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com

Cancer Letters 271 (2008) 260–271

www.elsevier.com/locate/canlet

Effect of tumor suppressor gene PTEN on the resistance

to cisplatin in human ovarian cancer cell lines and
related mechanisms
HuiJuan Wu a,b,1, Yang Cao a,1, Danhui Weng a, Hui Xing a, Xiaohong Song a,
Jianfeng Zhou a, Gang Xu a, Yunping Lu a, Shixuan Wang a, Ding Ma a,*
a
Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan 430030, PR China
b
Department of Gynecological Oncology, Cancer Hospital, Tianjin Medical University, Tianjin 300060, PR China

Received 31 March 2008; received in revised form 31 March 2008; accepted 11 June 2008

Abstract

Purpose The aim of this study was to explore role of PTEN gene in chemosensitivity to cisplatin in human ovarian can-
cer cells and related mechanisms.
Method A PTEN-targeted short hairpin RNA (shRNA) expression vector and a wild-type sense PTEN plasmid were
constructed, human ovarian cisplatin-sensitive cancer cell line OV2008 and its resistant variant C13* cells were transfected
with PTEN shRNA or wild-type PTEN plasmid, respectively, and cells were then treated with cisplatin. Next, AKT activ-
ity was regulated with co-transfection of antisense or sense AKT plasmid in OV2008 /PTENshRNA cells or C13*/p-PTEN
cells, respectively. Effects of transfection of above vectors on cell growth, apoptosis and expression of PTEN and AKT
were evaluated.
Results Expression of PTEN in OV2008 cells was significantly higher than that in C13* cells. Transfection of PTEN
shRNA into OV2008 cells remarkably down-regulated expression of PTEN and up-regulated expression of phospho-
AKT protein, with transfected cells being resistant to cisplatin. Overexpression of PTEN by transfection with sense PTEN
obviously enhanced cisplatin-induced apoptosis of C13* cells. Furthermore, decreased AKT activity could increase cis-
platin-induced apoptosis in OV2008/PTENshRNA cells; while, transfection of pcDNA3.1-AKT plasmid into C13*/p-
PTEN cells resulted in increased activity of AKT, with cisplatin-induced apoptosis being inhibited significantly.
Conclusions PTEN might reverse chemoresistance to cisplatin in human ovarian cancer cells through inactivation of the
PI3K/AKT cell survival pathway and may serve as a potential molecular target for the treatment of chemoresistant ovar-
ian cancer.
Ó 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: PTEN; AKT; Ovarian cancer; Cisplatin; Multidrug resistance

1. Introduction
E-mail addresses: [email protected] (S. Wang), dma@
tjh.tjmu.edu.cn, [email protected] (D. Ma). Ovarian cancer is the fifth leading cause of cancer
1
These authors contributed equally to this work. deaths in women and the leading cause of death

0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2008.06.012
 HuiJuan Wu et al. / Cancer Letters 271 (2008) 260–271 261

from gynecologic malignancies characterized by function as a tumor suppressor, this gene product
rapid progression, late metastases and poor progno- was recently reportedly to enhance the sensitivity
sis [1,2]. Only about 25% of ovarian cancers are of cancer cells to anticancer agents [15–17].
diagnosed at an early stage. Approximately 60% In this study, we constructed PTEN shRNA and
of cases are diagnosed after the cancer has spread, wild-type sense PTEN plasmids and focused on the
when the 5-year survival rate is close to 30%. The role of PTEN in chemoresistance in ovarian cancer
vast majority of cases frequently have no symptoms cisplatin-sensitive cell line OV2008 and its resistant
until the cancer has spread extensively. Although variant C13* and effects of wild-type PTEN gene
cisplatin-centered chemotherapy which is the cur- on reversing cisplatin resistance in human ovarian
rently preferred treatment modality in human ovar- cancer cells as well as the related mechanisms by
ian cancer can strongly cut down the mortality and which PTEN enhances the sensitivity of ovarian
lengthen the survival time of patients, a major cancer to cisplatin-induced apoptosis.
obstacle in ovarian cancer treatment is the develop-
ment of drug resistance making tumor cells refrac- 2. Materials and methods
tory to therapy [3].
Cisplatin (cis-diamminedichloroplatinum, DDP) 2.1. Cell lines and cell culture
is a platinum-based compound that forms intra-
and inter- strand adducts with DNA [4,5] and is A cisplatin-sensitive ovarian cancer cell line
one of the first-line chemotherapeutic agents for (OV2008) and its resistant variant (C13*) were gifts
the treatment of ovarian cancer. However, the resis- from Dr. Rakesh Goel from the Ottawa Regional
tance developed by ovarian cancer cells to cisplatin Cancer Center, Ottawa, Canada. Cells were main-
results in failure in the therapy of ovarian cancer, tained in RPMI-1640 complete medium supple-
and the molecular mechanisms leading to cisplatin mented with 2 mM L-glutamine and 10% FBS at
chemoresistance are poorly understood. Possible 37 °C in a humidified atmosphere containing 5%
mechanisms of acquired resistance to cisplatin CO2 [18].
include reduced accumulation of the drug [6],
increased levels of glutathione and metallothionein 2.2. Chemicals and antibodies
[7] and enhanced DNA repair [8]. It is now widely
accepted that the apoptotic capacity of cancer cells Cisplatin was obtained from QiLu Pharmaceuti-
is pivotal in determining in the response to chemo- cal Manufacture (Shandong, China). RPMI-1640,
therapeutic agents [9]. At the same time, apoptosis fetal bovine serum, Lipofectamine 2000 reagent
is the cellular underpinning of cisplatin-induced cell and TRIzol reagent were purchased from Life
TM

