| Natural anti-tumor drugs are an important branch of anti-cancer drugs and occupy an extremely important position in clinical anti-cancer treatment.In this study,a traditional Chinese medicine,the Laggera pterodonta(DC.)Benth was selected as research object which show antipyretic anti-inflammation and antitussive effects,as well as anti-tumor potential.The small molecule compounds in it were separated and purified to screen the substances with the activity of promoting tumor cell apoptosis and inhibiting tumor cell growth.And its mechanism of anti-tumor effect in vitro was explored.The purpose of the study was to provide a research basis for the new anti-tumor drugs possessed with high-efficiency,unique target.The following results were obtained.1.Using silica gel,gel,high-performance liquid chromatography and other chromatographic separation techniques,7 candidate compounds,including 4 flavonoids and 3 sesquiterpenes,were isolated from the Laggera pterodonta(DC.)Benth.Identifying by NMR,MS,etc,the seven compounds are 5?-olide,Isocostic acid,3H-Naphth[1,8a-b]oxirene-7-acetic acid,Artemetin,3,6,7,4'-Tetra-O-methyl-5,3'-dihydroxyflavone,5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3,6,7-trimethoxy-4H-chromen-4-one,4',5-Dihydroxyflavone.2.The in vitro antitumor activities of the above seven compounds isolated from the Laggera pterodonta(DC.)Benth were evaluated by MTT assay,and the inhibition rates were 89%,11.3%,4%,9.7%,16.1%,0%,2.9%,respectively.The results showed that 5?-olide had the best antitumor activity.3.The IC50 of 5?-olide to 5 tumor cells was tested with the MTT method and the IC50 of SMMC 7721,SW480,MDA-MB-231,T47 D and Hela were 3.36 mM/L,4.04 mM/L,11.8 mM/L,10.57 mM/L,14.31 mM/L,respectively.4.Treating by 5?-olide,the growth curve of SMMC 7721 and SW480 were detected.The result showed that the inhibitory activity of 5?-olide to SMMC 7721 and SW480 was verified under the concentration ?2 mM and ?24h.5.The nuclear morphology of SMMC 7721 was observed by fluorescence microscopy.After treated with 5?-olide at 8 ?M for 24 h,and the abnormal changes of the nuclei in SMMC 7721 cells were obvious.Some nuclei were crescent-shaped and had a typical apoptosis pattern.After staining by Hoechst/PI,it was found that with the increase of 5?-olide concentration and the extension of the action time,the number of bright blue cells increased.It indicated the increase of apoptosis.6.The effect of 5?-olide on the endogenous reactive oxygen of SMMC 7721 was detect by DCFH-DA method.The relation of apoptosis and ROS was determined with ROS clearance test.The results showed that bright green fluorescence of the cell was induced by the H2O2 and the 8 ?M 5?-olide.After added NAC to clear ROS,the cytotoxicity of 5?-olide on SMMC 7721 was significantly reduced.It indicated that 5?-olide induced the apoptosis of SMMC 7721 by triggering excessive ROS production.7.The effect of 5?-olide on mitochondrial membrane potential of SMMC 7721 was observed by testing JC-1.After labeled cell mitochondria with mito-tracker Red probe,the effects of 5?-olide on cell mitochondria morphology were observed under laser confocal scanning.The results showed that the cell of control group possessed stronger red fluorescence and stronger green fluorescence for FCCP.After treated with 5?-olide,the shape of mitochondria stained with mito-tracker Red were granular.It indicated that the mitochondrial membrane potential was reduced and the mitochondria was broke up by 5?-olide.8.Treated with 5?-olide,the protein changes related mitochondrial apoptosis pathway in SMMC 7721 were observed by Western blot.In the 5?-olide treatment group,it was verified that Bax,Bid,caspase 3 and PARP were up-regulated,Bcl-2 was down-regulated in the cell.9.The effects of 5?-olide on the physiological properties in the cells were examined by colony formation assay,cell scratching and transwell assay.After treatment with 5?-olide for 24 h,with the increase of 5?-olide concentration,the number of colonies formed by monoclonal cells decreased,and the healing of cell scratch was reduced,and its healing ability was gradually weakened,and the number of transwell cells migrating to the lower chamber was also reduced.Conclusion: Seven compounds were isolated from the Laggera pterodonta(DC.)Benth,among which 5?-olide possessed the best antitumor activity and inhibited the proliferation of various tumor cells,and the inhibiting activity on SMMC 7721 was most strongly.The 5?-olide can cause excessive production of ROS in SMMC 7721,decrease its mitochondrial membrane potential and up-regulate pro-apoptotic proteins Bax and Bid,and down-regulate the antiapoptotic protein Bcl-2.Finally,the caspases were activated to initiate the cascade reaction of apoptosis and promote apoptosis.This study revealed for the first time that 5?-olide can initiate apoptosis of cancer cells by activating the mitochondrial caspases pathway,promoting ROS production,inhibiting the formation of cell cloning,and inhibiting cell migration.This study provides a basis for the development of new antitumor drugs. |
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