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Reversal of the malignant phenotype of ovarian cancer A2780 cells through transfection with wild-type PTEN gene
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Cancer Letters 271 (2008) 205–214 www.elsevier.com/locate/canlet
Reversal of the malignant phenotype of ovarian cancer A2780 cells through transfection with wild-type PTEN gene Huijuan Wu a,b,1, Shixuan Wang a,1, Danghui Weng a, Hui Xing c, Xiaohong Song a, Tao Zhu a, Xi Xia a, Yanjie Weng a, Gang Xu a, Li Meng a, Jianfeng Zhou a,*, Ding Ma a,* a
Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China b Department of Gynecological Oncology, Cancer Hospital, Tianjin Medical University, Tianjin 300060, PR China c Department of Obsterics and Gynecology, Xiangfan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Xiangfan, Hubei 441021, PR China Received 18 April 2008; received in revised form 18 April 2008; accepted 2 June 2008
Abstract Objective: PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor gene identified on human chromosome 10q23. Substantial studies have demonstrated that PTEN can inhibit cell proliferation, migration and invasion of many cancer cells. The purpose of this study was to determine whether upregulation of PTEN gene by transfection wild-type PTEN gene to ovarian cancer cells can inhibit growth and migration and to explore the potential for PTEN gene therapy of ovarian cancers. Method: Wild-type and phosphatase-inactive (C124A) PTEN plasmids were transfected into ovarian epithelial cancer A2780 cells, and their effects on cell apoptosis, cell proliferation, cell migration and cell invasion were analyzed by flow cytometry analysis, TUNEL assay, MTT assay, wound-healing assay and transwell assay. Results: Both wild-type and mutant PTEN can upregulate the expression of PTEN gene dramatically; however, it is wild-type PTEN not phosphatase-inactive PTEN that can induce apoptosis and decrease cell migration, invasion and proliferation in ovarian cancer cells. Conclusion: These results demonstrated that PTEN had played an important role in the cell proliferation, cell migration and invasion dependent on its phosphatase activity. Enhanced expression of PTEN by gene transfer is sufficient to reverse the malignant phenotype of ovarian cancer cells and transfection of ovarian cancer cells with wild-type PTEN gene might be another novel approach for therapeutic intervention in ovarian cancer. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Ovarian cancer; PTEN; Apoptosis; Proliferation; Invasion; Migration
* Corresponding authors. Fax: +86 27 83663180 (J. Zhou); +86 27 83662681 (D. Ma). E-mail addresses: [email protected] (J. Zhou), dma@ tjh.tjmu.edu.cn (D. Ma). 1 These two authors are equally contributed.
1. Introduction Ovarian cancer is the fifth leading cause of cancer 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.2008.06.018
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from gynecologic malignancies characterized by rapid progression, late metastases and poor prognosis [1,2]. Only about 25% of ovarian cancers are diagnosed at an early stage. Approximately 60% of cases are diagnosed after the cancer has spread, when the 5-year survival rate is close to 30%. The vast majority of cases frequently have no symptoms until the cancer has spread extensively. To treat advanced ovarian cancer, surgical therapy and combination chemotherapy using a platinum analogue as the key drug are performed, but the long-term prognosis is still poor [3]. Hence, developing alternative strategies is a matter of urgency. Gene therapy might be a useful additive treatment for women with advanced cancer in order to prolong their time to progression. PTEN (phosphatase and tensin homologue deleted on chromosome 10, also called MMAC1 or TEP1) is a tumor suppressor gene identified on human chromosome 10q23 which encodes a lipid and protein dual-specific phosphatase [4–6]. It has been investigated that PTEN is the first tumor suppressor gene with activity of phosphatase, its protein product which was associated with cellular growth, apoptosis and tumorigenesis through its protein and lipid phosphatase activity antagonizes the action of phosphoinositide 3 kinase (PI3K) by dephosphorylating the signaling lipid phosphatidylinositol (3,4,5)-trisphosphate (PI[3,4,5]P3) to produce phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and results in a decreased phosphorylation of AKT which is one of the major direct downstream target of PI3K [7]. Indeed, the absence of functional PTEN in cancer cells leads to constitutive activation of downstream components of the PI3K pathway including the AKT; therefore, PTEN is named as ‘‘on–off switch” of PI3K/AKT pathway [8]. PTEN has been implicated in cellular processes such as cell proliferation, cell migration and spreading [9]. It is reported that PTEN is frequently deleted or mutated in a wide range of human cancers, including glioblastoma [10], melanoma [11], and prostate [12], breast [13], and endometrial cancers [14]. Recently, PTEN has been reported to inhibit growth and migration of many cancer cells by gene transfer [15]. However, the cellular mechanisms of PTEN function are still not completely understood. Recent genetic studies have shown that ovarian cancer contains frequent mutations in PTEN [16]. PTEN is mutated in a subset of late stage ovarian cancer patient samples and decreased PTEN expression accompanies the progression of ovarian cancer [15]. Gene therapy with PTEN would provide a suc-
cessful approach for treating human ovarian cancer. The precise function of PTEN in ovarian cancer progression has not been fully studied. Thus, to test the potential for PTEN gene therapy of ovarian cancers, we wanted to determine the function of PTEN in ovarian cancer in this study. To investigate the role of PTEN in ovarian cancer progression, we performed proliferation, cell migration and invasion assays by transfection ovarian cancer cells with exogenous PTEN plasmids and analyzed how overexpression of PTEN affects proliferation, migration and invasion in ovarian cancer cells. 2. Materials and methods 2.1. Cell lines and culture conditions Human ovarian epithelial carcinoma cell line A2780 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK), cells were maintained in RPMI-1640 complete medium supplemented with 2 mM L-glutamine and 10% FBS at 37 °C in a humidified atmosphere containing 5% CO2 [17]. 2.2. Chemicals and antibodies RPMI-1640, fetal bovine serum, Lipofectamine 2000 reagent, and TRIzol reagent were purchased from Life Technologies Inc. MTT, DMSO and G418 were obtained from Sigma (St. Louis, MO). Mouse anti-human PTEN polyclonal antibody was obtained from R&D System, Inc. Rabbit polyclonal anti-human phosphor-AKT (Ser473) antibody, total rabbit polyclonal anti-human AKT antibody, rabbit polyclonal anti-human caspase3 antibody, rabbit monoclonal anti-human PARP antibody, rabbit monoclonal anti-human MMP-9 antibody and rabbit monoclonal antihuman b-actin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). PCR primer and pEGFP-C1 plasmid were from Invitrogen Corp. (Invitrogen Corp. USA). TM
2.3. Construction of recombinant plasmids GFP expression plasmids based on pGZ21dxZ with full-length wild-type PTEN and the point mutant C124A (Cys124-Ala) PTEN which lost its phosphatase activity were generous gifts from Dr. Kenneth M. Yamada [18] (National Institute of Dental and Craniofacial Research, NIH, Bethesda,
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MD, USA). The plasmids were restrictively digested with HindIII and XbaI, then the full-length wildtype and mutant type PTEN mRNA were reclaimed and purified, and then cloned into pEGFP-C1 to form pEGFP-C1-WT-PTEN and pEGFP-C1C124A-PTEN, the pEGFP-C1 vector was treated as a control. 2.4. Plasmid transfections For transfection, A2780 cells were seeded in sixwell plates at 5 105/well and incubated in 5% CO2/95% air incubator at 37 °C. When cells were 80–90% confluent, cells were transfected using Lipofectamine 2000 transfection reagent according to the Manufacturer’s protocol. Recombinant plasmids (4 lg) were mixed with lipofectamine and pre-incubated for 20 min at room temperature in serum-free and antibiotic-free DMEM. A2780 cells transfected with pEGFP-C1-WT-PTEN, pEGFP-C1-C124APTEN and pEGFP-C1 empty vector were named as WT-PTEN/A2780, C124A-PTEN/A2780 and GFP/A2780. G418 (500 lg/ll) was applied to stable screening and isolating the resistant colonies. 2.5. RT-PCR analysis Total RNA were prepared using Trizol regent, following manufacturer’s instructions. Total RNA (2 lg) was reversed transcribed using M-MLV Reverse transcriptase (Promega, USA). cDNA was amplified by PCR using Taq DNA polymerase (Promega, USA). Primers were as follows: for PTEN expression, sense: 50 -ACGGGAA 0 GACAAGTTCATGTAC-3 , and antisense: 50 -TTT GAC GGCTCCTCTACTGT-30 (product size, 389 bp); for GAPDH (housekeeping gene) expression, sense 50 -ACGGATTTG GTCG TATTGGG30 and antisense 50 -TGATTTTGGAGGGATC TCGC-30 (product size, 180 bp). The RT-PCR cycle was as 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 94 °C, 30 s at 56 °C, 30 s at 72 °C, 28 cycles. PCR products were analyzed by 1.5% agarose gel electrophoresis in the presence of ethidium bromide for UV light transilluminator visualization. 2.6. Western blotting analysis Cells harvested were lysed in lysis buffer (50 mM Tris–HCl pH7.4, 150 mM NaCl, 1% Nonidet P 40, 1% Triton X-100, 0.2% SDS, 1% sodium deoxycho-
