Experimental study of etomidate inhibiting proliferation and inducing apoptosis in non-small cell lung cancer A549 cells through down-regulation of recombinant WW domain containing E3 ubiquitin protein ligase 2 expression
He Yuan
Duan Xiaofei
Dang Shajie
Wang Pei
Abstract：Objective To investigate the effects of etomidate(ETO)on the proliferation and apoptosis of non-small cell lung cancer(NSCLC)A549 cells and the mechanism in which recombinant WW domain containing E3 ubiquitin protein ligase 2(WWP2)plays a role.Methods The human normal lung epithelial cell line BESA-2B and the human NSCLC cell line A549 were treated with ETO at 0,1,2 and 3 mg/L,respectively,and cell viability was detected by the cell counting kit method;the A549 cells were randomly divided into the control group,the ETO(3 mg/L)group,the ETO+NC group and the ETO+WWP2-OE group,and the latter two groups were transfected with empty vector pcDNA3.1-NC or WWP2 overexpression vector pcDNA3.1-WWP2 after ETO treatment,respectively.Colony formation assay was performed to detect cell proliferation.TUNEL staining was performed to detect apoptosis.Quantitative real-time polymerase chain reaction was performed to detect mRNA expression of WWP2 in the cells of each group;and proteins directly interacting with ETO were selected from STITCH database.Western blot detected WWP2,proliferation,apoptosis and phosphatase and tensin homolog deleted on chromosome ten(PTEN)/phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)pathway related protein expression in each group of cells.Results ETO decreased A549 cell viability in a dose-dependent manner(F=147.923,P＜0.001),whereas it had no effect on the viability of normal lung epithelial BESA-2B cells(F=1.427,P=0.126).The number of colony formation was progressively reduced[0,1,2,3 mg/L ETO group were(898±38),(785±48),(635±36),(388±20)]and apoptosis was progressively increased[0,1,2,3 mg/L ETO group were(2.23±0.65)％,(7.63±0.35)％,(13.24±0.47)％,(18.93±0.36)％]in a dose-dependent manner after ETO treatment(0,1,2,3 mg/L)of A549 cells(F=218.732 and 352.786,both P＜0.001).The STITCH database predicted that ETO could interact with WWP2 and PTEN by upregulating WWP2 protein expression and downregulating PTEN protein expression.Compared with the control group,cells in the ETO group showed decreased mRNA and protein expression of WWP2,decreased number of colony formation,increased apoptosis rate,decreased proliferating cell nuclear antigen(PCNA),cell proliferation antigen Ki67,B-cell lymphoma-2(Bel-2)and PTEN protein expression,increased Bcl-2 associated X protein(Bax)and cysteinyl aspartate specific proteinase 3(Cleaved caspase-3)protein expression,and decreased phospho-PI3K(p-PI3K)/PI3K and phospho-Akt(p-Akt)/Akt ratios(all P＜0.01);compared with the ETO+NC group,cells in the ETO+WWP2-OE group showed elevated mRNA and protein expression of WWP2,increased number of colony formation,decreased apoptosis rate,increased expression of PCNA,Ki67,Bcl-2 and PTEN proteins,decreased expression of Bax and Cleaved caspase-3 proteins,and decreased p-PI3K/PI3K and p-Akt/Akt ratios were elevated(all P＜0.01).Conclusion ETO inhibits proliferation and promotes apoptosis in A549 cells,and its mechanism of action may be related to the regulation of WWP2 down-regulation and activation of PTEN/PI3 K/Akt pathway.
Keywords：Non-small cell lung cancerEtomidateRecombinant WW domain containing E3 ubiquitin protein ligase 2Apoptosis and phosphatase and tensin homolog deleted on chromosome ten/phosphatidylinositol-3-kinase/protein kinase B pathwayProliferationApoptosis
Publication Date：2024-11-08
Online Publishing Date：2026-09-14(First online date of this platform, not the publication date of the document)
Pages：5( 1640-1644 )