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Our previous study showed remarkable differences in the effect of R-sulforaphane (R-SFN) on the expression of CYPs 19, 1A1, 1A2, and 1B1 in ER(+) MCF7, ER( -) MDA-MB-231, and non-tumorigenic immortalized MCF10A (8). This study aimed to evaluate the effect of R-SFN on phase II enzymes induction and expression of AhR, Nrf2, and ERα in the same breast cell lines. The results showed increased expression of GSTP as a result of treatment with R-SFN in breast cancer cells. An increased NQO1 transcript and protein levels were found in all breast cells, with the most significant increase in MCF7 cells. Similarly, the enhancement of Nrf2 expression was noticed in all tested cells. AhR gene transcript and protein were decreased in MCF7 cells. In MDA-MB-231, increased AhR mRNA was not confirmed at the protein level. No differences were found in the expression of ERα. Overall, the results of the present study extended our earlier suggestions on the possible interference of R-SFN with estrogens homeostasis in breast cancer cells differing in ERα status, as well as in non-tumorigenic immortalized breast epithelial cells. While some of R-SFN effects might be beneficial and useful in breast cancer prevention, the others, particularly GSTP induction, may lead to adverse effects.

Glutathione S-transferase P1 (GSTP1), a phase II detoxifying enzyme, is overexpressed and plays an important role during breast cancer drug resistance. Tumor-associated macrophages (TAMs), representing most of the leukocyte population in solid tumors, are involved in cancer cell resistance to chemotherapy. Although GSTP1 exists in TAMs, whether GSTP1 in TAMs promotes drug resistance is still unclear. In the current study, we found a novel mechanism that GSTP1 in TAMs contributes breast cancer cell drug resistance. GSTP1 is aberrantly expressed in TAMs from breast cancer tissues of patients after chemotherapy than that without chemotherapy. Adriamycin (ADR) time-dependently induced the expression of GSTP1 in TAMs in vitro. Conditional medium of TAMs significantly inhibited ADR-induced cell death of MCF-7 breast cancer cells. Meanwhile, overexpression of GSTP1 in TAMs promoted the expression and release of interleukin-6 (IL-6) associated with reduced ADR-induced breast cell death, which was reversed by IL-6 antibody. Mechanistically, GSTP1 interacted with inhibitor of nuclear factor κB kinase β (IKKβ) to activate nuclear factor-κB (NF-κB) to induced the expression and release of IL-6 in TAMs. Moreover, IL-6 further upregulated GSTP1 through c-Jun, and ultimately mediated drug resistance in MCF-7 cells. Taken together, our data demonstrated for the first time that GSTP1 in TAMs promoted ADR-resistance in breast cancer by regulating interleukin-6 release., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ovarian cancer is one of the leading causes of mortality in women worldwide. Currently, paclitaxel is one of the most effective chemotherapies. However, resistance to paclitaxel is a major cause of therapy failure and the precise mechanism of paclitaxel resistance remains unclear. In this study, we demonstrated that the oxidative pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) promotes paclitaxel resistance. We showed that G6PD expression was higher in paclitaxel-resistant cancer cells than in their paclitaxel-sensitive counterparts. Furthermore, we demonstrated that suppressing G6PD using shRNA, or an inhibitor, either as single agents or in combination, sensitized paclitaxel-resistant cancer cells to paclitaxel treatment and thereby improving the therapeutic efficacy of paclitaxel. Interestingly, we found that the upregulation of G6PD in paclitaxel-resistant cells was due to the decreased expression of protein arginine methyltransferase 6 (PRMT6), which targets the promoter of G6PD. We further identified that G6PD promotes paclitaxel resistance by regulating the expression of glutathione S-transferase P1 (GSTP1), which confers resistance to chemotherapy by detoxifying several anticancer drugs. Taken together, our results suggest that G6PD is a novel potential target to overcome paclitaxel resistance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Glutathione S-transferase Pi (GSTP1) was originally identified as one of the cytosolic phase II detoxification enzymes and was also considered to function via its non-catalytic, ligand-binding activity. Autophagy is a self-protective mechanism of the cell to remove unnecessary or dysfunctional components, which plays a crucial role in balancing the beneficial and detrimental effects of immunity and inflammation. However, little is known about whether and how GSTP1 mediates autophagy via inhibiting LPS-induced inflammatory response. Here, we show that LPS-induced autophagy and autophagic flux blockade in THP-1 cells in a concentration- and time-dependent manner. Further, we found that the autophagy activation inhibited the activation of inflammatory signaling pathway and the release of inflammatory factors. However, inhibition of autophagy by 3-methyladenine or chloroquine significantly reduced the anti-inflammatory effect of GSTP1. In addition, our findings provide evidence that GSTP1 regulates autophagy through PI3K-Akt-mTOR pathway and inhibits LPS-induced inflammation. Overall, the current study provides an important reference for future applications of GSTP1 in the treatment of inflammatory diseases.

Liver coinfection by hepatitis B virus (HBV) and hepatitis D virus (HDV) can result in a severe form of hepatocellular carcinoma with poor prognosis. Coinfection with HDV and HBV causes more deleterious effects than infection with HBV alone. Clinical research has shown that glutathione S-transferase P1 (GSTP1), a tumor suppressor gene, is typically downregulated in liver samples from hepatitis-infected patients. In the present study, our data indicated that small HDV antigen (s-HDAg) could specifically bind to GSTP1 mRNA and significantly downregulate GSTP1 protein expression. For the human fetal hepatocyte cell line L-02, cells transfected with s-HDAg, along with decreased GSTP1 expression, there was a significant accumulation of reactive oxygen species (ROS) and increased apoptotic ratios. Restoring GSTP1 expression through silencing s-HDAg via RNAi or overexpressing exogenous GSTP1 could largely recover the abnormal cell status. Our results revealed a novel potential mechanism of HDV-induced liver injury and hepatocarcinogenesis: s-HDAg can inhibit GSTP1 expression by directly binding to GSTP1 mRNA, which leads to accumulation of cellular ROS, resulting in high cellular apoptotic ratios and increased selective pressure for malignant transformation. To our knowledge, this is the first study to examine s-HDAg-specific pathogenic mechanisms through potential protein-RNA interactions.

Objective: Breast cancer (BC) is a common malignant tumor in females. The combined assay of multiple molecular markers benefits the diagnosis and prognostic prediction. Human epidermal growth factor receptor 2 (HER2) facilitates the proliferation and differentiation of cancer cells through ligand binding. Ki67 is a tumor proliferation-related gene, whereas GSTP1 is a DNA repair-related gene. This study thus investigated the significance of HER2 and Ki67/GSTP1 gene combined assay in the diagnosis and prognosis of BC., Materials and Methods: A total of 86 breast tumor tissues and adjacent tissues were collected. Gene expression and protein levels of HER2 and Ki67 were quantified by real-time polymerase chain reaction (PCR) and Western blot, respectively. Methylation frequency of GSTP1 was analyzed by methylation-specific PCR. The correlation between HER2 and Ki67/GSTP1 and clinical/pathological features of BC was analyzed., Results: Gene and protein expression levels of HER2 and Ki67 in tumor tissues were increased (p < 0.05 compared with adjacent tissues). Methylation frequency of GSTP1 gene was 37.2%, which was significantly higher in breast tumor tissues than in adjacent tissues (12.79%, p < 0.05). HER2 expression was positively correlated with TNM stage, tumor size, and lymph node metastasis, and negatively correlated with tissue grade and estrogen receptor (ER)/progesterone receptor (PR) expression (p < 0.05). GSTP1 methylation was positively correlated with TNM stage and tumor size, and negatively correlated with ER/PR expression (p < 0.05)., Conclusions: HER2, Ki67, and GSTP1 methylation were correlated with clinical and pathological features of BC. The combined assay benefits the early diagnosis and prognostic prediction of cancer.

