GSTP1 Protein Expression Research Articles

Alpha-Lipoic Acid Reduces Cell Growth, Inhibits Autophagy, and Counteracts Prostate Cancer Cell Migration and Invasion: Evidence from In Vitro Studies

Alpha-lipoic acid (ALA) is a natural antioxidant dithiol compound, exerting antiproliferative and antimetastatic effects in various cancer cell lines. In our study, we demonstrated that ALA reduces the cell growth of prostate cancer cells LNCaP and DU-145. Western blot results revealed that in both cancer cells, ALA, by upregulating pmTOR expression, reduced the protein content of two autophagy initiation markers, Beclin-1 and MAPLC3. Concomitantly, MTT assays showed that chloroquine (CQ) exposure, a well-known autophagy inhibitor, reduced cells’ viability. This was more evident for treatment using the combination ALA + CQ, suggesting that ALA can reduce cells’ viability by inhibiting autophagy. In addition, in DU-145 cells we observed that ALA affected the oxidative/redox balance system by deregulating the KEAP1/Nrf2/p62 signaling pathway. ALA decreased ROS production, SOD1 and GSTP1 protein expression, and significantly reduced the cytosolic and nuclear content of the transcription factor Nrf2, concomitantly downregulating p62, suggesting that ALA disrupted p62-Nrf2 feedback loop. Conversely, in LNCaP cells, ALA exposure upregulated both SOD1 and p62 protein expression, but did not affect the KEAP1/Nrf2/p62 signaling pathway. In addition, wound-healing, Western blot, and immunofluorescence assays evidenced that ALA significantly reduced the motility of LNCaP and DU-145 cells and downregulated the protein expression of TGFβ1 and vimentin and the deposition of fibronectin. Finally, a soft agar assay revealed that ALA decreased the colony formation of both the prostate cancer cells by affecting the anchorage independent growth. Collectively, our in vitro evidence demonstrated that in prostate cancer cells, ALA reduces cell growth and counteracts both migration and invasion. Further studies are needed in order to achieve a better understanding of the underlined molecular mechanisms.

Expression Levels of Glutathione S-Transferase and Cytochrome P450 Isoenzymes in Nasal Polyp Tissue.

Nasal polyps are benign sinonasal masses composed of eosinophils and extracellular edema. Pathogenesis of the polyp formation is unclear but several studies strongly suggest a correlation with infection, inflammation and allergy conditions. Our aim is to investigate the potential link between allergy and nasal polyp in tissue level. Nasal polyp group included 60 patients whose diagnosis was confirmed with biopsy and the control group included 38 healthy patients. Tissue sample of the control group was taken from inferior turbinate mucosa under local anesthesia and nasal polyp tissue was collected from functional endoscopic sinus surgery. The glutathione S-transferase (GST) and cytochrome P450 (CYP) isoenzyme expressions of the tissue samples were investigated under light microscopy and graded by a senior pathologist. GSTP1 protein expression was significantly higher in tissue samples from nasal polyp group compared to that of control group (p < 0.05). However, CYP1A1, GSTM1 and GSTA1 isoenzymes were not different between the two groups (p > 0.05). We have found that GSTP1 isoenzyme was elevated in nasal polyp tissue compared to the control. The increase in protein expression of GSTP1 might have occured as a tissue response to the increased oxidative stress thus suggesting a role of GSTP1 in polyp formation.

GSTP1 Loss results in accumulation of oxidative DNA base damage and promotes prostate cancer cell survival following exposure to protracted oxidative stress.

