Glutathione S-transferase Pi Research Articles

Glutathione Transferase P1: Potential Therapeutic Target in Ovarian Cancer.

Chemotherapy resistance of ovarian cancer, regarded as the most lethal malignant gynecological disease, can be explained by several mechanisms, including increased activity of efflux transporters leading to decreased intracellular drug accumulation, increased efflux of the therapeutic agents from the cell by multidrug-resistance-associated protein (MRP1), enhanced DNA repair, altered apoptotic pathways, silencing of a number of genes, as well as drug inactivation, especially by glutathione transferase P1 (GSTP1). Indeed, GSTP1 has been recognized as the major enzyme responsible for the conversion of drugs most commonly used to treat metastatic ovarian cancer into less effective forms. Furthermore, GSTP1 may even be responsible for chemoresistance of non-GST substrate drugs by mechanisms such as interaction with efflux transporters or different signaling molecules involved in regulation of apoptosis. Recently, microRNAs (miRNAs) have been identified as important gene regulators in ovarian cancer, which are able to target GST-mediated drug metabolism in order to regulate drug resistance. So far, miR-186 and miR-133b have been associated with reduced ovarian cancer drug resistance by silencing the expression of the drug-resistance-related proteins, GSTP1 and MDR1. Unfortunately, sometimes miRNAs might even enhance the drug resistance in ovarian cancer, as shown for miR-130b. Therefore, chemoresistance in ovarian cancer treatment represents a very complex process, but strategies that influence GSTP1 expression in ovarian cancer as a therapeutic target, as well as miRNAs affecting GSTP1 expression, seem to represent promising predictors of chemotherapeutic response in ovarian cancer, while at the same time represent potential targets to overcome chemoresistance in the future.

Abstract B009: Multi-omics analysis of glutathione S-transferase P1 (GSTP1) knockdown model suggests role in pancreatic ductal adenocarcinoma (PDAC) cell metabolism

Abstract GSTP1 belongs to a large family of detoxification enzymes essential for cellular homeostasis in healthy cells. GSTP1 is overexpressed in many cancers and is associated with poor survival in PDAC patients. Studies in glioblastoma and breast cancer have suggested crucial roles of GST family members in metabolism through increased lactate dehydrogenase A and increased GAPDH activity, respectively. The mechanisms underlying the action of GSTP1 in PDAC metabolism and cell proliferation are not well studied. To begin characterizing the diverse functions of GSTP1 in cancer cells, we knocked down GSTP1 expression in three different PDAC cell lines. We revealed unique changes in the global transcriptomic, proteomic, and metabolomic signatures associated with GSTP1 knockdown compared to control cells through multi-omics techniques, real-time ATP production assays, and comprehensive hydrophilic metabolite and phospholipid panels. Through preliminary transcriptomic and proteomic analyses, we found 461 genes and 57 proteins differentially expressed in our GSTP1 knockdown MIA PaCa-2 cells. Among these, 41 were similarly upregulated or downregulated at the mRNA and protein levels. Pathway analysis of these changes revealed profound effects on energy production and lipid metabolism in GSTP1 knockdown cells. Hydrophilic metabolite and phospholipid panels and real-time ATP production assays supported our findings. Our results indicate that GSTP1 knockdown may directly influence mitochondrial dysfunction and lipid peroxidation pathways through associated reductions in ALDH7A1, GLUT3, and SLC7A11 levels. Continued research to further elucidate precise mechanisms and understand GSTP1’s role in driving cancer cell metabolism will aid in establishing GSTP1 targeting as a therapeutic opportunity for PDAC treatment. Citation Format: Jenna Duttenhefner, Katie M. Reindl. Multi-omics analysis of glutathione S-transferase P1 (GSTP1) knockdown model suggests role in pancreatic ductal adenocarcinoma (PDAC) cell metabolism [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B009.

Identification of Nrf2 Activators from the Roots of Valeriana officinalis.

