Inhibition Of Autophagy Enhanced Multi-drug Resistant Ovarian Cancer Cells Sensitivity To Radiotherapy And Chemotherapy In Vitro

Epithelial ovarian cancer (EOC) is one of the highly malignant gynecologicaltumors. It is estimated that21,990newly diagnosed cases and15,460relationaldeaths established in the United States in2011. Currently, platinum/paclitaxel-basedchemotherapy is the standard strategy for EOC surgical staging and post-operationtreatment. Although chemotherapy achieved success on EOC treatment, theprognosis is still poor for the presence of abdominal or pelvic recurrence and furtherdeveloping drug resistance. The emergence of drug resistance is an essential causeof high mortality rate of ovarian cancer. Therefore, how to improve the sensitivity oftumor cells to chemotherapeutic agents is currently one of problems urgently to besolved for EOC treatment.The activations of multiple genes and pathways are normally involved in tumordevelopment consequently, to obtain the resistance to chemotherapy andradiotherapy-induced cell death occurr. Generally, cell death includes necrosis,apoptosis and autophagic cell death. Apoptosis, also called type I programmed celldeath, is the most important mechanism of radiotherapy and other therapy modalitiesto tumors. Autophagic cell death is another significant regulatory mechanism of celldeath in addition to apoptosis, which plays an important role in the development ofcancer. Autophagy is an evolutionarily conserved metabolic process, and its majorfunction is the degradation of impaired intracellular proteins, excess or defectiveorganelles and the reusing and recycling proteins and amino acids. Apart from this,autophagy is also found to enhance the response to stress or pressure. Autophagyplays a remarkable role in cancer development and drug resistance, and exhibits twoopposite outcomes, induce either adaptive response or cell death depending ontumor types and environmental conditions. Therefore, to clarify the function andmechanisms of autophagy in cancer treatment is beneficial for the individualized treatment of different types of cancers to acquire considerable therapeutic effect,especially the cancers resistant to radio-chemotherapy.Objective: In this study, we investigated the function and mechanism ofautophagy in chemotherapy and ionizing radiation-induced cell death in humanovarian cancer cell line SKOV3and its drug-resistant phenotype SKVCR in order tooptimize cancer treatment strategies and complement experimental theoretical basisof new therapeutic targets.Methods:(1) Human ovarian cancer cell line SKOV3and multi-drugresistance phenotype cell line SKVCR were used in this study;(2) X-irradiation wasused, irradiation conditions were as follows: voltage180kV, current18.0mA,filtration plate0.25mm Cu and1.0mm Al, distance between target and X source60cm, dose rate0.40Gy/min;(3) VCR (vincristine), THP (pirarubicin) and VP-16(etoposide) were used for chemotherapy resistance experiments;(4) CCK8(cellcounting Kit-8) method and colony formation assay were used for cell survivaldetection;(5) Real-time PCR method was used for mRNA detection;(6) Westernblot was used for protein expression;(7) GFP-LC3Cell transfection and MDCstaining was used for cell autophagy detetion;(8) Hoechst33342staining was usedfor apoptosis detection;(9) Flow cytometry was used for apoptosis, autophagy andcell cycle detection.Results:1. The different concentrations of VCR, VP-16and THP inhibited the growthof SKOV3and SKVCR cells, at the same concentration of drug, the survival rate ofSKVCR cells was higher than that of SKOV3cells (P <0.05). P-gp expression inSKVCR cells was50times higher than that in SKOV3cells. Based on the results ofMDC staining, real-time PCR and Western blot tests, the apoptosis level was foundto be the same in two cell lines, but autophagy in SKVCR cells was significanthigher than that in SKOV3cells (P <0.05).2. The different concentration of VCR can inhibit the survival rate of SKOV3and SKVCR cells in time-and-dose-dependent manner;0.02μg/ml VCR had no effect on apoptosis in SKOV3cells, but autophagy rate increased2.63times;10μg/ml VCR induced apoptosis in SKVCR cells without any effect on autophagy (P <0.05).3.3MA、CQ、Rapamycin and ZVAD at the maximal non-effect concentrationwere used in SKOV3and SKVCR cells,3MA and CQ inhibited but Rapamycininduced