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The Study on Anti-cancer Activity of Chemical Constituents from Lactarius Vellereus and Suillus Granulatus and Its Mechanisms

Author: WangJie
Tutor: CaoZhiRan
School: Hebei University
Course: Pharmacology
Keywords: Lactarius vellereus Suillus granulatus Mitissimol D Anti-cancer activity immunity activiity Apoptosis MTT
CLC: R96
Type: Master's thesis
Year: 2010
Downloads: 96
Quote: 2
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Abstract


Aim To screen the twelve chemical constituents from higher fungi for anti-cancer activities in vitro. Furthermore the related mechanisms and the immunity toxicity of Mitissimol D which was distracted from Lactarius vellereus were investigated. Then to establish a basis for developing new anticancer drugs from this kind of compound.Methods1 The cytotoxicity of the twelve chemical constituents of higher fungi in five human tumor cell lines was investigated by MTT [3-(4-5-dimethylthiazol-2-yl)-2 ,5-diphenyltetrazolium bromide] colorimetric assay;2 Morphological changes of HeLa cells induced by Mitissimol D were observed by invert microscope, light microscope and fluorescent microscope. DNA ladder was observed using agarose gel electrophoresis. Flow cytometry was used to detect the effect of Mitissimol D on cell cycle and apoptotic rate;3 The proliferatory effet of Mitissimol D against T and B lymphocytes was also tested by MTT assay. The effect of Mitissimol D on IL-2 secreted by spleen cells was detected with ELISA.Results1 Among the twelve chemical constituents, Mitissimol D showed high cytotoxicity . For A549、Bel-7402、SMMC7721 and HeLa cells, Mitissimol D has a more inhibitive activity. The IC50 of 72h for the four kinds of cells are all less than 20μg/mL. Furthermore the inhibitory rate increased by the contact time in the range of the test, the inhibitory rate also increased with increases in the agent concentration. For HCT-8, the inhibitive activity was not significant;2 The HeLa cells were observed under the inverted microscope-falling off instead of adherence and volume shrinked. Marked morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentations were found clearly using Giemsa staining. Gel electrophoresis found the appearance of DNA ladder. The result of FCM demomstrated that it could significantly induce the apoptosis of HeLa cells. Cell apoptotic rates were 2.17%,4.1%,18.75% at 24h, 5.3%,5.5%,23.3% at 48h , 20.22%,10.43%,32.14% at 72h at Mitissimol D on centrations of 1,10,100μg/mL respectively,all significantly higher than those of the control group1.74% ,3.58% and 4.98%(P <0.05或P <0.01).Meantime Mitissimol D could block the cell cycle into S and G2 phase;3 The proliferative activity of spleen lymphocytes were decreased with Mitissimol D being combined with ConA or LPS. It could also decrease the IL-2 level secreted by spleen cells stimulated by ConA.Conclusion1 Mitissimol D exhibits strong time-dose-dependent cytotoxicity in four human tumor cell lines;2 The mechanism could be related with cell cycle block in S and G2 phases. Mitissimol D could availably inhibit the growth of HeLa cells and induce apoptosis. It has the potential to be a candidate for anticancer drug;3 Mitissimol D could down-modulate function of T and B lymphocytes in mice.The potential mechanism maybe related with the interference with the expression of cytokines.

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