Various age-related chronic diseases have been linked to oxidative stress. The cellular antioxidant response pathway is regulated by the transcription factor nuclear erythroid factor 2. Therefore, plant-derived nuclear erythroid factor 2 activators might be useful therapeutics to stimulate the body's defense mechanisms. Our study focused on the discovery of potent nuclear erythroid factor 2 activators from medicinal plants. Initially, a variety of medicinal plant extracts were screened for nuclear erythroid factor 2 activity using a nuclear erythroid factor 2 luciferase reporter cell line. Among these, Valerian (Valeriana officinalis) root was identified as a potent candidate. Sequential extraction and bioassay-guided fractionation led to the isolation of four nuclear erythroid factor 2-active compounds, which were structurally identified by NMR and LC/HRMS as the known compounds isovaltrate, valtrate, jatamanvaltrate-P, and valerenic acid. These four compounds were then tested in relevant biological assays. Firstly, their effects on the expression of glutathione S-transferase, glutamate-cysteine ligase catalytic subunit, glutathione peroxidase, and heme oxygenase 1 were determined in HepG2 cells. Glutathione S-transferase P1 and glutamate-cysteine ligase catalytic subunit were upregulated by isovaltrate, valtrate, and jatamanvaltrate-P, while heme oxygenase 1 was upregulated by isovaltrate, jatamanvaltrate-P, and valerenic acid. The four compounds also increased the levels of glutathione and its metabolite, CysGly. As glutathione aids in the detoxification of hydrogen peroxide, cytoprotective effects of these four nuclear erythroid factor 2 activators against hydrogen peroxide toxicity were investigated, and indeed, the compounds significantly improved cell survival. This study provides evidence that four valepotriates from the roots of V.officinalis are activators of nuclear erythroid factor 2-mediated antioxidant and detoxification pathways. Our data might expand the medical use of this plant beyond its current application as a sleep aid.

Ethyl Acetate Extract of Marine Algae, Halymenia durvillei, Provides Photoprotection against UV-Exposure in L929 and HaCaT Cells.

Halymenia durvillei is a red alga distributed along the coasts of Southeast Asian countries including Thailand. Previous studies have shown that an ethyl acetate fraction of H. durvillei (HDEA), containing major compounds including n-hexadecanoic acid, 2-butyl-5-hexyloctahydro-1H-indene, 3-(hydroxyacetyl) indole and indole-3-carboxylic acid, possesses high antioxidant and anti-lung cancer activities. The present study demonstrated that HDEA could protect mouse skin fibroblasts (L929) and human immortalized keratinocytes (HaCaT) against photoaging due to ultraviolet A and B (UVA and UVB) by reducing intracellular reactive oxygen species (ROS) and expressions of matrix metalloproteinases (MMP1 and MMP3), as well as increasing Nrf2 nuclear translocation, upregulations of mRNA transcripts of antioxidant enzymes, including superoxide dismutase (SOD), heme oxygenase (HMOX) and glutathione S-transferase pi1 (GSTP1), and procollagen synthesis. The results indicate that HDEA has the potential to protect skin cells from UV irradiation through the activation of the Nrf2 pathway, which leads to decreasing intracellular ROS and MMP production, along with the restoration of skin collagen.

Association of GSTP1 and PLCE1 gene polymorphisms with primary esophageal cancer

To assess the association of polymorphisms of glutathione S-transferase P1 (GSTP1) and phospholipase C epsilon-1 (PLCE1) genes with the susceptibility of primary esophageal cancer and their interaction with environmental factors. 162 patients with primary esophageal cancer and 162 healthy controls were recruited in this cross-sectional study. Basic information such as gender, age, history of smoking and alcohol consumption and family history of esophageal cancer were collected. Single nucleotide polymorphisms at A105G locus of GSTP1 gene and rs3765524, rs2274223 and rs3781264 loci of PLCE1 gene were detected. A logistic regression model was established to analyze the risk factors of esophageal cancer and the interaction among the factors. The proportions of individuals with smoking history, family history of esophageal cancer and hot diet in esophageal cancer group were higher than those in the control group (P<0.05). Conditional Logistic regression analysis showed that smoking, family history of esophageal cancer and GG genotype at the rs2274223 locus of PLCE1 gene were the risk factors for esophageal cancer (P<0.05), and AG/GG genotypes at the A105G locus of GSTP1 gene were the protective factors for esophageal cancer (P<0.05). In the two-factor interaction model, both AA genotype at A105G locus of GSTP1 gene and GG genotype at rs2274223 locus of PLCE1 gene had an interaction with smoking, and the risk of esophageal cancer has increased by 83.6% and 85.7%, respectively (P<0.05). AA genotype at A105G locus of GSTP1 gene, GG genotype at rs2274223 locus of PLCE1 gene and smoking constituted the best three-factor interaction model, and the risk of esophageal cancer has increased by 244.0% (P<0.05). Four-factor interaction model analysis showed that the risk of esophageal cancer among individuals with AA genotype at A105G locus of GSTP1 gene, GG genotype at rs2274223 locus of PLCE1 gene, smoking and family history of esophageal cancer has increased by 264.4% (P<0.05). The AG and GG genotypes at the A105G locus of GSTP1 gene are protective factors for esophageal cancer, and the GG genotype at rs2274223 locus of PLCE1 gene is a risk factor, both of them may interact with smoking and affect the susceptibility to esophageal cancer.

