Relationship Between TCM Syndrome And Pathogenesis In Pancreatic Cancer And The Potential Anti-cancer Mechanism Of Qingyihuaji Formular

ObjectiveTo study the relationship between traditional Chinese medicine(TCM) syndrome and TCM pathogenesis in pancreatic cancer.And to explore the potential mechanism of Qingyihuaji formula(QYHJ) in the treatment of pancreatic cancer.MethodsPartⅠ:Three subcutaneous transplanted panc02 tumor models with different TCM syndrome types,including spleen deficiency,damp-heat and blood stasis,in the nude mice were established.The effect of each TCM syndrome on subcutaneous transplanted tumor were evaluated and the response of anti-pancreatic cancer TCM formular named QYHJ on subcutaneous transplanted tumor with defferent TCM syndrome were also studied.PartⅡ:The expression changes of SKI mRNA and protein in pancreatic carcinoma subcutaneous tumor treated with QYHJ were detected by real-time PCR and western blot.Then the expression of SKI and related TGF-βsignaling pathway components in four human pancreatic cancer cell lines,including SW1990,BxPC3, PANC-1and PC3 were also analysed by semi-quantitative real-time RT-PCR. SW1990 and BxPC3 cell lines were chosen for the following RNAi study.Three different small interfering RNA for human SKI(siRNA1 siRNA2 and siRNA3) and one negative siRNA were designed.Then,the primer pairs were annealed and subcloned into the BamH I(GGATCC) and EcoRI(GAATTC) sites of pSIH1-H1-copGFP shRNA Vector.The recombinant vectors were identified by PCR and automated sequencing analysis and then transfected into 293T cells by Lipofectamine^TM 2000 Transfection Reagent.Real-time PCR were used for selection of the most effective SKI silencing sequence.Next,according to the instruction of SBI Lentivector Expression System,a lentiviral expression construct and pPACK packaging plasmid mix were co-transfected into 293T cells.Then viral particles were collected and the titer were determined.The recombinant lentivector were used for infecting target cells(SW1990 and BxPC3).The expression of SKI after SKI RNAi or negative RNAi were identified by real-time PCR and western blotting.Cell growth,cell cycle distribution,migration and invasion in vitro were detected respectively by WST,FCM with PI staining,wound healing assay and in vitro invasion assay.To examine the effects of SKI RNAi on tumor growth,we performed the in vivo assay using an subcutaneous transplanted tumor model in the nude mice. in vivo metastasis model were also established through tail vein injection with tumor cells and the effect of SKI expression status on metastasis were also evaluated.Next, molecular basis underlying the involvement of SKI in the phenotypes of human pancreatic cancer cells were explored.Firstly,the induction of specific TGFβ-responsive targeted genes were detected by RT-PCR and real-time PCR.Then the expression of TGFβsignaling pathway components and proliferative-,invasive-and metastatic-related genes after SKI RNAi were also examined by RT-PCR and western blot.Finally,using a selective TβR-I kinase inhibitor,SD-431542,we evaluate the effect of SKI RNAi on in vitro proliferative and invasive potential after blocking TGF-βsignaling.After determination of the function and its potential molecular mechanism,we studied the effect of QYHJ on subcutaneous transplanted tumor with defferent SKI expression status.ResultsPartⅠ:The incidence of tumor with different TCM syndrome type were all 100%,however,subcutaneous transplanted tumor grew slowly than that with no TCM syndrome,especial in damp-heat group(compared with control group,the tumor weight inhibitory rate decreased 31.7%).The volume and weight of tumor with spleen deficiency and damp-heat syndrome decreased after administration of QYHJ.But this didn't happened in blood stasis group. PartⅡ:Expression of SKI mRNA and protein in SW1990 subcutaneous transplanted tumor after treatment of QYHJ decreased 39.6%and 41.3%of that in the untreated respectively.The levels of SKI mRNA in BxPC3,PANC-1 and PC3 were 0.9-,1.4- and 0.7-fold respectively in comparison to that in SW1990.The expresssion of SKI related TGF-βsignaling pathway components,including TβRⅡ, TβRⅠ,smad2,smad3,smad4 and smad7 were