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CHAPTER THREE
IN VITRO CELL STUDIES
RESULTS
3.0. INTRODUCTION
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Hitherto the erroneous activation and inactivation of signalling pathways are
characteristic in human cancers. The connectivity of these pathways remains a
central tenet of the prostate biology. The ‘sense’ in which they operate will provide
insights into disease and novel avenues for the development of direct, non-cytotoxic
interventions. The Protein Kinase C (PKC) superfamily continues to be a major
element in prostate neoplasia as a family of targets for classes of tumour promoters
and lipid-dependent.
The complexity of PKC involvement in critical regulatory processes within different
cell-systems has long been apparent. However, certain principles controlling the
activity of different members of the PKC family have only emerged recently. The
domain-specific locations of the autophosphorylation sites on PKC’s are indicative
of distinct roles Amongst the almost twelve members of the family, the novel PKC-
is organizing stochastic signalling pathways with c-Raf-1/PKC- displaying
characteristic morphological properties of malignant transformation (Cacace AM et.
al. 1996, Hamilton M. 2001). PKC- has been implicated in the traffic of β1-integrins
from an intracellular recycling compartment through vimentin oligomerisation
(Ivaska J 2005).
Inappropriate activity of this protein, alone, appears to be sufficient to promote cells
transformation from the benign to neoplastic phenotype. In androgen dependent
LNCaP cells, overexpression of PKC- contribute to androgen independent
expansion in absence of testicular androgens (Wu D 2002 Henttu P 1998). PKC-
has been linked to anti-apoptotic programmes, but the molecular details of the
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downstream pathway(s) remain largely unknown. The oncogenic potential of the
PKC- in response to a variety of stimuli appears to regulate transcription of protooncogene, transcription factors, metabolic pathways, apoptosis, and actin
cytoskeleton rearrangement (Cacace AM et. al. 1993, Li Y. 1996, Ricky D. 2002,
Franklin RB 2000, 1997).
Therefore, objectives of this initial part of the overall study was to investigate the
expression patterns of both RNA and protein level of the PKC- isoenzyme in human
prostate epithelial cell lines, and its relationship with the malignancy. Consequently,
identification of sites and patterns of PKC- gene expression can provide important
indications about this particular gene function. Thereafter, PKC- expression was
analysed to determine its association with tumour suppression and/or tumour
progression.
3.1. RESULTS
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3.1.1. RNA Expression level of the PKC- is increased in human prostate cancer
cells.
RT-PCR analysis was performed to detect the mRNA for PKC- in both benign and
malignant prostate epithelial cell lines. The result was shown in the following figure
(Figure 3.1.1.):
480 bp
1
2
3
4
5
6
7
8
Figure 3.1.1. RT-PCR analysis was performed to detect PKC- mRNA in both benign and malignant
prostate cell lines. Lane 1: DNA size Marker. Lane 2: Positive control HeLa cells. Lane 3: benign
PNT2 cells. Lane 4: malignant LNCaP cells. Lanes 5-7: highly malignant prostate cancer cell lines
PC-3, PC3M and DU 145. Lane 8: Negative control.
To further study the mRNA levels of PKC- expressed in benign and malignant
prostate epithelial cells, Northern Blotting hybridisation and Real-Time RT-PCR
were employed to make quantitative assessment of the mRNA levels.
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3.1.2. Western Blot analysis of the levels of PKC- protein expression in benign
and malignant prostate epithelial cell lines
Protein extracts prepared from different cell lines were subjected to Western blot
analysis to measure the levels of PKC- protein (Figure 3.1.2.A.). Liver Hela cell
line was used as appositive control and the β-actin antibody was used to correct the
possible loading artefact. Western blot analysis detected a clear PKC- band with a
size of 80 kDa (pointed by the arrow in the figure) in all cell lines examined.
Quantitative analysis of the levels in different cell lines was obtained by measuring
the intensities of the bands on the blot. The relative levels of PKC- protein
expressed in each cell line was shown in Figure 3.1.2.B.
A
80kDa
PKC-
β-Actin
HeLa
PNT2
LNCaP
PC 3
PC3M DU145
4.5
B
4
3.5
3
2.5
2
1.5
1
5
Figure 3.1.2. A: Western blot analysis of protein expressed in prostate epithelial cell lines. Liver Hela
cells was used as positive control and the antibodies against the constitutively expressed β-actin
protein was used to correct the possible loading artifacts. B: The bands of PKC- protein in each cell
lines was measured by densitometry as described in method section. The relative levels shown here is
the mean (± SD) of three separate measurements.