Activity-Based Proteomic Identification of the S-Thiolation Targets of Ajoene in MDA-MB-231 Breast Cancer Cells.

Garlic is a medicinal plant and spice that has been used for millennia for its health-promoting effects. These medicinal properties are associated with low molecular weight organosulfur compounds, produced following the crushing of garlic cloves. One of these compounds, ajoene, is proposed to act by S-thioallylating cysteine residues on target proteins whose identification in cancer cells holds great promise for understanding mechanistic aspects of ajoene's cancer cell cytotoxicity. To this end, an ajoene analogue (called biotin-ajoene, BA), containing a biotin affinity tag, was designed as an activity-based probe specific for the protein targets of ajoene in MDA-MB-231 breast cancer cells. BA was synthesized via a convergent "click" strategy and found to retain its cytotoxicity against MDA-MB-231 cells compared to ajoene. Widespread biotinylation of proteins was found to occur via disulfide bond formation in a dose-dependent manner, and the biotin-ajoene probe was found to share the same protein targets as its parent compound, ajoene. The biotinylated proteins were affinity-purified from the treated MDA-MB-231 cell lysate using streptavidin-coated magnetic beads followed by an on-bead reduction, alkylation, and digestion to liberate the peptide fragments, which were analyzed by liquid chromatography tandem mass chromatography. A total of 600 protein targets were identified, among which 91% overlapped with proteins with known protein cysteine modification (PCM) sites. The specific sites were enriched for those susceptible to S-glutathionylation (-SSG) (16%), S-sulfhydration (-SSH) (20%), S-sulfenylation (-SOH) (22%), and S-nitrosylation (-SNO) (31%). As target validation, both ajoene and a dansylated ajoene (DP) were found to S-thiolate the pure recombinant forms of glutathione S-transferase pi 1 (GSTP1) and protein disulfide isomerase (PDI), and the ajoene analogue DP was found to be a more potent inhibitor than 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). Pathway analysis elucidated that ajoene targets functional and signaling pathways that are implicated in cancer cell survival, specifically cellular processes, metabolism, and genetic information processing pathways. The results of this study provide mechanistic insights into the character of the anti-cancer activity of the natural dietary compound ajoene.

Prostate Imaging Reporting and Data System score (PI-RADS) and Glutathione S-transferase P1 methylation status (GST-P1) in the diagnosis of prostate cancer patients with borderline PSA values

Objectives. The objective of this study was to evaluate the potential use of Prostate Imaging – Reporting and Data System version 2 (PI-RADS) in combination with Glutathione S-transferase P1 (GST-P1) expression for an improved diagnosis of prostate cancer, in patients with inconclusive values of prostate-specific antigen (PSA). Materials and Methods. The study was conducted on 80 patients for whom PSA values were evaluated and were found to be inconclusive (4-10 ng/ml). These patients underwent imagistic evaluation (PI-RADS), followed by transurethral prostate biopsy, with the evaluation of GST-P1 expression and histopathological examination (for diagnosis confirmation). Results. By combining the results of PI-RADS and GST-P1 the capacity of the tests to correctly identify healthy subjects, with an area under curve of 0,832 (95% CI 0.732–0.907), with a sensitivity of 73,25% and a specificity of 77,78%. Conclusions. PI-RADS lesions and GST-P1 methylation testing when PSA levels are in a “grey zone”, provide a better specificity and sensitivity by comparison through single testing. Testing patients with inconclusive PSA levels allows for a more accurate diagnosis and less over-diagnosis by non-invasive procedures, such as repeated biopsies.

NBDHEX re-sensitizes adriamycin-resistant breast cancer by inhibiting glutathione S-transferase pi.

Adriamycin is a novel chemotherapeutic agent of great benefit for treating breast cancer. However, adriamycin -resistance remains a major obstacle. The vital Glutathione transferase P1 (GSTPi) inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX) has recently shown antitumor activity in various cancers. In this study, we analyzed the effect of NBDHEX and adriamycin combination against breast cancer in vitro and in vivo. CCK-8 assay was performed to test cell viability. The location and expression level of GSTpi was determined by immunofluorescence and Western blot in cells and immunohistochemistry staining in tissues. The enzyme activity test was applied to detect the effect of NBDHEX on the activity of GSTpi. The apoptosis related proteins' expression was tested using Western blot. The phosphorylation sites of GSTpi were detected by mass spectrometry. Antitumor effects of single treatment or co-administration of adriamycin and NBDHEX were evaluated in nude mice. NBDHEX treatment inhibited GSTpi enzyme activity and co-administration of adriamycin and NBDHEX promoted apoptosis of adriamycin-resistance breast cancer cell. Moreover, drug combination of NBDHEX and adriamycin significantly enhanced tumor growth inhibition compared with single agent. NBDHEX serves as a good candidate for combination with adriamycin, offering new insights for breast cancer treatment.

