Sulforaphane Represses Carcinogenesis Of Colorectal Cancer Through The ERK/Nrf2-UDP Glucuronosyltransferase 1A Metabolic Axis

Background and purposeColorectal cancer(CRC)is one of the common malignant tumors of the digestive system.The long pre-cancerous state of CRC provides an opportunity to prevent the occurrence and development of CRC.Heterocyclic amines(HAs)is food-borne carcinogens of colorectal cancer and the detoxification of heterocyclic amines is highly dependent on UDP glucuronosyltransferase 1A(UGT1 A)-mediated glucuronidation Sulforaphane(SFN),a phytochemical,possesses antioxidant,anti-inflammatory and anticarcinogenic effects on the prevention of CRC.Our previous studies revealed that SFN upregulates the expression of UGT1A,thus increasing the catabolic metabolism of heterocyclic amines.However,the specific mechanism is not clear.The aim of the present study was to investigate the regulatory mechanism of SFN-induced UGT1A upregulation and provide novel understanding on the basic research and chemoprevention of CRCMethods1.In the present study,the viability and proliferation of CRC cells(HT-29 and SW480)treated with SFN were measured by MTT,colony formation and EdU assays.The motility of cells was determined by wound healing and Transwell assays.2.Flow cytometry was used to detect the cell cycle arrest and apoptosis of cells treated with different concentrations of SFN3.Different concentrations of SFN were used to treat cells.The expressions of p-ERK,ERK.Nrf2 and UGT1A were detected by quantitative real-time PCR(qRT-PCR)and Western blotting.Cellular immunofluorescence technology is used to observe the expression and localization of Nrf24.Nuclear factor erythroid-2 related factor 2(Nrf2)short hairpin RNA(shRNA)and negative control shRNA lentiviruses were used for cell transfection.Cells were treated with medium with or without SFN and divided into 6 groups:NC shRNA group,NC shRNA+10μmol/L SFN group,NC shRNA+20 μmol/L SFN group,Nrf2 shRNA group,Nrf2 shRNA+10 μmol/L Group and Nrf2 shRNA+20μmol/L SFN group.qRT-PCR and western blotting were employed to verify the role of Nrf2 in SFN-induced UGT1A after treating cells with medium containing or without SFN5.HT-29 and SW480 cells were divided into a control,an SFN and a PD98059[an extracellular signal-regulated kinase(ERK)inhibitor]+SFN group.Western blotting detected the protein levels of Nrf2 and UGT1A.Intracellular levels of reactive oxygen species(ROS)were detected using a reactive oxygen assay kit6.Nude mice tumorigenicity assay were designed to observe the effects of different concentrations of SFN on subcutaneous tumor formation in nude miceResults1.SFN inhibits the proliferation activity,cell colony forming ability,and cell migration ability of HT-29 cells and SW480 cells in a dose-dependent and time-dependent manner(P<0.05)2.SFN induces G0/M1 phase arrest and inhibits the apoptosis of CRC cells(P<0.05).3.SFN activates the ERK pathway,induces Nrf2 nuclear translocation,and upregulates the expression of UGT1A(P<0.05).4.SFN up-regulated the expression of UGT1A in the NC group,but the induction of SFN in the Nrf2 shRNA group was significantly reduced(P<0.05).5.Pretreatment with PD58059 reversed the SFN-induced subcellular translocation of Nrf2 and the expression of UGT1A(P<0.05)6.Sulforaphane inhibited tumor formation of CRC cells subcutaneously in nude mice in a dose-dependent and time-dependent manner(P<0.05)Conclusions1.Sulforaphane inhibited the proliferation of CRC cells,inhibited the cell cycle,promoted apoptosis and reduced the migration of CRC cells in a dose-dependent and time-dependent manner.2.SFN induced the expression of UGT1A by promoting the nuclear translocation of Nrf2.3.SFN may induce the expression of UGT1A through the ERK/Nrf2 signaling pathway.