death, which is associated with expression of specific Technologies Inc. (Carlsbad, California, USA).
‘‘death” genes and down-regulation of ‘‘survival” MTT, DMSO and G418 were obtained from Sigma
counterparts [10]. Failure of cancer cells to maintain Chemical Inc. (St. Louis, Missouri, USA). Mouse
a balance in the expression of these genes in favor of anti-human PTEN polyclonal antibody was
apoptotic cell death may be an important factor of obtained from R&D System Inc. (Minneapolis,
chemoresistance [11]. Recent studies also demon- Minnesota, USA). Rabbit anti-human phospho-
strated that the acquired drug resistance of ovarian AKT (Ser473), total AKT and b-actin antibodies
cancer cells is related to alteration in apoptosis [12]. were obtained from Santa Cruz Biotechnology
More and more studies have shown that some (Santa Cruz, California, USA), PCR primers and
activation of oncogenes and inactivation of tumor pEGFP-C1 plasmid were from Invitrogen Corpora-
suppressor genes may contribute to the acquired tion (Carlsbad, California, USA).
drug resistance of cancer cells by inhibiting apopto-
sis [3,9,13,14]. In recent years, the identification of 2.3. Construction of the plasmids
molecules involved in the regulation and execution
of apoptosis, and their alteration in ovarian cancer, Eukaryotic expression plasmid pGenesil-1 con-
have provided new insights into the molecular basis taining U6 shRNA promoter and the cDNA of
of ovarian cancer chemoresistance. PTEN is a GFP and karamycin resistance gene was purchased
recently identified tumor suppressor gene that plays from Genesil Corporation (Genesil Corp, Wuhan,
important roles not only in suppressing cancer but China). shRNAs corresponding to PTEN and
also regulating apoptosis. In addition to its original AKT were designed with the web tools on Ambion
262 HuiJuan Wu et al. / Cancer Letters 271 (2008) 260–271

homepage (Ambion, USA) according to Tuschl’s The PCR primers for full-length AKT were
principle [19]. The following gene-specific sequences designed with Oligo software. The sequence of the
were used successfully: PTEN: 50 -ACGGGAAGA upper primer was 50 -TCCTGCATGTCCTGC
CAAGTTCATG-30 ; AKT: 50 -CACCTTTGTCATA TGCCCTGAG-30 and the lower primer sequence
CGCTGC-30 , and then the designed oligonucleo- was 50 -CAGCGGTGATGGCAGCGAGCGTGC-
tides to generate shRNAs were synthesized by Invit- 30 . PCR conditions were as follows: 5 min for pre-
rogen, USA. Additional restriction (SalI) site was denaturation at 95 °C, then 94 °C for 30 s, 55 °C
added to the tail of the DNA sequences for identifi- for 30 s and 72 °C for 30 s for 30 cycles, and a final
cation. The double-stranded shRNA molecules were extension at 72 °C for 10 min. The PCR product
prepared by brief boiling and slow cooling and then (1500 bp) was purified and restrictively digested
the DNA sequences were cloned into the BamHI/ with HindIII and XbaI, and then cloned into
HindIII restriction site of the pGenesil-1 vector. pcDNA3.1 to form pcDNA3.1-AKT; the
The control vectors were constructed by insertion pcDNA3.1 vector was treated as a control.
of a sequence containing random DNA fragment.
All the inserted sequences were verified by DNA 2.4. Transfection of the ovarian cancer cells
sequencing. The recombinant plasmids were named
pGenesil-PTENshRNA, pGenesil-AKTshRNA, For transfection, OV2008 and C13* were seeded
pGenesil-PTEN-Con and pGenesil-AKT-Con, in six-well plates at 5  105/well and incubated in
respectively. The sequences for representative shR- 5% CO2 95% air incubator at 37 °C. When cells were
NAs are shown in Fig. 1A. 80–90% confluent, cells were transfected using Lipo-
GFP expression plasmid based on pGZ21dxZ fectamine 2000 transfection reagent according to the
with full-length wild-type PTEN was a generous gift manufacturer’s protocol. Recombinant plasmids
from Dr Kenneth M. Yamada (National Institute of (4 lg) were mixed with lipofectamine and pre-incu-
Dental and Craniofacial Research, NIH, USA) [20]. bated for 20 min at room temperature in serum-free
The plasmid was restrictively digested with HindIII and antibiotic-free DMEM. OV2008 cells transfec-
and XbaI, then the full-length wild-type PTEN ted with pGenesil-PTENshRNA and pGenesil-
mRNA was reclaimed and purified, and then cloned PTEN-Con were named as OV2008/PTENshRNA
into pEGFP-C1 to form pEGFP-C1-PTEN; the and OV2008/p-Con. C13* cells transfected with
pEGFP-C1 vector was treated as a control. pEGFP-C1-PTEN and pEGFP-C1 were named as

