Survey
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ORIGINAL STUDY Survivin Status Affects Prognosis and Chemosensitivity in Epithelial Ovarian Cancer Lifeng Chen, MD,* Lizhi Liang, MD,Þ Xiaojian Yan, MD,* Naihua Liu, PhD,þ Lihua Gong, MD,* Shishi Pan, MD,* Feng Lin, MD,* Qian Zhang, MD,* Hongqin Zhao, MD,* and Feiyun Zheng, MD* Objective: The objective of this study was to explore the clinical significance of survivin expression in epithelial ovarian cancer (EOC) and the effect of survivin small hairpin RNA (shRNA) on survivin expression, apoptosis, and chemosensitivity in the human ovarian cancer cell line OVCAR3. Methods: A retrospective review of 90 consecutive EOC patients with a median follow-up time of 51 months was conducted. Survivin expression was examined by immunohistochemistry. OVCAR3 cells were transfected in vitro with survivin shRNA. Survivin mRNA expression levels were detected using reverse transcriptionYpolymerase chain reaction. Flow cytometry was applied to determine survivin protein expression levels and cell apoptotic rates. The MTT method was used to examine the effects of survivin shRNA on chemosensitivity in OVCAR3 cells. Results: Positive cytoplasmic expression of survivin was associated with advanced International Federation of Gynecology and Obstetrics (FIGO) stage, nonmucinous type, high grade, and recurrence. Positive survivin expression was also associated with platinum resistance (r = 0.306, P = 0.003). Statistical results indicated that FIGO stage (hazard rate = 1.649, P = 0.047) and cytoplasmic expression of survivin (hazard rate = 1.734, P = 0.010) were independent prognostic factors. Survivin mRNA and protein levels were lower in OVCAR3S (ovarian cancer cells transfected with a survivin recombinant vector) cells at 24 hours after transfection as compared with controls. The flow cytometric analysis revealed that survivin shRNA induced accumulation of cells in the G0/Gl phase, with a decrease in G2/M phase cells following 24 hours of culture as compared with a nontransfected group (P G 0.01). Furthermore, survivin shRNA increased the sensitivity of OVCAR3 cells to paclitaxel 15-fold (P G 0.05), whereas it had no significant effect on cisplatin (P 9 0.05). Conclusions: In addition to FIGO stage, cytoplasmic survivin protein expression is an independent molecular marker for predicting EOC prognosis. Sequence-specific shRNA targeting survivin can effectively suppress survivin expression, enhance apoptosis, and increase the sensitivity of ovarian cancer cells to paclitaxel but not to cisplatin. Key Words: ovarian neoplasms, survivin, RNA interference, short hairpin RNA Abbreviations: EOC, epithelial ovarian cancer, IC50, 50% cell growth inhibition, shRNA, small hairpin RNA, OVCAR3WT, wild-type human ovarian cancer cells, OVCAR3S, ovarian cancer cells transfected with a survivin recombinant vector, OVCAR3L, ovarian cancer cells transfected with a liposome, OVCAR3B, ovarian cancer cells transfected with a blank vector *Department of Gynecology, the First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang; †Department of Gynecology, Cancer Center, Sun Yat-Sen University, Guangzhou, Guangdong; and ‡Department of Pharmacy, Wenzhou Medical College, Wenzhou, Zhejiang, China. Copyright * 2013 by IGCS and ESGO ISSN: 1048-891X DOI: 10.1097/IGC.0b013e31827ad2b8 256 Address correspondence and reprint requests to Xiaojian Yan, MD, Department of Gynecology, the First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China. E-mail: [email protected]. The main financial support was from Zhejiang Provincial Education Department (grant Y201016398) and from Guangdong Natural Science Foundation (grant 2003-31746). The authors declare no conflicts of interest. International Journal of Gynecological Cancer & Volume 23, Number 2, February 2013 Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited. International Journal of Gynecological Cancer & Volume 23, Number 2, February 2013 Survivin Status in Epithelial Ovarian Cancer Received July 15, 2012, and in revised form October 23, 2012. Accepted for publication October 24, 2013. (Int J Gynecol