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late, 5 mM iodoacetamide, proteinase inhibitor cocktail, 2 mM PMSF). Protein (60 lg) was denatured in SDS sample buffer at 100 °C for 10 min and separated by electrophoresis on a 10% SDS– PAGE gel, then transferred to a nitrocellulose membranes. The membrane was blocked in TBST (25 mM Tris–HCl, pH 7.5, 137 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20) with 5% non-fat milk for 1 h at 37 °C, and then incubated with the primary antibodies (mouse anti-PTEN, 1:2000; rabbit anti-AKT, 1:500; rabbit anti-phospho-AKT, 1:1000; rabbit anti-caspase 3, 1:500; rabbit antiPARP, 1:200; rabbit anti-MMP-9, 1:500; rabbit anti-actin, 1:500) in blocking buffer overnight at 4 °C. The membrane was washed three times in TBST and incubated with alkaline phosphatase– conjugated goat IgG (1:500) for 2 h at room temperature and then visualized with NBT/BCIP/buffer (1:1:50). Protein loading was assessed by blotting of the same membrane with an antibody against b-actin. 2.7. Flow cytometric analysis and TUNEL assay Cell apoptosis was evaluated by flow cytometry (FCM) according to the methods described by Ishibashi and Lippard [19]. Cells were harvested with 0.2% trypsin, washed in cold PBS, fixed in 70% ice-cold ethanol and then stained with propidium iodide (PI) overnight at 20 °C. On the following day, the stained cell were run on a FACSort flow cytometery (Becton-Dickinson, Franklin Lake, USA) and evaluated using the CELLQuest software system (BD Biosciences, USA) to calculate the percentage of sub-G1 (hypodiploid) apoptotic cells and the percentage of cells in different cell cycle. At the same time, nuclear morphology of apoptotic cells was determined using the in situ Cell Death Detection kit from Roche Diagnostic (Mannheim, Germany) following the manufacturer’s instruction. The apoptotic cells (brown staining) were counted under a microscope. 2.8. Cell proliferation assays Cell proliferation was determined by the MTT assay. Briefly, cells were cultured in 96-well plates the day before experiment at a density of 5 103/ well, and then were incubated for 1, 2, 3, 4, 5, 6 and 7 days, then 20 ll 3-(4,5 dimethylthiazol-2-yl)2,5-diphenyl-tetrazoliumbromide (MTT) (5 mg/ml, Sigma Chemical Co.) was added to the wells, which
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was followed by incubation at 37 °C for 4 h, aspirating the supernatants, adding 100 ll DMSO and incubating at 37 °C for an additional 20 min. The absorbance of the wells was then read with a microplate reader at a test wavelength of 570 nm and a reference wavelength of 630 nm. Appropriate controls lacking cells were included to determine background absorbance. Response to drug treatment was assessed by standardizing treatment groups to untreated control.
different fields of each well were counted with two wells per treatment. The mean values were obtained from three replicate experiments and were subjected to the t-test. 2.11. Statistical analysis All experiments were repeated at least thrice. The data were analyzed with the software package SPSS 12.0. P value less than 0.05 were considered significant statistically.
2.9. Wound-healing assay Post-transfected A2780 cells were seeded into 35mm dishes at 1.5 105 cells/cm2 and allowed to grow to 90% confluence in their recommended media. Medium was replaced by corresponding media with 0.1% BSA for 24 h. The confluent cell monolayer was scraped with a sterile pipette tip with a constant diameter. For each dish, 3–5 wounds were made, and three sites of regular wounds were selected and marked. Wounded monolayers were then washed three times with PBS to remove cell debris. Cells were permitted to migrate into the area of clearing for 24 h. Immediate after wounding and at the end of the experiment (after 24 h), wounds were photographed and semiquantitative measurements were taken of control and treated wounds. A mean wound width was determined, and the average width of wound closure was calculated as described previously [20]. 2.10. Cell invasion assays The A2780 cells were harvested by trypsinization and washed in RPMI medium without serum. The cells were suspended in RPMI 1640 medium at 5 105 cells/ml. Prior to preparing the cell suspension, the dried layer of matrigel matrix was rehydrated with serum-free RPMI 1640 medium for 2 h at 37 °C. The rehydration solution was carefully removed, 0.75 ml RPMI 1640 medium containing 10% FBS was added to each well as a chemoattractant, and 0.5 ml (2.5 105 cells) of cell suspension was added to each well. The plates were incubated for 48 h at 37 °C. The invaded cells on the bottom surface of the membrane were fixed and stained by sequentially transferring them through wells of a second 24-well plate containing the three solutions from the Diff-QuikÒ staining kit (Dade Behring Inc.). The cells were enumerated by taking photomicrographs at 200 magnification. The cells in three
3. Results 3.1. Effect of WT-PTEN and C124A-PTEN plasmids transfection on PTEN expression To investigate whether the WT-PTEN and C124APTEN plasmids increased the expression of PTEN gene, WT-PTEN/A2780 (A2780 cells transfected with wild-type PTEN plasmid), C124A-PTEN/A2780 (A2780 cells transfected with mutant PTEN plasmid), GFP/A2780 (A2780 cells transfected with pEGFP-C1 plasmid) and A2780 cells were analyzed for the expression of PTEN mRNA and protein by RT-PCR and Western blot analysis. The RT-PCR and Western blot product levels were normalized to b-actin and GAPDH expression. PTEN mRNA expression in WT-PTEN/A2780 and C124A-PTEN/ A2780 cells (1.71 ± 0.15 and 1.75 ± 0.09) were significantly higher than those of GFP/A2780 and A2780 cells (1.07 ± 0.07 and 1.06 ± 0.07), respectively (Fig. 1A). (t = 5.300, P = 0.034; t = 11.963, P = 0.007; t = 7.869, P = 0.016; t = 22.421, P = 0.002, n = 4) (Fig. 1A); The expression of PTEN protein of WT-PTEN/A2780 and C124A-PTEN/A2780 cells (1.21 ± 0.04 and 1.22 ± 0.02) were also obviously higher compared with those of GFP/A2780 and A2780 cells (0.74 ± 0.03 and 0.74 ± 0.02) (P < 0.05) (Fig. 1B). These results demonstrated that PTEN plasmid transfection were an effective and pragmatic approach to increase the expression of PTEN gene. 3.2. Effect of PTEN plasmids transfection on A2780 cell proliferation To examine whether PTEN gene is able to inhibit the proliferation of A2780 cells, WT-PTEN/A2780, C124APTEN/A2780, GFP/A2780 and A2780 cells were incubated for 1, 2, 3, 4, 5, 6 and 7 days, and the growth rates were calculated by MTT assay. In WT-PTEN/A2780 group, the cell proliferation rate decreased gradually and reached its lowest level 7 days after incubation (Fig. 2). No significant changes in cell proliferation rate were observed for either C124A-PTEN/A2780, GFP/A2780 cells or A2780 cells.
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Fig. 2. Effect of PTEN transfection on cell proliferation of A2780 cells. WT-PTEN/A2780, C124A-PTEN/A2780, GFP/A2780 and nontreated A2780 cells were incubated for 1, 2, 3, 4, 5, 6 and 7 days. Cell growth was measured using absorbance as described in Section 2. Points, averages of three independent experiments; bars, SD.
Fig. 1. Expression levels of PTEN mRNA and protein of the cells transfected with PTEN plasmids (WT-PTEN/A2780 and C124APTEN/A2780) or empty EGFP vector (GFP/A2780) and A2780 cells. (A) The expression of PTEN mRNA (389 bp PCR product) was detected by semiquantitative RT-PCR, GAPDH (180 bp PCR product) was co-amplified as the internal control. (B) The expression of PTEN protein was detected by Western blot analysis in total cell extracts. b-actin was reprobed to confirm equal protein loading. The experiment was repeated 3 times.
