GSTP1 Expression Research Articles

IASPP-Mediated ROS Inhibition Drives 5-Fu Resistance Dependent on Nrf2 Antioxidative Signaling Pathway in Gastric Adenocarcinoma.

Inhibitor for the apoptosis-stimulating protein of p53 (iASPP) has been reported to be correlated with 5-fluorouracil (5-Fu) resistance in renal cell carcinoma. Here, we uncover mechanisms of iASPP-Nrf2-ROS regulation of 5-Fu resistance which are important for the development of alternative treatment strategies for gastric adenocarcinoma treatment. We analyzed iASPP and Nrf2 through TCGA RNA-seq data, UALCAN analysis, and cBioPortal datasets. Intracellular ROS generation was determined by 2',7'-dichloro-fluorescin diacetate staining. Transwell was used to evaluate the invasion. The expression of iASPP, Nrf2, HO-1, and GSTP1 was tested using western blot. We found that iASPP KD led to an apparent 5-Fu-induced ROS accumulation in MGC803 and SCG790 cells. Accompanied by iASPP KD, Nrf2 was markedly decreased. iASPP-induced ROS inhibition relies on Nrf2, and due to both knocked down iASPP and Nrf2, the level of ROS did not show an obvious difference with Nrf2 KD solely. Similarly, iASPP KD failed to enhance the Nrf2 KD-mediated ROS accumulation after 5-Fu treatment, suggesting that iASPP-induced antioxidative effects related to 5-Fu resistance are partially dependent on Nrf2. Also, the combination of iASPP KD and Nrf2 KD did not show any synergistic effect on apoptosis after 5-Fu treatment in MGC803 and SCG790 cells. Further studies revealed that iASPP KD or Nrf2 KD could decrease the expression of HO-1 and GSTP1. Our data highlight that iASPP plays a crucial role in the inhibition of 5-Fu-induced apoptosis resistance by removing ROS accumulation in gastric adenocarcinoma, and that the removal of ROS induced by iASPP is Nrf2 signaling dependent.

Curcumin Regulates ERCC1 Expression and Enhances Oxaliplatin Sensitivity in Resistant Colorectal Cancer Cells through Its Effects on miR-409-3p.

Background Oxaliplatin (L-OHP) resistance is a major obstacle to the effective treatment of colorectal cancer. The resistance mechanism(s) of colorectal tumors to L-OHP may be related to the regulation of ERCC1 by cancer-expressed miRNAs, but no in-depth studies on the miRNAs that affect drug resistance have been performed. Curcumin (Cur) can reverse the drug resistance of cancer cells, but its effects on ERCC1 expression and miRNA profiles in colorectal cancer have not been studied. Methods To study the regulation effect of curcumin on ERCC1 expression and its effects on miRNAs, the L-OHP-resistant colorectal cancer cell line HCT116/L-OHP was established. MTT assays were used to evaluate cell proliferation. Flow cytometry was used to investigate apoptotic induction. Western blot and RT-PCR analysis were used to evaluate the expression of drug-associated ERCC1, Bcl-2, GST-π, MRP, P-gp, and survivin. Results HCT116//L-OHP cell lines were successfully established. The combination of L-OHP and curcumin could reduce L-OHP resistance in vitro. In addition, combination therapy inhibited the expression of ERCC1, Bcl-2, GST-π, MRP, P-gp, and survivin at the mRNA and protein level. Curcumin was found to inhibit ERCC1 through its ability to modulate miR-409-3p. Conclusion Curcumin can overcome L-OHP resistance in colorectal cancer cells through its effects on miR-409-3p mediated ERCC1 expression.

Silencing of lncRNA PART1 inhibits proliferation, invasion and migration of breast cancer cells and promotes the efficacy of cisplatin in breast cancer cells.

LncRNAs have proved to be related to the progression of multiple cancers. The present study aimed to investigate the effect of PART1 on the proliferation, invasion and migration of breast cancer cells and the efficacy of cisplatin in these cells. The expression of lncRNA PART1 in tissues and cells were detected by RT-qPCR analysis which was also used to verify the transfection effects. The cell proliferation, invasion and migration of breast cancer cells were respectively analyzed by CCK-8 assay, transwell assay and wound healing assay. The cell apoptosis was determined by flow cytometry analysis. The detection of CDK2, cyclinE1, P21, MMP3, MMP10, MMP13, Bcl2, Bax, cleaved caspase-3, caspase-3, MDP1, MRP1, GST-π and ABCB1 expression was performed by Western blot analysis. The results revealed that PART1 was increased in breast cancer tissues and cells, silencing of PART1 significantly inhibited cell proliferation, invasion and migration by regulating the expression of relative proteins. In addition, silencing of PART1 obviously improved the sensitivity of breast cancer cells to cisplatin, promoted cell apoptosis, and decreased the expression of breast cancer resistance proteins. In conclusion, silencing of PART1 inhibited proliferation, invasion and migration of breast cancer cells and promoted the efficacy of cisplatin in these cells. Therefore, PART1 may be considered as a novel therapeutic target in breast cancer.