Cancer 2013;23: 256Y263) ovarian cancer (EOC) is the leading cause of death E pithelial from gynecologic malignancy. Over the past 3 decades, important advances in EOC treatment have been made. However, the majority of patients succumb to the disease. The 5-year survival rate is still about 30%.1 Despite the activity of initial chemotherapy with a combination platinum-based regimen in advanced ovarian cancer, most patients with advanced disease will ultimately have a relapse because of the development of drug resistance. Second- or third-line chemotherapeutic agents have been shown to have a relatively high response rate in shortterm therapy, but the long-term effects are poor because of acquired drug resistance. Chemotherapeutic agents also have severe toxicities. It is necessary to identify new methods or novel agents for the treatment of ovarian cancer. Survivin is a member of the IAP (inhibitors of apoptosis protein) family. It is undetectable in normal adult tissues, but highly expressed in several types of cancer.2,3 Survivin expression in the primary tumor has been associated with a worse prognosis, resistance to anticancer agents,4,5 and metastasis.6 Furthermore, attenuation of survivin expression can induce apoptosis and sensitize cancer cells to conventional chemotherapeutics. The inhibition of apoptosis by survivin is associated with the mitotic spindle assembly checkpoint.7,8 Therefore, survivin may be a potential target for EOC therapy. However, the prognostic significance of survivin in ovarian cancer is still unknown; more data are needed to confirm its value.9Y12 A variety of survivin antagonists, such as antisense oligonucleotides, ribozymes, small interfering RNAs (siRNAs),13 dominant negative mutants, and cyclin-dependent kinase inhibitors, have been tested.14 Gene-specific inhibitors of survivin have become a new trend as novel anticancer targets because of their wide spectrum and minimum toxicity. In this study, we explored whether survivin plays an important role in ovarian cancer and whether silencing survivin could enhance therapeutic efficacy. We investigated the expression of survivin in 90 Chinese female patients with untreated EOC and then determined clinical pathological parameters and the impact on chemosensitivity, recurrence, and prognosis. We used an in vitro RNA interference technique to explore the effects of survivin small hairpin RNA (shRNA) on survivin expression, apoptosis, and chemosensitivity in human ovarian cancer cell line OVCAR3 to broaden the list of available therapeutic modalities in the treatment of ovarian cancer. This would allow us to screen out high-risk cases and modify our treatment strategy to improve chemotherapeutic efficacy. MATERIALS AND METHODS Clinical Data and Immunohistochemistry A retrospective review of 90 consecutive EOC patients from January 1993 to December 2000 at the Cancer Center of Sun Yat-Sen University was conducted. The study was approved by the Ethical Review Committee of Sun Yat-Sen University Cancer Center. Inclusion criteria were as follows: (1) no patients were treated with preoperative chemotherapy; (2) the first surgeries were performed in this cancer center; (3) the pathological results were diagnosed as EOC; (4) the adjuvant therapy administered after surgery included platinumbased chemotherapy; (5) follow-up data were available. All cases were followed up to November 2004. The median follow-up time was 51 months. The median age was 50 years (range, 22Y75 years). Platinum sensitivity refers to the diseasefree or treatment-free interval more than 6 months after primary platinum treatment. Platinum resistance refers to platinumresistant recurrence (tumor progression within 6 months of completion of platinum-based therapy), persistent disease (clinically measurable disease with best response as stable disease at the completion of planned first-line therapy), and refractory disease (progressive disease during platinum therapy). A rabbit polyclonal antisurvivin antibody was used in the immunohistochemical analysis. Two investigators independently scored all of the samples. In discordant cases, a