| Background and Objection:Colorectal cancer(CRC)is one of the most common digestive malignant tumors,as well as the third most common malignant tumor worldwide.Every year,almost 2 million new patients are diagnosed as colorectal cancer,and approximate 700 thousands colorectal cancer patient are dead worldwide.Thus,colorectal cancer is also the forth most common tumor types that cause the death of patients,which seriously endanger people's life and healthy.In the recent years,the morbidity of colorectal cancer has been increasing rapidly,rising by more than 200 thousands new cases per year from 1990 to 2012,especially in the Western countries(55%).In the decades,with the rapid development of economy in our country,people life-style changed greatly,especially in the developed cities,which significantly increasing the incidence of colorectal cancer.In current studies,we found that approximate 50%of patients with colorectal cancer would suffer from tumor metastasis.In addition,up to about 90%of colorectal cancer related-death were caused by tumor metastasis.Owing to the characteristic of high incidence,metastasis and death rate of colorectal cancer,it is necessary to clarify the molecular mechanism of tumorgenesis and development of colorectal cancer,which will provide more help for colorectal cancer prevention and therapy.Methods1.Ethanol induces inhibition of cell proliferation and promotion of migration by activating TGF-?/SMAD signaling pathwaya.The current study sample included 100 male patients diagnosed with primary CRC between January 2008 and December 2013 by the Department of Pathology in Zhujiang Hospital of Southern Medical University in Guangdong,China.88 health controls,free of colorectal cancer history at the time when the case was diagnosed,were randomly selected from the cohort of outpatients.We restricted our analysis to population-based cases,not selected on the basis of family history.Study participants completed a self-administered risk-factors questionnaire regarding sociodemographic information,medical and lifestyle histories via telephone at the time of enrollment.b.After short-term treated with two different concentrations of ethanol,the CCK-8 and EdU incorporation assay were performed to detect the cell proliferation.In addition,the cell-cycle and apoptosis analysis were also applied to find out the causing of abnormal cell proliferation.Moreover,transwell and wound-healing assay were performed to detect the effect of short-term ethanol-treated to cell motility and migration in vitro.c.Animal studies were applied to perform tumor growth and metastasis assay.Colorectal cancer cells were injected subcutaneously on the back of BALB/c nude mice aged 4 to 5 weeks to detect the formation and proliferation of tumor.Cells were also injected into nude mice through the tail vein to observe their lung homing potential.d.Western blot,immunofluorescence,real-time PCR and ELISA assay were performed to determine the molecular mechanism underlying ethanol inducing inhibition of cell proliferation and promotion of cell migration.2.Ethanol promotes colorectal cancer EMT and metastasis by inducing cytoplasmic localization of RUNX3 proteina.The current study sample included 63 patients diagnosed with primary CRC between January 2013 and December 14 by the Department of Pathology in Zhujiang Hospital of Southern Medical University in Guangdong,China.Western blot assay was applied to determine the relationship between alcohol consumption and the abnormal expression of RUNX3 in colorectal cancer patients.b.Real-time PCR was applied to detect the expression of RUNX3 transcription after treated with short-term ethanol.Immunofluorescence assay was performed to observe the subcellular distribution of RUNX3 protein.In addition,western blot assay was also applied to respectively detect the expression of RUNX3 protein in cytoplasm and nuclear.c.By using the RUNX3 over-expression vector,product of which will specifically locate in nuclear.CCK-8 assay was applied to detect the effect of nuclear RUNX3 protein on short-term ethanol-induced cell proliferation inhibition.Transwell assay,as well as wound-healing assay,was performed to find out the role of nuclear RUNX3 protein in the promotion of cell migration induced by short-term ethanol treatment.Western blot and immunofluorescence assay were also applied to detect the expression of P21,Snail and EMT-related protein.d.Immunofluorescence assay was applied to observe the co-localization of RUNX3 and Smad2 or p-Smad2 protein,Pearson correlation coefficient was provided by the software of Olympus company.Co-immunoprecipitation was applied to confirm the effect of ethanol on the combination of RUNX3 and Smad2 or p-Smad2.Result1.Ethanol induces inhibition of cell proliferation and promotion of migration by activating TGF-?/SMAD signaling pathwayAccording to the analysis of patients' pathological data and life-style information,we found that alcohol consumption was related to the morbidity of colorectal cancer,which was negatively related to the T class,dose-dependently.In addition,alcohol consumption was also positively related to the N class,but without dose-dependence.Gain-and loss-of function assay in vitro revealed that short-term ethanol treated would dose-dependently inhibit the proliferation of colorectal cancer cells by inducing cell-cycle arrest but not apoptosis.Short-term ethanol treated would also increase the motility and migration potential of colorectal cancer cells by promoting the initiation of epithelial to mesenchymal transition,which played a vital role in tumor metastasis.Further studies also determined that short-term ethanol treated would inhibit the proliferation and promote the lung homing potential of cancer cells in vivo.Short-term ethanol treated would activate the TGF-?/SMAD signaling pathway by up-regulating the transcription of TGF-?1 genes,and the secretion of TGF-?1 protein.The activation of TGF-?/SMAD signaling pathway would subsequently promote the expression of P21,Snail and EMT-related protein.2.Ethanol promotes colorectal cancer EMT and metastasis by inducing cytoplasmic localization of RUNX3 proteinAccording to the analysis of the expression of RUNX3 protein in clinical tissue samples and patients' life-style information,we found that alcohol consumption was positively related to the expression of RUNX3.Further studies revealed that short-term ethanol would decrease the transcription of RUNX3 gene,and inducing a cytoplasmic mislocalizetion of RUNX3 protein in NCM460,SW480 and HCT116 cell lines.The effects of short-term ethanol treated on the EMT,motility and migration of cells were weakened by transfection with RUNX3 over-expression vector,the product of which would specifically locate in the nuclear of cells.Short-term ethanol treatment induced cytoplasmic localization of RUNX3 protein by promoting the dissociation of RUNX3 and p-Smad2 but not Smad2.ConclusionAlcohol consumption is related to the incidence and development of colorectal cancer.Short-term ethanol would inhibit cell proliferation,as well as promoting cell migration,by activating the TGF-?/SMAD signaling pathway.In addition,short-term ethanol treatment would induce the cytoplasmic mislocalization of RUNX3 protein by promoting the dissociation of RUNX3 and p-Smad2,which would increase the sensitivity of cells to TGF-?/SMAD signaling pathway,promoting cells epithelial to mesenchymal transition and metastasis. |
PDF Full Text Request