As shown in figure 3.1.2.B, the levels of PKC- protein was increased in the
malignant cells. When the level of PKC- protein expressed in the benign PNT2 cells
was set at 1, its level in the weakly malignant cell line LNCaP was increased to 1.3,
further increased to 1.6 in the highly malignant cell line PC3. The level of PKC-
protein expressed in the PC3M cells is 2.3 times of that in the benign PNT2 cells,
and the highest level of PKC- expression was detected in the highly malignant
Du145 cells which expressed 3.8 times of more PKC- protein than the benign PNT2
cells.
3.1.3. Suppression of PKC- expression by RNA interference
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To study the biological significance of the increased PKC- expression in prostate
cancer cells and particularly to investigate whether the elevated PKC- expression
plays an important role in promoting the malignant progression of prostate cancer
cells, RNA interference was used to establish transfectant clones, in which the
expression of PKC- in the highly malignant prostate cancer cells was blocked. The
effect of suppressing PKC- expression on tumourigenicity of the recipient cells was
assessed by comparing the malignant characteristics between the control and the
PKC--suppressed cells.
To suppress PKC- expression by RNAi, two short interference molecules was
synthesized as described in the method section. Before performing stable DNA
transfection, transient transfection of highly malignant Du-145 and PC3M cells with
two selected RNAi molecules either singly or jointly to identify the most effective
way to inhibit the PKC- expression. The levels of PKC- expression in different cell
lines were assessed by Western blotting analysis.
The results from Western blot analysis of the transient transfection of RNAi
molecule-1 was shown in Figure 3.1.3.i.A. and 3.1.3.i.B. When the short RNAi
molecule-1 was transfected into Du-145 cells and PC3M cells respectively, it
produced a very small reduction 40% and 45% respectively in the level of PKC-
protein expression.
A
siRNA/PKC-
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DU 145
DU 145
Molecule 1
B
siRNA/PKC-
PC3 M
PC3 M
Molecule 1
Figure 3.1.3.i. A. Western blotting analysis showing the PKC- reduction in transient transfectant DU
145 cells by siRNA molecule 1. B. The PC3M cells transient transfectant with siRNA molecule 1 the
relative protein PKC- reduction as was verified by Western blotting analysis
The results from Western blot analysis of the transient transfection of RNAi
molecule-2 were shown in Figure 3.1.3.ii.A and 3.1.3.ii.B. When the short RNAi
molecule-2 was transfected into Du-145 cells and PC3M cells respectively, it
produced some reduction (Vasso: give exactly the relative levels) in the level of
PKC- protein expression.
A
siRNA/PKC-
DU 145
DU 145
Molecule 2
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B
siRNA/PKC-
PC3 M
PC3 M
Molecule 2
Figure 3.1.3.ii. Western blotting analysis was verified the relative PKC- protein reduction levels
with siRNA molecule 2 in both DU 145 cells (A) and PC3M cells(B).
Most effective suppression of PKC- expression was achieved by the transfection of
both RNAi-1 and RNAi-2 molecules in a jointly manner in both PC3m and Du145
cells. The levels of the control cells established by transfecting the parental PC3M
cells with plasmid DNA alone and 10 separate RNAi transfected clones isolated from
the joint transfection of molecules 1 and 2 were shown Figure 3.1.3.iii.
siRNA/PKC-
DU 145
DU 145
Molecule
1/2
siRNA/PKC-
PC3 M
PC3 M
Molecule 1/2
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Amongst the 10 transfectant clones, the levels of PKC- expression were reduced by
40-85% (or 15% to 60% of the level detected in the control cells) in comparison with
the control transfectants.
The levels of the control cells established by transfecting the parental PC3M cells
with plasmid DNA alone and 10 separate RNAi transfected clones isolated from the
joint transfection of molecules 1 and 2 were shown Figure x (Vasso: if possible, give
the Western blot figures in the following space):
As shown in Figure 2.3.7, the RNAi transfectant clone (PC3M/PKC-RNAi-1),
which was originated from PC3M cells, exhibited a 6-fold reduction in the level of
PKC-, in comparison of the that in parental cells.
PKC-
-Actin
PC3M
Control cells
PKC--1/Si
Figure 3.1.3. Western blot analysis of PKC- expression in patrental, control and siRNA-transfectant
cells. The possible loading artifacts were corrected by incubating the same blot with antibody against
-Actin.
The levels of the control cells established by transfecting the parental Du145 cells
with plasmid DNA alone and 10 separate RNAi transfected clones isolated from the
joint transfection of molecules 1 and 2 were shown Figure y (Vasso: give the
Western blot figures in the following space):
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Amongst the 10 transfectant clones, the levels of PKC- expression were reduced by
?-?% (or ?% to ?% of the level detected in the control cells), in comparison with the
control transfectants.
