GST Isozymes Research Articles

The role of tissue persistent organic pollutants and genetic polymorphisms in patients with benign and malignant kidney tumors

This study aimed to explore whether there is an association between environmental exposure to POPs and kidney tumor induction, and whether blood POP concentrations reflect kidney tissue concentrations. POP derivatives were determined in blood, tumor tissue, tumor surrounding tissue, and perirenal fat tissue samples taken from patients who underwent surgery for renal tumors. A voluntary control group was recruited for blood and urine samples as well. Urinary excretions of o,o′-dityrosine, chlorotyrosine, nitrotyrosine, and 8-OHdG were measured in the same patients. The possible role of genetic polymorphisms in CYP1A1, GST isozymes P, M, and T, and hOGG1 genes on the predisposition to renal cancer was investigated. Some POPs have been found to be associated with kidney cancer, as evidenced by their significantly high ORs. 8-OHdG levels were significantly higher compared to the control group. The GSTT1 null polymorphism can be a risk factor for malignant but not for benign kidney tumors.

GSTs, MRP and Apoptototic Markers in DLD-1 Human Colon Cancer Cell Line Before and After 5-FU Treatment

Multidrugresistance is an important factor limiting the effect of chemotherapy on cancer treatment. Disorders of drug transport and apoptosis, deterioration of redox homeostasis are among the main mechanisms that lead to multidrug resistance. The aim of this study was to determine the effect of 5-FU on GST isozymes, drug resistance proteins and apoptotic proteins before and after 5-Flourouracil application on DLD-1 colon cancer cell line. The cytotoxic effect of 5-FU was measured by WST-1test and, the efficiency of drug application was, also, proved by double staining via Hoechst 33342 with Propidium iodide. Next, the expression levels of GST isozymes, drug resistance proteins and apoptotic proteins were determined by immunocytochemistry. The cytotoxic effect of 5-FU at different doses on DLD-1 colon cancer cell line was determined by WST-1 method. MRP-2, 3, 6, 7 of drug resistance proteins; GSTA1, GSTM1, GSTT1, GSTZ1, GSTK1 and GSTO1 of GST proteins; bcl-2, caspase-3, p38, and p53, which are apoptotic proteins, have higher expression in the drug-treated DLD-1 cell line. GSTS1, MDR-1 and MRP-1expressions were not immunocytochemically different. It was determined that there is a direct correlation between the level of cytotoxicity and applied drug concentration. The cytotoxic effect of the drug increased with the increase in the dose of the drug. In this study, as first in the literature, the expression levels of some apoptotic markers, GST isozymes and drug resistance proteinswere evaluated togetherand except GSTS1, MDR-1 and MRP-1, they were all upregulated with respect to the control group after 5-FU administration.

Implications of glutathione-S transferase P1 in MAPK signaling as a CRAF chaperone: In memory of Dr. Irving Listowsky.

Glutathione-S transferase P1 (GSTP1) is one of the glutathione-S transferase isozymes that belong to a family of phase II metabolic isozymes. The unique feature of GSTP1 compared with other GST isozymes is its relatively high expression in malignant tissues. Thus, clinically, GSTP1 serves as a tumor marker and as a refractory factor against certain types of anticancer drugs through its primary function as a detoxifying enzyme. Additionally, recent studies have identified a chaperone activity of GSTP1 involved in the regulation the function of various intracellular proteins, including factors of the growth signaling pathway. In this review, we will first describe the function of GSTP1 and then extend the details onto its role in the mitogen-activated protein kinase signal pathway, referring to the results of our recent study that proposed a novel autocrine signal loop formed by the CRAF/GSTP1 complex in mutated KRAS and BRAF cancers. Finally, the possibilities of new therapeutic approaches for these cancers by targeting this complex will be discussed.

Identification of 3-Bromo-1-Ethyl-1H-Indole as a Potent Anticancer Agent with Promising Inhibitory Effects on GST Isozymes.

