| Background and Purpose Oxymatrine,an alkaloid active ingredient,is extracted from Sophora flavescens Ait.It shows strong anti-inflammatory anticancer activities,but how oxymatrine exhibits anticarcinogenic effects in human colorectal cancer is still unclear.In this study we aimed to explain the exactmechanism by which oxymatrine exhibits anticarcinogenic effects in human colorectal cancer and provide experimental basis for the treatment of colorectal cancer with traditional Chinese medicine monomer.Method1.We cultured of colorectal cancer cells in vitro,observed the growth state of cells,and used the cells in the logarithmic phase for the experiment.Essay grouping:(1)experimental group:the colorectal cancer cells were dealed with different concentrations of oxymatrine(mg/ml)(0.25,0.5,0.75,1.0,1.25,1.5,1.75,2);(2)control group: the colorectal cancercells without oxymatrine;(3)blank control group:only cell culture medium without the colorectal cancer cells.Dealed with different concentrations of the oxymatrine(mg/ml)(0.25,0.5,0.75,1.0,1.25,1.5,1.75,2.0)for 24 h,the cytotoxic effect of oxymatrine on SW480,RKO,HCT116 were assessed with MTT assay.Treated with different concentrations of the oxymatrine(mg/ml)(0.25,0.5,0.75,1.0)for 24 h,migration and invasion was examined by wound healing assay and Matrigel invasion assay,respectively.The morphological changes of the cells before and after the intervention were observed by inverted phase contrast microscope.RKO cells were divided into four groups,the final concentration of oxymatrine intervention was 0,0.25,0.5,0.75(mg/ml).The protein expression of E-cadherin,N-cadherin,Snail and P65 protein in each group was detected by Western blot assay(Blotting Western).2.Design and synthesis of P65 sh RNA with high interference efficiency by using gene interference technique.The experiment was divided into four groups,namely,control group,oxymatrine group(final concentration 0.25mg/ml),P65 sh RNA group,oxymatrine(final concentration 0.25mg/ml)+ P65 sh RNA group.The protein expression of E-cadherin,N-cadherin,Snail protein in each group was detected by Western blot assay(Blotting Western).The morphological changes of the cells were observed by inverted phase contrast microscope.migration and invasion was examined by wound healing assay and Matrigel invasion assay,respectively.Result1.MTT assay showed that oxymatrine can inhibit the proliferation of colorectal cancer cells,when the oxymatrine intervention concentration in the range of 0~2mg/ml,with the increase of the concentration of oxymatrine intervention,the colorectal cancer cells proliferation inhibition was more obvious.Treated with different concentrations of oxymatrine(mg/ml)(0,0.25,0.5,0.75),the migration distance in each group were 263.00±25.51,170.25±46.38,103.87±33.90,75.46±20.19.In the Transwell chamber invasion assay,the number of cells in 0mg/ml group,0.25mg/ml group,0.5mg/ml group and 0.75mg/ml group through the upper chamber were: 166±8.89,130.65±10.23,93.60±9.21,62.28±7.07.Without intervention of oxymatrine,colorectal cancer cells were elongated spindle mesenchymal cells,loosely connected.Treated with oxymatrine,cancer cells wereepithelioid morphology,tight junctions,almost invisible pseudopodia.Western blot assay showed that the protein expression of E-cadherin in 0mg/ml group,0.25mg/ml group,0.5mg/ml group and 0.75mg/ml group were 0.30±0.10,0.48±0.19,0.72±0.13,1.06±0.44,respectively;The protein expression of N-cadherin in 0mg/ml group,0.25mg/ml group,0.5mg/ml group and 0.75mg/ml group were 0.50±0.10,0.33±0.09,0.25±0.08,0.18±0.05,respectively;The protein expression of Snail in 0mg/ml group,0.25mg/ml group,0.5mg/ml group and 0.75mg/ml group were0.93±0.04、0.71±0.12、0.62±0.10、0.35±0.07,respectively;The protein expression of P65 in 0mg/ml group,0.25mg/ml group,0.5mg/ml group and 0.75mg/ml group were 0.75±0.03,0.40±0.08,0.34±0.08,0.20±0.06,respectively.2.Successfully designed and synthesized a P65 sh RNA with high interference efficiency.The expression of E-cadherin protein in the control group,oxymatrine group,P65 sh RNA group,oxymatrine +P65sh RNA group were:0.16±0.01,0.23±0.02,0.33±0.03,0.5±0.06;The expression of N-cadherin protein in the control group,oxymatrine group,P65 sh RNA group,oxymatrine+P65sh RNA group were: 0.60±0.04,0.46±0.07,0.32±0.06,0.20±0.05;The expression of Snail protein in the control group,oxymatrine group,P65 sh RNA group,oxymatrine +P65sh RNA group were: 0.93±0.04、0.71±0.12、0.62±0.10、0.35±0.07.In summary,the expression level of N-cadherin and Snail protein in P65 sh RNA groupwas lower than that of oxymatrine group,oxymatrine+P65sh RNA group was lower than oxymatrine group and P65 sh RNA group,and the expression level of E-cadherin was on the contrary.Inverted phase contrast microscope shown cells in control group were elongated spindle shaped mesenchymal like cells and loosely connected cells.Cells in oxymatrine group or P65 sh RNA were more connected closely than the control group.Compared with oxymatrine group or P65 sh RNA group,cells in oxymatrine +P65sh RNA group were more closely connected,almost invisible pseudopodia.The migration distance in each group were 297.31±65.91,180.00±50.24,116.84±36.89,59.09±15.36.In the Transwell chamber invasion assay,the number of cells in each group through the upper chamber were:143.67±10.97,119.33±20.23,97.67±18.77,50.33±8.50.conclusion1.Oxymatrine exerts anticancer effects on colorectal cancer cells,it has the ability to suppress the proliferation,migration and invasion of colorectal cancer cells.2.Oxymatrine could affect the epithelial-mesenchymal transition associated proteins in colorectal cancer cells.The ability of suppress the migration and invasion of colorectal cancer cells is associated with inhibiting the epithelial-mesenchymal transition.3.Oxymatrine could affect the NF-?B pathway related proteins.Oxymatrine suppresses the activation of the NF-κB signaling pathway and inhibits colorectal cancermigration and invasion by modulating epithelial-mesenchymal transition. |
PDF Full Text Request