Fig. 1. Construction and identification of the recombinant plasmids. (A) The shRNA oligonucleotide sequence design. The arrow shows
the sites of restrictive digestion. (B) The plasmid pGenesil-PTEN or pGenesil-con with restrictive digestion show two bands. (C) The
plasmid pEGFP-CI-PTEN with restrictive digestion showed two bands in the gel (intervened DNA was 1.3 kb, the vector was 4.7 kb).
pEGFP-C1 showed a single band (4.7 kb) in the gel.
 HuiJuan Wu et al. / Cancer Letters 271 (2008) 260–271 263

C13*/p-PTEN and C13*/p-Con, respectively. G418 4 °C. The membrane was washed three times in
(500 lg/ll) was applied to stable screening and iso- TBST and incubated with alkaline phosphatase-
lating the resistant colonies, which were followed conjugated goat IgG (1:500) for 2 h at room temper-
by transfected with pGenesil-AKTshRNA plasmid ature and then visualized with NBT/BCIP/buffer
and pcDNA3.1-AKT full-length plasmid, at the (1:1:50). Protein loading was assessed by blotting
same time corresponding empty vector was transfec- the same membrane with an antibody against b-
ted as control. The cells which were co-transfected actin.
with two different plasmids were termed as OV2008/
PTENshRNA/p-Con, OV2008/PTENshRNA/ 2.7. Cell viability assay with MTT
AKTshRNA, C13*/p-PTEN/pcDNA3.1 and
C13*/p-PTEN/p-AKT cells. The cytotoxic effect of cisplatin was determined
with the MTT assay. Briefly, cells were cultured in
2.5. RT-PCR 96-well plates the day before experiment at a density
of 5  103/well, cells were then treated with different
Total RNA was prepared using Trizol reagent, concentrations of cisplatin for 24 h, then 20 ll
following manufacturer’s instructions. Total RNA of 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetra-
(2 lg) was reverse transcribed using M-MuLV zoliumbromide (MTT) (5 mg/ml, Sigma Chemical
Reverse Transcriptase (Promega, USA). cDNA Co.) was added to the wells, which was followed by
was amplified by PCR using Taq DNA polymerase incubation at 37 °C for 4 h, the supernatants were
(Promega, USA). Primers were as follows: for aspirated, 100 ll DMSO was then added and incu-
PTEN expression, sense: 50 -ACGGGAAGAC bated at 37 °C for an additional 20 min. The absor-
AAGTTCATGTAC-30 , and antisense: 50 -TTTGAC bance of the wells was then read with a microplate
GGCTCCTCTACTGT-30 (product size, 389 bp); reader at a test wavelength of 570 nm and a reference
For GAPDH (housekeeping gene) expression, wavelength of 630 nm. Appropriate controls lacking
sense: 50 -ACGGATTTGGTCGTATTGGG-30 and cells were included to determine background absor-
antisense: 50 -TGATTTTGGAGGGATCTCGC-30 . bance. Response to drug treatment was assessed by
(product size, 180 bp). The RT-PCR cycle was as standardizing treatment groups to untreated control.
follows: for PTEN, 30 s at 94 °C, 45 s at 51.6 °C,
30 s at 72 °C, 28 cycles; for GAPDH: 30 s at 2.8. Flow cytometry
94 °C, 30 s at 56 °C, 30 s at 72 °C, 28 cycles. PCR
products were analyzed with 1.5% agarose gel elec- OV2008 and C13* were treated with 7.5 lmol/l
trophoresis in the presence of ethidium bromide for cisplatin for 6, 12, 18, 24, 30 h and then were har-
UV light transilluminator visualization. vested and fixed in ice-cold 70% ethanol and stored
at 20 °C overnight. Other cells transfected with
2.6. Western blot analysis different plasmids were treated with 7.5 lmol/l cis-
platin for 24 h and then were harvested and fixed
Cells harvested were lysed in lysis buffer in ice-cold 70% ethanol and stored at 20 °C over-
(50 mmol/l Tris–HCl, pH 7.4, 150 mmol/l NaCl, night. All samples were then washed once in PBS
1% Nonidet P 40, 1% Triton X-100, 0.2% SDS, and resuspended in a solution containing propidium
1% sodium deoxycholate, 5 mmol/l iodoacetamide, iodide (5 mg/ml) and RNase A (0.5 mg/ml) in PBS
proteinase inhibitor cocktail, 2 mmol/l PMSF). Pro- for an additional 30 min. Cells were sorted using a
tein (40 lg) was denatured in SDS sample buffer at FACScan (BD Biosciences, USA) and analyzed
100 °C for 10 min and separated by electrophoresis with CellQuest version 3.3 software. The apoptotic
on a 10% SDS–PAGE gel, then transferred to a cells were calculated after FACS analysis.
nitrocellulose membrane. The membrane was
blocked in TBST (25 mmol/l Tris–HCl, pH 7.5, 2.9. Image analysis
137 mmol/l NaCl, 2.7 mmol/l KCl and 0.05%
Tween 20) with 5% non-fat milk for 1 h at 37 °C, The images of PCR and Western blot products
and then incubated with the primary antibodies were collected by Uvp Grab-it image system
(mouse anti-PTEN, 1:2000; rabbit anti-AKT, (UVP, Cambridge, UK) and analyzed by Gelworks
1:500; rabbit anti-phospho-AKT, 1:1000; rabbit ID advanced V4d system (UVP, Cambridge, UK).
anti-b-actin, 1:500) in blocking buffer overnight at The absorbency difference (A) between the absor-
264 HuiJuan Wu et al. / Cancer Letters 271 (2008) 260–271

