Mechanism Of Programmed Cell Death Induced By Acid Cleaning-Realgar Nanoparticles In Human Breast Cancer Cells

GLOBAR CANCER OBSERVATORY reported that there were an estimated 18.1 million new cases of cancer and 9.6 million deaths from cancer worldwide in 2018.In both sexes combined,lung cancer is the most commonly diagnosed cancer?11.6%of the total cases?,closely followed by human breast cancer?11.6%?,prostate cancer?7.1%?,and colorectal cancer?6.1%?for incidence.Among females,breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death.There were367900 new cases of breast cancer?accounts for 8.6%of the total cases?and97972 deaths in China in 2018.Breast cancer is by far the most common cancer in women worldwide.Surgery,chemotherapy,radiotherapy,endocrine,traditional Chinese medicine and molecular targeted comprehensive therapy have become the standard mode of breast cancer treatment.Many studies show that Chinese medicine plays an important role in the treatment of breast cancer.Realgar,which first recorded in the“Shen Nong's herbal classic”of the Eastern Han Dynasty,and comes from the mineral sulfide realgar family,an arsenic-containing Chinese traditional mineral drug.It's major component is As₂S₂.It is a common medicine for internal treatment and external use in traditional Chinese medicine.Modern medical research has found that realgar has an good clinical effectiveness for the treatment of some varieties of refractory or relapsed tumor or cancer.Previous studies have shown that Realgar and Realgar Nanoparticles Processed can inhibit MCF-7 cells proliferation and induce apoptosis.In this paper,based on the previous study,the programmed death of breast cancer cells induced by acid cleaning-Realgar Nanoparticles and its mechanism were further studied.Objectives:To invisitigate the anti-cancer effects of acid cleaning-Realgar Nanoparticles in human breast cancer cell lines MCF-7,MDA-MB-231,MDA-MB-435S and human hepatic epithelial cells L-02,and further elucidated its underlying mechanisms.Methods:MCF-7 cells,MDA-MB-231 cells,MDA-MB-435S cells and L-02 cells were treated with different concentrations and different times of acid cleaning-Realgar Nanoparticles,and CCK-8 method was used to evaluate the cell proliferation.The half maximal inhibitory concentration(IC₅₀)was calculated to design the concentration of the following test.Hoechst33258fluorochrome staining were employed to observe cell morphological changes and to explore whether drug can induce the cells apoptosis.The apoptosis rate was detected with Annexin V-FITC/PI double staining assay.The PI staining assay was used to detect the cell cycle arrest.Ultramicroscope structure of autophagosome was confirmed under transmission electron microscope.The expression of apoptosis and autophagy related protein were detected by Western blot to explore the drug's underlying mechanisms.Results:Acid cleaning-Realgar Nanoparticles can inhibit significantly cell proliferation of three breast cancer cells and L-02 cells in a concentratin and dosage dependent manner compared with the control group.The MDA-MB-435S cells was regarded as the research object to investigate the effect of Acid cleaning-Realgar Nanoparticles on programed death and its mechanism in human breast cancer.No typical morphological changes of cell apoptosis such as apoptotic body were observed by Hoechst33258fluorochrome staining.Annexin V-FITC/PI double staining assay showed that there was no significant difference between the apoptosis rate of the drug groups and the control group.Western blot was performed to detect the cleaved-PARP.After treatment,the protein expression level have no significant difference with the control group.It is proved that the drug induce MDA-MB-435S cells apoptosis is not obvious.The PI staining assay showed that the drug can induce the cell cycle arrest.Using transmission electron microscope,autophagylysosomes were observed in cells after treatment.Besides,a dosage and time dependent increase of the protein level of the LC?/?ratio as well as decrease of the protein level of P62 were detected by Western blot,Which showed that the acid cleaning-Realgar Nanoparticles can induce autophygy.But the protein level of p-PI3K,p-Akt,p-mTOR,Beclin1and Bcl-2 have no significant difference with the control group,which showed that the drug induce autophagy probably through PI3K-Akt-mTOR-independent pathway.Conclusion:Acid cleaning-Realgar Nanoparticles can inhibit the proliferation of three breast cancer,the drug probably induce MDA-MB-435S cells autophagic death via PI3K-Akt-mTOR-independent pathway.