The Effect And Mechanism Of Ganetespib,an Inhibitor Of HSP90,on Epithelial Mesenchymal Transition Of A549 Cells

ObjectiveLung cancer is one of the most common cancer and the leading cause of human death in the world.Metastasis is a major cause of death in patients with lung cancer,and epithelial mesenchymal transition is an important factor in the initiation of tumor metastasis.Multiple studies have shown that the acquisition of tumor cell migration and invasion characteristics is mediated by the process of epithelial mesenchymal transition(EMT).The mesenchymal transition of tumor epithelial cells promotes cell adhesion and loss of cell polarity,which leads to the migration and invasion of tumor cells.Heat shock protein 90(HSP90)is an ATP-dependent molecular chaperone that is highly expressed in cancer cells and it works by facilitating folding,assembly,and stabilization of a variety of proteins.HSP90 plays a crucial role in maintaining the stability and homeostasis of tumor proteins,helping cancer cells survive in unsuitable environmental conditions and participating in the survival and progression of tumors.Studies have shown that HSP90 is involved in oncogenic signaling pathways,including epithelial-mesenchymal transition(EMT),and is a key molecule in tumor genesis,development,metastasis and drug resistance.Inhibition of HSP90 reduces many of its client proteins associated with the oncogenic signaling cascade,so the development of HSP90 inhibitors is a promising strategy for the treatment of lung cancer.Ganetespib is a second-generation HSP90 inhibitor that has a potent antitumor effect against a variety of cancers.The aim of this study was to investigate the effects of Ganetespib,an inhibitor of HSP90,on transforming growth factor-β(TGF-β)-induced EMT in A549 cells,and to explore the relevant signaling molecular mechanisms,so as to provide experimental basis for the research of targeting EMT and further research on inhibiting lung cancer cell metastasis.Methods1.EMT model construction of A549 cellsTGF-β at a concentration of 5μg/L was selected to stimulate A549 cells to construct the epithelial mesenchymal transition model.2.To determine the appropriate intervention time and concentration of GanetespibA549 cells were treated with different concentrations of Ganetespib,and the viability of A549 cells were detected by CCK-8 method,and the expression of Hsp90 in A549 cells was detected by Western blot.3.Detection of the intervention effect of inhibiting HSP90 on TGF-β-induced EMT of A549 cells and the related signal molecular changesExperimental design groups: the Control group,the DMSO group,the TGF-βgroup,the Ganetespib group,the TGF-β-Ganetespib group.(1)CCK-8 method was used to detect the effects of various treatment factors on A549 cell viability(2)Cell morphological changes were observed under a microscope(3)The EMT-related protein of E-cad,vimentin and related signal molecule NFκB(p65),HSP70 and MMP9 after inhibiting HSP90 were detected by Western blot.(4)The cell migration ability was detected by scratch assay,and the scratch widths at 0h and 24 h were recorded and analyzed.4.Changes of related indexes after inhibition of NFκB by BAY11-7082The experimental design was divided into three groups: blank control group,TGF-β group,Ganetespib group,TGF-β+Ganetespib group,BAY11-7082 group,and TGF-β+BAY11-7082 group.The expressions of E-cad,vimentin,HSP90,NFκB(p65),Hsp70 and TGF-β were detected by Western blot.5.Statistical AnalysisSPSS 27.0 statistical analysis was carried out on the experimental data,the results were shown mean ± standard deviation((?)±S).Two independent samples t-test or one way ANOVA were used to compare the mean of each group.LSD-t test was pairwise comparison between groups.Detection level α=0.05.Results1.The EMT model of A549 cells was constructedMorphologically,the cells in TGF-β treatment group were more elongated and the spacing between cells became larger,showing the characteristics of spindle-like stromal morphology.In contrast,A549 cells in the control group were closely arranged and showed a pebble-like epithelioid morphology.The results of western blot showed that compared with blank control group,the expression of E-cad was decreased and the expression of vimentin was increased in TGF-β group(P<0.05).In addition,the expression of Hsp90,NFκB(p65)and TGF-β in TGF-β group was increased,while the expression of Hsp70 was decreased(P<0.05).2.Intervention effect of Ganetespib on the EMT model of A549 cells(1)The changes of cell morphologyThe microscopic results showed that,compared with the control group,the cell morphology of TGF-β group became elongated and showed a long spindle shape,while the cell morphology of DMSO group did not change significantly.Compared with TGF-β group,the cells in TGF-β+Ganetespib group became shorter,rounded,and the spacing between cells became smaller,which was similar to the pebble-like cell morphology of control group.(2)The changes of EMT related proteinCompared with the control group,the expression of E-cad was decreased and the expression of vimentin was increased in TGF-β group(P<0.05).After the application of Ganetespib,an inhibitor of HSP90,the expression of E-cad was increased and the expression of vimentin was decreased in the Ganetespib group and TGF-β+Ganetespib group compared with the TGF-β group(P<0.05).(3)The changes of cell migration abilityThe scratch width of TGF-β group at 24 h was significantly narrower than that at0 h.At 24 h,compared with the control group,the scratch width in TGF-β group was significantly narrower(P<0.05).Compared with TGF-β group,the scratch width in both Ganetespib group and TGF-β+ Ganetespib group was wider(P<0.05).3.Participation and changes of NFκB(p65),HSP70,MMP9 and other related signal molecules during Ganetespib interventionCompared with control group,the expression of NFκB(p65)was increased,the expression of HSP70 was decreased,and the expression of MMP9 was increased in TGF-β group(P<0.05).After application of Ganetespib,compared with TGF-β group,the expression of NFκB(p65)was decreased,the expression of HSP70 was increased,and the expression of MMP9 protein was decreased in TGF-β+Ganetespib group(P<0.05).4.The changes of related indexes after inhibition of NFκB by BAY11-7082Compared with TGF-β group,the expression of HSP90 was decreased the expression of NFκB(p65)was decreased,the expression of HSP70 was increased,and the expression of TGF-β was decreased in Ganetespib group,TGF-β+Ganetespib group,BAY11-7082 group and TGF-β+ BAY11-7082 group(P<0.05).ConclusionTGF-β-induced EMT model of A549 cells was accompanied by increased expression of HSP90,NFκB and decreased expression of HSP70.The HSP90 inhibitor Ganetespib can inhibit TGF-β-induced EMT in A549 cells,and this process may be regulated by HSP90/NFκB signal.