ABSTRACT
OBJECTIVE： To study the anti-tumor effect of anemoside B4 （AB4） on hepatocellular carcinoma Huh-7 and tumor-bearing nude mice and its mechanism. METHODS： Huh-7 cells were divided into AB4 treatment group， negative control group （constant volume of culture medium） and positive control group （5-fluorouracil 50 μmol/L）. The inhibitory rate of Huh-7 cell proliferation was tested by MTT after treated with 0， 3， 6， 12， 25， 50， 100， 200， 400， 800， 1 600 μmol/L AB4 for 12， 24， 36， 48 h； IC50 were calculated. The number of clone cells was evaluated by clone formation tests after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h. The apoptosis rate of Huh-7 cells was tested by Annexin Ⅴ-FITC/PI double staining after treated with 50 μmol/L AB4 for 24 h. The expression of apoptotic proteins such as Bcl-2， Bax， Caspase-3， Cleaved Caspase-3 and Cleaved PARP were tested by Western blot after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h. Tumor-bearing nude mice were randomly divided into negative control group （normal saline）， positive control group （5-fluorouracil 50 mg/kg）， AB4 lwo-dose， medium-dose and high-dose groups （25， 50， 100 mg/kg）， with 3 mice in each group. They were given relevant medicine intraperitoneally， once a day， for consecutive 19 d. The growth of tumor was observed， and anti-tumor rate was also calculated. HE staining was used to observe the morphology of tumor cells. RESULTS： The inhibition rate of AB4 on Huh-7 cell proliferation increased with the increase of concentration of AB4， but it did not increase significantly after reaching 50 μmol/L； it increased with the increase of time， but it did not increase significantly after 24 h. The IC50 of AB4 was （56.76±1.756） μmol/L. Compared with negative control group， the number of clone cells was decreased significantly after Huh-7 cells were treated with 50 μmol/L AB4 for 24 h， while the protein expression of Bcl-2 was decreased significantly （P＜0.05）； the apoptotic rate， the protein expression of Bax， Caspase-3， Cleaved Caspase-3 and Cleaved PARP were increased significantly （P＜0.05 or P＜0.01）， there was no statistical significance， compared with positive control group. Compared with negative control group， the volume of tumor was decreased significantly in AB4 low-dose， medium-dose and high-dose groups， positive control group （P＜0.05）； the outline of tumor cells become more and more blurred； there were nuclear pyknosis， unclear nucleoplasm and nuclear fragmentation； its anti-tumor rates were 25.93％， 39.15％， 46.26％， 42.83％， respectively. CONCLUSIONS： AB4 inhibits Huh-7 cells and tumor-bearing mice， the mechanism of which may be associated with up-regulating the proportion of Bax/Bcl-2， activating Caspase-3， cracking PARP and inducing apoptosis.
ABSTRACT
Background and purpose:A causal relationship between inflammation and cancer has been extensively investigated. Nuclear factor-kappa B (NK-?B) is a key regulator in inflammation-associated cancer, and is activated by a variety of stimuli. This paper studied the anti-inflammatory effect of NK-104 on NF-?B activated by TNF-? in hepatocellular carcinoma cell line (Huh 7). Methods:The cell proliferation was examined by the WST-8 assay. The NF-?B and I?B? expressions were evaluated by western blotting. IL-8 and its product were quantitated by ELISA.Results:TNF-?(1 ng/ml) strongly induced the expression of NF-?B by approximately 1.8-fold compared with the control in the nuclei of Huh 7 cells, the induced NF-?B expression was significantly suppressed by the addition of pitavastatin at 0.1 ?mol/L (P 0.05). Subsequently, the addition of TNF-? significantly increased IL-8 production, which could be reversed by the addition of NK-104.Conclusions:These results suggest that NK-104 at low dose (0.1 ?mol/L) inhibits NF-?B activation and decreases IL-8 production induced by TNF-?. It is therefore expected to play a role in the new strategy for the treatment of hepatocellular carcinoma.