Wogonin Prevents Colorectal Cancer Tumorigenesis By Regulating P53 Nuclear Translocation

ObjectiveColonrectal cancer(CRC)as the third diagnosed and the fourth cause of malignant cancer related death,about one million people were killed every 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 CRC.Wogonin,a flavonoid extracted from labiatae plants root,has anti-tumor,anti-inflammatory,anti-oxidant,and antibacterial activities.Previous studies show that wogonin inhibit proliferative ability of cancer cells through apoptosis,moreover,wogonin suppress migration and invasiveness of tumor cells by Wnt/p-catenin pathways.P53 is a kind of cancer suppressor gene,it cytoplasmic localization will inhibit autophage,it nuclear localization,however,will promote autophage.Whether wogonin prevents CRC through p53 remains unclear and the impact of genomic status on cellular response to wogonin has not been illustrated yet.In this study,the anti-tumor efficacy of wogonin was addressed in vitro and in vivo.Methods1.Effects of wogonin on colon cancer cell viability,autophagy and apoptosisHCT116(a MSI cell line)and HT29 cells(a MSS cell line)were treated with different concentration of wogonin(0-80 μM)for 72 h to determine cell viability by MTT assay.Moreover,colony-formation was performed to assess the colony formation ability of wogonin on colorectal cancer cells.Furthermore,the role of wogonin on autophagy and apoptosis was evaluated on colorectal cancer cells as detected by using Flow cytometry and Western blot.2.Regulation of wogonin on ROS in colorectal cancer cells.After treatment with wogonin for 2 hrs,cells were collected and ROS were detected by flow cytometry using CM-H2DCFDA dye,the percentage of CM-H2DCFDA fluorescence were used to indicate ROS level in colorectal cancer cells.3.Regulation of wogonin on ER-stress and DNA-damage in colorectal cancer cells.Cells were treated with indicated concentrations of wogonin for 72 hrs.Cell lysates for protein blot analysis were prepared using RIPA buffer,and the effect of wogonin on ER-stress and DNA-damage in two cell lines were detected by Western blot.Moreover,gene expression of DDIT3 and XBP1 were performed by using Real-time PCR.In addition,comet assay was used to analyze the effect of wogonin on DNA-damage in colorectal cancer cells.4.Effects of wogonin on p53 nuclear translocation in colorectal cancer cells.Cells were treated with indicated concentration wogonin for 72 hrs,then cytoplasmic and nuclear protein were isolated.Western blot was used to detect phosphor-p53 at S15,S315 and S376(closely related with p53 nuclear translocation).Furthermore,immunofluorescence was used to evaluate the effect of wogonin on p53 nuclear translocation in colorectal cancer cells.5.Chemoprevention effect of wogonin on AOM/DSS-induced colon cancer animal modelWe used AOM/DSS induced colitis-induced colorectal cancer in C57BL/6 mice to explore the chemopreventive effect of wogonin on CRC in vivo.Upon AOM injection for 3 days,mice were treated for 50 mg/kg and 100 mg/kg wogonin until the end of experiment(20 weeks).After AOM injection for one week,mice were free access to 1.5%DSS water for a week,then provided regular water for next two weeks.The DSS exposure was repeated total three cycles until the end of experiment.During the experiment,body weights were recorded.At the termination of experiment,the colon tumor number and colon length were count.Tumor tissues were collected for western blot analysis of autophagy,apoptosis,ER-stress and DNA-damage related proteins.Results1L Wogonin induced cell death in colorectal cancer cellsTo evaluate the effect of wogonin on cytotoxicity,we performed MTT in HCT116 and HT29 cells.The IC50 of wogonin on HCT116 and HT29 cells were 70.14μM and 117.90 μM,respectively.Moreover,colony-forming assay also confirmed that wogonin inhibit colony formation capacity of colorectal cancer cells in a dose-dependent manner.In order to further illustrate the potential mechanism of wogonin induced cell proliferation inhibition,we investigate the effect of wogonin on pathways leading to cell death such as autophagic cell death and apoptosis.Results showed that wogonin could inhibit autophagy,but induce cell apoptosis in HCT116 cells.However,wogonin could induce cell autophagy instead of apoptosis in HT29 cells.2.Wogonin induced ROS in HCT116 cells and inhibited ROS in HT29 cellsTo evaluate the effect of wogonin on oxidative status in colorectal cancer cells,ROS was detected by flow cytometry.Wogonin elevated ROS level in HCT116 cells.When cells were pre-treated with NAC(ROS scavenger),NAC abolished the ROS elevation in HCT116 cells.Wogonin,however,inhibited ROS generation in HT29 cells.3.Effects of Twogonin on ER stress and DNA damage in colorectal cancer cellsExcess ROS damage to essential biomolecules,such as proteins and DNA,eventually accumulated unfolded protein or misfolded protein in endoplasmic reticulum(ER stress)and DNA damage loci.WB analysis showed that ER-stress and DNA-damage pathway were activated in HCT116 cells;whereas ER-stress andDNA-damage pathway were inhibited in HT29 cell.And comet assay,a gold-standard method for DNA damage assessment,was used to further detect the impact of wogonin on DNA damage.We found that wogonin dose-dependently induced genotoxic stress as demonstrated by the formation of comets in HCT116 cells,there was no same case occurred in HT29 cells.4.Wogonin modulated nuclear translocation of p-p53To investigate how p-p53 shuffle was affected by wogonin,we studied the cytoplasm/nucleus translocation of p-p53 by western blot and immunofluorescence.For total protein,p-p53(ser315)and p-p53(ser376)was increased in both cytoplasmic and total protein in HCT-116 cells.Immunofluorescence indicated that p-p53(ser15)was accumulated in cytoplasm after wogonin treatment,suggesting that wogonin could retained p-p53 in cytoplasm.In HT29 cells,p-p53(ser315)and p-p53(ser376)in both cytoplasmic and total protein were decreased.Immunofluorescence also supported that observation that wogonin promoted p-p53(ser15)nuclear import.These findings indicate that wogonin regulate p-p53 shuffle between nuclear and cytoplasm in CRC.5.Chemoprevention effect of wogonin on AOM/DSS-induced colon cancer animal modelThe AOM-DSS colon cancer animal model was used to evaluate the chemopreventive effect and toxicity of wogonin in vivo.After consecutive 20 weeks of wogonin administration by orally gavage,there was no significant body weight loss in mice.Tumor multiplicity with size of<3 and>3 mm were 55.55%and 11.11%in mice treated with 100 mg/kg wogonin compared with model group 33.34%and 46.67%,respectively,meanwhile,wogonin also prevented the shorten length of colon caused by AOM/DSS exposure.To figure out the effect of wogonin on autophagy and apoptosis in vivo,western blot was performed to detect proteins related to autophagy and apoptosis.After 1OOmg/kg wogonin treatment,autophagy was inhibited and apoptosis was induced.Consistent with observations in HCT116 cells,wogonin activated ER-stress and DNA-damage pathway in tumor tissue,compared to that in control group.ConclusionIn this study,we investigate the effect of wogonin on colitis-associated colon cancer in vivo and underlying mechanisms.Wogonin prevented colon cancer through converging ROS/DNA damage signaling,and ER stress pathway to affect p53 translocation.Consequently,wogonin induced autophagy or apoptosis in CRC.In addition,wogonin prevented AOM/DSS induced colitis-associated colon cancer.Our findings suggested wogonin protected against CRC through multifaced pathways.And these observations provided wogonin as a promising chemopreventive agent for colitis-associated colon cancer.