Study on the Functional Role of Glutathione Peroxidase 3 as a Tumor Suppressor in Prostate Cancer

Abstract

Prostate cancer is the most frequently diagnosed cancer in Western men, and more men have been diagnosed at younger ages in recent years. A high-fat Western-style diet is a known risk factor for prostate cancer. Dietary fat increases oxidative stress and levels of reactive oxygen species (ROS) that interfere with cellular processes. Among the antioxidant enzymes, glutathione peroxidase 3 (GPx3) is an essential component of the cellular detoxification system. GPx3 is involved in protecting cells from oxidative damage, and down-regulated levels of expression have been found in prostate cancer samples. However, it remains unknown whether GPx3 expression can regulate the development of prostate cancers and the role and mechanism of GPx3 in prostate tumorigenesis has never been directly tested. In addition, little is known about the effect of GPx3 expression on tumor invasion in prostate cancer. In this study, to study the functional role of GPx3 as a tumor suppressor in prostate cancer, I evaluated the expression and function of GPx3 during prostate cancer tumorigenesis in transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model and PC-3 human prostate cancer cells (Figure I). Chapter I study evaluated the association between dietary animal fat and expression of antioxidant GPx3, in the early stages of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Six-week-old male nontransgenic and TRAMP mice were placed on high animal-fat (45% Kcal fat) or control (10% Kcal fat) diets and sacrificed after 5 or 10 weeks. The histopathological score increased with age and high-fat diet consumption. TRAMP mice fed a high-animal fat diet showed decreased GPx3 expression both at the mRNA and protein levels. GPx3 decreased both at the mRNA and protein levels in mouse prostate. GPx3 mRNA expression decreased (~36.27% and ~23.91%, respectively) in the anterior and dorsolateral prostate of TRAMP mice fed a high-fat diet compared to TRAMP mice fed a control diet. Cholesterol treatment increased PC-3 human prostate cancer cell proliferation, decreased GPx3 mRNA and protein levels, and increased H2O2 levels in culture medium. Moreover, increasing GPx3 mRNA expression by troglitazone (TGZ) in PC-3 cells decreased cell proliferation and lowered H2O2 levels. Overall, these results suggest that dietary fat enhances prostate cancer progression, possibly by suppressing GPx3 expression and increasing proliferation of prostate intraepithelial neoplasia (PIN) epithelial cells. In Chapter II, I evaluated the tumorigenic role of the GPx3 in vivo by using GPx3 deficient TRAMP mice. TRAMP / GPx3 (–/–) KO mice were generated by cross-mating of TRAMP mutant mice and GPx3 mutant mice. Prostate cancer incidence and progression were determined in TRAMP, TRAMP / GPx3 (+/–) HET, and TRAMP / GPx3 (–/–) KO mice at 8, 16, and 20 weeks of age. GPx3 expression was decreased in TRAMP mice during progression of prostate cancer in a fashion that parallels observations in human prostate cancer. GPx3 was not detected in GPx3 KO mice both in mRNA and protein levels. Disruption of GPx3 expression in TRAMP mice increased the genitourinary tract weights and the histopathological scores in each lobe with increased proliferation rates. Moreover, deletion of one (+/–) or both (–/–) alleles of GPx3 gene resulted in increase in prostate cancer incidence with activated Wnt/β-catenin pathway. These results provide the first in vivo molecular genetic evidence that GPx3 does indeed function as a tumor suppressor during prostate carcinogenesis. Chapter III study aimed to determine the inhibitory effect and mechanism of TGZ on cell growth, migration, and invasion using prostate cancer cell line PC-3. TGZ is a synthetic peroxisome proliferator-activated receptor γ (PPARγ) ligand that exhibits potential antitumor effects on a broad range of cancers, including prostate cancer. Cell migration and invasion were assessed with wound healing assay and transwell assay, respectively. The expression levels of mRNA and protein were determined by quantitative reverse transcription-polymerase chain reaction and western blotting. TGZ dose-dependently inhibited cell migration and invasion of PC-3 cells. In addition, TGZ increased the mRNA and protein levels of E-cadherin and GPx3 in PC-3 human prostate cancer cells. In addition, GW9662, a PPARγ antagonist, attenuated the increased mRNA and protein levels of E-cadherin and GPx3, suggesting that PPARγ-dependent pathway was involved. Taken together, these results suggest that the anti-migration and anti-invasion effect of TGZ on PC-3 prostate cancer cells is, at least in part, mediated through upregulating E-cadherin and GPx3. I also conclude that PPARγ could be used as a potential therapeutic target for the prevention and treatment of prostate cancer cell invasion and metastasis. These findings further support the important role that GPx3 plays as a tumor suppressor provide an insight into disease pathogenesis, and indicate that it may serve as a substrate for translational investigations in prostate cancer. Additionally, identification of mechanisms that cooperate with loss of GPx3 to promote tumorigenesis may provide additional therapeutic targets.