Glutathione S-transferase Pi Research Articles

Peroxiredoxin-1 as a molecular chaperone that regulates glutathione S-transferase P1 activity and drives mutidrug resistance in ovarian cancer cells

Ovarian cancer is among the most prevalent gynecological malignancies around the globe. Nonetheless, chemoresistance continues to be one of the greatest obstacles in the treatment of ovarian cancer. Therefore, understanding the mechanisms of chemoresistance and identifying new treatment options for ovarian cancer patients is urgently required. In this study, we found that the mRNA and protein expression levels of PRDX1 were significantly increased in cisplatin resistant A2780/CDDP cells. Cell survival assays revealed that PRDX1 depletion substantially increased ovarian cancer cell sensitivity to cisplatin, docetaxel, and doxorubicin. Additionally, PRDX1 significantly increased GSTP1 activity, resulting in multidrug resistance. Biochemical experiments showed that PRDX1 interacted with GSTP1 through Cysteine 83, which regulated GSTP1 activity as well as chemotherapy resistance in ovarian cancer cells. Our findings indicate that the molecular chaperone activity of PRDX1 is a promising new therapeutic target for ovarian cancer.

Glutathione transferase P1 is modified by palmitate.

Glutathione transferase P1 (GSTP1) is a multi-functional protein that protects cells from electrophiles by catalyzing their conjugation with glutathione, and contributes to the regulation of cell proliferation, apoptosis, and signalling. GSTP1, usually described as a cytosolic enzyme, can localize to other cell compartments and we have reported its strong association with the plasma membrane. In the current study, the hypothesis that GSTP1 is palmitoylated and this modification facilitates its dynamic localization and function was investigated. Palmitoylation is the reversible post-translational addition of a 16-C saturated fatty acid to proteins, most commonly on Cys residues through a thioester bond. GSTP1 in MCF7 cells was modified by palmitate, however, GSTP1 Cys to Ser mutants (individual and Cys-less) retained palmitoylation. Treatment of palmitoylated GSTP1 with 0.1 N NaOH, which cleaves ester bonds, did not remove palmitate. Purified GSTP1 was spontaneously palmitoylated in vitro and peptide sequencing revealed that Cys48 and Cys102 undergo S-palmitoylation, while Lys103 undergoes the rare N-palmitoylation. N-palmitoylation occurs via a stable NaOH-resistant amide bond. Analysis of subcellular fractions of MCF7-GSTP1 cells and a modified proximity ligation assay revealed that palmitoylated GSTP1 was present not only in the membrane fraction but also in the cytosol. GSTP1 isolated from E. coli, and MCF7 cells (grown under fatty acid free or regular conditions), associated with plasma membrane-enriched fractions and this association was not altered by palmitoyl CoA. Overall, GSTP1 is modified by palmitate, at multiple sites, including at least one non-Cys residue. These modifications could contribute to regulating the diverse functions of GSTP1.

Kindlin-2 maintains liver homeostasis by regulating GSTP1–OPN-mediated oxidative stress and inflammation in mice

Hepatocyte plays a principal role in preserving integrity of the liver homeostasis. Our recent study demonstrated that Kindlin-2, a focal adhesion protein that activates integrins and regulates cell-extracellular matrix interactions, plays an important role in regulation of liver homeostasis by inhibiting inflammation pathway; however, the molecular mechanism of how Kindlin-2 knockout activates inflammation is unknown. Here we show that Kindlin-2 loss largely downregulates the antioxidant glutathione S-transferase P 1 (GSTP1) in hepatocytes by promoting its ubiquitination and degradation via a mechanism involving protein-protein interaction. This causes overproduction of intracellular reactive oxygen species (ROS) and excessive oxidative stress in hepatocytes. Kindlin-2 loss upregulates osteopontin in hepatocytes partially due to upregulation of ROS and consequently stimulates overproduction of inflammatory cytokines and infiltration in liver. The molecular and histological deteriorations caused by Kindlin-2 deficiency are markedly reversed by systemic administration of an antioxidant N-acetylcysteine in mice. Taken together, Kindlin-2 plays a pivotal role in preserving integrity of liver function.

Abstract PR013: First-in-human dose-expansion study of NBF-006, a novel investigational siRNA targeting GSTP, in patients with KRAS-mutated non-small cell lung cancer

