Research On Bufalin Inhibits Liver Cancer Cell MHCC97H Metastasis

BackgroundHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide byannual incidence and the third leading cause of cancer mortality.Currently, surgery remainsthe first choice for HCC clinical therapy. However, the prognosis for HCC is poor, and the5-year survival rate worldwide is less than5%, mainly because of a high potential forintra-hepatic and distant metastasis and recurrence even after surgical resection. Hence, itis important to elucidate the molecular mechanism for metastasis and novel treatmentmethods and agents are required.Increasing evidence indicates the metatisis of HCC is a complexphenomenon,involving a series of extracellular molecules such as extracellularproteases,cytokines, angiogenetic factors, and growth factors,cell adhesionmolecules.Among them,Cdc42is one of Ras-related GTP-binding proteins that play a keyrole in cell migration and regulate the assembly and disassembly of the actin cytoskeletonin response to extracellular signals and.GTPases act as molecular switches by cyclingbetween an active GTP-bound state and an inactive GDP-bound state.GTPase activatingproteins (GAPs) stimulate the endogenous GTPase activity of these proteins to hydrolyzebound GTP,leading to the inactive GDP-bound state. Conversely, guanine nucleotideexchange factors (GEFs) accelerate the intrinsic GDP/GTP exchange to cause formation ofthe active GTP-bound protein.Bufalin, a major bufadeinolide-like cardiac glycoside isolated from the skin andparotid venom glands of the toad,the molecular formula of which is C24H34O4with arelative molecular weight386.5. Bufalin performs diverse functions,such as to increasevasoconstriction,vascular resistance and blood pressure.In terms of its anti-tumor activity,bufalin has been shown to induce apoptosis and differentiation of tumor cells.In this study,we aimed to investigate the inhibitory effect of bufalin on HCCmetastasis and the detail molecular mechanism,especially the role of Cdc42in this process.As a result,we identified that the anti-invasive and anti-metastasis effect of bufalin ismediated by down-regulation of Cdc42. Furthermore, the potential downstream molecularmechanisms of Cdc42-mediated invasion were investigated.Our results indicate that bufalin might be used therapeutically to suppress invasion and metastasis of HCCObjectiveTo investiagate the effects of bufalin on the mirgration and invasion of MHCC97Hcells.In order to illuminate the molecule mechanism between the inhibitory effect ofbufalin on HCC metastasis and the Cdc42-mediated signaling pathway both in vitro and invivo.Providing experiment evidence for find an effective medicine and target in thetreatment of HCC.Methods(1) Effect on the migration and invasion ability of MHCC97H cells treated bybufalin.MHCC97H cells were treated with different concentration of bufalin,then scrapemigration assay was used to qualitatly analysis the change of migration ability ofit.Subsequently,the transwell migration assay and the transwell invasion assay were used todetermine the anti-invasive and anti-migrate effects of bufalin in quantitation.(2) Methods of observing the change of Cdc42protein and related signal pathwaywith HCC.The protein level of Cdc42in MHCC97H cells in the presence or absence ofbufalin was determined by western blot. In order to estimate the value of Cdc42in theanti-invasive effect of bufalin in MHCC97H cells, a specific siRNA for Cdc42was used tosilenced Cdc42expression. The inhibitor of ERK(PD98059),the JNK inhibitor (SP-600125)and P38MAPK inhibitor (SB203580) were performed to study the relation between bufalinand MAPK.Cytoplasmic and nuclear proteins were stepwise extracted to observe thechange of NF-κBp65.Expression of P53and down-stream molecules were determined bywesterblot.The protein level of COX-2was determined by western blot,while the level ofMMPs family were determined by Elisa.