Background: Glutathione S-transferase pi 1 (GSTP1) is a cytosolic detoxifying enzyme that protects cells against deleterious effects of oxidative stress. Deregulated expression of GSTP1 protein and aberrant promoter methylation of GSTP1 gene were reported in various human tumors and were shown to be involved in the molecular pathway for cancer development., Aims and Methods: In this study, we aimed to determine the expression status of GSTP1 in relation to its gene promoter methylation in Moroccan population of 30 bladder cancer (BC) patients and in two noncancerous bladder tissues used as controls. GSTP1 expression was assessed by immunohistochemistry and GSTP1 gene promoter methylation status was studied by methylation-specific PCR (MS-PCR)., Results: Glutathione S-transferase pi 1 was expressed in the two normal tissues. In BC cases, GSTP1 expression was strong in 23.33% (7/30), moderate in 60% (18/30), and weak in 13.33% (4/30) of cases, while GSTP1 was not expressed in one cancer case (3.33%). Variability of GSTP1 expression does not correlate with high-grade cancer or invasive-stage (p > 0.05). No GSTP1 gene promoter methylation was detected in all control and cancer cases., Conclusion: Our data suggest that GSTP1 expression is not associated with BC development, limiting its use as a biomarker for BC management in Morocco. Moreover, difference in GSTP1 expression among BC cases is not due to GSTP1 promoter methylation., (© 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Aims: To identify experimental conditions that induce preneoplastic cells positive for glutathione S-transferase P-form (GST-P) in the rat liver by new approaches, and analysis of the mechanism of cancer initiation based on the findings., Main Methods: The experimental protocols employed to induce GST-P + preneoplastic cells in rat liver were as follows. Protocol 1: adult rats were fed basal diet containing 2-acetylaminofluorene (AAF, 0.02% by wt) and high concentrations of N-acetyl-l-cysteine (0.5%) over 10 weeks. Protocol 2: rats were subjected to partial hepatectomy (2/3PH), followed by an AAF (0.04%) diet for two more weeks. Vibratome-prepared liver sections were then immunostained for GST-P., Key Findings: GST-P was inducible in the rat liver in response to the strong carcinogenic stress by AAF in the two experimental protocols. When examined immunocytochemically with vibratome sections, the biliary tracts of hepatocytes, GST-P + single hepatocytes and foci were heavily positive for the marker enzyme in addition to ordinary cytosolic staining of preneoplastic cell populations. The biliary tracts of hepatocytes were severely injured, and the excretory portions of GST-P + single hepatocytes were significantly injured., Significance: The cytotoxic action of AAF that give rise to the GST-P + single hepatocytes was suggested to be an injury to the excretory pump(s) and the duct of hepatocytes. A new physiological mechanism was hypothesized for the induction of preneoplastic cell populations in the rat liver instead of a genetic mechanism., (Copyright © 2018. Published by Elsevier Inc.)

Troxerutin (TXER) a rutin derivative is known for its anticancer effect against hepatocellular carcinoma (HCC). As part of large study, recently we have shown TXER interact with genetic material and its anti-mutagenic property. In the present study we have explored its possible mode of action in HCC. Since TXER alone did not show significant anticancer effect on Huh-7 cells, in vitro biochemical assays were performed for determining anticancer efficacy of TXER + metal complex using transition metals such as Cu, Zn, and Fe. The anticancer efficacy of TXER + Cu on Huh-7 cells were evaluated using MTT assay, DCFDA, JC-1 staining, comet assay, cell cycle analysis, immunocytochemistry, and Western blotting. Non-toxic nature of TXER was analyzed on primary rat hepatocytes. The in vivo efficacy of TXER was tested in N-nitrosodiethylamine initiated and γ-benzene hexachloride and partial hepatectomy promoted rat liver cancer. Liver markers, transition metal levels, histopathological examination, and expression levels of GST-P, 8-OHdG and Ki-67 were studied to assess the in vivo anticancer effect of TXER. We observed that TXER + Cu induced extensive cellular death on Huh-7 cells through generating free radicals and did not possess any toxic effect on normal hepatocytes. The in vivo studies revealed that TXER possess significant anti-cancer effect as assessed through improved liver markers and suppressed GST-P, 8-OHdG, and Ki-67 expression. TXER treatment reduced the hepatic Cu level in cancer bearing animals. Current study brings the putative mechanism involved in anti-cancer effect of TXER, further it will help to formulate phytoconstituents coupled anti-cancer drug for effective treatment of HCC., (© 2017 Wiley Periodicals, Inc.)

Purpose: We previously demonstrated the anti-inflammatory and antioxidant effects of exenatide. We now hypothesized that exenatide also increases the plasma concentration of interleukin-1 receptor antagonist (IL-1RA), an endogenous anti-inflammatory protein, and modulates the nuclear factor erythroid 2‒related factor‒Kelchlike ECH-associated protein 1‒antioxidant response element (Nrf-2‒Keap-1‒ARE) system to induce key antioxidant enzymes to suppress inflammatory and oxidative stress., Methods: Twenty-four patients with obesity and type 2 diabetes receiving combined oral and insulin therapy were randomly assigned to receive either exenatide 10 μg or placebo twice a day for 12 weeks., Results: Exenatide increased IL-1RA concentration by 61% (from 318 ± 53 to 456 ± 88 pg/mL; P < 0.05). Exenatide treatment also suppressed Keap-1 protein (P < 0.05) and increased messenger RNA expression of NQO-1, glutathione S-transferase PI, heme oxygenase-1, and p21 and increased NAD(P)H dehydrogenase [quinone] 1 protein (P < 0.05) in mononuclear cells., Conclusions: Because IL-1RA protects, maintains, and stimulates β-cell function in humans and Nrf-2‒Keap-1‒ARE protects β cells in animals with experimental diabetes, these actions of exenatide may contribute to a potential protective effect on β cells in diabetes.

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined., (© 2017 Wiley Periodicals, Inc.)

Purpose: Oxidative damage of lens tissue contributes to the formation of age-related cataract. Pi-class glutathione-S-transferase (GSTP1) plays a role in the removal of oxidative adducts by transferring them to glutathione. To assess epigenetic regulation of GSTP1 and its potential role in age-related nuclear cataract (ARNC) pathogenesis, we evaluated GSTP1 mRNA expression, methylation, and chromatin modifications in lenses from ARNC patients., Methods: The mRNA and protein of lens GSTP1 were assayed by relative quantitative real-time polymerase chain reaction (qRT-PCR) and Western blots. Methylation of the GSTP1 promoter was determined by bisulfite genomic sequencing. Chromatin modification was detected by chromatin immunoprecipitation. DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities were also assayed by enzyme-linked immunosorbent assay (ELISA)-like reaction. To assess the effect of DNA methylation on the mRNA expression of GSTP1, human lens epithelium HLE-B3 cells were treated with the demethylation compound 5-aza-dC, followed by qRT-PCR assay., Results: GSTP1 mRNA and protein levels were significantly reduced in lens epithelium and cortex of ARNC cases versus age-matched controls. The changes corresponded to hypermethylation of the GSTP1 promoter CpG islands. The loss of GSTP1 mRNA and protein and the increased DNA promoter methylation might be correlated with the severity of the ARNC. ARNC lenses also had lower acetylation of histone proteins H3, H4, and lower methylation of H3K4, and higher methylation of H3K9. Histone modifications were not correlated with the severity of the ARNCs. DNMT and HDAC were elevated in lenses from ARNCs compared with controls. Demethylation treatment of HLE-B3 cells with 5-aza-dC enhanced the expression of GSTP1., Conclusions: Epigenetic alteration of GSTP1 regulates its expression in lens epithelial and cortical tissues. These changes likely contribute to the pathogenesis of ARNC.

Quercetin and phenylpropanoids are well known chemoprotective compounds identified in many plants. This study was aimed at determining their effects on activation of Nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response element (Nrf2-ARE) signalling pathway and expression of its important downstream effector phase II detoxification enzyme glutathione-S-transferase P1 (GSTP1) in BJ foreskin fibroblasts and skin HaCaT keratinocytes. Cell lines and their corresponding Nrf2-ARE luciferase reporter cells were treated by ginger phenylpropanoids and quercetin for 10 h and the level of Nrf2 activity was subsequently determined. Both, ginger phenylpropanoids and quercetin, significantly increased the level of Nrf2 activity. Subsequent western blot analyses of proteins showed the increased expression level of glutathione-S-transferase P1 (GSTP1) in BJ cells but not in HaCaT cells. Such phenomenon of unresponsive downstream target expression in HaCaT cells was consistent with previous studies showing a constitutive expression of their GSTP1. Thus, while both ginger phenylpropanoids and quercetin have the property of increasing the level of Nrf2 both in HaCaT and in BJ cells, their effects on its downstream signalling were mediated only in BJ cells.