CRISPR-Generated Nrf2a Loss- and Gain-of-Function Mutants Facilitate Mechanistic Analysis of Chemical Oxidative Stress-Mediated Toxicity in Zebrafish.

The transcription factor Nrf2a induces a cellular antioxidant response and provides protection against chemical-induced oxidative stress, as well as playing a critical role in development and disease. Zebrafish are a powerful model to study the role of Nrf2a in these processes but have been limited by reliance on transient gene knockdown techniques or mutants with only partial functional alteration. We developed several lines of zebrafish carrying different null (loss of function, LOF) or hyperactive (gain of function, GOF) mutations to facilitate our understanding of the Nrf2a pathway in protecting against oxidative stress. The mutants confirmed Nrf2a dependence for induction of the antioxidant genes gclc, gstp, prdx1, and gpx1a and identified a role for Nrf2a in the baseline expression of these genes, as well as for sod1. Specifically, the 4-fold induction of gstp by tert-butyl hydroperoxide (tBHP) in wild type fish was abolished in LOF mutants. In addition, baseline gstp expression in GOF mutants increased by 12.6-fold and in LOF mutants was 0.8-fold relative to wild type. Nrf2a LOF mutants showed increased sensitivity to the acute toxicity of cumene hydroperoxide (CHP) and tBHP throughout the first 4 days of development. Conversely, GOF mutants were less sensitive to CHP toxicity during the first 4 days of development and were protected against the toxicity of both hydroperoxides after 4 dpf. Neither gain nor loss of Nrf2a modulated the toxicity of R-(-)-carvone (CAR), despite the ability of this compound to potently induce Nrf2a-dependent antioxidant genes. Similar to other species, GOF zebrafish mutants exhibited significant growth and survival defects. In summary, these new genetic tools can be used to facilitate the identification of downstream gene targets of Nrf2a, better define the role of Nrf2a in the toxicity of environmental chemicals, and further the study of diseases involving altered Nrf2a function.

Abstract 578: Brg1 Protects Cardiomyocytes Against Oxidative Damage Through Activation of the Nrf2 Signaling Pathway in Acute Myocardial Infarction

Background: Brahma-related gene 1 (Brg1), the core ATPase subunit of a large chromatin remodeling complex, plays critical role in the regulation of gene expression during cardiac growth, differentiation. In adults, Brg1 is turned off in cardiomyocytes and reactivated by cardiac stress. How Brg1 in myocardial infarction (MI) is poorly understood. The Keap1-Nrf2-ARE pathway plays an important role in the development of MI. However, by which Nrf2 activation is mediated in MI still remains to be determined. Methods and results: In vivo, adult male C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery for MI model. Our data demonstrated that in peri-infarct zone, the protein of Brg1 was significantly increased 7 days after MI compared with the sham group, accompanied by Nrf2 nuclear translocation, and upregulation of the expressions of NQO1, GSTP1, HO1. We further revealed that with adenoviral intramyocardial injection, the Brg1 overexpression reduced the percentage myocardial infarct, improved cardiac dysfunction, decreased the relative fluorescence intensity of ROS with fluorescent probe DHE in MI mice. Conversely, shRNA-mediated knockdown of Brg1 enlarged the percentage myocardial infarct, exacerbated cardiac dysfunction, increased the relative fluorescence intensity of ROS. More importantly, the Brg1 overexpression significantly induced Nrf2 translocation from cytoplasm to nuclear and upregulated NQO1, GSTP1 and HO1 expressions. Whereas Brg1 knockdown decreased the level of Nrf2 protein in the nucleus, suppressed NQO1,GSTP1 and HO1 expressions. In vitro, Oxygen-Glucose deprivation(OGD) on neonatal cardiomyocytes was established. The effects of Brg1 on OGD and Nrf2 activation were observed with gain- and loss-of-function approaches to regulate Brg1 expression. The results were consistent with in vivo study. Moreover, the down-regulation of Nrf2 by brusatol inhibited the increase of these antioxidative genes induced by Brg1 overexpression. Conclusions: This study indicated that Brg1-induced cardiac protection was partially mediated through transcription activator Nrf2. The Brg1-Nrf2-ARE pathway may represent a novel therapeutic target for preventing cardiac dysfunction in patients with MI.

