| Background and PurposeColorectal cancer is one of most common tumors in worldwile.In recent years,the incidence of colorectal cancer shows an increasing trend.To clarify the etiology and molecular mechanisms of colorectal cancer,to improve its early diagnosis and therapeutic effect,to predict the early diagnosis and prognosis judgment are the focus of colorectal cancer research.Recently,more and more evidences showed that microRNAs have endogenous regulating function,which play a regulatory role in ontogeny,cell proliferation or apoptosis,cell differentiation,expression of viral replication,reproduction and tumors to a certain degree.A large number of experiments showed that the specifically expressed microRNAs are involved in the regulation of cancer development.Thus it has become a new hot spot in the research of molecular oncology.miRNA-200 family are hot molecules in recent studies,which include miRNA-200a.miRNA-200b?miRNA-200c?miRNA-141 and miRNA-429.Recently,lots of researches on the relation of miRNA-200 family with tumor development are studied,but the report about colorectal cancer is relatively rare.PI3K protein family participates in cell proliferation,differentiation,apoptosis and the regulation of glucose transport,and other cellular functions.The activation of PI3K leads to the production of second messenger PIP3 on plasma membrane.PIP3 combines with AKT,and leads to AKT activation.The activated AKT,also called p-AKT,regulates cell functions through causing the phosphorylation of enzymes,kinase and transcription factors.AKT play a role of resistance to apoptosis through multiple ways of target protein phosphorylation.AKT can inhibit the activity of protein hydrolysis enzyme Caspase-9 and thus prevent the activation of apoptotic cascade.PI3K-AKT signal transduction pathway plays an important role in cancer occurrence,development,treatment and outcome,and has become the new target for diagnosis and treatment of tumor.Recently,several studies have shown that microRNAs can directly or indirectly regulate PI3K-AKT pathway.A research shows that the expression of miRNA-200c can induce colorectal cancer stem cell features and the activity of PI3K-AKT pathway.Hence the correlation ship between colorectal cancer and molecular mechanism of miRNA-200 family,PI3K-AKT signaling pathway needs further research.Catalpol is one of the main active ingredients in rehmannia,which is also effective active ingredient in the function of nourishing Yin,with a variety of pharmacological effects such as diuretic,laxative,lowering blood sugar,protecting liver and anti-aging.Recent studies have found that catalpol had anticancer effect.In the current study,the purpose of this study was to observe the catalpol's effect in colorectal cancer cells,then to investigate its mechanism in colorectal cancer cell line and determine its therapeutic value in treating colorectal cancer.Method1.HCT-116 cell was cultured at various concentrations(0,25,50 and 100 ?g/ml)of catalpol.After 24-,48-,and 72-h treatment,20?l of MTT solution was added into each well and the mixture liquor was incubated at 37? for 4h.2.HCT-116 cell was treated with catalpol(0,25,50 and 100 ?g/ml)for 48h,and then Caspase-3 and Caspase-9 was determined by Caspase-3 and Caspase-9 Activity Assay Kit to investigate the effects of catalpol on Caspase-3 and Caspase-9 activities of HCT-116 cells.3.HCT-116 cell was treated with catalpol(0,25,50 and 100 ?g/ml)for 48h,and then the cells were measured with Annexin V-FITC/PI Apoptosis Detection kit to study the capacity of catalpol induced apoptosis of HCT-116 cell,and then measure the quantification of catalpol-induced apoptosis ratio in HCT-116 cells using flow cytometry.4.HCT-116 cell was treated with catalpol(0,25,50 and 100 ?g/ml)for 48h,and then nucleic morphology of cells was investigated by DAPI staining on fluorescence microscope,to observe the effects of catalpol on the nucleic morphology of HCT-116 cell.5.HCT-116 cell was treated with catalpol(0,25,50 and 100 ?g/ml)for 48h,and then the expression of PI3K,p-AKT and AKT protein were measured by Western Blot,to investigate the effects of catalpol on PI3K-AKT signal pathway.6.HCT-116 cell was treated with catalpol(0,25,50 and 100 ?g/ml)for 48h.With the purpose of explore the effects of catalpol on miRNA-200 expression of HCT-116 cell.Total RNA was extracted from HCT-116 cell using Trizol.miRNA-200 was performed using the High Capacity cDNA Reverse Transcription kit with ABI 7500 quantitative PCR system according to the manufactures instructions.7.To carefully verify the mechanism of catalpol on proliferation and apoptosis in human colorectal cancer HCT-116 cells,PI3K inhibitor(LY294002,5 ?M)was added into HCT-116 