Glutathione S-transferase Pi Gene Research Articles

Downregulation of VPS13C promotes cisplatin resistance in cervical cancer by upregulating GSTP1

Cisplatin resistance remains a major obstacle limiting the effectiveness of chemotherapy in cervical cancer. However, the underlying mechanism of cisplatin resistance is still unclear. In this study, we demonstrate that vacuolar protein sorting 13 homolog C (VPS13C) deficiency promotes cisplatin resistance in cervical cancer. Moreover, through an RNA sequencing screen, VPS13C deficiency was identified as negatively correlated with the high expression of glutathione S-transferase pi gene (GSTP1). Mechanistically, loss of VPS13C contributes to cisplatin resistance by influencing the expression of GSTP1 and inhibiting the downstream c-Jun N-terminal kinase (JNK) pathway. In addition, targeting GSTP1 with the inhibitor NBDHEX effectively rescued the cisplatin resistance induced by VPS13C deficiency. Overall, our findings provide insights into the underlying mechanisms of VPS13C in cisplatin resistance and identify VPS13C as a promising candidate for the treatment of chemoresistance in cervical cancer.

A study of association between presence or absence of GSTT1 and GSTM1 and/or single nucleotide polymorphism in FABP2 and GSTP1 with incidence of diabetes type 2: A case-control study.

To assess the association of single nucleotide polymorphisms in fatty acid binding protein-2 (rs1799883) and glutathione S-transferase pi (rs1695) genes with presence/absence of glutathione S-transferase mu and glutathione S-transferase theta genes in type 2 diabetes. The cross-sectional case-control study was conducted at Institute of Molecular Biology and Biotechnology during March till September 2019 and comprised type 2 diabetes patients and non-diabetic controls from two districts in southern Punjab. Polymerase chain reaction, polymerase chain reaction-restriction fragment length polymorphism and tetra-primer amplification refractory mutation system-polymerase chain reaction were applied to investigate glutathione S-transferase theta, mu and pi genes as well as fatty acid binding protein-2, as appropriate. The association of single nucleotide polymorphisms in all genes with the disease were studied either individually or in various combinations. Data was analysed using Minitab 18. Of the 448 subjects, 248(55.4%) were patients and 200(44.6%) were controls. Overall there were 213(47.5%) males and 235(52.5) were females, and 141(31.5%) were aged 30-46 years. The presence of rs1799883 in fatty acid binding protein-2 (p=0.03) and the absence of glutathione S-transferase mu gene (p<0.001) had significant association with type 2 diabetes, while the presence of glutathione S-transferase theta and rs1695 in glutathione S-transferase pi genes were not associated with the disease. Individuals with glutathione S-transferase mu gene null and Ileu/Ileu or Ileu/Val genotype of rs1695 in glutathione S-transferase pi gene have potential to develop type 2 diabetes in their lifetime (p<0.05). The presence of rs1799883 in fatty acid binding protein-2 and the absence of glutathione S-transferase mu gene were found to play significantly in the development of type 2 diabetes.

Прогнозирование врожденной расщелины губы и нёба при планировании беременности с применением генетических маркеров в регионе с экотоксикантами