Glutathione S-transferase genetic polymorphisms and fluoride-induced reproductive toxicity in men with idiopathic infertility.

Male infertility caused by idiopathic oligoasthenospermia (OAT) is known as idiopathic male infertility. Glutathione S-transferase (GST) and fluoride may play important roles in idiopathic male infertility, but their effects are still unknown. Our study examined the relationship between GST polymorphisms and fluoride-induced toxicity in idiopathic male infertility and determined the underlying mechanism. Sperm, blood, and urine samples were collected from 560 males. Fluoride levels were measured by a highly selective electrode method, and GST genotypes were identified using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Semen parameters, DNA fragmentation index (DFI), mitochondrial membrane potential (MMP), and oxidative stress (OS) biomarkers were statistically assessed at the P < 0.05 level. Compared with healthy fertile group, semen parameters, fluoride levels, OS biomarkers, sex hormone levels, and MMP and DFI levels were lower in the idiopathic male infertility group. For glutathione S-transferase M1 (GSTM1[-]) and glutathione S-transferase T1 (GSTT1[-]) or glutathione S-transferase P1 (GSTP1) mutant genotypes, levels of semen fluoride, OS, MMP, and DFI were considerably higher, and the mean levels of sperm parameters and testosterone were statistically significant in GSTM1(+), GSTT1(+), and GSTP1 wild-type genotypes. Both semen and blood fluoride levels were associated with oxidative stress in idiopathic male infertility patients. Elevated fluoride in semen with the genotypes listed above was linked to reproductive quality in idiopathic male infertility patients. In conclusion, GST polymorphisms and fluorine may have an indicative relationship between reproductive quality and sex hormone levels, and OS participates in the development of idiopathic male infertility.

Sesquiterpene lactones from Elephantopus scaber exhibit cytotoxic effects on glioma cells by targeting GSTP1

Sesquiterpene lactones possess excellent anti-tumor activity in multiple cancer cell lines, including glioma, the most common type of malignant brain tumor with high mortality. However, the detailed mechanism of this type of constituent, especially the potential target for anti-glioma effect, is still unclear. Here, we collected 52 sesquiterpene lactones from Elephantopus scaber Linn. for network pharmacology analysis. The results demonstrated that the targets of the active components were markedly enriched on the pathways in cancer, which were closely related to cell proliferation regulation. Next, the Gene Expression Omnibus (GEO) and DisGeNET were analyzed by bioinformatics, and 429 glioma-related targets were obtained. Furtherly, 34 common targets of compounds and glioma were revealed, and they were significantly enriched in MAPK signaling pathway. Subsequently, we constructed a common target-compound network, and glutathione S-transferase Pi 1 (GSTP1) had the highest degree value, which explained its significance in the network. Therefore, we speculated that the compounds might exert an anti-glioma effect by targeting GSTP1. To verify the above results, we obtained part of sesquiterpene lactones isolated from E. scaber in our laboratory and evaluated their activities against glioma U87 cells. Among these sesquiterpene lactones (1–27), compounds 1 (elephantopinolide A), 2 (cis-scabertopin) and 3 (elephantopinolide F) exhibited the strongest inhibitory effect, and the IC50 values were 4.22 ± 0.14 µM, 4.28 ± 0.21 µM and 1.79 ± 0.24 µM, respectively. The results from molecular docking, cellular thermal shift assay (CETSA), as well as RT-PCR and Western blot analysis suggested that the compounds exerted an inhibitory effect by targeting GSTP1. Meanwhile, the compounds also activated JNK/STAT3 signaling pathway. Furthermore, we found that 1, 2 and 3 could suppress cell proliferation and also induce mitochondrial dysfunction as well as oxidative stress, eventually leading to cellular apoptosis. Taken together, this study revealed that sesquiterpene lactones from E. scaber could be a promising therapeutic strategy for the treatment of glioma by targeting GSTP1.

