Glutathione S-transferase Mu 3 Research Articles

Echinatin suppresses cutaneous squamous cell carcinoma by targeting GSTM3-mediated ferroptosis

BackgroundCutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers for which effective drugs are urgently needed. Echinatin, a natural compound extracted from Glycyrrhiza plants, has shown promising antitumour effects. However, the efficacy and the direct target of echinatin in cSCC remain unclear. PurposeThis study conducted a systematic investigation of the antitumour effects of echinatin on cSCC and the underlying mechanisms involved. Study design and methodsThree cSCC cell lines, a xenograft model, and a UV-induced cSCC mouse model were used to investigate the potential protective effects of echinatin. The interactions between echinatin and glutathione S-transferase mu3 (GSTM3) and between echinatin and peroxiredoxin-2 (PRDX2) were evaluated by a proteome microarray assay, pull-down LC‒MS/MS analysis, surface plasmon resonance, and molecular docking. The potential mechanisms of GSTM3-mediated echinatin activity were analysed by using western blotting, lentivirus infection and small interfering RNA (siRNA) transfection. ResultsIn this study, we found that echinatin inhibited the proliferation and migration of cSCC cells but had no cytotoxic effect on primary human keratinocytes. Furthermore, echinatin significantly inhibited tumour growth in vivo. Mechanistically, our data showed that echinatin could directly bind to GSTM3 and PRDX2. Notably, echinatin inhibited GSTM3 and PRDX2 levels by promoting their proteasomal degradation, which led to the disruption of ROS production. We then revealed that echinatin increased mitochondrial ROS production by inhibiting GSTM3. Moreover, echinatin triggered ferroptosis by inhibiting GSTM3-mediated ferroptosis negative regulation (FNR) proteins. In addition, echinatin regulated GSTM3-mediated ROS/MAPK signalling. ConclusionEchinatin has good antitumour effects both in vitro and in vivo. Moreover, our findings indicate that GSTM3 and PRDX2 could function as viable targets of echinatin in cSCC. Consequently, echinatin represents a novel treatment for cSCC through the targeting of GSTM3-mediated ferroptosis.

GSTM3 enhances radiosensitivity of nasopharyngeal carcinoma by promoting radiation-induced ferroptosis through USP14/FASN axis and GPX4

BackgroundRadiotherapy is a critical treatment modality for nasopharyngeal carcinoma (NPC). However, the mechanisms underlying radiation resistance and tumour recurrence in NPC remain incompletely understood.MethodsOxidised lipids were assessed through targeted metabolomics. Ferroptosis levels were evaluated using cell viability, clonogenic survival, lipid peroxidation, and transmission electron microscopy. We investigated the biological functions of glutathione S-transferase mu 3 (GSTM3) in cell lines and xenograft tumours. Co-immunoprecipitation, mass spectrometry, and immunofluorescence were conducted to explore the molecular mechanisms involving GSTM3. Immunohistochemistry was performed to investigate the clinical characteristics of GSTM3.ResultsIonising radiation (IR) promoted lipid peroxidation and induced ferroptosis in NPC cells. GSTM3 was upregulated following IR exposure and correlated with IR-induced ferroptosis, enhancing NPC radiosensitivity in vitro and in vivo. Mechanistically, GSTM3 stabilised ubiquitin-specific peptidase 14 (USP14), thereby inhibiting the ubiquitination and subsequent degradation of fatty acid synthase (FASN). Additionally, GSTM3 interacted with glutathione peroxidase 4 (GPX4) and suppressed GPX4 expression. Combining IR treatment with ferroptosis inducers synergistically improved NPC radiosensitivity and suppressed tumour growth. Notably, a decrease in GSTM3 abundance predicted tumour relapse and poor prognosis.ConclusionsOur findings elucidate the pivotal role of GSTM3 in IR-induced ferroptosis, offering strategies for the treatment of radiation-resistant or recurrent NPC.

