Download Polyethyleniminemediated PUMA gene delivery to orthotopic oral

ORIGINAL ARTICLE
POLYETHYLENIMINE-MEDIATED PUMA GENE DELIVERY TO
ORTHOTOPIC ORAL CANCER: SUPPRESSION OF TUMOR
GROWTH THROUGH APOPTOSIS INDUCTION IN SITU AND
PROLONGED SURVIVAL
Cheng-Chang Yeh, MS,1 Hsiao-Ling Hsieh, PhD,2 Jihjong Lee, DVM,3 Yi-Hua Jan, MS,2
Tsung-Ching Lai, MS,2 Chi-Yuan Hong, DMD, PhD,4 Michael Hsiao, DVM, PhD,2
Mark Y. P. Kuo, DDS, PhD1,4
1
Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei, Taiwan
Genomics Research Center, Academica Sinica, Taipei, Taiwan. E-mail: [email protected]
3
Graduate Institute of Veterinary Medicine, College of Biological Resources and Agriculture,
National Taiwan University, Taipei, Taiwan
4
Department of Dentistry, National Taiwan University Hospital, National Taiwan University, Taipei, Taiwan
2
Accepted 16 June 2010
Published online 24 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/hed.21555
Contract grant sponsor: National Science Council; contract grant
sponsor: Academia Sinica.
The first 3 authors contributed equally to this work.
C 2010 Wiley Periodicals, Inc.
V
used, resulting in positively charged polyplexes that
interact with negatively charged cell surfaces via adsorptive endocytosis.2 Gene transfer using PEI is significantly more potent in cells bearing mitotic
activity, thus contrasting with adenoviruses, which
are less dependent on cell cycle stage at the time of
transfection. This higher efficiency of gene transfer
using PEI in proliferative cells could reveal particularly interesting results in heterogeneous tumor tissues, such as head and neck tumor tissue, containing
both proliferating tumor cells and differentiated nonproliferating stromal cells.
PUMA was initially identified as a gene activated by
p53 in cells undergoing p53-induced apoptosis.3,4 It is
known to interact with Bcl-2, and function to induce
cytochrome c release, thereby activating caspase-9 and
caspase-3.4,5 Exogenous expression of PUMA resulted in
rapid and complete apoptosis in a variety of cancer cell
lines, regardless of the p53 genotype.3–5 Expression of
PUMA also inhibits cell growth, reducing colony formation even more efficiently than wild-type p53 or Bax.3
Importantly, studies of PUMA knockout mice indicate
that PUMA is an essential mediator of p53-dependent
and -independent apoptosis in vivo.6 Ito et al7 demonstrated that direct injection of PUMA DNA with lipofectamine 2000 into subcutaneous U87-MG malignant
gliomas in nude mice efficiently suppressed the growth
of subcutaneous tumors. It has been shown previously
that PUMA induces apoptosis and chemosensitization
in head and neck SCC and esophageal cancer cell lines
more efficiently compared with p53.8,9 Therefore,
PUMA-based gene therapy can potentially be used as a
new strategy for anticancer therapy.
In the present study, we assessed PEI as a nonviral vector system for gene delivery, to investigate the
effect and mechanism of PUMA gene transfer on
Polyethylenimine-mediated PUMA Gene Delivery
HEAD & NECK—DOI 10.1002/hed
Abstract: Background. PUMA (a p53 up-regulated modulator of apoptosis) is induced by p53 tumor suppressor and
other apoptotic stimuli. It was found to be a principal mediator
of cell death in response to diverse apoptotic signals, implicating PUMA as a likely tumor suppressor.
Methods. In this study, we examined the efficacy of targeted PUMA gene therapy in human oral cancer (SAS) cells
using polyethylenimine (PEI)-mediated transfection for gene
delivery.
Results. Exogenous expression of PUMA in SAS cells
resulted in apoptosis with cytochrome c release, activation of
caspase-3 and -9, and cleavage of PARP. Gene delivery of
PEI/PUMA in SAS xenografts induced apoptosis and resulted in
significant reductions (60%) of tumor growth in vivo. Furthermore, we have shown that PEI-mediated PUMA gene therapy
prolonged survival of animals with orthotopic SAS oral cancers.
