The Effect Of Cisplatin On The Proliferation,Apoptosis And Invasion In The Ovarian Cancer Cell Line SKOV3with The β-catenin Silence

Epithelial ovarian cancer is a gynecological malignancy with the highestmortality rate. As it is insidious in onset,most ovarian cancer presents as stageIII or IV disease, its five-year survival rate is less than30%after initialdiagnosis. At the present, the most challenging problem about the treatment ofthe ovarian cancer is the chemotherapy-resistance. Increasingly research isfoucusing on looking for new therapeutic method to improve the treatmenteffectiveness and five-year survival rate.The aberrant of Wnt/β-catenin signaling pathway has been confirmed tobe involved in various kinds of human cancer, such as colorectal carcinoma,breast carcinoma, ovarian cancer, laryngocarcinoma and so on. Its capabilityof being the potential therapeutic target makes it the hot spot of molecularbiology, cell biology and antitumor drug domain. Accumulating evidencesuggests a role for Wnt signalling in ovarian tumorigenesis in the absence ofgenetic mutations. With the exception of the endometrioid histotype, Wntpathway mutations are rare in ovarian cancer, suggesting alternativemechanisms for Wnt pathway activation in EOC. But the pertinentdownstream mechanisms are not fully understood.Accumulation of non-junctional β-catenin is used as a surrogate markerfor activation of the Wnt/β-catenin pathway. Rask et al found high expressionof β-catenin in cytoplasm in poorly differentiated stage III serous EOC byco-immunoprecipitationthe, but not in the normal ovary,demonstrating that theWnt/β-catenin pathway was aberrantly activated. We supposed that wether theovarian cancer cell biological behavior (for example of the proliferation,apoptosis) could change when β-catenin protein expression was silenced?Verm et al using SiRNA in vitro to silence the expression of β-catenin, found that the proliferation of the colon carcinoma cells was supressed, and theability of colon carcinoma cells for forming tumor was lowered in NODmouse. They found no expression of β-catenin by Western blot assay. Abovethat, we can conclude that β-catenin can be the target point of tumor targettherapy. There are some evidences that β-catenin plays a role in drugresistance. It can activate the downstream target genes (such as MDR1、c-myc、MRP2、cyclinD), thus cause cancer cells resistent to drug sensitivity. Inthis study, we will silence the expression of β-catenin to observe the change ofproliferation, apoptosis, and invasion of the ovarian cancer cell-SKOV3.Objective: By silencing the expression of β-catenin, the key protein ofWnt/β-catenin signaling pathway, to observe the impact，induced by casplatin,on the proliferation, apoptosis, and invasive power of the ovarian cancercell-SKOV3. We aim to provide new thread for resistance inversion of ovariancancer.Methods:1Using β-catenin-RNAi-LV, β-catenin-negative-LV to transfect SKOV3, todetect the expression state of β-catenin by Western blot assay.2We had six groups in this study: SKOV3, SKOV3-neg, SKOV3-RNAi,SKOV3-cDDP, SKOV3-neg-cDDP, SKOV3-RNAi-cDDP.3MTT was used to detect the inhibition of proliferate of SKOV3,SKOV3-neg, SKOV3-RNAi esposing to different concentration ofcisplatin.4Using Flow cytometry to detect the apoptosis rate of each group cells.5The invasion and migretion ability of each group cells was evaluated by theTranswell chamber test in vitro.6Statistical Methods: Data were evaluated using SPSS13.0statisticalsoftware, each assay was performed at least three times. The measurementdata were expressed as mean±standard deviation. For Homogeneity ofvariance test, single factor analysis of variance (One-Way ANOVA) wasused for data analysis. α=0.05is size of test. When P＜0.05, the differencewas statistically significant; when P＞0.05, there was no statistically significant difference.Result:1Weastern blot assay showed that the β-catenin expression level inSKOV3-RNAi was significantly lower than that of SKOV3andSKOV3-neg (P＜0.05).2There were a significant differences between SKOV3-RNAi andSKOV3-neg or SKOV3(P＜0.05). The significant cisplatin concentration,that significantly inhibit the proliferation rates of SKOV3, SKOV3-negand SKOV3-RNAi, is12.5μg/ml.3The Flow cytometry results showed that the apoptosis rate ofSKOV3-RNAi, induced by cisplatin, is significantly higher than that ofSKOV3-neg or SKOV3(P＜0.05), and there was no significant differencebetween the apoptosis rate of SKOV3-neg and SKOV3(P＞0.05).4The number of the SKOV3-RNAi-cDDP that migrated in the under of theTranswell chamber filter membrane was (17.33±2.08), was statisticallyless than either that of SKOV3-RNAi(19.67±1.25) or SKOV3(41.67±4.04)(P＜0.05). The difference between SKOV3-RNAi and SKOV3wassignificant(P＜0.05). The difference between SKOV3-RNAi-cDDP andSKOV3-cDDP(36.33±2.52) was significant(P＜0.05). The differencebetween SKOV3-cDDP and SKOV3was significant(P＜0.05).5The number of the SKOV3-RNAi-cDDP that invaded in the under of thefilter membrane coverd with Matrigel was(6.33±1.528), was statisticallyless than either that of SKOV3-RNAi(19.67±1.25) orSKOV3-neg-cDDP(22.33±2.517)(P＜0.05). The difference betweenSKOV3-RNAi and SKOV3was significant(P＜0.05). The differencebetween SKOV3-RNAi-cDDP and SKOV3-cDDP(24.67±2.081) wassignificant(P＜0.05). The difference between SKOV3-cDDP and SKOV3was statistic(P＜0.05).Conclusion:By silencing β-catenin protein in the ovarian cancer cell line SKOV3,SKOV3-RNAi compared with SKOV3and SKOV3-neg, cisplatin can better significantly inhibit the proliferation and invasion in SKOV3-RNAi, andpromote the apoptosis in SKOV3-RNAi, demonstrating that wnt/β-cateninsignaling pathway is involved in the drug resistance mechanism in thechemotherapy of ovarian cancer.