STAT3 signaling in pancreatic ductal adenocarcinoma: a candidate therapeutic target.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor prognosis which is lethal in over 90% of cases despite the standard therapies. Mainly activated by Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3) is a key transcription factor, capable of exerting the expression of multitude of genes involved in survival. Moreover, STAT3 activity is regulated by the interleukin 28 receptor α (IL28RA) and glutathione s-transferase mu-3 (GSTM3), up-regulation of both contributes to the invasiveness of pancreatic cancer cells. In this regard, STAT3 overactivity has an important pathogenic role in the development of PDAC as it is associated with enhanced cell proliferation, survival, angiogenesis, and metastasis. STAT3-associated expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 3 and 9 are implicated in the angiogenic and metastatic behavior of the PDAC. Multitude of evidence underline the protective role of STAT3 inhibition against PDAC both in cell cultures and in tumor grafts. However, specific inhibition of STAT3 was not feasible until recently, when a selective potent chemical STAT3 inhibitor, termed N4, were developed and it turned out to be highly effective against PDAC in vitro, as well as in vivo. This review aims to discuss the most recent advances in our understanding of STAT3 role in the pathogenesis of PDAC and its therapeutic applications.

Single-cell analysis of cellular heterogeneity and interactions in the ischemia-reperfusion injured mouse intestine

Nine major cell populations among 46,716 cells were identified in mouse intestinal ischemia‒reperfusion (II/R) injury by single-cell RNA sequencing. For enterocyte cells, 11 subclusters were found, in which enterocyte cluster 1 (EC1), enterocyte cluster 3 (EC3), and enterocyte cluster 8 (EC8) were newly discovered cells in ischemia 45 min/reperfusion 720 min (I 45 min/R 720 min) group. EC1 and EC3 played roles in digestion and absorption, and EC8 played a role in cell junctions. For TA cells, after ischemia 45 min/reperfusion 90 min (I 45 min/R 90 min), many TA cells at the stage of proliferation were identified. For Paneth cells, Paneth cluster 3 was observed in the resting state of normal jejunum. After I 45 min/R 90 min, three new subsets were found, in which Paneth cluster 1 had good antigen presentation activity. The main functions of goblet cells were to synthesize and secrete mucus, and a novel subcluster (goblet cluster 5) with highly proliferative ability was discovered in I 45 min/R 90 min group. As a major part of immune system, the changes in T cells with important roles were clarified. Notably, enterocyte cells secreted Guca2b to interact with Gucy2c receptor on the membranes of stem cells, TA cells, Paneth cells, and goblet cells to elicit intercellular communication. One marker known as glutathione S-transferase mu 3 (GSTM3) affected intestinal mucosal barrier function by adjusting mitogen-activated protein kinases (MAPK) signaling during II/R injury. The data on the heterogeneity of intestinal cells, cellular communication and the mechanism of GSTM3 provide a cellular basis for treating II/R injury.

GLUL gene knockdown and restricted glucose level show synergistic inhibitory effect on the luminal subtype breast cancer MCF7 cells' proliferation and metastasis.

The glutamine synthetase path is one of the most important metabolic pathways in luminal breast cancer cells, which plays a critical role in supplying glutamine as an intermediate in the biosynthesis of amino acids and nucleotides. On the other hand, glycolysis and its dominant substrate, glucose, are the most critical players in cancer metabolism. Accordingly, targeting these two critical paths might be more efficient in luminal-type breast cancer treatment. MCF7 cells were cultivated in media containing 4.5, 2, and 1 g/L glucose to study its effects on GLUL (Glutamate Ammonia Ligase) expression. Followingly, high and low glucose cell cultures were transfected with 220 pM of siGLUL and incubated for 48 h at 37 ºC. The cell cycle progression and apoptosis were monitored and assessed by flow cytometry. Expression of GLUL, known as glutamine synthetase, was evaluated in mRNA and protein levels by qRT-PCR and western blotting, respectively. To examine the migration and invasion capacity of studied cells exploited from wound healing assay and subsequent expression studies of glutathione-S-transferase Mu3 (GSTM3) and alfa-enolase (ENO1). Expression of GLUL significantly decreased in cells cultured at lower glucose levels compared to those at higher glucose levels. siRNA-mediated knockdown of GLUL expression in low glucose cultures significantly reduced growth, proliferation, migration, and invasion of the MCF7 cells and enhanced their apoptosis compared to the controls. Based on the results, GLUL suppression down-regulated GSTM3, a main detoxifying enzyme, and up-regulated Bax. According to the role of glycolysis as a ROS suppressor, decreased amounts of glucose could be associated with increased ROS; it can be considered an efficient involved mechanism in this study. Also, increased expression of Bax could be attributable to mTOR/AKT inhibition following GLUL repression. In conclusion, utilizing GLUL and glycolysis inhibitors might be a more effective strategy in luminal-type breast cancer therapy. See also Figure 1(Fig. 1).