Indole-based heterocyclic compounds play important roles in pharmaceutical chemistry due to their unexpected biological and pharmacological properties. Herein, we describe novel biological properties (antioxidant, antimicrobial and anti-cancer) of 3- bromo-1-ethyl-1H-indole (BEI) structure. BEI was synthesized from 1-Methyl-2-phenylindole and N-bromosuccinimide and was characterized by using 1H and 13C NMR. Cytotoxicity was determined by MTT assay. Apoptosis analysis of BEI was determined by Arthur™ image-based Cytometer. Different methods were applied to assess the antioxidant activity of BEI. Molecular docking studies were conducted to determine the interactions of bonding between GST isozymes and BEI. According to the antioxidant and antimicrobial activity assays, BEI compound showed reduced total antioxidant activity compared to the Trolox standard, whereas it showed moderate antimicrobial activity against Aspergillus niger and Phytophora eryhtrospora. Notably, the BEI compound demonstrated substantial selective cytotoxicity for the first time towards cancer cell lines, and there existed a significant decrease in the percentage of live cells treated with BEI, in comparison to the control ones. Interestingly, BEI exhibited a promising glutathione S-transferase isozymes inhibition. The results of this study suggest that BEI seems to be a promising molecule to be used in the design of new anti-cancer agents that provide superiority to present commercial anti-cancer drugs.

GSTM1 polymorphism in Oncotype DX assay is a potential predictive factor for taxane-based neoadjuvant chemotherapy in estrogen receptor-positive Chinese breast cancer patients

Background: Investigations of the 21-gene Oncotype DX assay in neoadjuvant settings for breast cancer patients are currently underdeveloped. It is still unclear whether any individual gene of the 16 cancer-related genes can be used to predict response to taxane-based neoadjuvant chemotherapy. Methods: Here, we performed TaqMan RT-PCR reactions to profile the expression patterns of these genes in 66 Chinese ER-positive breast cancer patients’ tumor tissues who underwent taxane-based neoadjuvant chemotherapies. Only GSTM1 has distinct non-expression/expression polymorphism, and we observed an almost 2.5-fold increment in the possibility of achieving pathological complete response (pCR) was defined as no histological evidence of residual invasive cancer both in the breast and in the axilla. in GSTM1 non-expression patients. We then analyzed the expression levels of 4 main GST isozymes (GSTA1, GSTT1, GSTP1 and GSTT1) and docetaxel cytotoxicity in various breast cancer cell lines (MCF7, MDA-MB-231, BT474, and SKBR3). Furthermore, we knocked down 4 GST isozymes with siRNA against each isoform in resistant MDA-MB-231. We then observed the changes of docetaxel sensitivity though WST-1 assay, in addition to apoptosis by Hoechst stain and TUNEL assay. Results: Docetaxel IC50 values ranging from 33.0 nM (MDA-MB-231) to 7.20 µM (resistant MDA-MB-231) revealed a close correlation with the expression levels of 4 main GST isozymes. Significantly, depletion of GSTM1 and GSTA1 but not GSTP1 nor GSTT1, suppressed cell viability by 174% and 155% respectively, and was able to induce obvious apoptosis in resistant MDA-MB-132. Conclusions: Taken together, our results suggest that GSTM1 polymorphism could be useful in the prediction of taxane-based neoadjuvant chemotherapy in ER-positive Chinese breast cancer patients, and that GSTM1 and GSTA1 expression are linked to docetaxel resistance.

GSTM1 polymorphism in Oncotype DX assay is a potential predictive factor for taxane-based neoadjuvant chemotherapy in estrogen receptor-positive Chinese breast cancer patients.