Abstract Background Glutathione S-transferase Pi (GSTP) has an important role in detoxification and anti-oxidative damage response. Additionally, GSTP has a chaperone function which regulates key oncogenic pathways such as the KRAS and JNK. Since alternative or redundant pathways could compensate for the inhibition of a single kinase, targeting multiple pathways could lead to more effective therapy. NBF-006 is a novel drug product comprised of GSTP siRNA encapsulated within a proprietary lipid nanoparticle. It is designed to deliver siRNA to localized and metastatic lung tumors. As GSTP downregulation leads to inhibition of KRAS signaling through modulation of both MAPK and PI3K pathways, NBF-006 is being developed to treat KRAS mutant tumors. NBF-006-001 is a first-in-man dose escalation (Part A) and expansion (Part B) study evaluating safety, tolerability, PK, and preliminary efficacy of NBF-006 administered intravenously over 70 minutes, QW4 followed by a 2-week rest period. Part A of the study enrolled 21 patients with advanced non-small cell lung cancer (NSCLC), pancreatic, or colorectal cancer. No DLTs or treatment-related SAEs were observed. NSCLC disease control rate of 55% (6/11) was observed in heavily pre-treated patient population, warranting further investigations. Results from advanced KRAS-mutated NSCLC patients enrolled in NBF-006-001 are reported here. Results Thirty-four KRAS-mutated NSCLC patients received treatment at 0.6 mg/kg [n=12], 1.2 mg/kg [n=11], or 1.6 mg/kg [n=11]. NBF-006 was well tolerated with no treatment-related grade 4-5 AEs, SAEs, or DLTs. Two grade 3 events were assessed as possibly related to study treatment, anemia and bilateral pleural effusions (both at 0.6 mg/kg). The most common (>10 % of patients) NBF-006-related AEs were Grade 1 or 2 infusion related reactions (low grade IRRs) (20.6 %) and nausea (11.8 %). There was no dose relationship in the number of AEs or any observed AEs. Low grade IRRs (n=8 in seven patients), occurred in the first cycle of therapy and were generally well-managed. Only one patient discontinued study therapy due to recurring IRR. Substantial cytokine elevation was only noted in this patient. No clinically meaningful complement activation nor ADA response has been observed. PK analysis indicated a mean terminal half-life of 41 (± 13) hours with no accumulation. Cmax and AUC demonstrated dose-proportionality up to 1.2 mg/kg and no apparent increase in exposure at 1.6 mg/kg. Patients had received 1-7 prior lines of therapy (average 2.6; 1 line [n=10], 2 lines [n=9], and ≥ 3 lines [n=15] respectively). Two patients (treated at 1.2 and 1.6 mg/kg) experienced a durable PR, 14 patients experienced SD. One patient with PR and one patient with a mixed response stayed on study for >12 months. Conclusion The expansion part of the NBF-006-001 study confirmed the favorable safety profile of NBF-006 and demonstrated early signs of antitumor activity in KRAS-mutated NSCLC. The results suggest that further clinical development of NBF-006, alone or in combination with standard therapy, is warranted. Citation Format: Hirva Mamdani, Alberto Chiappori, Lyudmila Bazhenova, Alexander Spira, Wade Iams, Minal Barve, Anthony W Tolcher, Eli Gabayan, Andrae Vandross, Annie Huynh, Yael Cohen-Arazi, Zachary Albaugh, Joachim Gullbo, Sonya Zabludoff. First-in-human dose-expansion study of NBF-006, a novel investigational siRNA targeting GSTP, in patients with KRAS-mutated non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR013.

Abstract C103: Preclinical and clinical safety and tolerability evaluation of NBF-006, a novel siRNA inhibitor of glutathione-s-transferase P (GSTP) encapsulated in a lipid nanoparticle (LNP) for treatment of advanced non-small cell lung cancer (NSCLC)

Abstract Glutathione S-transferase Pi (GSTP) has an important role in detoxification and anti-oxidative damage responses. Additionally, GSTP has a chaperone function that regulates key oncogenic pathways such as the KRAS and JNK pathways (E Laborde, 2010). Since alternative or redundant pathways could compensate for the incomplete inhibition of a single kinase, targeting multiple pathways could lead to more effective therapy. NBF-006 is a novel drug product containing a GSTP siRNA encapsulated within a proprietary lipid nanoparticle (LNP). It has been designed to deliver siRNA to tumors of the lung and common sites of metastatic spread (liver and marrow). As GSTP downregulation leads to inhibition of KRAS signaling through modulation of both MAPK and PI3K pathways, NBF-006 is being developed to treat KRAS mutant tumors. Preclinical studies have demonstrated activity across KRAS mutations, as well as efficacy in orthotopic non-small cell lung cancer (NSCLC) models. Immune toxicities have been recognized as safety concerns associated with LNP therapeutics. The safety assessment of NBF-006 was conducted both preclinically and clinically. In vitro preclinical toxicity studies included hemolysis in hRBC samples, cytokine release in hPBMCs, and TLR activation in human THP1 cells. GLP-compliant general toxicity studies were conducted in both mice and cynomolgus monkeys, IV administration, once a week, for 5 weeks. Complement splits were measured in the monkey study to assess any immune-related responses. Safety pharmacology parameters were also conducted in monkeys. The mouse was slightly more sensitive than monkeys to NBF-006, and the highest non-seriously toxic dose level in mice was 20 mg/kg/week, which yielded a good safety margin to proceed for a Phase I clinical trial. The highest dose level tested in monkeys was of 18 mg/kg/week and was well tolerated by the monkeys. Immunological assessments were conducted during Phase 1 dose escalation and expansion in patients with NSCLC, pancreatic cancer, or colorectal cancer. NBF-006 was administered by IV infusion over 70 minutes, once a week, for 4 weeks followed by a 2-week rest period. Clinical symptoms and reactions were monitored, and lab tests were conducted according to the protocol, including CBC, complement split products (Bb, C3a, C5a, and CH50), cytokines (IL-6, IL-1β, IFNg, and TNFα), and anti-drug antibodies against the LNPs. Dose-dependent increases in C3a and Bb of 1.2 and 1.6 mg/kg were observed following NBF-006 administration at doses ranging from 0.15 to 1.6 mg/kg. The increases were transient at either 10 minutes or 1 hour after the end of the infusion. Due to lack of corresponding increases in C5a or decreases in CH50, transient elevations of C3a and Bb were not considered clinically significant. In summary, NBF-006 was well tolerated in both animal species and patients: 20 mg/kg/week was the non-seriously toxic dose level in mice; the highest dose level of 18 mg/kg/week tested in monkeys was the NOAEL; and 1.6 mg/kg/week was the highest dose level evaluated in humans. Citation Format: Jian Liu, Cima Cina, Douglas Kornbrust, Sonya Zabludoff. Preclinical and clinical safety and tolerability evaluation of NBF-006, a novel siRNA inhibitor of glutathione-s-transferase P (GSTP) encapsulated in a lipid nanoparticle (LNP) for treatment of advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C103.

