in ANT-1, GHD und Hlac79. Kontaminierte Zellen zeigten
hierbei höhere Expressionmuster als native Zellen. Neben TLRs wurde nach Einüssen auf c-Myc, NF-κB und EpCAM gesucht. EpCAM ist ein gut beschriebenes
und in vielen Tumorentitäten stark ausgeprägtes Antigen. Dementsprechend wurde
EpCAM auch in allen untersuchten Kulturen gefunden, mit höchsten Leveln in Ha-
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German Summary
Cat, Hlac78 und PCI-13. Interessanterweise wird EpCAM in kontaminierten Zellen
weniger stark exprimiert. NF-κB wird in allen Zelllinien exprimiert. Mycoplasmen
haben hierbei einen positiven Anreiz für die Expression des Wachstumsfaktors.
Diskussion
Es ist unbestritten, dass Entzündungsprogzesse, hervorgerufen durch chronischen
Alkohol- oder Tabakkonsum, die Entstehung und das Wachstum von HNSCC beeinussen. Dabei sind die genauen Mechanismen dieser Entzündungsprozesse noch
weitgehend im Dunklen. Klar ist, dass Prozesse des unspezischen und spezischen
Immunsystems Hand in Hand gehen. Unsere Arbeitsgruppe hat bisher zeigen können, dass insbesondere TLRs eine grosse Rolle spielen. Sie sind in der Lage, Proliferationsfaktoren wie c-Myc zu aktivieren, durch welche letztendlich das Tumorwachstum stimuliert wird. Im Zentrum dieser Regelkreise steht der Transkriptionsfaktor
NF-κB. NF-κB wirkt hierbei in Abhängigkeit vom Zellkontext unterschiedlich. Wir
konnten zeigen, dass alle unsere Agenzien in der Lage sind, NF-κB zu inhibieren,
und dass dies auch eine Auswirkung auf die vorgeschalteten Zellmoleküle und das
Zytokinprol der Tumorzellen hat. Daraus resultierend führt eine Inhibierung von
NF-κB auch zu einer verminderten Zellproliferationsrate der untersuchten Tumorzellen. Besonders hervorzuheben ist, dass uns in dieser Arbeit erstmals geglückt ist,
EPs 7630 als einen bis dato noch unbeschriebenen Inhibitor von NF-κB zu beschreiben. Abgesehen von den oben genannten Einussgrössen wie Alkohol und Tabak sind
Mikroorganismen des Oropharyngealtraktes Unterhalter chronischer Entzündungsmechanismen. Diesen Zusammenhang untersuchten anhand von Mycoplasma spp.
in diesem Zusammenhang. Der Eekt dieses kleinsten Bakteriums auf Tumorzellen
ist relativ einfach zu simulieren, da Mycoplasma ein häuger Grund für Kontamination in Zellkulturlinien darstellt. Die Proteinanalyse dieser Proben zeigte, dass
Mycoplasmen nicht nur eine Einussgrösse im Bereich der chronisch entzündlichen
Prozesse des Tumorwachstums darstellen. Vielmehr wird diese Kontamination bei
Zellkulturen oft billigend in Kauf genommen, was aufgrund der nachweislich veränderten Expressionmuster zu vermeiden ist. Traditionelle Therapieoptionen von
HNSCC wie die chirurgische oder strahlentherapeutische Bekämpfung des Tumors
sind immer noch mit hohen Rezidivraten und einer schlechten Überlebenszeit vergesellschaftet. Im Bemühen um eine Verbesserung der Heilungschancen von Patienten mit HNSCC rückt eine spezische Immuntherapie daher immer mehr in den
Vordergrund. Der epidermale Wachstumsfaktor-Rezeptor (EGF-R) ist in diesem Zusammenhang gut beschrieben. Hohe Expressionraten von EGF-R gehen mit einer
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German Summary
niedrigen Ansprechrate auf die Therapie und einer allgemein schlechteren Prognose
einher. Die Einführung des spezischen EGF-R-Antikörpers Cetuximab zeigte uneinheitliche Resultate. War die Ansprache bei manchen Patienten gut, so war bei
anderen Patienten dahingegen eine Wirkung zunächst nicht (non-responder) oder
erst nach erfolgter Kombinationstherapie mit anderen Chemotherapeutika nachzuweisen. Vor dem Hintergrund unserer bisherigen Ergebnisse postulierten wir, dass
diese non-responder möglicherweise die TLR3-Signalwege nutzen, um das Immunsystem zu unterwandern. Im Zentrum dieser Mechanismen steht NF-κB, durch dessen
Aktivierung die Selbsterhaltungsprozesse des Tumors verstärkt werden. Da NF-κB
aber auch eine wichtige Rolle in physiologischen Regelkreisen spielt, ist eine reine Inhibierung dieses Moleküls ohne Achtung des zellspezischen Kontextes nicht
möglich. Zwar konnten wir zeigen, dass jeder unserer Metaboliten in der Lage ist,
NF-κB wirksam zu inhibieren und sich dieses auch in den Wachstumsraten der einzelnen Zelllinien widerspiegelt. Weitere Studien müssen aber darlegen, dass diese
Wirkungen auch in komplexeren Modellen bestehen. Von einigen unserer Metaboliten wie Dexamethason oder Celecoxib ist bekannt, dass sie im Zusammenspiel
onkologischer Kombinationstherapien vielfältig einsetzbar sind. So kann man davon
ausgehen, dass aufgrund der fehlenden Spezität von NF-κB-Inhibitoren eine Monotherapie mit einem einzelnen Metaboliten unwahrscheinlich ist. Auch wenn eine
zellkontextspezische NF-κB-Inhibierung eventuell erreicht werden könnte, stehen
technische Probleme wie die Art der Applikation und das Erreichen von lokal wirksamen Wirkstokonzentrationen einer denitiven Therapie (bis jetzt) noch im Weg.