Association of glutathione s-transferase pi (GSTP1) gene polymorphism in unexplained infertile women

15–20 million people in India are affected by infertility.Among them, 34% of the couples are shown to have unexplained infertility (UEI). Both genetic and environmental factors influence UEI. Data from various studies show that oxidative stress plays an important role in unexplained infertility. The genes of the phase II detoxification enzyme, Glutathione s-transferase family are upregulated in humans as a defense mechanism opposing the adverse effects of oxidative stress and play important role during pregnancy. Since all investigations for infertility work-up are normal evaluating the causes for UEI will have impact on the treatment protocol. In this study the association between GSTP1 variations and unexplained infertility are discussed. To compare the association of GSTP1 polymorphism in women with unexplained infertility and a control group.: This is a case control study with 70 normal ovulatory women who conceived within 12 months of contraceptive free intercourse, and with no history of miscarriage were recruited in the control group and 70 women with unexplained infertility were recruited as study group. All participants included in the study were between 28 and 38 years of age. The association of GSTP1 polymorphism were studied using real-time PCR with the Light Cycler instrument (Roche Applied Science).: GSTP1 variant allele frequencies were 0.39 and 0.41for control and cases respectively. The results of this study indicate GSTP1 genetic polymorphisms did not show a significant association with unexplained infertile group.

Praeruptorin B inhibits osteoclastogenesis by targeting GSTP1 and impacting on the S-glutathionylation of IKKβ

Osteoporosis a common disease in postmenopausal women which contains significant impact on the living quality of women. With the aging of the population, the number of patients suffer from osteoporosis has shown a significant increase. Given the limitations of clinical drugs for the treatment of osteoporosis, natural extracts with small side effects have a great application prospect in the treatment of osteoporosis. Praeruptorin B (Pra-B), is one of the main components found in the roots of Peucedanum praeruptorum Dunn and exhibits anti-inflammatory effects. However, there is no research on the influence of Pra-B on osteoporosis. Here, we showed that Pra-B can dose-dependently suppress osteoclastogenesis without cytotoxicity. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL)-induced the nuclear import of P65 was inhibited by Pra-B, which indicated the suppressive effect of Pra-B on NF-κB signaling. Further, Pra-B enhanced the expression of Glutathione S-transferase Pi 1 (GSTP1) and promoted the S-glutathionylation of IKKβ to inhibit the nuclear translocation of P65. Moreover, in vivo experiments showed that Pra-B considerably attenuated the bone loss in ovariectomy (OVX)-induced mice. Collectively, our studies revealed that Pra-B suppress the NF-κB signaling targeting GSTP1 to rescued RANKL-induced osteoclastogenesis in vitro and OVX-induced bone loss in vivo, supporting the potential of Pra-B for treating osteoporosis in the future.

Identification of plasma proteins associated with oesophageal cancer chemotherapeutic treatment outcomes using SWATH-MS

Oesophageal adenocarcinoma (OAC) is an aggressive cancer with a five-year survival of <15%. Current chemotherapeutic strategies only benefit a minority (20‐30%) of patients and there are no methods available to differentiate between responders and non-responders. We performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on albumin/IgG-depleted and non-depleted plasma samples from 23 patients with locally advanced OAC prior to treatment. Individuals were grouped based on tumour regression (TRG) score (TRG1/2/3 vs TRG4/5) after chemotherapy, and differentially abundant proteins were compared. Protein depletion of highly abundant proteins led to the identification of around twice as many proteins. SWATH-MS revealed significant quantitative differences in the abundance of several proteins between the two groups. These included complement c1q subunit proteins, C1QA, C1QB and C1QC, which were of higher abundance in the low TRG group. Of those that were found to be of higher abundance in the high TRG group, glutathione S-transferase pi (GSTP1) exhibited the lowest p-value and highest classification accuracy and Cohen's kappa value. Concentrations of these proteins were further examined using ELISA-based assays. This study provides quantitative information relating to differences in the plasma proteome that underpin response to chemotherapeutic treatment in oesophageal cancers. SignificanceOesophageal cancers, including oesophageal adenocarcinoma (OAC) and oesophageal gastric junction cancer (OGJ), are one of the leading causes of cancer mortality worldwide. Curative therapy consists of surgery, either alone or in combination with adjuvant or neoadjuvant chemotherapy or radiation, or combination chemoradiotherapy regimens. There are currently no clinico-pathological means of predicting which patients will benefit from chemotherapeutic treatments. There is therefore an urgent need to improve oesophageal cancer disease management and treatment strategies. This work compared proteomic differences in OAC patients who responded well to chemotherapy as compared to those who did not, using quantitative proteomics prior to treatment commencement. SWATH-MS analysis of plasma (with and without albumin/IgG-depletion) from OAC patients prior to chemotherapy was performed. This approach was adopted to determine whether depletion offered a significant improvement in peptide coverage. Resultant datasets demonstrated that depletion increased peptide coverage significantly. Additionally, there was good quantitative agreement between commonly observed peptides. Data analysis was performed by adopting both univariate as well as multivariate analysis strategies. Differentially abundant proteins were identified between treatment response groups based on tumour regression grade. Such proteins included complement C1q sub-components and GSTP1. This study provides a platform for further work, utilising larger sample sets across different treatment regimens for oesophageal cancer, that will aid the development of ‘treatment response prediction assays’ for stratification of OAC patients prior to chemotherapy.