3.3. Effect of PTEN plasmids transfection on A2780 cell cycle and cell apoptosis It has been shown previously by others that transfection of WT-PTEN into cells induces cell cycle arrest as well as apoptosis [21,22]. To determine the role of the PTEN gene transfection in cell cycle distribution and cell apoptosis in A2780 cells, WT-PTEN/A2780, C124APTEN/A2780, GFP/A2780 and A2780 cells therefore were stained with PI and subjected to flow cytometry analysis to analysis cell cycle and apoptosis. As shown in Fig. 3A, expression of wild-type PTEN gene led to a substantial increase in the G0/G1 population, as compared with that of the vector control{(71.18 ± 4.34)% vs. (51.91 ± 2.88)%} (P < 0.05). The cell cycle arrest caused by PTEN expression depended on its phosphatase activity, as very little effect on the cell cycle profile was observed when the phosphatase-inactive PTEN (C124A) was expressed. We also found from Fig. 3B, up to
32.05% of WT-PTEN/A2780 cells displayed sub-G1 DNA content, which was an indication of DNA fragmentation and often associated with apoptotic cell death [23]. In contrast, only 1.77% of A2780 cells or 1.59% of GFP/ A2780 cells exhibited sub-G1 DNA content (P < 0.05). We also found that expression of wild-type PTEN, but not its phosphatase-inactive (C124A) PTEN, led to a substantial upregulation in the amount of cells undergoing apoptosis. TUNEL assay showed similar results. In WTPTEN transfected A2780 cells, the morphologic alterations characteristic of apoptosis, including nuclear condensation and fragmentation, were detected (data not shown). These results confirmed that PTEN expression led to G0/G1 cell cycle arrest and cell apoptosis dependent on its phosphatase activity in A2780 ovarian epithelial cancer cells. 3.4. Effect of PTEN plasmids transfection on migration of A2780 cells Cell migration is a characteristic feature of carcinoma cells [24]. PTEN has been reported to be implicated in the regulation of cell migration [25]. To test whether PTEN gene play an important role in regulation of cell migration in A2780 cells, a wound-healing assay was carried out to test the cell migration. Confluent and quiescent monolayers of WT-PTEN/A2780, C124A-PTEN/A2780, GFP/ A2780 and A2780 cells were wounded. Recovery of these monolayer cells depends on their migration during wound-healing. Photographs were taken directly at 24 h after wounding to observe the changes of migration and then the distance of cell migration to the wound area were measured (Fig. 4A) [26]. The WT-PTEN/A2780 cells had
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Fig. 3. Effect of PTEN transfection on cell cycle and apoptosis of A2780 cells by flow cytometric analysis. Flow cytometry was used to analyze the changes of the propidium iodide-stained A2780 cells. (A) PTEN-mediated G0/G1 arrest in A2780 cells was analyzed by determining the cell cycle distribution by FACS and percentage of G0/G1 phase cells was presented. (B) The number of apoptotic cells was counted with a FACSort flow cytometer according to the methods described in Section 2. (C) Apoptotic cells were detected by TUNEL assay (brown staining).
a much slower wound-healing rate when compared with those of C124A-PTEN/A2780, GFP/A2780 and A2780 cells (P < 0.05) (Fig. 4B). These results demonstrated that cell migration was down-regulated by expression of phosphatase-active form of PTEN but not by PTEN with an phosphatase-inactive PTEN gene.
44.7 ± 2.1 and 45.0 ± 3.0) (P < 0.05) (Fig. 5B). These results also showed that no significant change was observed after transfection with the phosphatase-inactive mutant of PTEN (C124A), indicating that the phosphatase activity of PTEN is required for inhibition of cell invasion.
3.5. Effect of PTEN plasmids transfection on invasion of A2780 cells
3.6. Effect of PTEN plasmids transfection on the expression of total AKT and phospho-AKT (Ser473) protein in A2780 cells
To investigate the role of PTEN gene in tumor cell invasion in A2780 cells, we measured the ability of different A2780 cells to migrate through matrigel-coated filters in a modified transwell assay (Fig. 5A), The number of invaded cells of WT-PTEN/A2780 cells (24.3 ± 2.5) were obviously much fewer than those of C124A-PTEN/ A2780, GFP/A2780 and A2780 cells (43.7 ± 3.8,
To determine the molecular mechanisms by which PTEN may contribute to the inhibited proliferation and migration as well as suppressed invasion. Total cellular protein extracts were prepared and subjected to Western blot analysis using antibodies against total AKT, phosphoAKT (Ser473), caspase-3, PARP[poly(ADP-ribose) poly-
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Fig. 4. Effect of PTEN on migration of A2780 cells in vitro. (A) Confluent monolayers of A2780 cells were wounded. Photographs were taken directly (t = 0 h) and 24 h after wounding (t = 24 h). (B) Quantification of the endothelial wound repair. The distance of cell migration to the wound area was measured in A2780 cells 24 h after wounding. Values are means ± SD from three independent experiments.
merase-1] and MMP-9 and b-actin. The result had shown that the overexpression of wild-type PTEN gene in WTPTEN/A2780 cells resulted in a markedly suppressed expression of phospho-AKT and MMP-9 protein (Fig. 6). There was no difference was in the expression of total AKT, caspase-3 and PARP in different treated A2780 cells.
4. Discussion The incidence of ovarian cancer has been increasing in recent years. Although the combination cytoreductive surgery with cisplatin-centered chemotherapy dramatically lengthened the survival time of patients and cut-down the mortality of ovarian cancer, ovarian cancer has the worst diagnosis and prognosis of any gynecological cancer due to many factors, including the poor understanding of precursor lesions and tumor progression mecha-
nisms, and the usually late-stage presentation of this aggressive disease [27]. The current major research focus is identifying an effective therapeutic treatment of ovarian cancer. PTEN was discovered by three different groups in 1997 [4]. It codifies a 403-amino acid dual-specific phosphatase with putative tumor suppressing abilities. PTEN protein has the activity of protein phosphatase and lip phosphatase. Loss of PTEN expression has been detected in a wide range of human cancers, including human brain, breast, prostate, endometrial and ovarian cancers [28] and PTEN has already proven itself to be a remarkable molecule. It is now known that PTEN plays important role not only in inducing cell cycle detention and cell apoptosis, but also in regulation of cell adherence, cell migration, cell differentiation and chemoresistance [29,30]. Therefore, in this study
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Fig. 5. Effect of PTEN on invasion of A2780 cells in vitro. (A) Cell invasion through matrigel basement membrane extract was analyzed using a modified transwell assay as described in Section 2. Cells that invaded to the lower surface of the membrane were fixed and stained. (B) Mean cell counts from at least 10 fields and four experiments are shown. The number of invaded cells of WT-PTEN/A2780 cells (24.3 ± 2.5) were obviously much fewer than those of C124A-PTEN/A2780, GFP/A2780 and A2780 cells (43.7 ± 3.8, 44.7 ± 2.1 and 45.0 ± 3.0) (P < 0.05).
we transfected A2780 cells with wild-type and phosphatase-inactive PTEN to inhibit the expression of PTEN gene, and determined their effects on cell apoptosis, cell cycle distribution and cell proliferation as well as cell migration and invasion. Expansion of tumor cell populations can be the result of unchecked cell division or a consequence of cell accumulation secondary to avoidance of programmed cell death (apoptosis) or both [27]. Tumor cell survival is one of the major reasons for tumor expansion and progression. In our study, upregulation of PTEN mRNA was verified by RT-PCR in WT-PTEN/A2780 and C124A-PTEN/A2780 cells using b-actin as an internal normalization standard, and the upregulation of PTEN proteins was reconfirmed by Western blotting analysis. The results suggested that transfection with PTEN was efficient to increase PTEN expression in ovarian cancer cell lines. We further found that stable expression of wild-type PTEN markedly suppressed the growth rate of A2780 cells as compared with mock cells. Next, we wondered whether these inhibitory functional properties endowed on wild-type PTEN-overexpressing cells were linked to cell cycle arrest and a greater rate of apoptosis, therefore, we attempted to perform flow cytometry analysis to analysis the cell cycle and apoptosis of A2780 cells and found that a marked increase in the G0/G1 and sub-G1 (apoptotic cells) population in WT-PTEN/A2780 cells which showed significant growth suppression. In contrast, C124A-PTEN/ A2780 cells which were overexpressing the phosphatase-inactive mutant of PTEN showed the rate of proliferation and G0/G1 population as well as apoptotic ratio was comparable to those exhibited by mock cells. Taken together, these findings suggested that
the growth-inhibitory activity of exogenous PTEN is mediated by two mechanisms, apoptosis and/or arrest at the G1 phase of the cell cycle, at the same time, the above results also demonstrated that phosphatase activity of PTEN is critical for its role as a tumor suppressor gene. To investigate the molecular mechanisms by which PTEN may contribute to enhanced apoptosis and cell cycle arrest, we detected the protein expression levels of some molecules which were involved in various sig-
Fig. 6. Wild-type PTEN transfection reduced the expression of phospho-AKT (Ser473) and MMP-9 protein in A2780 cells analyzed by Western blot analysis. Protein was extracted from WT-PTEN/A2780, C124A-PTEN/A2780, GFP/A2780 and A2780 cells. An amount of 60 lg of each sample was resolved by gel electrophoresis, transferred to a membrane and probed with antibodies specific to total AKT, phospho-AKT (Ser473), caspase-3, PARP and MMP-9, as indicated. The b-actin was used as an internal control.