Glutathione S-transferase Mu 3 is associated to in vivo fertility, but not sperm quality, in bovine

In the dairy breeding industry, pregnancy of dairy cows is essential to initiate milk production, so that high fertility rates are required to increase their productivity. In this regard, sperm proteins that are indicative of sperm quality and/or fertility have become an important target of study. Glutathione S-transferase Mu 3 (GSTM3) has been established as a fertility and sperm quality parameter in humans and pigs and, consequently, it might be a potential biomarker in cattle. For this reason, the present work aimed to determine if GSTM3 could predict sperm quality and in vivo fertility in this species. Sperm quality was assessed with flow cytometry and computer-assisted sperm analysis. Immunoblotting and immunofluorescence analysis were performed to determine the presence and localisation pattern of sperm GSTM3. This enzyme was found to be present in bovine sperm and to be localised along the sperm tail and the equatorial segment of the head. No significant associations between sperm GSTM3 and sperm quality parameters were observed, except a negative association with morphologically abnormal sperm having a coiled tail. In addition, and more relevant, higher levels of GSTM3 in sperm were seen in bulls showing lower in vivo fertility rates. In conclusion, our data evidenced the presence of GSTM3 in bovine sperm. Moreover, we suggest that, despite not being associated with sperm quality, GSTM3 might be an in vivo subfertility biomarker in cattle sperm, and that high levels of this protein could be an indicative of defective spermatogenesis and/or epididymal maturation.

P-049 Sperm GSTM3: a potential molecular biomarker for sperm quality and male (in)fertility

Abstract Study question Is sperm Glutathione S-transferase Mu 3 (GSTM3) associated to sperm physiological status and male (in)fertility? Summary answer GSTM3 is expressed in human sperm, and is associated to sperm quality and fertility, which suggests it as a potential molecular biomarker for male (in)fertility. What is known already GSTM3 is an antioxidant enzyme expressed in sperm cells that is essential for an appropriate mitochondrial function, plasma membrane stability and oxidative regulation. Previous evidence suggested sperm GSTM3 to be useful as an infertility prognosis and diagnosis tool in mammalian species. In humans, expression of sperm GSTM3 has been found to be altered in mitochondrial-impaired sperm and in sperm from patients with unilateral varicocele. However, neither has this enzyme been related to semen alterations nor has it been reported to be associated to male infertility. Study design, size, duration A total of 34 semen samples from healthy, fertile men and men referred for infertility evaluation were collected, analysed and subsequently cryopreserved. Samples were classified as fertile having normal sperm parameters (NSP; n = 10); infertile with asthenoteratozoospermia (AT; n = 8) or oligoasthenoteratozoospermia (OAT; n = 8); or with idiopathic infertility (n = 8). Finally, frozen-thawed sperm samples were assessed for DNA fragmentation, and the presence, localization and relative content of GSTM3. Protein expression of GSTM3 in sperm was compared between groupss. Participants/materials, setting, methods All samples were initially evaluated by conventional sperm analysis (ejaculate volume and pH, sperm concentration, motility and morphology). Protein expression profile of GSTM3 was determined through immunoblotting and immunolocalization assays. Sperm GSTM3 was quantified using an enzyme-linked immunosorbent assay. Spearman's rank-order correlation coefficients between GSTM3 levels and sperm quality parameters were calculated. Statistical differences between groups were determined by Kruskal-Wallis followed by Mann-Whitney U tests. Main results and the role of chance GSTM3 was found to be present in human sperm cells, and to be localized along the tail. Sperm GSTM3 was positively and significantly correlated with sperm quality parameters (concentration [Rs=0.51], morphology [Rs=0.42], total [Rs=0.60] and progressive motility [Rs=0.58]; P < 0.05). Sperm GSTM3 levels were compared between sperm quality groups (NSP, AT and OAT). Sperm GSTM3 levels were found to be significantly higher (P < 0.05) in men with NSP than in those with AT or OAT (62.1, 28.1 and 20.6 ng GSTM3/mg total protein, respectively). Finally, levels of GSTM3 in sperm were compared between fertile and infertile men. Whereas fertile and idiopathic infertile men showed similar sperm GSTM3 levels (63.7 vs. 60.0 ng GSTM3/mg total protein, respectively, P > 0.05), infertile men with an altered seminogram had significantly lower (P < 0.05) levels (24.4 ng GSTM3/mg total protein). Limitations, reasons for caution The limited sample size of the present study warrants further research and clinical trials with larger sample sets before implementing this biomarker as a molecular diagnostic tool in fertility clinics. Wider implications of the findings The use of sperm GSTM3 as a novel biomarker in fertility clinics may be translated into cost-effective, non-invasive, time-saving and accurate diagnosis of men (in)fertility. Trial registration number N/A

