| By using various kinds of methods of isolation and purification, researchers of the Center of Biology Venom of Zhengzhou University have seperated Fractions A and B from Buthus Martensii Venom whose antitumor effects have not been studied. In the present study the antitumor activity of Fractions A and B in vitro and their mechanism were investigated.MTT colormetric method and growth-inhibiting test and the colony formation inhibiting assay were applied in order to obersve the antineoplastic effects of Fractions A and B on the tumor cell lines, such as poor differentiation human gastric mucinous adenocarcinoma, MGC-803, and human mammary gland medullary carcinoma, BCaP-37. The results of MTT colormetric method showed that Fraction A had obvious cytotoxic effect on MGC-803 cells. The correlation between dose and response wassignificant (r=0.984, P<0.02). Cells treated with Fraction A for 48h, the fifty percent inhibitory concentration (IC50) was 25.119mg/L. Within the concentration between, Fraction A of 5-15mg/L exhibited significant growth inhibitory effect on MGC-803 cells, and the dose-response correlation was obvious. Fraction A had no apparent cytotoxic effect on BCaP-37 cells, but Fraction B had obvious cytotoxic effect both on MGC-803 cells and BcaP-37 cells (P<0.01). The dose-response correlations were significant (r=0.989 and 0.998, P<0.01). Tumor cells were treated with B for 48h, The IC50 of MGC-803 and Bcap-37 cells were 16.386mg/L and 17.179mg/L respectively. In growth-inhibitory test, from 5mg/L to 15mg/L, Fractions A and B exhibited significant growth inhibitory effects on MGC-803 cells(P<0.0l), having obvious dose-response correlation (r=0.994 and 0.990, P<0.01). The IC50 were 12.823mg/L and 6.368mg/L respectively. In the colony formation inhibitory assay, Fractions A and B exhibited significant colony formation inhibitory effect on MGC-803 cells (P<0.01) and had obvious dose-response correlation (r=0.997 and 0.987, P<0.02). The IC50 were 5.636mg/L and 11.290mg/L respectively.In order to investigate the mechanism of the cytotoxic and growth inhibiting function of Fractions A and B on MGC-803 cells and BcaP-37 cells, Flow Cytometry (FCM) was applied to test thechanges of cell cycle, intracellular Ca2+ content, the levels of the expression of P53, P16 and PCNA. MGC-803 cells treated with Fraction A were arrested in G0/Gj phase in the cell cycle, the number of cells in G0/Gl and S phase was significantly increased and reduced, respectively (P<0.05). Compared with the control group, the expressions of P53 and P16 were reduced (P<0.02), the intracellular Ca2+ concentration was increased significantly (P<0.02 or P<0.001). The expression of PCNA had no significant changes. Fraction B could arrest MGC-803 cells in G0/G[ phase, the number of cells in S phase was reduced (P<0.02). The expressions of P53, P16 and PCNA were also reduced (P<0.02). The intracellular Ca2+ concentration was increased significantly (P<0.02 or P<0.001). For BCap-37 cells, Fraction B could arrest them in G0/G1 phase, decrease the number of cells in S phase, reduce the expression of PCNA and increase the intracellular Ca2+ concentration significantly (PO.02 or P<0.001). But the expression of P53 and P16 had no obvious changes. Conclusion:1. Fraction A has significant cytotoxic effect on MGC-803 cells, but not on BCaP-37 cells. Fraction B has strong cytotoxic and growth inhibitory effect on MGC-803 cells and BCaP-37 cells.2. Fraction A can arrest MGC-803 cell cycle in G0/G1 phase andreduce the expressions of P53 and P16. The intracellular Ca2+ concentration is significantly increased. The expression of PCNA has significant changes when high dose of Fraction A was added to MGC-803 cells.3. Fraction B can arrest MGC-803 cells cycle in G0/G1 phase and significantly reduce the expressions of PCNA, P16 and P53. The intracellular Ca2+ concentration is significantly increased. For BCaP-37 cells, Fraction B can arrest them in G0/G1 phase in cell cycle and significantly reduce the expression of PCNA and increas... |
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