Epigenetic silencing of glutathione S-transferase π (GSTP1) is a hallmark of transformation from normal prostatic epithelium to adenocarcinoma of the prostate. The functional significance of this loss is incompletely understood. The present study explores the effects of restored GSTP1 expression on glutathione levels, accumulation of oxidative DNA damage, and prostate cancer cell survival following oxidative stress induced by protracted, low dose rate ionizing radiation (LDR). GSTP1 protein expression was stably restored in LNCaP prostate cancer cells. The effect of GSTP1 restoration on protracted LDR-induced oxidative DNA damage was measured by GC-MS quantitation of modified bases. Reduced and oxidized glutathione levels were measured in control and GSTP1 expressing populations. Clonogenic survival studies of GSTP1- transfected LNCaP cells after exposure to protracted LDR were performed. Global gene expression profiling and pathway analysis were performed. GSTP1 expressing cells accumulated less oxidized DNA base damage and exhibited decreased survival compared to control LNCaP-Neo cells following oxidative injury induced by protracted LDR. Restoration of GSTP1 expression resulted in changes in modified glutathione levels that correlated with GSTP1 protein levels in response to protracted LDR-induced oxidative stress. Survival differences were not attributable to depletion of cellular glutathione stores. Gene expression profiling and pathway analysis following GSTP1 restoration suggests this protein plays a key role in regulating prostate cancer cell survival. The ubiquitous epigenetic silencing of GSTP1 in prostate cancer results in enhanced survival and accumulation of potentially promutagenic DNA adducts following exposure of cells to protracted oxidative injury suggesting a protective, anti-neoplastic function of GSTP1. The present work provides mechanistic backing to the tumor suppressor function of GSTP1 and its role in prostate carcinogenesis.

Epigenetic alterations are involved in the overexpression of glutathione S-transferase π-1 in human colorectal cancers.

Glutathione S-transferase π-1 (GSTP-1) is a member of the glutathione S-transferase enzyme superfamily, which catalyzes the conjugation of electrophiles to glutathione during the process of detoxification. In this study, the epigenetic alterations of GSTP-1 expression in human colorectal cancers and the underlying mechanisms were investigated. In 10 colon cancer patients, proteomic analysis revealed that expression of GSTP-1 protein was higher in tumor tissues than in paired adjacent normal tissues. Likewise, in 7 of 10 colon cancer patients, GSTP-1 protein expression was more than 1.5-fold higher in tumor tissues than in adjacent normal tissues, as determined by western blotting. Immunohistochemical data confirmed that GSTP-1 protein was expressed at higher levels in colon cancer tissues compared to normal mucosa. GSTP-1 enzyme activity was closely correlated with GSTP-1 protein expression in colon cancer patients. Consistent with this, GSTP-1 mRNA, protein and activity levels were higher in the colorectal cancer cell lines Caco-2, HCT-116, HT-29, SNU-407 and SNU-1033 compared to the normal colon cell line FHC. Methylation-specific PCR results indicated that the high levels of GSTP-1 in human colorectal cancer cell lines were likely due to the lower degree of promoter methylation in colon cancer cell lines compared to the normal colon cell line, consistent with findings in colon cancer patients. Moreover, the levels of specific activator-protein complexes and histone marks were higher in human colorectal cancer cells compared to the normal human colon cell line, whereas the repressor protein complexes exhibited the opposite pattern. Furthermore, chromatin immunoprecipitation assays demonstrated that expression levels of the transcription factors AP-1 and SP-1 were correlated with the upregulation of GSTP-1 expression in colorectal cancer cells. Finally, knockdown of GSTP-1 promoted the sensitivity of SNU-407 cells to the anticancer agent 5-fluorouracil. These data indicate that GSTP-1 may serve as a clinically useful biomarker of colon cancer and a target for anti-colon cancer drugs.

Significance of GSTP1 for predicting the prognosis and chemotherapeutic efficacy in esophageal squamous cell carcinoma

Glutathione S-transferases (GSTs) have been reported to be activated in several types of cancers, including esophageal squamous cell carcinoma (ESCC). The aim of the present study was to investigate whether GSTP1 protein expression is a useful predictor of the clinical outcome or drug resistance in ESCC. Immunohistochemistry was conducted with 75 ESCC resected specimens using a monoclonal antibody against GSTP1. The patients were divided into two groups according to the degree of GSTP1 staining, and the relationship between the GSTP1 level and the clinicopathological features was examined. Seventy-five patients were divided into low (grade 1, n=36) and high (grade 2, n=39) GSTP1 expression groups. The overall survival was significantly worse in the grade 2 patients than in the grade 1 patients (5‑year survival rate, 78.5 vs. 51.2%; p=0.027). Cox proportional hazard analysis revealed that macroscopic type 3 or 4 disease (p=0.001), lymph node metastasis (p=0.010), and high GSTP1 expression (p=0.029) were independent predictors of a poor prognosis. With regard to the subgroup analysis among the 31 patients undergoing adjuvant chemotherapy, the grade 2 patients had a worse prognosis than did the grade 1 patients (5‑year survival rate, 45.0 vs. 81.8%; p=0.081). This tendency was not observed in the subgroup without adjuvant chemotherapy (5‑year survival rate, 51.7 vs. 59.9%; p=0.979). In conclusion, the GSTP1 expression is a good predictor of prognosis, and it may be closely related to the chemotherapeutic efficacy of 5-FU plus cisplatin in ESCC patients.