bency volume of the target gene and that of internal DNA was 400 bp and vector was 4.9 kb) (Fig. 1B). The
control was used as the relatively absorbency of the restriction digest product of pEGFP-C1 was a single band
target gene expression. (4.7 kb), and the pEGFP-C1-PTEN had two bands (inter-
vened DNA was 1.3 kb, and vector was 4.7 kb) (Fig. 1C).
DNA sequencings were done at the same time. Sequenc-
2.10. Statistical analysis
ing showed that all the vectors were exactly corrected.

All experiments were repeated at least three 3.2. Expression of PTEN mRNA and protein in OV2008
times. The data are expressed as mean ± SD. and C13* cells
P < 0.05 was considered statistically significant. Sta-
tistical analysis was done by using SPSS 11.5 for The expression of PTEN in OV2008 and C13* was
Windows by Student’s t-test. examined with RT-PCR and Western blot analysis. The
expression of PTEN mRNA in OV2008 and C13* cell
3. Results lines was detectable and relative A were 1.02 ± 0.05 and
0.45 ± 0.03. The expression of PTEN protein in OV2008
3.1. Identification of the recombinant plasmids and C13* cell lines was assayed and relative A were
1.02 ± 0.07 and 0.55 ± 0.03. The expression of PTEN
The transformed Escherichia coli were cultured and mRNA and protein in OV2008 was significantly higher
amplified in the media containing kanamycin. Plasmid than that in C13* (P < 0.05, Fig. 2A and B). These results
DNAs were digested with restriction enzymes and then demonstrated that the expression levels of PTEN mRNA
analyzed by electrophoresis. The restriction digest prod- in these cells were positively correlated with those of
ucts of pGenesil-PTENshRNA and pGenesil-PTEN-Con PTEN protein. The cytotoxic effect of cisplatin on
were separated into two bands in the gel (short strand OV2008 and C13* cells was determined with MTT assay.

Fig. 2. Expression of PTEN and cisplatin-induced cytotoxicity and apoptosis in OV2008 and C13* cells. (A) Expression of PTEN mRNA
(389 bp PCR product) was detected with RT-PCR. GAPDH (180 bp PCR product) was co-amplified as the internal control. (B)
Expression of PTEN protein was detected with Western blot analysis in total cell extracts. b-actin was reprobed to confirm equal protein
loading. (C) Cell viability was assessed with MTT assay after treatment with cisplatin for 24 h. (D) Apoptotic cells were counted with flow
cytometry after treatment with cisplatin for 6, 12, 18, 24, 30 h.
 HuiJuan Wu et al. / Cancer Letters 271 (2008) 260–271 265

