Anti-cancer Activity Evaluation And Mechanism Study On Triptolide And FTY720

Hepatocellular carcinoma(HCC)is a highly prevalent malignant tumor in China,which severely threatened the health of people in our country.Currently,surgical resection and liver transplantation are the most frequently used curative therapy for HCC.However,HCC usually dose not present obvious symptom at early stage.Most patients have lost the chance of curative surgery when diagnosed.Systemic therapy for HCC is extremely limited.The only effective systemic therapy drug for HCC is sorafenib.Unfortunately,sorafenib only improved median survival of advanced stage HCC patients for 2 to 3 months.Therefore the cost-effectiveness ratio of this molecular targeted drug is unsatisfactory.The prognosis of HCC is poor,overall 5 year survival is rather low.Facing the dilemma of HCC treatment,especially in the situation where most systemic therapy drugs are proven insufficient,the research and development of novel anti-HCC drugs become an urgent scientific problem.Traditional Chinese medicine has the history of thousands years.With recent advances in the research on active component of herbal medicine,small molecular compounds derived from traditional medicine have provided new insights and approaches for anti-HCC drug research and development.This thesis focused on two active compounds:triptolide(TPL)from Tripterygium wilfordii Hook.f.,and dongchongxiacao fungi Ischaria sinclairii derived chemical modified compound FTY720.We evaluated these immuno-active compounds for their anti-HCC activity,and investigated potential related mechanism.Our results indicated that TPL showed potent anti-HCC activity through inducing intrinsic apoptosis.Our further study revealed that TPL inhibited the expression of OGT which is the key enzyme for protein O-G1cNAcylation in HCC cells.The inhibition of OGT subsequently reduced the level of protein O-G1cNAcylation.Since modification by O-GlcNAc is crucial for the stability of cancer-promoting transcription factor Sp1,TPL-mediated decrease of O-G1cNAcylation induced protesomal degradation of Sp1.The down-regulation of Sp1-regulated pro-survival factors such as NF-κB,HSF1 and anti-apoptotic proteins finally promoted the apoptosis of HCC cells.Besides,the inhibition of OGT and Sp1 resulted in genomic instability of HCC cells and this could be another mechanism by which TPL induced p53-independent apoptosis.We also evaluated in vivo activity of TPL in nude mice subcutaneous xenograft tumor and orthopic liver HCC model.Our results showed that TPL has potent anti-HCC activity in vivo.The drug is well tolerated in animal models and no significant adverse effect was observed.In the study of anti-HCC activity of FTY720,we found that molecules in sphingosine 1-phosphate(S1P)signaling pathway,including sphingosine kinase 1/2(SphK1/2)and S1P receptor 1(S1PR1)were significantly up-regulated in HCC cells with high potentiality of metastasis.The kinases catalyzing the production of SIP,SphK1 and Sphk2,are also activated in this cell line.Interestingly,the activation of S1 PR1-regulated pathways involved in migration of HCC cells are also observed and may be associated with high motility of HCC cells.FTY720 inhibited SphK1,S1PR1 and their downstream signaling pathways such as Src,FAK and JAK/STAT3,and subsequently reduced migration of HCC cells.Our results also showed that FTY720 induced apoptosis and necrosis via endoplasmic reticulum stress(ER stress).At the same time,ER stress upregulated autophagy reflux through ER stress-mediated Akt/mTOR inhibition.The autophagy induced by FTY720 was protective against cell death,indicating combination of FTY720 and autophagy inhibition may enhance anti-HCC activity of FTY720.In summary,our study revealed that TPL and FTY720 have potent anti-HCC activity with well tolerance.These compounds have the potentiality as candidate drug against HCC and may help improve the outcome of HCC patients.Moreover,TPL and FTY720 have both immunosuppressive and anti-HCC activity.Considering their previous application in clinical transplantation,our results may also provide new insight for postoperative therapy of liver transplantation for HCC.