third investigator scored the samples, and the final intensity score was determined by majority vote. Cytoplasmic staining for survivin was scored as positive when a staining reaction was noted in more than 10% of the tumor cells and as negative when it was noted in less than 10% of the tumor cells, as described previously.15 Statistical Analysis Continuous variables were reported as the mean (SD) or the median and first and third quartiles. Categorical variables were described using proportions. Group differences were determined using a t test, analysis of variance, Kruskal-Wallis test, or W2 test as appropriate. The survival of ovarian cancer patients with positive and negative survivin expression was compared by Kaplan-Meier method and log-rank test. A univariate analysis by W2 test was applied to assess prognostic significance of the different variables. To compare the effect of survivin expression, we determined the associations of multiple variables with prognosis using Cox multivariate regression models. The proportional hazards assumption for all confounding variables was tested visually using log-negativelog survival plots. All statistical analyses were performed using the Statistical Package for the Social Sciences software program version 13.0 (SPSS, Inc, Chicago, IL), and P G 0.05 was accepted as statistically significant. Reagents and Instruments The human ovarian cancer cell line OVCAR3 was obtained from China Medical University, Institute of Cell Biology. The survivin shRNA expression vector Mu6/survivin * 2013 IGCS and ESGO Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited. 257 International Journal of Gynecological Cancer Chen et al & Volume 23, Number 2, February 2013 plasmids were acquired from Prof Luo CQ of Guangdong Medical College. The pEGFPC2 plasmids were purchased from Clonetech (Palo Alto, CA). The Plasmid Extraction Kit was purchased from Qiagen (Valencia, CA). Lipofectamine TM2000 was purchased from Invitrogen (Carlsbad, CA). MTT was purchased from Sigma (St Louis, MO). A reagent kit was purchased from Promega (Madison, WI). The rabbit polyclonal antibody against human survivin was purchased from Santa Cruz (Santa Cruz, CA). twice, fixed in 1 mL of 0.1% formaldehyde for 30 minutes, and then incubated with 0.5 Kg survivin rabbit antiYhuman polyclonal antibody for 30 minutes after the cell membranes were broken. Irrelevant isotype-matched mAb was used as a negative control. Goat antiYrabbit immunoglobulin GYfluorescein isothiocyanate was then added at a final dose of 5 Kg, and the mixture was incubated for 30 minutes at room temperature away from light. The cells were then analyzed using FCM (Beckman coulter Epics ELITE). Cells and Culture Conditions Cell Cycle and Apoptosis Human ovarian cancer cell line OVCAR3 was grown in Dulbecco modified Eagle medium (GIBCO) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 Kg/mL streptomycin and incubated at 37-C in a humidified chamber supplemented with 5% CO2. Cell Transfection and Optimization In a 24-well plate, human ovarian cancer cell line OVCAR3 cells were seeded at a density of 3 105 cells/mL in growth medium with free fetal bovine serum before transfection. To determine the optimal conditions necessary for higher-efficiency cationic lipid-mediated cell transfection, 4 experimental groups were established: wild-type human ovarian cancer cells (OVCAR3WT), ovarian cancer cells transfected with a survivin recombinant vector (OVCAR3S), ovarian cancer cells transfected with a liposome (OVCAR3L), and ovarian cancer cells transfected with a blank vector (OVCAR3B). The ratios of pEGFPC2 plasmid to liposome were 1:1, 1:2, 1:3, and 1:4, respectively. The transfection efficiencies of the 4 groups were identified by flow cytometry (FCM) and fluorescence microscopy 24 hours after transfection to identify the optimization ratios. OVCAR3 cells were then trypsinized and plated in 24 wells at 3 105 cells per well 1 day before transfection. Twenty-four hours later, Mu6/survivin plasmids were mixed with liposome at a 1:2 ratio (which was asserted by the last step). The mixture was incubated for 20 minutes