3.3. Cisplatin-induced cytotoxicity and apoptosis in Fig. 4A and B). The PTEN, AKT and p-AKT protein
OV2008 and C13* cells expression was then detected with Western blot analysis.
After transfection with pGenesil-PTENshRNA, the ratio
The levels of cytotoxicity were indicated as the concen- of PTEN protein/b-actin protein was 0.45 ± 0.02,
tration that inhibits the response by 50%, IC50. The IC50 0.95 ± 0.02 and 0.94 ± 0.03 in OV2008/PTENshRNA,
in OV2008 and C13* cells were (7.5 ± 0.3) lmol/l and OV2008/p-Con and OV2008 cells, respectively (Fig. 4C).
(13.0 ± 0.3) lmol/l, respectively, and the resistance factor The expression of PTEN protein in OV2008/
which was the ratio of IC50 value of the resistance cells to PTENshRNA cells was significantly lower than those in
the IC50 of the sensitive cells was 1.73, which showed that OV2008/p-Con and OV2008 cells (P < 0.05), at the same
the OV2008 cells were more sensitive to cisplatin-induced time, the expression of p-AKT protein in OV2008/
apoptosis than C13* cells (P < 0.05, Fig. 2C). Cisplatin PTENshRNA cells (0.53 ± 0.02) was obviously higher
treatment also resulted in a time-dependent increase in than those in OV2008/p-Con and OV2008 cells
the apoptosis of OV2008 and C13* cells, while the per- (0.37 ± 0.01, 0.38 ± 0.02)(P < 0.05). However, the expres-
centages of apoptotic cells in OV2008 cell lines were at sion of AKT protein was similar in OV2008/
least twofold higher than those in C13* cells at the same PTENshRNA, OV2008/p-Con and OV2008 cells
time (Fig. 2D). (P > 0.05) (Fig. 4E). The PTEN, AKT and p-AKT pro-
tein expression was also examined with Western blot anal-
3.4. Effects of recombinant plasmids on cisplatin-induced ysis in C13* cells (Fig. 4D). The PTEN protein expression
cytotoxicity and apoptosis in OV2008 and C13* cells in C13*/p-PTEN cells (0.94 ± 0.04) was remarkably
higher than those in C13*/p-Con (0.36 ± 0.01) and C13*
To test whether the PTEN plays a key role in cisplatin- cells (0.37 ± 0.01) (P < 0.05). The p-AKT protein expres-
induced apoptosis, we selectively inhibited the PTEN sion in C13*/p-PTEN cells (0.94 ± 0.07) was obviously
expression through PTEN shRNA transfection in lower than those in C13*/p-Con (1.66 ± 0.10) and C13*
OV2008 cells and enhanced PTEN expression through cells (1.68 ± 0.14); However, the difference of AKT pro-
pEGFP-C1-PTEN plasmid transfection in C13* cells, tein expression in these three cells is not statistically signif-
and then treated with 7.5 lmol/l cisplatin for 24 h. After icant (P > 0.05, Fig. 4F).
24 h of cisplatin treatment, only 26.79 ± 2.53% of the cells
underwent apoptosis in OV2008/PTENshRNA cells. In 3.6. AKT activity is required for PTEN-mediated apoptosis
contrast, as many as 51.66 ± 2.66% and 47.42 ± 2.28%
of the OV2008/p-Con and OV2008 cells underwent apop- To further investigate the role of AKT in PTEN-med-
tosis (P < 0.05) (Fig. 3A and C). In the meantime, nearly iated apoptosis, OV2008/PTENshRNA cells were then
41.65 ± 0.87% cells underwent apoptosis in pEGFP-C1- transfected with pGenesil-AKTshRNA plasmid, C13*/p-
PTEN transfected C13* cells and displayed a significant PTEN cells were transfected with sense pcDNA3.1-AKT
difference compared with pEGFP-C1 transfected and plasmid, and treated thereafter with cisplatin (7.5 lmol/
untransfected cells (P < 0.05) (Fig. 3B and D). The results l, 24 h). In OV2008/PTEN-shRNA cells, the effectiveness
suggested that PTEN is required for cisplatin-induced of co-transfected with pGenesil-AKTshRNA was evident
apoptosis in human ovarian carcinoma cells. The cyto- by decreased AKT and p-AKT protein content, respec-
toxic effect of cisplatin was also determined with MTT tively (P < 0.05) (Fig. 5A). Subsequent cisplatin treatment
assay. As revealed in Fig. 3E, the level of toxicity induced remarkably enhanced apoptotic cell death compared with
by cisplatin in OV2008/PTENshRNA cells was OV2008/PTENshRNA cells and OV2008/PTENshRNA/
8.1 ± 0.2 lmol/l, which was significantly higher than p-Con cells 39.19 ± 2.99% vs 26.79 ± 2.53%,
those of OV2008 and OV2008/p-Con cells, and the level 28.36 ± 2.43% (Fig. 5C and E). In the meantime, the effec-
of sensitivity in OV2008 cells was similar to that of tiveness of co-transfected with pcDNA3.1-AKT plasmid
OV2008/p-Con cells. On the other hand, the levels of was evident by increased AKT and p-AKT protein con-
IC50 of C13*, C13*/p-Con and C13*/p-PTEN cells tent (P < 0.05) (Fig. 5B). The apoptotic ratio of C13*/p-
induced by cisplatin were 12.1 ± 0.2 lmol/l, PTEN/p-AKT cells was 25.57 ± 1.42% which was obvi-
12.2 ± 0.3 lmol/l and 7.5 ± 0.2 lM, respectively, and ously lower than those of C13*/p-PTEN cells
the differences were statistically significant (Fig. 3F) 41.65 ± 0.87% and C13*/p-PTEN/pcDNA3.1 cells
(P < 0.05). 39.29 ± 2.39% (P < 0.05) (Fig. 5D and F). These results
suggested that PTEN facilitates cisplatin-induced apopto-
3.5. Effects of recombinant plasmids on expression of sis in an AKT-dependent manner. The sensitivity of co-
PTEN, AKT and p-AKT protein transfected cells to cisplatin was also detected with MTT
assay. As shown in Fig. 6, co-transfection of OV2008/
OV2008 cells transfected with pGenesil-PTENshRNA PTENshRNA cells with pGenesil-AKTshRNA made the
and C13* cells transfected with pEGFP-C1-PTEN express cells more sensitive to cisplatin than OV2008/
a red-shifted variant of wild-type GFP (as shown in PTENshRNA cells and OV2008/PTENshRNA/p-Con
266 HuiJuan Wu et al. / Cancer Letters 271 (2008) 260–271