BackgroundInvestigations of the 21-gene Oncotype DX assay in neoadjuvant settings for breast cancer patients are currently underdeveloped. It is still unclear whether any individual gene of the 16 cancer-related genes can be used to predict response to taxane-based neoadjuvant chemotherapy.MethodsHere, we performed TaqMan RT-PCR reactions to profile the expression patterns of these genes in 66 Chinese ER-positive breast cancer patients’ tumor tissues who underwent taxane-based neoadjuvant chemotherapies. Only GSTM1 has distinct non-expression/expression polymorphism, and we observed an almost 2.5-fold increment in the possibility of achieving pathological complete response (pCR) was defined as no histological evidence of residual invasive cancer both in the breast and in the axilla. in GSTM1 non-expression patients. We then analyzed the expression levels of 4 main GST isozymes (GSTA1, GSTT1, GSTP1 and GSTT1) and docetaxel cytotoxicity in various breast cancer cell lines (MCF7, MDA-MB-231, BT474, and SKBR3). Furthermore, we knocked down 4 GST isozymes with siRNA against each isoform in resistant MDA-MB-231. We then observed the changes of docetaxel sensitivity though WST-1 assay, in addition to apoptosis by Hoechst stain and TUNEL assay.ResultsDocetaxel IC50 values ranging from 33.0 nM (MDA-MB-231) to 7.20 µM (resistant MDA-MB-231) revealed a close correlation with the expression levels of 4 main GST isozymes. Significantly, depletion of GSTM1 and GSTA1 but not GSTP1 nor GSTT1, suppressed cell viability by 174% and 155% respectively, and was able to induce obvious apoptosis in resistant MDA-MB-132.ConclusionsTaken together, our results suggest that GSTM1 polymorphism could be useful in the prediction of taxane-based neoadjuvant chemotherapy in ER-positive Chinese breast cancer patients, and that GSTM1 and GSTA1 expression are linked to docetaxel resistance.

Glutathione S‑transferase isozyme alpha 1 is predominantly involved in the cisplatin resistance of common types of solid cancer.

The roles of glutathione S‑transferase pi 1 (GSTP1), glutathione S‑transferase mu 2 (GSTM2) and glutathione S‑transferase alpha 1 (GSTA1) in cisplatin (DDP)‑resistance of solid cancer cells (A549/DDP, SKOV3/DDP and SGC7901/DDP) were compared following expression downregulation with small interfering RNAs (siRNAs). DDP cytotoxicity was reflected by its half maximal inhibition concentration (IC50) calculated from data using a Cell Counting Kit‑8 assay; cell apoptosis was examined using flow cytometry and Hoechst 33342 staining. Higher activities of GST were detected in the cytosol of DDP‑resistant cells, compared with those in the parental DDP‑susceptible cells. The silencing efficacy of each positive siRNA was supported by western blot analysis. GSTP1 silencing resulted in a 4‑fold sensitization of SGC7901/DDP cells to DDP cytotoxicity, but negligible sensitization of SKOV3/DDP and A549/DDP cells. GSTM2 silencing sensitized SKOV3/DDP and A549/DDP cells to DDP cytotoxicity by ~2‑fold, but did not sensitize SGC7901/DDP cells. Notably, GSTA1 silencing enhanced DDP cytotoxicity in SGC7901/DDP cells by 6‑fold, in A549/DDP cells by 5‑fold and in SKOV3/DDP cells by 2‑fold. The combined actions of positive siRNAs and DDP increased the percentages of apoptotic cells in the DDP‑resistant solid cancer cells compared with the combined actions of DDP and the negative siRNAs. The present findings indicated that GSTA1 is a predominant GST isozyme associated with DDP resistance of SGC7901/DDP, A549/DDP and SKOV3/DDP cells; GSTA1‑specific inhibitors may be general sensitizers of SGC7901/DDP, A549/DDP and SKOV3/DDP cells to DDP cytotoxicity through the promotion of cell apoptosis.