Abstract 2657: GSTP mediated S-glutathionylation of GRP78 attributes to multiple myeloma resistance

Abstract The proteasome inhibitor bortezomib (Btz) is used in the treatment of multiple myeloma (MM), but its efficacy is restricted by the wide-spread occurrence of resistance. MM cells have the highest rate of secretion of proteins with disulfide bonds and are exquisitely dependent upon redox balance and accurate protein folding. Our results showed that some important redox enzymes, including glutathione transferase P (GSTP) are upregulated in Btz-resistant cells. While GSTP was originally described as a phase II detoxification enzyme, our laboratory has ascribed a thiolase activity catalyzing the forward S-glutathionylation reaction, a post-translational modification (PTM) through reversible addition of glutathione to thiolate anions of cysteines in target proteins. Although primarily cytosolic, GSTP has been reported in the nuclear and mitochondrial compartments. We recently showed that GSTP is localized at high concentrations in the endoplasmic reticulum (ER) and regulates a number of redox active proteins through S-glutathionylation; these include glucose-regulated protein (GRP78). Proteomic analysis revealed that both cys41 and cys 420 of GRP78 were subject to S-glutathionylation and this PTM altered the activity of GRP78, enhancing its holdase activity, while concomitantly decreasing its foldase activity. This alters protein folding and contributes to the Btz-resistant phenotype of MM. Our data also support the principle that drugs that target the ER protein folding machinery significantly alter local redox conditions. Thapsigargin (Thg) is an inhibitor of SERCA, interfering with the functional activities of calcium-dependent chaperones, while tunicamycin (TuM) inhibits N-linked glycosylation. Btz resistant cells showed marked levels of cross-resistance (2-10 fold) to both. Linking such results with GSTP/redox, the absence of GSTP (from GSTP knockout mice) makes both bone marrow derived dendritic and mouse embryonic fibroblast cells more sensitive to Btz, ThG and TuM. The nature of the cross-resistance patterns and the fact that the presence of GSTP in drug naïve cells also confers resistance provides a rational platform for studying redox-based mechanisms as causative in resistance. Moreover, inhibition of GSTP with Telintra restored Btz sensitivity, decreased S-glutathionylation and increased expression of markers of UPR induced apoptosis. We conclude that altered GSTP expression and S-glutathionylation of key ER-localized proteins are significant contributors to drug response and resistance in MM treated with Btz. Citation Format: Zhiwei Ye, Jie Zhang, Kenneth D. Tew, Danyelle M. Townsend. GSTP mediated S-glutathionylation of GRP78 attributes to multiple myeloma resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2657.

Transcriptional Activation of Gstp1 by MEK/ERK Signaling Confers Chemo-Resistance to Cisplatin in Lung Cancer Stem Cells.

Lung cancer management remains a challenge due to its asymptomatic and late presentation when it is metastatic. The clinical response to the first-line platinum-based chemotherapy in patients with advanced lung cancer is disappointing due to the development of chemoresistance. Chemoresistance is a complex phenomenon. Mechanistic research using experimental models has yielded limited clinical results to help increase understanding for overcoming resistance. While the role of lung CSCs in conferring multidrug resistance has been postulated, experimental evidence remains associative and lacks in depth mechanistic inquisition. In the present study, using mouse and human lung adenocarcinoma cell lines and their respective paired CSC derivative cell lines that we generated, we identified cancer stem cell component of lung adenocarcinoma as the source that confers multidrug resistance phenotype. Mechanistically, Gstp1 confers cisplatin resistance in mouse and human lung CSC models, both in vitro and in vivo. Further, transcriptional activation of Gstp1 expression by MEK/ERK signaling underlies cisplatin resistance in lung CSC cells. Moreover, we show that GSTP1 expression is a poor diagnostic and prognostic marker for human lung adenocarcinoma, thus is of high clinical relevance. Taken together, we have provided mechanistic understanding of the lung CSC in mediating chemoresistance.