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naling pathways in A2780 cells. Relative to total AKT content, WT-PTEN/A2780 cells contained lower levels of AKT that was phosphorylated on Ser473, one of the two phosphorylation sites on AKT that results in its activation. In contrast, no difference was observed between the A2780 cells in their levels of caspase-3 and PARP. Therefore, the AKT signaling pathway appears to be specifically suppressed in wild-type PTEN-expressing A2780 cells. PI3K/PIP3 pathway is important in controlling a diverse array of cellular functions including cell survival, cell cycle progression, motility, differentiation in response to extracellular stimuli. AKT, as one of the major direct down stream target 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 phosphorylating several substrates, such as Bad, HIF-1a, NFrB. Resistance to apoptosis may contribute to tumorigenesis of many malignancies and insensitivity of ovarian cancer to standard therapies. Cell migration is a key cellular feature of tumor progression because it regulates metastasis. Both tumor cell migration and invasion through the basement membranes are crucial steps in the multi-stage process that leads to metastasis formation [31]. Ovarian cancer is a highly metastatic cancer, and therefore cell migration and invasion are particularly important processes of ovarian cancer [16]. Thus, we investigated whether that PTEN was involved in cell migration and invasion to understand ovarian cancer progression. In our study, wild-type PTEN-overexpressing A2780 cells showed significantly decreased the rate of cell migration and invasion and obviously reduced MMP-9 expression compared with the mock cells. Since phosphatase-inactive mutant C124A PTEN did not inhibit cell migration and invasion which suggests that the phosphate domain is necessary for inhibiting migration and invasion. Cell migration requires cell invasion, the process of cellular-induced degradation of the basal membrane through membrane penetration. A common feature of the invasive process is the degradation of the surrounding ECM by an array of proteolytic enzymes expressed by the infiltrating tumor cells [32]. Matrix metalloproteinases, capable of degrading almost all ECM components, have been suggested to play an important role in mediating tumor invasion [33]. Therefore, the elevated expression of various MMPs is strongly associated with
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the invasive phenotype. Our data have demonstrated the expression of MMP-9 protein was significantly inhibited by the overexpression of wild-type PTEN. These observations raise the interesting possibility of exploring the precise intracellular signaling mechanism of control of MMP-9 expression by the tumor suppressor gene PTEN. It is reported that FAK, one of substrates of the PTEN protein, modulates cell migration and invasion [16] and is also involved in the activation of MMPs [34]. Thus, wild-type PTEN might inhibit the expression of MMP-9 protein by decreasing the expression of FAK protein. Further work is required to verify the mechanism by which PTEN inhibit cell migration and invasion of ovarian cancer cells. In conclusion, these results suggest that PTEN have played an important role in the cell proliferation and migration as well as cell invasion in ovarian cancer cells. Overexpression of PTEN can reverse the malignant phenotype of ovarian cancer cell. PTEN gene may be a novel target for treatment of patients with ovarian cancer. Conflict of Interest None. Acknowledgments This work was supported by Grants from the National Natural Science Foundation of China (Nos. 30571950, 30528012, and 30371657, 30672227) and the ‘‘973” Program of China (No. 2002CB513100). References [1] H. Naora, D.J. Montell, Ovarian cancer metastasis: integrating insights from disparate model organisms, Nat. Rev. Cancer 5 (2005) 355–366. [2] X. Ma, S. Wang, J. Zhou, H. Xing, G. Xu, B. Wang, G. Chen, Y.P. Lu, D. Ma, Induction of apoptosis in human ovarian epithelial cancer cells by antisurvivin oligonucleotides, Oncol. Rep. 14 (2005) 275–279. [3] W.P. McGuire, W.J. Hoskins, M.F. Brady, P.R. Kucera, E.E. Partridge, K.Y. Look, D.L. Clarke-Pearson, M. Davidson, Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer, N. Engl. J. Med. 334 (1996) 1–6. [4] J. Li, C. Yen, D. Liaw, K. Podsypanina, S. Bose, S.I. Wang, J. Puc, C. Miliaresis, L. Rodgers, R. McCombie, S.H. Bigner, B.C. Giovanella, M. Ittmann, B. Tycko, H. Hibshoosh, M.H. Wigler, R. Parsons, PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer, Science 275 (1997) 1943–1947.
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[5] P.A. Steck, M.A. Pershouse, S.A. Jasser, W.K. Yung, H. Lin, A.H. Ligon, L.A. Langford, M.L. Baumgard, T. Hattier, T. Davis, C. Frye, R. Hu, B. Swedlund, D.H. Teng, S.V. Tavtigian, Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers, Nat. Genet. 15 (1997) 356–362. [6] D.M. Li, H. Sun, TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta, Cancer Res. 57 (1997) 2124–2129. [7] S. Hafizi, F. Ibraimi, B. Dahlback, C1-TEN is a negative regulator of the Akt/PKB signal transduction pathway and inhibits cell survival, proliferation, and migration, FASEB J. 19 (2005) 971–973. [8] I. Vivanco, C.L. Sawyers, The phosphatidylinositol 3-Kinase AKT pathway in human cancer, Nat. Rev. Cancer 2 (2002) 489–501. [9] M. Tamura, J. Gu, K. Matsumoto, S. Aota, R. Parsons, K.M. Yamada, Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN, Science 280 (1998) 1614–1617. [10] S.I. Wang, J. Puc, J. Li, J.N. Bruce, P. Cairns, D. Sidransky, R. Parsons, Somatic mutations of PTEN in glioblastoma multiforme, Cancer Res. 57 (1997) 4183–4186. [11] P. Guldberg, P. thor Straten, A. Birck, V. Ahrenkiel, A.F. Kirkin, J. Zeuthen, Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma, Cancer Res. 57 (1997) 3660–3663. [12] P. Cairns, K. Okami, S. Halachmi, N. Halachmi, M. Esteller, J.G. Herman, J. Jen, W.B. Isaacs, G.S. Bova, D. Sidransky, Frequent inactivation of PTEN/MMAC1 in primary prostate cancer, Cancer Res. 57 (1997) 4997–5000. [13] E. Rhei, L. Kang, F. Bogomolniy, M.G. Federici, P.I. Borgen, J. Boyd, Mutation analysis of the putative tumor suppressor gene PTEN/MMAC1 in primary breast carcinomas, Cancer Res. 57 (1997) 3657–3659. [14] H. Tashiro, M.S. Blazes, R. Wu, K.R. Cho, S. Bose, S.I. Wang, J. Li, R. Parsons, L.H. Ellenson, Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological malignancies, Cancer Res. 57 (1997) 3935–3940. [15] T. Schondorf, U.J. Gohring, G. Roth, I. Middel, M. Becker, N. Moser, M.M. Valter, M. Hoopmann, Time to progression is dependent on the expression of the tumour suppressor PTEN in ovarian cancer patients, Eur. J. Clin. Invest. 33 (2003) 256–260. [16] Q. Meng, C. Xia, J. Fang, Y. Rojanasakul, B.H. Jiang, Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway, Cell. Signal. 18 (2006) 2262–2271. [17] E. Asselin, G.B. Mills, B.K. Tsang, XIAP regulates Akt activity and caspase-3-dependent cleavage during cisplatininduced apoptosis in human ovarian epithelial cancer cells, Cancer Res. 61 (2001) 1862–1868. [18] M. Tamura, J. Gu, T. Takino, K.M. Yamada, Tumor suppressor PTEN inhibition of cell invasion, migration, and growth: differential involvement of focal adhesion kinase and p130Cas, Cancer Res. 59 (1999) 442–449. [19] T. Ishibashi, S.J. Lippard, Telomere loss in cells treated with cisplatin, Proc. Natl. Acad. Sci. USA 95 (1998) 4219–4223. [20] E.J. Battegay, Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects, J. Mol. Med. 73 (1995) 333–346.