Crystal structure of the delta-class glutathione transferase in Musca domestica

Among the various glutathione transferase (GST) isozymes in insects, the delta- and epsilon-class GSTs fulfill critical functions during the detoxification of insecticides. We crystalized MdGSTD1, the major delta-class GST isozyme in the housefly (Musca domestica), in complex with glutathione (GSH) and solved its structure at a resolution of 1.4 Å. The overall folding of MdGSTD1 resembled other known delta-class GSTs. Its substrate binding pocket was exposed to solvent and considerably more open than in the epsilon-class GST from M. domestica (MdGSTE2). However, their C-terminal structures differed the most because of the different lengths of the C-terminal regions. Although this region does not seem to directly interact with substrates, its deletion reduced the enzymatic activity by more than 70%, indicating a function in maintaining the proper conformation of the binding pocket. Binding of GSH to the GSH-binding region of MdGSTD1 results in a rigid conformation of this region. Although MdGSTD1 has a higher affinity for GSH than the epsilon class enzymes, the thiol group of the GSH molecule was not close enough to serine residue 9 to form a hydrogen-bond with this residue, which is predicted to act as the catalytic center for thiol group deprotonation in GSH.

Glutathione S-Transferase P-Mediated Protein S-Glutathionylation of Resident Endoplasmic Reticulum Proteins Influences Sensitivity to Drug-Induced Unfolded Protein Response.

S-glutathionylation of cysteine residues, catalyzed by glutathione S-transferase Pi (GSTP), alters structure/function characteristics of certain targeted proteins. Our goal is to characterize how S-glutathionylation of proteins within the endoplasmic reticulum (ER) impact cell sensitivity to ER-stress inducing drugs. We identify GSTP to be an ER-resident protein where it demonstrates both chaperone and catalytic functions. Redox based proteomic analyses identified a cluster of proteins cooperatively involved in the regulation of ER stress (immunoglobulin heavy chain-binding protein [BiP], protein disulfide isomerase [PDI], calnexin, calreticulin, endoplasmin, sarco/endoplasmic reticulum Ca2+-ATPase [SERCA]) that individually co-immunoprecipitated with GSTP (implying protein complex formation) and were subject to reactive oxygen species (ROS) induced S-glutathionylation. S-glutathionylation of each of these six proteins was attenuated in cells (liver, embryo fibroblasts or bone marrow dendritic) from mice lacking GSTP (Gstp1/p2-/-) compared to wild type (Gstp1/p2+/+). Moreover, Gstp1/p2-/- cells were significantly more sensitive to the cytotoxic effects of the ER-stress inducing drugs, thapsigargin (7-fold) and tunicamycin (2-fold). Within the family of GST isozymes, GSTP has been ascribed the broadest range of catalytic and chaperone functions. Now, for the first time, we identify it as an ER resident protein that catalyzes S-glutathionylation of critical ER proteins within this organelle. Of note, this can provide a nexus for linkage of redox based signaling and pathways that regulate the unfolded protein response (UPR). This has novel importance in determining how some drugs kill cancer cells. Contextually, these results provide mechanistic evidence that GSTP can exert redox regulation in the oxidative ER environment and indicate that, within the ER, GSTP influences the cellular consequences of the UPR through S-glutathionylation of a series of key interrelated proteins. Antioxid. Redox Signal. 26, 247-261.

Acquisition of Multidimensional NMR Data on GST-Fused Proteins

With the advances in biological sciences, there is a prevalent demand for large quantities of biologically active polypeptides. High throughput screenings in fields such as proteomics, genomics and bioinformatics give a large variety of proteins that could potentially have therapeutic, diagnostic or industrial purposes and recombinant proteins have become the preferred way of production.Glutathione S-Transferase (GST) is not only a very commonly used affinity tag to increase expression yields, but is also known to enhance the solubility of the protein of interest making it a valuable tool in the pursuit of purifying recombinant proteins.Multidimensional NMR spectroscopy is a popular technique to elucidate the 3D structure of proteins in solution. However, obtaining the structural information of peptides and small proteins can be challenging. We have developed a new method, which facilitates the acquisition of NMR based structural information on protein(s) of interest by using the GST-tagged target protein. Our results show that the carrier protein GST surprisingly does not seem to have an effect on the quality of NMR data of its associated fusion partner. It is well known that GST isozymes exist as dimers, but there has been little research on the state of GST-fused proteins. Our results suggest that the GST-tag and the fused partner can be observed as two entities in multidimensional NMR spectra. Furthermore, small angle X-ray scattering (SAXS) supports that GST-fused proteins are predominantly dimers in solution. We predict the loss of signals in the 1H-15N HSQC spectrum corresponding to the GST carrier is primarily due to decrease in the T2 relaxation rate.