Glutathione S-transferase pi (GSTP), a phase II gene downstream of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant-responsive element (ARE)/electrophile response element (EpRE) transcription pathway, plays a key role in both the signaling and detoxification response to Se-organic compounds with thiol peroxidase activity. We here investigated the role of GSTP on the Nrf2 activation response of cells challenged with a new class of diselenides derived from the basic structure of diphenyl diselenide [(PhSe)2]. These diselenides, and particularly 2,2'-diselenyl dibenzoic acid (DSBA), behave as mild thiol peroxidases leading to a moderate generation of H2O2 and NOx, and signaling of stress-activated and survival-promoting MAPKs, which ultimately control the mitochondrial pathway of apoptosis. Used in murine embryonic fibroblasts (MEFs) and HepG2 human hepatocarcinoma cells to produce submaximal conditions of stress, the diselenide compounds stimulated Nrf2 nuclear translocation and then the transcription of the same Nrf2 gene as well as of GSTP and other phase II genes. This resulted in a higher degree of protection against H2O2 cytotoxicity (hormetic effect). Diselenide toxicity increased in GSTP knockout MEFs by a higher generation of NOx and stress activated protein kinase (SAPK)/JNK activation. A lowered hormetic potential of these cells was observed in association with an abnormal expression and nuclear translocation of Nrf2 protein. Immunoprecipitation and affinity purification experiments revealed the existence of an Nrf2/GSTP complex in MEFs and HepG2 cells. Covalent oligomers of GSTP subunits were observed in DSBA-treated HepG2 cells. In conclusion, GSTP gene expression influences the Nrf2-dependent response to hormetic diselenides. Mechanistic interpretation for this GSTP-dependent effect may include a direct and redox-sensitive interaction of GSTP with Nrf2 protein., (Copyright © 2015 Elsevier Inc. All rights reserved.)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive type of blood malignancy, deriving from T-cell progenitors in the thymus, and comprises 10-15% of pediatric and 25% of adult primary ALL cases. Despite advances, 20% of pediatric and the majority of adult patients with T-ALL succumb to mortality from resistant or relapsed disease, and the survival rate for patients with resistant or relapsed T-ALL remains poor. Alterations in the expression of Forkhead box protein M1 (FoxM1) have been detected in several types of cancer, and the inhibition of FoxM1 has been investigated as therapeutic strategy in cancer. The present study investigated the effects of the inhibition of FoxM1 by thiostrepton in human T-ALL Jurkat cells. The cells were treated with different concentrations of thiostrepton, either alone or in combination with doxorubicin. Cell viability was measured using CCK-8 assays and the cell cycle distribution, apoptosis and cell-associated mean fluorescence intensity of intracellular doxorubicin were assessed using flow cytometric analysis. The mRNA and protein expression levels were detected by reverse transcription-quantitative polymerase chain reaction and western blot analyses. The inhibition of FoxM1 by thiostrepton significantly decreased the proliferation of the Jurkat cells proliferation in a time- and dose-dependent manner. Cell arrest at the G2/M phase, and apoptosis was significantly increased in the thiostrepton-treated Jurkat cells. Thiostrepton reduced the half maximal inhibitory concentration of doxorubicin in the Jurkat cells, and significantly enhanced the cytotoxicity of doxorubicin within the Jurkat cells by enhancing doxorubicin-induced apoptosis and increasing the accumulation of intracellular doxorubicin. Furthermore, the inhibition of FoxM1 by thiostrepton enhanced doxorubicin-induced apoptosis, possibly through a caspase-3-dependent pathway, and increased the accumulation of intracellular doxorubicin, possibly through downregulating the expression of glutathione S-transferase pi. Collectively, the results of the present study suggested that targeting FoxM1 with thiostrepton resulted in potent antileukemia activity and chemosensitizing effects in human T-ALL Jurkat cells.

The aim of the present study was to investigate the correlation between the expression of DNA (cytosine‑5)‑methyltransferase 1 (DNMT1), glutathione S‑transferase‑P1 (GSTP1) and adenomatous polyposis coli (APC), and the methylation status of GSTP1 and APC in prostate cancer (PCa) and benign prostatic hyperplasia (BPH), and to examine its clinical significance. Immunohistochemistry and reverse transcription‑polymerase chain reaction (RT‑PCR) was used to detect the expression of DNMT1, GSTP1 and APC in 56 samples of PCa tissue and 10 samples of BPH tissue. Methylation‑specific‑PCR was used to detect the methylation status of the CpG island promoters of GSTP1 and APC. The positive rate of expression of DNMT1 in poorly‑differentiated PCa, moderately‑differentiated PCa, well‑differentiated PCa and BPH was 86.7%, 70.6%, 55.6% and 30.0%, respectively (P<0.05); for GSTP1, the positive rate was 13.3%, 29.4%, 44.4% and 90.0%, respectively (P<0.05); and for APC, the positive rate was 23.3%, 47.6%, 55.6% and 70.0%, respectively (P<0.05). The correlation coefficient for the association between the expression of DNMT1 and GSTP1 was ‑0.891 (P<0.05). Between the expression of DNMT1 and APC, the correlation coefficient was ‑0.721 (P<0.05). GSTP1 and APC were hypermethylated in the majority of PCa tissue samples. The positive rate of methylation of these genes in poorly‑differentiated PCa was 83.3% and 73.3%, respectively. By contrast, hypomethylation (or demethylation) was observed in BPH samples, in which the positive rate of methylation was 10.0% and 20.0%, respectively (P<0.05). The increased expression of DNMT1, and the reduced expression of GSTP1 and APC have an important role in the development of PCa. Due to the high expression of DNMT1 mRNA, it is likely that the hypermethylation of CpG islands contributed to the silencing of GSTP1 and APC in PCa tissues.

Chemoresistance is a poor prognostic factor in breast cancer and is a major obstacle to the successful treatment of patients receiving chemotherapy. However, the precise mechanism of resistance remains unclear. In this study, a pair of breast cancer cell lines, MCF-7 and its adriamycin-resistant counterpart MCF-7/ADR was used to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We applied nanoflow liquid chromatography (nLC) and tandem mass tags (TmT) quantitative mass spectrometry to distinguish the differentially expressed proteins (DEPs) between the two cell lines. Bioinformatics analyses were used to identify functionally active proteins and networks. 80 DEPs were identified with either up- or down-regulation. Basing on the human protein-protein interactions (PPI), we have retrieved the associated functional interaction networks for the DEPs and analyzed the biological functions. Six different signaling pathways and most of the DEPs strongly linked to chemoresistance, invasion, metastasis development, proliferation, and apoptosis. The identified proteins in biological networks served to resistant drug and to select critical candidates for validation analyses by western blot. The glucose-6-phosphate dehydrogenase (G6PD), gamma-glutamyl cyclotransferase (GGCT), isocitrate dehydrogenase 1 (NADP+,soluble)(IDH1), isocitrate dehydrogenase 2 (NADP+,mitochondrial) (IDH2) and glutathione S-transferase pi 1(GSTP1), five of the critical components of GSH pathway, contribute to chemoresistance.

Breast cancer is the most common malignancy in women worldwide. Environmental factors such as xenobiotic exposure and lifestyle and nutrition play a key role in its etiology. This study was designed to evaluate the age-related changes in the expression of major xenobiotic-metabolizing enzymes (XMEs) in the rat liver and the mammary gland in the dimethylbenz(a)anthracene-induced breast cancer model. The influence of dietary lipids on the ontogeny of XMEs was also evaluated. mRNA and protein levels of phase I (CYP1A1, CYP1A2, and CYP1B1) and phase II (NAD(P)H:quinone acceptor oxidoreductase 1 and GSTP1) enzymes were analyzed, as well as their regulation by AhR and Nrf2, respectively. Results showed differences in the phase I enzymes expression, whereas little changes were obtained in phase II. High corn oil and olive oil diets differentially influenced the expression of age-related changes, suggesting that the different susceptibility to xenobiotic exposure depending upon the age may be modulated by dietary factors., (© 2014 Wiley Periodicals, Inc.)

