Influence Of Autophagy Inhibitors Combined With EGFR Inhibitors In Triple-negative Breast Cancer Cells

Purpose and background: Breast cancer is the first female malignancy in the world,triple negative breast cancer is a special subtype of breast cancer,which is characterized by estrogen receptor,progesterone receptor and epidermal growth factor receptor 2 negative expression.Triple negative breast cancer is generally considered as an aggressive disease associated with aggressive,easy distant metastasis,and poor outcomes.Evidences reported that the incidence rate of triple negative breast cancer in China accounted for about 17% of the world.Triple negative breast cancer lacks of endocrine therapy and HER-2 targeted therapy,and the main treatment currently is chemotherapy.The high expression of epidermal growth factor receptor is associated with the high metastasis rate and poor prognosis.However,targeted therapy for EGFR is not ideal because of the resistance of EGFR molecular targeted therapy.The ways to enhance the sensitivity of targeted therapy has become a hot research topic.Evidences reported that autophagy expressed abnormal in tumors with the high expression of EGFR.Autophagy is a unique physiological and pathological phenomenon of eukaryotes,which can be activated by a variety of external stimuli(such as radiotherapy,chemotherapy,targeted therapy).In the process of tumor formation,autophagy can provide the necessary nutrition and energy for the tumor to profect the tumor tissue from the treatment of injury,which is an important mechanism of tumor resistance.Thus,blocking the level of autophagy during targeting therapy can improve the therapeutic sensitivity.Previous studies have shown that blocking the abnormal expression of autophagy levels in non-small cell lung cancer can significantly enhance the sensitivity of non-small cell lung cancer cells to gefitinib treatment.However,in triple-negative breast cancer,autophagy and EGFR targeted therapy research has not been reported.Methods: All the cells were treated with 3-Methyladenine/bafilomycin A1 and/or gefitinib.The effect of autophagy inhibitor and gefitinib on the growth of the cells was evaluated by MTT and colony formation.Cell cycle distrbitation was detected by flow cytometry.Western blot analysis was used to detect the DNA damage and apoptosis related proteins.ROS assay was used to determine the relationship between apoptosis and mitochondrial apoptosis induced by the combination therapy.The expression of Cleaved-CASP 3 in the transplanted tumor was detected by immunohistochemistry.Results: MTT assay showed the IC50 in groups of GE+3MA and GE+BAF were(4.1±0.2)?mol/L and(3.8±0.3)?mol/L,which is a stronger inhibitory effect than gefitinib alone that is(7.0±0.2)?mol/L on MDA-MB-468(P<0.05).Similarly,the IC50 in groups of GE+3MA and GE+BAF were(9.7±0.1)?mol/L and(7.7±0.2)?mol/L,which is a stronger inhibitory effect than gefitinib alone that is(14.7±0.1)?mol/L on MDA-MB-231(P<0.05).Clonal formation experiments showed that,compared with the control group,the expression of MDA-MB-468 cells in combination with gefitinib and autophagy inhibitor was(29.85 ± 5.54)% and(33.58 ± 6.31)%,respectively,significantly lower than that of Gefitinib(63.46 ± 8.32)%(Z =-2.309,P <0.017).The percentage of clones in MDA-MB-231 cells was(22.63 ± 6.27)% and(31.54 ± 5.81)%,respectively,which was significantly lower than that of the gefitinib group(49.35 ± 6.56)%(Z =-2.309,P <0.017).The expression of DNA damage-related proteins was induced and cell cycle was arrested at G0 / G1 phase by gefitinib combined with 3MA or BAF in the triple negative breast cancer cell lines MDA-MB-468 and MDA-MB-231 cells.After exposure to gefitinib combined with 3MA or BAF,the ROS was found to increase in the groups with gefitinib combined with 3MA or BAF.Western blot analysis showed that mitochondrial apoptosis-related protein Cyto C expression was significantly increased.Nude mice transplanted tumor model showed that autophagy inhibitor combined with gefitinib significantly inhibited the growth of MDA-MB-468 cell tumor.Conclusions: Autophagy inhibitor may enhance the sensitivity of gefitinb in MDA-MB-468 and MDA-MB-231 cells through down-regulation of the Akt pathway and the mitochondrial apoptosis pathway.