GSTP1 c.313A > G mutation is an independent risk factor for neutropenia hematotoxicity induced by anthracycline-/paclitaxel-based chemotherapy in breast cancer patients

BackgroundThe link between glutathione S-transferase P1 (GSTP1) c.313A > G polymorphism and chemotherapy-related adverse events remains controversial. The goal of this study was to assess how this variant affected the toxicity of anthracycline-/paclitaxel-based chemotherapy in patients with breast cancer.MethodsThis study retrospectively investigated pharmacogenetic associations of GSTP1 c.313A > G with chemotherapy-related adverse events in 142 breast cancer patients who received anthracycline and/or paclitaxel chemotherapy.ResultsThere were 61 (43.0%), 81 (57.0%), 43 (30.3%), and 99 (69.7%) patients in the T0-T2, T3-T4, N0-N1, and N2-N3 stages, respectively. There were 108 (76.1%) patients in clinical stages I–III and 34 (23.9%) patients in clinical stage IV. The numbers of patients with luminal A, luminal B, HER2 + , and triple-negative breast cancer (TNBC) were 10 (7.0%), 77 (54.2%), 33 (23.2%), and 22 (15.5%), respectively. The numbers of patients who carried GSTP1 c.313A > G A/A, A/G, and G/G genotypes were 94 (66.2%), 45 (31.7%), and 3 (2.1%), respectively. There were no statistically significant differences in the proportion of certain toxicities in patients with A/G, G/G, and A/G + G/G genotypes, except for neutropenia, in which the proportion of patients with A/G + G/G (χ2 = 6.586, P = 0.035) genotypes was significantly higher than that with the AA genotype. The logistic regression analysis indicated that GSTP1 c.313A > G mutation (A/G + G/G vs. A/A genotype) (adjusted OR 4.273, 95% CI 1.141–16.000, P = 0.031) was an independent variable associated with neutropenia.ConclusionsThe findings of this study indicate that the GSTP1 c.313A > G mutation is an independent risk factor for neutropenia hematotoxicity in breast cancer patients induced by anthracycline-/paclitaxel-based chemotherapy.

Abstract 3734: Progressive spreading of CpG methylation in the CpG island of Glutathione S-Transferase pi 1 (GSTP1) alleles across transitions from precursor to invasive prostate cancer

Abstract Glutathione S-transferase pi 1 (GSTP1) is expressed at low levels in normal prostate luminal cells and becomes induced in most proliferative inflammatory atrophy lesions (PIA). GSTP1 becomes silenced in prostatic intraepithelial neoplasia (PIN) and prostate adenocarcinoma (CaP) via CpG island promoter hypermethylation. However, the specific methylation patterns in CaP and its precursors have not been investigated. We used bisulfite genomic sequencing to examine methylation of 39 CpG sites in the GSTP1 promoter. Radical prostatectomy specimens were selected from 32 subjects that underwent treatment for CaP. Serial sections were subjected to laser capture microdissection for enrichment of epithelial cells from benign, PIA, PIN, and CaP regions. Isolated DNA was bisulfite converted. Sequences corresponding to the GSTP1 promoter CpG island were amplified with nested PCR, and PCR products were cloned. 10 independent clones were sequenced and analyzed with a Java program, DNAMethylMap, to determine the methylation pattern of 39 CpG sites of the GSTP1 promoter in each lesion. The extent of methylation on an individual allele was classified as negative (&amp;lt;10% CpGs), mild (10-25%), moderate (25-50%), or high (&amp;gt;50%). 212 clones from 24 normal epithelium regions, 327 clones from 37 PIA regions, 167 clones from 18 PIN regions, and 202 clones from 23 CaP regions were sequenced, totaling 34863 CpG sites. Normal and PIA lesions were mostly unmethylated with 0.52% and 1.3% of total CpG sites methylated, respectively. Methylated PIA lesions were adjacent to CaP or PIN regions significantly more often than unmethylated PIA lesions (χ2, p = 0.012). PIN and CaP lesions had greater methylation with 24% and 51% of total CpG sites methylated, respectively. PIN lesions generally showed partial methylation with 28.7% of alleles in PIN having mild/moderate methylation density compared to 5.5% in PIA and 11% in CaP. PIA and PIN lesions were enriched for methylation changes at 6 CpG sites that aligned with AP1 and SP1 binding sites. Among 16 PIN and CaP lesions with an overall intermediate level of methylation, 5 of the lesions had clones that clustered into either fully methylated or unmethylated. One patient with 3 CaP lesions displayed significant heterogeneity in methylation patterns: full, moderate, or no methylation across all clones in a given lesion. The results demonstrate that methylation density in the GSTP1 CpG island from PIN was intermediate between normal prostate epithelium/PIA and CaP lesions. The observed methylation in PIA and PIN was enriched at binding sites of key transcription factors, AP1 and SP1. The results are consistent with gradual spreading of DNA methylation centered at the transcription factor binding sites in the putative precursor lesions, with subsequent spreading of methylation across the entire CpG island in transition to CaP. Citation Format: Harshath Gupta, Hitoshi Inoue, Yasutomo Nakai, Masashi Nakayama, William G. Nelson, Angelo M. De Marzo, Srinivasan Yegnasubramanian. Progressive spreading of CpG methylation in the CpG island of Glutathione S-Transferase pi 1 (GSTP1) alleles across transitions from precursor to invasive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3734.

