The Anti-tumor Effects And Mechanisms Of Osthole In Hepatocellular Carcinoma

Part1The Anti-tumor Effects of Osthole in Hepatocellular CarcinomaObjective：To study the anti-hepatocelluar carcinoma effects of osthole. Methods：The inhibitions of osthole on proliferation of HCC cells were analyzed by MTT assays,cell cycle distribution and apoptosis were determined by flow cytometry in vitro. HCCtumor models were established in mice by subcutaneously injection of SMMC-7721orHepa1-6cells and the effects of osthole on tumor growths in vivo and the drug toxicity,absorption and distribution were studied. Results：1) Osthole displayed a dose-andtime-dependent inhibition of the HCC cell proliferation in vitro, including human HCCcell lines SK-HEP-1, SMMC-7721, HepG-2and murine HCC cell line Hepa1-6.Osthole also inhibited the proliferation of normal hepatic cells, but the IC50was higherthan HCC cells, which indicated the cytotoxicity of osthole was selective.2) Ostholeinduced apoptosis and caused cell cycle arrest in G2pahse of SMMC-7721andHepa1-6cells.3) Osthole could suppress HCC tumor growth in vivo, and intraperitonealinjection was the best way of drug dilivery.4) Osthole could transport into the tussueswith abundant blood stream (including tumor) through simple passie diffusion.Conclusion：Osthole is a natural compound with anti-hepatocelluar carcinoma effectand low toxicity.Part2The Mechanisms of Osthole Inducing Apoptosis inHepatocellular CarcinomaObjective：To explore the mechanisms of osthole inducing apoptosis in HCC.Methods：The pathway-focused gene expression profiling of apoptosis using Real-TimePCR, and the protein levels of Bax, Bcl-2, Caspase, PARP, CyclinB, NF-κB and PI3K/Akt were measured by western blot and EMSA analysis. The inhibition of theSMMC-7721cell proliferation with combination treatment of osthole and TRAIL wasanalyzed by MTT assays. Results：1) Osthole could up-regulate the expression of28genes in the pathway-focused gene expression profiling of apoptosis, and TNF receptor(21.43%) and TNF ligand (17.86%) family were the highest proportion among them.2)Osthole could up-regulate the Bax and down-regulate the Bcl-2expression, so the ratioof Bax/Bcl-2was increased. Osthole also promoted the Caspase-3, Caspase-8andCaspase-9activities and the PARP cleavage, as well as the down-regulation of CyclinB1in HCC cells.3) Osthole displayed a dose-and time-dependent inhibition of theNF-κB activations.4) Osthole could down-regulate the key proteins (p110, p85andp-Akt) in PI3K/Akt signaling pathway, while the t-Akt was not significantly changed.Insulin could decrease the effect of osthole down-regulating p110and p-Akt, whileLY294002further promoted this effect.5) Poliferation of SMMC-7721cells wasdramatically inhibited by the combination treatment of osthole and TRAIL, which alsoexhibited a synergistic inhibition with low concentrations of osthole. Conclusion: Themechanisms of osthole inducing apoptosis in HCC were through multi-targets andmulti-paths. One of mechanisms was through the inhibition of NF-κB activation anddown-regulation of CyclinB via inhibiting PI3K/Akt pathway, resulting in cell cyclearrest in G2pahse. The another mechsnism was through up-regulation of Bax andactivation of Caspase-9, or through activation of Caspase-8through death domainreceptors, reaulting in Caspase-3activation and PARP cleaveage, which leads to theapoptosis of HCC cells.Part3The Effects of Osthole on Anti-Tumor Immune ResponseObjective：To study the effects of osthole on the anti-tumor immune responses inthe HCC murine model. Methods：The effects of osthole on proliferation of spleniclymphocyte were analyzed by MTT assays. The lymphocyte subsets of spleen weredetected by flow cytometry and the serum levels of IL-2and TNF-α were measured byELISA in HCC murine model by subcutaneously injection of Hepa1-6. Results:1)Osthole promoted the proliferation of splenic lymphocyte under un-stimulated or ConA-and LPS-stimulated conditions.2) The weights and coefficients of thymus were not changed; the weights and coefficients of spleens were increased after osthole treatmentin the HCC murine model.3) Osthole could elevate the proportion and number ofmacrophages, CD4+T and CD8+T cells in the spleen, and keep the ratio of CD4+/CD8+at normal levels, as well as improve the proportion of CD4+T and CD8+T cells intumor tissue too. The proportion, number, activation and maturation of B cells in thespleen were not changed.4) Osthole promoted the activation of CD4+T and CD8+Tcells in the spleen and elevated the proportion of CD8+effector T cells.5) Ostholedecreased the proportion of CD4+CD25+Foxp3+regulatory T cells in the spleen in theHCC murine model.6) Osthole could increase the levels of IL-2and TNF-α in theserum of the HCC murine model. Conclusion: Osthole could enhancethe T cellmediated anti-tumor immune responses in the HCC murine model.