The Chemoprevention Of Kaempferol On Colorectal Cancer

ObjectiveColorectal cancer(CRC)is the third incidence of malignant tumor in worldwide,second only to lung cancer and breast cancer,killing about one million people a year.Although mortality declined,the incidence continues to rise.So "prevention beforehand" is put forward,namely to prevent diseases is the most important thing for control colorectal cancer.Kaempferol is a kind of polyphenol,which is rich in a variety of fruits and vegetables,as well as traditional Chinese medicine,and generally was regarded as antioxidants compound.In recent years,more and more researchers focus on the effect of kaempferol on the research of reducing risk of chronic disease,especially in cancer research.Epidemiological studies have shown that kaempferol is inversely proportional intake and cancer.In vitro studies have shown that kaempferol can regulate tumor cell apoptosis,angiogenesis,inflammation,and transfer the relevant multiple signal transduction pathways.It is worth noting that kaempferol,on the one hand,kaempferol induce cancer cell apoptosis,but on the other hand,kaempferol seems to protect normal cell viability,in some cases,which make it could become a potential cancer chemoprevention agent.In addition,chromatin epigenetic changes also becomes the key to induce colorectal cancer etiology,mainly including tumor suppressor gene promoter regions CpG island methylation and selective histone modification.However,there is no literature reported the effect of kaempferol on colitis related colon cancer.Based on the current research,this article will be the first use of AOM/DSS mice model to evaluate chemoprevention of kaempferol on colorectal cancer,and verify its effect of colorectal tumor suppressor methylation regulation and the mechanism of anti-inflammatory in vitro.Methods1.Regulation of kaempferol on colon cancer cell viability and DACT2 mRNA expression levelThe commercial colonrectal cancer cell lines,HCT-116 and HT-29 cells,were treated with different concentration of kaempferol(0-150 μM)for 5 days to verified treatment concentration and time for following experiment by MTT assay.The gene expression level of DACT2 in the two cell lines were determined by RT-PCR.2.Regulation effect on DACT2 epigenetic alterations in colorectal cancer cell by kaempferolBased on the drug concentration determined by MTT assay,cells were treated with different kaempferol concentration for 5 days,and the expression of methyl-transferase and histone deacetylase were detected by western blotting in HCT-116 and HT-29 colorectal cancer cell.The demethylation effect of CpG island DACT2 promoter in the two cell lines by kaempferol were verified by MSP.3.Inhibition of kaempferol on DACT2 downstream Wnt/β-catenin signalingWnt/β-catenin signaling pathway key genes and proteins expression levels were determined by RT-PCR and Western blot assay in HCT-116 and HT-29 cell lines after kaempferol treatment.We further detect apoptosis and cell cycle proportion of kaempferol untreated and treated groups using flow cytometry.In addition,migration of two cell lines were tested by wound healing.Finally,we tested matrix metalloproteinases(MMPs)level of cell culture supernatants.4.Anti-inflammation and antioxidation effect of kaempferolWe use commercially LS174T and Caco-2 colorectal cancer cell lines as the research model in this part.The cell viability was determined by MTT method.The cell lysate of LS174T and Caco-2 colon cancer cell were blot on PVDF membrane for determined the expression of NF-κB,pNF-κB,COX-2,IκBα,pIκBα which were related to inflammation.We detect Nrf2 protein,a controller on antioxidant signaling,expression by immunofluorescence.5.Chemoprevention effect of kaempferol on AOM/DSS-induced colon cancer animal modelAOM/DSS were used to induce colitis-associated of colon cancer in C57BL/6 mice.3 days after AOM injection,75 mg/kg and 150 mg/kg kaempferol were treated for mice until the end of experiment.1 week after AOM injection,1.5%DSS in water was free drinking for a week,after which provide regular water two week,which is total 3 cycles until the end of experiment.We record weight change in the whole process of experimental and collect animal plasma for inflammatory factor levels testing and mainly organs including colorectal tissue.The colon tumor number and colon length were count,then colon were Fixed in the paraformaldehyde for H&E staining and PCNA and β-catenin protein immunohistochemistry or immunohistofluorescence.The mRNA expression of DACT2 and other gene of Wnt signaling pathway were detected by RT-PCR.Results1.Regulation of kaempferol on colon cancer cell viability and DACT2 mRNA expression level5 μM kaempferol showed significant inhibition in HT-29 cells after 5 days treatment.The IC50 of kaempferol on HCT-116 and HT-29 cells were 192.7 μM and 57.88 μM respectively.According to the MTT results,kaempferol treatment concentrations were identified as 0,1.25,2.5,5 μM and the time of treatment is 5 days.In addition,mRNA of DACT2,a tumor suppressor was significantly increased by kaempferol.2.Regulation effect on DACT2 epigenetic alterations in colorectal cancer cell by kaempferolThe results showed that methylation transferase and histone deacetylase expression were inhibited by kaempferol in HCT-116 and HT-29.Meanwhile,DACT2 gene methylation ratio was also reduced in the two cell lines.3.Inhibition of kaempferol on DACT2 downstream Wnt/β-catenin signalingThe results show that Axinl,β-catenin,Dvl3,LRP6,Wnt 5a,key genes of Wnt signaling,were suppressed by kaempferol and with a dose dependent.At the protein level,Wnt 5 a/b,Naked2,LPR6,Dvl2,Dvl3 protein expression was significantly suppressed only in HCT-116 colon cancer cells.In addition,kaempferol can arrest cell cycle in G1 phase and induce cell apoptosis.At the same time,kaempferol inhibit two cell migration and the secretion of MMPs.4.Anti-inflammation and antioxidation effect of kaempferolHigh concentrations of kaempferol significantly inhibit LS174T and Caco-2 colorectal cancer cell proliferation.The NF-κB and inflammation related protein expression levels were also significantly suppressed.In addition,the Nrf2 was induced by 100μM kaempferol in LS174T cells.5.Chemoprevention effect of kaempferol on AOM/DSS-induced colon cancer animal modelIn AOM/DSS induced colitis-induced colonrectal cancer animal model,the results show that kaempferol can significantly reduce the incidence of colorectal cancer compare to model group,and colorectal length of kaempferol treatment groups animal significantly longer than the model,but there is no significant difference in animal body weight.In addition,inflammatory cytokines IL-1β,IL-6,TNF-α of two dose kaempferol plasma has been decreased,of which IL-1β of 75 mg/kg kaempferol treatment animal is significantly suppressed.HE staining showed that kaempferol can reduce AOM/DSS inducing colorectal inflammatory cells infiltration,and can decrease PCNA protein expression.ConclusionIn this study,we research kaempferol effect on colitis-associated colon cancer in vivo for the first time,and the results show that kaempferol has a potential treatment for colon cancer.Kaempferol chemo-prevent colon cancer may through regulating DNMTs and HDACs protein expression,thus reduce colorectal cancer suppressor DACT2 gene expression level.With recovery DACT2 gene expression,Wnt signaling pathways were suppressed,which involved in tumor cell cycle,cell apoptosis,and cell migration.In addition,kaempferol reduced body inflammation and enhance the body’s antioxidant ability.Therefore,this research can provide theoretical basis for colorectal cancer chemoprevention.