Abstract 614: Increased oxidative DNA damage in human prostate cancer and its relationship to GSTP1 expression

Abstract The pi-class glutathione S-transferase (GSTP1) has been described as a ‘caretaker’ gene, as it actively protects the cell from oxidative genome damage mediated by carcinogens and electrophilic compounds. Loss of GSTP1 expression via promoter hypermethylation is one of the most common epigenetic alterations observed in human prostate cancer. Silencing of GSTP1 can increase prostate epithelial cells more vulnerable to both oxidant and electrophile carcinogens and causes genetic aberrations leading to increased risk of tumor progression. Observations suggest that increased reactive oxygen species (ROS) causes DNA damage in cells, as measured by 8-hydroxy-2′-deoxyguanosine (8-OHdG), contribute to increased incidence of prostate cancer. However there are no studies which demonstrate correlation between GSTP1 protein/activity and DNA damage in prostate cancer. We took a panel of 24 human prostate specimens obtained after surgery which comprised of matched primary tumor and adjacent nontumorous (benign) prostate tissues. Oxidative DNA damage, GSTP1 protein expression and its activity, and GSTP1 promoter methylation was assessed. The 8-OHdG levels in DNA of prostate cancer tissues ranged from 0.307 to 0.609ng/μg DNA, whereas the levels in benign tissue range from 0.156 to 0.322ng/μg DNA, respectively. 8-OHdG and GSTP1 promoter methylation levels were higher in adenocarcinoma compared to their benign counterparts, which positively correlated with the loss of GSTP1 activity. The GSTP1 protein activity of prostate cancer tissues ranged from 738.2 to 1754.4ng/mg protein, whereas the levels in benign tissue range from 1728.6 to 2580.4ng/mg protein. The loss of GSTP1 expression in cells might increase their vulnerability to cytotoxicity and reactive oxygen species (ROS)-mediated DNA damage. In further experiments, human prostate carcinoma LNCaP cells, which contains a silenced GSTP1 gene, was genetically modified to constitutively express high levels of GSTP1. Introduction of GSTP1 lowered endogenous ROS levels in LNCaP cells; treatment with H2O2 significantly reduced ROS and 8-OHdG in LNCaP-GSTP1 cells, compared to parental cell line. Furthermore, treatment with green tea polyphenols to LNCaP cells caused re-expression of GSTP1 which protected the cells from H2O2-mediated DNA damage compared to pretreated cells. These results suggest that oxidative stress-induced DNA damage may be an important measure for primary prevention of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 614. doi:1538-7445.AM2012-614

GSTP1 DNA Methylation and Expression Status Is Indicative of 5-aza-2′-Deoxycytidine Efficacy in Human Prostate Cancer Cells

