| Objective : To investigate the inhibitory effect of Aikete injection (AKT) on human hepatic cancer cell line SMMC-7721 and Hepatic cancer H22 . To study the mechanism of anti-hepatocarcinoma of Aikete injection which is mainly composed of Naphthoquinones derivatives.Methods: MTT assay and growth curve assay were used to assess the inhibitory effect of Aikete injection on proliferation of SMMC-7721 cells. SMMC-7721 cells were treated with AKT at the dose 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6mg·L-1 in MTT assay to analyze the inhibitory effect on proliferation of SMMC-7721 cells and calculated IC50 after treated for 48 hours; After treated with AKT at the dose 1, 2, 4mg·L-1, 0.05 mg·L-1 Adriamycin (ADM) as positive control for 7 days, growth curve of SMMC-7721 cells was completed; Hepatocarcinoma H22 model in mice was performed to study anti-hepatocarcinoma activity of AKT in vivo which were injected by intraperitoneal injection for 6 times, cyclophosphamide (CTX) as positive control by intraperitoneal injection just for once. After 11 days, the weight of tumor,spleen and thymus were observed to calculate the percentage of inhibition on tumor, and complete the growth curve of tumor by measuring length and width of tumor. Apoptosis-related protein (Caspase-3, Bcl-2 and Bax) were checked by immunohistochemical staining (IHC) . Its apotosis-inducing effect was approved by morphological observation by Giemsa staining and Hoechst 33258 fluorescent staining. The expressions of Bax and Bcl-2 protein, apoptosis rates and cell cycle were analyzed by flow cytometry (FCM). The expression of bax mRNA and bcl-2 mRNA were analyzed by RT-PCR.Results: After treated with AKT for 48 hours, MTT assay showed AKT caninhibit the proliferation of SMMC-7721 cells and had obvious dose-effect relationship. IC50 of AKT to SMMC-7721 cells was 2.325mg·L-1. The dose-effect and time-effect relationship were described in growth curve. The experiment in vivo showed AKT of 0.5, 1, 2mg·kg-1 had significant inhibitory effect on tumor and dose-related effect on mice with hepatocarcinoma, the inhibitory rate of tumor ranged from 34.37%(P<0.05) to 57.99 %(P<0.01). Cells shrinkage, nuclear pyknosis, chromatin condensation which were the characteristics of cell apoptosis were observed by Hoechst 33258 and Giemsa staining. FCM showed Cells in S phase decreased, cells in G2/M phase increased, the cell cycle was arrested in G2/M phase. In groups of 2mg·L-1 and 4mg·L-1, the sub-G1 peak was detected, the sub-G1 peaks at the dose 4mg·L-1 was obvious. The percentage of apoptosis increased. The expression of Bcl-2 and Bax protein was down-regulated, while the index of Bcl-2/Bax was decreased in the AKT group. RT-PCR showed that AKT could down-regulate the expression of bcl-2 mRNA and bax mRNA, and decreased the index of bcl-2 mRNA/bax mRNA. It was found that AKT could down-regulate the expression of Bcl-2 protein and up-regulate the expression of Bax and Caspase-3 protein in Hepatocarcinoma H22. Conclusion: AKT could inhibit proliferation of SMMC-7721 cells in vitro and the growth of Hepatocarcinoma H22 in vivo. It may induce Hepatocarcinoma cells apoptosis through arresting cell cycle in G2/M phase, down-regulating the expression of bcl-2 mRNA and Bcl-2 protein, decreasing the index of Bcl-2/Bax, and up-regulating the expression of Caspase-3 protein.
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