Potential of Teucrium chamaedrys L. to modulate apoptosis and biotransformation in colorectal carcinoma cells

Ethnopharmacological relevanceTeucrum chamaedrys L. is one of the known medicinal plants, useful for treatment of various health problems, especially digestive. In this study, we investigated methanol, ethyl-acetate and acetone extracts of T. chamaedrys in respect to their anticancer properties in SW480 colorectal cancer cells. Materials and methodsCytotoxicity and proapoptotic potential were assessed by MTT cell viability assay and AO/EB double staining. Molecular mechanisms of induced apoptosis were determined by monitoring Fas receptor protein expression through immunofluorescence, Caspase 8 and 9 activity, as well as concentrations of O2.- spectrophotometrically. Additionally, mRNA expression of biotransformation enzymes (CYP1A1, CYP1B1, GSTP1) and membrane transporters (MRP1 and MRP2) involved in drug resistance were investigated by qPCR method. Qualitative analysis of individual phenolic compounds was performed by reversed phase HPLC-MS analysis. ResultsMethanol extract shows the best cytotoxicity and selectivity compared to ethyl-acetate and acetone extracts, mainly causing apoptosis of SW480 cells, without affecting normal HaCaT keratinocytes. The increased expression of Fas receptor protein and caspase 8 activity indicate that the death receptor-mediated pathway plays a crucial role in the observed apoptosis. The increased caspase 9 activity and O2.- concentration suggest that mitochondria are also involved in the apoptosis. T. chamaedrys methanol extract inhibits mRNA expression of CYP1A1, CYP1B1, GSTP1, MRP1 and MRP2 in SW480 cells. ConclusionsInduction of apoptosis and inhibition of CYP1A1, CYP1B1, GSTP1, MRP1 and MRP2 mRNA expression implies that T. chamaedrys can serve as a valuable source of bioactive compounds as dietary supplements or selective anticancer agents, with the ability to induce apoptosis and modulate drug resistance in colorectal cancer cells.

Expression levels of MRP1, GST-π, and GSK3β in ovarian cancer and the relationship with drug resistance and prognosis of patients.

Expression levels of multidrug resistance-associated protein 1 (MRP1), glutathione S-transferase π (GST-π) and glycogen synthase kinase-3β (GSK3β) were investigated in ovarian epithelial cancer and the relationship with the primary drug resistance of patients with ovarian cancer to chemotherapy. One hundred and twenty-one ovarian cancer tissue samples from patients who underwent ovarian cancer resection from January 2013 to June 2015 in Liaoning Cancer Hospital and Institute, Cancer Hospital of China Medical University were enrolled in the Experimental group, while 58 ovarian tissue samples from patients with fallopian tube obstruction but with no ovarian cancer who received surgical treatment for blocked fallopian tube were included in the Control group. After the detection of the expression levels of MRP1, GST-π, and GSK3β mRNA by RT-PCR and the analysis of related clinical pathological factors, patients in the Experimental group were divided into the Chemotherapy-sensitive and Chemotherapy-resistant groups according to the chemotherapy efficacy. Additionally, with the mean expression levels of MRP1, GST-π, and GSK3β in ovarian cancer tissues as the boundaries, the expression levels of the three genes in the Experimental group were classified into high expression and low expression. Ovarian cancer tissues had much higher expression levels of MRP1, GST-π, and GSK3β mRNA than normal ovarian tissues (P<0.05). The expression levels of MRP1, GST-π, and GSK3β mRNA in the Chemotherapy-sensitive group were significantly lower than those in the Chemotherapy-resistant group (P<0.05). Patients with high expression of MRP1, GST-π, and GSK3β mRNA had a much lower 3-year survival rate than patients with low expression of the genes (P<0.05). Highly expressed in patients with ovarian cancer, MRP1, GST-π, and GSK3β mRNA play an important role in the development and drug resistance of ovarian cancer, which ensures this study is of positive clinical guiding significance in developing proper treatment for ovarian cancer and evaluating the efficacy of chemotherapy.

The Relation between Polymorphisms in Exon 5 and Exon 6 of GSTP1 Gene and the Risk of Lung Cancer in Iranian People

Objective:The GSTP1 gene, which is located on chromosome 11q13, consists of 7 exons and 6 introns. There are two polymorphisms in GSTP1 that have been exposed to a transposition for codon 105 (Ile/Val) and 114 (Ala/Val) in exons 5 and 6, which have been studied previously in relation to lung cancer. Since the level of GSTP1 expression in lung tissues and other human epithelial tissues is high, GSTP1Val-105 polymorphism is recognized as a sensitive factor for tobacco-related cancers, especially lung cancer.Methods:One hundred and twenty tissue block samples of patients with lung cancers and 120 peripheral blood samples of the control group were obtained from two referral cancer centers in Tehran, Iran, from 2011 to 2016. Genomic DNA was extracted from tissue blocks and buffy coat of study cases to detect SNP of GSTP1 gene using Tetra-primer ARMS-PCR.Results:There was a notable correlation between the incidence of lung cancer and variant Val105 (P-value=0.001; OR=2/6; 95% CI=1.49-4.53) and Ile105 (P-value=0.003; OR=0.41; 95% CI=0.23-0.73). The odds ratio for lung cancer in the homozygous Ile105/Ile105 genotype was 3.56 times higher than that of individual with heterozygous Ile105/Val105 (P-value<0.001; OR=3/56; 95% CI=1.826-6.934) genotype, that was statistically significant. Furthermore, the results showed that there was no significant correlation between Ala114/Val114 genotypes and lung cancer. The BC (P-value=0.007; OR=0.16; 95% CI=0.04-0.61) and AA (P=0.001) genotypes were statistically significant (P-value <0.05); and for those who had AA genotype, the odds ratio was almost six times higher than those with BC genotype.Conclusions:The study of GSTP1 polymorphisms indicated that unlike the polymorphism in exon 5, the GSTP1 exon 6 polymorphism correlated with the lung cancer risk in the select group of Iranian people. Likewise, the potential use of this genetic polymorphism as a lung cancer predictor is confirmed.