Solange nach immer neuen NF-κB-Inhibitoren wie EPs 7630 gesucht und deren Wirkungsweise analysiert wird, besteht berechtigte Honung nach innovativen Ansätzen
für eine eektive und wirksame Therapie von HNSCC.
103
Acknowledgements
I would like to thank Prof. Dr. Barbara Wollenberg for granting me entrance into
her lab and work group. Her provision of the topic has not only broadened my
conception of research but also left me with so many positive experiences otherwise.
A very special acknowledgement is due to Dr. Ralph Pries. This holds not only
true for his commitment to my topic and his scientic prowess but also for endless
professional and general guidance throughout the years.
My appreciation goes to the entire lab sta and my mostly female fellows for keeping
a most pleasant and friendly atmosphere at all times. I would especially like to
mention Brigitte Wollmann for her introduction into the world of cell culture and
immunohistochemistry. She and Ewelina Szymanski provided their superbly skilful
support in many tedious experiments.
I thank Priv.-Doz. Dr. Andreas Lubienski for his review of this manuscript.
I am grateful to Dr. Willmar Schwabe GmbH & Co. KG for the generous donation
of EPs 7630 extracts.
My family and friends I would like to thank for moral support during times of
procrastination and doubts. I especially thank Marie Douthitt for careful review of
the manuscript.
Last but denitely not least I owe my deepest gratitude to my parents for everything.
104
Curriculum Vitae
Personal Information
Christian Paul Meyer, MD
* 27/12/1981 in Münster/Westf., GER
Employment
since 03/2011
Department of Urology
University Hospital Hamburg-Eppendorf, Hamburg, GER
09/200903/2011
Department of General Surgery
Kantonsspital Baden, Baden, CH
Final Year Electives
06/200807/2008
Cardiology and Endocrinology
Cleveland Clinic Foundation, Cleveland, USA
04/200805/2008
Rheumatology
Rheumaklinik Bad Bramstedt, Bad Bramstedt, GER
02/200803/2008
Colorectal/Hepatobiliary/Transplant Surgery
Addenbrooke's Hospital, Cambridge, UK
12/200701/2008
Orthopedic Surgery and Traumatology
King's College Hospital, London, UK
08/200711/2007
Neurosurgery
University Hospital UK S-H, Lübeck, GER
105
Curriculum Vitae
Academic Studies
03/200508/2009
Doctoral Program and Postgraduate Studies
Department of Otorhinolaryngology, University of Lübeck
10/200411/2008
Graduate Studies
University of Lübeck Medical School
10/200209/2004
Undergraduate Studies
Ruhr-University Bochum Medical Scool
Civilian Service
09/200106/2002
KSHG (Catholic Student Community)
Münster/Westf., GER
Education
08/199206/2001
Advanced Education
Gymnasium Augustinianum, College Preparatory High School,
Greven, GER
08/199806/1999
High School Exchange Year
Northland High School, Columbus, USA
Degrees and Qualications
09/2009
MD, Educational Commission for Foreign Graduates Certicate
06/2009
United States Medical Licensing Exam Step 2 CS
11/2008
Final German Medical State Exam
09/2007
United States Medical Licensing Exam Step 2 CK
04/2006
United States Medical Licensing Exam Step 1
09/2004
Preliminary German Medical Exam
06/2001
German High School Diploma
06/1999
American High School Diploma
106
Curriculum Vitae
Publications
Meyer C,
Pries R, Wollenberg B Established and novel NF-κB inhibitors lead to
downregulation of TLR3 and the proliferation and cytokine secretion in HNSCC,
Oral Oncol. 2011 Jul 9.[Epub ahead of print]
Meyer C, Pries R, Wollenberg B Inuence of Mycoplasma spp.
on the expression of
upstream and downstream targets of NF-κB in HNSCC, Submitted for publication.
Ditz C, Brunswig K,
Meyer C,
Reusche E, Tronnier V, Nowak G Intracranial
melanotic schwannoma: A case report of recurrence with extra- and intradural manifestation two decades after initial diagnosis and treatment, Cen Eur Neurosurg.
2010 Apr 22. [Epub ahead of print]
Meyer C,
Pries R, Wollenberg B Role of transcription factor NF-κB for the tu-
morigenesis of head and neck squamous cell carcinoma, Meeting abstract for the
77th annual congress of the German association of otorhinolaryngology, Mannheim,
2006 (German Abstract).
Fähndrich J,
Meyer C, Niepagenkemper R, Schulze J, Siegers CP Benzene as the
causing agent of secondary acute myeloid leukemia? - A case report, SchleswigHolsteinisches Ärzteblatt 03/2005, pp. 56-58 (Publication in German).
107