Carnosic acid (CA), a diterpene found in the rosemary (Rosmarinus officinalis), has been reported to have a neuroprotective effect. Glutathione S-transferase (GST) P (GSTP) is a phase II detoxifying enzyme that provides a neuroprotective effect. The aim of this study was to explore whether the neuroprotective effect of CA is via an upregulation of GSTP expression and the possible signaling pathways involved. SH-SY5Y cells were pretreated with 1 μM CA followed by treatment with 100 μM 6-hydroxydopamine (6-OHDA). Both immunoblotting and enzyme activity results show that CA also induced protein expression and enzyme activity of GSTP. Moreover, CA significantly increased the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/Akt, the nuclear translocation of p65, but not mitogen-activated protein kinases (p < 0.05). Pretreatment with LY294002 (a PI3K/Akt inhibitor) suppressed the CA-induced phosphorylation of IκB kinase (IKK) and IκBα, p65 nuclear translocation, and nuclear factor-kappa B (NF-κB)-DNA binding activity as well as GSTP protein expression. Furthermore, CA attenuated 6-OHDA-induced caspase 3 activation, and cell death was reversed by GSTP siRNA or LY294002 treatment. Additionally, male Wistar rats with lesions induced by 6-OHDA treatment in the right striatum responded to treatment with CA, which significantly reversed the reduction in GSTP protein expression that resulted from lesioning. We suggest that CA prevents 6-OHDA-induced apoptosis through an increase in GSTP expression via activation of the PI3K/Akt/NF-κB pathway. Therefore, CA may be a promising candidate for use in the prevention of Parkinson's disease.

Purpose: The DOCUMENT multicenter trial in the United States validated the performance of an epigenetic test as an independent predictor of prostate cancer risk to guide decision making for repeat biopsy. Confirming an increased negative predictive value could help avoid unnecessary repeat biopsies., Materials and Methods: We evaluated the archived, cancer negative prostate biopsy core tissue samples of 350 subjects from a total of 5 urological centers in the United States. All subjects underwent repeat biopsy within 24 months with a negative (controls) or positive (cases) histopathological result. Centralized blinded pathology evaluation of the 2 biopsy series was performed in all available subjects from each site. Biopsies were epigenetically profiled for GSTP1, APC and RASSF1 relative to the ACTB reference gene using quantitative methylation specific polymerase chain reaction. Predetermined analytical marker cutoffs were used to determine assay performance. Multivariate logistic regression was used to evaluate all risk factors., Results: The epigenetic assay resulted in a negative predictive value of 88% (95% CI 85-91). In multivariate models correcting for age, prostate specific antigen, digital rectal examination, first biopsy histopathological characteristics and race the test proved to be the most significant independent predictor of patient outcome (OR 2.69, 95% CI 1.60-4.51)., Conclusions: The DOCUMENT study validated that the epigenetic assay was a significant, independent predictor of prostate cancer detection in a repeat biopsy collected an average of 13 months after an initial negative result. Due to its 88% negative predictive value adding this epigenetic assay to other known risk factors may help decrease unnecessary repeat prostate biopsies., (Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Relatively little progress has been made in determining the in vivo regulation of glutathione S-transferase P (GSTP), particularly the human enzyme hGSTP1, despite being identified as a significant factor in carcinogenesis and development of drug resistance in tumor cell lines. Here, we report the characterization of a transgenic reporter mouse that reveals how hGSTP1 is regulated in vivo by chemopreventive agents. Basal expression was found in crypts and villi of the small and large intestine, bronchiolar epithelial cells, the epidermis and hair follicles, gall bladder epithelium, choroid plexus, and biliary epithelium. Expression was induced in different tissues by the antioxidant chemopreventive agents ethoxyquin and butylated hydroxyanisole. However, genetic deletion of the Nrf2 transcription factor, which directs central genetic programs of detoxification and protection against oxidative stress, increased rather than attenuated GSTP1 expression. In vitro investigations with mouse embryonic fibroblasts revealed factors, in addition to Nrf2, that control the expression of GSTP1, offering further insights into regulation. The new reporter mouse described here provides a useful tool to gain deeper insights into the mechanisms of action of chemopreventive compounds and other environmental agents., (©2014 American Association for Cancer Research.)

Objective: To construct the vectors of human glutathione S-transferase A1 (GSTA1), P1 (GSTP1), T1(GSTT1) genes and express in Escherichia coli (E. coli)., Methods: Human GSTA1, GSTP1 and GSTT1 gene whole length cDNAs were amplified by RT-PCR and then subcloned into pET-28a(+) vectors. The proteins were expressed in E. coli BL21(DE3). After purified by Ni2+ affinity chromatography, the enzymatic activities of GSTs were measured with 1-chloro-2,4 -dinitrobenzene (CDNB) as substrate., Results: The correct GSTA1, GSTP1 and GSTT1 genes were cloned. And soluble GSTA1, GSTT1, GSTP1 proteins were expressed in E.coli. After purification, GSTA1, GSTT1 and GSTP1 showed good enzymatic activities, which were 17.55, 0.02, 18.75 μmol·min-1·mg-1, respectively., Conclusion: The expression plasmids for GSTA1, GSTT1 and GSTP1 have been constructed and the recombinant proteins are expressed successfully.

Spirogyra neglecta, a freshwater green alga, is a local food in the northern and northeastern parts of Thailand. This investigation explored the anticarcinogenicity of S neglecta and its possible cancer chemopreventive mechanisms in rats divided into 14 groups. Groups 1 and 10 served as positive and negative control groups, respectively. Groups 1-9 were intraperitoneally injected with diethylnitrosamine (DEN) once a week for 3 weeks. Groups 10-14 received normal saline instead. One week after the last DEN injection, groups 2-5 were administered for 9 consecutive weeks various doses of S neglecta extract (SNE) and dried S neglecta (SND), mixed with basal diet. Groups 6-9 and 11-14 similarly were administered various doses of SNE and SND starting from the first week of the experiment. Administration of SNE and SND was not associated with formation of glutathione-S- transferase placental form (GST-P) positive foci in rat liver. SNE and SND during initiation phase significantly reduced the number of GST-P positive foci in rats injected with DEN. The number of GST-P also diminished in groups treated with SNE and SND after injection with DEN, except for the low dose extract group. SNE showed stronger anticarcinogenic potency than SND. Furthermore, SNE also decreased the number of Ki-67 positive cells. However, the numbers of TUNEL-positive cells in the liver of the SNE-treated groups were not statistically different from the controls. The GST activity in 50 mg/kg bw of SNE and 1% of SND groups was significantly increased as compared to the positive control. In conclusion, Spirogyra neglecta (Hassall) Kutzing showed cancer chemopreventive properties at the early stages of diethylnitrosamine-induced hepatocarcinogenesis in rats. Possible inhibitory mechanisms include enhancement of the activities of some detoxifying enzymes and/or suppression of precancerous cells.

Background: Methylation of tumor suppressor genes has been investigated in all kinds of cancer. Tumor specific epigenetic alterations can be used as a molecular markers of malignancy, which can lead to better diagnosis, prognosis and therapy. Therefore, the aim of this study was to evaluate the association between gene hypermethylation and expression of fragile histidine triad (FHIT), glutathione S-transferase P1 (GSTP1) and p16 genes and various clinicopathologic characteristics in primary non-small cell lung carcinomas (NSCLC)., Materials and Methods: The study included 28 primary non-small cell lung carcinomas, where an additional 28 tissue samples taken from apparently normal safety margin surrounding the tumors served as controls. Methylation-specific polymerase chain reaction (MSP) was performed to analyze the methylation status of FHIT, GSTP1 and p16 while their mRNA expression levels were measured using a real-time PCR assay with SYBR Green I., Results: The methylation frequencies of the genes tested in NSCLC specimens were 53.6% for FHIT, 25% for GSTP1, and 0% for p16, and the risk of FHIT hypermethylation increased among patients with NSCLC by 2.88, while the risk of GSTP1 hypermethylation increased by 2.33. Hypermethylation of FHIT gene showed a highly significant correlation with pathologic stage (p<0.01) and a significant correlation with smoking habit and FHIT mRNA expression level (p<0.05). In contrast, no correlation was observed between the methylation of GSTP1 or p16 and smoking habit or any other parameter investigated (p>0.05)., Conclusions: RESULTS of the present study suggest that methylation of FHIT is a useful biomarker of biologically aggressive disease in patients with NSCLC. FHIT methylation may play a role in lung cancer later metastatic stages while GSTP1 methylation may rather play a role in the early pathogenesis.