Abstract C101: Preclinical and clinical pharmacokinetics (PK) of NBF-006, a novel siRNA inhibitor of glutathione-s-transferase P (GSTP) encapsulated in a lipid nanoparticle (LNP), for treatment of advanced non-small cell lung cancer (NSCLC)

Abstract Background: GSTP has an important role in detoxification and anti-oxidative damage response. Additionally, GSTP has a chaperone function that regulates multiple key oncogenic pathways such as KRAS and JNK. Since alternative or redundant pathways could compensate for the inhibition of a single kinase, targeting multiple pathways could lead to more effective therapy. NBF-006 is a drug product comprised of GSTP siRNA encapsulated within a novel LNP. It is designed to deliver siRNA to localized and metastatic lung tumors. Unlike other well-established drug modalities, there is a general need for increased understanding of the PK of RNA therapeutics. For NBF-006, we present preclinical PK data in healthy mice and monkeys, prediction of human doses, and phase 1 clinical PK data. Methods: Preclinical PK studies were performed by intravenous bolus injection of NBF-006 once weekly for 1 month at 5, 20, and 40 mg/kg in mice and 6, 12, and 18 mg/kg in cynomolgus monkeys. Human drug exposures were predicted based upon simple allometry. For the phase 1 clinical study, a total of 48 advanced NSCLC, pancreatic, or colorectal cancer patients received 4 weekly doses of NBF-006 by infusion at 0.15, 0.3, 0.6, 1.2 or 1.6 mg/kg followed by a 2-week rest period. In all studies, serial blood samples were collected at weeks 1 and 4. Plasma concentrations of siRNA were determined by a hybridization ELISA. Non-compartmental PK analysis (NCA) was performed with the resulting concentration-time data. Results: In mice, terminal elimination half-life (t1/2) of plasma siRNA ranged between 3.69 to 9.04 hr, clearance (CL) ranged between 3.61 and 5.81 mL/hr/kg, and volume of distribution (Vd) ranged between 23.5 and 57.4 mL/kg. In cynomolgus monkeys, t1/2 ranged between 8.22 and 28.2 hr, CL ranged between 1.38 and 3.97 mL/hr/kg, and Vd ranged between 31.7 to 83.3 mL/kg. Exposure (maximal plasma concentration, Cmax, and area under the curve, AUC) increased with increasing dose in both species and was generally dose proportional. Following weekly administration of NBF-006, no apparent accumulation or reduction of siRNA exposure was observed. Human PK exposures were well-predicted within 2-fold accuracy at clinically studied doses. Clinically, NCA demonstrated consistent slow CL and low Vd with a mean t1/2 of 41 (± 13) hours. Exposure showed dose proportionality from 0.15 to 1.2 mg/kg. At 1.6 mg/kg, exposure was not statistically different than that of 1.2 mg/kg. No accumulation risk potential was identified following the current dosing regimen. Moreover, exposure was not reduced after repeat dosing in any dose group. Conclusions: Preclinical PK profiling enabled initial characterization of the in vivo disposition of NBF-006 and prediction of human PK and doses. In patients, repeated administration of NBF-006 was well-tolerated up to 1.6 mg/kg. Exposure appeared to be dose proportional through 1.2 mg/kg. Neither exposure reduction nor accumulation was observed. Citation Format: Michael Hall, Zhihong O'Brien, Sonya Zabludoff. Preclinical and clinical pharmacokinetics (PK) of NBF-006, a novel siRNA inhibitor of glutathione-s-transferase P (GSTP) encapsulated in a lipid nanoparticle (LNP), for treatment of advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C101.

Gene methylation status in focus of advanced prostate cancer diagnostics and improved individual outcomes.

