氧化白藜芦醇通过PI3K/Akt信号通路抑制非小细胞肺癌上皮间充质转化的机制OA

Objective:To investigate the mechanisms by which oxyresveratrol(OXY)inhibits epithelial-mesenchymal transition(EMT)in non-small cell lung cancer(NSCLC)through the phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway.Methods:Cell counting kit-8(CCK-8)assays were used to determine the survival rates of A549 and H1299 cells treated with different concentrations of OXY,and appropriate concentrations(0,30,60,90 μmol·L-1)were selected.The effects of OXY on the proliferation of A549 and H1299 cells were evaluated using 5-ethynyl-2'-deoxyuridine(EdU)assays and colony formation assays.Wound healing assays and Transwell invasion assays were performed to assess the effects of OXY on cell migration and invasion.Western blot(WB)was used to detect the expression levels of Snail,E-cadherin,N-cadherin,and Vimentin in A549 and H1299 cells.Network pharmacology and molecular docking were applied to predict the mechanism of action of OXY,and WB was used to evaluate the effects of OXY on proteins in the PI3K/Akt signaling pathway.Rescue experiments were conducted using the PI3K/Akt signaling pathway agonist 740Y-P.Under activation of the PI3K/Akt pathway,the effect of OXY on proliferation,migration,and invasion phenotypes,as well as on the expression levels of PI3K/Akt pathway-related proteins and EMT markers(Snail,E-cadherin,N-cadherin,and Vimentin),were examined.Results:In the forward experiments,CCK-8 assay results showed that,compared with the control group,the survival rates of NSCLC cells in the OXY-treated groups(20-120 μmol·L-1)were significantly decreased(P＜0.05).The half-maximal inhibitory concentration(IC50)values of A549 and H1299 cells after 48 h of OXY treatment were 113.6 μmol·L-1 and 92.53 μmol·L-1,respectively.Therefore,concentrations of 0,30,60,90 μmol·L-1 were selected as the gradient for subsequent phenotypic and mechanistic studies.Compared with the control group,the proliferation rate,colony number,migration rate,and invasion number of NSCLC cells in the OXY groups(30,60,and 90 μmol·L-1)were significantly decreased(P＜0.01,P＜0.05).WB results showed that,compared with the control group,the protein expression levels of Snail,N-cadherin,and Vimentin in NSCLC cells of the OXY groups were significantly decreased(P＜0.05),whereas E-cadherin expression was significantly increased(P＜0.01).Network pharmacology and molecular docking results indicated that OXY could act on the PI3K/Akt signaling pathway and exhibited good binding affinity with PI3K and Akt proteins.Further WB results showed that,compared with the control group,there were no statistically significant differences in the expression levels of PI3K and Akt proteins in NSCLC cells of the OXY groups,whereas the expression levels of phosphorylated PI3K(p-PI3K)and phosphorylated Akt(p-Akt)were significantly decreased(P＜0.05).In the rescue experiments,compared with the control group,the proliferation rate,colony number,migration rate,and invasion number of NSCLC cells in the 740Y-P group(15 μmol·L-1)were significantly increased(P＜0.01).Compared with the control+OXY group(90 μmol·L-1),these indices in the 740Y-P+OXY group(15 μmol·L-1+90 μmol·L-1)were also significantly increased(P＜0.01).WB results showed that,compared with the control group,there were no statistically significant differences in the expression levels of PI3K and Akt proteins in the 740Y-P group.However,the expression levels of p-PI3K,p-Akt,Snail,N-cadherin,and Vimentin were significantly increased(P＜0.05),while E-cadherin expression was significantly decreased(P＜0.01).Compared with the control+OXY group,there were no statistically significant differences in PI3K and Akt protein expression in the 740Y-P+OXY group.However,the expression levels of p-PI3K,p-Akt,Snail,N-cadherin,and Vimentin were significantly increased(P＜0.05),while E-cadherin expression was significantly decreased(P＜0.05).Conclusion:OXY inhibits the PI3K/Akt signaling pathway and suppresses the EMT process,thereby exerting anti-metastatic effects in NSCLC.