Fig. 3. Effects of pGenesil-PTENshRNA and pEGFP-C1-PTEN on cisplatin-induced apoptosis and sensitivity to cisplatin of OV2008 cells
and C13* cells after treatment with cisplatin. Apoptosis was detected with flow cytometry. Cytotoxicity of cisplatin was determined with
MTT assay and reported as concentration that inhibits response by 50% (IC50). (A) Pictures were representative results from cytometry of
propidium iodine-stained OV2008 cells with different treatment. (B) Pictures were representative results from cytometry of propidium
iodine-stained C13* cells with different treatments. (C) The apoptotic ratio of OV2008/PTENshRNA was significantly lower than those of
OV2008 and OV2008/p-Con cells. (D) The apoptotic ratio of C13*/p-PTEN cells was remarkably higher than those of C13* and C13*/p-
Con cells. (E) The level of IC50 of OV2008/PTENshRNA induced by cisplatin was significantly higher than those of OV2008 and OV2008/
p-Con cells. (F) The level of IC50 of C13* and C13*/p-Con cells induced by cisplatin was remarkably higher than that of C13* cells.

cells, while the IC50 of C13*/p-PTEN/p-AKT cells was expressions of ‘‘cell death” and ‘‘cell survival” genes
obviously higher than those of C13*/p-PTEN cells and may be an important factor of chemoresistance. In
C13*/p-PTEN/pcDNA3.1 cells (P < 0.05). In brief, addition, many studies have also shown that many
AKT played an important role in the PTEN-mediated chemotherapeutics could induce cancer cell under-
reversal of cisplatin resistance.
going apoptosis by different pathway [21].
PTEN (phosphatase and tensin homolog deleted
4. Discussion on chromosome ten), also known as MMAC1 and
TEP1, was discovered by three different groups in
Ovarian carcinoma is the leading cause of death 1997 [22]. PTEN is located in chromosome 10, a
from gynecological cancer; chemoresistance remains region undergoing frequent somatic deletion in
a major therapeutic problem. Up to now, more and tumors. It comprises of 9 exons and 8 introns and
more investigations have demonstrated that failure codifies a 403-amino acid dual-specific phosphatase
of cancer cells to maintain a balance between with putative tumor suppressing abilities. A struc-
 HuiJuan Wu et al. / Cancer Letters 271 (2008) 260–271 267

Fig. 4. Effects of PTEN plasmids on the expression of PTEN, AKT and p-AKT protein. (A) Fluorescent imaging of the OV2008 cells
transfected with pGenesil-PTENshRNA (100). (B) Fluorescent imaging of the C13* cells transfected with pEGFP-C1-PTEN (200). (C)
Representative Western blots depicting changes in the expression of PTEN, AKT and p-AKT protein following transfection in OV2008
cells. (D) Representative Western blot displaying changes in the expression of PTEN, AKT and p-AKT protein following transfection in
C13* cells. (E) Quantification of PTEN, AKT and p-AKT content to b-actin (expressed as fold/folds of internal control) in OV2008 cells.
(F) Quantification of PTEN, AKT and p-AKT content to b-actin (expressed as fold/folds of internal control) in C13* cells.