Freezability biomarkers in the epididymal spermatozoa of swamp buffalo

As an alternative to ejaculated semen, epididymal spermatozoa (ES) can be recovered and cryopreserved for use in artificial insemination and other assisted reproductive technologies. However, variabilities in the sperm response to freeze-thaw procedures challenge its inherent value. Therefore, the present study aims to clarify the freezability phenomena in the swamp buffalo ES, where pieces of literature do not abound. Here, we isolated ES from swamp buffaloes using abattoir-derived, post-mortem caudal epididymis by slicing-flushing technique. Following cryopreservation by slow-freezing, ES samples were classified into high (HF) and low freezability (LF) based on their post-thaw total and progressive motilities. Conventional sperm parameters and proteins of interest, such as glutathione S-transferase Mu 3 (GSTM3) and ATP synthase beta subunit (ATP1B1), were assessed and compared between HF and LF. Computer-assisted sperm analysis revealed that nearly all motion and kinematic parameters significantly differed among freezability groups except for wobble, linearity, and straightness. Moreover, intracellular reactive oxygen species production was evident in both HF and LF after fluorescence staining, with the latter having considerably greater malondialdehyde levels than the former. Immunohistochemical labeling demonstrated that both GSTM3 and ATP1B1 proteins were present in the ES and the epididymal tubular epithelium. Although the GSTM3 relative amounts, as analyzed through Western blot, were significantly higher in LF than HF in association with lipid peroxidation, no significant differences were observed in the case of ATP1B1. Such variations in motility, motion and kinematics, oxidative stress status, and specific sperm proteins suggest their potential utility in distinguishing freezability phenotypes in swamp buffalo ES.

GSTM3 deficiency impedes DNA mismatch repair to promote gastric tumorigenesis via CAND1/NRF2-KEAP1 signaling

Gastric cancer (GC) is one of the most severe gastric diseases worldwide. However, the molecular basis that drives tumorigenesis and progression is not completely understood, which hinders the efficacy and development of therapeutic options. Glutathione-S-transferases (GSTs) are a group of phase II detoxification enzymes that maintain redox homeostasis; however, their roles in cancers are not well defined. Here, we revealed that the expression of GST family members is significantly impaired in GC tissues. Glutathione-S-transferase mu 3 (GSTM3), a member of GST family, is dramatically downregulated in cancerous tissues and has been identified as an independent prognostic factor in GC associated with tumor differentiation, inhibiting GC cell proliferation and migration in vitro and in vivo. Mechanistically, GSTM3 is transcriptionally activated by NRF2/KEAP1 signaling. As a feedback loop, GSTM3 binds to Cullin-associated and neddylation-dissociated 1 protein (CAND1), an exchange factor for integrating Kelch-like ECH-associated protein 1 (KEAP1) into Cul3-RING ubiquitin ligases (CRL3), to disrupt nuclear factor-erythroid factor 2-related factor 2 (NRF2)/KEAP1 binding and prevent NRF2 ubiquitination and degradation, leading to its activation. A deficiency in glutathione S-Transferase Mu 3 (GSTM3) reduces DNA mismatch repair (MMR) gene expression and increases mutagenesis via CAND1/NRF2 binding. Importantly, GSTM3/NRF2 and KEAP1 were negatively and positively associated with the genomic signature for microsatellite instability, respectively. Clinically, GSTM3, NRF2, and MutS homolog 6 (MSH6) were positively correlated in the GC specimens. This study uncovered a reciprocal regulation between GSTM3 and NRF2 and established a functional and clinical link between GSTM3-NRF2/KEAP1 and MMR during GC cell proliferation and progression, thus providing potential therapeutic targets for GC.