DNA methylation plays an important role in carcinogenesis and the reversibility of this epigenetic modification makes it a potential therapeutic target. To date, DNA methyltransferase inhibitors (DNMTi) have not demonstrated clinical efficacy in prostate cancer, with one of the major obstacles being the inability to monitor drug activity during the trial. Given the high frequency and specificity of GSTP1 DNA methylation in prostate cancer, we investigated whether GSTP1 is a useful marker of DNMTi treatment efficacy. LNCaP prostate cancer cells were treated with 5-aza-2′-deoxycytidine (5-aza-CdR) either with a single high dose (5–20 µM), every alternate day (0.1–10 µM) or daily (0.005–2.5 µM). A daily treatment regimen with 5-aza-CdR was optimal, with significant suppression of cell proliferation achieved with doses of 0.05 µM or greater (p<0.0001) and induction of cell death from 0.5 µM (p<0.0001). In contrast, treatment with a single high dose of 20 µM 5-aza-CdR inhibited cell proliferation but was not able to induce cell death. Demethylation of GSTP1 was observed with doses of 5-aza-CdR that induced significant suppression of cell proliferation (≥0.05 µM). Re-expression of the GSTP1 protein was observed only at doses of 5-aza-CdR (≥0.5 µM) associated with induction of cell death. Treatment of LNCaP cells with a more stable DNMTi, Zebularine required at least a 100-fold higher dose (≥50 µM) to inhibit proliferation and was less potent in inducing cell death, which corresponded to a lack of GSTP1 protein re-expression. We have shown that GSTP1 DNA methylation and protein expression status is correlated with DNMTi treatment response in prostate cancer cells. Since GSTP1 is methylated in nearly all prostate cancers, our results warrant its testing as a marker of epigenetic therapy response in future clinical trials. We conclude that the DNA methylation and protein expression status of GSTP1 are good indicators of DNMTi efficacy.

Abstract 5706: High-throughout library screening identifies phytochemical inducers of phase II metabolism enzymes GSTP1 and NQO1 in human lung cells

Abstract Many phytochemicals possess anti-oxidant and cancer-preventive properties, some putatively through phase II metabolism-mediated mutagen/oxidant quenching. However, in studies to date, most candidate agents are not active in human lung cells. In vitro and in vivo screening studies for discovery are clearly needed for new, more potent chemopreventive agents for lung cancer. Here we applied an mRNA-specific gene expression-based high-throughout screening approach to identify phytochemical inducers of phase II metabolism enzymes GSTP1 and NQO1 in primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBEC). We have screened 800 compounds in the MicroSource Natural Products Library for inducers of GSTP1 and NQO1 expression, and identified 2, 3-dihydroxy-4-methoxy-4′-ethoxybenzophenone, triacetylresveratrol, ivermectin, sanguinarine sulfate, and daunorubicin as new inducers for GSTP1 and NQO1 mRNA expression. Consistent with this effect, the induction of NQO1 protein expression by immunoblot was found for all these agents in both HBEC and NHBE cells at the same concentration that induced NQO1 mRNA expression. However, GSTP1 protein expression did not significantly change with any selected agents. Proliferation/viability assays showed that HBEC and NHBE were significantly inhibited by ivermectin, sanguinarine sulfate, and daunorubicin. These data demonstrate the feasibility of high-throughput screening for inducers of NQO1. Moreover, the data identify 2, 3-dihydroxy-4-methoxy-4′-ethoxybenzophenone and triacetylresveratrol as the most potent and low toxicity inducers [≥3-6-fold, at the protein level], implying they are potential new agents for further in vitro and preclinical in vivo testing for prevention of oxidant-related disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5706.

Abstract A69: High-throughput library screening identifies phytochemical inducers of phase II metabolism enzymes GSTP1 and NQO1 in human lung cells

Abstract Many phytochemicals possess cancer-preventive properties, some putatively through phase II metabolism-mediated mutagen/oxidant quenching. However, in studies to date, most candidate agents are not active in human lung cells. In vitro and in vivo screening studies for discovery are clearly needed for new, more potent chemopreventive agents for lung cancer. Here we applied a gene expression-based high-throughput screening approach to identify phytochemical inducers of phase II metabolism enzymes GSTP1 and NQO1 in primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBEC). We have screened 800 compounds in the MicroSource Natural Products Library for inducers of GSTP1 and NQO1 expression, and identified 2, 3-dihydroxy-4-methoxy-4′-ethoxybenzophenone, triacetylresveratrol, ivermectin, sanguinarine sulfate, and daunorubicin as new inducers for GSTP1 and NQO1 mRNA expression. Consistent with this effect, the induction of NQO1 protein expression by immunoblot was found for all these agents in both HBEC and NHBE cells at the same concentration (1.0 µM, final concentration) that induced NQO1 mRNA expression. However, GSTP1 protein expression did not significantly change with any selected agents. Proliferation/viability assays showed that HBEC and NHBE were significantly inhibited by ivermectin, sanguinarine sulfate, and daunorubicin. These data demonstrate the feasibility of high-throughput screening for inducers of NQO1. Moreover, the data identify 2, 3-dihydroxy-4-methoxy-4′-ethoxybenzophenone and triacetylresveratrol as most potent and low toxicity inducers [12-fold, and 10-fold, respectively, at the protein level], implying they are potential new agents for further in vitro and preclinical in vivo testing for prevention of tobacco-related lung cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A69.