Objective. To predict the risk of congenital cleft lip and palate during pregnancy planning in patients residing in a polluted region using genetic markers. Materials and methods. This study included 45 women with children who had congenital cleft lip and palate. The control group comprised 119 donors of the Republican Blood Transfusion Station without cleft lip and palate who resided in the Republic of Bashkortostan. DNA was isolated from peripheral blood using standard phenol-chloroform extraction protocol. Polymorphic variants were assessed using polymerase chain reaction with locus-specific oligonucleotide primers. We analyzed the prevalence of genotypes and alleles of polymorphic loci in genes encoding glutathione S-transferase mu 1 (GSTM1) and glutathione S-transferase Pi 1 (GSTP1) to predict congenital cleft lip and palate. Results. The analysis of prevalence of genotypes and alleles of the glutathione S-transferase genes demonstrated that parents carrying deletions in the GSTM1 gene were more likely to have children with cleft lip and palate (OR = 2.65 (CI 95% 1.10–6.41), p = 0.028). We observed a significant association when comparing the allele frequencies. The frequency of deletion was 60.0% in parents with children who had congenital cleft lip and palate and 36.13% in the control group. The odds ratio in the parent group was 2.65 (CI 95% 1.42–4.95), p = 0.003. Significant differences were obtained in the additive model (OR = 2.21 (CI 95% 1.05–4.67), p = 0.038). The presence of the G allele more than doubled the odds of having a baby with congenital cleft lip and palate (OR = 2.21 (CI 95% 1.05–4.67), p = 0.038). Conclusion. Genotyping of the polymorphic locus rs1695 in the GSTP1 gene, as well as identification of GSTM1 gene deletions and AG or GG genotypes of the rs1695 locus in the GSTP1 gene can be used to predict the risk of congenital cleft lip and palate in a baby. Key words: congenital cleft lip and palate, pollutants, risk assessment during pregnancy planning, genetic markers

Detoxification Genomics in Children with β-Thalassemia Major: Pilot Study of Glutathione S Transferase M1, Pi &amp; Methyltetrahydrofolate Reductase Gene Polymorphisms Combinations in β- Thalassemia Major

Background: Children with β-thalassemia major differ regarding age at presentation, transfusion requirements and unpredictable timing, rate and severity of hemolytic crisis. The blood transfusions are associated with iron overload. Glutathione S transferase M1 (GSTM1) null mutation was reported to be associated with myocardial iron overload with low body iron. Aim of the Work: To investigate if children with β-thalassemia major have more than a detoxification enzyme defect. Materials and Methods: GSTM1, glutathione S transferase Pi (GSTPi) and methyltetrahydrofolate reductase (MTHFR) polymorphism were studied among 97 children with β-thalassemia major in a cross-sectional study. Results: The studied cohort comprised 24 (24.7%) girls and 73 (75.3%) boys. Mean hemoglobin was 5.9+/- 0.7gm%, serum iron was 145.69 +/- 58.6 mcg% and total iron binding capacity was 222.58 +/-50.66 mcg%. Of them, 68 (70.1%) demonstrated single or multiple polymorphisms (43 had GSTM1, 20 GSTPi and 32 with MTHFR polymorphisms respectively), while 29 (29.2%) did not demonstrate any polymorphism. There was no correlation between type, number of polymorphisms and clinical phenotype. Sample size and cross- sectional nature of our study did not allow genotype-phenotype correlation. Most of studied children express GSTM1, GSTPi and MTHFR gene polymorphism which was not consistent among them. Conclusion: Children with β-thalassemia major may have one or more than a detoxification/ regeneration potential enzyme gene GSTM1, GSTPi and MTHFR polymorphism. Every child with β-thalassemia major has unique genetic detoxification and regeneration abilities. The detected detoxification defects might explain the lack of predictability of occurrence of hemolytic attacks and their severity. More studies are needed to highlight impact of detoxification and regeneration genomics in β-thalassemia.

Predictive value of clinical toxicities of chemotherapy with fluoropyrimidines and oxaliplatin in colorectal cancer by DPYD and GSTP1 gene polymorphisms

