The Effect And Related Mechanism Of Ornithogalum Saponins On Autophagy And Apoptosis In Colorectal Cancer Cells

Research bjective:Ornithogalum caudatum Ait(OCA)has the obvious antitumor effects.Saponins are active components.Here we objectively oberserved the mechanism of action of Ornithogalum caudatum Ait Saponins(OCA-TS)on human CRC cell lines,focusing on exploring the role of autophagy,apoptosis and cell cycle control in cytotoxic anticancer effects.Methods:1.(1)Under the sterile conditions,the each group mice were injected subcutaneously LoVo human colon cancer cells suspension(1×10~7/0.1ml,0.1ml/single)in the right axillary.When the tumor volume reached 100 mm3,the tumor bearing nude mice were randomly divided into 3 groups.Three groups of mice were treated for 7consecutive days and the weights were recorded daily.After the last treatment,the mice were dissected to excise and weigh the tumors.(2)General tissue morphology and histopathological changes were visualized by hematoxylin and eosin(H&E)staining eventually.Immunohistochemical detection was performed after sequence of actions.2.We used MTT and Colony formation assay to analyze the cellular proliferation.Flow cytometry assays,Annexin-V-FITC/PI,Ad-mCherry-GFP-LC3Bfluorescence assay and Transmission electron microscopy were used to investigate cell cycle,apoptosis and autophagy.Results:1.The growth of the tumor was slow and limited,tumor size decreased almost twice in OCA-TS-treated animals,The pathological features of tumors were observed by HE stained and IHC staining using VDAV-1 antibody,which plays an important role in mediating autophagic signaling in mitochondria.Under microscope,the HE staining results we observed indicated that multifocal necrosis area in treatment group were significantly less than that of the control2.(1)OCA-TS suppressed proliferation,in a time-or concentration-dependent manner in human CRC cells with low cytotoxicity on normal cells of colon.In comparison with 24 hours of treatment,the cytotoxic effect of 48and72 hours was most evident,with an half-maximal cell inhibitory concentration(IC50)respectively:after 48hours of OCA-TS leaf action,IC50:RKO,1.28mg/ml;Caco-2,0.96mg/ml;HCT116,0.96mg/ml;LoVo,0.96mg/ml;after 48 hours of OCA-TS bulb action,IC50:RKO,1.17mg/ml;Caco-2,1.00mg/ml;HCT116,0.97mg/ml;Lo Vo,0.64mg/ml.The rate of colon formation was reduced,with the action concentration Increasing.(2)OCA-TS leaded to G1 cell cycle arrest and promoted the apoptosis of LoVo cells,the total apoptosis rate was close to 50%,compared with positive drug control group(p(27)0.01).(3)OCA-TS apparently induced autophagy in Lo Vo cells and increased autophagic flux by the up-regulation of Beclin 1 and LC3-II.Punctate structure,representing autophagosomes,could visualise the formation of LC3-II.Thus,after transiently transfected with GFP-LC3,cells were treated with OSW-1 for different times.Conclusions:1.The growth of the tumor was slow and limited,tumor size decreased almost twice in OCA-TS-treated animals 2.OCA-TS suppressed proliferation,in a time-or concentration-dependent manner in human CRC cells with low cytotoxicity on normal cells of colon.The result also shows that the total saponins extracted from Ornithogalum bulb and leaf exhibited antitumor activity 3.OCA-TS leaded to G1 cell cycle arrest and promoted the apoptosis of LoVo cells by preventing the mitochondrial apoptosis pathway and regulating the expression of the apoptotic regulatory proteins Bcl-2 and Bax.4.In addition,OSW-1 apparently induced autophagy in LoVo cells and increased autophagic flux by the up-regulation of Beclin 1 and LC3-II.