The Inhibition Effects And Mechanisms Of Haishengsu On Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. The current treatment options for liver cancer, including liver resection surgery, intra-arterial chemotherapy and radiation therapy, remain disappointing. Scientists are working hard, however, to address this problem.As carcinogenesis is closely related to the control of tumor cell proliferation, and apoptosis, the induction of apoptotic cell death may be the important therapeutic strategy for live cancer treatment. Meanwhile, angiogenesis plays a critical role in tumor growth, invasion and metastasis. Live cancer is rich in blood supply, its high mortality rate is mainly a result of vascular invasion and intra-hepatic metastases occurring during the early stage of cancer formation.Haishengsu, which is extracted from Tegillarca granosa Linnaeus, is an novel antineoplastic agent. Our previous studies showed that Haishengsu can inhibit the growth of human erythroleukemia K562 cell, lung adenocarcinoma epithelial A549 cell line, renal cell carcinoma OS-RC-2 cell, and suppress Ehrlich ascites carcinoma in mice. The aim of the present study is to investigate the apoptotic effect and anti-angiogenesis actions of Haishengsu on hepatocellular carcinoma cells.First, we investigated the inhibitory effect of Haishengsu on the growth of hepatocellular carcinoma cells in vitro and its effect on the apoptosis pathway. MTT metabolism assay showed that Haishengsu treatment for 72h significantly inhibited the human hepatoma BEL-7402 cell proliferation in a dose dependent manner. Electron microscopy analysis was performed to bserve apoptosis of BEL-7402 cells induced by HSS.In these cells, the typical condensed chromatin pulled away from the nuclear envelope and fragmented into several micronuclei.In addition, the nuclear volume decreased markedly and a considerable increase in vacuolisation was observed.FACS analysis showed that treatment with 100μg/ml of Haishengsu for 48h could induce 29.63±1.09% apoptosis. Hoechst 33258 staining also showed that Haishengsu caused condensed nuclei and apoptotic bodies.These data indicated that Haishengsu could induce BEL-7402 cells apoptosis. To reveal its mechanism of apoptosis induction, RT-PCR was performed to test Fas mRNA levels in BEL-7402 cells. Fas mRNA was upregulated by Haishengsu, which indicated that Fas receptor pathway may involve in Haishengsu-induced apoptosis. Western blot assay showed that haishengsu activated caspase-8 and caspase-3.Pretreatment with z-IETD-fmk markedly decreased the activation of caspase-8 and caspase-3.These data suggested that haishengsu may induce BEL-7402 apoptosis via Fas signaling pathway.Next, based on the antiapoptotic effects of Haishengsu, we measured the antitumor activity of haishengsu in null mice inoculated with BEL-7402 cells. Daily feed with 62.5 125 and 250mgkg-1 Haishengsu for 25 days yielded tumor inhibition of about 50%,58% and 60% respectively, relative rate of tumor proliferation was 45%,42% and 32% respectively. This indicated that HSS significantly inhibited tumor growth in vivo.HE staining showed that apoptotic bodies and necrosis appeared in tumor in Haishengsu treated groups.Electron microscopy analysis showed that haishengsu caused the typical apoptotic morphology in tumor. Haishengsu increased Fas mRNA level in tumor, as assessed by in-situ hybridization. Immunohistochemistry assay showed that haishengsu increased caspase-8 and caspase-3 in mice turmor. These results confirmed that haishengsu induced BEL-7402 apoptosis via Fas singnaling pathway.Finally, the antiangiogenic properties of haishengsu were investigated with several in vitro and in vivo models. Possible mechanisms underlying its antiangiogenic activity were also assessed. Haishengsu dose-dependently inhibited proliferation of human umbilical vein endothelial cells (HUVEC).In tube formation and cell migration assays using HUVEC cells, haishengsu significantly inhibited formation of intact tube networks and reduced the number of migratory cells. In the chick chorioallantoic membrane assay, haishengsu reduced new vessel formation compared with the vehicle control. Microvessel density (MVD) in sections of the tumor was reduced after Haishengsu treatment. The levels of VEGF, and MMP-2 were also significantly decreased.These data suggested that Haishengsu could directly inhibit vescular endothelial cells and exert anti-angiogenic effect via downregulation of VEGF and MMP-2.Collectively, Haishengsu could inhibit hepatocellular carcinoma and its effect associated at least in part with Fas singnaling pathway and the antiangiogenesis.