[21] H. Sun, R. Lesche, D.M. Li, J. Liliental, H. Zhang, J. Gao, N. Gavrilova, B. Mueller, X. Liu, H. Wu, PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway, Proc. Natl. Acad. Sci. USA 96 (1999) 6199–6204. [22] S. Persad, S. Attwell, V. Gray, M. Delcommenne, A. Troussard, J. Sanghera, S. Dedhar, Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells,Proc. Natl. Acad. Sci. USA 97 (2000) 3207–3212. [23] N. Sima, S. Wang, W. Wang, D. Kong, Q. Xu, X. Tian, A. Luo, J. Zhou, G. Xu, L. Meng, Y. Lu, D. Ma, Antisense targeting human papillomavirus type 16 E6 and E7 genes contributes to apoptosis and senescence in SiHa cervical carcinoma cells, Gynecol. Oncol. 106 (2007) 299–304. [24] S. Wu, L. Meng, S. Wang, W. Wang, L. Xi, X. Tian, G. Chen, Y. Wu, J. Zhou, G. Xu, Y. Lu, D. Ma, Reversal of the malignant phenotype of cervical cancer CaSki cells through adeno-associated virus-mediated delivery of HPV16 E7 antisense RNA, Clin. Cancer Res. 12 (2006) 2032–2037. [25] M.P. Myers, I. Pass, I.H. Batty, J. Van der Kaay, J.P. Stolarov, B.A. Hemmings, M.H. Wigler, C.P. Downes, N.K. Tonks, The lipid phosphatase activity of PTEN is critical for its tumor suppressor function, Proc. Natl. Acad. Sci. USA 95 (1998) 13513–13518. [26] L. Zhao, G. Xu, J. Zhou, H. Xing, S. Wang, M. Wu, Y.P. Lu, D. Ma, The effect of RhoA on human umbilical vein endothelial cell migration and angiogenesis in vitro, Oncol. Rep. 15 (2006) 1147–1152. [27] S.V. Nicosia, W. Bai, J.Q. Cheng, D. Coppola, P.A. Kruk, Oncogenic pathways implicated in ovarian epithelial cancer, Hematol. Oncol. Clin. North Am. 17 (2003) 927–943. [28] A. Di Cristofano, P.P. Pandolfi, The multiple roles of PTEN in tumor suppression, Cell 100 (2000) 387–390. [29] L.C. Cantley, B.G. Neel, New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway, Proc. Natl. Acad. Sci. USA 96 (1999) 4240–4245. [30] X. Yan, M. Fraser, Q. Qiu, B.K. Tsang, Over-expression of PTEN sensitizes human ovarian cancer cells to cisplatininduced apoptosis in a p53-dependent manner, Gynecol. Oncol. 102 (2006) 348–355. [31] C. Jimenez, R.A. Portela, M. Mellado, J.M. Rodriguez-Frade, J. Collard, A. Serrano, A.C. Martinez, J. Avila, A.C. Carrera, Role of the PI3K regulatory subunit in the control of actin organization and cell migration, J. Cell Biol. 151 (2000) 249–262. [32] D. Koul, R. Parthasarathy, R. Shen, M.A. Davies, S.A. Jasser, S.K. Chintala, J.S. Rao, Y. Sun, E.N. Benvenisite, T.J. Liu, W.K. Yung, Suppression of matrix metalloproteinase-2 gene expression and invasion in human glioma cells by MMAC/PTEN, Oncogene 20 (2001) 6669–6678. [33] M.J. Park, M.S. Kim, I.C. Park, H.S. Kang, H. Yoo, S.H. Park, C.H. Rhee, S.I. Hong, S.H. Lee, PTEN suppresses hyaluronic acid-induced matrix metalloproteinase-9 expression in U87MG glioblastoma cells through focal adhesion kinase dephosphorylation, Cancer Res. 62 (2002) 6318–6322. [34] T.T. Sein, A.A. Thant, Y. Hiraiwa, A.R. Amin, Y. Sohara, Y. Liu, S. Matsuda, T. Yamamoto, M. Hamaguchi, A role for FAK in the Concanavalin A-dependent secretion of matrix metalloproteinase-2 and -9, Oncogene 19 (2000) 5539–5542.
Cancer Letters 271 (2008) 205–214 www.elsevier.com/locate/canlet
Reversal of the malignant phenotype of ovarian cancer A2780 cells through transfection with wild-type PTEN gene Huijuan Wu a,b,1, Shixuan Wang a,1, Danghui Weng a, Hui Xing c, Xiaohong Song a, Tao Zhu a, Xi Xia a, Yanjie Weng a, Gang Xu a, Li Meng a, Jianfeng Zhou a,*, Ding Ma a,* a
Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China b Department of Gynecological Oncology, Cancer Hospital, Tianjin Medical University, Tianjin 300060, PR China c Department of Obsterics and Gynecology, Xiangfan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Xiangfan, Hubei 441021, PR China Received 18 April 2008; received in revised form 18 April 2008; accepted 2 June 2008
Abstract Objective: PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumor suppressor gene identified on human chromosome 10q23. Substantial studies have demonstrated that PTEN can inhibit cell proliferation, migration and invasion of many cancer cells. The purpose of this study was to determine whether upregulation of PTEN gene by transfection wild-type PTEN gene to ovarian cancer cells can inhibit growth and migration and to explore the potential for PTEN gene therapy of ovarian cancers. Method: Wild-type and phosphatase-inactive (C124A) PTEN plasmids were transfected into ovarian epithelial cancer A2780 cells, and their effects on cell apoptosis, cell proliferation, cell migration and cell invasion were analyzed by flow cytometry analysis, TUNEL assay, MTT assay, wound-healing assay and transwell assay. Results: Both wild-type and mutant PTEN can upregulate the expression of PTEN gene dramatically; however, it is wild-type PTEN not phosphatase-inactive PTEN that can induce apoptosis and decrease cell migration, invasion and proliferation in ovarian cancer cells. Conclusion: These results demonstrated that PTEN had played an important role in the cell proliferation, cell migration and invasion dependent on its phosphatase activity. Enhanced expression of PTEN by gene transfer is sufficient to reverse the malignant phenotype of ovarian cancer cells and transfection of ovarian cancer cells with wild-type PTEN gene might be another novel approach for therapeutic intervention in ovarian cancer. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Ovarian cancer; PTEN; Apoptosis; Proliferation; Invasion; Migration
* Corresponding authors. Fax: +86 27 83663180 (J. Zhou); +86 27 83662681 (D. Ma). E-mail addresses: [email protected] (J. Zhou), dma@ tjh.tjmu.edu.cn (D. Ma). 1 These two authors are equally contributed.