Graphene-triphenyl phosphate (TPP) co-exposure in the marine environment: Interference with metabolism and immune regulation in mussel Mytilus galloprovincialis

Both immune regulation and endocrine systems are great challenges to marine organisms, and effective protocols for determining these adverse outcome pathways are limited, especially in vivo. The increasing usage of graphene nanomaterials can lead to the frequent exposure to marine organisms. Triphenyl phosphate (TPP), an organophosphate flame retardant, is frequently detected in natural environments. In this study, the combined toxic effects of co-exposure to graphene and TPP was investigated in Mytilus galloprovincialis using computational toxicology and multi-omics technology. Noticeably, graphene could disturb the membrane stability and increase the tissue accumulation of TPP. The adsorption behavior of TPP on graphene could inhibit the surface activity of graphene. In the digestive gland, transcriptomics analysis revealed the down-regulated genes in graphene + TPP treatment, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sorbitol dehydrogenase (SORD), glutathione s-transferase mu 3 (GSTM3) and 4-aminobutyrate aminotransferase (ABAT), were mainly associated with oxidative stress and energy metabolism. Moreover, metabolic responses indicated that graphene + TPP could cause disturbances in energy metabolism and osmotic regulation marked by differentially altered ATP, glucose and taurine in mussels. These data underline the need for further knowledge on the potential interactions of nanomaterials with existing contaminants in marine organisms.

Whole-proteome analysis of mesonephric-derived cancers describes new potential biomarkers

Mesonephric carcinomas (MEs) and female adnexal tumors of probable Wolffian origin (FATWO) are derived from embryologic remnants of Wolffian/mesonephric ducts. Mesonephric-like carcinomas (MLCs) show identical morphology to ME of the cervixbut occur in the uterus and ovary without convincing mesonephric remnants. ME, MLC, and FATWO are challenging to diagnose due to their morphologic similarities to Müllerian/paramesonephric tumors, contributing to a lack of evidence-based and tumor-specific treatments. We performed whole-proteomic analysis on 9 ME/MLC and 56 endometrial carcinomas (ECs) to identify potential diagnostic biomarkers. Although there were no convincing differences between ME and MLC, 543 proteins showed increased expression in ME/MLC relative to EC. From these proteins, euchromatic histone lysine methyltransferase 2 (EHMT2), glutathione S-transferase Mu 3 (GSTM3), eukaryotic translation elongation factor 1 alpha 2 (EEF1A2), and glycogen synthase kinase 3 beta were identified as putative biomarkers. Immunohistochemistry was performed on these candidates and GATA3 in 14 ME/MLC, 8 FATWO, 155EC, and normal tissues. Of the candidates, only GATA3 and EHMT2 were highly expressed in mesonephric remnants and mesonephric-derived male tissues. GATA3 had the highest sensitivity and specificity for ME/MLC versus EC (93% and 99%)but was absent in FATWO. EHMT2 was 100% sensitive for ME/MLC & FATWObut was not specific (65%). Similarly, EEF1A2 was reasonably sensitive to ME/MLC (92%) and FATWO (88%)but was the least specific (38%). GSTM3 performed intermediately (sensitivity for ME/MLC and FATWO: 83% and 38%, respectively; specificity 67%). Although GATA3 remained the best diagnostic biomarker for ME/MLC, we have identified EHMT2, EEF1A2, and GSTM3 as proteins of interest in these cancers. FATWO's cell of origin is uncertain and remains an area for future research.

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.