Background: Glutathione S-transferase P1 (GSTP1) is an important phase II enzyme involved in detoxification of carcinogens. GSTP1 gene overexpression has been observed in a variety of human cancers but there are no studies in plasma cell disorders. The aim of this study was to examine GSTP1 mRNA expression level in multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). In addition, we have determined GSTP1 polymorphic variants in order to estimate MM risk and their relationship with the expression level. Results were also correlated with laboratory parameters and clinical outcome., Methods: Bone marrow mononuclear cells from 125 patients with plasma cell disorders were studied. Peripheral blood samples of 110 age and sex matched healthy controls were also evaluated. Real-Time Quantitative RT-PCR and PCR-RFLP assays were used., Results: Upregulation of GSTP1 was observed in 37.7% MM and in 22.6% MGUS patients. A significant increase of GSTP1 expression in MM with respect to MGUS was detected (p=0.0427). Most MM patients that achieved complete remission had low transcription levels (77.8%) compared to those who did not reach this condition (44.4%) (p=0.0347). GSTP1 heterozygous carriers showed reduced expression compared to those with homozygous wild type genotype (p=0.0135)., Conclusion: Our findings suggest, for the first time, a role for GSTP1 expression in development and/or progression of plasma cell disorders, and a probable influence of functional capacity of the enzyme on clinical outcome. These results and those of the literature support GSTP1 as an interesting tumor marker and a potential therapeutic target., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

Because induction of phase II detoxification enzyme is important for chemoprevention, we study the effects of Indigofera suffruticosa Mill, a medicinal herb, on the expression of π class of glutathione S-transferase (GSTP) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in rat Clone 9 liver cells. Both water and ethanolic extracts of I. suffruticosa significantly increased the expression and enzyme activities of GSTP and NQO1. I. suffruticosa extracts up-regulated GSTP promoter activity and the binding affinity of nuclear factor erythroid 2-related factor 2 (Nrf2) with the GSTP enhancer I oligonucleotide. Moreover, I. suffruticosa extracts increased nuclear Nrf2 accumulation as well as ARE transcriptional activity. The level of phospho-ERK was augmented by I. suffruticosa extracts, and the ERK inhibitor PD98059 abolished the I. suffruticosa extract-induced ERK activation and GSTP and NQO-1 expression. Moreover, I. suffruticosa extracts, especially the ethanolic extract increased the glutathione level in mouse liver and red blood cells as well as Clone 9 liver cells. The efficacy of I. suffruticosa extracts in induction of phase II detoxification enzymes and glutathione content implies that I. suffruticosa could be considered as a potential chemopreventive agent., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Exposure to high levels of manganese (Mn) results in a neurological condition termed manganism, which is characterized by oxidative stress, abnormal dopamine (DA) signaling, and cell death. Epidemiological evidence suggests correlations with occupational exposure to Mn and the development of the movement disorder Parkinson's disease (PD), yet the molecular determinants common between the diseases are ill-defined. Glutathione S-transferases (GSTs) of the class pi (GSTπ) are phase II detoxification enzymes that conjugate both endogenous and exogenous compounds to glutathione to reduce cellular oxidative stress, and their decreased expression has recently been implicated in PD progression. In this study we demonstrate that a Caenorhabditis elegans GSTπ homologue, GST-1, inhibits Mn-induced DA neuron degeneration. We show that GST-1 is expressed in DA neurons, Mn induces GST-1 gene and protein expression, and GST-1-mediated neuroprotection is dependent on the PD-associated transcription factor Nrf2/SKN-1, as a reduction in SKN-1 gene expression results in a decrease in GST-1 protein expression and an increase in DA neuronal death. Furthermore, decreases in gene expression of the SKN-1 inhibitor WDR-23 or the GSTπ-binding cell death activator JNK/JNK-1 result in an increase in resistance to the metal. Finally, we show that the Mn-induced DA neuron degeneration is independent of the dopamine transporter DAT, but is largely dependent on the caspases CED-3 and the novel caspase CSP-1. This study identifies a C. elegans Nrf2/SKN-1-dependent GSTπ homologue, cell death effectors of GSTπ-associated xenobiotic-induced pathology, and provides the first in vivo evidence that a phase II detoxification enzyme may modulate DA neuron vulnerability in manganism., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Aberrant gene expression is a hallmark of cancer. Quantitative reverse-transcription PCR (qRT-PCR) is the gold-standard for quantifying gene expression, and commonly employs a house-keeping gene (HKG) as an endogenous control to normalize results; the choice of which is critical for accurate data interpretation. Many factors, including sample type, pathological state, and oxygen levels influence gene expression including putative HKGs. The aim of this study was to determine the suitability of commonly used HKGs for qRT-PCR in prostate cancer., Methods: Prostate cancer (LNCaP, 22Rv1, PC3, and DU145) and normal (PWR1E and RWPE1) cell lines were cultured in air and hypoxia. The performance of 16 HKGs was assessed using Normfinder and coefficient of variation. In silico promoter analysis was performed to identify putative hypoxia response elements (HREs). The impact of the endogenous control on expression levels of HIF1A and GSTP1 was investigated by qRT-PCR in cell lines and tissue specimens respectively., Results: Hypoxia altered expression of several HKGs: IPO8, B2M, and PGK1. The most stably expressed HKGs were ACTB, PPIA, and UBC. Both UBC and ACTB showed constitutive expression of HIF1A in air and hypoxia, while PGK1 falsely implied a sixfold hypoxia-induced down-regulation. In prostate tumors, UBC and PGK1 both revealed down-regulation of GSTP1 relative to matched benign, whereas ACTB showed variability., Conclusions: This study demonstrates that no universal endogenous control exists for gene expression studies, even within one disease type. It highlights the importance of validating expression of intended HKGs between different sample types and environmental exposures., (Copyright © 2012 Wiley Periodicals, Inc.)

There is a lack of understanding of the casual mechanisms behind the observation that some breast adenocarcinomas have identical morphology and comparatively different cellular growth behavior. This is exemplified by a differential response to radiation, chemotherapy, and other biological intervention therapies. Elevated concentrations of the free radical nitric oxide (NO), coupled with the up-regulated enzyme nitric oxide synthase (NOS) which produces NO, are activities which impact tumor growth. Previously, we adapted four human breast cancer cell lines: BT-20, Hs578T, T-47D, and MCF-7 to elevated concentrations of nitric oxide (or high NO [HNO]). This was accomplished by exposing the cell lines to increasing levels of an NO donor over time. Significantly, the HNO cell lines grew faster than did each respective ("PARENT") cell line even in the absence of NO donor-supplemented media. This was evident despite each "parent" being morphologically equivalent to the HNO adapted cell line. Herein, we characterize the HNO cells and their biological attributes against those of the parent cells. Pairs of HNO/parent cell lines were then analyzed using a number of key cellular activity criteria including: cell cycle distribution, DNA ploidy, response to DNA damage, UV radiation response, X-ray radiation response, and the expression of significant cellular enzymes. Other key enzyme activities studied were NOS, p53, and glutathione S-transferase-pi (GST-pi) expression. HNO cells were typified by a far more aggressive pattern of growth and resistance to various treatments than the corresponding parent cells. This was evidenced by a higher S-phase percentage, variable radioresistance, and up-regulated GST-pi and p53. Taken collectively, this data provides evidence that cancer cells subjected to HNO concentrations become resistant to free radicals such as NO via up-regulated cellular defense mechanisms, including p53 and GST-pi. The adaptation to NO may explain how tumor cells acquire a more aggressive tumor phenotype.

Objectives: Glutathione S-transferase (GST) isoenzymes play important roles in resistance to cell apoptosis and carcinogenesis. We aimed to establish the relationship between GST expression and the prognosis of upper urinary tract urothelial carcinoma (UTT-UC) in Taiwan., Methods: This study retrospectively reviewed 46 patients with pathologically confirmed UUT-UC at Kaohsiung Medical University Hospital. In each patient, expression of GSTT1 and GSTP1 was compared between urothelial carcinoma and normal urothelial cells by Western blotting., Results: GSTP1 expression in the UUT-UC cells was significantly higher than that in normal urothelial cells (1.6 fold, p<0.001). Expression of GSTT1 was significantly associated with the invasiveness of the carcinoma (p=0.006)., Conclusions: In UUT-UC, GSTP1 might be a potential tumor marker, whereas high GSTT1 expression could be used as an indicator of cancer progression. This study is the first to demonstrate potential applications of different GST isoenzymes for biomolecular analysis of UUT-UCs in Taiwan.