A trial to find appropriate animal models of dichloropropane-induced cholangiocarcinoma based on the hepatic distribution of glutathione S-transferases.

It has been reported that 1,2-Dichloropropane (DCP) induced cholangiocarcinoma (CCA) in offset color proof-printing workers. However, exposure to DCP by inhalation or gavage for 2 year did not induce CCA in mice and rats. The present study mapped the hepatic distribution of GST, which is known to activate dihalogenated alkanes, and proliferative and fibrotic changes in bile ducts in various species to find the most appropriate animal model of DCP-induced CCA. First, 12 each of C57BL/6J mice, Balb/cA mice, F344 rats, Syrian hamsters, and guinea pigs were divided into four equal groups and exposed to DCP at 0, 300, 1,000, or 3,000 ppm 8 hours/day for 7 days. Second, 32 Balb/cA mice and 32 Syrian hamsters were each divided into four equal groups and exposed to DCP at 0, 200, 400, and 800 ppm 6 hours/day for 14 days. After the last exposure, the animals were decapitated, and the livers were dissected out for histopathological evaluation. Immunostaining was conducted to determine the distribution of GSTT1, GSTM1, and GSTPi, as well as the expression of proliferation marker Ki67. GSTT1, GSTM1, and GSTPi were expressed in both hepatocytes and bile duct cells in all control and exposed animals. There was no clear difference in the expression of Ki67 between the exposed groups and the control. No fibrotic changes were observed in any species or strains examined. Expression of GSTT1 or other GST isozymes might not explain the difference in sensitivity of hepatocytes and the bile duct to DCP between humans and rodents.

Glutathione S-transferase GSTM 1, null genotype may be associated with susceptibility to age-related cataract.

Age-related cataract (ARC) is the leading cause of visual disability and reversible blindness all over the world. The different expressions of GST isozymes among animals may explain the variations in the cataract formation caused by oxidative stress. In this study, we evaluated the distribution of GST gene polymorphisms in ARC patients and the possible associations between the presence of ARC and GST gene polymorphisms. The epidemiological data was collected by a standard questionnaire and blood samples were obtained from 130 ARC patients and 159 healthy controls. Data about smoking habits of the groups was recorded. Real-time polymerase chain reaction-based methods were used to detect genetic polymorphisms. The GSTM 1 null genotype was found to carry an increased risk for developing ARC (OR: 1.84, 95% CI: 1.13-2.99). The frequency of the GSTT 1 null genotype was not significantly different among the ARC patients and the controls (OR: 1.0, 95% CI: 0.64-1.6). The GSTP 1 Val/Val genotype was also not significantly different among the ARC patients and control groups (OR: 1.06, 95% CI: 0.50-2.23). GSTM 1 null genotype was highly frequent in non-smokers (OR: 3.25, 95% CI: 1.66-6.35) and moderately frequent in smokers (OR: 2.50, 95% CI: 1.28-4.86). Also, carrying the combined genotypes of GSTM 1 null, GSTT 1 and GSTP 1 105-Val allele was seen to have an increased risk of developing ARC (OR: 2.91, 95% CI: 1.31-6.44). This data may provide evidence that GSTM 1 gene polymorphisms may be associated with genetic susceptibility to develop ARC. Larger studies are warranted to verify these findings.

The effect of resveratrol and its methylthio-derivatives on the Nrf2-ARE pathway in mouse epidermis and HaCaT keratinocytes.