Conclusions. Taken together, these results indicated that
PUMA gene therapy via PEI delivery could be a promising method
C 2010 Wiley
for the treatment of oral squamous cell carcinoma. V
Periodicals, Inc. Head Neck 33: 878–885, 2011
Keywords: PEI; PUMA; oral cancer; apoptosis; gene therapy
Oral
cancer, most commonly squamous cell carcinoma (SCC), is the leading cause of cancer-related
deaths in India and other South Asian countries. Polyethylenimine (PEI) is a highly water soluble, positively charged, synthetic polymer that has been used
successfully for gene delivery both in vitro and in
vivo.1 Complexes of PEI with DNA are efficiently
taken up by cells when relatively high N/P ratios are
Correspondence to: M. Hsiao and M. Kuo
878
June 2011
human oral SCC cells in vitro and in an oral SCC
xenograft severe combined immunodeficiency (SCID)
mouse model.
MATERIALS AND METHODS
The human oral squamous cell line SAS was obtained from the Japanese
Collection of Research Bioresources (Tokyo, Japan).
The cell line was cultured in 5% CO2 at 37 C in Dulbecco modified Eagles’ medium (DMEM; Gibco/BRL,
Gaithersburg, MD), supplemented with penicillin
(100 U/mL), streptomycin (100 U/mL), and 10% fetal
bovine serum (Gibco/BRL). pCEP4-PUMA was
obtained from Dr. Bert Vogelstein (Johns Hopkins
School of Medicine, Baltimore, MD). pCMV-DsRed2
and pCMV-Luc were purchased from Clontech Laboratories (Mountain View, CA).
Cell Culture and Plasmids.
Transfection and Flow Cytometry. DNA (5 lg) and
3.75 lg of branched PEI (Sigma–Aldrich, St. Louis,
MO) were mixed in 250 lL of OPTI-MEM and incubated at room temperature for 15 minutes before adding to the cells. Fresh culture medium was added to
the cells 3 hours post-transfection. SAS cells were
transfected with pCMV-DsRed2 and analyzed using a
fluorescent activated cell sorter (FACS, Becton Dickinson, San Jose, CA) with CellQuest software (Becton
Dickinson, Mountain View, CA). Cells were fixed in
ice-cold methanol/phosphate-buffered saline (PBS)
(2:1, vol/vol) at indicated time points and resuspended
in 500 lL of PBS containing 50 lg/mL propidium
iodide (PI) and 20 lg/mL DNase-free RNase A. Cell
cycles were analyzed after 30 minutes of staining on
flow cytometry.
Cells
were fixed in 4% paraformaldehyde, permeabilized in
methanol and stained using TdT-mediated dUTP nick
end labeling (TUNEL) reaction mixture (Boehringer
Mannheim, Indianapolis, IN). Cells were counterstained with PI (1 lg/mL) and visualized under a fluorescence microscope. Apoptotic cells on the paraffinembedded sections were determined using an in situ
cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany). Genomic DNA of the
transfected cells was extracted using Wizard Genomic
DNA Purification Kit (Promega, Madison, WI) in
accord with the manufacturer’s instructions.
TUNEL Assay and Genomic DNA Extraction.
Cells were harvested in lysis buffer (20 mM Tris-HCl at pH 7.4, 150
mM NaCl, 0.5% Nonidet P-40, 1 mM ethylenediaminetetraacetic acid [EDTA], 50 lg/mL leupeptin, 30 lg/
mL aprotinin, 1 mM phenylmethylsulfonyl fluoride
[PMSF]) at indicated times. The cytosolic fraction was
extracted in cytosolic protein extraction buffer (50
mM Tris-HCl, pH 4.5, 5 mM EDTA, 10 mM EGTA,
Western Blot and Luciferase Assay.
Polyethylenimine-mediated PUMA Gene Delivery
0.3% of 2M-E, 5 lg/mL leupeptin, 5 lg/mL aprotitin,
10 lg/mL soybean trypsin inhibitor, 1 mM PMSF).