Automated approach for the evaluation of glutathione-S-transferase P1-1 inhibition by organometallic anticancer compounds

A novel automated method based on sequential injection analysis (SIA), a non-segmented flow injection technique, was developed to evaluate glutathione S-transferase P1-1 (GST P1-1) activity in the presence of organometallic complexes with putative anticancer activity. The assay is based on the reaction of L-glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) in the presence of GST P1-1 to afford the GS-DNB conjugate and the reaction may be monitored by an increase in absorbance at 340 nm. A series of ruthenium, iron, osmium and iridium complexes were evaluated as GST P1-1 inhibitors by evaluating their half-maximal inhibitory concentration (IC50). An iridium compound displays the lowest IC50 value of 6.7 ± 0.7 µM and an iron compound displays the highest IC50 value of 275 ± 9 µM. The SIA method is simple to use, robust, reliable, and efficient and uses fewer reagents than batch methods and each analysis takes only 5 minutes.

Target molecular-based neuroactivity screening of Panax ginseng through network pharmacology integrated molecular docking

Panax ginseng is a medicinal herb that has therapeutic effects against neuronal damage. The main goal of the present study is to investigate the therapeutic potential of P. ginseng in treating neuronal damage through in silico analysis. The literature review was conducted to create a ingredients-target database for P. ginseng. The database was essential in identifying the targets related to neuronal damage, which was followed by the construction of protein-protein interaction network by the STITCH database. The enrichment analysis was performed using Cytoscape (ClueGO) software. The molecular docking analysis was ran using AutoDock and Discovery Studio. The literature search revealed the presence of 66 compounds in P. ginseng, which subsequently resulted in the retrieval of 102 potential targets of these compounds. They were screened for their association with neuronal damage. Three proteins, namely, Glutathione S-transferase P1, HSP90, and Mitogen-activated protein kinases (MAPK8), were found to be in prime interaction with the relative compounds. Subsequently, 91 Gene ontology terms were found, of which four genes were associated with neuronal damage. Molecular docking analysis also revealed good binding affinities of various compounds of P. ginseng to the selected target, MAPK8. In conclusion, the biological processes linked to the therapeutic action of P. ginseng involve the positive regulation of JUN kinase activity, chaperone-mediated protein folding, and MyD88-dependent toll-like receptor signaling pathway.

Melatonin modulates Nrf2 activity to protect porcine pre-pubertal Sertoli cells from the abnormal H2 O2 generation and reductive stress effects of cadmium.