BackgroundFluoropyrimidines and platinum are still widely used for colorectal cancer (CRC) management. Several studies have reported that mutations of dihydropyrimidine dehydrogenase (DPYD) and glutathione S-transferase pi-1 (GSTP1) polymorphisms are related to chemotherapy-related adverse events. In the present study, we purposed to assess the impact of DPYD and GSTP1 variants on the toxicity of adjuvant chemotherapy risk among the Hakka population, minimize adverse events, and to maximize therapy outcome for individualized treatment.MethodsGenotyping was examined in 104 patients diagnosed with CRC cases and receiving fluoropyrimidine and platinum drug-based chemotherapy regimen by direct sequencing of DPYD and GSTP1 polymorphisms. Three DPYD variants including *2A, *5A, *9A, and GSTP1 c.313A>G were analyzed and clinical outcomes were assessed.ResultsThe data suggest that the incidence of DPYD*5A, DPYD*9A, and GSTP1 c.313A>G variants were 38.4%, 24%, and 32.7%, respectively. DPYD*2A variant was not found. A total of 23 patients (22.1%) suffered severe vomiting and 19 patients (18.3%) suffered severe anemia. DPYD*5A polymorphism was found significantly associated with grade 3/4 ulceration (p = 0.001). GSTP1 was determined to be an independent risk factor for severe vomiting and skin ulceration (p = 0.042 and p = 0.018, respectively). Patients with GSTP1 c. 313A>G mutant type contributed to a higher risk for grade severe toxicity compared with wild genotype (p = 0.027). Nevertheless, no significant difference was found between patients with DPYD*2A, *5A, and *9A for chemotherapeutic toxicity.ConclusionsThe results demonstrated that GSTP1 polymorphisms were useful predictors of severe events. Screening of single-nucleotide polymorphisms of GSTP1 in colorectal cancer patients before chemotherapy may help to realize personalized therapy.

Glutathione S-transferase pi 1 variant and squamous cell carcinoma susceptibility: a meta-analysis of 52 case-control studies

BackgroundThere are several meta-analyses on the genetic relationship between the rs1695 polymorphism within the GSTP1 (glutathione S-transferase pi 1) gene and the risk of different SCC (squamous cell carcinoma) diseases, such as ESCC (oesophageal SCC), HNSCC (head and neck SCC), LSCC (lung SCC), and SSCC (skin SCC). Nevertheless, no unified conclusions have been drawn.MethodsHerein, an updated meta-analysis was performed to evaluate the probable impact of GSTP1 rs1695 on the susceptibility to different SCC diseases under six genetic models (allele, carrier, homozygote, heterozygote, dominant, and recessive). Three online databases, namely, PubMed, WOS (Web of Science), and Embase (Excerpta Medica Database), were searched.ResultsInitially, we obtained a total of 497 articles. Based on our selection criteria, we eventually included 52 case-control studies (9763 cases/15,028 controls) from 47 eligible articles. As shown in the pooling analysis, there was no difference in the risk of overall SCC disease between cases and controls [allele, Pa (P value of association test) = 0.601; carrier, Pa = 0.587; homozygote, Pa = 0.689; heterozygote, Pa = 0.167; dominant, Pa = 0.289; dominant, Pa = 0.548]. Similar results were obtained after stratification by race (Asian/Caucasian), genotyping, control source, and disease type (ESCC/HNSCC/LSCC/SSCC) (all Pa > 0.05).ConclusionThe rs1695 polymorphism within the GSTP1 gene is not associated with the risk of overall SCC or a specific SCC type, including ESCC, HNSCC, LSCC, and SSCC.

Overexpression of long noncoding RNA LINC01419 in esophageal squamous cell carcinoma and its relation to the sensitivity to 5-fluorouracil by mediating GSTP1 methylation.

Background:Genome-wide sequencing investigations have identified numerous long noncoding RNAs (lncRNAs) among mammals, many of which exhibit aberrant expression in cancers, including esophageal squamous cell carcinoma (ESCC). Herein, this study elucidates the role and mechanism by which LINC01419 regulates the DNA methylation of glutathione S-transferase pi 1 (GSTP1) in relation to ESCC progression and the sensitivity of ESCC cells to 5-fluorouracil (5-FU).Methods:LINC01419 and GSTP1 levels were quantified among 38 paired ESCC and adjacent tissue samples collected from patients with ESCC. To ascertain the contributory role of LINC01419 in the progression of ESCC and identify the interaction between LINC01419 and GSTP1 promoter methylation, LINC01419 was overexpressed or silenced, and the DNA methyltransferase inhibitor 5-Aza-CdR was treated.Results:Data from the GEO database (GSE21362) and the Cancer Genome Atlas displayed elevated levels of LINC01419 and downregulated levels of GSTP1 in the ESCC tissues and cells. The silencing of LINC01419 led to decreased proliferation, increased apoptosis, and enhanced sensitivity to 5-FU in ESCC cells. Notably, LINC01419 could bind to the promoter region of the GSTP1 gene, resulting in elevated GSTP1 methylation and reduced GSTP1 levels via the recruitment of DNA methyltransferase among ESCC cells, whereby ESCC progression was stimulated accompanied by reduced ESCC cell sensitivity to 5-FU. GSTP1 demethylation by 5-Aza-CdR was observed to reverse the effects of LINC01419 overexpression in ESCC cells and the response to 5-FU.Conclusion:Highly expressed LINC01419 in ESCC promotes GSTP1 methylation, which ultimately acts to promote the event of ESCC and diminish the sensitivity of ESCC cells to 5-FU, highlighting a novel potential strategy to improve 5-FU-based chemotherapy in ESCC.

