Oridonin Promotes Autophagy And Apoptosis Of Triple Negative Breast Cancer Cells Through MAPK-ERK1/2 Pathway

Objective: In this study,triple negative breast cancer(TNBC)cell lines MDA-MB-231 and BT549 were taken as research objects to explore the anti-tumor effects of Oridonin on triple negative breast cancer cells,including the possible mechanisms of cell proliferation,apoptosis,autophagy and ERK1/2 signal transduction pathway.Methods:(1)CCK-8 assay was used to detect the viability of MDA-MB-231 and BT549 cells under the intervention of different concentrations of Oridonin,and the IC50 values of the two cells under the action of Oridonin were calculated.(2)Ed U staining and colony formation assay were used to further observe the changes of proliferation ability of MDA-MB-231 and BT549 cells under the intervention of different concentrations of Oridonin.(3)Flow cytometry was used to detect the changes of MDA-MB-231 and BT549 cells apoptosis under the intervention of Oridonin.(4)After transfecting TNBC cells with autophagy double-labeled adenovirus(m RFP-GFP-LC3),the change of autophagosome level was observed by adding Oridonin.(5)Western blot experiment was used to observe the autophagy of triple negative breast cancer cells MDA-MB-231 and BT549 and the changes of the expression levels of apoptosis proteins p62,Bcl-2,Cleved-Caspase3,Beclin1 and LC3-II/I under the intervention of Oridonin.(6)MDA-MB-231 and BT549 cells were treated with 3-methyladenine(3MA)and Oridonin or not,and the changes of apoptosis level were detected,and the changes of expression levels of Bcl-2,LC3-II/I and p-ERK1/2 proteins were detected by Western blot experiment.(7)Cell proliferation and apoptosis were observed after combined with ERK1/2 pathway inhibitor(FR180204)and Oridonin.Results:(1)Oridonin could inhibit the viability of MDA-MB-231 and BT549 cells in vitro.CCK-8 results showed that Oridonin could significantly inhibit the in vitro viability of MDA-MB-231 and BT549 cells in a concentration-dependent manner compared with the control group after treatment with different concentrations of Oridonin for 24hours(P<0.05).(2)Ed U staining and plate colony formation assay found that the growth ability of tumor cells MDA-MB-231 and BT549 after treatment with Oridonin was significantly lower than that of the control(P<0.05).(3)Flow cytometry showed that Oridonin could induce the apoptosis of MDA-MB-231 and BT549 cells in vitro compared with the control group,and it was observed that higher concentration of Oridonin could induce the apoptosis of MDA-MB-231 and BT549 cells in a dose-dependent manner(P<0.05).(4)After transfection of autophagy double-labeled adenovirus,the number of red fluorescence(autophagy lysosome)in Oridonin treatment group was higher than that in control group(P<0.05).It was confirmed that ORI could promote the occurrence of autophagy lysosome.(5)Western blot results showed that Oridonin inhibited the protein levels of p62 and Bcl-2 in two TNBC cells(P<0.05),and increased the levels of Cleved-Caspase 3,Beclin1 and LC3-II(P<0.05),which confirmed that Oridonin had the effect of promoting apoptosis in TNBC cells.Autophagy inhibitor 3MA can reverse the apoptosis promoted by Oridonin(P<0.05),further confirming the correlation between Oridonin autophagy and apoptosis,and Oridonin can regulate and increase cell apoptosis by promoting autophagy.(6)In triple-negative breast cancer MDA-MB-231 and BT549 cells,we found that the phosphorylation level of ERK1/2(P44/42)decreased(P<0.05)under the action of Oridonin,and the expression of p-ERK1/2increased(P<0.05)after adding 3MA.We think that Oridonin may inhibit ERK1/2 pathway.(7)After the addition of ERK1/2 inhibitor FR180204,combined with Oridonin could synergistically inhibit proliferation and promote the increase of apoptosis rate of triple negative breast cancer cells compared with Oridonin alone(P<0.05).Conclusion: Through this experiment,we verified that Oridonin can inhibit the proliferation of TNBC cells and promote apoptosis and autophagy.The addition of autophagy inhibitor can reverse the apoptosis induced by Oridonin,indicating that Oridonin may promote apoptosis through autophagy.At the same time,it was found that the level of ERK1/2(P44/42)phosphorylation decreased under the action of Oridonin,which could be reversed by adding autophagy inhibitor.We think that Oridonin can regulate autophagy and promote apoptosis by inhibiting MAKP-ERK1/2 pathway.Oridonin combined with ERK1/2 inhibitor FR180204 can synergistically inhibit cell proliferation and further promote the increase of apoptosis.These results provide new insights into the anticancer effect of Oridonin.