The cytosolic fraction was further extracted by being
passed through a 27-gauge needle several times before
ultracentrifugation at 55,000 revolutions/minute for 30
minutes at 4 C. Protein concentration was determined
using BCA protein assay reagent (Thermo Fisher Scientific, Rockford, IL) and bovine serum albumin (BSA)
as a standard (BioRad, Hemel Hempstead, UK).
Around 30 lg of the lysates were subjected to 12.5%
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Hybond C super, Amersham,
Arlington Heights, IL). The membrane was then
probed with primary antibodies against p53 (1:1000,
DO-1; Santa Cruz Biotechnology, Santa Cruz, CA),
Bax (1:250; BD Transduction Laboratories, Franklin
Lakes, NJ), PUMA (1:1000; ProSci-Inc, Poway, CA),
cytochrome c (1:1000; BD Biosciences Pharmingen,
San Diego, CA), caspase-9, caspase-3, or PARP
(1:1000; Cell Signaling Technology, Danvers, MA)
overnight, followed by the addition of goat anti-mouse
or anti-rabbit horseradish peroxidase-linked secondary antibodies (1:10,000) (Jackson ImmunoResearch
Laboratories, West Grove, PA). Chemiluminescence
was detected using an ECL kit from Amersham. The
luciferase activity was measured using luciferin (Promega) and a TD-20/20 Luminometer (Turner BioSystems, Sunnyvale, CA).
SAS cells (1 106) were
injected in their flank area of 6-week-old SCID mice.
SCID mice bearing SAS subcutaneous tumors were
injected with 20 lg of pCMV-Luc DNA complexed
with PEI, using PEI/DNA molar ratios of 0.25 and
0.75. Animals were killed 48 hours later, and tumor
masses were analyzed for luciferase activity. For evaluation of the tumor sizes, 1 106 SAS cells were
injected in their flank area of 6-week-old SCID mice.
On the fifth day, the injected mice were divided randomly into 3 groups (5 mice in each group) and
treated every 2 days by intratumoral injection, either
with 20 lg plasmids (pCEP4-PUMA or pCMV-Luc)
complexed with 15 lg PEI or PEI only in 100 lL of a
5% glucose solution. Mouse body weight and the tumor size were measured every 3 days with a caliper.
The volumes of tumors were calculated using the
equation volume ¼ length (width)2 0.5. At day 38
after treatment, the mice were sacrificed and the tumor masses were isolated and weighed. For survival
studies, 1 105 SAS cells were injected into the buccal pouch in the left cheek of SCID mice, to study the
effect on survival. Twenty-four mice were randomized
into 4 groups (No DNA, 5 Vector Control, 1
PUMA, and 5 PUMA) 1 week later to receive a subsequent treatment as described. Animals were
assayed for survival after 180 days. The experiment
was repeated twice with similar results.
In Vivo Tumor Model.
HEAD & NECK—DOI 10.1002/hed
June 2011
879
FIGURE 1. Evaluation of the transfection efficiency in SAS cells with PEI/DNA complexes. (A) Luciferase reporter gene expression is
a function of the PEI/DNA ratio. SAS cells were transfected with pCMV-Luc (5 lg per well) and 0 to 20 lg of 25 kDa PEI. The data
are shown as RLU of the luciferase activity per lg of total cellular protein (mean SD, obtained from 3 experiments). pCMV-DsRed2
transfection in SAS, Ca9-22, and HSC3 cells (PEI/DNA weight ratio of 0.75) was visualized using fluorescent microscopy (B) and the
transfection efficiencies were evaluated using flow cytometry analysis (C). PEI, polyethylenimine; RLU, relative light unit; DsRed2, red
fluorescent protein reporter gene. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Statistical Analysis. For the measurement of tumor
size, Kruskal–Wallis tests were used to analyze the
significance of the effect of gene therapy. The data
were expressed as mean values SD. ANOVA was
used to evaluate statistical significance with p < .05
as statistically significant. Survival differences
between groups were compared and their statistical
significance analyzed using the log-rank test.
and HSC3, as measured by both fluorescence microscopy (Figure 1B) and flow cytometry (Figure 1C).