The different expression of tumor suppressors, RASSF1A, RUNX3, and GSTP1, in patients with alcoholic steatohepatitis (ASH) vs non-alcoholic steatohepatitis (NASH)

As the fifth most common cancer and the second leading cause of cancer related deaths worldwide, hepatocellular carcinoma (HCC) causes up to one million deaths annually. Alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis (NASH) are becoming the two major risk factors because both may develop liver fibrosis and hepatocellular carcinoma (HCC) if left untreated. However, compared with 3–10% of patients with ASH may progress to HCC annually, about only 0.5% NASH patients may progress to HCC annually. The present study is to clarify the protein expression differences of tumor suppressor genes (TSGs) between ASH and NASH. In liver biopsied specimens from NASH and ASH patients, using an immunofluorescence method and morphometrically quantitating the fluorescence intensity, we studied the protein expression within hepatocytes cytoplasm of candidate TSGs including RUNX3, GSTP1, and RASSF1A. Compared with the control group of patients, the expression levels of all three proteins were upregulated in the ASH group of patients (p < .001 in all molecules). While RUNX3 was upregulated, GSTP1 and RASSF1 did not change in the NASH group of patients. The most important finding is that compared with the ASH group of patients, the expression levels of all three TSG proteins, RUNX3, GSTP1, and RASSF1, were significantly lower in the NASH group of patients (p < .001 in all three molecules). These results confirmed our previous finding that there are significant differences of many molecules including TSGs that changed in NASH compared to ASH. Thus, we conclude that there are significantly different TSGs and pathways involved during the pathogenesis of HCC development in NASH compared to ASH that may help to develop different strategies for prevention and treatment of NASH and ASH patients.

Aloe polysaccharide protects skin cells from UVB irradiation through Keap1/Nrf2/ARE signal pathway

The aim of this study was to investigate if aloe polysaccharide (AP) has the repairing effect on ultraviolet b (UVB) injured nerve cells. The study applied BALB/c female mice as animal model, and NFG-activated PC12 cells as cell model of skin nerve. The cell viability was detected by MTT assay, and cell apoptosis was detected by TUNEL (TdT-mediated dUTP nick-end labeling) and Annexin-V/PI assay, and cell-cycle status in different groups were observed via flow cytometry (FCM). Enzyme-linked immunosorbent assay (ELISA) was applied to analyze oxidative stress and anti-oxidative ability in each group. Real-time PCR and western blot were used to detect the expression levels of Bax, Bcl-2, Caspase-3, Cyclin D1, Keap1, Nrf2, GCLC, and GSTP1. The results showed obvious inhibition of cell viability and cell-cycle progression and promotion of cell apoptosis by UVB irradiation through inducing oxidative stress. In AP treated groups, cell viability and proliferation could be markedly improved and cell apoptosis inhibited with higher anti-oxidative capability and up-regulated expression of Keap1, Nrf2, GCLC, and GSTP1. It suggested that AP was able to repair UVB induced injury on NGF activated skin neural cell PC12, probably through Keap1/Nrf2/ARE signal pathway.

