Soy Isoflavones Promote Apoptosis Of The Human Prostate Cancer DU-145Cells Through The Activation Of PPARγ

Prostate cancer is one of the common malignant toumors of the reproductive system inthe older men. Dietary and nutritional factors play an important role in the tumorigenesis andprogression of prostate cancer，the protective effects of soybeans and their products onprostate cancer have been attracted more attention. Epidemiological studies havedemonstrated that soy and its product intake is inversely associated with the risk of prostatecancer. Soy isoflavones as one kind of the phytochemicals existed in soy belong to thepolyphenols, and include two main ingredients genistein and daidzein. Many experimentalstudies in vitro have demonstrated that soy isoflavones exerted anti-cancer effects through theinhibition of tumor cell proliferation,invasion and metastasis as well as the induction ofapoptosis of tumor cells,therefore soy isoflavones may be the main active substance thatcontribute to the anti-cancer effects of soy and its products. It is well known that apoptosis is aprogrammed cell death regulated by multiple genes and induction of apoptosis is one of thestrategies for the control of tumor growth.A large number of studies have shown thatapoptosis of prostate cancer cells induced by soy isoflavones was associated with the changesof apoptosis-related genes,such as Bax,Bcl-2,P53,Akt and NF-κB,etc.However,the molecularmechanisms underlying the expression of these genes modulated by soy isoflavones remainunclear.Peroxisome proliferator-activated receptors(PPARs) have been identified as one of themembers of the nuclear receptors superfamily. Once activated by a corresponding ligand forPPARs,PPARs acted as transcription factors and regulated many important biologicalprocesses through modulation the expression of target genes by binding to the peroxisomeproliferator responsive element in the promoter area of target genes. PPARs include threesubtypes:PPARα,PPARβ and PPARγ.PPARγ has been attracted attention because of its tightcorrelation with obesity,insulin resistance,atherosclerosis,diabetes and other metabolicdiseases. Recent studies have demonstrated that PPARγ was widely expressed in many kinds of tumor cells originated from different organs,so the role of PPARγ in the genesis andprogression of tumor attracted more concern. Many exogenous ligands for PPARγ such asrosiglitazone, troglitazone belonging to the anti-diabetic drug of the thiazolidinedione (TZD)family, exerted anti-cancer effects through inhibition of cell proliferation and metastasis aswell as induction of apoptosis, so that PPARγ is considered to be a new therapeutic target fortumor,and PPARγ agonists may be the potential drugs for cancer chemotherapy treatment.Since soy isoflavones were comfirmed as natural ligands for PPARγ in2003,the roles ofPPARγ signaling pathway in the hypolipidemic and anti-diabetic effects of soy isoflavonesattracted more attention recently.It has been demonstrated that genistein decreased lipid andglucose through the activation of PPARγ. However,the relationship between the PPARγsignaling pathway and the anti-cancer effects of soy isoflavones in prostate cancer has notbeen fully understood yet.In the present study,the effects of genistein and daidzein,the two main components of soyisoflavones,and PPARγ agonist rosiglitazone(as a posistive control)alone or in combinationwith GW9662，a selective inhibitor of PPARγ on the transcriptional activity of PPARγ, cellapoptosis and the expression of apoptosis-related genes such as bax, bcl-2, p53and PTEN inhuman prostate cancer DU-145cells were examined by the methods of luciferase reportergene assay, immunofluorescence staining, TUNEL staining, flow cytometry labeling withFITC-Annexin V/PI, western blot and Caspase3activity assay,so as to investigate thepro-apoptotic effects of soy isoflavones in prostate cancer and its correlation with the PPARγsignaling pathway.The main results and conclusions were summarized as the following:1. The PPRE-driven luciferase reporter gene assay showed that DU-145cells transfectedwith PPRE-TK-Luc plasmid after treatment with genistein,daidzein,and rosiglitazonerespectively resulted in a significant increase of the luciferase activities(P＜0.05), and theseenhanced effects of genistein, daidzein, and rosiglitazone on the luciferase activities could bereversed by GW9662significantly(P＜0.05).2. Immunofluorescence staining of PPARγ showed that the green fluorescence of PPARγpositive staining mainly distributed in the cytoplasm, but rarely in the nucleus of DU-145cells in the control group,the green fluorescence in DU-145cells after treatment withgenistein, daidzein, and rosiglitazone respectively for48hours,distributed markedly in both the cytoplasm and the nucleus. The distribution of green fluorescence in DU-145cells ofgenistein, daidzein, and rosiglitazone in combination with GW9662-treated groups weresimilar to that of the control group.It suggested that genistein,daidzein,and rosiglitazonepromoted nuclear translocation of PPARγ,but this promotion effect was inhibited byGW9662.3. The TUNEL staining assay showed that there were not any apoptotic cells in DU-145cells of the control group,there was a certain number of apoptotic cells with blue-greenfluorescence labeled nucleus in DU-145cells after treatment with genistein, daidzein, androsiglitazone respectively for48hours, but when genistein, daidzein, and rosiglitazone treatedin combination with GW9662, the numbers of the apoptotic cells decreased than the numbersof treated alone.4. The quantitative assay of apoptotic cells by flow cytometry showed that the rate ofapoptotic cells in DU-145cells treated with genistein, daidzein, and rosiglitazone respectivelyfor48h,increased significantly as compared with the control group(P＜0.05).But whendaidzein, and rosiglitazone treated in combination with GW9662, the rate of apoptotic cells inDU-145cells decreased obviously than that of treated alone(P＜0.05).5. The results of Western blot showed that the expression of PTEN, p53and bax proteinin DU-145cells treated with genistein, daidzein, and rosiglitazone alone respectively for48h,increased significantly as compared with the control group(P＜0.05),but the expression ofbcl-2protein decreased significantly(P＜0.05).These effects of genistein, daidzein, androsiglitazone could be reversed by GW9662significantly(P＜0.05).6. The results of caspase3activity test showed that the activity of caspase3in DU-145cells treated with genistein, daidzein, and rosiglitazone alone respectively for48h,increasedsignificantly as compared with the control group(P＜0.05), but the increased caspase3activityinduced by genistein,daidzein or rosiglitazone could be significantly inhibited by GW9662(P＜0.05).The above results suggest that human prostate cancer DU-145cells express PPARγ,genistein and daidzein,the two main components of soy isoflavones can promote nucleartranslocation of PPARγ in DU-145cells, increase the transcription activity of PPARγ andresult in significant changes of the expression of apoptosis-related genes such as PTEN, bax,bcl-2and p53as well as the activity of Caspase3, thus induce apoptosis of DU-145cells,but these effects induced by genistein or daidzein can be reversed significantly by GW9662,aselective inhibitor of PPARγ.Taken together,the current results demonstrate that the activationof PPARγ is one of the molecular mechanism underlying the induction of apoptosis inducedby soy isoflavone in prostate cancer cells.