Study On The Regulation Of Arsenic Trioxide Combined With Tetrandrine On Triple-negative Breast Cancer Cells And Stem Cells

ObjectiveIn this study,through systematic and comprehensive retrieval of relevant databases,a randomized controlled trial of Chinese herbal medicine for the treatment of triple nagetive breast cancer was carried out.The curative effect of traditional Chinese medicine for the treatment of TNBC was systematically evaluated,and the evidence of evidence-based medicine was provided for the clinical efficacy of traditional Chinese medicine for the treatment of TNBC.The TNBC stem cells were cultured in the experiment,the mechanisms of arsenic trioxide combined with tetrandrine on TNBC cell lines and stem cells were researched in this study.Through the study of the biological characteristics of breast cancer stem cells by ATO and Tet,to identify the related signal transduction pathways and action sites of the TNBC stem cells and provided the experimental basis for the treatment of TNBC by ATO combined with Tet.MethodsComprehensive retrieval of relevant databases,the randomized controlled trial(RCT)of Chinese herbal medicine for the treatment of TNBC was carried out.The quality of the included test was evaluated by the internationally recognized evaluation criteria,and the curative effect of Chinese herbal medicine for the treatment of TNBC was systematically evaluated.MDA-MB-231 cell lines was cultured in vitro and MTT method was used to explore the combined effect of ATO and Tet in different concentrations.The optimal drug ratio for the combination treatment was determined by using the median-effect principle to guide the subsequent experiments.The morphological changes of cells after ATO and Tet were observed.Cell apoptosis and cell cycle distribution were detected by flow cytometry.The inductively coupled plasma mass spectrometry(ICP-MS)was used to observe whether Tet could change the concentration of arsenic in cells,and to explore whether Tet could reduce the toxicity of arsenic.By detecting the changes in drug resistance related proteins,topoisomerase,and glutathione transferase,to observe whether the synergistic effects of ATO and Tet were related to the change of drug resistance.By observing the protein related to the absorption of arsenic,to find whether tetrandrine could increase the sensitivity of the TNBC cell line MDA-MB-231 to ATO.By detecting the expression of CD44 and CD24 in each group,to observe whether ATO combined with Tet could affect the invasion and metastasis of TNBC cells and its mechanism.MDA-MB-231 stem cells were enriched by serum-free suspension culture,and were induced by culture medium containing serum.The expression of CD44 and CD24 were detected by flow cytometry.The common stem cell markers Sox2 and Oct4 were detected by qPCR,Transwell chamber assay was used to detect the invasive ability.CCK-8 assay was used to detect the proliferation ability.CCK-8 method was used to explore the combined effect of ATO and Tet in different concentrations to MDA-MB-231 stem cells.The optimal drug ratio for the combination treatment was determined by using the median-effect principle to guide the subsequent experiments.The morphological changes of cells after ATO and Tet were observed.Cell apoptosis and cell cycle distribution were detected by flow cytometry.Western blot method was used to detect the signal pathway,and the effect mechanism of ATO and Tet on the growth regulation of TNBC stem cells was observed.ResultsThe system evaluation results showed that Chinese herbal medicine had advantages in improving the symptoms of patients with TNBC.It could increase the overall survival,increase the disease-free survival,increase the KPS score and decrease the recurrence rate.It was found that different concentrations of Tet,ATO showed good inhibitory effect on MDA-MB-231 cells,The combination treatment was superior than the single drug alone.The synergistic effect of 48h was best at Tet(1.5μg/mL)and ATO(1.25μg/mL).Tet and ATO alone or combined could promote the apoptosis of MDA-MB-231 cells,and the combined effect of the two drugs was better than the single drug.The combination of ATO and Tet could block the cycle of MDA-MB-231 cells in G2/M period.Combination of ATO and Tet compared with the single drug,the morphology of MDA-MB-231 cells was more obvious,which showed that the cell volume was reduced,the nucleus was condensed,and a large number of cells disintegrated and died.Tet and ATO alone or combined could reduce the proportion of CD44+CD24-in MDA-MB-231 cell lines.Tet and ATO alone or combined could reduce the expression of AQP9,MDR1,MRP1,Topo Ⅱ protein,and the combination of two drugs was superior to single drug.Tet and ATO alone or combined could reduce the expression of GST-π,The expression of MRP2 protein was increased,but the combined effect of two drugs was not significantly different from that of single drug.After adding Tet,the content of arsenic in cells was decreased compared with ATO alone.The serum free suspension culture was used to enrich the breast cancer stem cells and induced differentiation in the serum containing medium.The proportion of CD44+CD24-in MDA-MB-231 cells and MDA-MB-231 stem cells was high.There was no significant difference in the expression of Oct4 and Sox2 genes in the two kinds of cells,but the invasion ability of MDA-MB-231 stem cells was much higher than that of the primary cells.Serum free enriched MDA-MB-231 cells still could maintain slow proliferation.It was found that different concentrations of Tet,ATO showed good inhibitory effect on MDA-MB-231 stem cells,with a significant relationship with dose and anction time.The combination treatment was superior than the single drug alone.The synergistic effect of 48h was best at Tet(1.5μg/mL)and ATO(2.5μg/mL).Tet and ATO alone or combined could promote the apoptosis of MDA-MB-231 stem cells,and the combined effect of the two drugs was better than the single drug.The combination of ATO and Tet could block the cycle of MDA-MB-231 stem cells in G0/G1 period.Combination of ATO and Tet compared with the single drug,the morphology of MDA-MB-231 cells was more obvious,which showed that the cell volume was reduced,the nucleus was condensed,and a large number of cells disintegrated and died.ATO combined with Tet could reduce the expression of GLI1 and SMO protein in Hedgehog signaling pathway and increase the expression of PTEN protein.ConclusionsThe system evaluation results showed that Chinese herbal medicine had advantages in improving the symptoms of patients with TNBC,increasing the overall survival and disease-free survival,decreasing the recurrence rate.However,more high-quality studies with larger sample sizes are needed to validate these findings.ATO and Tet showed good inhibitory effects on MDA-MB-231 cell lines and MDA-MB-231 stem cells.The combined effect of two drugs was better than that of single action.Its mechanism was related to inducing cell apoptosis,blocking cell cycle,changing drug resistance,reducing GLI1,SMO and increasing the expression of PTEN protein.Tet could reduce the content of arsenic in cells,and its mechanism might be to promote arsenic excretion in cells by decreasing the expression of AQP9 protein.It was concluded that the combination of two drugs may have synergistic and attenuated effects.CD44+CD24-was highly expressed in MDA-MB-231 cells.MDA-MB-231 stem cells had potential of redifferentiation and strong invasiveness.