Association of GSTP1 and P16 promoter methylation with the risk of HBV-related hepatocellular carcinoma: a meta-analysis.

BackgroundStudy on the relationship between glutathione-S-transferase Pi 1 (GSTP1) and P16 promoter region methylation and the risk of hepatitis B virus-related hepatocellular carcinoma (HBV-related HCC) has produced inconsistent results.ObjectivesTo assess the correlation between GSTP1 and P16 promoter methylation frequency and HBV-related HCC susceptibility.MethodsAll relevant studies were identified by searching PubMed, Embase, Web of Science, and China National Knowledge Infrastructure literature databases before December, 2017. The OR and the corresponding 95% CI were calculated to investigate the risk of GSTP1 and P16 promoter methylation rate and HBV-related HCC. Sensitivity analysis was performed and publication bias was estimated using the Begg’s and Egger’s test.ResultsOur meta-analysis identified the relationships of GSTP1 (six studies including 213 HBV-related HCC tumor tissues) and P16 (nine studies with 287 HBV-related HCC tumor tissue) promoter methylation with HCC risk. Compared with normal liver tissue and cirrhosis, the pooled ORs of GSTP1 promoter region methylation in HBV-related HCC cancer tissues were 6.05 (95% CI =1.20–30.52) and 5.21 (95% CI =2.19–12.41), respectively. Compared with paracancerous tissue, normal liver tissue, cirrhosis, and chronic hepatitis B as controls, the pooled ORs of P16 promoter region methylation in HBV-related HCC cancer tissues were 7.18 (95% CI =2.31–22.33), 24.89 (95% CI =3.38–183.03), 5.92 (95% CI =1.78–19.68), and 12.12 (95% CI =0.75–196.50).ConclusionIn summary, our meta-analysis found strong associations between GSTP1 and P16 gene promoter methylation and an increased HBV-related HCC susceptibility. Moreover, GSTP1 and P16 methylation in promoter region could obviously increase the risk of HBV-related HCC in patients with cirrhosis, indicating that these would be promising biomarkers for early clinical diagnosis of HBV-related HCC.

High GSTP1 inhibits cell proliferation by reducing Akt phosphorylation and is associated with a better prognosis in hepatocellular carcinoma.

Glutathione S-transferase (GST) family members promote carcinogenesis and cancer progression. We assessed GST pi 1 (GSTP1) mRNA and protein levels in hepatocellular carcinoma (HCC) using genome databases and tissue microarray (TMA) technology. We found that in cancerous tissues, GSTP1 mRNA was down-regulated in genome databases, and immunohistochemical staining of GSTP1 in 237 HCC cases varied from negative to strongly positive. GSTP1 levels correlated negatively with tumor size and serum alpha-fetoprotein (AFP) in HCC patients, and higher GSTP1 levels associated with longer overall survival (OS) and disease-free survival (DFS). We also found that GSTP1 overexpression restrained HepG2 and Huh7 liver cancer cell proliferation in vivo and in vitro. GSTP1 arrested the cell cycle at G1/S by up-regulating p21 and p27 and down-regulating p-Akt. Interrupting GSTP1 gene expression promoted liver cancer cell proliferation and increased the percentage of cells in S phase by decreasing levels of p21 and p27 and increasing p-Akt. These results suggest high GSTP1 levels provide a better prognosis through suppression of tumorigenesis in HCC.

