The Role Of Autophagy And Apoptosis In The Process Of Drug-Resistanceadriamycin In Breast Cancer

Objective:To investigate the effects of autophagy and apoptosis in the process of drug-resistanceadriamycin in breast cancer.Methods:1. We establish human multidrug-resistant breast tumor cell line(MCF-7/ADM) by high doses of intermittent induction. The multidrug resistance of MCF-7/ADM was detected by MTT assay.2. The expression of autophagy were determined by real-time PCR, western blot and fluorescence-microscopy in MCF-7 and MCF-7/ADM.3. Flow cytometry assay was used to detect the apoptosis expressions of MCF-7 and MCF-7/ADM. And the expression of XIAP protein was detected by real-time PCR and Western-blot.4. MCF-7/ADM cell line was classified into two groups. Each group was divided into the control group and experimental group. In the experimental group, autophagy inhibitor 3-Methyladenine(3-MA) and apoptosis inhibitor Z-VAD were added. The reversal resistance activity of each group was detected.Results:1.The drug-resistance line MCF-7/ADM of breast cancer was successfully established. IC50 of MCF-7 and MCF-7/ADM cell survival were 1.0971μg/mL and 175.8921μg/mL respectively.The drug resistance fold was 160.2. The results of autophagy experimental in vitro were summarized as follows:(1) The results of real-time PCR showed that compared with the sensitive cell line MCF-7, the expressions of autophagy related genes ATG7, ATG12 and BECLIN1 were up-regulated in MCF-7/ADM cells (P<0.05).(2) Western blot assay showed that the LC3II/LC3 I conversion ratio of breast cancer resistant line MCF-7/ADM was increased compared with the sensitive cell line MCF-7 and the expression of SQSTM1/p62 protein was decreased.(3) Fluorescence microscope showed that compared with the sensitive cell line MCF-7, the EGFP-LC3 puncta of MCF-7/ADM was significantly increased, the number of the positive autophagic cells was increased by 13.5%(P<0.05). The number of positive autophagic cells was 4% in the control group.3. Flow cytometry assay showed that the apoptosis rate significantly increased by 50% in MCF-7/ADM, compare with control group (P<0.05). The real-time PCR and Western blot results showed that the expression of anti-apoptosis protein XIAP was down-regulated in MCF-7/ADM.4. We detected the reversal activity of two different inhibitors on MCF-7/ADM by MTT assay, results showed that after the autophagy inhibitor 3-MA and apoptosis inhibitor Z-VAD were added, IC50 was decreased than before. The reversal folds were 1.16 and 1.2 (P<0.05).Conclusion:To inhibit autophagy and the caspase pathway of apoptosis could partly reverse the drug resistance of adriamycin of breast cancer.