Deciphering Glutathione S-Transferase P1 Inhibition Mechanisms for Overcoming Cancer Chemoresistance: Insights From Computational Analysis.

Combined expression of multidrug resistance protein (MRP) and glutathione S-transferase P1-1 (GSTP1-1) in MCF7 cells and high level resistance to the cytotoxicities of ethacrynic acid but not oxazaphosphorines or cisplatin

Interactions of α, β-unsaturated aldehydes and ketones with human glutathione S-transferase P1-1

Glutathione S-transferase P1 (GSTP1) has gradually become a promising target for cancer prevention and treatment. However, subtle variations in GSTP1 can lead to the occurrence of single nucleotide polymorphisms (SNPs). The correlation between specific genotypes of GSTP1 and the clinical outcome of the disease has been extensively investigated, demonstrating a significant area of research in this field. However, their impact on the responses to GSTP1 inhibitor treatment remains to be elucidated. Among the various SNPs of GSTP1, I105V polymorphisms is the most widely studied. In this study, a silico model of GSTP1 I105V polymorphism was successfully established to predict the changes of binding model and binding affinity between GSTP1 I105(WT) or GSTP1 V105 and ethacrynic acid via molecular docking and molecular dynamics, and ultimately further evaluated for its anticancer effects. The result demonstrated that the binding capacity of ethacrynic acid decreases with the I105V mutation of GSTP1, indicating the changes in its anticancer activities. Cancer cells expressing GSTP1 V105 may exhibit greater tolerance to ethacrynic acid-induced toxicity compared to other genotypes. In summary, this study provides the first evidence that the GSTP1 I105V polymorphism may impact cancer cell sensitivity to its inhibitor through theoretical prediction. Furthermore, a comprehensive understanding of the correlation between GSTP1 I105V polymorphisms and responses to GSTP1 inhibitor treatment would offer valuable insights for future drug development targeting GSTP1 in cancer-related diseases.

Arsenic trioxide (ATO) as a single agent is used for treatment of acute promyelocytic leukemia (APL) with minimal toxicity, but therapeutic effect of ATO in other types of malignancies has not been achieved. We tested whether a combination with ethacrynic acid (EA), a glutathione S-transferase P1-1 (GSTP1-1) inhibitor, and a reactive oxygen species (ROS) inducer will extend the therapeutic effect of ATO beyond APL. The combined apoptotic effects of ATO plus ethacrynic acid were tested in non-APL leukemia and lymphoma cell lines. The role of ROS, GSTP1-1, glutathione (GSH), and Mcl-1 in apoptosis was determined. The selective response to this combination of cells with and without GSTP1-1 expression was compared. ATO/EA combination synergistically induced apoptosis in myeloid leukemia and lymphoma cells. This treatment produced high ROS levels, activated c-jun-NH(2)-kinase (JNK), and reduced Mcl-1 protein. This led to the decrease of mitochondrial transmembrane potential, release of cytochrome c, and subsequently, to activation of caspase-3 and -9. Induction of apoptosis in leukemia and lymphoma cells expressing GSTP1-1 required high ethacrynic acid concentrations to be combined with ATO. Silencing of GSTP1 in leukemia cells sensitized them to ATO/EA-induced apoptosis. In a subgroup of B-cell lymphoma, which does not express GSTP1-1, lower concentrations of ethacrynic acid and its more potent derivative, ethacrynic acid butyl-ester (EABE), decreased intracellular GSH levels and synergistically induced apoptosis when combined with ATO. B-cell lymphoma cells lacking GSTP1-1 are more sensitive than myeloid leukemia cells to ATO/EA-induced apoptosis.

