The Mechanism Of Autophagy Induced By Ionizing Radiation In The Multidrug Resistance Ovarian Cancer Cells

Multidrug resistance (multidrug resistance, MDR) is that tumor cells have resistant to a kind of anticancer drugs in chemotherapy, but the change of cells' structure and mechanism result in cross-resistance to other anticancer drug. MDR is usually found in the process of cancer chemotherapy and have been a major obstacle in the treatment of malignant tumors. Autophagy, a type II programmed cell death, is a way of protein degradation, and has an important role for the maintenance of homeostasis. Since autophagy is mainly given to drug resistance of tumor cells by protecting residual or metastatic tumor cells to evade apoptosis, effectively inhibited autophagy may be an effective way to treat cancer to survive in the resistant tumor cells.It has been found that depending on tumor type, radiation therapy programs and kinds of drugs, radiation therapy can promote or reverse multidrug resistance in chemotherapy. In addition there is some indirect evidence that the autophagy may participate in the regulation of MDR. However, whether radiation can induce autophagy and the relationship between MDR and autophagy, especially whether radiation-induced autophagy can reverse MDR, has important clinical value. In this study, with cell and molecular biology methods, used human ovarian cancer cell line SKOV3 and its resistant SKVCR2.0,we detect the effect of ionizing radiation to autophagy, and to explore possible molecular mechanism, for MDR reversal by radiation-induced autophagy.1. SKOV3, SKVCR2.0 cell lines drug sensitivityMTT assay is used to detect drug sensitivity in human ovarian cancer cell line SKOV3 and SKVCR2.0 on the VCR, VP-16, DDP drug. With different concentrations of VCR, VP-16, DDP after 48h, cell viability of SKVCR2.0 was significantly higher than SKOV3 (P<0.05). The results showed that the SKVCR2.0 cells induced by vincristine show the characteristics of multi-drug resistance.2. Autophagy and related gene expression of SKOV3 and SKVCR2.0 cell linesBy MDC staining, Real-Time PCR and Western blot, we detect autophagy and related gene expression levels in SKOV3 and SKVCR2.0. In SKVCR2.0, cytoplasmic area corresponding autophagy spots can be seen clearly positive fluorescent particles MDC. MDC-positive cells was significantly increased. LC3II(MAP1LC3B) mRNA and protein expression were significantly higher than that of SKOV3 cells, indicating that in cell lines SKVCR2.0 autophagy are significantly higher than that of human ovarian cancer SKOV3 cells (P<0.01).In order to explore both of autophagy-related genes differentially expressed in cell lines, we studied two cell lines autophagy-related gene changes. Real-Time PCR is used to detect Beclinl, Bcl-2, Bax, DRAM. Tigar gene mRNA expression, using Western Blot test Beclinl, Tigar, DAPK1 protein expression. The results showed that compared with SKOV3 cells,in SKVCR2.0 cells Beclinl, Bax. Tigar, DRAM can be found increased expression; and Bcl-2 decreased expression. The results show that autophagy level in SKVCR2.0 compared with SKOV3 cells is increased, autophagy in SKVCR2.0 may be a survival mechanism.3. SKVCR2.0 cells to VCR, VP-16, DDP drug sensitivity after ionizing radiationMTT assay after ionizing radiation shows that different concentrations of VCR. VP-16, DDP effect after 48h, SKVCR's cell viability in corresponding drug concentrations was significantly lower than control, fractionated irradiation group survival rate have decreased significantly (P<0.05). The DDP treatment group no significant change in cell viability.4 Autophagy and its related genes expression with different methods of ionizing radiation on ovarian cancer cell lines SKVCRWe detected the incidence of autophagy and MAP1LC3B mRNA expression and its encoded protein with ionizing radiation (OGy, lOGy* 1,2Gy* 5) on SKVCR, the results show that MDC-positive cells SKVCR2.0 treated with ionizing radiation was significantly increased, and LC3Ⅱhave increased clearly in split doses group of radiation than single-dose radiation therapy.The results showed that ionizing radiation can increase the occurrence of cellular autophagy on SKVCR2.0, and autophagy after split dose irradiation increased more obviously.We further detected after exposure to ionizing radiation Beclinl, Bcl-2, Bax, p53, TP53INP1, DRAM, Tigar, DAPK1 gene mRNA. and Beclinl, Akt/mTOR/S6k, Tigar, DAPK1 protein expression. The results showed that, Beclinl expression increased after irradiation, fractionated irradiation group (2Gy/day* 5) increased more significantly than the single irradiation group (10Gy), indicating Beclinl expression increases in autophagy induced by ionizing radiation. After irradiation the expression of Bcl-2 and bax was significantly decreased, suggesting that Bcl-2 reduced in the incidence of autophagy. Compared with the control,Akt/mTOR/S6k pathway related, genes and proteins (Akt, mTOR, p-mTOR, P70 S6 Kinase, p-P70 S6 Kinase) expression were decreased, fractionated irradiation group (2Gy/day×5) decreased more significantly than the single irradiation group (10Gy). The results shows that ionizing radiation in particular the split dose exposure can induce cellular autophagy on SKVCR cell lines and this has some relevance with the Akt/mTOR/ S6k pathway...