Expression level and DNA methylation status of glutathione‐S‐transferase genes in normal murine prostate and TRAMP tumors

Glutathione-S-transferase (Gst) genes are downregulated in human prostate cancer, and GSTP1 silencing is mediated by promoter DNA hypermethylation in this malignancy. We examined Gst gene expression and Gst promoter DNA methylation in normal murine prostates and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors. Primary and metastatic tumors were obtained from TRAMP mice, and normal prostates were obtained from strain-matched WT mice (n = 15/group). Quantitative real-time RT-PCR was used to measure GstA4, GstK1, GstM1, GstO1, and GstP1 mRNA expression, and Western blotting and immunohistochemical staining was used to measure GstM1 and GstP1 protein expression. MassARRAY Quantitative Methylation Analysis was used to measure DNA methylation of the 5' CpG islands of GstA4, GstK1, GstM1, GstO1, and GstP1. TRAMP-C2 cells were treated with the epigenetic remodeling drugs decitabine and trichostatin A (TSA) alone and in combination, and Gst gene expression was measured. Of the genes analyzed, GstM1 and GstP1 were expressed at highest levels in normal prostate. All five Gst genes showed greatly reduced expression in primary tumors compared to normal prostate, but not in tumor metastases. Gst promoter methylation was unchanged in TRAMP tumors compared to normal prostate. Combined decitabine + TSA treatment significantly enhanced the expression of 4/5 Gst genes in TRAMP-C2 cells. Gst genes are extensively downregulated in primary but not metastatic TRAMP tumors. Promoter DNA hypermethylation does not appear to drive Gst gene repression in TRAMP primary tumors; however, pharmacological studies using TRAMP cells suggest the involvement of epigenetic mechanisms in Gst gene repression.

Promoter Methylation and Differential Expression of π-Class Glutathione S-Transferase in Endometrial Carcinoma

Pi-class glutathione S-transferase (GSTP1), located on chromosome 11q13, codes for a phase II metabolic enzyme that detoxifies reactive electrophilic intermediates. The protein also interacts with steroid hormones in the human body. The role of GSTP1 in endometrial carcinoma has not been reported. In this study, we aimed at determining the expression of GSTP1 in relation to the epigenetic and genetic changes of the gene in endometrial carcinoma. The GSTP1 protein and mRNA expression was assessed by immunohistochemistry on tissue microarray and quantitative real-time reverse transcriptase-polymerase chain reaction, respectively. Its methylation status was studied by methylation-specific polymerase chain reaction and bisulfite sequencing. Possible mutations in coding region of GSTP1 were assessed by cDNA sequencing. Ninety-seven cases of endometrial carcinoma with available tissue blocks and clinical data were studied. Our results showed that 68.0% (66 of 97) of the cases showed reduced protein expression while 64% (16 of 25) showed reduced mRNA expression; 30.9% (30 of 97) of the cases demonstrated methylated alleles in at least one of the six methylation-specific polymerase chain reaction reactions. The methylation status significantly correlated with reduced protein expression (P = 0.008) and reduced mRNA expression (P = 0.003). Methylation at non-CpG sites including CpCpG trinucleotides and CpT dinucleotides were also observed. cDNA sequencing did not reveal genetic alterations in coding region of the gene. The extent of myometrial invasion was found to be significantly correlated with both the methylation status (P = 0.009) and the protein expression (P = 0.036) of the GSTP1 gene. We postulated that hypermethylation of the GSTP1 gene promoter region may act as a dynamic regulation mechanism contributing to reduced GSTP1 expression, which is associated with myometrial invasion potential of the endometrial carcinoma.