Prostate cancer (PCa) is the most prevalent type of male genitourinary tumor, remains the second leading cause of deaths due to cancer in the United States in men. The aim of this study was to perform an integrative epigenetic analysis to explore the epigenetic abnormalities involved in the development and progression of PCa, and present advanced diagnostics and improved individual outcomes. Genome-wide DNA methylation profiles obtained from The Cancer Genome Atlas (TCGA) were analyzed and a diagnostic model was constructed. For validation, we employed profiles from the Gene Expression Omnibus (GEO) and methylation data derived from clinical samples. Gene set enrichment analysis (GSEA) and the Tumor Immune Estimation Resource (TIMER) were employed for GSEA and to assess immune cell infiltration, respectively. An accurate diagnostic method for PCa was established based on the methylation level of Cyclin-D2 (CCND2) and glutathione S-transferase pi-1 (GSTP1), with an impressive area under the curve (AUC) value of 0.937. The model's reliability was further confirmed through validation using four GEO datasets GSE76938 (AUC =0.930), GSE26126 (AUC =0.906), GSE112047 (AUC =1.000), GSE84749 (AUC =0.938) and clinical samples (AUC =0.980). Notably, the TIMER analysis indicated that hypermethylation of CCND2 and GSTP1 was associated with reduced immune cell infiltration, higher tumor purity, and an increased risk of tumor progression. In conclusion, our study provides a robust and reliable methylation-based diagnostic model for PCa. This model holds promise as an improved approach for screening and diagnosing PCa, potentially enhancing early detection and patient outcomes, as well as for an advanced clinical management for PCa in the framework of predictive, preventive and personalised medicine.

Abstract C108: Enhanced growth inhibition in lung cancer cells through NBF-006 combination therapy with chemotherapy or KRAS inhibitors

Abstract Background: Glutathione-S-transferase P (GSTP) is strongly up-regulated in many cancer types, specifically KRAS-mutant lung and is also a known regulator of oncogenic MAPK and PI3K pathways. In cancer cells, overexpression of GSTP regulates apoptosis by inhibiting Jun-p53 complex in JNK pathway. NBF-006 is a novel lyophilized lipid nanoparticle formulation, encapsulating a small interfering ribonucleic acid (siRNA) designed to inhibit the expression of GSTP in lung tumors. NBF-006 has been very well tolerated with early signs of efficacy in NSCLC patients in a phase 1 dose escalation and expansion study. As NBF-006 is well tolerated and unlikely to have drug-drug interactions, combination strategies are currently being developed. Due to the detoxifying role of GSTP, combination with chemo agents used to treat NSCLC may increase the sensitivity of cancer cells resulting in apoptosis and/or reduced resistance. As GSTP and KRAS enhance tumor growth and survival, inhibition of GSTP and KRAS may be synergistic. Methods: Anti-tumor activity of GSTP siRNA (NDT-05-1040), when combined with chemo agents, KRAS inhibitors or immune cells was assessed in a panel of KRAS mutant NSCLC lines. For chemo agents and KRAS inhibitors, NDT-05-1040 combinations was evaluated using Combenefit software and Loewe model. Results: Combination of NDT-05-1040 with chemotherapy drugs including paclitaxel, gemcitabine, cisplatin, and pemetrexed controlled tumor cell growth significantly better than either NBF-006 alone or the chemo treatment alone. In A549, H1373, H358, and H1792 lines, at low doses paclitaxel combined with NDT-05-1040 and resulted in synergistic cell killing (Loewe scores >20). Gemcitabine & Cisplatin combination with NDT-05-1040 in H1373 cells further improved cell killing by 5-fold (Loewe scores >20). Pemetrexed combined with NDT-05-1040 and led to additive cell killing in A549 (Loewe score >10). Combination of NDT-05-1040 with KRASG12C inhibitors, Sotorasib & Adagrasib were conducted in H358, H1222, and MiaPaca-2. Cell viability assays demonstrated that the combination resulted in strong synergistic, anti-proliferative effects at low doses in H358 cells (the greatest Loewe scores for Adagrasib and Sotorasib: 53 & 26, respectively). Conclusion: Results from the current studies suggest that NBF-006, a very well tolerated monotherapy, can be used in combination with other drugs currently indicated for KRAS-mutant NSCLC to achieve more potent antitumor effects. Citation Format: Cima Cina, Akinori Sugiyama, Murali Kuracha, Nazia Abbasi, Dominic Medina, Hiroyuki Tanaka, Sonya Zabludoff. Enhanced growth inhibition in lung cancer cells through NBF-006 combination therapy with chemotherapy or KRAS inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C108.

Adaptive changes in tumor cells in response to reductive stress

Reductive stress is characterized by an excess of cellular electron donors and can be linked with various human pathologies including cancer. We developed melanoma cell lines resistant to reductive stress agents: rotenone (ROTR), n-acetyl-L-cysteine, (NACR), or dithiothreitol (DTTR). Resistant cells divided more rapidly and had intracellular homeostatic redox-couple ratios that were shifted towards the reduced state. Resistance caused alterations in general cell morphology, but only ROTR cells had significant changes in mitochondrial morphology with higher numbers that were more isolated, fragmented and swollen, with greater membrane depolarization and decreased numbers of networks. These changes were accompanied by lower basal oxygen consumption and maximal respiration rates. Whole cell flux analyses and mitochondrial function assays showed that NACR and DTTR preferentially utilized tricarboxylic acid (TCA) cycle intermediates, while ROTR used ketone body substrates such as D, L-β-hydroxybutyric acid. NACR and DTTR cells had constitutively decreased levels of reactive oxygen species (ROS), although this was accompanied by activation of nuclear factor erythroid 2–related factor 2 (Nrf2), with concomitant increased expression of the downstream gene products such as glutathione S-transferase P (GSTP). Further adaptations included enhanced expression of endoplasmic reticulum proteins controlling the unfolded protein response (UPR). Although expression patterns of these UPR proteins were distinct between the resistant cells, a trend implied that resistance to reductive stress is accompanied by a constitutively increased UPR phenotype in each line. Overall, tumor cells, although tolerant of oxidative stress, can adapt their energy and survival mechanisms in lethal reductive stress conditions.