Polymorphisms of genesencoding enzymes of the xenobiotic detoxification system and the risk of infectious endocarditis

Infectioue endocarditis (IE) is frequently associated with the use of narcotic drugs, glucocorticosteroids, and cytostatics that are metabolized in the body by enzymes of the xenobiotic detoxification system (XDS). This work was aimed at elucidating association between IE and mononucleotide polymorphisms of genes encoding XDS enzymes. 46 IE patients and 114 subjects without cardiovascular diseases (controls) underwent genotyping for polymorphic loci of cytochrome P450 1A1 gene I462V (CYP1A1), I105VandA114V gene of glutathione-S-transferase Pi1 (GSTP1) using allele-specific PCR. The study revealed association of CYP1A1 I462VandGSTP1 I105V with IE while IE proved unrelated to GSTP1 A114V polymorphism. Combination of homozygous variant I462I of the CYP1A1 gene and heterozygous variant I105V/ of the GSTP1 gene was associated with the 9-fold increase of the risk of IE in the subjects practicing intravenous druginjections or having congenital and acquired heart failure or implanted valve prostheses. These findings suggest the necessity of further studies on the role of XDS in pathogenesis of IE and other infectious diseases.

Glutathione S-transferase pi expression regulates the Nrf2-dependent response to hormetic diselenides

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.

HapMap-based study on the association between MPO and GSTP1 gene polymorphisms and lung cancer susceptibility in Chinese Han population.

Myeloperoxidase (MPO) and glutathione S-transferase pi 1 (GSTP1) are important carcinogen-metabolizing enzymes. The aim of this study was to investigate the association between the common polymorphisms of MPO and GSTP1 genes and lung cancer risk in Chinese Han population. A total of 266 subjects with lung cancer and 307 controls without personal history of the disease were recruited in this case control study. The tagSNPs approach was used to assess the common polymorphisms of MOP and GSTP1 genes and lung cancer risk according to the disequilibrium information from the HapMap project. The tagSNP rs7208693 was selected as the polymorphism site for MPO, while the haplotype-tagging SNPs rs1695, rs4891, rs762803 and rs749174 were selected as the polymorphism sites for GSTP1. The gene polymorphisms were confirmed using real-time PCR, cloning and sequencing. The four GSTP1 haplotype-tagging SNPs rs1695, rs4891, rs762803 and rs749174, but not the MPO tagSNP rs7208693, exhibited an association with lung cancer susceptibility in smokers in the overall population and in the studied subgroups. When Phase 2 software was used to reconstruct the haplotype for GSTP1, the haplotype CACA (rs749174+rs1695 + rs762803+rs4891) exhibited an increased risk of lung cancer among smokers (adjust odds ratio 1.53; 95%CI 1.04-2.25, P=0.033). Furthermore, diplotype analyses demonstrated that the significant association between the risk haplotype and lung cancer. The risk haplotypes co-segregated with one or more biologically functional polymorphisms and corresponded to a recessive inheritance model. The common polymorphisms of the GSTP1 gene may be the candidates for SNP markers for lung cancer susceptibility in Chinese Han population.

English

The Mother and Child Environmental Cohort (MACE) study piloted in South Africa in 2010 to 2011, collected genetic, biochemical and clinical data from pregnant females residing in south and north Durban. We evaluated birth outcomes and the influence of GSTM1pos→GSTM1null and theGSTP1 (Ile105Val; AA→AG/GG) polymorphisms on the extent of DNA damage and with biomarkers [glutathione (GSH) and malondialdehyde (MDA)] related to oxidative stress in mothers with different levels of pollutant exposure. There was no significant difference in adverse birth outcomes or genotype distribution between mothers from the exposed and lower exposed areas. Mean GSH and comet tail length did not differ significantly between GSTM1pos and GSTM1null genotypes. When stratified by genotype, mean MDA levels was higher among GSTM1 null mothers compared to the GSTM1pos mothers (p = 0.01). When each of the genotypes was stratified by exposure, mean GSH concentration was significantly higher in north Durban for theGSTM1pos, GSTM1null and GSTP1AG+GG genotypes (p < 0.05), and mean comet tail length was significantly increased in south Durban among participants with the GSTM1pos, GSTM1null, and the GSTP1AG+GG genotypes. The expression of GSTM1 and GSTP1 polymorphic genotypes may lead to varying susceptibility to the adverse effects of pollutants by modifying the response to oxidative stress. Key words: Glutathione S-transferase Mu 1 (GSTM1), glutathione S-transferase pi gene(GSTP1), oxidative stress, birth cohort, glutathione, gene polymorphism, DNA damage.

