The Effect And Molecular Mechanism Of Pancreatic Stellate Cell On Chemoresistance In Pancreatic Cancer

Objective To explore the role and potential molecular mechanism of pancreatic stellate cell(PSC)and the HGF/c-Met biological axis on gemcitabine(GEM)chemoresistance of pancreatic cancer(PC),and to provide a promising breakthrough for targeted therapeutic strategy of tumor microenvironment in pancreatic cancer.Methods PSC-conditioned medium(PSC-CM)and PCC-conditioned medium(PCC-CM)were collected and preserved.SW1990 and PANC-1 cells were treatment with PSC-CM and chemotherapy drugs GEM.Then we observed the chemoresistant phenotypes change of PCCs.CCK-8 assay was used to analyze the changes of the half maximal inhibitory concentration(IC50 values)and the growth inhibitions of PCCs.Annexin V-FITC/ PI double staining flow cytometry was used to detect cells apoptosis rate of PCCs induced by GEM.Western Blot was used to analyse the activated level of apoptosis protein caspase 8 and caspase 3.Real-time quantitative PCR(qRT-PCR),Western Blot and cell immunofluorescence assay were used to assess the changes of epithelial-to-mesenchymal transition(EMT)molecular markers E-Cadherin and Vimentin.Hepatocyte growth factor(HGF)expression in PSC and PCCs was determined via using enzyme linked immunosorbent assay(ELISA),qRT-PCR and Western Blot.Both PCCs were cultured with PSC-CM,and then detected the level of HGF in PSC-CM at different time points.PSC and two kinds of PCCs were co-cultured via Transwell technique,and then detected the changes of HGF level in culture medium by ELISA.SW1990 and PANC-1 cells were treatment with exogenous recombinant human HGF protein(rhHGF).qRT-PCR and Western Blot were used to re-examine the changes of E-Cadherin and Vimentin expression.ELISA was used to analyse the effect of HGF neutralizing antibody AMG102 on HGF level in PSC-CM.qRT-PCR and Western Blot were used to analyze the change of activated c-Met(p-c-Met)level of PCCs upon PSC-CM and rhHGF exposure.The application of AMG102 and c-Met receptor inhibitor PHA 665752 could block the HGF/c-Met axis,the changes of p-c-Met level were detected by Western Blot,and observed the effect of both on GEM chemoresistance and EMT molecular markers expression.The effect of PSC-CM and rhHGF on PI3K/Akt signaling pathway of PCCswas determined by using Western Blot.Specific inhibitor LY294002 application blocked the PI3K/Akt signaling,the changes of phosphorylated Akt(p-Akt)level were determined by Western Blot,and re-examined the effect of PSC-CM and LY294002 on EMT and chemoresistance.Results 1.PSC enhances GEM chemoresistance of PCCs.Compared with the PCCs in normal culture medium,the CCK-8 results showed that the IC50 values of SW1990 and PANC-1 cells cutured in PSC-CM were significantly increased,the difference was statistically significant(P<0.01),the growth inhibitions of both PCCs in response to GEM were significantly decreased,the difference was statistically significant(P<0.01).Flow cytometric analysis results showed that PSC-CM significantly decreased the total apoptosis rate of PCCs induced by GEM,the difference was statistically significant(P<0.01).Western Blot revealed that GEM-induced intracellular caspase 8 and caspase 3activation products were profoundly decreased when they collaborated with PSC-CM.2.PSC promotes phenotypic changes of PCCs consistent with EMT.qRT-PCR,Western Blot and immunofluorescence staining results revealed that PSC-CM could down-regulatethe expression of E-Cadherin and up-regulate Vimentin expression in PCCs,the difference was statistically significant(P<0.01).3.PSC highly expressed HGF.qRT-PCR and Western Blot results showed that PSC highly expressed HGF,and both PCCs highly expressed c-Met,the difference was statistically significant(P<0.01).ELISA results also showed that the level of HGF in PSC-CM was higher than that in PCC-CM,the difference was statistically significant(P<0.01).The presence of PCCs decreased HGF levels in PSC-CM in a time-dependent manner.And AMG102 could effectively decrease the level of HGF in PSC-CM,the difference was statistically significant(P<0.01).Furthermore,PCCs could promote PSC secret HGF,the difference was statistically significant(P<0.01).Western Blot results showed that both PSC-CM and rhHGF could promote the p-c-Met level of PCCs.4.HGF/c-Met axis is the key pathway of EMT in PCCs.qRT-PCR and Western Blot showed that rhHGF promoted EMT of PCCs,the difference was statistically significant(P<0.01).Western Blot results showed that AMG102 and PHA 665752 not only effectively antagonized increased p-c-Met expression induced by PSC-CM,but also attenuated EMT induced by activated HGF/c-Met axis.5.c-Met activation contributed to GEM chemoresistance in PC.Compared with the PCCs cutured in PSC-CM,the IC50 values of PCCs cultured in PSC-CM including PHA 665752 were significantly decreased,the difference was statistically significant(P<0.01),and the growth inhibitions were significantly increased,the difference was statistically significant(P<0.01).Flow cytometric analysis results showed that the total apoptosis rate of PCCs cultured in PSC-CM including PHA 665752 was significantly increased,the difference was statistically significant(P<0.01).Western Blot also showed that the level of GEM-induced intracellular cleaved caspase 8 and cleaved caspase 3 products were profoundly increased,compared with the level of PCCs cutured in PSC-CM.6.HGF/c-Met axis activation induces EMT and enhances GEM resistance via a PI3K/Akt-dependent pathway.Western Blot showed that both PSC-CM and rhHGF promoted Akt phosphorylation of PCCs,and LY294002 could attenuate above Akt phosphorylation.It also could help PHA 665752 inhibit EMT induced by PSC-CM.Moreover,CCK-8 results showed that LY294002 could reverse the increased IC50 values and the decreased growth inhibitions induced by PSC-CM,the difference was statistically significant(P<0.01).Conclusion PSC highly expressed HGF,combined with the c-Met receptor in pancreatic cancer cells by paracrine mechanisms,then activated the HGF/c-Met axis and led to PI3K/Akt signaling pathway activation in tumor cells,inducing EMT of PCCs and inhibiting cancer cell apoptosis via decreasing the activated level of intracellular caspase,which may be contributed to the enhanced GEM chemoresistance of PCCs.Therefore,the targeted study on the interaction between PSC and PCCs may inspire a new direction for overcoming chemoresistance of PC.Targeted therapy of the HGF/c-Met axis and its downstream signaling pathway in combination with GEM may improve the therapeutic efficacy of PC patients.