1. Introduction Ovarian cancer is the fifth leading cause of cancer 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.2008.06.018
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from gynecologic malignancies characterized by rapid progression, late metastases and poor prognosis [1,2]. Only about 25% of ovarian cancers are diagnosed at an early stage. Approximately 60% of cases are diagnosed after the cancer has spread, when the 5-year survival rate is close to 30%. The vast majority of cases frequently have no symptoms until the cancer has spread extensively. To treat advanced ovarian cancer, surgical therapy and combination chemotherapy using a platinum analogue as the key drug are performed, but the long-term prognosis is still poor [3]. Hence, developing alternative strategies is a matter of urgency. Gene therapy might be a useful additive treatment for women with advanced cancer in order to prolong their time to progression. PTEN (phosphatase and tensin homologue deleted on chromosome 10, also called MMAC1 or TEP1) is a tumor suppressor gene identified on human chromosome 10q23 which encodes a lipid and protein dual-specific phosphatase [4–6]. It has been investigated that PTEN is the first tumor suppressor gene with activity of phosphatase, its protein product which was associated with cellular growth, apoptosis and tumorigenesis through its protein and lipid phosphatase activity antagonizes the action of phosphoinositide 3 kinase (PI3K) by dephosphorylating the signaling lipid phosphatidylinositol (3,4,5)-trisphosphate (PI[3,4,5]P3) to produce phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and results in a decreased phosphorylation of AKT which is one of the major direct downstream target of PI3K [7]. Indeed, the absence of functional PTEN in cancer cells leads to constitutive activation of downstream components of the PI3K pathway including the AKT; therefore, PTEN is named as ‘‘on–off switch” of PI3K/AKT pathway [8]. PTEN has been implicated in cellular processes such as cell proliferation, cell migration and spreading [9]. It is reported that PTEN is frequently deleted or mutated in a wide range of human cancers, including glioblastoma [10], melanoma [11], and prostate [12], breast [13], and endometrial cancers [14]. Recently, PTEN has been reported to inhibit growth and migration of many cancer cells by gene transfer [15]. However, the cellular mechanisms of PTEN function are still not completely understood. Recent genetic studies have shown that ovarian cancer contains frequent mutations in PTEN [16]. PTEN is mutated in a subset of late stage ovarian cancer patient samples and decreased PTEN expression accompanies the progression of ovarian cancer [15]. Gene therapy with PTEN would provide a suc-
cessful approach for treating human ovarian cancer. The precise function of PTEN in ovarian cancer progression has not been fully studied. Thus, to test the potential for PTEN gene therapy of ovarian cancers, we wanted to determine the function of PTEN in ovarian cancer in this study. To investigate the role of PTEN in ovarian cancer progression, we performed proliferation, cell migration and invasion assays by transfection ovarian cancer cells with exogenous PTEN plasmids and analyzed how overexpression of PTEN affects proliferation, migration and invasion in ovarian cancer cells. 2. Materials and methods 2.1. Cell lines and culture conditions Human ovarian epithelial carcinoma cell line A2780 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK), cells were maintained in RPMI-1640 complete medium supplemented with 2 mM L-glutamine and 10% FBS at 37 °C in a humidified atmosphere containing 5% CO2 [17]. 2.2. Chemicals and antibodies RPMI-1640, fetal bovine serum, Lipofectamine 2000 reagent, and TRIzol reagent were purchased from Life Technologies Inc. MTT, DMSO and G418 were obtained from Sigma (St. Louis, MO). Mouse anti-human PTEN polyclonal antibody was obtained from R&D System, Inc. Rabbit polyclonal anti-human phosphor-AKT (Ser473) antibody, total rabbit polyclonal anti-human AKT antibody, rabbit polyclonal anti-human caspase3 antibody, rabbit monoclonal anti-human PARP antibody, rabbit monoclonal anti-human MMP-9 antibody and rabbit monoclonal antihuman b-actin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). PCR primer and pEGFP-C1 plasmid were from Invitrogen Corp. (Invitrogen Corp. USA). TM
2.3. Construction of recombinant plasmids GFP expression plasmids based on pGZ21dxZ with full-length wild-type PTEN and the point mutant C124A (Cys124-Ala) PTEN which lost its phosphatase activity were generous gifts from Dr. Kenneth M. Yamada [18] (National Institute of Dental and Craniofacial Research, NIH, Bethesda,
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MD, USA). The plasmids were restrictively digested with HindIII and XbaI, then the full-length wildtype and mutant type PTEN mRNA were reclaimed and purified, and then cloned into pEGFP-C1 to form pEGFP-C1-WT-PTEN and pEGFP-C1C124A-PTEN, the pEGFP-C1 vector was treated as a control. 2.4. Plasmid transfections For transfection, A2780 cells were seeded in sixwell plates at 5 105/well and incubated in 5% CO2/95% air incubator at 37 °C. When cells were 80–90% confluent, cells were transfected using Lipofectamine 2000 transfection reagent according to the Manufacturer’s protocol. Recombinant plasmids (4 lg) were mixed with lipofectamine and pre-incubated for 20 min at room temperature in serum-free and antibiotic-free DMEM. A2780 cells transfected with pEGFP-C1-WT-PTEN, pEGFP-C1-C124APTEN and pEGFP-C1 empty vector were named as WT-PTEN/A2780, C124A-PTEN/A2780 and GFP/A2780. G418 (500 lg/ll) was applied to stable screening and isolating the resistant colonies. 2.5. RT-PCR analysis Total RNA were prepared using Trizol regent, following manufacturer’s instructions. Total RNA (2 lg) was reversed transcribed using M-MLV Reverse transcriptase (Promega, USA). cDNA was amplified by PCR using Taq DNA polymerase (Promega, USA). Primers were as follows: for PTEN expression, sense: 50 -ACGGGAA 0 GACAAGTTCATGTAC-3 , and antisense: 50 -TTT GAC GGCTCCTCTACTGT-30 (product size, 389 bp); for GAPDH (housekeeping gene) expression, sense 50 -ACGGATTTG GTCG TATTGGG30 and antisense 50 -TGATTTTGGAGGGATC TCGC-30 (product size, 180 bp). The RT-PCR cycle was as 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 94 °C, 30 s at 56 °C, 30 s at 72 °C, 28 cycles. PCR products were analyzed by 1.5% agarose gel electrophoresis in the presence of ethidium bromide for UV light transilluminator visualization. 2.6. Western blotting analysis Cells harvested were lysed in lysis buffer (50 mM Tris–HCl pH7.4, 150 mM NaCl, 1% Nonidet P 40, 1% Triton X-100, 0.2% SDS, 1% sodium deoxycho-
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late, 5 mM iodoacetamide, proteinase inhibitor cocktail, 2 mM PMSF). Protein (60 lg) was denatured in SDS sample buffer at 100 °C for 10 min and separated by electrophoresis on a 10% SDS– PAGE gel, then transferred to a nitrocellulose membranes. The membrane was blocked in TBST (25 mM Tris–HCl, pH 7.5, 137 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20) with 5% non-fat milk for 1 h at 37 °C, and then incubated with the primary antibodies (mouse anti-PTEN, 1:2000; rabbit anti-AKT, 1:500; rabbit anti-phospho-AKT, 1:1000; rabbit anti-caspase 3, 1:500; rabbit antiPARP, 1:200; rabbit anti-MMP-9, 1:500; rabbit anti-actin, 1:500) in blocking buffer overnight at 4 °C. The membrane was washed three times in TBST and incubated with alkaline phosphatase– conjugated goat IgG (1:500) for 2 h at room temperature and then visualized with NBT/BCIP/buffer (1:1:50). Protein loading was assessed by blotting of the same membrane with an antibody against b-actin. 2.7. Flow cytometric analysis and TUNEL assay Cell apoptosis was evaluated by flow cytometry (FCM) according to the methods described by Ishibashi and Lippard [19]. Cells were harvested with 0.2% trypsin, washed in cold PBS, fixed in 70% ice-cold ethanol and then stained with propidium iodide (PI) overnight at 20 °C. On the following day, the stained cell were run on a FACSort flow cytometery (Becton-Dickinson, Franklin Lake, USA) and evaluated using the CELLQuest software system (BD Biosciences, USA) to calculate the percentage of sub-G1 (hypodiploid) apoptotic cells and the percentage of cells in different cell cycle. At the same time, nuclear morphology of apoptotic cells was determined using the in situ Cell Death Detection kit from Roche Diagnostic (Mannheim, Germany) following the manufacturer’s instruction. The apoptotic cells (brown staining) were counted under a microscope. 2.8. Cell proliferation assays Cell proliferation was determined by the MTT assay. Briefly, cells were cultured in 96-well plates the day before experiment at a density of 5 103/ well, and then were incubated for 1, 2, 3, 4, 5, 6 and 7 days, then 20 ll 3-(4,5 dimethylthiazol-2-yl)2,5-diphenyl-tetrazoliumbromide (MTT) (5 mg/ml, Sigma Chemical Co.) was added to the wells, which
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was followed by incubation at 37 °C for 4 h, aspirating the supernatants, adding 100 ll DMSO and incubating at 37 °C for an additional 20 min. The absorbance of the wells was then read with a microplate reader at a test wavelength of 570 nm and a reference wavelength of 630 nm. Appropriate controls lacking cells were included to determine background absorbance. Response to drug treatment was assessed by standardizing treatment groups to untreated control.
different fields of each well were counted with two wells per treatment. The mean values were obtained from three replicate experiments and were subjected to the t-test. 2.11. Statistical analysis All experiments were repeated at least thrice. The data were analyzed with the software package SPSS 12.0. P value less than 0.05 were considered significant statistically.