Therapeutic effect of Jin Long capsule combined with neoadjuvant chemotherapy on invasive breast cancer and the expression change of multidrug resistance proteins

Objective: To investigate the therapeutic effect of Jin Long Capsule (JLC) combined with neoadjuvant chemotherapy on the invasive breast cancer, and to explore the mechanism of JLC in inhibiting multidrug resistance of breast cancer. Methods: 200 patients were divided into experimental group and control group (100 cases per group). The control group used TEC regimen for neoadjuvant chemotherapy. And the experimental group was treated with TEC regimen combined with oral JLC. According to the Miller & Payne grading system (MP), the efficacy of neoadjuvant chemotherapy was evaluated based on histopathological changes of breast cancer after neoadjuvant chemotherapy. Adverse effect was evaluated according to the classification criteria of the National Cancer Institute of the United States-The Common Terminology Criteria for Adverse Events (CTCAE) version 3.0. The expression of P-glycoprotein (P-gp), glutathione thiol transferase (GST)-π and topoisomerase Ⅱα (TopoⅡα) in breast cancer tissues before and after neoadjuvant chemotherapy were detected by immunohistochemical staining. Results: There were 83 effective cases (83%) in the experimental group, which was higher than that in the control group (65.0%, P<0.05). The incidence of leukopenia, gastrointestinal reactions and alopecia in grade 3 to 4 of the experimental group were lower than those of the control group (all P<0.05). The positive rates of P-gp, GST-π and TopoⅡα expression in the control group were 65.0% (65/100), 61.0% (61/100) and 69.0% (69/100), respectively, and they were 80.6% (75/93), 78.5% (73/93) and 37.6% (35/93) after chemotherapy. The positive rates of P-gp and GST-π expression were significantly higher than those before chemotherapy (both P<0.05), whereas the positive rate of TopoⅡα expression was significantly lower than that before chemotherapy (P<0.05). In the experimental group, the positive rates of P-gp, GST-π and TopoⅡα expression before chemotherapy were 62.0% (62/100), 63.0% (63/100) and 69.0% (69/100), respectively, while after chemotherapy, they were 68.2% (60/88), 67.0% (59/88) and 63.6% (56/88). There was no significant difference in the positive rates and expression intensity of P-gp, GST-π and TopoⅡα before and after the chemotherapy (P>0.05). Conclusion: Jin Long Capsule (JLC) can inhibit multidrug resistance, improve the efficacy of neoadjuvant chemotherapy, and reduce adverse reactions of breast cancer.

Gene expression patterns and related enzymatic activities of detoxification and oxidative stress systems in zebrafish larvae exposed to the 2,4-dichlorophenoxyacetic acid herbicide

The present study aims to assess the effects of 2,4-D herbicide on biotransformation and oxidative stress status of zebrafish larvae. Animals were exposed to a range of sublethal concentrations (0.02–0.8 mg/L) and biomarkers at transcriptomic level and biochemical level were assessed. Chemical analysis with showed that the bioaccumulation of 2,4-D in 96 hpf zebrafish larvae were increased in a concentration-dependent manner. This herbicide induced significant effects at both gene expression and enzymatic activities levels after at 96 hpf. Results of mRNA expression showed a differential transcription regulation with all target genes depending on the tested concentrations. The mRNA level of gsr and cyp1a were up regulated at the highest dose of herbicide (0.8 mg/L). The gene expression of gstp1 showed an up regulation at lower dose (0.02 mg/L) and a down regulation at the highest dose (0.8 mg/L) of 2,4-D. A significant induction of EROD activity and inhibition of GST activity were noted in groups exposed to 0.8 mg/L of 2,4-D. Considering the antioxidant defenses, the activity of CAT was increased in larvae exposed to 0.8 mg/L of herbicide and GPx activity was induced at lower doses of 2,4-D (0.02 and 0.051 mg/L). Moreover, peroxidative damage, assessed as MDA content, was markedly increased in larvae exposed to high 2,4-D concentration. Overall, the present study data indicate that bioaccumulation of 2,4-D in 96 hpf zebrafish larvae and alterations in detoxification and oxidative stress related parameters, likely associated with ROS production, which may endanger the embryo-larval stages development of fish.

Glutathione S-transferase enzyme activity and protein expression in patients with recurrent tonsillitis and idiopathic tonsillar hypertrophy.

The palatine tonsil is a significant part of the secondary immune system. Tonsillitis and idiopathic tonsillar hypertrophy (ITH) are the most common pathologies of this component. Although there are studies on their pathogenesis, there is insufficient study of the role of antioxidant agents. Glutathione S-transferase (GST) isozymes contribute to the antioxidation reactions in the tissue via the glutathione pathway. The purpose in this study was to reveal the levels of the GST enzyme activity and protein expression of GSTP1 and GSTA1 isozymes in patients with tonsillitis and tonsil hypertrophy, and to investigate their role in the pathogenesis of these diseases. Sixteen patients with recurrent tonsillitis and 5 patients with ITH and were included in the study. Cytosolic extracts were prepared from post-tonsillectomy tissues of both patient groups and GST enzyme activities were measured. The expression of GSTP1 was found to be significantly higher than GSTA1 in tissue samples of patients with ITH and recurrent tonsillitis (P<0.001). Increased GST activity and GSTP1 isozyme expression were shown in patients with recurrent tonsillitis compared to the idiopathic tonsillar hypertrophy study group. There was a positive correlation between the expressions of GSTP1 (P=0.040; r=0.47). Increased GST activity and GSTP1 isozymes were demonstrated histologically in the pathogenesis of ITH and recurrent tonsillitis. We believe that the data of changes in antioxidant capacity, obtained from studies with more extensive and larger samples, would support our findings.