PUMA Induces Apoptosis in Various Cancer Cell
PEI/DNA weight ratios were screened in search for
the optimal transfection condition and lowest cytotoxicity. As shown in Figure 1A, the best transfection efficiency was achieved with a PEI/DNA weight ratio of
0.75 (PEI nitrogen/DNA phosphate ratio equivalent to
6.25). No cytotoxicity was found below a weight PEI/
DNA of 1.0 (data not shown). Oral cancer cells were
also transfected with pCMV-DsRed2 plasmid to evaluate the transfection efficiency using flow cytometry.
The best transfection efficiency was observed in SAS
(40%) compared with the transfections in Ca9-22
PUMA has been shown to mediate the apoptotic response to p53 in colorectal cancer cells.10 We
were interested in whether the PUMA gene can
induce apoptosis in SAS cells. As shown in Figure 2A,
PUMA-transfected SAS cells showed nuclear chromatin condensation and fragmentation with a positive
TUNEL signal. The percentage of cells undergoing
apoptosis was about 37 5.7% in SAS, and apoptosis
was only rarely found among untreated cells (Figure
2B). Similarly, significant apoptosis was also observed
in the other 2 oral cell lines, Ca9-22 and HSC as
shown in Figure 2B. Agarose gel electrophoresis of
chromosomal DNA from PUMA-transfected cells
showed a ladder-like pattern of DNA fragments consisting of multiples of about 180–200 base pairs (Figure 2C). To investigate whether PUMA-transfected
cells show cell-cycle perturbations prior to apoptosis,
the PEI/PUMA-transfected cells were analyzed by
flow cytometry. As shown in Figure 2D, the sub-G1
population increased from 1.99% in the control to
Polyethylenimine-mediated PUMA Gene Delivery
HEAD & NECK—DOI 10.1002/hed
RESULTS
Effects of Different PEI/DNA Ratios on the
Transfection Efficiency in SAS Cells. A range of
880
Lines.
June 2011
FIGURE 2. PUMA induces apoptosis in SAS cells. (A) SAS cells transfected with 5 lg pCEP4-PUMA and 3.75 lg of 25 kDa PEI for
24 hours were visualized by fluorescence microscopy after staining with PI or TUNEL. Note apoptotic cells with nuclear condensation,
fragmentation, and positive TUNEL signal. (B) The percentage of 3 oral cancer cell lines transfected by PEI-pCFP-PUMA undergoing
apoptosis are expressed as the means of 3 independent experiments, and the asterisks indicate that the p values calculated by comparing
with the vector alone are <.01. (C) SAS cells were transfected with 5 lg pCEP4-PUMA at a PEI/DNA ratio of 0.75 for 0 to 48 hours. DNA
from cells exposed at different time points was extracted and electrophoresed in a 1.5% agarose gel containing ethidium bromide. (D) Flow
cytometric analysis of DNA content in SAS cells transfected with PUMA cDNA. Cells were transfected with 5 lg pCEP4-PUMA at the indicated time points. After treatment, cells were fixed and stained with PI, and the cell cycle distribution was examined by flow cytometry.
(E) Flow cytometric analysis of DNA content in Ca9-22 and HSC3 cells transfected with PUMA cDNA. Cells were transfected with 5 lg of
either vector alone or pCEP4-PUMA at 24 hours. After treatment, cells were fixed and stained with PI, and the cell cycle distribution was
examined by flow cytometry. PUMA, p53 up-regulated modulator of apoptosis; PEI, polyethylenimine; PI, propidium iodide; TUNEL, TdTmediated dUTP nick end labeling. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
45.61% in cells transfected with PEI/PUMA for 24
hours, with corresponding declines in G2/M populations. Similarly, a PUMA-induced sub-G1 increase
was also observed in the other 2 oral cancer cell lines,
Ca9-22 and HSC3, as shown in Figure 2E.