at room temperature and then added to the cells. Cells were collected 24 hours later to assess the down-regulation of survivin. In addition, a blank plasmid and liposome were used as negative control groups. Reverse TranscriptionYPolymerase Chain Reaction Analysis Total RNA from transfected cells was isolated using the RNeasy Kit according to the manufacturer’s instructions. Reverse transcription was performed at 42-C for 60 minutes using oligo(dT). Sequences for polymerase chain reaction (PCR) amplification used in this study were as follows: survivin, forward primer 5¶-CAG ATT TGA ATC GCG GGA CCC-3¶, reverse primer 5¶-CCA AGT CTG GCT CGT TCT CAG-3¶; GAPDH(internal control), forward primer 5¶-GTA GAG GCA GGG ATG ATG TTC-3¶, reverse primer 5¶-GAG TTA GCT CAC TCA TTA GGC-3¶. The specificity of PCR products was checked on agarose gel. Fluorescence-Activated Cell Sorting Survivin Protein Expression Briefly, after expressing either survivin shRNA or an empty vector, cells were washed in phosphate-buffered saline 258 Cells were selected after transfection at 12, 24, 36, or 48 hours and then fixed with 70% ice-cold ethanol overnight. RNA enzyme was added at a final concentration of 50 Kg/mL. One hour later, cells were resuspended in phosphate-buffered saline and incubated with propidium iodide according to the manufacturer’s instructions. Cells were analyzed by fluorescenceactivated cell sorting, and the data were analyzed using the Multicycle software (San Diego, CA). MTT Assay Cells (4 103) were seeded into two 96-well plates after transfection for 24 hours and subsequently were treated with 20, 15, 10, or 5 Kmol/L paclitaxel and 165, 82, 41, or 20 Kmol/L cisplatin. Control cells were treated with 0.1% dimethyl sulfoxide in culture medium. After treatment for 68 hours, the cells were incubated with MTT reagent (0.5 mg/mL; [3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide]) at 37-C for 4 hours. The resulting formazan crystals were solubilized by the addition of 150 KL dimethyl sulfoxide to each well. The optical densities at 540 and 655 nm were measured, and the cell inhibition rate was determined based on the following formula: cell inhibition rate (%) = (1 j absorbance of the treated wells / absorbance of the blank control wells) 100%. RESULTS Survivin Expression and Its Correlation With Clinical Parameters in EOC To investigate the status of survivin in EOC, we examined the expression of survivin in 90 EOC tissues by immunohistochemistry (Figs. 1AYD). The positive rate of survivin expression was 60%. Among the 90 patients, 40 (44.44%) relapsed, and the median survival time was 46 months. The 1-, 3-, and 5-year survival rates of all patients were 84.54%, 79.75%, and 73.62%, respectively. Table 1 shows the distribution of cytoplasmic survivin positivity according to clinical pathological characteristics. Positive survivin staining was associated with a lower 5-year survival rate when compared with negative staining (56.64% vs 94.2%, P = 0.003). Cytoplasmic survivin expression was associated with advanced International Federation of Gynecology and Obstetrics (FIGO) stage (72.92% vs 45.24%, P = 0.007), nonmucinous type (66.67% vs 40.74%, P = 0.022), high grade (74.47% vs 47.62% vs 40.91%, P = 0.012), and recurrence (90% vs 36%, P G 0.001). Survivin status had no relationship with age, family tumor history, blood type, and preoperative CA-125 (P 9 0.05). A univariate analysis showed * 2013 IGCS and ESGO Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited. International Journal of Gynecological Cancer & Volume 23, Number 2, February 2013 Survivin Status in Epithelial Ovarian Cancer FIGURE 1. Survivin immunostaining in EOC. A, Serous cystadenocarcinoma (negative). B, Adenocarcinoma (positive). C, Endometrioid carcinoma (positive). D, Mucinous carcinoma (positive) (original magnification 400). that the factors associated with 5-year survival rate were FIGO stage, pathological grade, survivin protein expression (Fig. 2), and recurrence (P G 0.05) (Table 2). Both nonrecurrence (n = 50) and platinum-sensitive recurrent EOCs (n = 18) were classified as the chemotherapy-sensitive