Allelic variants of glutathione S-transferase P1-1 differentially mediate the peroxidase function of peroxiredoxin VI and alter membrane lipid peroxidation

The dual-functioning antioxidant enzyme peroxiredoxin VI (Prdx6) detoxifies lipid peroxides particularly in biological membranes, and its peroxidase function is activated by glutathione S-transferase Pi (GSTP). The GSTP gene is polymorphic in humans, with the wild-type GSTP1-1A (Ile105, Ala114) and three variants: GSTP1-1B (Ile105Val, Ala114), GSTP1-1C (Ile105Val, Ala114Val), and GSTP1-1D (Ile105, Ala114Val). The focus of this study was to determine the influence of these polymorphisms on Prdx6 peroxidase function. Using extracellular generation of OH radicals and fluorescence (DPPP dye) detection, we found a fast (∼300s) onset of lipid peroxidation in membranes of MCF-7 cells transfected with a catalytically inactive Y7F mutant of GSTP1-1 and either GSTP1-1B or GSTP1-1D. However, this effect was not detected in cells expressing either GSTP1-1A or GSTP1-1C. Imaging of DPPP-labeled MCF-7 cells showed fluorescence localized in the plasma membrane, but intensity was substantially diminished in the GSTP1-1A- and GSTP1-1C-expressing cells. Moreover, in the Y7F mutant of GSTP1-1A-, GSTP1-1B-, and GST1-1D-expressing cells OH generation resulted (after 36h) in plasma membrane-permeability-related cell death, whereas GSTP1-1A- and GSTP1-1C-expressing cells had significantly better survival. We used FRET analyses to measure in vitro binding of purified GSTP1-1 allelic variant proteins to purified recombinant Prdx6. The affinities for Prdx6 binding to GSH-loaded GSTP1-1's either mirrored their observed peroxidase activities (using phospholipid hydroperoxide as a substrate), GSTP1-1A>GSTP1-1C (KD=51.0 vs 57.0nM), or corresponded to inactivation, GSTP1-1B (GSTP1-1D) (KD=101.0 (94.0) nM). In silico modeling of the GSTP1-1–Prdx6 heterodimer revealed that the sites of GSTP1-1 polymorphism (Ile105 and Ala114) are in close proximity to the binding interface. Thus, there is a hierarchy of effectiveness for polymorphic variants of GSTP1-1 to regulate Prdx6 peroxidase function, a feature that may influence human population susceptibilities to oxidant stress.

Glutathione-S-transferase pi 1(GSTP1) gene silencing in prostate cancer cells is reversed by the histone deacetylase inhibitor depsipeptide

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.

Pharmacogenetic Study in Rectal Cancer Patients Treated With Preoperative Chemoradiotherapy: Polymorphisms in Thymidylate Synthase, Epidermal Growth Factor Receptor, GSTP1, and DNA Repair Genes