Polyethylenimine-mediated PUMA Gene Delivery
PUMA Induces Cytochrome c Release and Caspase
Activation in Oral Cancer Cell Lines. To investigate
whether a mitochondrial pathway is involved in
PUMA-induced apoptosis in SAS cells, we examined
the cytochrome c levels in cytosol. As shown in
HEAD & NECK—DOI 10.1002/hed
June 2011
881
FIGURE 3. PUMA-induced apoptotic gene expression in oral cancer cells. SAS (A) or Ca9-22 (B) PUMA regulates apoptotic gene
expression. SAS cells were transfected with 5 lg pCEP4-PUMA at the indicated time points. After treatment, total cellular proteins or
cytosolic fraction for cytochrome c were extracted and subjected to Western blot analysis. b-Actin was used as an internal control. The
experiment was repeated 3 times with similar results.
Figure 3, PEI/PUMA caused release of cytochrome c
into the cytosol in SAS cells because cytochrome c
release was detected in the cytosolic fraction. We
have also shown the cleavage of procaspase-9 and
procaspase-3 in cells following PUMA gene transfer.
Immunoblotting demonstrated that both caspases
were cleaved into the characteristic active fragments
in a time-dependent manner after PUMA transfection
(Figure 3A). Similarly, cleavage of PARP and activation
of caspase-3 and 9 were also observed in PUMA-transfected Ca9-22 oral cancer cell line (Figure 3B).
PEI/vector control group (p < .001; Figure 5A). At 14
days, the volume of PEI/vector control-treated SCC
increased to >2.6-fold the size measured on the first
day of DNA injection. However, the volume of SCC
injected with pCEP4-PUMA was significantly reduced
(Figure 5B). More apoptotic cells were also observed in
tumor cryosections from PUMA-transfected cells stained
with TUNEL compared with the control cells, as shown
in Figure 5C.
Introduction of PEI/PUMA on Orthotopic SAS Oral
Apoptosis In Vivo. To analyze the efficiency of gene
delivery after direct injection of PEI/DNA complexes
into tumors, SCID mice bearing SAS subcutaneous
tumors were injected with 20 lg of pCMV-Luc DNA
complexed with PEI, using PEI/DNA molar ratios of
0.25 and 0.75. Animals were killed 48 hours later,
and tumor masses were analyzed for luciferase activity. A higher level of gene expression was observed in
the tumors that were injected with PEI/pCMV-Luc of
0.75 (see Figure 4). pCMV-DsRed2 was used as a reporter to show that the nuclei of transfected tumor
cells were along a needle track (white arrows) and in
parts of a heterogeneous tumor (Figure 4B). We have
therefore used 20 lg of DNA complexed with PEI at a
ratio of 0.75 for our further in vivo experiments. The
ability of the PEI/PUMA complex to suppress the
growth of SAS subcutaneous tumors in SCID mice
was then assessed. Treatment of tumor-bearing mice
with the PEI/PUMA complex significantly inhibited
tumor growth, compared with tumor growth in the
Cancers Results in Prolonged Survival. For a more
clinically relevant study, we used an intraoral tumor
model for PUMA gene therapy. As we have empirically determined the PEI/DNA for the in vitro experiments, this ratio of 0.75 with 20 lg DNA was used as
1 PEI/PUMA for our in vivo experiments; 5 PEI/
PUMA was then chosen arbitrarily to evaluate the
possible survival effect of a higher dose of PEI/PUMA
in vivo. In the 5 PEI/PUMA treatment group, there
were 2 (40%) mice still alive at the end of the experiment (180 days). In contrast, all the mice treated
with PEI or 5 PEI/control DNA died before day 90
(Figure 5D). The median survival times in the 2 control groups were 65 (PEI) and 68 (5 PEI/control
DNA) days. In comparison, the median survival times
for mice treated with 1 and 5 of PEI/PUMA were
87.2 and 147.2 days, respectively. The mean survival
time of SAS-bearing mice injected with 5 PEI/
PUMA was significantly longer than that of mice
injected with 5 PEI/control DNA. The p values for
5 VC, 1 PUMA, and 5 PUMA compared with the
‘‘No DNA’’ group are .23545, .00507, and .00025,
respectively.