Resveratrol is the most extensively studied stilbene derivative. We previously showed that methylthiostilbenes were more effective inhibitors of CYP1A1 and 1B1 activity than resveratrol. In this study, we investigated whether resveratrol and its methylthio-substituted derivatives, i.e. 3-M-4′-MTS (S2), 3,5-DM-4′-MTS (S5) and 3,4,5-TM-4′-MTS (S7) could activate Nrf2 signaling in the mouse epidermis and in human keratinocytes. Western blot analysis showed translocation of Nrf2 from the cytosol to the nucleus in both models. All of the tested stilbenes increased GST activity, but resveratrol was the most effective inducer. Moreover, only resveratrol increased the protein level of GSTP in the mouse epidermis. GSTM was enhanced in HaCaT cells after the treatment with derivatives S2 and S5. The same effect was observed for GSTP in the case of compound S2. Resveratrol and its derivatives reduced the NQO2 protein level in HaCaT cells. Thus, it is possible that increased expression of GSTP or GSTM and GST activity was linked with NQO2 inhibition in these cells. The results of this study indicate that resveratrol and its methylthioderivatives activate Nrf2 not only in the mouse epidermis, but also in human keratinocytes. Upregulating GST isozymes might be particularly important for deactivating chemical carcinogens, such as PAH.

Glyceryl trinitrate metabolism in the quail embryo by the glutathione S-transferases leads to a perturbation in redox status and embryotoxicity

Exposure of stage 9 quail (Coturnix coturnix japonica) embryos to glyceryl trinitrate (GTN) induces malformations that were associated in previous studies with an increase in protein nitration. Increased nitration suggests metabolism of GTN by the embryo. The goals of this study were to characterize the enzymes and co-factors required for GTN metabolism by quail embryos, and to determine the effects of in ovo treatment with N-acetyl cysteine (NAC), a precursor of glutathione (GSH), on GTN embryotoxicity. GTN treatment of quail embryos resulted in an increase in nitrite, a decrease in total GSH, and an increase in the ratio of NADP(+)/NADPH, indicating that redox balance may be compromised in exposed embryos. Glutathione S-transferases (GSTs; EC 2.5.1.18) purified from the whole embryo (K(m) 0.84 mM; V(max) 36 μM/min) and the embryonic eye (K(m) 0.20 mM; V(max) 30 μM/min) had GTN-metabolizing activity (1436 and 34 nmol/min/mg, respectively); the addition of ethacrynic acid, an inhibitor of GST activity, decreased GTN metabolism. Peptide sequencing of the GST isozymes indicated that alpha- or mu-type GSTs in the embryo and embryonic eye had GTN metabolizing activity. NAC co-treatment partially protected against the effects of GTN exposure. Thus, GTN denitration by quail embryo GSTs may represent a key initial step in the developmental toxicity of GTN.

Structural analysis of an epsilon-class glutathione transferase from housefly, Musca domestica

Glutathione transferases (GSTs) play an important role in the detoxification of insecticides, and as such, they are a key contributor to enhanced resistance to insecticides. In the housefly (Musca domestica), two epsilon-class GSTs (MdGST6A and MdGST6B) that share high sequence homology have been identified, which are believed to be involved in resistance against insecticides. The structural determinants controlling the substrate specificity and enzyme activity of MdGST6s are unknown. The aim of this study was to crystallize and perform structural analysis of the GST isozyme, MdGST6B. The crystal structure of MdGST6B complexed with reduced glutathione (GSH) was determined at a resolution of 1.8Å. MdGST6B was found to have a typical GST folding comprised of N-terminal and C-terminal domains. Arg113 and Phe121 on helix 4 were shown to protrude into the substrate binding pocket, and as a result, the entrance of the substrate binding pocket was narrower compared to delta- and epsilon-class GSTs from Africa malaria vector Anopheles gambiae, agGSTd1-6 and agGSTe2, respectively. This substrate pocket narrowing is partly due to the presence of a π-helix in the middle of helix 4. Among the six residues that donate hydrogen bonds to GSH, only Arg113 was located in the C-terminal domain. Ala substitution of Arg113 did not have a significant effect on enzyme activity, suggesting that the Arg113 hydrogen bond does not play a crucial role in catalysis. On the other hand, mutation at Phe108, located just below Arg113 in the binding pocket, reduced the affinity and catalytic activity to both GSH and the electrophilic co-substrate, 1-chloro-2,4-dinitrobenzene.