group, with a total of 68 cases. Platinum-resistant (n = 11), persistent (n = 2), and refractory (n = 9) recurrent EOCs were regarded as a chemotherapy-resistant group, totaling 22 cases. Positive survivin expression was associated with platinum resistance based on the Spearman test (r = 0.306, P = 0.003). Cox proportional hazards regression model analysis indicated that FIGO stage (hazard rate = 1.649, P = 0.047) and cytoplasmic expression of the survivin protein (hazard rate = 1.734, P = 0.010) were independent poor prognostic factors in all patients (Table 3). Survivin shRNA Silenced Survivin Expression in OVCAR3 Cells To regulate the expression of survivin, we transfected the survivin shRNA expression vector Mu6/survivin and control plasmids into OVCAR3 cells. Cells were collected 24 hours after transfection for analysis by reverse transcription (RT)YPCR and FCM. Reverse transcriptionYPCR results TABLE 1. Correlations between survivin expression and clinicopathological parameters in 90 EOCs Survivin Parameter FIGO stage I + II III + IV Histology Mucinous Nonmucinous Grade G1 G2 G3 Recurrence Yes No Total Negative Positive P 42 48 23 13 19 35 0.007 27 63 16 21 11 42 0.022 22 21 47 13 11 12 9 10 35 0.012 40 50 4 32 36 18 0.000 FIGURE 2. Five-year survival after a median follow-up time of 51 months in 90 EOCs according to survivin expression. * 2013 IGCS and ESGO Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited. 259 Chen et al International Journal of Gynecological Cancer TABLE 2. Univariate analysis of the prognostic factors of EOC (n = 90) Characteristics No. 5-y Survival Patients Rate, % W2 P Age, y G50 46 77.68 Q50 44 69.74 1.12 0.2893 Family tumor history No 80 69.96 Yes 10 100.00 2.86 0.0907 Blood type A 27 68.82 B 24 66.23 AB 7 71.43 O 32 83.40 2.89 0.4089 Preoperative CA-125 Unknown 12 67.46 G35 16 100.00 35Y200 17 85.23 9200 45 63.44 7.39 0.605 FIGO stage IYII 42 84.97 IIIYIV 48 63.63 4.09 0.0431 Histology Serous 34 73.09 Mucinous 27 81.27 Endometrioid 4 100.00 Adenocarcinoma 25 61.44 3.38 0.2736 Grade G1 22 100.00 G2 21 75.96 G3 47 60.15 8.92 0.0116 Postoperative residual tumors diameters, cm e2 78 76.29 92 12 47.34 2.93 0.0869 Recurrence Yes 40 45.25 No 50 94.52 22.78 0.0000 Survivin Negative 36 94.20 Positive 54 56.64 9.07 0.0026 Bold numbers indicate statistical significance. showed that the intensity of OVCAR3S was weaker than that in control groups (Fig. 3A), indicating that survivin shRNA down-regulated survivin expression in OVCAR3 at the RNA level. Flow cytometry results showed that survivin protein in OVCAR3S decreased as compared with the control groups (Fig. 3B). 260 & Volume 23, Number 2, February 2013 Silencing Survivin Induced Cell Apoptosis and Cell Cycle Arrest in OVCAR3 Cells To evaluate the effect of survivin shRNA on OVACR3 cells, we examined cell apoptosis and cell cycle distribution after silencing survivin. The apoptotic rates of OVCAR3 cells at 12, 24, 36, and 48 hours after transfection were 20.7%, 31.9%, 39.0%, and 46.7%, respectively, and were significantly higher than those of control groups (Fig. 3C). Flow cytometric results showed that 24 hours after culture, survivin shRNA induced the accumulation of cells in the G0/Gl phase with a decrease in cells in the G2/M phase as compared with nontransfected groups (P G 0.01) (Table 4). Silencing Survivin Enhanced the Sensitivity of Paclitaxel But Not Cisplatin Treatment in OVCAR3 Cells We performed a sensitivity analysis of the effect of paclitaxel and cisplatin after silencing survivin in OVACR3 cells by MTT assay. For OVCAR3WT, OVCAR3L, OVCAR3B, and OVCAR3S, the IC50 of paclitaxel was 0.305 (SD, 0.032) Kmol/L, 0.157 (SD, 0.031) Kmol/L, 0.175 (SD, 0.010) Kmol/L, and 0.019 (SD, 0.001) Kmol/L, respectively, and the IC50 of cisplatin was 9.4100 (SD, 0.796) Kmol/L, 6.675 (SD, 1.739) Kmol/L, 6.930 (SD, 1.273) Kmol/L, and 7.862 (SD, 0.081) Kmol/L, respectively. Survivin shRNA increased the sensitivity of OVCAR3 cells to paclitaxel 15-fold (P G 0.05) but did not have a significant effect on cisplatin (P 9 0.05) (Fig. 3D). DISCUSSION The effect of drug resistance on the long-term