Several studies have been performed to evaluate the usefulness of neoadjuvant treatment using oxaliplatin and fluoropyrimidines for locally advanced rectal cancer. However, preoperative biomarkers of outcome are lacking. We studied the polymorphisms in thymidylate synthase, epidermal growth factor receptor, glutathione S-transferase pi 1 (GSTP1), and several DNA repair genes to evaluate their usefulness as pharmacogenetic markers in a cohort of 128 rectal cancer patients treated with preoperative chemoradiotherapy. Blood samples were obtained from 128 patients with Stage II-III rectal cancer. DNA was extracted from the peripheral blood nucleated cells, and the genotypes were analyzed by polymerase chain reaction amplification and automated sequencing techniques or using a 48.48 dynamic array on the BioMark system. The germline polymorphisms studied were thymidylate synthase, (VNTR/5'UTR, 2R G>C single nucleotide polymorphism [SNP], 3R G>C SNP), epidermal growth factor receptor (Arg497Lys), GSTP1 (Ile105val), excision repair cross-complementing 1 (Asn118Asn, 8092C>A, 19716G>C), X-ray repair cross-complementing group 1 (XRCC1) (Arg194Trp, Arg280His, Arg399Gln), and xeroderma pigmentosum group D (Lys751Gln). The pathologic response, pathologic regression, progression-free survival, and overall survival were evaluated according to each genotype. The ∗3/∗3 thymidylate synthase genotype was associated with a greater response rate (pathologic complete remission and microfoci residual tumor, 59% in ∗3/∗3 vs. 35% in ∗2/∗2 and ∗2/∗3; p=.013). For the thymidylate synthase genotype, the median progression-free survival was 103 months for the ∗3/∗3 patients and 84 months for the ∗2/∗2 and ∗2/∗3 patients (p=.039). For XRCC1 Arg399Gln SNP, the median progression-free survival was 101 months for the G/G, 78 months for the G/A, and 31 months for the A/A patients (p=.048). The thymidylate synthase genotype and XRCC1 Arg399Gln polymorphism might help to identify Stage II-III rectal cancer patients with a better outcome after preoperative concomitant chemoradiotherapy.

Lack of association of GSTT1 and GSTP1 genes methylation and their expression profiles with risk of NAFLD in a sample of Iranian patients

Reactive oxygen species can affect many cellular functions through protein oxidation or initiation of the lipid peroxidation cascade that can lead to non-alcoholic fatty liver disease (NAFLD), characterized by significant lipid deposition in the hepatocytes of patients with no history of excess alcohol intakes. The present study aimed to analyze the methylation status of the antioxidative stress genes GSTT1 (glutathione S-transferase theta-1) and GSTP1 (glutathione S-transferase pi-1), and their expression profiles, in a sample population of patients with NAFLD living in South-East Iran. Peripheral blood samples were obtained from 80 NAFLD patients and 80 healthy controls. Promoter methylation of the GSTT1 and GSTP1 genes were analyzed by methylation-specific polymerase chain reaction (MS-PCR). Expression profiles of these genes were also examined by quantitative real-time PCR analysis. Promoter methylation of the GSTT1 gene was detected in 86.2% of cases and in 91.2% of controls and, of the GSTP1 gene, in 88.8 and 87.5% of cases and controls, respectively. Promoter methylation of GSTT1 and GSTP1 was not statistically different in cases compared with healthy controls. Similarly, mRNA expression levels showed no statistically significant variations between healthy individuals and patients with NAFLD. Our findings indicate no association between methylation status and expression profiles of GSTT1 and GSTP1 genes and NAFLD. This is the first report to assess such associations in a sample of the Iranian population.

Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease

BackgroundTumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis.Materials and methodsWe prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF.ResultsWe found a hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly significantly correlated with positive Her2/neu status (p = 0.003). Elevated serum CA15.3 was strongly correlated with methylated APC and CTC detection (both p = 0.000). Methylated ESR1 failed to exhibit significant correlations with any of the above mentioned parameters. The presence of CTC in peripheral blood was significantly associated with methylated APC (p = 0.012) and methylated GSTP1 (p = 0.001).ConclusionThe detection of methylated APC and GSTP1 DNA in serum correlated with the presence of CTC in the blood of breast cancer patients. Both methylated DNA and CTC correlated with a more aggressive tumor biology and advanced disease.