Piperlongumine based nanomedicine impairs glycolytic metabolism in triple negative breast cancer stem cells through modulation of GAPDH & FBP1

BackgroundTriple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and exhibits high rate of chemoresistance, metastasis, and relapse. This can be attributed to the failure of conventional therapeutics to target a sub-population of slow cycling or quiescent cells called as cancer stem cells (CSCs). Therefore, elimination of CSCs is essential for effective TNBC treatment. PurposeResearch suggests that breast CSCs exhibit elevated glycolytic metabolism which directly contributes in maintenance of stemness, self-renewability and chemoresistance as well as in tumor progression. Therefore, this study aimed to target rewired metabolism which can serve as Achilles heel for CSCs population and have far reaching effect in TNBC treatment. MethodsWe used two preclinical models, zebrafish and nude mice to evaluate the fate of nanoparticles as well as the therapeutic efficacy of both piperlongumine (PL) and its nanomedicine (PL-NPs). ResultsIn this context, we explored a phytochemical piperlongumine (PL) which has potent anti-cancer properties but poor pharmacokinetics impedes its clinical translation. So, we developed PLGA based nanomedicine for PL (PL-NPs), and demonstrated that it overcomes the pharmacokinetic limitations of PL, along with imparting advantages of selective tumor targeting through Enhanced Permeability and Retention (EPR) effect in zebrafish xenograft model. Further, we demonstrated that PL-NPs efficiently inhibit glycolysis in CSCs through inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by modulating glutathione S-transferase pi 1 (GSTP1) and upregulation of fructose-1,6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis. We also illustrated that inhibition of glycolysis results in overall tumor regression in two preclinical models. ConclusionThis study discusses novel mechanism of action by which PL acts on CSCSs. Taken together our study provides insight into development of PL based nanomedicine which could be exploited in clinics to achieve complete eradication of TNBC by targeting CSCs.

Inhibition of human glutathione transferase by catechin and gossypol: comparative structural analysis by kinetic properties, molecular docking and their efficacy on the viability of human MCF-7 cells.

Glutathione transferase Pi (GSTP1) expression is increased in many cancer types and is associated with multidrug resistance and apoptosis inhibition. Inhibitors of GSTP1-1 have the potential to overcome drug resistance and improve chemotherapy efficacy as adjuvant agents. This study investigated the effects of catechin and gossypol on human glutathione transferase Pi (GSTP1-1) activity and their cytotoxic effects on breast cancer cells (MCF-7) individually and in combination with tamoxifen (TAM). Gossypol effectively inhibited the enzyme with an IC50 value of 40μM, compared to 200μM for catechin. Gossypol showed stronger inhibition of GSTP1-1 activity (Ki = 63.3 ± 17.5μM) compared to catechin (Ki = 220 ± 44μM). Molecular docking analysis revealed their binding conformations to GSTP1-1, with gossypol binding at the subunit interface in an un-competitive manner and catechin showing mixed non-competitive inhibition. Gossypol had severe cytotoxic effects on both MCF-7 cells and normal BJ1 cells, while catechin had a weak cytotoxic effect on MCF-7 cells only. Combination therapy with TAM resulted in cytotoxicity of 27.3% and 35.2% when combined with catechin and gossypol, respectively. Gossypol showed higher toxicity to MCF-7 cells, but its strong effects on normal cells raised concerns about selectivity and potential side effects.

Arylcoumarin and novel biscoumarin derivatives as potent inhibitors of human glutathione S-transferase

A series of arylcoumarin derivatives and two novel biscoumarin derivatives were investigated for their human recombinant glutathione S-transferase P1-1 (GSTP1-1) enzyme inhibitory activities for the first time. 4-(3,4-Dihydroxyphenyl)-6,7-dihydroxycoumarin (compound 24) was observed to be the most active coumarin derivative (IC50: 0.14 µM). The inhibition was found to be time-dependent and irreversible. Hypothetical binding modes of the ten most active compounds were calculated by molecular docking. Ligand efficiency indices (LEI) were estimated to better understand the binding performance of the coumarin derivatives. Extensive structure-activity relationship studies showed that hydroxy substitution on both the coumarin and the aryl ring enhanced the biological activity and the position of hydroxy group on the coumarin ring is critical for the binding pose and the activity. Top three ligands were subjected to molecular dynamics simulations and MM/PBSA for further investigation. Binding mode of compound 24 suggested that its high inhibitory activity might be attributed to its position between Tyr7 and the cofactor, glutathione (GS-DNB). Exhibiting favorable druglikeness profiles and pharmacokinetics based on ADME studies, compound 5 and 24 can be considered as potential drug leads in future studies for further development. Communicated by Ramaswamy H. Sarma

BPDE-DNA adduct formation and alterations of mRNA, protein, and DNA methylation of CYP1A1, GSTP1, and GSTM1 induced by benzo[a]pyrene and the intervention of aspirin in mice.