Bioavailability of the diterpenoid 14-deoxy-11,12-didehydroandrographolide in rats and up-regulation of hepatic drug-metabolizing enzyme and drug transporter expression

Background14-Deoxy-11,12-didehydroandrographolide (deAND) is the second most abundant diterpenoid in Andrographis paniculata (Burm. f.) Nees, a traditional medicine used in Asia. To date, the biological activity of deAND has not been clearly investigated. PurposeIn this study, we intended to examine the modulatory effect of deAND on hepatic drug metabolism as well as its bioavailability. Study designdeAND prepared from A. paniculata was orally given to Sprague-Dawley rats and changes in plasma deNAD were determined by HPLC-MS. Modulation of deAND on drug-metabolizing enzyme and drug transporter expression as well as the possible mechanism involved was examined in primary rat hepatocytes. ResultsAfter a single oral administration of 50 mg/kg deAND to rats, the maximum plasma concentration (Cmax), time to reach the Cmax, area under the curve (AUC0–24h), mean retention time, and half-life (t1/2) of deAND were 2.65 ± 0.68 μg/ml, 0.29 ± 0.15 h, 6.30 ± 1.66 μg/ml•h, 5.55 ± 2.52 h, and 3.56 ± 1.05 h, respectively. The oral bioavailability was 3.42%. In primary rat hepatocytes treated with up to 10 μM deAND, a dose-dependent increase was noted in the expression of cytochrome P450 (CYP) 1A1/2, CYP2C6, and CYP3A1/2; UDP-glucuronosyltransferase (UGT) 1A1, NAD(P)H:quinone oxidoreductase (NQO1), π form of GSH S-transferase (GSTP), multidrug resistance-associated protein 2, p-glycoprotein, and organic anion transporter protein 2B1. Immunoblotting assay and EMSA revealed that deAND increases the nuclear translocation and DNA binding activity of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), and nuclear factor erythroid-derived 2-related factor 2 (Nrf2). Knockdown of AhR and Nrf2 expression abolished deAND induction of CYP isozymes and UGT1A1, NQO1, and GSTP expression, respectively. ConclusionThese results indicate that deAND quickly passes through enterocytes in rats and effectively up-regulates hepatic drug-metabolizing enzyme and drug transporter expression in an AhR-, PXR-, and Nrf2-dependent manner.

Melatonin and Docosahexaenoic Acid Decrease Proliferation of PNT1A Prostate Benign Cells via Modulation of Mitochondrial Bioenergetics and ROS Production.

Prostate cancer development has been associated with changes in mitochondrial activity and reactive oxygen species (ROS) production. Melatonin (MLT) and docosahexaenoic acid (DHA) have properties to modulate both, but their protective role, mainly at early stages of prostate cancer, remains unclear. In this study, the effects of MLT and DHA, combined or not, on PNT1A cells with regard to mitochondria bioenergetics, ROS production, and proliferation-related pathways were examined. Based on dose response and lipid accumulation assays, DHA at 100 μM and MLT at 1 μM for 48 h were chosen. DHA doubled and MLT reduced (40%) superoxide anion production, but coincubation (DM) did not normalize to control. Hydrogen peroxide production decreased after MLT incubation only (p < 0.01). These alterations affected the area and perimeter of mitochondria, since DHA increased whereas MLT decreased, but such hormone has no effect on coincubation. DHA isolated did not change the oxidative phosphorylation rate (OXPHOS), but decreased (p < 0.001) the mitochondrial bioenergetic reserve capacity (MBRC) which is closely related to cell responsiveness to stress conditions. MLT, regardless of DHA, ameliorated OXPHOS and recovered MBRC after coincubation. All incubations decreased AKT phosphorylation; however, only MLT alone inhibited p-mTOR. MLT increased p-ERK1/2 and, when combined to DHA, increased GSTP1 expression (p < 0.01). DHA did not change the testosterone levels in the medium, whereas MLT alone or coincubated decreased by about 20%; however, any incubation affected AR expression. Moreover, incubation with luzindole revealed that MLT effects were MTR1/2-independent. In conclusion, DHA increased ROS production and impaired mitochondrial function which was probably related to AKT inactivation; MLT improved OXPHOS and decreased ROS which was related to AKT/mTOR dephosphorylation, and when coincubated, the antiproliferative action was related to mitochondrial bioenergetic modulation associated to AKT and ERK1/2 regulation. Together, these findings point to the potential application of DHA and MLT towards the prevention of proliferative prostate diseases.