Seminal GSTM3, CRISPLD2, and RARRES1 diagnose sperm functional alterations

Sperm functional alterations are considered the main cellular mechanisms of male infertility, observed in 25% of infertile men. We have previously shown, using a shotgun proteomics approach, that Glutathione S-transferase Mu3 (GSTM3), Cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) and Retinoic acid receptor responder protein 1 (RARRES1) seminal proteins are associated with sperm abnormalities. Current limitations in the quantitative aspect of a shotgun proteomics approach demand these results be validated in a larger cohort. In this study, thus, we sought to evaluate, using a confirmatory method, GSTM3, CRISPLD2 and RARRES1 levels in seminal plasma in association with sperm mitochondrial activity and DNA fragmentation. Prospective study. 197 normozoospermic men were recruited for this study. Semen was collected by masturbation following 2 to 5 days of ejaculatory abstinence. An aliquot was used for semen analysis, another for sperm functional evaluation, and the remaining semen volume was centrifuged for separation of seminal plasma. Sperm functional data were used to separate the experimental groups: High (control, n=27) and Low (study, n=27) sperm mitochondrial activity, and Low (control, n=29) and High (study, n=29) sperm DNA fragmentation. Seminal plasma of all samples was utilized to evaluate the levels of GSTM3, CRISPLD2 and RARRES1 by Western blotting. Data were normalized using total protein (Ponceau S staining) and multiplied by the ejaculate volume, to obtain the total protein levels in the ejaculate. Groups were compared using an unpaired Student’s t test (α=5%). GSTM3 was observed as two different bands (27 kDa, its expected molecular mass, and 38 kDa). Total levels of both bands were twice as high in samples with sperm mitochondrial alterations. CRISPLD2 was observed as three different bands (17, 28 and 38 kDa, expected molecular mass = 51 kDa), suggesting its digestion by seminal plasma proteases during semen liquefaction. Total seminal levels of the 17 and 28 kDa bands were decreased in samples with high sperm DNA fragmentation (1.6-fold and 2.6-fold decrease, respectively). Likewise, RARRES1 was identified as two bands (50 and 62 kDa, expected molecular mass = 33 kDa), suggesting post-translational modifications and/or dimer formation. The 50 kDa band was 1.9-fold decreased in the seminal plasma of men with sperm DNA fragmentation. Seminal plasma proteins directly reflect sperm functional alterations and, thus, male infertility. Seminal plasma proteins may be used as markers of functional alterations. In this study, we confirmed that this is the case for GSTM3 (sperm mitochondrial activity), and CRISPLD2 and RARRES1 (sperm DNA fragmentation).

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

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. 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. 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. 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.

Evaluation of genetic polymorphisms in glutathione S-Transferase theta1, glutathione S-Transferase mu1, and glutathione S-Transferase mu3 in oral leukoplakia and oral squamous cell carcinoma with deleterious habits using polymerase chain reaction

Context:Oral squamous cell carcinoma (OSCC) is the sixth most common cancer in the world. As per previous studies, most patients who develop oral cancer are elderly males who are heavy users of tobacco and alcohol; however, the incidence is increasing in younger individuals and in those who neither smoke nor drink. Many of the genes that code for the detoxification enzymes are polymorphic with abnormal activity profiles.Aims and Objectives:The aim of this study was to evaluate the risk of development of oral leukoplakia (OLP) and OSCC in glutathione S-transferase polymorphisms genes in the east coast of Andhra Pradesh population with tobacco consumption habit and habit-free controls using polymerase chain reaction (PCR-restriction fragment length polymorphism).Materials and Methods:This study included 15 patients each with histologically proven epithelial dysplasia and OSCC and compared with age- and gender-related controls with no tobacco habits in any form. A volume of 2 ml of blood sample was collected into presterilized vials containing ethylenediaminetetracetic acid from each individual under aseptic conditions. DNA extraction was done from whole blood, and PCR was performed. Statistical analysis was performed using Chi-square test and odds ratio (OR) and 95% confidence intervals (CIs).Results:The results are suggestive that glutathione S-transferase mu1 (GSTM1) null was associated with increased risk of OLP (OR = 5.5, 95% CI = 1.14–26.41, P = 0.021) and OSCC (OR = 11, 95% CI = 1.99–60.5, P = 0.021). Glutathione S-transferase theta1 (GSTT1) null genotype was associated with increased risk of OLP (OR = 2.154, 95% CI = 0.74–26.672, P > 0.99) and OSCC (OR = 2.154, 95% CI = 0.74–26.672, P > 0.99). The glutathione S-transferase mu3 (GSTM3) AB + BB genotypes appear to be risk factors for OSCC (OR = 1.31, 95% CI = 0.31–5.58, P = 0.7) although statistically insignificant.Conclusion:Hence to conclude, because of small sample size in the present study, statistically insignificant results were found and this study failed to observe the relationship between GSTM3 and GSTT1 polymorphism and risk of developing OSCC and positive relationship was observed with GSTM1 polymorphism and risk of developing OSCC.