Carcinogen detoxifying genes may be involved in pathogenesis of head and neck cancer (HNC). CYP1A1 is phase I enzyme that converts carcinogens into water soluble compounds which are easily excreted from body. GSTs constitute phase II detoxification enzymes that recognize these highly electrophilic compounds and detoxify them. Abnormal expression of these genes can potentially lead to cancer initiation. In present study, we analyzed protein expression of these genes in a total of 192 HNC patients and noncancerous healthy control serum samples screened for GSTs specific activity by ELISA. Furthermore, expression of these molecules was also determined in 49 HNC tissues/ adjacent control tissue by immunohistochemistry with specific antibodies. Mean serum GSTs specific activity was found to be 7.7 (+11.5)U/L in HNC patients and 11.4 (+7.5)U/L in controls. Significant decrease (P < 0.05) in GSTs specific activity was observed in HNC patients compared with controls (P < 0.001). Data for immunohistochemistry showed that CYP1A1 and GSTT1 was down expressed whereas GSTP1 was over expressed in HNC tissues compared with adjacent normal control tissues. Results of immunohistochemistry revealed 63 % HNC tissues had weak, 27 % moderate and 10 % strong staining for CYP1A1. For GSTT1, 27 % HNC tissues had no staining, 49 % weak staining, 16 % moderate and 8 % strong staining. Similarly for GSTP1, percentages for weak, moderate and strong staining were 6 %, 12 % and 82 % respectively. These reduced proteins observed in cancer patients highlight a potential breach on DNA repair mechanism when compared with control. Thus altered expression of these detoxifying molecules may collectively contribute to HNC development in Pakistani population.

Drug-induced toxic epidermal necrolysis (TEN) is a rare but potentially lethal bullous disease whose complex pathomechanisms remain uncertain. The aim of the study was an exploratory attempt to assess TEN pathobiology using a combination of immunohistochemistry and proteomics. Skin blister fluid (BF) and serum were collected in a patient in the early TEN stage at day (D) +4 of evolution and three days later (D +7). Intravenous cyclosporine A (CsA) treatment was initiated since D +4. Immunohistochemistry was performed on skin blister biopsies. In addition, proteomic analyses compared the BF protein content before and at the issue of the 3-day CsA treatment. Proteins were selected according to their prominent differential abundance in BF between D+4 and D+7, when influenced by lesional skin cells, but not in serum. Among 300 proteins, four were considered. Glutathione transferase π was related to oxidative stress in TEN epidermis. The monocyte differentiation antigen CD14 and myeloperoxidase indicated macrophage activation. The proinflammatory S100-A8 protein probably originated from activated keratinocytes and/or macrophages. These proteomic findings were in line with immunohistochemistry and supported the prominent involvement of keratinocytes and macrophages in TEN pathomechanism. As opposed to CD14, other proteins were mainly present in BF at D+7, confirming that CsA expressed little effect, if any, on the activity of keratinocytes and macrophages in the present TEN patient. Of note, the present exploratory study using proteomic analyses in a single TEN case supports a pathogenic hypothesis without establishing any firm conclusion.

Many phytochemicals possess antioxidant and cancer-preventive properties, some putatively through antioxidant response element-mediated phase II metabolism, entailing mutagen/oxidant quenching. In our recent studies, however, most candidate phytochemical agents were not potent in inducing phase II genes in normal human lung cells. In this study, we applied a messenger RNA (mRNA)-specific gene expression-based high throughput in vitro screening approach to discover new, potent plant-derived phase II inducing chemopreventive agents. Primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBECs) were exposed to 800 individual compounds in the MicroSource Natural Products Library. At a level achievable in humans by diet (1.0 μM), 2,3-dihydroxy-4-methoxy-4'-ethoxybenzophenone (DMEBP), triacetylresveratrol (TRES), ivermectin, sanguinarine sulfate, and daunorubicin induced reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1 (NQO1) mRNA and protein expression in NHBE cells. DMEBP and TRES were the most attractive agents as coupling potency and low toxicity for induction of NQO1 (mRNA level, ≥3- to 10.8-fold that of control; protein level, ≥ two- to fourfold that of control). Induction of glutathione S-transferase pi mRNA expression was modest, and none was apparent for glutathione S-transferase pi protein expression. Measurements of reactive oxygen species and glutathione/oxidized glutathione ratio showed an antioxidant effect for DMEBP, but no definite effect was found for TRES in NHBE cells. Exposure of NHBE cells to H(2)O(2) induced nuclear translocation of nuclear factor erythroid 2-related factor 2, but this translocation was not significantly inhibited by TRES and DMEBP. These studies show that potency and low toxicity may align for two potential NQO1-inducing agents, DMEBP and TRES.

Aim: Platinum agents have shown to be effective in the treatment of colorectal cancer. We assessed whether single nucleotide polymorphisms (SNPs) in GSTP1, ERCC1 Asn118Asn and ERCC2 Lys751Gln might predict the overall survival in patients receiving oxaliplatin-based chemotherapy in a Chinese population., Methods: SNPs of GSTP1, ERCC1 Asn118Asn and ERCC2 Lys751Gln in 335 colorectal cancer patients were assessed using TaqMan nuclease assays., Results: At the time of final analysis on Nov. 2011, the median follow-up period was 37.7 months (range from 1 to 60 months). A total of 229 patients died during follow-up. Our study showed GSTP1 Val/Val (HR=0.44, 95% CI=0.18-0.98), ERCC1 C/C (HR=0.20, 95% CI=0.10-0.79) and ERCC2 G/G (HR=0.48, 95% CI=0.19-0.97) to be significantly associated with better survival of colorectal cancer. GSTP1 Val/Val, ERCC1 C/C and ERCC2 G/G were also related to longer survival among patients with colon cancer, with HRs (95% CIs) of 0.41 (0.16-0.91), 0.16 (0.09-0.74) and 0.34 (0.16-0.91), respectively., Conclusion: GSTP1, GSTP1, ERCC1 Asn118Asn and ERCC2 Lys751Gln genotyping might facilitate tailored oxaliplatin-based chemotherapy for colorectal cancer patients.

Unlabelled: Overdose of acetaminophen (APAP), the active ingredient of Tylenol, is the leading cause of drug-induced acute liver failure in the United States. As such, it is necessary to develop novel strategies to prevent or manage APAP toxicity. In this report, we reveal a novel function of the liver X receptor (LXR) in preventing APAP-induced hepatotoxicity. Activation of LXR in transgenic (Tg) mice or by an LXR agonist conferred resistance to the hepatotoxicity of APAP, whereas the effect of LXR agonist on APAP toxicity was abolished in LXR-deficient mice. The increased APAP resistance in LXR Tg mice was associated with increased APAP clearance, increased APAP sulfation, and decreased formation of toxic APAP metabolites. The hepatoprotective effect of LXR may have resulted from the induction of antitoxic phase II conjugating enzymes, such as Gst and Sult2a1, as well as the suppression of protoxic phase I P450 enzymes, such as Cyp3a11 and Cyp2e1. Promoter analysis suggested the mouse Gst isoforms as novel transcriptional targets of LXR. The suppression of Cyp3a11 may be accounted for by the inhibitory effect of LXR on the PXR-responsive transactivation of Cyp3a11. The protective effect of LXR in preventing APAP toxicity is opposite to the sensitizing effect of pregnane X receptor, constitutive androstane receptor, and retinoid X receptor alpha., Conclusion: We conclude that LXR represents a potential therapeutic target for the prevention and treatment of Tylenol toxicity., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Aims: This study investigated the association between glutathione S-transferase Pi (GST Pi) expression, histopathology and overall survival in 468 patients after resection of stage C colonic adenocarcinoma., Methods and Results: Data were drawn from a prospective hospital registry of consecutive bowel cancer resections with a minimum follow-up of 5 years. Nuclear and cytoplasmic GST Pi expression, assessed by both intensity of staining and percentage of stained cells at both the central part of the tumour and the invasive tumour front, were evaluated retrospectively by tissue microarray immunohistochemistry on archival specimens. The most effective measure of GST Pi expression was the percentage of immunostained nuclei in central tumour tissue, where >40% stained was associated significantly with high grade, invasion beyond the muscularis propria, involvement of a free serosal surface or apical node, and invasion into an adjacent organ or structure. After adjustment of other predictors, GST Pi expression remained independently prognostic for reduced overall survival (hazard ratio 1.4, P = 0.002)., Conclusions: In patients with clinicopathological stage C colonic cancer, GST Pi expression is associated with features of tumour aggressiveness and with reduced overall survival. Further appropriately designed studies should aim to discover whether GST Pi can predict response to adjuvant chemotherapy., (© 2011 Blackwell Publishing Limited.)