Polyethylenimine-mediated PUMA Gene Delivery
HEAD & NECK—DOI 10.1002/hed
Injection of PEI/DNA Complexes into Subcutaneous
Tumors Suppresses Tumor Growth and Induces
882
June 2011
FIGURE 4. Determination of PEI/DNA transfection efficiency in vivo and protein expression after PEI/DNA injection in xenografts in
SCID mice. (A) Subcutaneous tumors were used to analyze the efficiency of 0.25 and 0.75 PEI/DNA weight ratios with 20 lg of
pCMV-Luc compared with the control without DNA. Mice were sacrificed 48 hours after transfection, and luciferase activity in the homogenized xenografts was assessed. Data are shown as RLU of the luciferase activity per lg of total cellular protein (mean SD,
obtained from triplicate experiments). The experiment was repeated 3 times with similar results. (B) Transient expression of the
DsRed2 reporter gene in SAS xenografts in SCID mice resulting from direct injection of PEI/DNA complexes. SAS xenografts were
injected with 20 lg pCMV-DsRed2 or control plasmid with PEI at a weight ratio of 0.75. Histochemical detection shows red fluorescence in the nuclei of transfected tumor cells along a needle track and of a group of transfected tumor cells in a heterogeneous portion
of a tumor. PEI, polyethylenimine; SCID, severe combined immunodeficiency; RLU relative light unit; DsRed2, red fluorescent protein
reporter gene. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
DISCUSSION
PEI derivatives are potent polycationic nonviral vectors that form stable complexes with plasmid DNA
for gene therapy in vitro and in vivo.11 This gene
delivery tool has shown its potential in treatment of
pancreatic cancer,12 ovarian cancer,13,14 hepatoma,13,15
and head and neck cancer.16 The PEI/DNA ratio seems
to play a crucial role in gene delivery and cytotoxicity.
In our experiments, DNA complexes with 25 kDa PEI
at a nitrogen/phosphate ratio of 5.7 (0.75: 1 w/w ratio
of PEI:DNA) showed the highest transfection efficiency
and the lowest cytotoxicity. The fact that efficiency of
PEI gene transfer is higher in proliferating cells could
reveal particularly interesting results in heterogeneous
tumor tissues, such as head and neck tumor tissue,
containing both proliferating tumor cells and differentiated, nonproliferating stromal cells. Moreover, we
found that significant levels of reporter gene expression
were detected only within tumor tissue, and were localized predominantly at the injection site. Therefore,
direct intratumoral injection of PEI/DNA complexes
may result in safe and efficient gene delivery to oral
cancers.
Previous studies showed that PUMA is an essential mediator of p53-dependent and -independent apoptosis in vivo. Gene knockouts in human colorectal
cancer cells showed that PUMA was required for apo-
Polyethylenimine-mediated PUMA Gene Delivery
ptosis induced by p53, hypoxia, and DNA-damaging
agents.17–20 Furthermore, PUMA deficiency protects
lymphocytes from p53-independent apoptotic stimuli
such as cytokine withdrawal or exposure to dexamethasone, staurosporine, or phorbol esters.6,21
PUMA gene therapy could therefore be used independently from p53 activity because apoptosis
induced by PUMA overexpression does not rely on
the p53 genotype of the cells.3–5 Recent study has
also shown that PUMA-induced mitochondria autophagy may contribute to apoptosis.22 Activation of
PUMA has also been suggested to be involved in the
endoplasmic reticulum stress-induced apoptosis pathway.23 We show here that exogenous expression of
PUMA in oral cancer cells resulted in apoptosis
through a mitochondrial pathway, as evidenced by cytochrome c release from mitochondria to cytoplasm and
consequent activation of caspase-9, caspase-3, and
cleavage of PARP. The number of cells expressing the
reporter gene after gene transfer was comparable to
the number observed undergoing apoptosis in our
study. We also showed that PEI/PUMA gene transfer
in SAS xenografts led to apoptotic tumor cell death
and a significant inhibition in tumor growth in animals injected with PEI/PUMA. PEI complexed with a
