| Objective: Triple negative breast cancer has a higher malignant degree than other types in breast cancer.The effect of drug treatment is not ideal.The proportion of primary drug resistance is high and the time of secondary drug resistance is short.Triple negative breast cancer always shows a worse prognosis.Therefore,triple negative breast cancer is a great challenge for clinical treatment for a long time.There is always a lack of effects of treatment drugs for triple negative breast cancer.Overexpression of anti-apoptotic proteins and disturbance of apoptotic signal transduction are important reasons for drug resistance in cancer cells.In addition,for multidrug resistance,cancer stem cell-mediated drug resistance and tumor microenvironment are also important factors.The clinical treatment of multidrug resistant in triple negative breast cancer is facing enormous challenge.Traditionally,the main idea of overcoming drug resistance was to design drugs for different targets of apoptotic barriers in order to restore the apoptotic ability of cancer cells.However,due to the complexity of apoptotic mechanism and the inherent heterogeneity of cancer cells,there has been no significant breakthrough in this area.Recent studies have shown that besides apoptosis,cancer cells can also die through reserve death mechanisms such as programmed necrosis,and the obstacles set up by cancer cells to avoid apoptosis do not affect programmed necrosis.Bufalin is one of the main active ingredients extracted from the dry secretions of the retroauricular glands and skin glands of toad.Its anti-tumor effect has been reported in many literatures.In our previous work,bufalin were loaded with biotinylated chitosan nanoparticles to prepare macromolecular drug particles with an average diameter of 171.6 nm,which enable the macromolecular drug particles to maximize the permeability and retention effect of solid tumors in vivo and significantly inhibited breast cancer cells in nude mice.During the previous work,we occasionally found that bufalin could induce programmed necrosis of breast cancer cells.This necrosis was not dependent on caspase,so it was not inhibited by z-VAD-fmk,an inhibitor of Caspase.It also had special ultrastructural characteristics: early disintegration of cell membranes,disappearance of a large number of mitochondrial cristae structures in the cytoplasm,low electron density vacuole-like structures and autophagy but no obvious changes in the nucleus.Based on the research results of domestic and the previous research findings of our group,this topic attempts to clarify:(1)whether bufalin can effectively kill triple negative breast cancer cells,especially drug resistant triple negative breast cancer cells;(2)whether bufalin can indeed induce programmed necrosis;(3)what is the molecular mechanism,by which bufalin can induce programmed necrosis of breast cancer cells;(4)whether bufalin can restore the chemosensitivity of drug resistant cells;(5)whether bufalin can synergize and enhance the effect of chemosensitivity.Method: The main research methods in this study included:(1)MTT assay for cell viability;(2)AV/PI double staining method for cell survival;(3)Hoechst 33342/PI double staining method for fluorescence microscopy for nuclear morphology and cell survival;(4)ROS detection kit for intracellular ROS content;(5)PI staining method for cell death ratio;(6)transmission electron microscopy for cell morphology;(7)JC-1 staining was used to detect the changes of mitochondrial membrane potential;(8)Real-time PCR,Western blot and immunohistochemistry were used to detect the expression of genes in mRNA and protein levels;(9)in vivo tumor implantation and drug intervention experiments was performed in mice,including tumor implantation,observation,measurement and dissection;(10)inhibitors used in the experiment mainly included programmed necrosis inhibitor Nec-1 and apoptotic inhibitor z-VAD-fmk,reactive oxygen species inhibitor NAC,phospholipase A2 inhibitor bromoenol lactone,lipoxygenase inhibitor AA861,mitochondrial respiratory chain complex I inhibitor Rotenone,complex III inhibitor Antimycin and ATP synthase inhibitor Oligomycin;(11)Image J was used for gray value determination;(12)Graphpad was used for image editing;(13)SPSS22.0 was used for data statistics,P < 0.05 indicated significant differences.Results: MTT assay showed that bufalin had a dose/time dependent killing effect on triple negative breast cancer cell lines MDA-MB-231,MDA-MB-231/ADR and MDA-MB-231/DOC.Through further study of MDA-MB-231/ADR cells,we found that the killing effect of bufalin was achieved by inducing programmed necrosis.The specific manifestations were as follows:(1)programmed necrosis was dominant in dead cells observed by Hoechst 33342/PI double staining;(2)cell death could be significantly inhibited by programmed necrosis inhibitor Nec-1;(3)intracellular reactive oxygen species(ROS)increased significantly,NAC as an inhibitor for ROS could significantly reduce the proportion of cell death;(4)under transmission electron microscopy,necrotic morphological changes could be observed,including: 1)the nucleus was swollen but intact,without nuclear pyknosis and fragmentation;2)the integrity of cell membrane was lost,and apoptotic bodies were not formed by "germination or bubbles";and 3)a large number of vacuoles was found in the cytoplasm;4)autophagic vacuoles with bilayer membrane structure formed in the cells;5)the swelling of mitochondria could be found.In order to explore the possible mechanism of bufalin-induced programmed necrosis,we carried out further research.The results indicated that after the treatment of bufalin on MDA-MB-231 and MDA-MB-231/ADR cells:(1)the ROS content in cells was significantly increased,and the increase of ROS was dose-dependent with the concentration of bufalin;(2)the mitochondrial dysfunction was characterized by a significant decrease in mitochondrial membrane potential under JC-1 detection;(3)PLA2/LOXs inhibitors could not inhibit the increase of ROS content in cells;(4)antimycin,an inhibitor of respiratory chain complex III,and oligomycin,an inhibitor of ATP synthase,could not inhibit the increase of ROS,but rotenone,an inhibitor of mitochondrial respiratory chain complex I,could inhibit the increase of ROS.We also found that non-toxic dose of bufalin could reverse MDA-MB-231/ADR and MDA-MB-231/DOC multidrug resistance in triple negative breast cancer cells and restore the chemosensitivity of drug resistant cells.Bufalin could reduce the expression of multidrug resistance proteins P-gp,BCRP and MRP1 in drug-resistant cell lines,which might be the way to reverse drug resistance.After the tumorigenesis of MDA-MB-231/ADR cells planted in the axillary of mice,through the experiment of the drug intervention of bufalin or doxorubicin or combination drugs,it was found that bufalin could effectively inhibit the growth of MDA-MB-231/ADR,and the therapeutic effect of combination with doxorubicin was more remarkable.It was found that bufalin could significantly reduce the expression of multidrug resistance proteins P-gp,BCRP and MRP1 by detecting the proteins in tumors from the mice.Conclusion: 1.Bufalin could effectively kill triple negative breast cancer cells and their drug resistant cells,and this killing was achieved by inducing programmed cell necrosis.2.Bufalin could destroy the mitochondrial function of triple-negative breast cancer cells,lead to electron leakage during the transmission of electrons from respiratory chain,which increase intracellular reactive oxygen species content,and finally induce programmed cell necrosis.3.Bufalin could reverse drug resistance by reducing the expression of multidrug resistance proteins in drug resistant triple negative breast cancer cells.Bufalin could synergistically sensitize and adriamycin-resistant triple negative breast cancer cells in vivo. |
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