2.9. Wound-healing assay Post-transfected A2780 cells were seeded into 35mm dishes at 1.5 105 cells/cm2 and allowed to grow to 90% confluence in their recommended media. Medium was replaced by corresponding media with 0.1% BSA for 24 h. The confluent cell monolayer was scraped with a sterile pipette tip with a constant diameter. For each dish, 3–5 wounds were made, and three sites of regular wounds were selected and marked. Wounded monolayers were then washed three times with PBS to remove cell debris. Cells were permitted to migrate into the area of clearing for 24 h. Immediate after wounding and at the end of the experiment (after 24 h), wounds were photographed and semiquantitative measurements were taken of control and treated wounds. A mean wound width was determined, and the average width of wound closure was calculated as described previously [20]. 2.10. Cell invasion assays The A2780 cells were harvested by trypsinization and washed in RPMI medium without serum. The cells were suspended in RPMI 1640 medium at 5 105 cells/ml. Prior to preparing the cell suspension, the dried layer of matrigel matrix was rehydrated with serum-free RPMI 1640 medium for 2 h at 37 °C. The rehydration solution was carefully removed, 0.75 ml RPMI 1640 medium containing 10% FBS was added to each well as a chemoattractant, and 0.5 ml (2.5 105 cells) of cell suspension was added to each well. The plates were incubated for 48 h at 37 °C. The invaded cells on the bottom surface of the membrane were fixed and stained by sequentially transferring them through wells of a second 24-well plate containing the three solutions from the Diff-QuikÒ staining kit (Dade Behring Inc.). The cells were enumerated by taking photomicrographs at 200 magnification. The cells in three
3. Results 3.1. Effect of WT-PTEN and C124A-PTEN plasmids transfection on PTEN expression To investigate whether the WT-PTEN and C124APTEN plasmids increased the expression of PTEN gene, WT-PTEN/A2780 (A2780 cells transfected with wild-type PTEN plasmid), C124A-PTEN/A2780 (A2780 cells transfected with mutant PTEN plasmid), GFP/A2780 (A2780 cells transfected with pEGFP-C1 plasmid) and A2780 cells were analyzed for the expression of PTEN mRNA and protein by RT-PCR and Western blot analysis. The RT-PCR and Western blot product levels were normalized to b-actin and GAPDH expression. PTEN mRNA expression in WT-PTEN/A2780 and C124A-PTEN/ A2780 cells (1.71 ± 0.15 and 1.75 ± 0.09) were significantly higher than those of GFP/A2780 and A2780 cells (1.07 ± 0.07 and 1.06 ± 0.07), respectively (Fig. 1A). (t = 5.300, P = 0.034; t = 11.963, P = 0.007; t = 7.869, P = 0.016; t = 22.421, P = 0.002, n = 4) (Fig. 1A); The expression of PTEN protein of WT-PTEN/A2780 and C124A-PTEN/A2780 cells (1.21 ± 0.04 and 1.22 ± 0.02) were also obviously higher compared with those of GFP/A2780 and A2780 cells (0.74 ± 0.03 and 0.74 ± 0.02) (P < 0.05) (Fig. 1B). These results demonstrated that PTEN plasmid transfection were an effective and pragmatic approach to increase the expression of PTEN gene. 3.2. Effect of PTEN plasmids transfection on A2780 cell proliferation To examine whether PTEN gene is able to inhibit the proliferation of A2780 cells, WT-PTEN/A2780, C124APTEN/A2780, GFP/A2780 and A2780 cells were incubated for 1, 2, 3, 4, 5, 6 and 7 days, and the growth rates were calculated by MTT assay. In WT-PTEN/A2780 group, the cell proliferation rate decreased gradually and reached its lowest level 7 days after incubation (Fig. 2). No significant changes in cell proliferation rate were observed for either C124A-PTEN/A2780, GFP/A2780 cells or A2780 cells.
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Fig. 2. Effect of PTEN transfection on cell proliferation of A2780 cells. WT-PTEN/A2780, C124A-PTEN/A2780, GFP/A2780 and nontreated A2780 cells were incubated for 1, 2, 3, 4, 5, 6 and 7 days. Cell growth was measured using absorbance as described in Section 2. Points, averages of three independent experiments; bars, SD.
Fig. 1. Expression levels of PTEN mRNA and protein of the cells transfected with PTEN plasmids (WT-PTEN/A2780 and C124APTEN/A2780) or empty EGFP vector (GFP/A2780) and A2780 cells. (A) The expression of PTEN mRNA (389 bp PCR product) was detected by semiquantitative RT-PCR, GAPDH (180 bp PCR product) was co-amplified as the internal control. (B) The expression of PTEN protein was detected by Western blot analysis in total cell extracts. b-actin was reprobed to confirm equal protein loading. The experiment was repeated 3 times.
3.3. Effect of PTEN plasmids transfection on A2780 cell cycle and cell apoptosis It has been shown previously by others that transfection of WT-PTEN into cells induces cell cycle arrest as well as apoptosis [21,22]. To determine the role of the PTEN gene transfection in cell cycle distribution and cell apoptosis in A2780 cells, WT-PTEN/A2780, C124APTEN/A2780, GFP/A2780 and A2780 cells therefore were stained with PI and subjected to flow cytometry analysis to analysis cell cycle and apoptosis. As shown in Fig. 3A, expression of wild-type PTEN gene led to a substantial increase in the G0/G1 population, as compared with that of the vector control{(71.18 ± 4.34)% vs. (51.91 ± 2.88)%} (P < 0.05). The cell cycle arrest caused by PTEN expression depended on its phosphatase activity, as very little effect on the cell cycle profile was observed when the phosphatase-inactive PTEN (C124A) was expressed. We also found from Fig. 3B, up to
32.05% of WT-PTEN/A2780 cells displayed sub-G1 DNA content, which was an indication of DNA fragmentation and often associated with apoptotic cell death [23]. In contrast, only 1.77% of A2780 cells or 1.59% of GFP/ A2780 cells exhibited sub-G1 DNA content (P < 0.05). We also found that expression of wild-type PTEN, but not its phosphatase-inactive (C124A) PTEN, led to a substantial upregulation in the amount of cells undergoing apoptosis. TUNEL assay showed similar results. In WTPTEN transfected A2780 cells, the morphologic alterations characteristic of apoptosis, including nuclear condensation and fragmentation, were detected (data not shown). These results confirmed that PTEN expression led to G0/G1 cell cycle arrest and cell apoptosis dependent on its phosphatase activity in A2780 ovarian epithelial cancer cells. 3.4. Effect of PTEN plasmids transfection on migration of A2780 cells Cell migration is a characteristic feature of carcinoma cells [24]. PTEN has been reported to be implicated in the regulation of cell migration [25]. To test whether PTEN gene play an important role in regulation of cell migration in A2780 cells, a wound-healing assay was carried out to test the cell migration. Confluent and quiescent monolayers of WT-PTEN/A2780, C124A-PTEN/A2780, GFP/ A2780 and A2780 cells were wounded. Recovery of these monolayer cells depends on their migration during wound-healing. Photographs were taken directly at 24 h after wounding to observe the changes of migration and then the distance of cell migration to the wound area were measured (Fig. 4A) [26]. The WT-PTEN/A2780 cells had
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Fig. 3. Effect of PTEN transfection on cell cycle and apoptosis of A2780 cells by flow cytometric analysis. Flow cytometry was used to analyze the changes of the propidium iodide-stained A2780 cells. (A) PTEN-mediated G0/G1 arrest in A2780 cells was analyzed by determining the cell cycle distribution by FACS and percentage of G0/G1 phase cells was presented. (B) The number of apoptotic cells was counted with a FACSort flow cytometer according to the methods described in Section 2. (C) Apoptotic cells were detected by TUNEL assay (brown staining).
a much slower wound-healing rate when compared with those of C124A-PTEN/A2780, GFP/A2780 and A2780 cells (P < 0.05) (Fig. 4B). These results demonstrated that cell migration was down-regulated by expression of phosphatase-active form of PTEN but not by PTEN with an phosphatase-inactive PTEN gene.
44.7 ± 2.1 and 45.0 ± 3.0) (P < 0.05) (Fig. 5B). These results also showed that no significant change was observed after transfection with the phosphatase-inactive mutant of PTEN (C124A), indicating that the phosphatase activity of PTEN is required for inhibition of cell invasion.
3.5. Effect of PTEN plasmids transfection on invasion of A2780 cells
3.6. Effect of PTEN plasmids transfection on the expression of total AKT and phospho-AKT (Ser473) protein in A2780 cells
To investigate the role of PTEN gene in tumor cell invasion in A2780 cells, we measured the ability of different A2780 cells to migrate through matrigel-coated filters in a modified transwell assay (Fig. 5A), The number of invaded cells of WT-PTEN/A2780 cells (24.3 ± 2.5) were obviously much fewer than those of C124A-PTEN/ A2780, GFP/A2780 and A2780 cells (43.7 ± 3.8,
To determine the molecular mechanisms by which PTEN may contribute to the inhibited proliferation and migration as well as suppressed invasion. Total cellular protein extracts were prepared and subjected to Western blot analysis using antibodies against total AKT, phosphoAKT (Ser473), caspase-3, PARP[poly(ADP-ribose) poly-
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Fig. 4. Effect of PTEN on migration of A2780 cells in vitro. (A) Confluent monolayers of A2780 cells were wounded. Photographs were taken directly (t = 0 h) and 24 h after wounding (t = 24 h). (B) Quantification of the endothelial wound repair. The distance of cell migration to the wound area was measured in A2780 cells 24 h after wounding. Values are means ± SD from three independent experiments.
merase-1] and MMP-9 and b-actin. The result had shown that the overexpression of wild-type PTEN gene in WTPTEN/A2780 cells resulted in a markedly suppressed expression of phospho-AKT and MMP-9 protein (Fig. 6). There was no difference was in the expression of total AKT, caspase-3 and PARP in different treated A2780 cells.