Estrogen receptor 1, Glutathione S-transferase P1, Glutathione S-transferase M1, and Glutathione S-transferase T1 Genes with Dysmenorrhea in Korean Female Adolescents

Dysmenorrhea is the most common gynecologic complaint among adolescent females. We investigated the association between genetic polymorphisms and dysmenorrhea. A total of 202 postmenarcheal Korean female adolescents 16-17 yr old participated in this study. Genotyping for glutathione S-transferase mu 1 (GSTM1), glutathione S-transferase theta 1 (GSTT1), glutathione S-transferase pi 1 (GSTP1), and estrogen receptor 1 (ESR1) was performed using PCR-based methods. The PP+Pp genotype of the ESR1 gene was more frequent than pp genotypes in subjects with dysmenorrhea than in subjects without dysmenorrhea (odds ratio=2.440; 95% confidence interval, 1.036-5.753; P=0.040) using an unadjusted univariate logistic regression analysis. The relationship between dysmenorrhea and ESR1 gene polymorphisms remained significant after adjustment for premenstrual syndrome, years elapsed after menarche, and family history of dysmenorrhea. No significant difference was observed between subjects with dysmenorrhea and subjects without dysmenorrhea for polymorphisms of GSTM1, GSTT1, and GSTP1 genes. Our results suggest that ESR1 gene polymorphisms may be associated with dysmenorrhea.

Formamide as a denaturant for bisulfite conversion of genomic DNA: Bisulfite sequencing of the GSTPi and RARβ2 genes of 43 formalin-fixed paraffin-embedded prostate cancer specimens

Analysis of methylated DNA, which refers to 5-methycytosine (5mC) versus cytosine (C) at specific loci in genomic DNA (gDNA), has received increased attention in epigenomics, particularly in the area of cancer biomarkers. Many different methods for analysis of methylated DNA rely on initial reaction of gDNA with concentrated acidic sodium bisulfite to quantitatively convert C to uracil (U) via sulfonation of denatured, single-stranded gDNA under conditions where 5mC is resistant to analogous sulfonation leading to thymine (T). These methods typically employ polymerase chain reaction (PCR) amplification after bisulfite conversion, thereby leading to readily detectable amounts of amplicons where T and C are measured as surrogates for C and 5mC in the original unconverted gDNA. However, incomplete bisulfite conversion of C in gDNA has been reported to be a common source of error in analysis of methylated DNA. Incomplete conversion can be revealed during the course of bisulfite sequencing, which is the generally accepted “gold standard” for analysis of methylated DNA. Previous bisulfite sequencing investigations of conventional predenaturation of gDNA with NaOH followed by the use of bisulfite containing added urea to maintain denaturation and thus mitigate incomplete conversion of C have been reported to give conflicting results. The current study describes a new approach where conventional predenaturation of gDNA with NaOH is instead achieved with formamide and maintains denaturation during subsequent sample handling and sulfonation. This formamide-based method was applied to 46 formalin-fixed/paraffin-embedded (FFPE) biopsy tissue specimens from well-characterized patients with primary prostate cancer. These specimens were representative of difficult-to-analyze samples due to the chemically compromised nature of the gDNA, which was recovered by modifying the protocol for a commercially available total RNA/DNA extraction kit (RecoverALL). An additional novel aspect of this study was analysis of CpG-rich promoter regions of two prostate cancer-related genes: glutathione S-transferase pi (GSTPi) and retinoic acid receptor beta2 (RARβ2). High-quality bisulfite sequencing results were obtained for both genes in 43 of 46 (93%) specimens. Detection of methylated GSTPi and RARβ2 genes was significantly associated with primary prostate cancer as compared with the benign prostate (Fisher’s exact test, P < 0.001). The sensitivity and specificity of detection of methylated GSTPi and RARβ2 genes were 86% and 100% and 91% and 100%, respectively. Moreover, the presence of either methylated gene was detected in primary prostate cancer with sensitivity and specificity of 100% and 100%, respectively. The results demonstrated a high degree of reliability of formamide-based denaturation and bisulfite conversion that should extend, generally, to FFPE and other types of samples intended for any analytical method predicated on bisulfite conversion. This pilot study also demonstrated the efficacy of determining methylation of these two genes with high sensitivity and specificity in FFPE biopsy tissue specimens. Moreover, the results showed a highly significant association of methylated GSTPi and RARβ2 genes with primary prostate cancer. Finally, this improved procedure for determining these two methylated genes may allow the detection of prostate cancer cells in core biopsy specimens with insufficient numbers of cells and poor morphology.