Study On The Mechanism Of Arsenous Acid Combined With Tetrandrine Against Breast Cancer

Objective and SignificanceIn recent years,the treatment of breast cancer with traditional Chinese medicine has made good progress in clinical treatment and basic research.Screening anti-cancer drugs from traditional Chinese medicine has become a hot topic at home and abroad.At present,studies have found that both arsenic and Tetrandrine(Tetra)have good anti-cancer effect.However,the toxicity of arsenic has caused some problems in its clinical application,which need to be solved urgently.According to the TCM mutual restraint theory of "Fangji killing realgar toxin",arsenic and tetrandrine,the effective ingredients of the two drugs,were used as combination in different breast cancer cell lines.From the perspective of inhibiting the proliferation of breast cancer cells,we intended to observe whether the combination of the two drugs has synergistic effect to reduce the toxicity?And the molecular mechanism of inhibition of proliferation induced by sodium arsenite(NaAsO2)combined with tetrandrine(Tetra)was further explored in terms of apoptosis,autophagy,necrosis and cell cycle arrest.The role of MAPK signaling pathway and reactive oxygen species(ROS)in the process of cell death induced by combined use of NaAsO2and Tetra was also explored.At the same time,we studied whether NaAsO2 combined with Tetra could induce differentiation of breast cancer cell,and further explored the specific molecular mechanism of differentiation induction.Last,we observed the effects of NaAsO2 combined with Tetra on CD4+CD25+Foxp3+regulatory T cells in human peripheral blood mononuclear cells(PBMC).This study was carried out in order to provide experimental basis for clinical application of NaAsO2 combined with Tetra in the treatment of breast cancer,it has important theoretical and practical significance for the promotion and application of traditional Chinese medicine.Research Methods(1)Effects of NaAsO2 combined with Tetra on proliferation,apoptosis,autophagy,necrosis and cell cycle arrest of breast cancer cells MDA-MB-231 and T47D cells were intervened with gradient concentration of NaAsO2,Tetra alone or in combination.Cell proliferation was detected by XTT method,apoptotic rate and cell cycle were detected by flow cytometry,and expression of autophagy-related proteins,mitochondrial apoptotic pathway-related proteins and MAPK signaling pathway proteins were detected by Western-Blot method.The effects of autophagy inhibitors Wortmannin,ROS inhibitor NAC,Trolox and MAPK on MDA-MB-231 proliferation inhibition rate,apoptosis rate,necrosis rate,cell cycle and autophagy marker protein LC3,Beclin-1 were detected.The changes of ROS level within cells after treatment of NaAsO2 combined with Tetra were detected.The effect of NaAsO2 combined with Tetra on cell migration was detected by Transwell experiment.(2)Differentiation of breast cancer cells induced by NaAsO2combined with Tetra MDA-MB-231,MCF-7 and T47D cells were intervened with gradient concentration of NaAsO2,Tetra alone or in combination.Flow cytometry was used to detect ICAM-1 immunofluorescence and Western-Blot method was used to detect the expression of ICAM-1,p-ERK,ERK and HER-2 cell differentiation-related proteins.The effects of ERK inhibitors on ICAM-1 immunofluorescence of three kinds of cells were detected,and the specific molecular mechanism of differentiation induced by combination of ERK inhibitors was preliminarily revealed.Western-Blot assay was used to detect the expression of ICAM-1,p-ERK,ERK and HER-2differentiated proteins in breast cancer xenograft nude mice treated with Arsenic Trioxide Inj ection and tetrandrine inj ection alone or in combination.(3)The effect of NaAsO2 combined with Tetra on CD4+CD25+Foxp3+ regulatory T cells in PBMC.Human peripheral blood mononuclear cells(PBMC)were isolated from human peripheral blood with lymphatic separators.The effects of drugs on PBMC proliferation were detected by XTT method,and the gradient concentration of NaAsO2,Tetra alone and combined with each other were used to interfere with PBMC cells.Flow cytometry was used to detect the proportion of CD4+CD25+Foxp3+regulatory T cells after drug intervention,and to explore the effect of NaAsO2 combined with Tetra on immune function.ResultsNaAsO2 and Tetra alone or in combination can inhibit the proliferation of MDA-MB-231 and T47D cells in a dose-dependent manner,and the combined effect is better than that of single drug.The sensitivity of MDA-MB-231 to Tetra was higher than that of T47D cells,and the inhibitory effect of combined drugs on the proliferation of MDA-MB-231 cells was better than that of T47D cells.NaAsO2 combined with Tetra can induce apoptosis of MDA-MB-231 cells and T47D cells.The apoptosis of MDA-MB-231 cells was dose-dependent and time-dependent,and also was caspase-independent.The combined treatment enhanced the apoptosis effect of MDA-MB-231 cells compared with when treated alone,and achieved the same apoptotic induction effect as tamoxifen(TAM).The apoptosis of T47D cells