The Anti-tumor Effects Of Lupeol In Hepatocellular Carcinoma

PartⅠlupeol induces apoptosis in hepatocellular carcinoma cells and inhibits tumor growth in vivoObjective: To study the anti-hepatocellular carcinoma effects of lupeol in vivo and in vitro, as well as to explore its cellular and molecular mechanisms. To explore the combined anti-tumor effects of lupeol and rTRAIL in HCC.Methods: (1) In vitro inhibitions of luepol on proliferation of HCC cells were analyzed by MTT assay, and the inhibitions of lupeol on transplantation tumor growth of human HCC cell line, SMMC7721, in nude mice were assayed; (2) Inverted microscope and flow cytometry were used to detect lupeol induced apoptosis and cell cycle inhibition by characteristic morphological changes and biochemical features, and the mechanism of apoptosis was explored by western blot analysis; (3) Inverted microscope and flow cytometry were used to detect combination treatment of lupeol and rTRAIL induced apoptosis and cell cycle inhibition by characteristic morphological changes and biochemical features, and to identify whether the combined effect is synergistic.Results: (1) The proliferation of human HCC cells were dramatically inhibited by lupeol in a concentration-dependent manner, and 48h after exposed to lupeol, the IC50 value was 40μmol/L-50μmol/L. Lupeol significantly inhibited the growth of transplanted tumors induced by SMMC7721 cells in nude mice. (2) Morphological features of apoptosis and cell cycle block induced by lupeol were observed by inverted microscope and flow cytometric analysis, and up-regulation of pro-apoptotic proteins, active caspase-3 and cleaved PARP, were indicated by western blot results. (3) Proliferation of human HCC cells were dramatically inhibited by combination use of lupeol and rTRAIL, and morphological features of apoptosis induced were observed by inverted microscope and flow cytometric analysis, but the combination effects was not synergistic.Conclusion: Lupeol showed significant inhibitory effects on the growth of HCC cells in vitro and inhibited the tumor growth in vivo. Lupeol and rTRAIL had combinational anti-tumor effect.PartⅡlupeol and PI3K inhibitor S14161 had synergistic anti-tumor effectsObjective: To improve the anti-tumor effects of lupeol; to explore its cellular and molecular mechanism of lupeol at low concentrations, and then to determine whether combination of lupeol with a PI3K inhibitor could synergistically promote anti-hepatocellular carcinoma effects.Methods: (1) The inhibitory effects on proliferation of human HCC cells induced by lupeol and its derivatives were detected by MTT assay. (2) Differences between inhibition effects on cell proliferation and induction of apoptosis induced by lupeol and one of its derivatives, H2, in low concentration range were detected by MTT assay and flow cytometric analysis, and molecular mechanisms were analyzed by RT-PCR and western blot. (3) Inhibitory effects of PI3K inhibitor, S14161, on cell proliferation of human HCC cells was detected by MTT assay. In vitro inhibitory effects on cell proliferation of human HCC cells induced by combination of appropriate concentrations of S14161 with low concentrations of lupeol were detected by MTT assay. Western blot analysis was used to study the molecular mechanism of combination therapy. (4) The inhibitions of combination use of lupeol and S14161 on transplantation tumor growth of human HCC cell line, SMMC7721, in nude mice were assayed.Results: (1) The anti-hepotocellular carcinoma effects of its derivatives, H1, T1 and T2, were some worse than lupeol, as IC50 of lupeol was about 40μmol/L-50μmol/L, while IC50 of H1, T1 and T2 were >120μmol/L, about 50μmol/L and 70μmol/L-80μmol/L, respectively. However, the anti-hepotocellular carcinoma effect of H2 was better than lupeol with an IC50 of less than 30μmol/L. (2) Flow cytometric analysis, RT-PCR and western blot results indicated that low concentrations of lupeol had little effects on cell apoptosis induction, intracellular molecule expression of pro-apoptotic proteins and key proteins in PI3K/Akt signaling pathway, while low concentrations of H2 induced cell apoptosis, up-regulated intracellular pro-apoptotic proteins and down-regulated of key proteins in PI3K/Akt signaling pathway. (3) IC50 of S14161 on HCC cells was approximately 4μmol/L, and 1μmol/L and 3μmol/L were chosen for later combination use. MTT results showed that lupeol combined with 1μmol/L or 3μmol/L of S14161 synergistically increased the anti-hepatocellularcarcinoma effects of lupeol, which was more obvious in low concentrations. (4) Western blot analysis indicated that 20μmol/L of lupeol combining with 1μmol/L or 3μmol/L of S14161 synergistically down-regulated the PI3K/Akt pathway in HCC cells. (5) Combination therapy of lupeol and S14161 synergistically inhibited tumor growth of transplanted tumors induced by SMMC7721 in nude mice.Conclusion: Lupeol and S14161 alone inhibited HCC cell proliferation dose-dependently,while low-dose combination have synergistic effects. Combination therapy improves the anti-hepatocellular carcinoma activity without toxic effects. Thus, lupeol combining with PI3K inhibitor may provide safer and more effective anti-tumor drug regimen. It is worthy of further investigation.Part III Detectable expression of IL-35 in CD4~+T cells from peripheral blood of chronic Hepatitis B patientsObjective: To detect whether IL-35 is expressed in CD4~+T cells from peripheral blood of chronic Hepatitis B (CHB) patients, providing theoretical basis for pathogenesis research and clinical treatment for CHB.Methods: Total RNA and total proteins were extracted from CD4~+ T cells purified from peripheral blood of CHB patients or healthy donors. RT-PCR and immunoprecipitation (IP) & western blot analysis were used to detect the expression of IL-35 in both mRNA and protein levels.Results: RT-PCR results showed that IL-35 mRNA expression could be detected in purified CD4~+T cells from CHB patients; IP & western blot results also indicated that IL-35 protein expression could be detected in purified CD4~+T cells of CHB patients. While IL-35 was undetectable in those of healthy donors.Conclusion: IL-35 secreted by peripheral CD4~+T cells of CHB patients may contribute to inhibition of anti-HBV specific immune responses, which may more clearly illustrate the pathogenesis of CHB. IL-35 may represent a new molecular target for clinical treatment of CHB.