Ethacrynic acid, a diuretic, inhibits glutathione S-transferase P1-1 (GSTP1-1) activity and induces cell death in malignant cells at high concentrations. To improve ethacrynic acid activity, ethacrynic acid oxadiazole analogs 6s and 6u were synthesized. Although both compounds have greater antiproliferative effects than ethacrynic acid in human HL-60 cells, 6u has a reduced ability to inhibit GSTP1-1 activity. The mechanisms of both 6s- and 6u-induced cell death as well as the role of GSTP1-1 in their actions were studied. Both 6s and 6u equally induced apoptosis in HL-60 cells due to the activation of caspase-3, -9, and -8, which was correlated with the downregulation of antiapoptotic proteins c-FLIP, Mcl-1, and XIAP. The caspase inhibitor Z-VAD-FMK blocked the reduction of XIAP, but not of c-FLIP and Mcl-1, in 6s-treated cells. The reduction of c-FLIP and Mcl-1 by 6s was not blocked by the proteasomal inhibitor MG132, but was correlated with inhibition of the phosphorylation of extracellular signal-regulated kinase (ERK) and eIF4E. Both 6s and 6u decreased the intracellular glutathione (GSH) levels. N-acetylcysteine blocked reduction in the levels of Mcl-1, c-FLIP, and intracellular GSH as well as apoptosis in HL-60 cells treated by either compound. Silencing of GSTP1-1 in K562 cells sensitized, but overexpression of GSTP1-1 in Raji cells blocked, apoptosis induction by either compound. GSH conjugation at the methylene group abrogated the ability of inducing apoptosis. These data suggest that the methylene group plays an important role in the downregulation of c-FLIP and Mcl-1 proteins and apoptosis induction, which is inactivated by GSTP1-1 by forming GSH conjugates.

A glutathione S-transferase (GST) was purified from an arsenic-resistant Chinese hamster ovary cell line, SA7. The SA7 GST was shown to catalyse the conjugation of glutathione and ethacrynic acid, a specific substrate for Pi class GST. Its N-terminal amino-acid sequence has 80% identical residues to that of rat GST P and human GST pi. Thus, the GST purified from SA7 cells belongs to the Pi family. Treatment with Cibacron Blue or ethacrynic acid, which are GST inhibitors, significantly decreased the resistance of SA7 cells to sodium arsenite. On the other hand, pretreatment of SA7N cells, a partial revertant of SA7 cells, with sublethal doses of sodium arsenite, cadmium acetate or zinc sulphate resulted in re-elevation of GST activities and the cells regained the arsenic resistance. The regained arsenic resistance was well correlated with the levels of GST pi which were induced dose-dependently by zinc sulphate. Heat-shock treatment (45 degrees C for 10 min) did not increase GST pi expression or arsenic resistance of SA7N cells. The results indicate that GST pi is possibly involved in the mechanism of arsenic detoxification.

Detoxification of 1-Chloro-2,4-dinitrobenzene in MCF7 Breast Cancer Cells Expressing Glutathione S-Transferase P1-1 and/or Multidrug Resistance Protein 1

Since ethacrynic acid (EA), an SH modifier as well as glutathione S-transferase (GST) inhibitor, has been suggested to induce apoptosis in some cell lines, its effects on a human colon cancer cell line DLD-1 were examined. EA enhanced cell proliferation at 20-40 microM, while it caused cell death at 60-100 microM. Caspase inhibitors did not block cell death and DNA ladder formation was not detected. Poly(ADP-ribose) polymerase, however, was cleaved into an 82-kDa fragment, different from an 85-kDa fragment that is specific for apoptosisis. The 82-kDa fragment was not recognized by antibody against PARP fragment cleaved by caspase 3. N-Acetyl-L-cysteine (NAC) completely inhibited EA-induced cell death, but 3(2)-t-butyl-4-hydroxyanisole or pyrrolidinedithiocarbamate ammonium salt did not. Glutathione (GSH) levels were dose-dependently increased in cells treated with EA and this increase was hardly affected by NAC addition. Mitogen-activated protein kinase (MAPK) kinase (MEK) 1, extracellular signal-regulated kinase (ERK) 1 and GST P1-1 were increased in cells treated with 25-75 microM EA, while c-Jun N-terminal kinase (JNK) 1 and p38 MAPK were markedly decreased by 100 microM EA. NAC repressed EA-induced alterations in these MAPKs and GST P1-1. p38 MAPK inhibitors, SB203580 and FR167653, dose-dependently enhanced EA-induced cell death. An MEK inhibitor, U0126, did not affect EA-induced cell death. These studies revealed that EA induced cell death concomitantly with a novel PARP fragmentation, but without DNA fragmentation. p38 MAPK was suggested to play an inhibitory role in EA-induced cell death.