induced by combined treatment was related to the activation of caspase-8,which may be induced by death receptor pathway.The results of cell cycle showed that MDA-MB-231 cells could be blocked in S phase by combination therapy.The combination of drugs can significantly inhibit cell migration.Cells treated with the combination of drugs,and then cultured in the normal condition without drugs,can also significantly reduce cell migration,which demonstrated a certain time lag effect.NaAsO2 combined with Tetra can induce autophagy of MDA-MB-231 cells.The autophagy induced by NaAsO2 inhibits the growth of cancer cells,which is characterized by autophagic cell death.The autophagy induced by the combination of drugs did not promote apoptosis,but promoted cell necrosis to a certain extent.When autophagic death induced by NaAsO2 combined with Tetra was strongly inhibited,S-phase blocked cells gradually transited to G2/M phase,and cell cycle showed a recovery trend to some extent.From the results of the changes of MAPK pathway protein level and the effect of MAPK inhibitors on cell survival rate,JNK,ERK and p38 expression were significantly up-regulated,but only JNK pathway was directly related to the inhibition of cell proliferation induced by combination of drugs.The apoptosis and autophagy of MDA-MB-231 cells induced by NaAsO2 combined with Tetra were not related to the activation of JNK pathway.The S-phase blockade of MDA-MB-231 cells induced by NaAsO2 may be related to the activation of JNK pathway.NaAsO2 combined with Tetra could induce the decrease of ROS level in MDA-MB-231 cells,and the decrease of ROS level was related to the inhibition of proliferation induced by combination of NaAsO2 and Tetra.The apoptosis,necrosis and autophagy of MDA-MB-231 cells induced by combination therapy may not be related to ROS level,but the cell cycle arrest induced by combination therapy may be related to the decrease of ROS level.In vitro,Tetra could induce the differentiation of MDA-MB-231,MCF-7 and T47D cells,NaAsO2 could induce the differentiation of MCF-7 cells,and NaAsO2 combined with Tetra could induce the differentiation of MDA-MB-231,MCF-7 and T47D cells.For MDA-MB-231 cells,the combination of NaAsO2 and MCF-7 cells can reduce the concentration of drugs needed to induce differentiation.For MCF-7 cells,the combination of NaAsO2 and NaAsO2 can enhance the differentiation induction effect of the two drugs when used alone.The differentiation of MDA-MB-231 and MCF-7 cells induced by NaAsO2 and Tetra alone or in combination is related to the activation of ERK signaling pathway and the inhibition of ERK signal.The induction effect of differentiation is also inhibited.The differentiation of MDA-MB-231 and MCF-7 cells induced by NaAsO2 and Tetra alone or in combination is also related to the down-regulation of HER2.In vivo experiments,sodium chloride arsenite injection and tetrandrine injection alone or in combination can induce the differentiation of breast cancer transplanted cells,and may be related to the up-regulation of ERK protein.There was no significant difference between the two drugs at three lower concentrations(0.5+0.5,0.25+0.25,0.125+0.125).Tetra could slightly reduce the percentage of CD4+CD25+Foxp3+ cells.The response of PBMC to NaAsO2 in different individuals is quite different.Some people's response is consistent with the results reported in the literature.That is,the proportion of CD4+CD25+Foxp3+Treg cells is increased,but the proportion in some people is basically unchanged.Combination of two drugs can also slightly reduce the proportion of CD4+CD25+Foxp3+Treg cells,but there is no statistical difference.Conclusion(1)NaAsO2 combined with Tetra can inhibit the proliferation,migration,apoptosis,autophagy and cell cycle arrest of breast cancer cells.Its inhibition of proliferation is related to the activation of JNK signaling pathway and the decrease of ROS level in cells.There is a certain relationship among autophagy,apoptosis,necrosis and cell cycle arrest induced by NaAsO2 combined with Tetra.(2)NaAsO2 combined with Tetra can induce the differentiation of MDA-MB-231,MCF-7 and T47D cells.The differentiation of MDA-MB-231 and MCF-7 cells induced by NaAsO2 is related to the activation of ERK pathway and the decrease of HER2 expression.Both sodium arsenite chloride injection and tetrandrine injection alone or in combination can induce the differentiation of breast cancer transplanted cells,which may be related to the up-regulation of ERK protein.(3)NaAsO2 combined with Tetra had no effect on PBMC proliferation at lower concentration.Tetra alone had a tendency to decrease the proportion of CD4+CD25+Foxp3+cells.PBMC responses to NaAsO2 were different in different individuals.The proportion of CD4+CD25+Foxp3+Treg cells was increased in some individuals,but almost unchanged some other individuals.The combination of Tetra also slightly decreased the proportion of CD4+CD25+Foxp3+Treg cells.It is preliminarily speculated that the combination therapy may play a role in reducing the proportion of CD4+CD25+Foxp3+ Treg cells.