Role And Mechanism Of Hedgehog Signaling Pathway In The Development And Progression Of Hepatocellular Carcinoma

[Background and Objective]Hepatocellular carcinoma(HCC)is one of the most common malignancies in ourcountry,and has the increased uptrend in incidence,recurring rate and death rate.Thehepatic resection remains the most effective treatment,but the prognosis of HCC isgenerally poor,due to the high post-operative recurrence and invasiveness of primary tumor.It is vital to explore new molecular markers for treatment strategies.Hedgehog(Hh)signaling pathway is a highly conserved system,which plays a crucial role in embryonictissue patterning,cell differentiation and proliferation.In vertebrate organisms,thesignaling pathway is initiated by the binding of ligands(Shh,Ihh,Dhh)to the membranousreceptor patched(Ptch)which in turn alleviates the suppression on smoothened(Smo),subsequently Smo triggers a series of intracellular events with resultant activation of thezinc-finger transcription effectors,glioma-associated oncogenes(Glil,Gli2,Gli3)transcription factor,which induces the expression of numerous target genes,such as Ptch-1,Hip,Gli1 and Wnt,that regulate proliferation,differentiation.Recent studies revealed thataberrantly persistent activation of Hh signaling pathway and overexpression of target geneslead to several malignant tumorigenesis,such as cancer of prostate,basal cell,pancreas,colon and stomach.However,the role of the pathway in pathogenesis of HCC is stillindistinct.[Aim]To investigate the effects of Hh signaling pathway on HCC and its possible mechanism,which not only furthered to detect the mechanisms of onset and developmentof HCC but provided the new scientific evidence of its molecular diagnosis and treatment.[Methods]1.Tissue microarray(TMA)contained HCC and corresponding tumor-adjacent livertissues were constructed.2.The expressions of Shh,Ihh,Ptch-1 and Gli-2 in HCC tissues and correspongdingadjacent-tumor liver tissues were detected by immunohistochemistry and the correlationbetween their expressions and clinicopathologic parameter was analyzed.3.The mRNA expressions of Hh signaling components in 5 hepatoma cell lines(L02,Hep3B,HepG2,SMMC-7721 and Huh-7)were detected by RT-PCR.4.After treated with different concentrations of KAAD-cyclopamine,a specificinhibitor of Hh signaling pathway,Methyl thiazolyl tetrazolium(MTT)assay was used toexamine the changes in the proliferation of SMMC-7721 cells,BrdU incorporation assaywas applied to measure DNA synthesis rates and flow cytometry(FCM)was employed toanalyze the cells cycle.5.The morphological changes of cells were observed by inverted microscope,theAnnexin V/PI and TUNEL(terminal deoxynucleotidyl transferase-mediated nick endlabeling of DNA fragmentation sites)were applied to detecte the apoptosis rate induced byKAAD-cyclopamine.The change of Caspase-3 relative activity was analyzed bycolorimetric assay.6.Wound healing assay,Transwell assay and Modified Boyden chamber techniquewere performed to determine the cells adhesion,motility and invasiveness.7.The expressions of the regulative genes,such as Cyclin D1,p21,Caspase-3,bcl-2,E-cadherin were detected by Western blot.8.Statistical analysis:Date were expressed as mean±SD.Statistical correlation ofdata was cheched for significance by the ANOVA and paired Student's t test.P＜0.05 wasconsidered significant. [Results]1.The TMA was constructed involving 88 spots,totally 44 HCC tissues andcorresponding tumor-adjacent tissues ranked lattice one-by-one.Those spots wereconfirmed that based on correct diagnosis in line with the original organization bymicroscope,and the structure of lesions were well-preserved.2.Hh signaling pathway activity enhanced significantly in HCC compared withcorresponding tumor-adjacent liver tissues.The expressions of Shh and Ptch-1 in HCCtissues were higher than the corresponding tumor-adjacent liver tissues(P＜0.05),but nocorrelation between expressions and clinicopathologic factors was found,including age,histological typing,tumor size,metastasize and HBV infection(P＞0.05).Neithersignificance different expression of Ihh between HCC and tumor-adjacent tissues,norrelationship between its expression and clinicopathologic factors was found.