The Role Of Chemokine SDF-1α In PSC-induced Chemoresistance Of Pancreatic Cancer

Although the cellular and molecular biology of pancreatic ductal adenocarcinoma (PDAC) have been extensively studied for more than several decades,but the treatment of PDAC remains to be an unsolved problem andexhibits the poorest prognosis among all cancers.One of the major reasons for poor prognosis ofpancreatic cancer is itshighly resistance to currently available chemotherapeutic agentsincluding gemcitabine (GEM) which is considered as the most effective first-line drugfor the treatment of advanced pancreatic cancer. BothGEM alone and GEM-based combination chemotherapy are not likely to achieve great success andmaybe accompanied by much higher toxicity according to clinical trials. Therefore, it is necessary to develop new therapeutic strategies.Targeted therapy in combination with conventional chemotherapy may be one ofeffective and feasible measures. For pancreatic cancer, the development of targeted drugs in combination with GEM is the focus of current research hotspot.The traditional researchesof pancreatic cancer arefocused on tumor cell itself, ignoring the effect of the tumor microenvironment, which may explain the poor prognosisof pancreatic cancer patients with currenttherapeutic strategies. Tumor microenvironment is a complex cellular ecosystem surrounding the tumor, which is closely related to the tumor growth, invasion, metastasis, and drug resistance.Pancreatic stellate cells (PSCs) are one of the most important stromal cells in the microenvironment of PDAC.In normal pancreatic tissues, PSCs are ofquiescent phonotype,while PSCs are activated and transform into Pa-PSCs (pancreatic cancer associated pancreatic stellate cells,Pa-PSCs) in PDAC tissues.The activatedPSCscan generate numerous soluble factors and extracellular matrix, resulting in the formation and proliferation of of fibrous connective tissue (desmoplasia), which is one of the most striking histological featureof PDAC.The interaction between PSCs and pancreatic cancer cells is a hot area of research in recent years, which open up a new research pattern of molecular biology of pancreatic cancer.In the present study, we first explored the differentially expressedcytokines betweenactivatedPSCs and quiescentPSCsby microarray. ChemokineSDF-lawas found to behighly expressed inactivatedPSCsand verified by traditional experimentalmethods.We further investigatedthe roleof SDF-lainPSCs-induced GEM chemoresistance ofpancreatic cancer cells.The following aspects were studied:Ⅰ. Screening the differentially expressed genes betweenactivatedPSCs and quiescentPSCs1.Activated Pa-PSCs were cultured using the outgrowth method and identified byimmunofluorescence staining for a-SMA and Vimentin.2.QuiescentPSCsinduced by all-trans retinoic acid (ATRA) were validated according to cell morphology and oil red Oand a-SMA staining.3.Differentially expressed genesbetween activated PSCs and quiescent PSCs were detected by the Human Transcriptome Array2.0chip.4.Functional annotation and GO analysis for differentially expressed genes were performedusing bioinformatic databases.5.The differential expression of SDF-la between activated PSCs and quiescent PSCs was verified by traditional experimentalmethods.Ⅱ. SDF-lacould mediate PSCs-induced GEM-chemoresistance of pancreatic cancer in Panc-1cells1.ExploringSDF-la and CXCR4expression in four primary cultured Pa-PSCsandfourpancreatic cancer cell lines.2.MTT assayconfirmed that GEM toxicity inPanc-1cells was reduced by PSC-CM.3.Hoechst staining and Annexin V-FITC/PI verified that PSC-CM SDF-lamediated the inhibition of GEM-induced apoptosis by PSC.Ⅲ. SDF-la could inhibitGEM-induced apoptosisvia the upregulation of IL-6expression in Panc-1cells1.Recombinant protein SDF-laincreased IL-6and IL-8mRNA levels in Panc-1cells and IL-61evel was elevated more significantly.2.CXCR4antagonist AMD3100could inhibit SDF-1α-induced IL-6protein and mRNA upregulation in Panc-1cells.3.Hoechst staining and Annexin V-FITC/PIshowed that the proportion of apoptotic cells in GEM+SDF-1α group was significantly decreased, compared with those in GEM group and GEM+SDF-1α+IL-6Ab group.Ⅳ.SDF-la stimulated IL-6expressionin Panc-1cellsthrough bothFAK-AKT andERKl/2signaling pathways1.Western blot showed that recombinant protein SDF-lapromoted FAK, AKT and ERK1/2phosphorylation. 2.FAK inhibitor PF573228reducedSDF-la-induced FAK and AKT phosphorylation,but didnot affect ERK1/2phosphorylation.3.Both FAK inhibitor PF573228and ERK1/2inhibitor PD98059couldreduce SDF-la-induced IL-6expression.Conclusion:In conclusion, this study suggested thatactivated PSCscouldpromote chemoresistance in pancreatic cancer cells. The mechanism included that:Pa-PSCs highly expressed SDF-la, activated theSDF-la/CXCR4axis by paracrine mechanisms and then led to FAK-AKT and ERK1/2signaling pathwaysactivationin Panc-1cells,resulting in IL-6upregulation.Finally, IL-6autocrine loopmediatedGEMchemoresistance inPanc-1cells.These results suggest thatthe study on theinteraction between PSCs and pancreatic cancer cell may bring breakthrough in pancreatic cancertherapeuticresearch. SDF-1α/CXCR4biological axis and its downstream signaling pathways in pancreatic cancer cells are important potential therapeutic targets for pancreatic cancer.