A novel functional polymorphism of GSTM3 reduces clear cell renal cell carcinoma risk through enhancing its expression by interfering miR-556 binding.

Dysregulation of glutathione-S-transferase M3 (GSTM3) has been related to clear cell renal cell carcinoma (ccRCC) in our former study. GSTM3 plays a pivotal role of detoxification and clearance of reactive oxygen species (ROS) in tumour tissues. This study aimed to examine: (1) the associations between GSTM3 single nucleotide polymorphisms (SNPs) and risk of ccRCC, and (2) the potential molecular mechanism accounting for its effects. 5 SNPs in 3'UTR of GSTM3 were initially genotyped in 329 cases and 420 healthy controls. A SNP-rs1055259 was found to be significantly associated with the susceptibility of ccRCC (OR = 0.59, 95% CI = 0.41-0.92; P = .019). The minor allele of rs1055259 (G allele) was associated with RCC risk. This SNP was predicted to affect microRNA (miR)-556 binding to 3'UTR of GSTM3 mRNA. To determine the functional impact, plasmid constructs carrying different alleles of rs1055259 were created. Compared to rs1055259 A-allele constructs, cells transfected with rs1055259 G-allele construct had higher transcriptional activity and were less responsive to miR-556 changes and gene expression. Elevated GSTM3 expression in G-allele cells was associated with ROS activity and ccRCC development. Taken together, this study indicated that a functional polymorphism of GSTM3 -rs1055259 reduced susceptibility of RCC in the Chinese population. It influenced GSTM3 protein synthesis by interfering miR-556 binding, subsequently suppressed ROS activity and ccRCC progression.

Oestrogen receptor-regulated glutathione S-transferase mu 3 expression attenuates hydrogen peroxide-induced cytotoxicity, which confers tamoxifen resistance on breast cancer cells.

PURPOSE: Glutathione S-transferase mu 3 (GSTM3) is an enzyme involving in the detoxification of electrophilic compounds by conjugation with glutathione. Higher GSTM3 mRNA levels were reported in patients with ERα-positive breast cancer who received only tamoxifen therapy after surgery. Thus, this study aimed to clarify the oncogenic characteristics of GSTM3 in breast cancer and the mechanism of tamoxifen resistance. METHODS: GSTM3 expression in human breast tumour tissues (n = 227) was analysed by RT-PCR and quantitative PCR. Western blot, promoter activity assays, and chromatin immunoprecipitation (ChIP) assays were used to investigate the mechanism of GSTM3 gene regulation. Hydrogen peroxide (H2O2)-induced cytotoxicity in breast cancer cells was detected by MTT assays and flow cytometry. The oncogenic characteristics of GSTM3 in MCF-7 cells were examined by siRNA knockdown in soft agar assays and a xenograft animal model. RESULTS: GSTM3 mRNA was highly expressed in ER- and HER2-positive breast cancers. Moreover, patients who received adjuvant Herceptin had increased GSTM3 mRNA levels in tumour tissue. Oestrogen-activated GSTM3 gene expression through ERα-mediated recruitment of SP1, EP300, and AP-1 complexes. GSTM3-silenced MCF-7 cells were more sensitive to H2O2, with significantly inhibited proliferation and colony formation abilities. Tamoxifen-resistant (Tam-R) cells lacking GSTM3 showed enhanced sensitivity to H2O2, but this result was contrary to that obtained after short-term tamoxifen exposure. The animal model suggested that GSTM3 silencing might suppress the tumourigenic ability of MCF-7 cells and increase tumour cell apoptosis. CONCLUSIONS: ROS production is one mechanism by which cancer drugs kill tumour cells, and according to our evidence, GSTM3 may play an important role in preventing breast cancer treatment-induced cellular cytotoxicity.

Low GSTM3 expression is associated with poor disease-free survival in resected esophageal squamous cell carcinoma.

BACKGROUND: Glutathione S-transferase mu 3 (GSTM3) plays a crucial role in tumor progression in various cancers. However, the relationship between GSTM3 expression and the clinical prognosis of esophageal squamous cell carcinoma (ESCC) has not been studied to date. We aimed to characterize the role of GSTM3 in predicting postoperative prognosis of ESCC patients. METHODS: In the retrospective study, GSTM3 mRNA levels in 184 ESCC tissues and matched 43 adjacent nontumorous tissues were measured by quantitative real-time PCR. GSTM3 protein levels in 247 ESCC tissues were measured by immunohistochemistry. RESULTS: Downregulation of GSTM3 occurred in 62.8 % of primary ESCC tissues compared with their nontumor counterparts. Patients with low GSTM3 expression tended to exhibit an increased rate of poor differentiation in both the mRNA cohort (p = 0.024) and protein cohort (p = 0.004). In the mRNA cohort, low GSTM3 expression was associated with unfavorable 3-year disease-free survival (DFS) (39.2 % vs. 57.4 %) and 5-year DFS (26.8 % vs. 45.1 %) (p = 0.023). The result was confirmed in the protein cohort. Patients with low GSTM3 expression had unfavorable 3-year disease-free survival (DFS) (18.7 % vs. 33.5 %) and 5-year DFS (5.3 % vs. 30.5 %) (p = 0.006). Cox multivariate analysis revealed that GSTM3 expression was an independent prognostic factor. CONCLUSIONS: The findings of the present study provide evidence that GSTM3 may function as a tumor suppressor in ESCC and represents a potential novel prognostic biomarker for disease-free survival for resected ESCC patients.

Glutathione S-Transferase Mu-3 Predicts a Better Prognosis and Inhibits Malignant Behavior and Glycolysis in Pancreatic Cancer.