(P＞0.05).Inaddition,positive expression of Gli2 was remarkably stronger in HCC than tumor-adjacentliver tissues(P＜0.01)and overexpression of Gli2 was significantly associated with HCChistologic differentiation and portal venous invasion(P＜0.05).3.All Hh pathway components were expressed in 5 HCC cell lines to different extent.Expression of Shh,Ptch,Smo and Gli2 mRNAs was robustly observed in hepatoma celllines,with predominance in SMMC-7721,but weaker expression in normal liver cell lines(L02)was noted,especially Gli2 was significantly over expressed in SMMC-7721 butalmost undetectable in L02.The enhanced expression of Gli1 was only observed in Hep3Band SMMC-7721.However,Ihh and Gli3 were expressed at similarly low levels in all celllines.4.Blockade of Hh signaling pathway in SMMC-7721 cells,which pathway wassignificantly activated,by using KAAD-cyclopamine,a specific antagonist of signaling.The antagonist inhibited the cell proliferation markedly.The inhibitory effect enhancedwith increasing concentration and action time of KAAD-cyclopamine,in a dose-andtime-dependent pattern.The FCM analysis showed that KAAD-cyclopamine deduced the synthesis of DNA in SMMC-7721 cells,and induced cell cycle G1/G0 phase arrest(P＜0.05).Additionally,up-regulation of p21 and down-regulation of cyclin D1 protein expressionswere detected by Western blot.5.Blockade of the Hh pathway reduced SMMC-7721 cells survival ability andincreased cells apoptosis.The treatment induced dramatic morphologic changes of cells,thecells transformed from a flat,elongate and adherent growth status into a rounded sharps,subsequently lost contact with neighboring cells and finally floated into medium.thetreated cells apoptosis rates increased significantly measureb by Annexin v/PI and TUNELassay(P＜0.01).Proapoptotic protein Caspase-3 expression down-regulated andanti-apoptotic gene bcl-2 expression up-regulated.6.The motility and invasive capacity of SMMC-7721 cells were greatly suppressed byKAAD-cyclopamine.Wound assay revealed significant reductions in wound closure by＞65% for treated cells and significantly descend in mean velocities compared with controlcells(P＜0.01).Transwell Invasion assay displayed that SMMC-7721 exhibited the stronginvasive potential to penetrate the basement membrane components Matrigel,but to asignificantly lesser extent after application of KAAD-cyclopamine(P＜0.01).Accordantly,the expressions of E-cadherin was up-regulated by KAAD-cyclopamine.[Conclusion]Overexpression of Hh signaling pathway in HCC compared with correspondingadjacent-tumor liver tissues,and the ectopic activation of Hh pathway was associated withhistologic differentiation and portal venous invasion of HCC.All of HCC cell linesexpressed Hh pathway to different extent,with predominance in SMMC-7721,which wereregarded as poorly differentiated.Blockade Hh pathway by KAAD-cyclopaminesignificantly inhibited the DNA synthesis and resultantly inhibited proliferation inSMMC-7721 cells.Moreover,blackade Hh signaling pathway induced apoptosis of andKAAD-cyclopamine attenuated invasiveness and motility of SMMC-7721 cells remarkably.The possibely machenism are Hh signaling pathway regulate the expression of genes, including CyclinD 1,p21,Caspase3,bcl-2 and E-cadherin.Taken together,Hh signaling pathway plays an important role in onset anddevelopment of human hepatocellular carcinama.The blockade of singaling inducedbroadly biological effects including cell growth and invasion inhibition,apoptosisenhancement in HCC.A deeper and full understanding of the function of Hh signalingpathway may provide signifieant insights into the pathogenesis,development andprognostic of hepatocellular carcinoma and a novel applicable strategy for diagnosis andtherapy.