Inhibition And Mechanism Research Of The Bcl-2 Inhibitor Sabutoclax On Human Breast Cancer Multidrug Resistance Cells

Objective:1.To study the effect of Sab on the proliferation,apoptosis and drug resistance of human breast cancer cell lines and drug-resistant cell lines,and to explore the antitumor effect of Sab on breast cancer resistant cells in vivo and in vitro.2.To study the cytotoxicity of Sab on breast cancer stem cells and to further explore the mechanism of Sab’s ability to overcome the resistance of breast cancer.3.The pan-Bcl-2 family inhibitor Sab combined with conventional chemotherapy drugs doxorubicin.To study the inhibitory effect and potential mechanism of combination therapy on chemotherapeutic resistant cells in breast cancer.Methods:1.After treatment of Sab,the difference of cytotoxic activity was measured between breast cancer cell lines and their corresponding drug-resistant cell lines by Cell Proliferation Assay(MTT).The drug resistant clones formation assay was used to evaluate the sensitivity of breast cancer cell lines and their corresponding drug-resistant cell lines to Sab.2.The apoptosis induced by Sab was detected by Annexin V/PI staining in MCF-7,Cal51 and drug-resistant cell lines MCF-7/A02 and CALDOX,Caspase-3/7 activity assay and Caspase-9 assay in drug-resistant cell lines MCF-7/A02 and CALDOX.Meanwhile,real-time quantitative PCR was used to detect the changes of m RNA levels of apoptotic pathway-related genes in drug-resistant breast cancer cell lines.We also investigated the protein expression of IL-6/STAT3 signal transduction pathway and related apoptotic protein were detected by Western blotting.3.Real-time quantitative PCR was used to detect the changes of m RNA levels of breast cancer stem cell genes in drug-resistant breast cancer cell lines before and after treatmen.The percentage of stem cells in drug-resistant cell lines MCF-7/A02 and CALDOX were detected by flow cytometry with CD44+/ CD24-and ALDH+ cell as the markers of breast cancer stem cell.To exposure Sab to MCF-7/A02 and CALDOX cells,we performed to compare changes in percentage of stem cell makers before and after treatment.4.The proliferation of stem cell of MCF-7/A02 and CALDOX treated by Sab was detected by mammosphere culture technique.The sensitivity of drug-resistant cells was measured by soft agar clone formation experiment.5.Immunohistochemistry was used to detect the changes of signal transduction pathway and related apoptotic proteins after treatment with Sab in breast cancer patients.6.MTT assay was used to detect the effect of Sab combined with doxorubicin on breast cancer drug resistant cell lines.Using Calcusyn software,the combination index(CI)was used to determine whether the combination of the two drugs had synergistic effect.Apoptosis induced by Sab combined with VP-16 on resistant breast cancer cells was detected by Annexin V/PI staining.Results:1.The results of MTT showed that Sab had a significant inhibitory effect both on sensitive and drug resistant cell lines and the inhibitory effect was in a concentration-dependent manner.Resistance index is only between 1.6 to 3.3,and resistance index of doxorubicin and other conventional cytotoxic chemotherapy drug was in the range of 40 to 60,which was much higher than the former.The results of clones formation assay showed that Sab had a long-term inhibitory effect on the cells in the above,which could effectively inhibit the proliferation of drug-resistant cells.2.Detection of breast cancer sensitive cells by Annexin V/PI staining was sensitive to cytotoxic chemotherapeutic drugs etoposide and resistant to drug-resistant cell lines.While the same dose of Sab on breast cancer sensitive and resistant cell lines have induced apoptosis.Caspase-3/7 activity test and Caspase-9 test assay to verify the above conclusions.PCR results showed that the m RNA levels of Bim and Bax were significantly increased.Western blotting showed that the expression level of Bim,Bax and PUMA increased and the expression of survivin decreased.The results of immunohistochemistry showed that the expression of Bcl-2 and survivin was decreased and the expression of Bax was increased after treatment with Sab.3.The m RNA levels of CD44,ALDH and ABCG2 were significantly decreased,and the m RNA level of CD24 did not change significantly.Breast cancer cell line stem cell subpopulation analysis showed that the proportion of CD44+/CD24-and ALDH+ cells in MCF-7/A02 and CALDOX cells was significantly higher than that in breast cancer sensitive cells.The percentage of CD44+/CD24-and ALDH+ cells decreased in a dose-dependent manner after administration of Sab in drug-resistant cell line.The expression of CD44 and ALDH in stem cells decreased after treatment with Sab and the expression of p AKT and p STAT3 was decreased after treatment with Sab,it means that the activity of IL-6 / STAT3 signal transduction pathway was inhibited.4.Sab inhibited the tumorigenic ability and mammosphere-forming ability of MCF-7/A02 and CALDOX cell lines in a dose-dependent manner.5.Drug combination experiment suggests that Sab combined with doxorubicin has synergistic effect on breast cancer resistant cell lines.Conclusion:1.Sab can effectively inhibit the growth and proliferation of breast cancer drug-resistant cells,and induce tumor cell apoptosis.2.Sab can increase the proapoptotic protein Bax,reduce the expression of anti-apoptotic protein survivin and thus promote its apoptosis affect the multidrug resistance of breast cancer cells.3.Sab can effectively inhibit the proportion of breast cancer stem cells in drug-resistant cell lines,thereby inhibiting the proliferation of drug-resistant cells and tumorigenic ability.4.Bcl-2 family inhibitor Sab can overcome the resistance of tumor cells,which combined with conventional cytotoxic chemotherapy drugs reveal synergistic effect.