Glutathione S-transferases (GSTs) are an important group of isoenzymes that play an essential role in the detoxification of carcinogens. In this way, GST P1-1 is considered a suitable and excellent targetable biomarker to differentiate cancer from normal cells. Ruthenium-, iron-, osmium- and iridium-based compounds are considered promising candidates for the next generation of metal anticancer drugs in order to overcome the side effects produced by the anticancer platinum drugs used in clinic. In this work, it was developed a miniaturized approach based on sequential injection analysis (SIA) for the determination of the half maximal inhibitory concentration (IC50) of GSTs. Beyond the advantages presented by SIA systems such as versatility, simplicity, robustness, reliability and efficiency, this specific one present two more important advantages allowing the use of three times less reagent solutions than in batch method and reducing the analysis time from 8 minutes to 5 minutes. The newly developed method was applied to 22 ruthenium, iron, osmium and iridium derivates compounds ethacrynic acid (EA) and flurbiprofen, a cyclooxygenase inhibitor, were shown to be potent GST P1-1 inhibitors. The iridium compound tested is the strongest inhibitor of GST P1-1 (IC50 = 6.7 ± 0.7 µM) in the present work, more effective than EA itself and the ruthenium analogue.

Development of an assay for cellular efflux of pharmaceutically active agents and its relevance to understanding drug interactions.

Chapter 20 Small-Molecule Inhibitors of Glutathione S-Transferase P1-1 as Anticancer Therapeutic Agents

To investigate the possible role of glutathione S-transferase P (GSTP) in carcinogenesis and cell proliferation, ethacrynic acid (EA) was used to inhibit GSTP in the human Jurkat T cell line. At lower doses (0-30 microM), EA led to a decreased rate of proliferation as assessed by the MTT assay. This was associated with a decreased DNA S+G2/M phase population and also a dose-dependent increase in apoptosis. At concentrations of EA > 30 microM, cells suffered non-specific cytotoxic injury and underwent necrosis. The total cell number fell over the time course of the experiment. A resistant subculture of cells which proliferated in the presence of EA at 30 microM was selected by continuous growth in the presence of EA. Although this had a higher basal rate of apoptosis than control cells, it also showed a significantly larger growth fraction as assessed by flow cytometry. GSTP is frequently overexpressed in human tumours and animal models of carcinogenesis, and is regarded as a marker of the 'drug-resistant phenotype' of initiated cells. Our findings suggest that the role of GSTP in models of chemical carcinogenesis and in tumours may be its permissive effect on cell cycle activity and downregulation of apoptosis, thus allowing expansion of a population of initiated cells.

Glutathione transferase P1-1 (GSTP1-1) plays crucial roles in cancer chemoprevention and chemoresistance and is a key target for anticancer drug development. Oxidative stress or inhibitor-induced GSTP1-1 oligomerization leads to the activation of stress cascades and apoptosis in various tumor cells. Therefore, bivalent glutathione transferase (GST) inhibitors with the potential to interact with GST dimers are been sought as pharmacological and/or therapeutic agents. Here we have characterized GSTP1-1 oligomerization in response to various endogenous and exogenous agents. Ethacrynic acid, a classic GSTP1-1 inhibitor, 4-hydroxy-nonenal, hydrogen peroxide, and diamide all induced reversible GSTP1-1 oligomerization in Jurkat leukemia cells through the formation of disulphide bonds involving Cys47 and/or Cys101, as suggested by reducing and nonreducing SDS-polyacrylamide gel electrophoresis analysis of cysteine to serine mutants. Remarkably, the electrophilic prostanoid 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) induced irreversible GSTP1-1 oligomerization, specifically involving Cys101, a residue present in the human but not in the murine enzyme. 15d-PGJ(2)-induced GSTP1-1 cross-linking required the prostaglandin (PG) dienone structure and was associated with sustained c-Jun NH(2)-terminal kinase activation and induction of apoptosis. It is noteworthy that 15d-PGJ(2) elicited GSTP1-1 cross-linking in vitro, a process that could be mimicked by other dienone cyclopentenone PG, such as Δ(12)-PGJ(2), and by the bifunctional thiol reagent dibromobimane, suggesting that cyclopentenone PG may be directly involved in oligomer formation. Remarkably, Δ(12)-PGJ(2)-induced oligomeric species were clearly observed by electron microscopy showing dimensions compatible with GSTP1-1 tetramers. These results provide the first direct visualization of GSTP1-1 oligomeric species. Moreover, they offer novel strategies for the modulation of GSTP1-1 cellular functions, which could be exploited to overcome its role in cancer chemoresistance.