red fluorescent protein reporter gene (DsRed2) into
SAS tumors that were established in SCID mice
HEAD & NECK—DOI 10.1002/hed
June 2011
883
FIGURE 5. PUMA gene transfer inhibits SAS oral cancer cell growth in vivo and prolongs animal survival. (A) Mice with established
subcutaneously implanted OSCCs (about 2.5 mm in diameter) were given intratumoral injections of PEI-PUMA plasmid or vector control. At each time point, animals were sacrificed and tumors were harvested. Data show mean tumor volume from 3 separate experiments with bars of SDs (n ¼ 5 mice for each group). (B) Excised tumors (from A) on day 3 after the last treatment. (C) The tumor
cryosections were prepared from B, and both Hoechst and TUNEL were used to evaluate the percentage of apoptotic cells in vectorand PUMA-transfected cells. (D) Kaplan–Meier survival curves of SCID mice with orthotopic SAS oral cancers after receiving PEI/
PUMA-directed gene therapy. A total of 10 SCID mice were injected for each group. Percentages of surviving animals after concentric
intratumoral injection using PEI complexed with no DNA (&), 5 vector control DNA (VC) (^), 1 pCEP4-PUMA (*), and 5 pCEP4PUMA (”) were recorded during the experiments to generate the survival curves. Mean survival time was determined. Although there
is no significant difference between mice treated with ‘‘No DNA’’ and ‘‘5 vector control’’, the p values calculated between ‘‘No DNA’’
versus ‘‘1 PUMA’’ and ‘‘No DNA’’ versus ‘‘5 PUMA’’ are <.01. Note that most of the Mock and PEI/VC DNA-injected mice died
within 65 days. OSCC, oral squamous cell carcinoma; PEI, polyethylenimine; TUNEL, TdT-mediated dUTP nick end labeling; SCID,
severe combined immunodeficiency. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
resulted in high transfection efficiency without
obvious cytotoxicity. Studies have implied that chemotherapeutic agents exert their cytotoxic effects mainly
by inducing apoptosis in tumor cells, and that the efficiency of antitumor agents is related to the intrinsic
propensity of the target tumor cells to respond to
these agents with apoptosis.24 Our results indicate
that PUMA can suppress oral cancer growth via induction of apoptosis in vivo. The apoptotic effect of PUMA
overexpression in oral cancer cells may contribute to
the prolonged survival of oral cancer-bearing mice
treated with PEI/PUMA. Although we showed beneficial effects of PUMA gene therapy in prolonging animal survival, single-agent therapy is unlikely to work
in patients. The combination therapies of PUMA delivery, chemotherapy, and/or radiation are currently
under investigation in our laboratory.
In conclusion, our study provides the rationale for
the use of a new form of anticancer gene therapy
through PEI-mediated PUMA gene transfer because
its use resulted in both a significant reduction in tumor burden and prolonged survival in a murine model
of human oral carcinoma. Studies to determine the
antitumor efficacy of PEI/PUMA combined with chemotherapeutic agents are currently under way.
Polyethylenimine-mediated PUMA Gene Delivery
HEAD & NECK—DOI 10.1002/hed
884
Acknowledgment. The authors thank Dr. Bert
Vogelstein for his generous gifts of pCEP4-PUMA
plasmid.
REFERENCES
1. Boussif O, Lezoualc’h F, Zanta MA, et al. A versatile vector for
gene and oligonucleotide transfer into cells in culture and in
vivo: polyethylenimine. Proc Natl Acad Sci U S A 1995;92:7297–
7301.
June 2011
2. Leonetti JP, Degols G, Lebleu B. Biological activity of oligonucleotide-poly(L-lysine) conjugates: mechanism of cell uptake. Bioconjug Chem 1990;1:149–153.
3. Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, is
induced by p53. Mol Cell 2001;7:683–694.
4. Yu J, Zhang L, Hwang PM, et al. PUMA induces the rapid apoptosis of colorectal cancer cells. Mol Cell 2001;7:673–682.
5. Han J, Flemington C, Houghton AB, et al. Expression of bbc3, a
pro-apoptotic BH3-only gene, is regulated by diverse cell death and
survival signals. Proc Natl Acad Sci U S A 2001;98:11318–11323.
