Based On The "TGFβ/smads" Pathway Of Pancreatic Stellate Cells, The Mechanism Of Cinobufacini's Inhibition Of Pancreatic Ductal Adenocarcinoma Was Explored

ObjectivesIt has been attested by series of clinical and experimental facts that special tumor microenvironment(matrix)of pancreatic cancer plays a pivotal role during the initiation and progression of the disease itself.Obviously,pancreatic stellate cells(PSCs),regarded as a promising research point,contribute a lot for the characterization of the microenvironment and can promote proliferation,migration,chemotherapy and radiotherapy resistance of pancreatic cancer cells.Cinobufacini,treating kinds of malignances,is a commonly used Chinese Medicine(CM)injection and,our previous clinical research has also proved that the injection may slow down pancreatic cancer progression and alleviate sufferings by patients.Based on analysis of possible ways of its antitumor effect,we make this hypothesis:cinofufacini could change the special microenvironment of pancreatic cancer by interfering in"TGFβ/smads"pathways in PSCs,such that excerts antitumor effects.So we are going to introduce cinofufacini into a indirect co-culcure system of pancreatic cancer cells and PSCs and observe changes of cytokine secretion,migration and invasion ability,and transcription and translation of collgen I and of pivotal molecules(p-smad2/3,etc.)in"TGFβ/smads" pathways.With all these,we hope to elucidate the specific influences of cinifufacini to panceatic cancer microenvironment on a cellular and molecular level,set a scientific fundamentation for CM pancreatic cancer treatment.MethodsPSCs and pancreatic cancer cell line SW1990 will be cultured,different concentrations of cinobufacini and gemcitabine will be added and kept for a period of time and then supernatants reaped after centrifugation.Indirect co-culture methods will then be introduced into the research utilizing supernatants as a culture media,with which is used to observe effects on two cell lines,respectively.Experiments includs cell proliferation assay(CCK-8 method),ELISA testing cytokines PDGF and TGFβ secreted by PSCs and SW1990,transwell assay for migration and invasion ability,western blot testing translation changes of smad2,smad3,p-smad2/3,smad7,collgen I and qRT-PCR for transcription changes of smad2,smad3,smad7,collgen I.Results1.The half maximal inhibitory concentration(IC50)of cinobufacini to pancreatic cancer cell line SW1990 is 0.18 mg/ml with its inhibitory effect going downward as concentration declines.And IC 50 of cinobufacini to pancreatic stellate cells is 25.62mg/ml,with its effects to PSC presented as a bidirectional one,i.e,cinobufacini promote proliferation at a low concentration(<20mg/ml),while repress proliferation at a high concentration(>20mg/ml).Meanwhile,IC50 of gemcitabine to SW1990 is 7.43μg/ml and to PSC is 1.67μg/ml,both of the effects are demonstrated concentration-dependent.2.PSC-SN3,PSC-SN2,PSC-SN1 and PSC-SNO represent serial numbers of supernatants of PSC culture media dealt with different concentrations of cinobufacini.The promoting effect on proliferation of the supernatants to SW1990 goes down as concentrations in the culture media of cinobufacini rise up.These effects are inverted after fetal bovine serum(FBS)is added to the supernatants,i.e,the supernatants containing 1%FBS enhanced proliferation of SW1990 as concentrations in the culture media of cinobufacini rise up,but the intensity is weaker than the corresponding group.3.With the above-mentioned methods,supernatants are named PCC-SN3,PCC-SN2,PCC-SN1 and PCC-SNO,representing different concentrations of cinobufacini in the culture media of SW1990(0.2μg/ml,0.04μg/ml,0.008μg/ml,Oμg/ml).No statistical differences are seen across the group about the effects of these supernatants on PSC4.No statistical difference is seen on the amount of TGFβ and PDGF secretion by pancreatic stellate cells and pancreatic cancer cells after treated with different concentrations of cinobufacini in ELISA.It is possible of cinobufacini to induce pancreatic stellate cells to secrete other different soluble cytokines in the culture supernatants,instead of TGFβand PDGF,to promote proliferation of pancreatic cancer cells.5.In the transwell experiment,pure pancreatic stellate cell culture supernatants could magnificently enhance the invasion and migration ability of pancreatic cancer cell,and after treated with cinobufacini,this kind of effect of the supernatants is gradually downregulated,with corresponding number of the transmembrane pancreatic cancer cell diminished in a dose-dependant manner.Cinibufacini in low concentration is not efficient to block the chemotaxsis of the pancreatic stellate cell secretion in the culture supernatants.In contrast,a medium to high concentration of cinobufacini could predominantly inhibit its invasion and migration of pancreatic cancer cell in the culture supnatants,rendering it weaker than the NORMAL group6.In the qRT-PCR experiment,the effects of cinobufacini on the transcription activity of smad2,smad3,smad7 exhibit as ascending of the generation of respective mRNAs with the elevating of cinobufacini concentration,while the transcription activity of Collagen I instead descends as the concentration of cinobufacini develops.Both of them function in a concentration-dependant manner.As for smad2/3,the improvement of its transcription activity may be related to a feedback regulation of the TGFβ/smads pathway.7.In the Western blot assay,cinofufacini up-regulated the expression of repressive molecules smad7,down-regulated phosphorylation levels of p-smad2/3,whereby repress the expression of collgen I,which is closely relative to fibrosis.As for smad2 and smad3,the levels of which descend at high intervention concentrations of cinobufacini(30mg/ml),while statistical differences are not seen amongst the other groups at a lower concentration.ConclusionsCinobufacini could,probably by intervening pancreatic stellate cells in the tumor microenvironment,slow down the progression of pancreatic cancer.The effects of cinobufacini are embodied in two aspects.Cinobufacini could regulate the secretion of soluble cytokines of pancreatic stellate cell,and thus impede the proliferation,invasion and migration of pancreatic cancer cell,but these effects have no relation to TGFβ and PDGF.Cinobufacini could inhibit the expression of collgen I by up-regulating repressive molecule smad7 and down-regulating phosphorylation of p-smad2/3 in the TGFβ/smads pathway,which is of pivotal importance in fibrosis of pancreas,thus brings transformation to the microenvironment of pancreatic cancer.These two mechanisms might work in synergy to slow down the progression of pancreatic cancer.