effectiveness of paclitaxel and cisplatin in cancer chemotherapy is a major problem. In fact, most deaths from cancer are attributable to acquired drug resistance in the clinic. It has been reported that patients with positive survivin expression are more likely to be chemoresistant and have higher mortality rates than those of negative expression.16 In our data set, the positive rate of survivin expression in female Chinese cancer tissue was 60%, which was in accordance with published results.10 The relationship between immunohistochemical assessment of cytoplasmic survivin status and clinicopathological parameters in ovarian cancer is inconsistent. Ferrandina et al9 did not show any relationship between survivin positivity rate and clinicopathological parameters in ovarian cancer. However, several studies have shown that positive survivin expression in ovarian cancer was associated with disease aggressiveness, unfavorable clinical outcomes, and chemotherapy resistance,11,12,17Y19 which is consistent with our results. Furthermore, recent research showed that serum survivin was positively correlated with advanced stage and poor diseasefree survival in ovarian cancer.20 Survivin has been proposed as a chemoresistance and radioresistance factor, and survivin inhibitors are intensively investigated for cancer therapy.21 Therefore, it is feasible that survivin inhibitors can be used for at least a subgroup of ovarian cancer patients. On the other hand, for the therapeutic application of siRNA technology, it may be critical to use an efficient gene delivery system for the transduction of siRNA into target cells. Therefore, we used the vector-based shRNA technique and survivin shRNA * 2013 IGCS and ESGO Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited. International Journal of Gynecological Cancer Survivin Status in Epithelial Ovarian Cancer & Volume 23, Number 2, February 2013 TABLE 3. Multivariate analysis of the prognostic factors of EOC (n = 90) 95% Confidence Interval for Exp(B) Survivin protein expression FIGO stage A SE Wald P Exp(B) Lower Upper 0.552 0.500 0.214 0.252 6.646 3.929 0.010 0.047 1.737 1.649 1.142 1.006 2.643 2.705 FIGURE 3. A, mRNA expression of survivin in OVCAR3 cells detected by RT-PCR. M: DNA marker; 1: OVCAR3WT, 2: OVCAR3L; 3: OVCAR3B (mU6 transfected) 24 h; 4: OVCAR3S (survivin shRNA transfected) 24 hours. B, Protein expression of survivin in OVCAR3 cells detected by FCM. C, The cell apoptotic rate in OVCAR3 cells examined by FCM. A, OVCAR3WT. B, OVCAR3L. C, OVCAR3S transfected 12 hours; D: OVCAR3S transfected 24 hours. E, OVCAR3S transfected 36 hours. F, OVCAR3S transfected 48 hours. D, The chemosensitivity in OVCAR3 cells among different groups. * 2013 IGCS and ESGO Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited. 261 International Journal of Gynecological Cancer Chen et al & Volume 23, Number 2, February 2013 TABLE 4. Results of cell apoptosis rate and cell cycle after silence survivin Groups OVCAR3WT OVCAR3L OVCAR3B OVCAR3S Cell No., 104 Apoptotic Rate, % G0/G1, % 30 30 30 30 4.9 (0.7) 5.6 (0.5) 5.9 (0.2) 31.9 (1.2) 60.6 (0.4) 61.5 (3.1) 61.9 (1.4) 72.0 (3.4)* S, % 26.7 27.1 26.3 24.9 (0.5) (2.5) (0.7) (3.5) G2/M, % 12.6 (0.2) 11.4 (0.6) 11.8 (0.7) 3.0 (2.5)* Values are mean (SD). *P G 0.05, compared with OVCAR3WT. expression vector Mu6/survivin plasmids to transfect OVCAR3 cells to further define the role of survivin in ovarian cancers. The results of RT-PCR and FCM in our study demonstrated that specific survivin shRNA obviously inhibited survivin mRNA and its corresponding protein expression in OVCAR3S cells as compared with OVCAR3B and OVCAR3L cells. Recent research on SKOV3 ovarian cancer cells showed similar results.22 Cell apoptosis plays a key role in the maintenance of homeostasis of multicultural organisms, which still represents one of the main prognostic indicators in neoplasia.23 Deregulated cell apoptosis leads to clinically important malignancies. Survivin participates in the complex network by regulating both programmed cell death and cell division.24 Previous reports found that survivin was associated with cell