| OBJECTIVE To investigate the inhibitory effect of nobiletin (5, 6, 7, 8, 3' 4'-hexamethoxyflavone) on hepatic cancer cells and its mechanism of action.METHODS The inhibitory effect of nobiletin on proliferation of SMMC-7721 cells was evaluated by MTT analysis, growth curve and clone-forming assay. SMMC-7721 were treated with nobiletin at the dose 2, 4, 8, 16, 32, 64, 128 mg·L-1 in MTT assay to analyze the inhibitory effect on proliferation of SMMC-7721 cells and IC50 was calculated; After treated with nobiletin at the dose 10mg·L-1, 20mg·L-1, 40 mg·L-1, 0.05 mg·L-1 Adriamycin as positive control for 7 days, growth curve of SMMC-7721 cells was completed. For clonogenic assay, cells were exposed to nobiletin at the dose 10mg·L-1, 20mg·L-1, 40 mg·L-1 for 24h, 0.05 mg·L-1 Adriamycin as positive control, the inhibitory rate of clone-forming was calculated. Its apotosis-inducing effect was approved by morphological observation under microscope (Giemsa staining and Hoechst 33258 fluorescent staining). The expression of apoptosis-related protein—Caspase-3 was analyzed by immunohistochemical SP method. The protein expressions of Bax and Bcl-2, apoptosis rates and cell cycle were analyzed by flow cytometric analysis. Hepatic cancer H22 model in mice was performed through subcutaneous inoculation of Right-Sidedness Axilla of KM mice. Different dosages of nobiletin (125, 250 and 500 mg·kg-1) were injected by intragastric gavage (ig) everyday. After 10 days, the weights, percent inhibition of tumor, spleen and thymus index were observed. And apoptosis-related proteins (Caspase-3, Bcl-2, Bax, COX-2) were analyzed by Immunohistochemical method (IHC).RESULTS After treated with nobiletin for 48 hours, MTT assay showed that nobiletin at the dose ranging from 2 mg·L-1 to 128 mg·L-1 had the inhibitory ratio ranging from 3% to 80%. IC50 of nobiletin to SMMC-7721 were 26.2 mg·L-1; the inhibitory rate of clone-forming ranged from 50% to 97% at the dose ranging from 10 mg·L-1～40 mg·L-1. The dose-effect and time-effect relationship were described in the growth curve and clone-forming assay. Under microscope, characteristic morphology typical for apoptosis was observed at the dose 20 mg·L-1 including cells shrinkage, vacuolus in cytoplasm, complete nuclear membrance, pyknosis, aniso chromatin, chromatin margination were observed. The expression of apoptosis-related protein—Caspase-3 was detected by IHC, and the expression quality was increased as concentration of nobiletin was increased. Cells in S phase decreased, cells in G2/M phase increased, the cell cycle was arrested in G2/M phase. In groups of 10 mg·L-1, 20 mg·L-1, 40 mg·L-1, the sub-G1 peak was detected by flow cytometry (FCM), the sub-Gi peaks at the dose 40 mg·L-1 was obvious. The percentage of apoptosis increased (P＜0.05), the protein expression of bax gene was up-regulated at the dose 20 mg·L-1,mg·L-1. The protein expression of Bcl-2 showed no significant difference (P＞0.05). And the Bcl-2/Bax were decreased at the dose 20 mg·L-1 and 40 mg·L-1 (P＜0.05). In vivo nobiletin of 500 mg·L-1 had significant inhibitory effect on H22 tumor growth, and showed dose-related effect in mice with hepatocarcinoma. And nobiletin had no significant effect on immune system. And nobiletin could down-regulating the expressions of Bcl-2, COX-2, while it can up-regulating the expressions of Bax and Caspase-3. CONCLUSION Nobiletin could inhibit the growth of SMMC-7721 cells in vitro, arrest cell cycle in G2/M phase, and induce cell apoptosis. And it could inhibit the growth of hepatic cancer H22 in vivo. |
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