The Effect Of Lycopene On The GSTP1of The Human Prostate Cancer Cell Lines

Prostate cancer (PCa) is the most common malignancy and the secondleading cause of cancer-related mortality in men of western countries. Theetiology of PCa involves several genetic and environmental factors. TheGlutathione S-transferase P1(GSTP1) is an important detoxifying enzymecatalyze the glutathione conjugation of a variety of electrophilicxenobiotics. However, most PCa fail to express GSTP1, and this silence ofGSTP1function is an carcinogenic event in prostate adenocarcinoma.Lycopene, which is naturally present in tomatoes and tomato products,is the most potent singlet oxygen quencher among the natural carotenoids.It can unable to synthesis by human and rely on an adequate consumptionfor their intake. Much of the recent interest in lycopene bioavailability is aresult of Giovannucci et al.’s publication associating35%reduction in PCarisk at1995. A number of epidemiological studies have shown an inverserelationship between dietary lycopene intake and the risk of PCa.Thus lycopene may be able to prevent or slow prostate cancer growththrough induction of GSTP1tumor suppressor gene. Therefore we conductthe effect of lycopene on GSTP1in human prostate cancer cell lines.Part1The epigenetic effect of lycopene on GSTP1of the humanProstate Cancer Cell linesPurpose: To evaluate the epigenetic effect of lycopene on the humanPCa cell lines, the androgen-dependent (LNCaP) and the androgen- independent (PC-3) were used.Method: To evaluate the effect of lycopene on the cell growth ofLNCaP and PC-3by MTS, cell apoptosis and cell cycle anylsis. Toevaluate the demethylation effect of lycopene on the promoter of GSTP1gene, and geneome ALU and LINE-1elements in LNCaP and PC-3byBSP. To evaluate the effect of lycopene on the mRNA of GSTP1andDNMTs of LNCaP and PC-3by qRT-PCR. To evaluate the effect oflycopene on the protein level of GSTP1and DNMTs of PC-3by WB andIF.Result: MTS, cell apoptosis and cell cycle assays demonstrated thatlycopene inhibited PCa cell proliferation by inducing G1arrest and cellapoptosis in vitro. Here, we also demonstrated that lycopene treatmentsignificantly decreased methylation level the GSTP1promoter, withincreased mRNA and protein level of GSTP1in androgen-independentPC-3cell line. In contrast, the same treatment with lycopene did notdemthylate GSTP1promoter and increased GSTP1expression inandrogen-dependent LNCaP cell line. PC-3also displayed downregulatedDNMT3A protein level, but not DNMT1and DNMT3B. Further, longrepeat elements LINE-1and short repeat element ALU did not showdemethylation when treated by lycopene. In LNCaP cells, lycopenetreatment did not affect all the detected DNMT protein expression, whilemethylation level of LINE-1and ALU was decreased.Conclusion: The results indicated lycopene inhibits PCa cellproliferation, and the protective effect of lycopene on prostate is partlythrough the epigenetic effect on GSTP1gene in the androgen-independentPC3cells line. Part2The effect of lycopene on GSTP1through Nrf2/AREsignal way in DU145cell linePurpose: To evaluate the effect of the Nrf2/ARE signal way in theinduction of GSTP1on human PCa DU145cell line by lycopene.Method: Using Dual-Luciferase reporter assay, to determine whetherlycopene treatment is capable of activating pARE-luc of GSTP1; to furtherexplore the role of Nrf2in ARE activtion by lycopene, cotransfectedDU145cells with the siRNA-Nrf2and pARE-luc. Using qRT-PCR andWestern blot, to evaluate the effect of lycopene on the mRNA and proteinexpression level of GSTP1in DU145cells; to further explore the role ofNrf2in the effect of lycopene on GSTP1expression, transfected eithersiRNA-Nrf2or Nrf2plasmid.Result: The potency of lycopene in ARE activation is significant,20μmol/L lycopene activated the pARE-luc reporter gene up to3-fold, aspotent as the antoxidant tBHQ (30μmol/L). However, stimulation wastotally inhibited in cells cotransfected with siRNA-Nrf2. After treated withlycopene72h, induced a significantly increase in the level of mRNA andprotein of GSTP1. As expected, an siRNA-Nrf2transfected was unable tosupport lycopene induction of GSTP1expression. Futhermore, in DU145cells transfected with a Nrf2plasmid, an significant increase in theinduction of GSTP1mRNA and protein expression level.Conclusion: The results indicated the effect of lycopene induction ofGSTP1mRNA and protein level depends on a functional Nrf2/ARE signalway in DU145cell line.