This study aimed to determine the relationship between the endogenous levels of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione-s-transferase-π (GST‑π) and topoisomerase IIα (TopoIIα) and intrinsic drug resistance in four human lung cancer cell lines, SK-MES-1, SPCA-1, NCI-H-460 and NCI-H-446, of different histological types. The expression of P-gp, MRP, LRP, GST-π and TopoIIα was measured by immunofluorescence, Western blotting and RT-PCR. Drug resistance to cisplatin, doxorubicin and VP-16 was determined using MTT assays. The correlation between expression of the resistance-related proteins and their roles in the resistance to drugs in these cancer cell lines was analyzed. We found that the endogenous levels of P-gp, MRP, LRP, GST-π and TopoIIα in the four cell lines varied. The level of GST-π in the SK-MES-1 cells was the highest, whereas the level of P-gp in the SPCA-1 cells was the lowest. The chemoresistance to cisplatin, doxorubicin and VP-16 in the four cell lines was different. The SPCA-1 cell line was most resistance to cisplatin; SK-MES-1 was most resistance to VP-16; whereas SK-MES-1 was most sensitive to doxorubicin. There was a positive correlation between GST-π expression and resistance to cisplatin, between TopoIIα expression and resistance to VP-16; and a negative correlation was noted between TopoIIα expression and resistance to doxorubicin. In summary, the endogenous expression of P-gp, MRP, LRP, GST-π and TopoIIα was different in the four human lung cancer cell lines of different histological types, and this variance may be associated with the variation in chemosensitivity to cisplatin, doxorubicin and VP-16. Among the related proteins, GST-π may be useful for the prediction of the intrinsic resistance to cisplatin, whereas TopoIIα may be useful to predict resistance to doxorubicin and VP-16 in human lung cancer cell lines.

Gene silencing by epigenetic mechanisms is frequent in prostate cancer (PCA). The link between DNA hypermethylation and histone modifications is not completely understood. We chose the GSTP1 gene which is silenced by hypermethylation to analyze the effect of the histone deacetylase inhibitor depsipeptide on DNA methylation and histone modifications at the GSTP1 promoter site. Prostate cell lines (PC-3, LNCaP, and BPH-1) were treated with depsipeptide; apoptosis (FACS analysis), GSTP1 mRNA levels (quantitative real-time PCR), DNA hypermethylation (methylation-specific PCR), and histone modifications (chromatin immunoprecipitation) were studied. Depsipeptide induced apoptosis in PCA cells, but not a cell cycle arrest. Depispeptide reversed DNA hypermethylation and repressive histone modifications (reduction of H3K9me2/3 and H3K27me2/3; increase of H3K18Ac), thereby inducing GSTP1 mRNA re-expression. Successful therapy requires both, DNA demethylation and activating histone modifications, to induce complete gene expression of epigenetically silenced genes and depsipeptide fulfils both criteria., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Association between long-term hormone replacement therapy (HRT) use and increased risk of breast cancer is still under debate. Functionally relevant genetic variants within the estrogen metabolic pathway may alter exposure to exogenous sex hormones and affect the risk of postmenopausal breast cancer. We investigated the associations of common polymorphisms in 4 genes encoding key proteins of the estrogen metabolic pathway, duration of HRT use and their interactions with breast cancer risk. We studied 530 breast cancer cases and 270 controls of the same age and ethnicity participating in a case-control study of postmenopausal women. Duration of HRT use was ascertained through a postal questionnaire. Genotyping was conducted for CYP1B1 (rs1056836), COMT (rs4680), GSTP1 (rs1695) and MnSOD (rs4880) polymorphisms by PCR-based RFLP and TaqMan® allelic discrimination method. Adjusted odds ratios and 95% confidence intervals were calculated using logistic regression analysis. HRT use was significantly associated with decreased breast cancer risk (p<0.001). None of the polymorphisms studied was associated with breast cancer risk. A significant interaction was observed between MnSOD 47T>C and HRT use (pinteraction=0.036); the risk of breast cancer associated with long-term vs. short-term HRT use was decreased in women homozygous for the wild-type allele and increased in women with at least one variant allele of the MnSOD 47T>C polymorphism. Our results suggest that MnSOD 47T>C polymorphism in interaction with long-term HRT use may modify the risk of breast cancer.

The present study aimed to study the mechanisms by which low dose arsenic trioxide (As(2)O(3)) reduces multidrug resistance. The potential influence of As(2)O(3) on cytotoxicity was examined by methyl thiazolyl tetrazolium (MTT) assay and the intracellular mean fluorescence intensity (MFI) of Adriamycin (ADM) was examined by flow cytometry. The gene expression of mdr1 mRNA was determined by RT-PCR. The change of cellular expression levels of drug resistant-related proteins, including P-gp, bcl-2, Topo-II, and GST-π, were measured by Western-blotting or immunocytochemistry assay. Data showed As(2)O(3) at non-cytotoxic concentration (2μM) significantly increased the cytotoxicity of ADM on K562/A02 cells. Cotreatment of As(2)O(3) and ADM significantly increased the ADM MFI than ADM alone (P<0.01). Following pretreatment of K562/A02 cells with As(2)O(3), the expression of Topo-II was increased while the expression of GST-π and bcl-2 was decreased. No obvious alternation of expression of mdr1 mRNA or P-gp was observed. Thus, low dose As(2)O(3) partially reduced drug resistance to ADM in K562/A02 cells via multiple mechanisms, which selectively inhibited the efflux pump GST-π but not P-gp, as well as modulated the expression of MDR-related proteins such as Topo-II and bcl-2, in line with previous studies. In conclusions: The effect of As(2)O(3) on reducing MDR may have wide clinical application in chemotherapy regimens for leukemia., (Crown Copyright © 2011. Published by Elsevier SAS. All rights reserved.)

The glutathione S-transferase P1 (GSTP1) protein plays several critical roles in both normal and neoplastic cells, including phase II xenobiotic metabolism, stress responses, signaling and apoptosis. Overexpression of GSTP1 has been observed in many types of cancer, including head and neck squamous cell carcinoma (HNSCC). However, the role of GSTP1 in HNSCC is not well understood. We investigated the role of GSTP1 in two HNSCC cell lines, HSC3 and SAS. Silencing of GSTP1 revealed that cancer cell proliferation was significantly decreased in both cell lines. In addition, the frequency of apoptotic cells increased following si-GSTP1 transfection of HSC3 and SAS cell lines. Growing evidence suggests that microRNAs (miRNAs) negatively regulate gene expression and can function as oncogenes or tumor suppressors in human cancer. Based on the results of web-based searches, miR-133α is a candidate miRNA targeting GSTP1. Down-regulation of miR-133α has been reported in many types of human cancer, including HNSCC. Transient transfection of miR-133α repressed the expression of GSTP1 at both the mRNA and protein levels. The signal from a luciferase reporter was significantly decreased at one miR-133α target site at the 3'UTR of GSTP1, suggesting that miR-133α directly regulates GSTP1. Our data indicate that GSTP1 may have an oncogenic function and may be regulated by miR-133α, a tumor suppressive miRNA in HNSCC. The identification of a novel oncogenic pathway could provide new insights into potential mechanisms of HNSCC carcinogenesis.

In the present study, protein lysates from microdissected glutathione S-transferase placental-form-positive (GST-P(+)) foci and hepatocellular carcinomas from livers of rats treated with N-diethylnitrosamine followed by phenobarbital at doses of 0 and 500 ppm in the diet for 10 and 33 weeks were analyzed using QSTAR Elite liquid chromatography with tandem mass spectrometry and iTRAQ technology. Among 75 proteins, a total of 27 and 50 proteins displaying significant quantitative changes comparing with adjacent normal-appearing liver tissue were identified in GST-P(+) foci of initiation control and promotion groups, respectively, which are related to transcription, protein folding, cytoskeleton filaments reorganization, cell cycle control, nuclear factor (erythroid-derived 2)-like 2 (NRF2)-mediated oxidative stress responses, lipid metabolism, glutathione metabolism, oxidative phosphorylation, and signal transduction. Furthermore, Ingenuity Pathway and bioinformatic analyses revealed that expression changes of genes encoding proteins with altered expression detected in GST-P(+) foci are likely to be controlled by c-myc, NRF2, aryl hydrocarbon receptor, nuclear factor kappa B, and hepatocyte nuclear factor 4 transcriptional factors. Coordinated overexpression of mitochondrial chaperons prohibitin (PHB) and prohibitin 2 (PHB2), septin 9 (SEPT9), neurabin 1, and other cytoskeletal and functional proteins in areas of GST-P(+) foci during initiation and/or promotion stages of rat hepatocarcinogenesis was associated with induction of cell proliferation and might be responsible for the neoplastic transformation of rat liver preneoplastic lesions. Newly discovered elevation of PHB, PHB2, and SEPT9 in GST-P(+) foci and tumors, imply that they might play important role in the onset of liver cancer and be of potential values in the studies of hepatocarcinogenesis.