(3) Bufalin inhibits HCC growth and metastasis in vivo.MHCC97-H cells (1×107/animal) in0.2ml of serum-free culture medium were injected s.c. into the upper leftflank region of nude mouse. When the subcutaneous tumor developed,it was removed andminced, and small tumor tissues were implanted into the liver of new recipient mice, Thirtynude mice were randomly divided into three groups. We injected two of the three groupswith bufalin(0.25mg/kg/day,0.5mg/kg/day) via the tail vein from next day after operationuntil a month. After35days, the animals were sacrificed under deep anesthesia and lungmetastatic lesions were conted by H＆E staining.The implanted tumor weight and volume were measued,then Cdc42level were determined by immunohistochemistry(IHC).Results(1) The result of scrape migration assay indicated that bufalin posses theanti-migration potency.Bufalin (50-400nmol/L) was able to significantly inhibitMHCC97H cancer cell migration and invasion in a concentration dependently mannermeasured by the transwell migration assay and the transwell invasion assay.(2)we confirmed that bufalin was able to decrease protein expression levels of Cdc42in MHCC97H cells in a concentration and time-dependent manner.We found that Cdc42expression decreased after48hours of20nmol/L Cdc42siRNA treatment; furthermore, theanti-invasive effect of bufalin was reinforced by pretreated with Cdc42siRNA,determinedby Transwell invasion assay. These results indicated that Cdc42siRNA and bufalin have asynergetic effect on anti-invasive of tumor cells.However, the scrambled siRNA controlshowed no difference compared with the control group.Bufalin induced activation of thec-Jun NH2-kinase (JNK) and P38mitogen-activated protein kinase(MAPK)phosphorylation,in contrast induced suppresses of the extracellular signal-regulatedkinase(ERK) phosphorylation.The inhibitor of ERK(PD98059) strengthened theantiinvasion ability of bufain,whereas the JNK inhibitor (SP-600125) and P38MAPKinhibitor (SB203580) could partially reversed it.Bufalin inhibited the NF-κBp65to enterthe cellular nucleus.Expression of P53and down-stream molecules Bax and P27wereup-regulated after treatment of bufalin.The protein expression level of COX-2obviouslydecreased treated with bufalin.Bufalin could decreased the secretion of MMP-2,but notinfluenced the secretion MMP-3and MMP-9,it indicated that MMP-2paly a important rolein bufalin induced anti-invasion and anti-metatisis ability.(3)Bufalin inhibits HCC growth and metastasis in vivo.The physical and psychologystatus of nude mice treated by bufalin were beter than control group,accompanied by andecrease in body weight in bufalin-treated group,maybe because of orthotopic transplantedtumor reverse the decrese in body weight,suggesting an inhibitory effect of bufalin oncachexia.We determined the inhibitory effect of bufalin in vivo on HCC tumor growth byexamining orthotopic transplanted tumor volume and nude mice body weights.The resultindicated that there was a significant decrease in tumor volume in bufalin-treated groupsuggesting an inhibitory effect of bufalin on HCC tumor growth. The necrosis degree oforthotopic transplanted tumor in bufalin-treated group was severe than in control group, observed under inverted microscope.To confirm the effect of bufalin on HCCmetastasis,we examined the metastasis in the lungs of nude mice. As expected, the lungmetastasis rate in control group was almost100%;The lung metastasis rate in control groupnude mice were much higher than in group treated with bufalin(P＜0.05). We found thatCdc42expression were lower in bufalin-induced group than that of control group.ConclusionBufalin can inhibit the migration and invasion ability of MHCC97H cells.Theexpression level of Cdc42protein is high in MHCC97H cells than other HCC cell lines anddecreased significantly after bufalin treatment.Bufalin could activted the JNK,P38MAPKsingal pathway,but attenuated the ERK singal pathway,inhibited the nuclear translocationof p65.Expression of P53and down-stream molecules Bax and P27were up-regulated.Theprotein expression level of COX-2decreased obviously after bufalin treatment.Bufalincould also downregulate the secretion of MMP-2,but not MMP-3and MMP-9,itindicated that MMP-2paly a important role in bufalin induced anti-invasion andanti-metatisis ability.We successfully constructed the orthotopic transplanted tumor modelLCI-D20, bufalin can inhibit the tumor growth and metastasis, and also decrease theCdc42level of orthotopic transplanted tumor.In sum,Bufalin could inhibit the invasion andmetastasis of HCC cell both in vitro and vivo and Cdc42is maybe the molecular target ofthe process.