Benzo[a]pyrene (B[a]P), one typical environmental pollutant, the toxicity mechanisms, and potential prevention remain perplexing. Available evidence suggests cytochrome P450 1A1 (CYP1A1) and glutathione S-transferases (GSTs) metabolize B[a]P, resulting in metabolic activation and detoxification of B[a]P. This study aimed to reveal the impact of B[a]P exposure on trans-7,8-diol-anti-9,10-epoxide DNA (BPDE-DNA) adduct formation, level of CYP1A1, glutathione S-transferase pi (GSTP1) and glutathione S-transferase mu1 (GSTM1) mRNA, protein and DNA methylation in mice, and the potential prevention of aspirin (ASP). This study firstly determined the BPDE-DNA adduct formation in an acute toxicity test of a large dose in mice induced by B[a]P, which subsequently detected CYP1A1, GSTP1, and GSTM1 at levels of mRNA, protein, and DNA methylation in the organs of mice in a subacute toxicity test at appropriate doses and the potential prevention of ASP, using the methods of real-time quantitative PCR (QPCR), western blotting, and real-time methylation-specific PCR (MSP), respectively. The results verified that B[a]P induced the formation of BPDE-DNA adduct in all the organs of mice in an acute toxicity test, and the order of concentration of which was lung > kidney > liver > brain. In a subacute toxicity test, following B[a]P treatment, mice showed a dose-dependent slowdown in body weight gain and abnormalities in behavioral and cognitive function and which were alleviated by ASP co-treatment. Compared to the controls, following B[a]P treatment, CYP1A1 was significantly induced in all organs in mice at mRNA level (P < 0.05), was suppressed in the lung and cerebrum of mice at protein level, and inhibited at DNA methylation level in the liver, lung, and cerebrum, whereas GSTP1 and GSTM1 at mRNA, protein, and DNA methylation levels showed organ-specific changes in mice following B[a]P treatment, which was generally alleviated by ASP intervention. In conclusion, B[a]P induced BPDE-DNA adduct formation in all organs in mice and altered the mRNA, protein, and DNA methylation levels in CYP1A1, GSTP1, and GSTM1 in an organ-dependent pattern, which could be related to the organ toxicity and mechanism of B[a]P. ASP intervention may be an effective measure to prevent B[a]P toxicity. The findings provide scientific evidence for further study on the organ toxicity and mechanisms of B[a]P.

Reconstructing hepatic metabolic profile and glutathione-mediated metabolic fate of acrylamide

The toxicity of acrylamide (AA) has continuously attracted wide concerns as its extensive presence from both environmental and dietary sources. However, its hepatic metabolic transformation and metabolic fate still remain unclear. This study aims to unravel the metabolic profile and glutathione (GSH) mediated metabolic fate of AA in liver of rats under the dose-dependent exposure. We found that exposure to AA dose-dependently alters the binding of AA and GSH and the generation of mercapturic acid adducts, while liver as a target tissue bears the metabolic transformation of AA via regulating GSH synthesis and consumption pathways, in which glutamine synthase (GSS), cytochrome P450 2E1 (CYP2E1), and glutathione S-transferase P1 (GSTP1) play a key role. In response to high- and low-dose exposures to AA, there were significant differences in liver of rats, including the changes in GSH and cysteine (CYS) activities and the conversion ratio of AA to glycidamide (GA), and liver can affect the transformation of AA by regulating the GSH-mediated metabolic pathway. Low-dose exposure to AA activates GSH synthesis pathway in liver and upregulates GSS activity and CYS content with no change in γ-glutamyl transpeptidase 1 (GGT1) activity. High-dose exposure to AA activates the detoxification pathway of GSH and increases GSH consumption by upregulating GSTP1 activity. In addition, molecular docking results showed that most of the metabolic molecules transformed by AA and GA other than themselves can closely bind to GSTP1, GSS, GGT1, N-acetyltransferase 8, and dimethyl sulfide dehydrogenase 1. The binding of AA-GSH and GA-GSH to GSTP1 and CYP2E1 enzymes determine the tendentiousness between toxicity and detoxification of AA, which exerts a prospective avenue for targeting protective role of hepatic enzymes against in vivo toxicity of AA.

Perturbations of Glutathione and Sphingosine Metabolites in Port Wine Birthmark Patient-Derived Induced Pluripotent Stem Cells.