Relationship Between the Altered Expression and Epigenetics ofGSTM3and Age-Related Cataract

Glutathione S-Transferase Mu 3 (GSTM3) protects the lens from oxidative stress that contributes to age-related cataract (ARC) formation. We examined the expression and epigenetics of GSTM3 in lens epithelial cells (LECs) and lens cortex of ARC, and investigated the potential role of molecular changes in ARC pathogenesis. This study included 120 ARCs and 40 controls. Expression of GSTM3, DNA methylation, and histone modification were assessed by quantificational real-time PCR, Western blot, bisulfite-sequencing PCR, pyrosequencing, and chromatin immunoprecipitation assay. Human lens epithelial (HLE) cell lines, SRA01/04 and HLEB3, were served as an in vitro model to observe the relationship between epigenetic status and GSTM3 expression. Potential transcription factors binding to GSTM3 promoter were detected by electrophoretic mobility shift assay. Expression of GSTM3 decreased in ARC lens tissues compared to that in the controls, which correlated with the hypermethylation of GSTM3 promoter. Lower level of GSTM3 was detected in HLEB3 than in SRA01/04, while HLEB3 displayed hypermethylation of GSTM3 and SRA01/04 did not. Compared to SRA01/04, HLEB3 displayed lower acetylated H3 and higher trimethylated H3K9 levels. After treatment with DNA methyltransferase inhibitor or histone deacetylase inhibitor, HLEB3 had an increased GSTM3 expression. Methylation of GSTM3 promoter abrogated the potential transcription factor binding. The GSTM3 expression declined in hydrogen peroxide-treated HLE cell lines. Expression of GSTM3 might be regulated by epigenetic changes in lens tissue. Hypermethylation in GSTM3 promoter and altered histone modification might have a role in the ARC formation. The results provided a potential strategy of ARC management by manipulating epigenetic changes.

Proteomic approaches for profiling negative fertility markers in inferior boar spermatozoa.

The ability to predict male fertility is of paramount importance for animal breeding industries and for human reproduction. Conventional semen analysis generally provides information on the quantitative parameters of spermatozoa, but yields no information concerning its functional competence. Proteomics have identified candidates for male fertility biomarkers, but no studies have clearly identified the relationship between the proteome and sperm fertility. Therefore, we performed a proteomic analysis to investigate small and large litter size boar spermatozoa and identify proteins related to male fertility. In this study, 20 proteins showed differential expression levels in small and large litter size groups. Nineteen of these proteins exhibited decreased expression in large litter size samples and increased expression in the small litter group. Interestingly, only one protein was highly expressed in the large litter size spermatozoa. We then identified signaling pathways associated with the differentially expressed protein markers. Glutathione S-transferase Mu3 and glutathione peroxidase 4 were related to the glutathione metabolic pathway and arginine vasopressin receptor 2 was linked to vasopressin R2/STAT. In summary, this is the first study to consider negative fertility biomarkers, and the identified proteins could potentially be used as biomarkers for the detection of inferior male fertility.