4. Discussion The incidence of ovarian cancer has been increasing in recent years. Although the combination cytoreductive surgery with cisplatin-centered chemotherapy dramatically lengthened the survival time of patients and cut-down the mortality of ovarian cancer, ovarian cancer has the worst diagnosis and prognosis of any gynecological cancer due to many factors, including the poor understanding of precursor lesions and tumor progression mecha-
nisms, and the usually late-stage presentation of this aggressive disease [27]. The current major research focus is identifying an effective therapeutic treatment of ovarian cancer. PTEN was discovered by three different groups in 1997 [4]. It codifies a 403-amino acid dual-specific phosphatase with putative tumor suppressing abilities. PTEN protein has the activity of protein phosphatase and lip phosphatase. Loss of PTEN expression has been detected in a wide range of human cancers, including human brain, breast, prostate, endometrial and ovarian cancers [28] and PTEN has already proven itself to be a remarkable molecule. It is now known that PTEN plays important role not only in inducing cell cycle detention and cell apoptosis, but also in regulation of cell adherence, cell migration, cell differentiation and chemoresistance [29,30]. Therefore, in this study
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Fig. 5. Effect of PTEN on invasion of A2780 cells in vitro. (A) Cell invasion through matrigel basement membrane extract was analyzed using a modified transwell assay as described in Section 2. Cells that invaded to the lower surface of the membrane were fixed and stained. (B) Mean cell counts from at least 10 fields and four experiments are shown. The number of invaded cells of WT-PTEN/A2780 cells (24.3 ± 2.5) were obviously much fewer than those of C124A-PTEN/A2780, GFP/A2780 and A2780 cells (43.7 ± 3.8, 44.7 ± 2.1 and 45.0 ± 3.0) (P < 0.05).
we transfected A2780 cells with wild-type and phosphatase-inactive PTEN to inhibit the expression of PTEN gene, and determined their effects on cell apoptosis, cell cycle distribution and cell proliferation as well as cell migration and invasion. Expansion of tumor cell populations can be the result of unchecked cell division or a consequence of cell accumulation secondary to avoidance of programmed cell death (apoptosis) or both [27]. Tumor cell survival is one of the major reasons for tumor expansion and progression. In our study, upregulation of PTEN mRNA was verified by RT-PCR in WT-PTEN/A2780 and C124A-PTEN/A2780 cells using b-actin as an internal normalization standard, and the upregulation of PTEN proteins was reconfirmed by Western blotting analysis. The results suggested that transfection with PTEN was efficient to increase PTEN expression in ovarian cancer cell lines. We further found that stable expression of wild-type PTEN markedly suppressed the growth rate of A2780 cells as compared with mock cells. Next, we wondered whether these inhibitory functional properties endowed on wild-type PTEN-overexpressing cells were linked to cell cycle arrest and a greater rate of apoptosis, therefore, we attempted to perform flow cytometry analysis to analysis the cell cycle and apoptosis of A2780 cells and found that a marked increase in the G0/G1 and sub-G1 (apoptotic cells) population in WT-PTEN/A2780 cells which showed significant growth suppression. In contrast, C124A-PTEN/ A2780 cells which were overexpressing the phosphatase-inactive mutant of PTEN showed the rate of proliferation and G0/G1 population as well as apoptotic ratio was comparable to those exhibited by mock cells. Taken together, these findings suggested that
the growth-inhibitory activity of exogenous PTEN is mediated by two mechanisms, apoptosis and/or arrest at the G1 phase of the cell cycle, at the same time, the above results also demonstrated that phosphatase activity of PTEN is critical for its role as a tumor suppressor gene. To investigate the molecular mechanisms by which PTEN may contribute to enhanced apoptosis and cell cycle arrest, we detected the protein expression levels of some molecules which were involved in various sig-
Fig. 6. Wild-type PTEN transfection reduced the expression of phospho-AKT (Ser473) and MMP-9 protein in A2780 cells analyzed by Western blot analysis. Protein was extracted from WT-PTEN/A2780, C124A-PTEN/A2780, GFP/A2780 and A2780 cells. An amount of 60 lg of each sample was resolved by gel electrophoresis, transferred to a membrane and probed with antibodies specific to total AKT, phospho-AKT (Ser473), caspase-3, PARP and MMP-9, as indicated. The b-actin was used as an internal control.
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naling pathways in A2780 cells. Relative to total AKT content, WT-PTEN/A2780 cells contained lower levels of AKT that was phosphorylated on Ser473, one of the two phosphorylation sites on AKT that results in its activation. In contrast, no difference was observed between the A2780 cells in their levels of caspase-3 and PARP. Therefore, the AKT signaling pathway appears to be specifically suppressed in wild-type PTEN-expressing A2780 cells. PI3K/PIP3 pathway is important in controlling a diverse array of cellular functions including cell survival, cell cycle progression, motility, differentiation in response to extracellular stimuli. AKT, as one of the major direct down stream target 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 phosphorylating several substrates, such as Bad, HIF-1a, NFrB. Resistance to apoptosis may contribute to tumorigenesis of many malignancies and insensitivity of ovarian cancer to standard therapies. Cell migration is a key cellular feature of tumor progression because it regulates metastasis. Both tumor cell migration and invasion through the basement membranes are crucial steps in the multi-stage process that leads to metastasis formation [31]. Ovarian cancer is a highly metastatic cancer, and therefore cell migration and invasion are particularly important processes of ovarian cancer [16]. Thus, we investigated whether that PTEN was involved in cell migration and invasion to understand ovarian cancer progression. In our study, wild-type PTEN-overexpressing A2780 cells showed significantly decreased the rate of cell migration and invasion and obviously reduced MMP-9 expression compared with the mock cells. Since phosphatase-inactive mutant C124A PTEN did not inhibit cell migration and invasion which suggests that the phosphate domain is necessary for inhibiting migration and invasion. Cell migration requires cell invasion, the process of cellular-induced degradation of the basal membrane through membrane penetration. A common feature of the invasive process is the degradation of the surrounding ECM by an array of proteolytic enzymes expressed by the infiltrating tumor cells [32]. Matrix metalloproteinases, capable of degrading almost all ECM components, have been suggested to play an important role in mediating tumor invasion [33]. Therefore, the elevated expression of various MMPs is strongly associated with
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the invasive phenotype. Our data have demonstrated the expression of MMP-9 protein was significantly inhibited by the overexpression of wild-type PTEN. These observations raise the interesting possibility of exploring the precise intracellular signaling mechanism of control of MMP-9 expression by the tumor suppressor gene PTEN. It is reported that FAK, one of substrates of the PTEN protein, modulates cell migration and invasion [16] and is also involved in the activation of MMPs [34]. Thus, wild-type PTEN might inhibit the expression of MMP-9 protein by decreasing the expression of FAK protein. Further work is required to verify the mechanism by which PTEN inhibit cell migration and invasion of ovarian cancer cells. In conclusion, these results suggest that PTEN have played an important role in the cell proliferation and migration as well as cell invasion in ovarian cancer cells. Overexpression of PTEN can reverse the malignant phenotype of ovarian cancer cell. PTEN gene may be a novel target for treatment of patients with ovarian cancer. Conflict of Interest None. Acknowledgments This work was supported by Grants from the National Natural Science Foundation of China (Nos. 30571950, 30528012, and 30371657, 30672227) and the ‘‘973” Program of China (No. 2002CB513100). References [1] H. Naora, D.J. Montell, Ovarian cancer metastasis: integrating insights from disparate model organisms, Nat. Rev. Cancer 5 (2005) 355–366. [2] X. Ma, S. Wang, J. Zhou, H. Xing, G. Xu, B. Wang, G. Chen, Y.P. Lu, D. Ma, Induction of apoptosis in human ovarian epithelial cancer cells by antisurvivin oligonucleotides, Oncol. Rep. 14 (2005) 275–279. [3] W.P. McGuire, W.J. Hoskins, M.F. Brady, P.R. Kucera, E.E. Partridge, K.Y. Look, D.L. Clarke-Pearson, M. Davidson, Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer, N. Engl. J. Med. 334 (1996) 1–6. [4] J. Li, C. Yen, D. Liaw, K. Podsypanina, S. Bose, S.I. Wang, J. Puc, C. Miliaresis, L. Rodgers, R. McCombie, S.H. Bigner, B.C. Giovanella, M. Ittmann, B. Tycko, H. Hibshoosh, M.H. Wigler, R. Parsons, PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer, Science 275 (1997) 1943–1947.
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