| Objective: To screen out the geographic population of cantharides which had the significantly activity on the hepatocellular carcinoma, we preliminary analyzed the bound cantharidin content in cantharides selected from Guizhou Province and surrounding regions, and to carry out the study on the effect of biological behavior on hepatocellular carcinoma cells of the bound cantharidin. In order to provide scientific research basis for the development and utilization of the bound cantharidin in excellent geographic population cantharides.Methods: The bound cantharidin of cantharides was extracted by water extraction. Cantharidin content in the cantharis were analyzed by using GC-MS.The sulforhodamine B(SRB) assay was employed to evaluate the inhibitory effect of the bound cantharidin on hepatocellular carcinoma cells Hep G2 in vitro,and screen out 3-4 excellent geographic populations with the better antitumor activity.Morphological alteration of hepatoma carcinoma Hep G2 cells was observed by phase contrast microscopy.Annexin V-FITC/PI staining was employed to detect the influence of the bound cantharidin from the excellent regional species on apoptosis of Hep G2 cells.The change of cell cycle was tested by PI markers combine with flow cytometry.Transmission electron microscopy was used to observed the changes of ultrastructural of hepatoma carcinoma Hep G2 cells.Western blotting assay was applied to measure the effect of the bound cantharidin on expression levels of Bax,Bcl-2 protein of Hep G2 cells.Results: The content of BCMP and BCML collected in Luodian County of Guizhou Province,which were higher than the same species collected from other geographic populations:375 mg·L﹣1 and 290 mg·L﹣1,respectively. The content of BCMP and BCML in Mao Jing Town followed, 262mg·L﹣1 and 199 mg·L﹣1,respectively.The bound cantharidin from different geographical species all have inhibitory effects on the proliferation of hepatocellular carcinoma cells Hep G2.,Moreover, the BCMP and BCML in the Maojing town of Guizhou Province have the most significantly inhibitory effects on the proliferation of hepatocellular carcinoma cells Hep G2, and the IC50 value were 0.78 mg·L﹣1 and 0.65mg·L﹣1respectively. The inhibitory effects on the proliferation of hepatocellular carcinoma cells Hep G2 of the BCMP and BCML in the Luo dian county of Guizhou Province followed, and the IC50 value were 0.88 mg·L﹣1 and 0.86mg·L﹣1respectively. The observation of phase contrast microscopy showed that, the BCMP and BCML extracted in Luodian County and Maojing Town at different concentration groups can cause the number of cancer cells reduce, cells morphology become irregular,cells shrinkage and smaller size. Flow cytometry showed that, compared with control,the apoptosis rates of Hep G2 cells were(9.77±1.33)% and(10.27±1.44)%(P<0.05),(22.16±1.35)% and(22.33±1.11)%(P < 0.05) after the BCMP and BCML in Maojing Town treatment for 24 h and 48 h,respectively. The apoptosis rates of Hep G2 cells were(7.60±1.05)% and(8.17±0.50)%,(18.70±2.26)% and(18.87±2.50)%(P<0.05) after the BCMP and BCML in Luodian County treatment for 24 h and 48 h,respectively. The results of cells cycle indicated that a decrease in S phase cells and an increase in G2/M phase cells after the BCMP and BCML in Maojing Town treatment on Hep G2 cells for 24 h,which appeared G2/M phase arrest.Meanwhile,it also displayed that a S phase cells,an increase in G2/M phase cells and a significant increase in G0/G1 phase cells,which appeared mainly G0/G1 phase arrest. Ultrastructure observation displayed that the endoplasmic reticulum and mitochondria in Hep G2 cells appeared swelling after the BCMP and BCML in Luodian County and Maojing Town treatment, the change of ultrastructure is more obvious with the extension of time. Western blotting assays showed that the BCMP and BCML in Maojing Town within a certain concentration range can increased the expression level of Bax protein,but decreased the expression level of Bcl-2 protein.Conclusions: 1. There is a wide range of variation of cantharidin content among the different kinds of cantharis collected from different regions.2. Among which, the BCMP and BCML in the Maojing town have the most significantly inhibitory effects on the proliferation of hepatocellular carcinoma cells Hep G2 than others,and the inhibitory effects of the BCMP and BCML in Luodian County followed.3. We found that the bound cantharidin of cantharis could have effects on biological behavior of Hep G2 cells proliferation and apoptosis, mainly to inhibit proliferation and induce apoptosis of Hep G2 cells,which the reason may be related to the change of cancer cells ultrastructure and the cycle arrest of cells, otherwise,it may also be related to the up-regulation of Bax expression,the down-regulation of Bcl-2 expression and the imbalance ratio of Bcl-2/Bax. |
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