cells,down-regulation of PI3K-AKT and then investage the mechanism of anticancer effect of catalpol.The expression of PI3K,p-AKT and AKT protein were measured by Western Blot,miRNA-200 was performed using the High Capacity cDNA Reverse Transcription kit with ABI 7500 quantitative PCR system.Results1.Effect of catalpol on the cell proliferationHCT-116 cells were treated with catalpol at different concentrations(0,25,50 and 100 ?g/ml)for 24-,48-,and 72-h,respectively.The absorbance of HCT-116 cells was detected with MTT assay.The cell proliferation of HCT-116 cell was inhibited by the treatment of catalpol,and this inhibition efficiency was in a dose-and time-dependent manner.These data suggested catalpol might markedly inhibit human colorectal cancer cell viability.2.The effects of catalpol on Caspase-3 and Caspase-9 activities of HCT-116 cellHCT-116 cells were treated with different doses of catalpol(0,25,50 and 100?g/ml)for 48h,a further increase in the activities of Caspase-3 and Caspase-9 at 50 and 100 ?g/ml catalpol concentrations compared with controls,respectively.3.Effects of catalpol induced apoptosis of HCT-116 cellTo observe the cell apoptosis using Annexin V-FITC/PI double dye notation after the treatment of catalpol in the HCT-116 cells for 48h,the apoptosis ratio was measured using flow cytometry assay.The apoptosis percentage was dramatically increased after 48 h treatment with 50 or 100 ?g/ml catalpol,respectively.4.Effects of catalpol on nucleic morphology of HCT-116 cellWe assessed the anticancer effect of catalpol on nucleic morphology of HCT-116 cells.Catalpol(50 or 100 ?g/ml)influence nucleic morphology of HCT-116 cells and significantly accelerated cell nucleic apoptosis of HCT-116 cells compared with those of the control group.5.Effects of catalpol on PI3K-AKT signal pathwayAfter treatment with catalpol(0,25,50 or 100 ?g/ml)for 48h,the expressions of PI3K,p-AKT and AKT proteins were analyzed by Western Blot.The expressions of PI3K and p-AKT proteins were obviously decreased compared with those of the control group.6.Effects of catalpol on miRNA-200 expression of HCT-116 cellTo further inquiry the mechanism of catalpol on proliferation and apoptosis in human colorectal cancer HCT-116 cells,we tested miRNA-200 expression in HCT-116 cells.The expression of miRNA-200 was increased after treatment with catalpol(0,25,50 and 100 ?g/ml)for 48 h compared with those of the control group.7.Down-regulation of PI3K-AKT on PI3K-AKT signal pathway and cell proliferation in catalpol treatmentTo carefully explore the mechanism of catalpol on proliferation and apoptosis in human colorectal cancer HCT-116 cells,PI3K inhibitor(LY294002,5 ?M)was added into HCT-116 cells.The result indicated that PI3K inhibitor effectively interdicted PI3K-AKT signal path,suppressed PI3K and p-AKT proteins expressions in HCT-116 cells compared with those of catalpol-treated(50 ?g/ml)group.Meanwhile,down-regulation of PI3K-AKT availably augmented the anti-proliferation effect of catalpol on HCT-116 cells compared with those of catalpol-treated(50 ?g/ml)group.8.Down-regulation of PI3K-AKT on miRNA-200 of HCT-116 cellsTo carefully verify the mechanism of catalpol on proliferation and apoptosis in human colorectal cancer HCT-116 cells,miRNA-200 expression in HCT-116 cells was detected after down-regulation of PI3K-AKT.Down-regulation of PI3K-AKT remarkably enhanced the miRNA-200 expression in HCT-116 cells compared with those treated with catalpol(50?g/ml)group.Conclusions1.The MTT assay showed the cell proliferation of HCT-116 cell was inhibited by the treatment of catalpol.2.Catalpol could improve the Caspase-3 and Caspase-9 activities of HCT-116 cells,indicated that catalpol has the ability of inducing apoptosis of HCT-116 cell.Annexin V-FITC/PI double dye notation discovered the apoptosis percentage was dramatically increased after 48h treatment with catalpol and we found catalpol significantly accelerated cell nucleic apoptosis of HCT-116 cells.3.After treatment with catalpol for 48h,the expressions of PI3K,p-AKT and AKT proteins in HCT-116 cells were obviously decreased,indicated that catalpol could inhibit PI3K-AKT signal pathway.The expression of miRNA-200 was increased after treatment with catalpol,demonstrated the anticancer effect of catalpol was possibly connected with miRNA-200 family.4.PI3K inhibitor(LY294002,5 ?M)was added into HCT-116 cells,the expressions of PI3K,p-AKT and AKT proteins were further decreased,and the miRNA-200 expression was remarkably enhanced.Thus the down-regulation of PI3K-AKT signal pathway and high miRNA-200 expression were possibly connected with anticancer effect of catalpol. |
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