6. Erlacher M, Michalak EM, Kelly PN, et al. BH3-only proteins
Puma and Bim are rate-limiting for gamma-radiation- and glucocorticoid-induced apoptosis of lymphoid cells in vivo. Blood
2005;106:4131–4138.
7. Ito H, Kanzawa T, Miyoshi T, et al. Therapeutic efficacy of
PUMA for malignant glioma cells regardless of p53 status. Hum
Gene Ther 2005;16:685–698.
8. Wang H, Qian H, Yu J, et al. Administration of PUMA adenovirus
increases the sensitivity of esophageal cancer cells to anticancer
drugs. Cancer Biol Ther 2006;5:380–385.
9. Sun Q, Sakaida T, Yue W, et al. Chemosensitization of head and
neck cancer cells by PUMA. Mol Cancer Ther 2007;6:3180–3188.
10. Wang P, Yu J, Zhang L. The nuclear function of p53 is required
for PUMA-mediated apoptosis induced by DNA damage. Proc
Natl Acad Sci U S A 2007;104:4054–4059.
11. Godbey WT, Wu KK, Mikos AG. Poly(ethylenimine) and its role in
gene delivery. J Control Release 1999;60:149–160.
12. Vernejoul F, Faure P, Benali N, et al. Antitumor effect of in vivo somatostatin receptor subtype 2 gene transfer in primary and metastatic pancreatic cancer models. Cancer Res 2002;62:6124–6131.
13. Lee CH, Ni YH, Chen CC, et al. Synergistic effect of polyethylenimine and cationic liposomes in nucleic acid delivery to human
cancer cells. Biochim Biophys Acta 2003;1611:55–62.
14. Poulain L, Ziller C, Muller CD, et al. Ovarian carcinoma cells
are effectively transfected by polyethylenimine (PEI) derivatives.
Cancer Gene Ther 2000;7:644–652.
Polyethylenimine-mediated PUMA Gene Delivery
15. Morimoto K, Nishikawa M, Kawakami S, et al. Molecular weightdependent gene transfection activity of unmodified and galactosylated polyethyleneimine on hepatoma cells and mouse liver. Mol
Ther 2003;7:254–261.
16. Dolivet G, Merlin JL, Barberi-Heyob M, et al. In vivo growth
inhibitory effect of iterative wild-type p53 gene transfer in
human head and neck carcinoma xenografts using glucosylated polyethylenimine nonviral vector. Cancer Gene Ther
2002;9:708–714.
17. Chen L, Jiang J, Cheng C, et al. P53 dependent and independent apoptosis induced by lidamycin in human colorectal cancer
cells. Cancer Biol Ther 2007;6:965–973.
18. Wu Y, Xing D, Liu L, et al. Regulation of Bax activation and
apoptotic response to UV irradiation by p53 transcription-dependent and -independent pathways. Cancer Lett 2008;271:
231–239.
19. Yamaguchi H, Chen J, Bhalla K, et al. Regulation of Bax activation and apoptotic response to microtubule-damaging agents
by p53 transcription-dependent and -independent pathways. J
Biol Chem 2004;279:39431–39437.
20. Yu J, Wang Z, Kinzler KW, et al. PUMA mediates the apoptotic
response to p53 in colorectal cancer cells. Proc Natl Acad Sci U
S A 2003;100:1931–1936.
21. Alves NL, Derks IA, Berk E, et al. The Noxa/Mcl-1 axis regulates susceptibility to apoptosis under glucose limitation in dividing T cells. Immunity 2006;24:703–716.
22. Yee KS, Wilkinson S, James J, et al. PUMA- and Bax-induced
autophagy contributes to apoptosis. Cell Death Differ
2009;16:1135–1145.
23. Li J, Lee B, Lee AS. Endoplasmic reticulum stress-induced apoptosis: multiple pathways and activation of p53-up-regulated
modulator of apoptosis (PUMA) and NOXA by p53. J Biol Chem
2006;281:7260–7270.
24. Kamesaki H. Mechanisms involved in chemotherapy-induced apoptosis and their implications in cancer chemotherapy. Int J
Hematol 1998;68:29–43.
HEAD & NECK—DOI 10.1002/hed
June 2011
885