Port Wine Birthmarks (PWBs) are a congenital vascular malformation on the skin, occurring in 1-3 per 1000 live births. We have recently generated PWB-derived induced pluripotent stem cells (iPSCs) as clinically relevant disease models. The metabolites associated with the pathological phenotypes of PWB-derived iPSCs are unknown, and so we aim to explore them in this study. Metabolites were separated by ultra-performance liquid chromatography and screened with electrospray ionization mass spectrometry. Orthogonal partial least-squares discriminant, multivariate, and univariate analyses were used to identify differential metabolites (DMs). KEGG analysis was used to determine the enrichment of metabolic pathways. A total of 339 metabolites was identified. There were 22 DMs, among which nine were downregulated-including sphingosine-and 13 were upregulated, including glutathione in PWB iPSCs, as compared to controls. Pathway enrichment analysis confirmed the upregulation of glutathione and the downregulation of sphingolipid metabolism in PWB-derived iPSCs as compared to normal ones. We next examined the expression patterns of the key molecules associated with glutathione metabolism in PWB lesions. We found that hypoxia-inducible factor 1α (HIF1α), glutathione S-transferase Pi 1 (GSTP1), γ-glutamyl transferase 7 (GGT7), and glutamate cysteine ligase modulatory subunit (GCLM) were upregulated in PWB vasculatures as compared to blood vessels in normal skin. Other significantly affected metabolic pathways in PWB iPSCs included pentose and glucuronate interconversions; amino sugar and nucleotide sugars; alanine, aspartate, and glutamate; arginine, purine, D-glutamine, and D-glutamate; arachidonic acid, glyoxylate, and dicarboxylate; nitrogen, aminoacyl-tRNA biosynthesis, pyrimidine, galactose, ascorbate, and aldarate; and starch and sucrose. Our data demonstrated that there were perturbations in sphingolipid and cellular redox homeostasis in PWB vasculatures, which could facilitate cell survival and pathological progression. Our data implied that the upregulation of glutathione could contribute to laser-resistant phenotypes in some PWB vasculatures.

Association of Glutathione S-Transferase P1 (GSTP1) Gene Polymorphism with the Response to Platinum Based Chemotherapy in Patients with Non-Small Cell Lung Cancer

Abstract Background Lung cancer is one of the most commonly diagnosed cancers and despite therapeutic advances, mortality remains high. Platinum based chemotherapy remains the mainstay of adjuvant or first line chemotherapy in non small cell lung cancer (NSCLC) treatment, pathophysiological features along with genetic background significantly influence therapeutic efficacy of platinum-based chemotherapy. GSTP1 gene is a member of the cytosolic superfamily of glutathione-S-transferases, which is responsible for phase II detoxification enzymes that convert various reactive metabolites to more water-soluble and less harmful forms by conjugating them with glutathione. The genetic polymorphism GSTP1 rs1695 is associated with decreased substrate specific catalytic activity and the reduced enzyme activity which may reduces platinum detoxification, thus promoting better response rates to platinum-based chemotherapy. Aim of the Work In the present study was aimed to investigate the association of GSTP1 gene polymorphism with the response to platinum based chemotherapy in patients with NSCLC in order to prevent the non necessary exposure to the toxic effect of chemotherapy. Patients and Methods The study was a retrospective cross sectional study conducted on 50 NSCLC patients who were recruited from the Oncology clinic in Ain Shams University Hospitals, twenty of them were responders to platinum based chemotherapy and the other 30 were not responders according to RECIST criteria. All subjects underwent full medical history focusing on tobacco smoking and family history of cancers, thorough clinical examination, histological diagnosis of NSCLC and TNM classification, radiological studies: computed tomography of chest and abdomen and laboratory investigations in the form of: complete blood picture (CBC), liver function tests and kidney function tests. Detection of the GSTP1 rs1695 (A/G) polymorphism by real-time PCR was done for both the responders and the non responders group. Results There was no statistically significant difference between the responders’ and non responders’ groups regarding the genotypic distribution (p &amp;gt; 0.05). However, the G allele was significantly higher in the responders’ group compared to the non responders’ group with p value &amp;lt; 0.05. Conclusion The present study did not confirm the presence of significant association between the genotypic frequencies of the GSTP1 rs 1695 among the responders and the non responders group, but despite that it showed that the A allele was significantly higher in the non responders’ group compared to the responders’ group. However, the G allele was significantly higher in the responders’ group compared to the non responders’ group. Further studies with higher sample size are needed to clarify the association.

Mechanistic insights into the inhibition of human placental glutathione S-transferase P1-1 by abscisic and gibberellic acids: An integrated experimental and computational study.

The interactions of the classic phytohormones gibberellic acid (gibberellin A3 , GA3 ) and abscisic acid (dormin, ABA), which antagonistically regulate several developmental processes and stress responses in higher plants, with human placental glutathione S-transferase P1-1 (hpGSTP1-1), an enzyme that plays a role in endo- or xenobiotic detoxification and regulation of cell survival and apoptosis, were investigated. The inhibitory potencies of ABA and GA3 against hpGSTP1, as well as the types of inhibition and the kinetic parameters, were determined by making use of both enzyme kinetic graphs and SPSS nonlinear regression models. The structural basis for the interaction between hpGSTP1-1 and phytohormones was predicted with the aid of molecular docking simulations. The IC50 values of ABA and GA3 were 5.3 and 5.0 mM, respectively. Both phytohormones inhibited hpGSTP1-1 in competitive manner with respect to the cosubstrates GSH and CDNB. When ABA was the inhibitor at [CDNB]f -[GSH]v and at [GSH]f -[CDNB]v , Vm , Km , and Ki values were statistically estimated to be 205 ± 16 μmol/min-mg protein, 1.32 ± 0.18 mM, 1.95 ± 0.25 mM and 175 ± 6 μmol/min-mg protein, 0.85 ± 0.06 mM, 1.85 ± 0.16 mM, respectively. On the other hand, the kinetic parameters Vm , Km , and Ki obtained with GA3 at [CDNB]f -[GSH]v and at [GSH]f -[CDNB]v were found to be 303 ± 14 μmol/min-mg protein, 1.77 ± 0.13 mM, 3.38 ± 0.26 mM and 249 ± 7 μmol/min-mg protein, 1.43 ± 0.07 mM, 2.89 ± 0.19 mM, respectively. Both phytohormones had the potential to engage in hydrogen-bonding and electrostatic interactions with the key residues that line the G- and H-sites of the enzyme's catalytic center. Inhibitory actions of ABA/GA3 on hpGSTP1-1 may guide medicinal chemists through the structure-based design of novel antineoplastic agents. It should be noted, however, that the same interactions may also render fetuses vulnerable to the potentially toxic effects of xenobiotics and noxious endobiotics.

