The Study On The Relationship Between Autophagy And The Sensitivity Of Cisplatin In Epithelial Ovarian Cancer Cell

Objective: Epithelial ovarian cancer （EOC） is one of the most common forms of ovarian cancer. Cisplatin （cDDP） is widely used as a first-line therapy for EOC, but cispatin resistance is one of the important factors which can cause a recurrence and poor prognosis.So looking forinnovative therapeutic methods to improve the cDDP effectiveness has been an urgent clinical demand. Autophagy plays a critical role in removing damaged, degenerative, aging and non-functional proteins and organelles and in cellular homeostasis. Many studies have indicated that the change of autophagic activity is closely related to the occurence and development of malignancy and drug resistance. However, the role of autophagy in anti-tumor therapeutic is till controversial. Beclin1gene, whichis confirmed as the first mammalian autophagy gene, is a well-known key regulator of mammalian autophagosome formation. Microtubule-associated protein1light chain3（MAP1LC3, LC3） is an autophagosomal ortholog of yeast Atg8. The LC3which is newly synthesized becomes cytosolic LC3-I after processing. After the ubiquitin-like processing, LC3-Iis conjugated to phosphatidylethanolamine of the autophagosomal membrane to form LC3-II. LC3-II is considered the only reliable autophagosomal marker protein. The amount of LC3-II and the proportion of LC3-II/LC3-I can show the number of autophagosomes and the activity of autophagy. In this present study, the autophagy specific inhibitor3-MA was added with cDDP to observe the change of cell proliferation, apoptosis and the autophagy protein expression on ovarian cancer cell strain SKOV3and its cispatine-resistance strain SKOV3/DDP, to explore the cisplatin sensitivity which was influenced by autophagy inhibition. Methods:1Ovarian cancer cell lines SKOV3and SKOV3/DDP were cultured;2The electron microscope was used to observe the formation of autophagosomes in SKOV3and SKOV3/DDP cells.3MTT assay was used to measure the proliferation inhibition rateof the SKOV3and SKOV3/DDP cells with cDDP. The SKOV3and SKOV3/DDP cells were exposed to cDDP （0μg/ml、0.625μg/ml、1.25μg/ml、2.5μg/ml、5μg/ml、10μg/ml、20μg/ml） for48h and calculate the half inhibition concentration (IC₅₀) of cisplatin in SKOV3and SKOV3/DDP cells.4MTT assay was used to measure the proliferation inhibition rateof the SKOV3and SKOV3/DDP cells with3-MA. The SKOV3and SKOV3/DDP cells were exposed to3-MA （0mmol/L、1.25mmol/L、2.5mmol/L、5mmol/L、10mmol/L、20mmol/L） for48h and calculate the dose ofnontoxic reaction （the drug concentration which can lead to less than10%cell proliferation inhibition rate,IC10） of3-MA in SKOV3and SKOV3/DDP cells.5MTT assay was used to measure the proliferation inhibition rateof the SKOV3and SKOV3/DDP cells with nontoxic doses of3-MA combined with different concentrations of cDDP （0μg/ml、0.625μg/ml、1.25μg/ml、2.5μg/ml、5μg/ml、10μg/ml、20μg/ml） for48h and calculate the IC₅₀of cDDP in SKOV3and SKOV3/DDP cells.6The SKOV3and SKOV3/DDP cells were divided into blank control group, cDDP group,3-MA group and3-MA+cDDP group. Flow cytometry was used to study the effect on apoptosis of SKOV3and SKOV3/DDP cells with cisplatin alone and in combination with3-MA.7The expression of microtubule-associated protein1light chain3（LC3） and the autophagy-related protein Beclin1of SKOV3and SKOV3/DDP cells which were exposed to different treatments were determinedby Western blotting.8Statistical methods: Data was evaluated using SPSS13.0statistical software, and data was expressed as mean±standard deviation （x±s）. The significance difference was determined using analysis of variance, LSD method and the t test. P <0.05was considered significant.Results:1By electron microscopy, more autophagosomes were observed in SKOV3/DDP cells than that in SKOV3cells. The autophagosomes werecharacterized by mitochondria, endoplasmic reticulum, organelle or the cytoplasm wrapped in double-layer or multi-layer membrane structures.2The inhibition effects of cDDP on the SKOV3and SKOV3/DDPcells were determined by MTT assays, getting IC₅₀value of4.47±0.08μg/ml for the SKOV3cells whereas IC₅₀value of17.71±0.15μg/ml for the SKOV3/DDP cells；IC₅₀value for the SKOV3/DDP cells was higherthan that for the SKOV3cells, and there was significant difference（P<0.05）.3The inhibition effects of3-MA on the SKOV3and SKOV3/DDPcells were determined by MTT assays, and3-MA decreased cell viability in a time-depentent manner. The IC10value for SKOV3cells was2.16mmol/L and for SKOV3/DDP cells was2.95mmol/L, so we chose2mmol/L for subsequent experiment.4MTT was used to detect the sensitivity of the SKOV3and SKOV3/DDP cells to cDDP which was used combined with3-MA. IC₅₀of cDDP for SKOV3and SKOV3/DDP cells was4.31±0.12μg/ml and8.85±0.11μg/ml. There was no influence of3-MA on cisplatin sensitivity ofSKOV3cells, but3-MA could improve the sensitivity of the SKOV3/DDP cells to cisplatin.5Flow cytometry results showed that the rate of apoptosis had nosignificant increase for the SKOV3and SKOV3/DDP cells exposed to3-MA（P>0.05）. When cDDP was used combined with3-MA, the apoptosis rate of SKOV3/DDP cells was increased significantly than that ofusing cDDP alone （P<0.05）, but the apoptosis rate of SKOV3cells had no obvious increase （P>0.05）. 6Western blotting results showed that the expression of LC3protein and Beclin1protein of SKOV3/DDP cells were significantly higher than that of SKOV3cells （P<0.05） and after the3-MA treatment the expression of LC3and Beclin1protein of SKOV3/DDP cells were significantly decreased （P<0.05）. The expression of LC3and Beclin1protein ofSKOV3/DDP cells was significantly increased after the cDDP treatment（P<0.05） and was significantly decreased comparing with the cDDP group when cisplatin was used combined with3-MA （P<0.05）.Conclusion:1The autophagic activity of SKOV3/DDP cells was significantly higher than that of SKOV3cells （P<0.05）. It suggests that the increasedof reactive autophagy activity is related to the formation of cisplatin-resistance.2The autophagy activity of SKOV3/DDP cells increased significantly after cDDP treamtent （P<0.05） and the apoptosis rate was significantly lower than that of SKOV3cells （P<0.05）. It indicates that the autophagy which was induced by cisplatin is related to the decline of apoptosis in SKOV3/DDP cells.3When cDDP was used combined with3-MA, the sensitivity of SKOV3/DDP cells on cDDP was enhanced and the apoptosis rate was increased （P<0.05）, which was not present in SKOV3cells. It indicates that inhibition of autophagy may partly reverse the cisplatin-resistance inovarian cancer.