Curcumin Inhibits HGF-Induced Invasion,Migration And EMT Via Repression C-Met/PI3K/AKT/mTOR Signaling Pathways In Lung Adenocarcinoma

It is well known that lung cancer is the leading cause of cancer-related deaths in the world.Tumor metastasis and drug resistance bring great challenge to the treatment of lung cancer,with a 5-year survival rate of<17%.Epithelial to mesenchymal transition(EMT)plays a crucial role in the malignant progression of lung cancer.It has been confirmed that Hepatocyte Growth Factor(HGF)participates in the process of EMT.HGF/c-Met signal pathway is closely associated with the occurrence,development,metastasis,prognosis of lung cancer,and is a highly potential target for lung cancer targeted therapies.Curcumin,the main active polyphenol extracted from the rhizomes of turmeric(curcuma longa),has multiple functions,including anti-inflammatory,antioxidant,anti-fibrotic and antitumor effects.It has been recently reported that curcumin could suppress cancer cell proliferation,invasion,metastasis and angiogenesis via multiple signaling pathways.Research about HGF induced EMT,especially in lung cancer,is rarely reported in the literature.Meanwhile,the role of curcumin in HGF-induced EMT and the underlying mechanisms remain largely unknown.We aimed to evaluate the potential mechanisms of curcumin in EMT stimulated by HGF(40ng/ml)in lung cancer in vitro.In the current study,we demonstrated curcumin inhibited HGF-induced cell migration and invasion,and significantly reversed EMT process,including up-regulation E-cadherin expression,down-regulation Vimentin expression.In the further study,we found curcumin repressed HGF-Induced the c-Met activation,along with repression the downstream signaling pathway AKT/mTOR phosphorylation.Meanwhile,we found that overexpression of c-Met promoted the process of EMT,which were both blocked by curcumin and SU11274,a specific small molecule inhibitor of c-Met.Meanwhile,both curcumin and SU11274 inhibited the downstream signaling pathway of c-Met activated by overexpression.We also found repression of c-Met/PI3K/AKT/mTOR pathway reversed HGF reduced migration,invasion and EMT in lung cancer cells,which was similar to the effect of curcumin.These findings suggest that curcumin inhibits HGF-induced EMT via repression c-Met mediated PI3K/AKT/mTOR signaling pathways in lung adenocarcinoma,and provides a new theoretical basis for the prevention and treatment of lung cancer.Part1 Study the effect of curcumin in HGF-induced invasion,migration and EMT in lung adenocarcinomaObjective To investigate the effect of curcumin in HGF-induced invasion,migration and EMT in lung adenocarcinoma.Method 1.Human lung adenocarcinoma cells A549 and PC9 were respectively divided into different groups:control group,HGF(40ng/ml)group,and HGF(40ng/ml)combined with curcumin(10,20,30μM)groups.After 48h of treatment,the difference of the cell morphology,proliferation rates,and migration ability were evaluated by light microscope,MTT method,Wound healing assay and Transwell assay.Western blot was performed to detect the expression of E-cadherin and Vimentin level.Result 1.HGF induced A549 and PC9 cells to present the typical morphological changes of EMT.It could be seen that cells acquired a spindle-shaped and fibroblast-like phenotype,and lost cell-to-cell contact.After HGF stimulation,the expression of E-cadherin was dramatically decreased and the expression of Vimentin was markedly increased in A549 and PC9 cells.Meanwhile,the cell proliferation,migration and invasion were significantly increased.2.Compared with HGF-treated group,curcumin dose-dependently blocked the HGF effects.Western blot showed the expression of E-cadherin was up-regulated and the expression of Vimentin was down-regulated by curcumin treatmentConclusion Curcumin inhibited HGF-induced invasion,migration and EMT in lung adenocarcinoma.Part2 Study the effect of curcumin in HGF/c-Met and its downstream PI3K/AKT/mTOR signaling pathways in lung adenocarcinomaObjective To investigate the role of curcumin in modulating HGF/c-Met and its downstream PI3K/AKT/mTOR signal pathways in lung adenocarcinoma cells and explore the underlying mechanisms.Method 1.Western blot was performed to investigate the expressions of c-Met phosphorylation,and the downstream signaling pathways AKT/mTOR phosphorylation induced by HGF with or without curcumin at different concentrations.2.A549 and PC9 cells were transfected with c-Met overexpression vector(pEZ/M98/neo-c-Met,designated as EX-Met)or the control vector(pEZ/M98/neo-control,designated as EX-NC)via Lipofectamine 2000 complying with the manufacturer’s protocol.Then curcumin(20μM)and SU11274(5μM)were used to interfere with the effect of overexpression of EX-Met transfected cells.The difference of the cell morphology,and migration ability were evaluated by light microscope,wound healing assay and Transwell assay.Western blot was used to examine the expression of the EMT marker proteins level.Then effect of curcumin(20μM)and SU11274(5μM)in c-Met/PI3K/AKT/mTOR were detected by Western blot in A549 and PC9 cells with c-Met overexpression.4.We also observed the effect of specific small molecule c-Met inhibitor,selective PI3K inhibitor and selective mTOR inhibitor in HGF-induced invasion,migration and EMT.Result 1.Western blot revealed that c-Met,AKT and mTOR were phosphorylated at 15min after HGF(40ng/ml)treatment.Curcumin significantly inhibited the phosphorylation of c-Met,along with dose-dependently repression the downstream signaling pathway AKT,mTOR phosphorylation.2.Compared with negative control vector,A549 and PC9 cells with c-Met overexpression demonstrated the typical mesenchymal phenotype,and promoted the process of EMT,which was manifested as E-cadherin down-regulation and Vimentin up-regulation.Meanwhile,the cell migration and invasion were significantly enhanced.Both curcumin and SU11274 blocked the effect of c-Met overexpression.3.Both curcumin and SU11274 inhibited c-Met,AKT,mTOR phosphorylation in A549 and PC9 cells with c-Met overexpression.4.SU11274,LY294002,Rapamycin all significantly inhibited HGF-induced EMT,and partly inhibited the c-Met/PI3K/AKT/mTOR signaling pathways.Conclusion Curcumin inhibited HGF/c-Met and its downstream PI3K/AKT/mTOR signaling pathways in lung adenocarcinoma,which may be the mechanisms of curcumin modulation HGF-induced invasion,migration and EMT in lung adenocarcinoma.