| Background: Hepatocellular carcinoma(HCC) is one of malignant tumors with high morbidity, high case-fatality rate and poor prognosis. Current clinical treatments to it including surgical resection, TACE, radiation and chemotherapy, immunotherapy, gene therapy,etc.. But these treatments were difficult to control the progress of this disease. Chemical drug treatment is currently the most used method, but chemotherapy drugs used have serious side effects to patients and their curative effect is not high. So to study new antitumor drug which have more antitumor activity and less adverse effects has been a research focus. Recently years, to extract effective antitumor components from natural products has become a new research trend. Grifola frondosa polysaccharide(GFP) is a kind of active ingredient which extracted from the fruiting body and mycelium of medicine edible species maitake. It has extensive pharmacological effects and the molecular mechanism of its strong antitumor effect is that it can induce tumor cell apoptosis. At the same time, vitamin C(VC) has been confirmed that it can induce osteosarcoma cell apoptosis and induce marrow stromal cells autophagy in vitro.Objective: In this study, we will make metrology analysis to the literatures of the an-titumor activity of GFP, and make the Meta analysis to research the role of apoptosis in tumor suppression effect of GFP at the same time, in order to master the research status of GFP inhibiting tumor. And on the basis of above studies, we will study the effect of combined use GFP with VC on cell proliferation of liver cancer SMMC-7721 cells to verify whether they have synergy effect on the antitumor effect and to illustrate the role of apoptosis and autophagy in the process of combination treatment to liver cancer cells. Then further research is to study the signal transduction pathways of apoptosis and autophagy in order to guide clinical practice medicine effectively and to provide the experimental basis to developing new ideas for clinicians treating liver cancer.Methods: In our study, literature metrology analysis and visual analysis to researches of bioactive activities of Grifola frondosa extracts were performed firstly in order to find the key points and difficulties of the application researches of the Grifola frondosa extracts in tumor treatment. Then the molecular mechanism of tumor suppression effect of GFP is analyzed bymaking system review and Meta analysis methods and the role of apoptosis in antitumor effect of GFP was explored. To GFP which has been extracted and routine medicine VC, MTT method was used to check the effect of single and combination use on liver cancer SMMC-7721 cell proliferation. Isobiologram analysis was used to study relationship of the two drugs in the process of combination treatment and to seek the best concentration of combination. Flow cytometry and Annexin V-FITC/PI double dye method were used to detect the cell cycle and cell apoptosis after treatment. The morphology and ultrastructure change of SMMC-7721 cell has been observed by inverted biological microscope and transmission electron microscope. Meanwhile, Hoechst33258 staining and MDC staining were used to identify the occurrence of apoptosis and autophagy by fluorescence microscope. Western blotting(WB) were used to detect the expression of apoptosis related proteins: Bax, Bcl-2, PARP and autophagy related proteins: Beclin-1, LC3, Akt and phospho-Akt protein after treatment. We determined whether cytochrome C was release through comparing the protein expression of cytochrome C in whole cell and in the cytoplasm. In addition,the activity of caspase-3, caspase-8 and caspase-9 of cell were detected in order to study the activity changes of apoptosis and autophagy on the level of molecular biology and to discusses what kind of signal transduction pathways of apoptosis and autophagy were followed by SMMC-7721 cells.Results: According to the results of literature analysis, we knew that the antitumor activity of Grifola frondosa polysaccharide is always the key point of research in a lot of researches of biological activities of Grifola frondosa extracts. And further Meta analysis results showed that the main mechanism of tumor suppression effect of GFP is it can induce tumor cell apoptosis Results of experiment show that Grifola frondosa polysaccharide and vitamin C both can inhibit the proliferation of liver cancer SMMC-7721 cells by time-dose dependent way. Small doses of GFP(< 0.6 mg/ml) and VC have synergies antitumor effect when we combined use of them. So the best combination of concentration we ultimately chose was combine 0.2 mg/ml GFP with VC 0.3 mM,and the inhibition rate of SMMC-7721 cell was 68.81% ±1.12 when we used this combination of concentration. It was higher than cell inhibition rate when we use single medication, the results was 16.03%±1.80( GFP) and 29.85 ±1.02(VC). The results of flow cytometry showed that SMMC-7721 cells had cell cycle block in G2/M phase after treatment. The cell apoptosis rate had rise to 64.45%±1.41 and it was 6.88 folds(GFP) and 2.24 folds(VC) when single drug was used respectively. The morphological changes of cells including chromatin edge set and cytoplasmic loose which reminder apoptosis and autophagic vacuoles with double membrane structure which reminder autophagy were both observed under optical microscope and transmission electron microscopy. After Hoechst33258 and MDC staining, cells in GFP, VC and combination use groupappeared visible typical nuclear changes of cell apoptosis and cells in VC and combination use group appeared autophagy particles which has enhance fluorescence, and these phenomena were more obvious in combined treatment group.The results of WB showed that after cells were treated by combination use GFP and VC,compared with the control group, the expression of promoting apoptosis proteins Bax was increased, the expression of anti-apoptotic protein Bcl-2 was decreased and the expression of the substrate of caspase-3(PARP) were enhanced. At the same time, there was no difference in the expression of cytochrome C in all cells, but the expression of cytochrome C in cytoplasm was enhanced. This proof hinted that cytochrome C was released into the cytoplasm. In addition, the activity of caspase-3, caspase-8 and caspase-9 were all enhanced in this process. It suggested that the levels of cell apoptosis was rose, and apoptosis followed mitochondria signal transduction pathway. Additionally, WB result showed that the expression of autophagy related protein Beclin-1 and autophagy molecular marker LC3-II were enhanced, and the proportion of LC3-II/LC3-I were rose. The expression of total Akt and phospho-Akt protein were enhanced. It suggested that the activity of cell autophagy was rose, but cell autophagy PI3K/Akt/mTOR/p70S6 K signal transduction pathway may not in this process.Conclusions: According to the results of literature analysis, the antitumor effect of Grifola frondosa polysaccharide always was the key research point in a lot of researches of the biological activities of Grifola frondosa extracts. And the results of Meta analysis showed that inducing tumor cell apoptosis was the main mechanism of tumor inhibition effect. The further experimental results indicated that Grifola frondosa polysaccharide have antitumor activity through inducing cancer cells to apoptosis and the combined application of small doses of Grifola frondosa polysaccharide and vitamin C has significant antitumor activity to liver cancer SMMC-7721 cells and both of them have synergy effect in this process. After cells were treated by drugs, apoptosis and autophagy are all activated. On the one hand, drugs induces cell apoptosis by internal mitochondria signal transduction pathway, on the other hand, drugs induces cell autophagy. |
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