ture with a dual-specificity protein phosphatase is be an effective way on reversing chemoresistance

located in the N-terminal of the molecule; therefore, of drug-resistant cancer cells [26–28] and PTEN
PTEN protein has the activity of protein phospha- has become a new potential target in sensitizing can-
tase and lip phosphatase. Loss of PTEN expression cer cells to chemotherapy.
has been detected in a wide range of human cancers, In this study, a pair of chemosensitive (OV2008)
including human brain, breast, prostate, endome- and chemoresistant (C13*) ovarian cancer cell lines
trial and ovarian cancers [23] and PTEN has already were used to investigate the possible roles of PTEN
proven itself to be a remarkable molecule. It is now in the regulation of cisplatin-mediated apoptosis
known that PTEN plays important roles not only in and its possible involvement in cisplatin resistance
cell cycle detention and apoptosis, but also in regu- in human ovarian epithelial cancer. Cisplatin-
lation of cell adherence, migration and differentia- induced apoptotic ratio in OV2008 cells was found
tion [24]. Recent researches have demonstrated to be remarkably higher than that in C13* cells.
that PTEN has the function of enhancing the sensi- In addition, it was found that the expression of
tivity of cancer cells to certain anticancer agents PTEN in cisplatin-sensitive ovarian cancer line
[25]. Gene therapy with PTEN has been found to OV2008 was significantly higher than that of its
268 HuiJuan Wu et al. / Cancer Letters 271 (2008) 260–271

Fig. 5. The effects of transient co-transfection of pGenesil-PTEN and pGenesil-AKT or pEGFP-C1-PTEN and pcDNA3.1-AKT on the
expression of PTEN, AKT and p-AKT protein and cisplatin-induced apoptosis in the OV2008 and C13* cells. (A) The panels’ shows
representative western blot depicting changes in PTEN, AKT and p-AKT content following co-transfection of pGenesil-PTENshRNA
and pGenesil-AKTshRNA. (B) The panels’ shows representative western blot depicting changes in PTEN, AKT and p-AKT content
following co-transfection of pEGFP-C1-PTEN and pcDNA3.1-AKT. (C) Cisplatin-induced apoptotic response to transient co-
transfection of pGenesil-PTENshRNA and pGenesil-AKTshRNA in the OV2008 cells after treatment with cisplatin. (D) Cisplatin-
induced apoptotic response to transient co-transfection of pEGFP-C1-PTEN and pcDNA3.1-AKT in the C13* cells. (E) Pictures were
representative results from cytometry of propidium iodine-stained OV2008 cells with different treatments. (F) Pictures were representative
results from cytometry of propidium iodine-stained C13* cells with different treatments.

parental cisplatin-resistant cell line C13*. These expression significantly inhibited cisplatin-induced
results strongly suggested that loss of PTEN might apoptosis and the cells were resistant to cisplatin
be involved in cisplatin resistance of ovarian cancer along with the up-regulation of p-AKT expression
cells and played an important role in chemoresis- in OV2008 cells. On the contrary, up-regulation of
tance of ovarian cancer cells. PTEN expression had been found to be an effective
The mechanism by which PTEN may contribute way to reverse resistance of C13* cells to cisplatin
to reversal of chemoresistance is unclear so far. We and sensitize C13* cells to cisplatin-induced apopto-
successfully transfected OV2008 cells with pGenesil- sis as reported [29]. At the same time, overexpres-
PTENshRNA plasmid and C13* cells with wild- sion of PTEN was accompanied with decreased
type sense PTEN plasmid by Lipofectamine 2000, expression of p-AKT protein. Therefore, it is sug-
and it was found that down-regulation of PTEN gested that combination chemotherapy with target-
 HuiJuan Wu et al. / Cancer Letters 271 (2008) 260–271 269