all detected in the four human pancreatic cancer cell lines,except for smad4 in BxPC3 and TβRⅡin PC3.Then SW1990 and BxPC3 pancreatic cancer cell lines were chosen for further SKI RNAi study.The expression of SKI mRNA in pSIH-siRNA1,pSIH-siRNA2 and pSIH-siRNA3 transfectants were 55.0%,54.0%and 45.0%respectively of that in pSIH-negative transfectant(P＜0.01).The pSIH-siRNA3 and pSIH-negative were selected for the subsequent studies.The recombinant lentivector were used for infecting SW1990 and BxPC3 cells.Then two stable transfection cells (SW1990/SKI RNAi and BxPC3/SKI RNAi) were created and SW1990/con RNAi and BxPC3/con RNAi were used as control.The expression of SKI mRNA and protein in SW1990/con RNAi and SW1990/SKI RNAi were 105%,123%and 46%, 30%respectively of that in parental cells.Similarly,the expression of SKI mRNA and protein in BxPC3/con RNAi and BxPC3/SKI RNAi were 94%,90%and 38%, 16%respectively of that in parental cells.SKI RNAi sensesized the response of SW1990 and BxPC3 cell to TGF-β1 and reduced the cell growth in vitro induced by TGF-β1 with different concentration.Flow cytometric(FCM) analyses revealed that the percentage of cells in G1-phase was dramatically increased from 40.4%to 62.9% in SW990 cell line and from 20.2%to 66.6%in BxPC3 cell line when SKI RNAi induced by TGF-β1.SKI RNAi transfectants were much higher migrative and invasive in comparison to parental or mock transfected cells.Tumors formed by SW1990/SKI RNAi and BxPC3/SKI RNAi cells were much smaller than those formed by parental and control vector-transfected cells at the end of the in vivo assays(P＜0.05).Tail vein injection of SKI RNAi BxPC3 into nude mice resulted in a significant increase of liver metastatic colonizations.Analysis of specific TGF-β-responsive target gene expression detected by real-time PCR showed that the expression of PAI-1 and CTGF mRNA were up-regulated dramatically and sustained for much longer time.Accompanied with PAI-1 and CTGF up-regulation,the expression of other TGF-β-responsive target genes also changed,with c-jun,JunB and p21 mRNA up-regulated and c-myc and CDC25A mRNA down-regulated.SKI RNAi had no influence on the expression of TGF-βsignaling pathway components, including TβRⅡ,TβRⅠ,smad2,smad3,smad4 and smad7,however,SKI RNAi resulted in a rapid and sustained increase in phosphorylated Smad3 in the two cells. The expression of proliferatory,invasive and metastatic related gene were also examined,with result of up-regulation of p21,MMP-2,MMP-9,VEGF and down-regulation of cyclin D1.Blocking TβR-I/ALK5 activity using a selective kinase inhibitor,SD-431542,strongly conteract the effect of G1-phase arrest and invasion promoting induced by SKI RNAi.After determination that down-regulation of SKI can result in decreased tumor growth,we examined the response of QYHJ on subcutaneous transplanted pancreatic cancer with different SKI expression status. The result showed that the tumor weight inhibitory rate of QYHJ on subcutaneous transplanted tumor formed by SW1990 or SW1990/con RNAi were 29.6%and 32.2%repectively,while it was 16.0%when the tumors were formed by SW1990/SKI RNAiconclusionTCM syndrome originating from tumor might be the relationship between tumor and TCM syndrome.The presence of TCM syndromes might be reaction of body to the existence of tumor.So the treatment of pancreatic cancer should aim at the essential TCM pathogenesis underlying the TCM syndrome.Down-regulation of SKI expression can inhibit the growth of pancreatic cancer both in vitro and in vivo. This effect is mainly achieved through increasing the level of phosphorylated smad3, enhancing the transcriptional activity induced by TGFβsignaling,thus restored the response of pancreatic cancer cell to the TGFβinduced cell cycle arrest.QYHJ inhibits growth of pancreatic cancer by down-regulating SKI expression.SKI is one of the potential targets of QYHJ in the treatment of pancreatic cancer Also,different expression status of SKI results in the difference of response of pancreatic cancer to QYHJ treatment.So SKI expression status detected before treatment might be used for predicting the response of pancreatic cancer to QYHJ treatment and screening for potential suitable population.