The Effect And Mechanism Of Antrodia Camphorata On HCC

[Objective]Worldwide, HCC is accounting for 9.2% of all newly diagnosed cance every year.Of all new patients with HCC,70% occur in Asia, with patients in China accounting for 55% of liver cancer deaths annually.Almost without exception, those who develop HCC each year die within 12 months, attesting to its rapid progression and serious prognosis.Therefore, a new effective anti-cancer agent is needed for HCC.Antrodia cinnamomea(AC) is a unique mushroom of Taiwan, ANCA-E-D is a dichloromethane extract of AC. Being an indigenous species,AC was traditionally used by Taiwan aborigines to treat food and drug intoxication,diarrhea,abdominal pain,skin itching,and improve the immune system and liver function.Recently,the pharmacology also confirmed that AC have several biological activities such as anti-cancer, antihepatotoxic, anti-inflammatory, antiviral, antioxidant.regulat immunity and neuroprotective activities. Traditional Chinese medicine Ganoderma lucidum and AC belong to the same Polyporaceae of fungus, have similar growth habit and sexual flavour, but is different in bioactive componennts. Especially the kinds of bioactive substances of AC are more rich than a single species of Ganoderma lucidum, and terpenoids and polysaccharide etc. composition content is higher in AC. Therefore, the antitumor and immunomodulating properties of AC has become a hot spot of research in recent years. We believe that AC is a potential anti-cancer medicine,due to its dual effect of antrodia righting tonic and anti-cancer detoxification. Research shows that, AC can inhibit lung cancer, colorectal cancer, ovarian cancer and other tumor growth. In this study, we use SMMC-7721 hepatocellular carcinoma cell line to study the effect of AC by in vivo and in vitro experiments, and to investigate its mechanisms by the gene chip technology.[Methods]1. The experiment in vivo(1) The Xenografts nude mice models were established by subcutaneous inoculation of 4x 105/cell SMMC-7721 cells into the right hind.(2) When HCC transplantation tumor became measurable, the nude mice were intraperitoneally received ANCA-E-D treatment (25mg/kg/d and 50mg/kg/d,21 d)(3) The tumor body, weight of Xenografts nude mice were measured; The serum content of ALT and AST were detected;The paraffin sections of Xenografts nude mice liver, kidney and tumor tissue were stained by hematoxylin-eosin(HE) and observed with microscope;The MVD and PCNA in tumor tissue were detected by IHC;The apoptosis of the tumor tissue were detected by TUNEL assay.2. The experiment in vitro(1) Teated with 20～80 mg/L ANCA-E-D for 24,48 and 72 hours, the proliferation inhibitory rate of SMMC-7721 cells were detected by MTT assay;(2) Treated with 20～60 mg/L ANCA-E-D for 24 hours, the SMMC-7721 cells were observed by Wright’s and Hoechst 33258 staining;(3) Treated with 20～80 mg/L ANCA-E-D for 24 hours,the SMMC-7721 cell cycle and apoptotic rates were detected by flow cytometry(FCM);(4) Treated with 20～60 mg/L ANCA-E-D for 24 hours, differentially expressed genes in SMMC-7721 cells were detected by Agilent Human 4x44K Gene Expression Microarrays.[Results]In vivo ANCA-E-D can significantly inhibit the volume and quality of transplanted tumor in nude mice and may reduce the angiogenesis and the expression of PCNA;In addition, ANCA-E-D can induce apoptosis of transplanted tumor cells in nude mice;We also find that the serum content of ALT and AST in nude mice had no significant changes after ANCA-E-D treated.In vitro the concentration of 20,40,60,80μg/ml ANCA-E-D could markedly inhibit the proliferation of SMMC-7721 cells after 24,48,72h.The SMMC-7721 cells displayed distinctive apoptotic character by Hoechst 33258 and Wright’s staining with ANCA-E-D.Flow cytometry indicated that the different concentrations of ANCA-E-D could induce apoptosis and influence the cell cycle process of SMMC-7721 cells, and there is a dose-dependent increase in apoptosis. After treated with 0,20,40,60 mg/L ANCA-E-D, there were 422 common up regulated genes and 377 common down regulated genes in the differentially expressed genes of SMMC-7721 cells.These differentially expressed genes involved in cell signal transduction, migration, adhesion, differentiation, apoptosis, metabolism, proliferation, angiogenesis, organ development and other aspects. Pathway analysis found that, these differentially expressed genes involved in TNF signaling pathway,Calcium signaling pathway,Pathways in cancer,PI3K-Akt signaling pathway,MAPK signaling pathway,Jak-STAT signaling pathway,Apoptosis and so on.[Conclusion]ANCA-E-D can inhibit the proliferation of human hepatocellular carcinoma cells SMMC-7721 in vivo and vitro, and the possible mechanism may relate to the induction of apoptosis,may be through influencing the expression of EDNRA, HRK, LCN2 and ANGPTL4, which affecting multiple signaling pathways including apoptosis to achieve.