As shown in Figure 2.3.10, both RNAi transfectant clones DU145/ PKC--2/Si and
DU145/ PKC--3/Si, which were originated from parental Du145 cells, exhibited a
x- and y- fold reduction respectively in the level of PKC-, in comparison of the that
in parental cells (Vasso: give exact numbers).
A
PKC-
-Actin
DU 145
Control
Cells
DU 145/ DU 145/
PKC--3/ PKC--2/
Si
Si
(Vasso: give the Western blot result Chart here;)
Figure 2.3.10. A: Western blot analysis of the levels of PKC- protein in parental Du145 cells,
control transfectants and in different Si transfectant clones. Normalisation of equal loading was
achieved by incubating -Actin antibody on the same blot. B: Relative levels of PKC- in parental
DU 145 cells, control transfectants and Si-transfectant clones.
3.1.4. The effect of suppressing the expression of PKC--3 on cell proliferation.
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To investigate the effect of suppressing the expression of PKC--3 on cell
proliferation, the in vitro proliferation assay was performed to assess the proliferation
rate of the control transfectants and the RNAi transfectant clone PC3M-PKC--1/Si
and DU 145-PKC--2/Si. To measure the proliferation rate of different cells, the
same number of cells from each cell line was cultured in triplicate and counted each
day for 15 (Vasso: 7 days would enough) days. The proliferation rats of each cells
was shown in Figure 3.1.4.
(Vasso: you need to change the following figure; using curves for the control
transfectants and Si clones of both PC3M and Du145. giving results of 7 day points
with mean ± SD of 3 measurements)
50000
40000
30000
20000
10000
0
DU 145/
RNAi
DU 145/PKC-2/RNAi
Figure 3.1.4. (Vasso: change this figure)The growth rate curves (7 days) of the PC-3M control
transfectants (Black), PC3M-PKC--1/Si (Blue), Du145 control transfectants (green) and DU 145PKC--2/Si (gray).
As showed in the figure, the number of Si transfectant clone DU 145/PKC--2/RNAi
cells at the end of the 7 days was x (Vasso: give number ± SD). The cells of the
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control transfectants DU 145/Vector cells was y (Vasso: give number ± SD); the
proliferation rate of DU 145/PKC--2/RNAi was significantly (Student T-test, P<?)
lower that that of the controll transfectant DU 145/Vector cells. The number of Si
transfectant clone PC3M-PKC--1/RNAi cells at the end of the 7 days was x (Vasso:
give number ± SD). The number of the control transfectants PC3M /Vector cells at
the end of assay was y (Vasso: give number ± SD); the proliferation rate of PC3MPKC--1/RNAi was significantly (Student T-test, P<?) lower that that of the control
transfectant PC-3M/Vector cells. Therefore, the proliferation assay results showed
that suppression of PKC- expression in both highly malignant prostate cancer cell
lines PC3M and Du145 greatly inhibited their proliferation rates in vitro.
3.3. DISCUSSION
Prostate cancer is a multistep process, accumulated by alterations in tumoursuppressor genes and dominant oncogenes. Increased activity of oncogenes defines
autonomous deregulation of cells growth and tumour formation. Autonomous
enhanced PKC- expression was associated with cancers of prostate, breast, skin,
kidney, thyroid, blood, brain, and colon (Cornford P 1999, Lavie Y 1998, Marks F
1995, Reddig PJ 2000, Engers R 2000, Knauf JA 1999, Mayne GC 1998, Sharif TR
1999, Perletti GP 1998). In the present study using human prostate epithelial cell
lines with a variety degree of malignancy we show by RT-PCR analysis that PKC-
is abundant expressed at the RNA level in all prostate epithelial cells. Its protein
expression level is increased in malignant cells, associated with malignant
progression in prostate cell lines. In particular, the DU 145 and PC3M malignant
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prostate cells appear the maximum protein expression level, and hence PKC- might
play an important role in prostate cancer development.
Although the protein expression level of PKC- is not related with the degree of the
malignancy. These results favouring the following hypothesis: First: PKC- through
its actin binding motif at the C2 domain can binds actin regulated cytoskeleton
rearrangement and cells morphogenesis suggesting that its expression appears to be
cell morphology and specific phenotype. Second, variety stimuli including fatty
acids, growth factors and hormones such as the androgen receptor can modulate
PKC’s function. The androgen receptor regulates PKC’s activity both in vivo and in
vitro (Gavrielides MV, 2006). Both PC3 and PC3M prostate malignant cells do not
expressed androgen receptor, and have been characterized as androgen unresponsive.