Ovarian cancer is primarily treated with platinum-based chemotherapy, with ROS generation implicated in cytotoxicity. We examined redox protein expression in ovarian tumors, focusing on the thioredoxin system, to determine the role it might play in mediating response to therapy. Nuclear and cytoplasmic expression of thioredoxin, thioredoxin reductase, thioredoxin-interacting protein, metallothionein, and glutathione S-transferase Pi was assessed, using standard immunohistochemical techniques, on a tissue microarray of 154 primary ovarian carcinomas obtained from patients subsequently treated with adjuvant platinum-based chemotherapy. Low cytoplasmic expression of thioredoxin (p=0.032) and negative nuclear expression of metallothionein (p=0.04) significantly correlated with better progression-free survival. When nuclear and cytoplasmic expression patterns were combined those patients with tumors with low cytoplasmic but high nuclear expression of thioredoxin exhibited better progression-free (p=0.003) and overall survival (p=0.004). This combination was, using multivariate analysis, an independent predictive factor for overall survival (p=0.034). Improved progression-free survival was also seen with negative expression of metallothionein, cytoplasmic and nuclear (p=0.038), and was independent of other clinical parameters (p=0.048). Such results support the suitability of using redox protein expression to predict response and, potentially, to alter treatment options accordingly., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective: The immunohistochemical staining characteristics of glutathione S-transferase (GST) alpha (GSTA), pi (GSTP), mu (GSTM4), and theta (GSTT1) and P53 were investigated in laryngeal squamous cell carcinoma (LSCC) cases and normal laryngeal tissue from 46 patients. The relationships between expression of the GST isoenzymes and some clinicopathologic features were also examined., Patients and Methods: For immunohistochemical studies, tissues from 46 patients with LSCC at the Dr. Lütfi Kirdar Kartal Education and Research Hospital were used. The relationship between expression of the GST isoenzymes and P53 in normal and tumour tissue was analyzed using the Wilcoxon signed rank test. The correlation between GST isoenzymes and P53 and clinicopathologic data was also examined using the Spearman rank test., Results: When the normal and tumour tissues of these cases were compared according to their staining intensity and percentage of positive staining, GSTA expression in normal cells was significantly higher than in tumour cells, and GSTP and P53 expression was higher in tumour cells (p < .05). GSTM and GSTT1 expression was higher in normal cells; however, the statistical significance was low (p > .05). There was no correlation between P53 and GST expression in patients with LSCC. When the immunohistochemical results of GST isoenzymes and P53 were correlated with the clinical parameters, GSTA expression was increased in poorly differentiated laryngeal tumour, but GSTM4 and GSTT1 expression was decreased (p < .05)., Conclusion: According to these results, GST-A, -P, and T1 and P53 were important in the diagnosis of LSCC.

Background: The cortical cytoskeleton network connects the actin cytoskeleton to various membrane proteins, influencing cell adhesion, polarity, migration and response to extracellular signals. Previous studies have suggested changes in the expression of specific components in prostate cancer, especially of 4.1 proteins (encoded by EPB41 genes) which form nodes in this network., Methods: Expression of EPB41L1, EPB41L2, EPB41L3 (protein: 4.1B), EPB41L4B (EHM2), EPB41L5, EPB49 (dematin), VIL2 (ezrin), and DLG1 (summarized as "cortical cytoskeleton" genes) as well as ERG was measured by quantitative RT-PCR in a well-characterized set of 45 M0 prostate adenocarcinoma and 13 benign tissues. Hypermethylation of EPB41L3 and GSTP1 was compared in 93 cancer tissues by methylation-specific PCR. Expression of 4.1B was further studied by immunohistochemistry., Results: EPB41L1 and EPB41L3 were significantly downregulated and EPB41L4B was upregulated in cancer tissues. Low EPB41L1 or high EPB41L4B expression were associated with earlier biochemical recurrence. None of the other cortical cytoskeleton genes displayed expression changes, in particular EPB49 and VIL2, despite hints from previous studies. EPB41L3 downregulation was significantly associated with hypermethylation of its promoter and strongly correlated with GSTP1 hypermethylation. Protein 4.1B was detected most strongly in the basal cells of normal prostate epithelia. Its expression in carcinoma cells was similar to the weaker one in normal luminal cells. EPB41L3 downregulation and EPB41L4B upregulation were essentially restricted to the 22 cases with ERG overexpression. Expression changes in EPB41L3 and EPB41L4B closely paralleled those previously observed for the extracellular matrix genes FBLN1 and SPOCK1, respectively., Conclusions: Specific changes in the cortical cytoskeleton were observed during prostate cancer progression. They parallel changes in the expression of extracellular matrix components and all together appear to be associated with oncogenic ERG overexpression. We hypothesize that these alterations may contribute to the increased invasivity conferred to prostate cancer cells by ERG deregulation.

Background/aim: Fas/FasL system is a major regulator of apoptosis. The mechanisms by which Fas mediates cisplatin resistance remain unclear. The aim of this study is to explore the effect of Fas over-expression on cisplatin resistance of small cell lung cancer cells and its possible mechanisms., Materials and Methods: Fas was over-expressed in H446/CDDP cells by infection with the adenoviruses containing Fas. Sensitivity of Fas-overexpressed H446/CDDP cells to cisplatin was evaluated using MTT assay. Expressions of Fas, GST-pi and ERCC1 were detected by RT-PCR and Western blot analysis. Apoptosis rate was examined by FACS., Results: Over-expression of Fas in H446/CDDP cells significantly decreased the expressions of GST-pi and ERCC1 at mRNA and protein levels, and increased the cell apoptosis. Furthermore, up-regulation of Fas significantly decreased the tolerance of H446/CDDP cells to cisplatin., Conclusion: Over-expression of Fas reverses drug resistance of H446/CDDP cells, possibly due to the increased cell sensitivity to apoptosis and the decreased expressions of GST-pi and ERCC1.

Chlorobenzene is a volatile organic compound (VOC) that is widely used as a solvent, degreasing agent and chemical intermediate in many industrial settings. Occupational studies have shown that acute and chronic exposure to chlorobenzene can cause irritation of the mucosa of the upper respiratory tract and eyes. Using in vitro assays, we have shown in a previous study that human bronchial epithelial cells release inflammatory mediators such as the cytokine monocyte chemoattractant protein-1 (MCP-1) in response to chlorobenzene. This response is mediated through the NF-kappaB signaling pathway. Here, we investigated the effects of monochlorobenzene on human lung cells, with emphasis on potential alterations of the redox equilibrium to clarify whether the chlorobenzene-induced inflammatory response in lung epithelial cells is caused via an oxidative stress-dependent mechanism. We found that expression of cellular markers for oxidative stress, such as heme oxygenase 1 (HO-1), glutathione S-transferase pi1 (GSTP1), superoxide dismutase 1 (SOD1), prostaglandin-endoperoxide synthase 2 (PTGS2) and dual specificity phosphatase 1 (DUSP1), were elevated in the presence of monochlorobenzene. Likewise, intracellular reactive oxygen species (ROS) were increased in response to exposure. However, in the presence of the antioxidants N-(2-mercaptopropionyl)-glycine (MPG) or bucillamine, chlorobenzene-induced upregulation of marker proteins and release of the inflammatory mediator MCP-1 are suppressed. These results complement our previous findings and point to an oxidative stress-mediated inflammatory response following chlorobenzene exposure.

Background: Overexpression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and glutathione-S-transferase pi (GSTpi) is associated with drug resistance in acute myeloid leukemia (AML). The short-term effects of drug exposure on their expression levels were investigated., Materials and Methods: HL-60 cells and drug-resistant sublines were cultured with or without daunorubicin (DNR) and cytarabine (Ara-C). At several time-points the expression levels of P-gp, BCRP and GSTpi were determined., Results: After exposure to Ara-C, P-gp mRNA rapidly increased in all the cell lines. P-gp protein was detected in the sensitive cells after 8 h exposure to Ara-C. GSTpi mRNA increased in the resistant cells, but no change in BCRP mRNA was observed. Exposure to DNR revealed rapidly increased P-gp and GSTpi mRNA in the resistant cells., Conclusion: Ara-C rapidly increases P-gp mRNA and protein expression in sensitive and resistant cells, and GSTpi mRNA in resistant cells, in vitro. This may be of clinical importance during AML induction chemotherapy.