Combined effect of hygienic and polygenic risk scores in children with allergic rhinitis.

Although the development of allergic rhinitis (AR) is associated with multiple genetic and hygienic environmental factors, previous studies have focused mostly on the effect of a single factor on the development of AR. This study aimed to investigate the combined effect of multiple genetic and hygienic environmental risk factors on AR development in school children. We conducted a cross-sectional study, comprising 1,797 children aged 9-12 years. Weighted environmental risk score (ERS) was calculated by using four hygienic environmental factors, including antibiotic use during infancy, cesarean section delivery, breast milk feeding, and having older siblings. Weighted polygenic risk score (PRS) was calculated by using four single nucleotide polymorphisms (SNPs), including interleukin-13 (rs20541), cluster of differentiation 14 (rs2569190), toll-like receptor 4 (rs1927911), and glutathione S-transferase P1 (rs1695). Multivariable logistic regression analysis was used. More than three courses of antibiotic use during infancy increased the risk of current AR (adjusted odd ratio [aOR], 2.058; 95% confidence interval [CI]: 1.290-3.284). Having older siblings, especially > 2 (aOR, 0.526; 95%Cl: 0.303-0.913) had a protective effect. High ERS ( > median; aOR, 2.079; 95%Cl: 1.466-2.947) and PRS ( > median; aOR, 1.627; 95%Cl: 1.117-2.370) increased the risk of current AR independently. Furthermore, children who had both high ERS and PRS showed a higher risk of current AR (aOR, 3.176; 95%Cl: 1.787-5.645). Exposure to multiple hygienic risk factors during infancy increases the risk of AR in genetically susceptible children.

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.

First-in-human dose-escalation study of NBF-006, a novel investigational siRNA targeting GSTP, in patients with non-small cell lung, pancreatic, or colorectal cancer.

3084 Background: Glutathione S-transferase Pi (GSTP) has an important role in detoxification and anti-oxidative damage response. Additionally, GSTP has a chaperone function which regulates key oncogenic pathways such as the KRAS and JNK pathways. Targeting multiple pathways could lead to more effective therapy than agents inhibiting single downstream kinase pathways (i.e., a MEK inhibitor), as alternative or redundant pathways could compensate for the inhibition of a single kinase. NBF-006 is a novel drug product containing a GSTP siRNA encapsulated within a lipid nanoparticle. It has been designed to deliver siRNA to tumors of the lung and common sites of metastatic spread (liver and marrow) which has been demonstrated in preclinical efficacy models. Methods: NBF-006-001 is a first-in-man (FIM) dose escalation study evaluating safety, tolerability, and PK of NBF-006 administered intravenously over 70 minutes, QW4 followed by a 2-week rest period in patients with advanced non-small cell lung (NSCLC), pancreatic (PANC), or colorectal cancer (CRC). Results: Twenty-one patients (11 NSCLC, 5 CRC, and 5 PANC) received treatment in a standard 3+3 dose escalation design (0.15 mg/kg [n=2], 0.3 mg/kg [n=3], 0.6 mg/kg [n=3], 1.2 mg/kg [n=6], and 1.6 mg/kg [n=7]). NBF-006 was well tolerated with no treatment-related grade 4-5 AEs, SAEs nor DLTs. Only one grade 3 event (WBC decrease) was assessed as possibly related. The most common NBF-006-related AEs were Grade 1 or 2 arthralgia, diarrhea, fatigue, and vomiting (11% each). There was no dose relationship in the number of AEs or any observed AEs. No meaningful complement activation, cytokine elevation nor ADA response has been observed to date (20 patients). One patient had 2 infusion related reactions (Grade 1 and 2). PK analysis indicated a terminal half-life ranging from 26 to 55 hours with no accumulation. Cmax and AUC demonstrated dose-proportionality up to 1.2 mg/kg and no additional increase in exposure at 1.6 mg/kg. NSCLC patients had an average and median number of prior lines of therapy of 4 and 3, respectively, with a range from 1-7 prior lines. One patient with NSCLC (treated at 1.6 mg/kg) experienced a durable partial response and progressed after ten months on the study. Four additional patients with NSCLC experienced stable disease, and two of these stayed on study treatment for &gt;6 months (both treated at 1.2 mg/kg). One patient remains on treatment. CRC and PANC patients had an average/median of 4 and 3 prior lines, respectively. In CRC and PANC, stable disease for 24 weeks was the best response (n=1 each). Conclusions: This FIM study with NBF-006 demonstrated a favorable safety profile with early signs of antitumor activity in NSCLC. No DLTs or treatment-related SAEs were observed. Dose expansion in NSCLC is ongoing and will not be presented. Clinical trial information: NCT03819387 .