Glutathione-S-transferase Pi1 (GSTP1) and multidrug resistance protein 1 (MRP1) are overexpressed in melanoma, a skin cancer notoriously resistant to all current modalities of cancer therapy. To investigate the involvement of these detoxifying enzymes in the drug resistance of melanoma, an inducible (Tet-On™ system) antisense (AS) RNA strategy was used to specifically inhibit GSTP1 expression in A375 cells, a human melanoma cell line expressing high levels of GSTP1 and MRP1. Stable transfectant clones were established and analysed for GSTP1 inhibition by AS RNA. The clone A375-ASPi1, presenting a specific 40% inhibition of GSTP1 expression in the presence of doxycycline, was selected. Lowering the GSTP1 level significantly increased (about 3.3-fold) the sensitivity of A375-ASPi1 cells to etoposide. Inhibitors of glutathione synthesis (BSO), GSTs (curcumin, ethacrynic acid), and also of MRPs (MK571, sulphinpyrazone) improved the sensitising effect of GSTP1 AS RNA. All these inhibitors had stronger sensitising effects in control cells expressing high GSTP1 level (A375-ASPi1 cells in the absence of doxycycline). In conclusion, GSTP1 can act in a combined fashion with MRP1 to protect melanoma cells from toxic effects of etoposide.

Although, Ras mutation are rare in human malignant gliomas, the Ras signaling pathway is frequently activated, particularly, in tumors with activated receptor tyrosine kinases, such as, epidermal growth factor receptor (EGFR). Glutathione S-transferase P1 (GSTP1), a major metabolizing and stress response signaling protein frequently overexpressed in human cancers, undergoes EGFR-dependent tyrosine phosphorylation, resulting in an enhancement of both its enzymatic and its cell signaling regulatory function. The EGFR-phosphorylated tyrosine 198 residue in human GSTP1 occurs within the consensus motif for high affinity binding to the SH2 domain of Grb2 (growth factor receptor-bound protein 2), an adaptor protein and critical downstream mediator of intracellular signal transduction from activated cell surface receptors via its SH2 domain. Here, we investigated, in glioma cells, the binding of GSTP1 to the Grb2 SH domain and its effect on Ras signaling and how this is impacted by EGFR. GSTP1 was co-immunoprecipitated with Grb2 in extracts of human glioblastoma (GBM) cells, following activation of EGFR with EGF, and EGFR-overexpressing GBM xenografts. Pull-down assays using biotinylated wild-type C-terminal GSTP1 peptides, containing the phospho-acceptor Y198 (Y198WT) showed it to form a complex with Grb2 and Sos1, when Y198 was phosphorylated by EGFR, and not when it was unphosphorylated. In contrast, its mutant peptide counterpart (Y198F) did not complex with Grb2 or Sos1. EGFR-induced Ras activation and ERK1/2 phosphorylation were significantly increased in wild-type GSTP1 but not GSTP1-Y198F transfectant cells. In human MGR3 GBM cells, both siRNA-mediated GSTP1 knockdown and GSTP1 inhibition with TLK199, a GSTP1 specific inhibitor, decreased ERK1/2 phosphorylation and activity markedly. The cell-permeable GSTP1-Y198 peptides also formed a complex with Grb2-Sos1 in UW228 human medulloblastoma cells with activated EGFR. Finally, following GSTP1 transfection, non-tumorigenic GSTP1-ve UW228 cells were transformed into highly tumorigenic cells that formed subcutaneous tumors in vivo. Our data show that, in human glioma cells, EGF-activated EGFR phosphorylates GSTP1 at Y198, facilitating formation (via Grb2-SH2 binding) of a GSTP1-Grb2-Sos1 complex and enhancement of Ras pathway and ERK1/2 activation. This EGFR-mediated GSTP1 phosphorylation-dependent enhancement of the Ras downstream signaling could contribute, in part, to the activated Ras signaling observed in the absence of structural Ras mutations and is a likely mechanism for the accelerated tumor growth characteristic of GBM and other tumors with elevated GSTP1 and aberrant EGFR signaling. Supported by NIH grants RO1 CA 153050, RO1CA127872, RO1 CA 112519, P50CA108786 and P30-CA14236. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2162. doi:1538-7445.AM2012-2162