| Hepatocellular carcinoma (HCC) is one of the most commonest primary cancer with amultifaceted molecular pathogenesis, it has poor recovery rate and poor prognosis, andduration of HCC patient is only few months, HCC has become the5th most prevalentmalignancy worldwide and the third leading cause of cancer-related death, mostimportantly, the incidence of HCC is increasing. Progression and development of HCCare very quick and delitescence, majority patient has been in advanced stage when theyare final diagnosed as HCC, more over it develops very quickly. Major motivations whichcause HCC include in hepatitis virus, flavacin, alcohol abuse and so on. Invasion andmetastasis are two fundamental properties, which determine the prognosis of the HCCpatients and lead to recrudescence and treatment failure. To this day,it is not unclearlyabout precise mechanism of HCC on invasion and metastasis.Many signaling pathways are thought to be involved in the development and invasion ofHCC, including the MAPK pathway, phosphatidylinositol-3kinase(PI3K)/AKT/mammalian target of rapamycin(mTOR) pathway, the Wnt/beta-cateninpathway, hepatocyte growth factor/c-MET pathway, Hedgehog/Gli signaling pathway andso on. Hedgehog/Gli signaling pathway is a highly conserved system, which controls axial organ allelotaxy and plays a crucial role in embryonal growth and development ofmammalian. This signaling pathway is also closely related with development andprogression of human cancer. The signaling pathway was found in nevoid basal cellcarcinoma syndrome at first, increasing evidences have demonstrated that the signalingpathway plays an important role in multiple tumor types, for example, basal cellcarcinoma, gastric cancer, pancreatic cancer, colon carcinoma, small cell lung cancer,prostate cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancers,HCC and so on.Hedgehog/Gli signaling pathway is activated abnormally in human HCC, which play akey role on promoting occurrence of HCC. Activated markers of the Hedgehog signalingpathway such as glioma-associated oncogene homolo(gGli),Patched(Ptch),Smoothened(Smo)are significantly up-regulated in the HCC tumor tissues, and the expression ofHip1, which inhibts this pathway, is lower in hepatic tissue of HCC than that in normalhepatic tissue. This pathway is also abnormally activated in some HCC cell lines, andpromotes proliferation of HCC cells, inhibites apoptosis of HCC cells. On the other hand,inhibitors of this pathway, KAAD-cyclopamin(KAAD-cyc) and Gli siRNA can decreaseproliferation of HCC cells, and promote apoptosis of HCC cells；Smo siRNA alsodepresses growth of HCC cells. Moreover, Hedgehog/Gli signaling pathway participatesin HCC stem cell signal transduction. This pathway plays an important role on themetastasis and migratory of many kinds of cells and the formation of vascularization, atthe same time, it also promotes the regeneration, metastasis and migratory of manycancers, including neuroglioma, oophoroma, gastric cancer, esophageal cancer, pancreaticcancer, prostate cancer and so on. However, the role and mechanism of this pathway oninvolving in human HCC invasion and metastasis are not clear.Traditional Chinese medicine play an importaant role in treatment of cancers.Paeoniflorin, a characteristic monoterpene glucoside, is one of the main effective components of the total glucosides of paeony (TGP) from the root of Raidix PaeoniaeAlba (PAR). PAR is known to have diverse pharmacologic activities, includinganti-inflammatory, antioxidant, anti-proliferative, analgesic, immunoregulatory andanticancer, for thousands of years, PAR has also been extensively used in China for thetreatment of liver diseases. Previous studies indicated that TGP had protective effectsfrom liver injury and hepatic fibrosis and inhibits the proliferation and apoptosis of HCCHepG2and SMMC-7721cells. But the effect of paeoniflorin on the metastasis andinvasion of human hepatoma has not been studied now.Above all, we investigated the role and mechanisms of the Hedgehog/Gli signalingpathway in invasion and metastasis of HCC in this study. Meanwhile, we also evaluatedthe effects of paeoniflorin