In addition the DU 145 cells restore androgen receptor function. Consequently PKC overexpression might related to androgen receptor status in DU 145 cells. Third, it
is plausible the DU 145 cells may have been derived from metastases that had grown,
possibly via a selection process, to be relatively homogenous for PKC- positive
cells.
Tumour development is the major cause of morbidity and mortality in patients with
prostate cancer. It is critical to identify oncogenes and understand how they are
responsible for inducing specific aspects of the malignant phenotype, and to allow
improvement of clinical detection and management. The inactivation of gene
function by reverse genetics is important for elucidating gene function and could also
have a great impact on the treatment of diseases initiated by aberrant gene
expression. Among the employed strategies, the recently discovery of RNA
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interference has offered an additional way to inhibit the expression of virtually any
gene in living cells. Therefore, to further investigate the biological significance of
PKC- elevated level in malignant prostate cell lines its expression was suppressed
by silence interference RNA technology.
PKC- unique structure is comprised from two distinct functional domains, the
regulatory and the catalytic domain. The regulatory domain is response to a second
messenger and is important for its intracellular targeting and interactions with other
proteins (Schechtman and Mochly-Rosen, 2001). The catalytic domain is responsible
for PKC- regulation and its subcellular distribution. In this study, the design siRNA
was silence at different points both the two PKC- domains. Transient transfectant
high malignant PC3M prostate cells and DU 145 androgen independent cells, with
one of the chosen siRNA targets only decline the PKC- protein level. It was not
plausible to clarify the reason. It is likely that the efficacy of RNAi depends on the
region where siRNA targets. Another possibility is that degradation rate of PKC- is
too slow to see the reduction of its amount by Western blotting during time period of
this investigation. Alternatively, a certain type of PKC may escape from the RNAi
pathway conserved in prostate epithelial cells.
Moreover, double transfection of the PC3M and DU 145 cells with the two siRNA
targets signify the maximum PKC- inhibition. Western blotting analysis with
phosphorylated PKC- antibody at position Ser729, shows that the enzyme abolishes
complete its functional activity. Phosphorylation of PKC- at Ser729 is related with
increased enzymatic activity and important for its proper intracellular localization
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and function. These results suggest that the siRNA technology is effective abolished
PKC- activity in PC3M and DU 145 cells.
Deregulated cell growth occurs as a result of perturbed signal transduction cellular
signals that modulate or alter cellular behaviour or function (Hunter, T. 2000). In
normal prostate gland, signalling needs to be precisely coordinated and integrated at
all times, whereas the properly regulated differentiated signals are critical for
preventing cells growth and oncogenesis. Therefore, to further analyse if the
complete reduction of PKC- activity triggers the malignant androgen independent
cellular growth the MTT proliferation assay was employed. The DU 145 and PC3M
stable transfectant with siRNA as well as the control cells were culturing under the
standard culture conditions as was described in section 2.1.3.i., and its growth
properties were monitoring for up to seven days. Present study results provides
several independent lines of evidence supporting the hypothesis that PKC-
expression may be sufficient to maintain prostate cancer growth and survival.
It has been reported that activation of PKC- in prostate malignant cell lines
modulate downstream effectors molecules such as mitogen-activated protein kinases,
c-Myc, and caveolin-1and contribute to malignant transformation (Wu D 2002a,
2002b). Force PKC- expression in prostate LNCaP cells and CWR22 xenografts
was sufficient to confer Bax conformational rearrangements that are important for
Bax oligomerization, mitochondrial integration, and cytochrome c release (McJilton
MA 2003. Considered these lines of results in their entirety were put forward the
notion that an association of PKC- with Bax may neutralize apoptotic signals
propagated through a mitochondrial death-signalling pathway.
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Furthermore, the complex signalling network operating downstream of PKC-
energetically advances the survival and proliferation of prostate cancer cells. Studies
in adherent prostate cancer cells demonstrated that a mutually reinforcing signalling
loop sustained by the activation of beta1 integrins, PKC-, and PKB/Akt (Wu D
2004). Activation of PKB/Akt is correlated with proliferation in human prostate
tumours as estimated by the expression of the cell proliferation antigen Ki67 (Ghosh
PM, 2005).
Present study results have shown that PKC- expression is elevated in malignant
metastasize prostate epithelial cells. The siRNA machinery can effectively reduce the
level of PKC- expression. Diminish PKC- expression significantly inhibits the
growth rate in DU 145 and PC3M malignant cells. In the light of recent reports that
the multiplicity of PKC- signals transduction force a variety of down stream
effectors driving to anchorage cells growth its overexpression may be an important
cause of malignant prostate cancer progression. However, the complicated, PKC-
signal transduction signalling pathways contain promising new targets of anticancer
treatment that otherwise remains elusive for this particular malignant disease
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