pathway including the AKT. Therefore, PTEN is

named as ‘‘on-off switch” of PI3K/AKT pathway
[30].
PI3K/AKT pathway is important in controlling a
diverse array of cellular functions including cell sur-
vival, cell cycle progression, motility and differenti-
ation in response to extracellular stimuli. AKT, as
one of the major direct downstream targets of
PI3K, is a key checkpoint to regulate crucial signal
transduction pathways and plays a critical role in
promoting cancer cell proliferation, survival and
angiogenesis and inhibiting apoptosis through phos-
phorylating several substrates, such as Bad, HIF-1a,
NF-rB. The fate of cancer cells in response to a che-
motherapeutic agent is a consequence of the overall
Fig. 6. The effects of transient co-transfection of pGenesil- apoptotic capacity of the cell. Resistance to apopto-
PTENshRNA and pGenesil-AKTshRNA or pEGFP-C1-PTEN sis may contribute to tumorigenesis of many malig-
and pcDNA3.1-AKT on the sensitivity of OV2008 cells and C13*
nancies and insensitivity of ovarian cancer to
cells to cisplatin. (*compared with control, P < 0.05).
standard therapies. Many extracellular stimuli have
been reported recently to inhibit drug-induced
ing PTEN expression up-regulation will draw a light apoptosis by phosphorylating AKT and result in
on the treatment of ovarian cancer. resistance of cancer cells to chemotherapeutic agents
To further investigate the role of AKT in PTEN- [31]. The activation of AKT requires the phosphor-
mediated chemoresistance of ovarian cancer, the ylation of AKT at both the Ser-473 and Thr-308
AKT expressions were inhibited or enhanced in sites, so the level of phospho-AKT (ser-473) can
the OV2008/PTENshRNA and C13*/p-PTEN cells stand for the activity of AKT [32].
by transfection with AKT shRNA plasmid or AKT The mechanisms by which PTEN contributes to
full-length plasmid, which were constructed by our reversal of chemoresistance are unclear so far. The
laboratory. Consequently, compared with the protein expression of AKT and phosphorylate
OV2008/PTENshRNA cells, the expression of AKT was examined in this study. It was found that
AKT and p-AKT protein was notably decreased the expression of phosphorylate AKT protein of
along with the elevation of the cisplatin-induced C13* cells transfected with wild-type PTEN was
apoptosis. Inversely, it was found that the AKT remarkably higher than tose of C13* cells transfec-
and p-AKT protein expression was obviously ted with empty plasmid and untransfected cells.
unregulated when the C13*/p-PTEN cells were However, there was no difference in AKT protein
transfected with AKT full-length plasmid. Similarly, expression in C13* cells. Our studies have shown
the cisplatin-induced apoptosis was remarkably that the overexpression PTEN in C13* cells results
inhibited in the C13*/p-PTEN/p-AKT cells after in a decreased phosphorylation of AKT. These find-
treatment with cisplatin. ings demonstrates that up-regulation of PTEN
PTEN is the first tumor suppressor gene with expression may lead to inactivation of PI3K/AKT
activity of phosphatase, its protein product which signal pathway, thereby to sensitize C13* cells to
was associated with cellular growth, apoptosis and cisplatin-induced apoptosis and reverse resistance
tumorigenesis through its protein and lipid phos- of C13* cells to cisplatin.
phatase activity antagonizes the action of phospho- In conclusion, inactivation or lower expression of
inositide 3 kinase (PI3K) by dephosphorylating the PTEN plays a crucial role in chemoresistance of
signaling lipid phosphatidylinositol (3,4,5)-trisphos- ovarian cancer, up-regulation of PTEN expression
phate (PI[3,4,5]P3) to produce phosphatidylinositol by transfection with wild-type PTEN plasmid could
4,5-bisphosphate, and results in a decreased phos- facilitate apoptosis and recover the sensitivity of the
phorylation of AKT which is one of the major direct chemoresistant ovarian cancer C13* cells through
downstream targets of PI3K. Indeed, the absence of the PI3K/AKT signal pathway. Further clarifica-
functional PTEN in cancer cells leads to constitutive tion of functional characterization is needed. The
activation of downstream components of the PI3K results in this paper will provide support for this
270 HuiJuan Wu et al. / Cancer Letters 271 (2008) 260–271

potentially novel gene therapy with PTEN in the Resveratrol inhibits proliferation, induces apoptosis, and
treatment of chemoresistant human ovarian cancer. overcomes chemoresistance through down-regulation of
STAT3 and nuclear factor-kappaB-regulated antiapoptotic
and cell survival gene products in human multiple myeloma
Acknowledgments cells, Blood 109 (2007) 2293–2302.
[14] J. Bai, J. Sui, A. Demirjian, C.M. Vollmer Jr., W. Marasco,
This work was supported by grants from the Na- M.P. Callery, Predominant Bcl-XL knockdown disables
tional Natural Science Foundation of China (No. antiapoptotic mechanisms: tumor necrosis factor-related
apoptosis-inducing ligand-based triple chemotherapy over-
30571950, 30528012, 30672227, 30600667, comes chemoresistance in pancreatic cancer cells in vitro,
30500596) and the ‘‘973” Program of China (No. Cancer Res. 65 (2005) 2344–2352.
2002CB513107). [15] Y. Saga, H. Mizukami, M. Suzuki, T. Kohno, M. Urabe, K.
Ozawa, I. Sato, Overexpression of PTEN increases sensitiv-
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