Melatonin (MLT) is a cytoprotective agent holding potential to prevent cadmium (Cd) toxicity and its impact in testicular function and fertility. In this study, we explored such potential in porcine pre‐pubertal Sertoli cells (SCs). Cd toxicity resulted in impaired SC viability and function, abnormal cellular H2O2 generation and efflux, and induction of reductive stress by the upregulation of Nrf2 expression and activity, cystine uptake and glutathione biosynthesis, glutathione‐S‐transferase P (GSTP) expression, and protein glutathionylation inhibition. Cd toxicity also stimulated the activity of cellular kinases (MAPK‐ERK1/2 and Akt) and NFkB transcription factor, and cJun expression was increased. MLT produced a potent cytoprotective effect when co‐administered with Cd to SCs; its efficacy and the molecular mechanism behind its cytoprotective function varied according to Cd concentrations. However, a significant restoration of cell viability and function, and of H2O2 levels, was observed both at 5 and 10 μM Cd. Mechanistically, these effects of MLT were associated with a significant reduction of the Cd‐induced activation of Nrf2 and GSTP expression at all Cd concentrations. CAT and MAPK‐ERK1/2 activity upregulation was associated with these effects at 5 μM Cd, whereas glutathione biosynthesis and efflux were involved at 10 μM Cd together with an increased expression of the cystine transporter xCT, of cJun and Akt and NFkB activity. MLT protects SCs from Cd toxicity reducing its H2O2 generation and reductive stress effects. A reduced activity of Nrf2 and the modulation of other molecular players of MLT signaling, provide a mechanistic rational for the cytoprotective effect of this molecule in SCs.

Biomarkers of Myocardial Injury and Remodeling in Heart Failure.

With its complicated pathophysiology, high incidence and prevalence, heart failure remains a major public concern. In hopes of improving diagnosis, treatment and prognosis, the utility of many different biomarkers is researched vigorously around the world. In this review, biomarkers of myocardial remodeling and fibrosis (galectin-3, soluble isoform of suppression of tumorigenicity 2, matrix metalloproteinases, osteopontin, interleukin-6, syndecan-4, myostatin, procollagen type I C-terminal propeptide, procollagen type III N-terminal propeptide, vascular endothelial growth factor, nitric oxidase synthetase and asymmetric dimethylarginine), myocyte injury (heart-type fatty acid-binding protein, glutathione S-transferase P1 and heat shock protein 60), as well as iron metabolism (ferritin, transferrin saturation, soluble transferrin receptor and hepcidin), are considered in terms of possible clinical applicability and significance. Our short review consists of a summary of the aforementioned cardiovascular biomarkers’ clinical relevance and perspectives.

Abstract 489: Glutathione Regulates Endothelial Cell Function And Vegfr2 Signaling

Under physiological and pathological conditions angiogenesis progresses in an idealized redox window. Mice heterozygous for the Glutamate cysteine ligase modifier (GCLm) subunit knockout have augmented ischemic remodeling. During occlusion, the ensuing ischemic milieu and accompanying vascular changes regulate the activity of vascular endothelial receptor 2 (VEGFR2). We hypothesized that the cellular GSH concentration regulates endothelial cell function via altered VEGFR2 signaling. To test this hypothesis, we first performed mouse aortic endothelial cell (MAEC) outgrowth assays on aortic segments dissected from mice homozygous (KO) and heterozygous (Het) for GCLm knockout as well as wildtype control (WT). Outgrowth area in the Het group was significantly higher than WT and KO at day 7 of outgrowth (2.15 fold, n=4, P&lt;0.001). MAECs were isolated and relative migration rates were determined using under-agarose migration assays. Het MAECs were 1.25 fold faster than WT (n=4 p&lt;0.001) while KO MAECs were slower, 0.90 fold (n=3, p&lt;0.05). Cell proliferation assays are currently underway. Concurrently, we inhibited glutathione reductase in primary human aortic endothelial cells using 2-AAPA. 2-AAPA (75 μm) alone didn’t activate VEGFR2 (determined by western blot for pY1175 VEGFR2). 2-AAPA in combination with H 2 O 2 (100 μM) induced a 5.5 fold (p&lt;0.01, n=4) increase in the relative amount of active VEGFR2 compared to vehicle treated HAECs. 2-AAPA with H 2 O 2 induced a significant increase in pY416 proto-oncogene c-Src (Src) 9.5 fold (p&lt;0.01, n=6), This effect was attenuated by the addition of DTT. Furthermore, Src kinase inhibitor 1 (SKI) (200 nM) attenuated the effect of 2-AAPA + H 2 O 2 on VEGFR2. While the VEGFR2 kinase inhibitor SU1948 inhibited activation of VEGFR2, it had no significant effect on the 2-AAPA + H 2 O 2 mediated activation of Src. Combined these data suggest Src mediates the 2-AAPA + H 2 O 2 activation of VEGFR2. Glutathione S-transferase P1 (GSTP1) catalyzes the enzymatic glutathionylation of proteins, The GSTP1 inhibitor TLK199 (50 μM) abrogated the effects of 2-AAPA + H 2 O 2 on VEGFR2 and experiments are underway to determine the glutathionylation status of Src in our system and its effect on the interaction of Src and VEGFR2.