Effects of shRNA Expression Vector-mediated Glutathione S-transferase P1 Gene Silencing on Prostate Cancer DU145 Cells

Objective: This study aims to explore the impact of silent GSTP1 gene on the proliferation activity, cell cycle and apoptotic rate of the androgen-independent prostate cancer cell strain DU145. Methods: Thirty subjects with benign prostatic hyperplasia (BPH) who had been subject to urinary surgical resection and pathological diagnosis, 30 subjects with radically cured prostate cancer (PCa), and another 30 subjects with hormone refractory prostate cancer (HRPC) were enrolled. The GSTP1 expression in the subjects was tested with the immunohistochemical S-P method, the GSTP1 expression in the blood serum of the patients with prostatic hyperplasia and prostate cancer was tested with Elisa, and the mRNA level of the GSTP1 gene after the DUl45 cells were transfected with shRNA was tested. Results: Four of the HRPC patients were complicated with bone metastasis, two with bladder invasion, and five with liver and lung metastasis. GSTP1 showed high expression in tissues with prostatic hyperplasia and hormone refractory prostate cancer and low expression in tissues with prostate cancer, and the differences were statistically significant (P 0.05). The GSTP1 mRNA content decreased significantly after shRNA554 intervention, with an IOD value of 67.1±8.7. The intervention effect was evidently lower than that of the carrier shRNA255 group (162.2±12.6) and the shRNA593 group (114.5±10.6) with statistically significant difference (P<0.01). Conclusion: The expression vector-mediated GSTP1-shRNA554 transfection can reduce the mRNA level of the GSTP1 gene of the androgen-independent prostate cancer DU145 cells and inhibit the proliferation activity of the in vitro cultivated DU145 cells.

Altered apoptosis and biotransformation signaling in HCT-116 colorectal carcinoma cells induced by Teucrium chamaedrys L. extract

The aim of this study was an investigation of pro-apoptotic activity of methanol extract from T. chamaedrys, a more detailed determination of the signal molecules activated in the process of apoptosis, and effects on mRNA expression of enzymes involved in biotransformation (CYP1A1 and GSTP1) and membrane transporter, MRP-2 in HCT-116 colorectal carcinoma cells. The results show pronounced proapoptotic activity of T. chamaedrys extract, due to activation of both extrinsic and intrinsic pathways. The death receptor associated signaling pathway was activated in HCT-116 following treatment by T. chamaedrys, via increased Fas receptor expression and activity of caspase 8. Activation of caspase 9 suggests that mitochondrial signalling also has an impact. The extract reduced mRNA expression of GSTP1 and MRP-2 genes, as one of the causes of multi drug resistance in cancer cells. Observed results offer the possibility for the use of T. chamaedrys extract in the context of cancer prevention and therapy.

Glutathione S-transferases genes variants and chemotherapy efficacy in gastrointestinal cancer patients: a meta-analysis based on 50 pharmacogenetic studies.

Background: The role of glutathione s-transferase genes (GSTP1, GSTM1 and GSTT1) variants and the GSTP1 expression level on chemotherapy efficacy of gastrointestinal cancer (GIC) patients were inconsistent.Methods: A meta-analysis about GSTP1, GSTM1 and GSTT1 variants and the GSTP1 expression level on chemotherapy efficacy of GIC patients was performed using data from PubMed, PMC, EMBASE, Web of Science, and Wanfang database.Results: Our meta-analysis enrolled 50 publications including 6518 patients. We found that patients with GIC harboring GSTP1 (IIe105Val) Val locus had higher objective response rates (ORR) than the IIe/IIe genotypic patients (odds ratio (OR) = 1.580, 95% confidence interval (CI) = 1.159-2.154, P = 0.004). Significant associations were found between the Ile105Val variant and overall survival of Caucasian GIC patients (IIe/Val vs. IIe/IIe: OR = 0.797 (0.674-0.944), P = 0.009). Caucasian GIC patients and gastric cancer patients with GSTT1 null genotype had worse response rates compared to GSTT1 present patients (OR = 0.530 (0.356-0.789), P = 0.002; OR = 0.643 (0.463-0.895), P = 0.009, respectively).Conclusion: This meta-analysis illustrates that GSTP1 IIe105Val and GSTT1 null/present variants could be useful predictors of chemotherapy efficacy in patients with gastrointestinal cancer.