cycle period and could control microtubule movement.25 Recently, it has been demonstrated that survivin overexpression in tumors is associated with poor prognosis because it may overcome the G1-S checkpoint, imposing progression of cells through the cell cycle and thus conferring a proliferative advantage.26 It can block cells in the G0/G1 phase and sharply reduce those in the G2/M phase.27 In our study, we found that the cell cycle of OVCAR3S cells changed significantly, as the G0/G1 phase cells increased and the G2/M phase cells decreased. Our data showed that silencing of survivin in OVCAR3 cells induced cell growth arrest in vitro, which confirmed previous reports.28 Combination chemotherapy of paclitaxel with platinum (TP) is one of the criterion standard chemotherapeutic agents to treat ovarian cancer.29,30 Paclitaxel strongly binds to the N-terminal region of A-tubulin and promotes stabilization of microtubules resulting in the inhibition of mitosis, which consequently induces cell death.31 Although TP increases overall survival rate, approximately 60% to 70% of advanced ovarian cancer patients had a relapse, mainly because of drug resistance.32 In this study, we aimed to evaluate whether survivin down-regulation improves the efficacy of paclitaxel and cisplatin against human ovarian cancer. To achieve this goal, 4 groups were treated with different concentrations of paclitaxel/cisplatin 24 hours after transfection. After 68 hours of incubation, an MTT cytotoxicity assay was performed. The results showed that survivin shRNA increased the sensitivity of OVCAR3 cells to paclitaxel 15-fold, supporting the conclusion that inhibition of survivin activation promotes paclitaxel-induced apoptosis in vitro, which is consistent with the study results of Zaffaroni et al.14 There are 3 reasons for enhancing OVCAR3 cell sensitivity to paclitaxel following transfection of survivin 262 shRNA: (1) survivin and paclitaxel both affect tubulin, exhibiting a competitive inhibition relationship. Once survivin expression is inhibited, it will significantly increase the sensitivity of OVCAR3 cells to paclitaxel. (2) Paclitaxel-induced apoptosis in cells proceeds via caspases 3 and 8. The downregulation of survivin also activates caspase, resulting in killing of cancer cells by a reduced dose of paclitaxel.33 (3) Survivin may bind to the activating factor of caspase during paclitaxel chemotherapy, thus obstructing the binding of the latter and inhibitors of apoptosis proteins and inhibiting the apoptosis of cells.34 The therapeutic effectiveness of cisplatin is thought to be mainly regulated by inhibition of DNA replication and/or delay of transcription through the formation of cisplatin-DNA adducts.35 Although our immunohistochemical findings indicate that the expression of survivin in ovarian cancer is strongly correlated with chemoresistance to cisplatin, the cellular experiment showed that survivin shRNA had no significant effect on sensitivity to cisplatin in OVCAR3 cells. Therefore, the role of survivin in chemotherapy with cisplatin requires further investigations. In summary, our results strongly support the hypothesis that survivin expression is a sensitive and valuable independent molecular prognostic indicator; positive survivin expression in EOC is associated with high chemoresistance and recurrence and reduced survival. Survivin may be used clinically for targeted therapy. We introduced an shRNAsurvivin vector into OVCAR3 cells, leading to a significant decrease in survivin expression. Furthermore, paclitaxelinduced apoptosis was significantly increased. These results suggest that shRNA targeting survivin may have therapeutic potential for the treatment of ovarian cancers, especially those with drug-resistant characteristics. To improve the overall survival of ovarian cancer patients, our results warrant further preclinical and clinical studies to assess the value of both survivin and shRNA in overcoming drug resistance. The detailed mechanism of action and extensive validation need to be further addressed. REFERENCES 1. 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