Background: Pancreatic cancer (PC) is a lethal malignancy with an extremely unfavorable 5-year survival rate and a high mortality rate. Glutathione S-transferase mu-3 (GSTM3) has been shown to exert different functions in the progression and development of various cancers, except for PC. This study aimed to explore the role of GSTM3 in the malignant behavior and metabolic aspects of PC, its clinical significance, and its possible molecular mechanism in pancreatic cancer. Methods: Tumor microarrays of pancreatic ductal adenocarcinoma (PDAC) were used to evaluate the clinicopathological variables and GSTM3 expression by immunohistochemical staining. Kaplan-Meier survival and Cox regression analyses were further performed to assess the prognosis. The effect of GSTM3 on PC aggressiveness was detected using overexpressing and silencing transfection methods. Western blot, RT-qPCR, CCK-8, and cell cycle assay were applied to evaluate the expression level and proliferation. A xenograft animal model was assessed. Reactive oxygen species (ROS) were measured using the laser confocal scanner and glycolysis was detected using an Agilent Seahorse kit. RNA sequencing was used to assess the underlying mechanism and the signaling pathway involved. Results: GSTM3 was relatively poorly expressed in PDAC tissues compared to para-tumoral tissues and a high level of GSTM3 indicated good overall survival. Functionally, overexpression of GSTM3 could significantly inhibit cell proliferation by delaying the G0/G1 transition, whereas the opposite results were found in the GSTM3 downregulation group. In addition, xenograft animal models further confirmed the effect on proliferation. Moreover, silencing of GSTM3 induced ROS accumulation and promoted glycolysis in PC, indicating its tumor suppressive effect, and vice versa when GSTM3 was upregulated. Finally, RNA sequencing results demonstrated that GSTM3 facilitates anti-tumorigenicity partly via the JAK-STAT signaling pathway in PC. Conclusion: GSTM3 inhibited tumor progression and altered the metabolic pattern in PC. This may be a potential predictive biomarker in PC and a prospective therapeutic target.

Effects of total parenteral nutrition on drug metabolism gene expression in mice.

Parenteral nutrition-associated liver disease (PNALD) is a liver dysfunction caused by various risk factors presented in patients receiving total parenteral nutrition (TPN). Omega-6 rich Intralipid® and omega-3 rich Omegaven® are two intravenous lipid emulsions used in TPN. TPN could affect the hepatic expression of genes in anti-oxidative stress, but it's unknown whether TPN affects genes in drug metabolism. In this study, either Intralipid®- or Omegaven®-based TPN was administered to mice and the expression of a cohort of genes involved in anti-oxidative stress or drug metabolism was analyzed, glutathione (GSH) levels were measured, and protein levels for two key drug metabolism genes were determined. Overall, the expression of most genes was downregulated by Intralipid®-based TPN (Gstp1, Gstm1, 3, 6, Nqo1, Ho-1, Mt-1, Gclc, Gclm, Cyp2d9, 2f2, 2b10, and 3a11). Omegaven® showed similar results as Intralipid® except for preserving the expression of Gstm1 and Cyp3a11, and increasing Ho-1. Total GSH levels were decreased by Intralipid®, but increased by Omegaven®. CYP3A11 protein levels were increased by Omegaven®. In conclusion, TPN reduced the expression of many genes involved in anti-oxidative stress and drug metabolism in mice. However, Omegaven® preserved expression of Cyp3a11, suggesting another beneficial effect of Omegaven® in protecting liver functions.

Glutathione-S-transferase: role in buffalo (Bubalus bubalis) sperm capacitation and cryopreservation.

In this study, glutathione-S-transferase Mu3 (GST) has been reported to play an important role in sperm capacitation, acrosome reaction, and fertilization. The freshly ejaculated buffalo spermatozoa were in vitro capacitated using heparin (10 µg/mL) or cryopreserved in egg yolk citrate extender. Glutathione-S-transferase was identified and characterized in terms of their isozymic forms, tyrosine phosphorylation, and immunolocalization patterns in cryopreserved buffalo spermatozoa in comparison with freshly ejaculated and in vitro capacitated spermatozoa. Two-dimensional gel electrophoresis, immunoblot, immunocytochemistry, and enzyme activity analyses were done to characterize GST in this study. Five and eight isozymic forms of GST were detected in cryopreserved and capacitated spermatozoa, respectively. Differential tyrosine phosphorylation of these enzymes was observed in cryopreserved and capacitated spermatozoa. The tyrosine phosphorylation of this enzyme involved cAMP protein kinase-A dependent and extracellular signal-regulated kinase independent pathways during in vitro capacitation of the spermatozoa. Differential immunolocalization patterns of GST were observed in freshly ejaculated, capacitated, and cryopreserved spermatozoa. Glutathione-S-transferase Mu3 enzyme activity was found to be significantly (P < 0.05) different in freshly ejaculated, capacitated, and cryopreserved spermatozoa. Activity of GST was significantly (P < 0.05) increased with the progression of capacitation. The cryopreserved spermatozoa showed significantly (P < 0.05) greater enzyme activity compared with fresh spermatozoa and was equal to 2-hour capacitated spermatozoa. The cryopreserved spermatozoa showed significant (P < 0.05) loss of GST enzyme protein. Tyrosine phosphorylated GST showed significantly (P < 0.05) greater activity compared with their dephosphorylated forms. The information generated in this study can be used to understand the molecular mechanism of the effects of GST on capacitation. Regulation of GST during sperm cryopreservation could be a good target to improve fertility of cryopreserved spermatozoa for their use in assisted reproductive technologies.