Proteome analysis of early lineage specification in bovine embryos

During mammalian embryo development, the zygote undergoes embryonic cleavage in the oviduct and reaches the uterus at the morula stage, when compaction and early lineage specification take place. To increase knowledge about the associated changes of the embryonic protein repertoire, we performed a comprehensive proteomic analysis of in vitro produced bovine morulae and blastocysts (six biological replicates), using an iTRAQ-based approach. A total of 560 proteins were identified of which 502 were quantified. The abundance of 140 proteins was significantly different between morulae and blastocysts, among them nucleophosmin (NPM1), eukaryotic translation initiation factor 5A-1 (EIF5A), receptor of activated protein kinase C 1 (GNB2L1/RACK1), and annexin A6 (ANXA6) with increased, and glutathione S-transferase mu 3 (GSTM3), peroxiredoxin 2 (PRDX2), and aldo-keto reductase family 1 member B1 (AKR1B1) with decreased abundance in blastocysts. Seventy-three percent of abundance altered proteins increased, reflecting an increase of translation activity in this period. This is further supported by an increase in the abundance of proteins involved in the translation machinery and the synthesis of ATP. Additionally, a complementary 2D saturation DIGE analysis led to the detection of protein isoforms, e.g. of GSTM3 and PRDX2, relevant for this period of mammalian development, and exemplarily verified the results of the iTRAQ approach. In summary, our systematic differential proteome analysis of bovine morulae and blastocysts revealed new molecular correlates of early lineage specification and differentiation events during bovine embryogenesis.

Genetic variation in the GSTM3 promoter confer risk and prognosis of renal cell carcinoma by reducing gene expression

Background:Glutathione S-transferase mu 3 (GSTM3) has been proven to be downregulated in renal cell carcinoma (RCC). We aimed to characterise the role of GSTM3 and its genetic predisposition on the occurrence and postoperative prognosis of RCC.Methods:The effect of GSTM3 on RCC aggressiveness was examined using transfection and silencing methods. Glutathione S-transferase mu 3 expression in renal tissues was examined by immunohistochemistry. The associations of rs1332018 (A-63C) and rs7483 (V224I) polymorphisms with RCC risk were examined using 400 RCC patients and 802 healthy controls. The factors contributing to postoperative disease-specific survival of RCC patients were evaluated using the Cox proportional hazard model.Results:Glutathione S-transferase mu 3 silencing increased the invasion and anchorage-independent growth of RCC cell lines. rs1332018 (AC+CC vs AA), which correlated with low expression of GSTM3 in kidney, was associated with RCC risk (odds ratio, 1.446; 95% confidence interval (CI), 1.111–1.882). rs1332018 variants and low GSTM3 expression significantly predicted unfavourable postoperative survivals of RCC patients (P<0.05). rs1332018 variants independently predicted a poor prognosis (hazard ratio, 2.119; 95% CI, 1.043–4.307).Conclusion:Glutathione S-transferase mu 3 may function as a tumour suppressor in RCC. rs1332018 genetic variants predispose the host to downregulating GSTM3 expression in kidney, facilitate carcinogenesis, and predict an unfavourable postoperative prognosis of RCC.

Oxidative stress contributes to liver damage in a murine model of alpha-1-antitrypsin deficiency

Alpha-1-antitrypsin deficiency is a genetic disorder resulting in the expression of misfolded mutant protein that can polymerize and accumulate in hepatocytes, leading to liver disease in some individuals. Transgenic PiZ mice are a well-characterized model, which express human alpha-1-antitrypsin mutant Z protein (ATZ protein) and faithfully recapitulate the human liver disease. Liver tissue expressing alpha-1-antitrypsin mutant Z protein exhibits inflammation, injury and replacement of damaged cells. Fibrosis and hepatocellular carcinoma (HCC) develop in aging PiZ mice. In this study, microarray analysis was performed comparing young PiZ (ZY) mice to wild-type (WY), and indicated that there were alterations in gene expression levels that could influence a number of pathways leading to liver disease. Redox-regulating genes were up-regulated in ZY tissue, including carbonyl reductase 3 (CBR3), glutathione S-transferase alpha 1 + 2 (GSTA(1 + 2)) and glutathione S-transferase mu 3 (GSTM3). We hypothesized that oxidative stress could develop in Z mouse liver, contributing to tissue damage and disease progression with age. The results of biochemical analysis of PiZ mouse liver revealed that higher levels of reactive oxygen species (ROS) and a more oxidized, cellular redox state occurred in liver tissue from ZY mice than WY. ZY mice showed little evidence of oxidative cellular damage as assessed by protein carbonylation levels, malondialdehyde levels and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG) staining. Aging liver tissue from PiZ older mice (ZO) had elevated ROS, generally lower levels of antioxidant enzymes than younger mice and evidence of cellular damage. These data indicate that oxidative stress is a contributing factor in the development of liver disease in this model of alpha-1-antitrypsin deficiency.