autophagy, and ZVAD had an effect on the activation of caspase-3andinduced apoptosis (P <0.05).4. SKOV3was treated with VCR alone or in combination with3MA, CQ,Rapamycin and ZVAD for24h:3MA and Rapamycin had no effect on the drugsensitivity to VCR; CQ combined with VCR enhanced VCR inhibition ofproliferation (P <0.05); ZVAD diminished the inhibition of0.02-0.2μg/ml VCR onSKOV3proliferation, and cell survival rate was higher than that in the VCR alonegroup (P <0.05). Based on the results of MDC staining, flow cytometry andWestern blot, VCR induced autophagy in SKOV3cells and had no effect onapoptosis, but treatment of3MA and CQ prior to VCR reduced the autophagy andincreased apoptosis induced by VCR (P <0.05), whereas pretreatment of ZVADinhibited apoptosis induced by VCR and promoted cell survival (P <0.05).5. SKVCR was treated with VCR alone or in combination with3MA, CQ,Rapamycin and ZVAD for24h:3MA/CQ combined with VCR significantlydecreased the cell survival rate and played a role in sensitizing chemotherapy, ascompared with VCR alone (P <0.05), but Rapamycin had no sensitizing effect (P>0.05)；ZVAD significantly increased the cell survival rate and diminished the drugsensibility to VCR in SKVCR cells (P <0.05). Based on the results of flowcytometry and Western blot, pretreatment of3MA and CQ reducedautophagy andincreased apoptosis induced by VCR(P <0.05),whereas pretreatment of ZVADdecreased apoptosis and autophagy (P <0.05).6. Radiosensitivity testing showed that ionizing radiation significantly reducedthe survival of SKOV3and SKVCR cells in a dose-dependent manner, whereasthere was no difference between single irradiation and fractionated irradiation; SKOV3cells radiosensitivity was higher than that in its drug-resistant cell lines (P <0.05).7. Effects of ionizing radiation on cell death: Hoechst staining showed thatirradiation induced SKOV3cell nuclear karyopyknosis, fragmentation, stainingenhancement and enrichment; Meanwhile flow cytometry further confirmed thatirradiation induced apoptosis in SKOV3and SKVCR cells (P <0.05). After ionizingradiation, the percentage of MDC staining positive cells and MAPLC3II/I ratiosignificantly increased in SKOV3and SKVCR cells (P <0.05), suggesting thationizing radiation can induce apoptosis and autophagy.8. Influence of modulators on radiation sensitivity: Apoptosis inhibitor ZVADhad no effect on the ionizing radiation-induced cell death in SKVCR and SKOV3cells. This suggests that SKVCR and SKOV3cell death induced by ionizingradiation may be apoptosis-independent; But autophagy inhibitor3MA significantlydecreased SKVCR survival and showed radiosensitization effect (P <0.05).9. Effects of modulators on cell death: ZVAD inhibited SKVCR and SKOV3apoptosis accompanied by autophagy inhibition as compared with that of ionizingradiation alone, while3MA reduced the percentage of MDC staining positive inSKVCR and SKOV3cells as compared with that of ionizing radiation alone,confirming that3MA could inhibit ionizing radiation-induced autophagy (P <0.05).Simultaneously,3MA inhibits ionizing radiation-induced apoptosis in SKOV3cells,but increased SKVCR cells apoptotic rate (P <0.05).10. Effects of ionizing radiation on cell cycle: Ionizing radiation induced G2/Marrest in SKVCR and SKOV3cells (P <0.05); a higher percentage of S phase cellswas found in SKVCR cells, and ionizing radiation induced significantly S delay;3MA decreased the percentage of S phase in cells which have lower sensitive toionizing radiation), but increased that of G1/M phase in which cells have highersensitive to ionizing radiation (P <0.05). The result shows that3MA can regulatecell cycle redistribution and might have synergistic effect of ionizing radiation. Conclusion:1. The high autophagic level in SKVCR consists of one of the importantmechanism of chemotherapy resistance.2. The inhibition of autophagy can increase apoptosis induced by VCR andenhance the chemosensitivity to VCR in SKVCR cells.3. Ionizing radiation can induce apoptosis and autophagy in ovarian cancer celllines, and autophagy prompts the resistance of SKVCR cells to ionizing radiation;3MA inhibits S phase delay and apoptosis induced by ionizing radiation, andenhances radiosensitivity through the inhibition of autophagy of SKVCR cells.Autophagy inhibitors may become the chemotherapy sensitizer for drug-resistant ovarian cancer in future.