The Studies Of Nitidine Chloride Of Antihepatocarcinoma Effect In Vitro

Nitidine chloride is a kind of benzophenanthrene alkaloid isolated from the foot of Zanthox-y lum nitidum (Roxb. )DC. The previous study primaryly showed that nitidine chloride had the action on the anti-flammation,analgesia,spasmolysis and the anti-tumor effect. Nitidine chloride showed good clinical effect of chronic cell leukemia. There is little information about the effect of nitidine chloride on study the hepatoma therapy. The present study was designed to investigate both the anti-hepatoma mechanisms of nitidine chloride and proliferations of human liver cell and human kidney cell in vitro.Objective:1. To evaluate the anti- hepatoma effect and mechanism of nitidine chloride in vitro.2 . To investigate the proliferations of nitidine chloride of human liver cell and human kidney cell in vitro.Methods:1. The effects of nitidine chloride on human hepatoma SMMC-7721 cell in vitro were assayed by MTT method.2. The inhibitory proliferations of human liver cell and human kidney cell in vitro were assayed by MTT method, the concentration of superoxide dismutase ( SOD),malondialdehyde (MDA) , and lactate dehydrogenase (LDH) were determined at different times in all groups. 3. The actue toxicity of mice was tested by tail vein injection.Results:1. At the concentration of(0.8-12.8)μg/mL , Nitidine chloride had slightly inhibitory effect on SMMC-7721 cell lines. It had remarkable dose-dependant inhibitory effect on cytotoxicity.2. Nitidine chloride (0,2,4,8 )μg/mL could arrest cell cycle at G2/M phase, which had accleration of cells of G2/M phase and reduction of cells at G0/G1,S phases. The results also showed that nitidine chloride could induce apoptosis of SMMC-7721 cells with the rate (18.9±0.23)%,(23.3±1.54)%,(28.3±3.83)%, respectively, while the rate in control group was (2.0±0.06) %. This suggested that inhibitory effect of nitidine chloride on the growth of tumor cells was probably related to its arresting cell cycle at G2/M phase and inducing apoptosis of tumor cells.3. The expressions of bcl-2, bax and p53 in the SMMC-7721 cells treated with nitidine chloride was evaluated by immunohistochemical staining. The results indicated that nitidine chloride could decrease the bcl-2 expression, and increase the expression of Bax. The level of p53 protein was significantly inceased(p<0.01). The morphological feature changes of apoptosis cells showed typical morphological features, such as nuclear fragmentation,chromatin condensation,cellular shrinkage and vacuolalization in cytoplasm were noted, apoptotic bodies could be found with transmission electron microscope(TEM).4. Nitidine chloride had slightly inhibitory effect on human liver cell L-02 and human kidney cell 293. Inhibitory rate was remarkable dose-dependant .5 The LD50 in mice by tail vein injection was 19.999 mg/kg, its 95% CI and 99% CI were (19.999±2.696) mg/kg and (19.999±3.534) mg/kg, respectively.Conclusions:Nitidine chloride has potent inhibitory effect on the growth of SMMC-7721 cells. Studies on anti-hepatoma effect of nitidine chloride show that its mechanisms of nitidine chloride anti-tumor activity are probably related to these actions: (1) inhibition of tumor cells directly. (2) Induction of cancer cells apoptosis by bcl-2 down-regulated and p53,bax up-regulated at protein level in vitro. (3) Nitidine chloride can arreste the cell cycle at G2/M phase and induce apoptosis of tumor cells. The result show that its toxic effects of liver cel1 and kidney cell is small, so systemic toxicity of mice is .