on invasion and metastasis of HCC and Hedgehog/Glisignaling pathway in order to explore the new target to inhibit the invasion and metastasisof HCC.OBJECTIVE1.To investigate the effect of Hedgehog/Gli signaling pathway on the invasion andmetastasis of HCC.2. To explore the mechanism by which Hedgehog/Gli signaling pathway mediates theinvasion and metastasis of HCC.3. To investigate the effects of paeoniflorin on the invasion and metastasis of HCC.4. To explore the mechanism by which Pae inhibites the invasion and metastasis of HCC.METHODS1. Immunohistochemistry were used to analyze the protein expression of Shh, Gli-1,p-ERK1/2and MMP-9in normal hepatic tissues and HCC hepatic tissues and in Bel-7402cells.2. The Spearman coefficient of correlation was used to examine the correlation between clinicopathologic variables and protein expression of Shh, Gli-1, p-ERK1/2and MMP-9.3. The Boyden chamber assay and wound-healing assay were established to detect andquantify invasion of Bel-7402, which is treated with Shh, KAAD-cyc, U0126, PD98059and Pae.4. Wound-healing assay were established to detect and quantify metastasis of Bel-7402,which is treated with Shh, KAAD-cyc, U0126, PD98059and Pae.5. Cell adhesion were established to detect and quantify adherency of Bel-7402, which istreated with Shh, KAAD-cyc, U0126, PD98059and Pae.6. MTT assay were used to detect cell proliferation of Bel-7402, which is treated withShh,KAAD-cyc, U0126, PD98059and Pae.7. Western blot were used to analyze the protein expression of sonic Shh, Gli-1, MMP-9,p-ERK1/2in Bel-7402cells which is treated with Shh, KAAD-cyc, U0126, PD98059andPae.RESULTS1.The positive ratios of Shh, nucleus Gli-1in non-metastasis are57.14%and44.90%,those in metastasis HCC hepatic tissues are81.08%and81.08%respectively, comparedwith non-metastasis HCC hepatic tissues, the protein expression of both Shh and Gli-1isnotably higher, the protein expression of both MMP-9and p-ERK1/2are also notablyhigher. But they is hardly expressed in normal hepatic tissues.2.The expression of Shh, nucleus Gli-1, p-ERK1/2and MMP-9was significantlycorrelated to the ability of the tumor to invade and metastasize (P=0.035,0.001,0.001,0.008respectively), the expression of Shh, nucleus Gli-1and p-ERK1/2was alsosignificantly correlated to the the tumor pathological grade(P=0.004,0.034,0.042respectively), nucleus Gli-1was also significantly correlated to p-ERK1/2and MMP-9(P=0.031, P=0.034respectively).3. Shh(0.5μg·mL-1) increased the adhesion, invasion and migration of Bel-7402cells,Shh(0.13μg·mL-1) also up-regulated the adherency and invasion of Bel-7402cells. 4. KAAD-cyc(1μmol·L-1,4μmol·L-1) inhibited the adhesion, invasion and migration ofBel-7402cells.5.Simultaneously, U0126(5μmol·L-1,10μmol·L-1) and PD98059(5μmol·L-1,10μmol·L-1),inhibitors of MAPK/ERK pathway, inhibited invasion and metastasis of Bel-7402cellsinduced by Shh.6.Shh(0.13μg·mL-1,0.5μg·mL-1) increased the expression of Gli-1 in nucleus and MMP-9,p-ERK1/2proteins in Bel-7402cells.7. KAAD-cyc(1μmol·L-1,4μmol·L-1) decreased the expression of Gli-1 in nucleus andMMP-9, p-ERK1/2proteins in Bel-7402cells.8. U0126(5μmol·L-1,10μmol·L-1) and PD98059(5μmol·L-1,10μmol·L-1) down-regulatedthe expression of p-ERK1/2and MMP-9induced by Shh in Bel-7402cells, howeverU0126and PD98059had no effects on the expression of Gli-1 in Bel-7402cells.9.Pae(6.25μmol·L-1,12.5μmol·L-1,25μmol·L-1) significantly reduced invasion, metastasisand adhesion of Bel-7402cells, decreased the protein expression of Gli-1, p-ERK1/2andMMP-9in Bel-7402cells.CONCLUSION1. Hedgehog/Gli signaling pathway mediates human HCC invasion and metastasis.2.The mechanism by which Hedgehog/Gli signaling pathway mediates human HCCinvasion and metastasis is correlated with up-regulating the protein expression of MMP-9via MAPK/ERK signaling pathway.3.Pae suppresses the invasion and metastasis of HCC.4.The mechanism by which paeoniflorin suppresses the invasion and metastasis of HCCis correlated with inhibiting Hedgehog/Gli signaling pathway, which down-regulating theprotein expression of MMP-9via MAPK/ERK signaling pathway. |
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