Overexpression of poplar GSTU51 confers selective tolerance to both mercury and methyl viologen but not to CDNB or cadmium in transgenic poplars

Glutathione S-transferases belong to a large ancient gene family and are thought to be one of the effective detoxification systems. To elucidate the function of the gene in poplar, a tau class gst gene (PatgGSTU51) was cloned from poplar cell suspension cDNA library and its expression was examined. The gene was not expressed in normal conditions, but significantly induced by toxic heavy metals like cadmium and inorganic mercury. However, the highest expression was observed when treated with its synthetic substrate, 1-chloro-2,4-dinitrobenzene (CDNB). Several transgenic poplar lines harboring a chimeric p35S-PatgGSTU51 were developed to understand its function in plant defense. Real-time quantitative PCR, western blot and cellular GST activities consistently showed the transgene was highly expressed in the transgenic lines. The transgenic lines showed increased tolerance to methyl viologen as well as to inorganic mercury but not to cadmium. Furthermore, they did not show any significant tolerance against CDNB, the electrophilic substrate of GST isozymes. Thus, the results suggest that, although PatgGSTU51 is induced by a number of stress agents, it confers a selective tolerance to the toxicity of heavy metal mercury over those of other stress agents and also to oxidation stress caused by MV. Tolerance to CDNB that induces the gene to high level may require such an extra supplemental gene action as vacuolar sequestration.

Occupation of the electrophilic substrate‐binding site of glutathione S‐transferase M1‐1 enhances its function as an activator of 1‐cysteine peroxiredoxin

Glutathione S‐transferase mu (GST‐mu) can form an in vitro heterodimer with 1‐cysteine peroxiredoxin, peroxiredoxin 6 (Prdx6), in the presence of glutathione (GSH), but the peroxidase specific activity of this complex is only 1.0 μmol/min/mg Prdx6, which is ~20% of that of the complex between the pi isozyme of GST and Prdx6 under the same conditions [L.A. Ralat, Y. Manevich, A.B. Fisher, R.F. Colman, Biochemistry 45 (2006) 360–372]. These results suggested that the interactions are different between Prdx6 and GST isozymes, which share an identity of less than 30%. Here, we show that Prdx6 can reduce hydrogen peroxide at 4.7 ± 0.3 μmol/min/mg Prdx6, as assayed using the GSH reductase/GSH/NADPH‐coupled GSH‐peroxidase method, after a 1‐hour incubation with a 1:1 molar ratio of GST‐mu at pH 8, containing saturating amounts of GSH and an electrophilic GST substrate analog, 2,4‐dinitrophenol. Interestingly, after this same incubation, GST‐mu lost all of its activity with respect to monobromobimane, a substrate that occupies a distinguishable site from 2,4‐dinitrophenol. These results suggest that occupation of the electrophilic site of GST‐mu may render a conformational change which optimizes its interaction with Prdx6, allowing the transfer of GSH to the inactive Cys‐47−sulfenic acid form of Prdx6, regenerating an active Prdx6, while inhibiting GST‐mu activity. Supported by NIH/NHLBI R01554495.

Amitriptyline may have a supportive role in cancer treatment by inhibiting glutathione S-transferase pi (GST-π) and alpha (GST-α)

A tricyclic anti-depressant, amitriptyline, is a highly prescribed drug for cancer patients for mood elevation but there are limited studies about the interaction of amitriptyline with glutathione S-transferases pi (GST-π) and glutathione S-transferases alpha (GST-α). GST isozymes have been implicated in chemotherapeutic drug resistance. We demonstrated that the concentration dependent inhibition of GST-π and GST-α by amitriptyline followed inverse hyperbolic inhibition curves with IC50 values of 5.54 and 8.32 mM, respectively.When the varied substrate was GSH, amitriptyline inhibited both isozymes competitively and similar Ki values were found for GST-π (Ki = 1.61 ± 0.17 mM) and GST-α (Ki = 1.45 ± 0.20 mM). On the other hand, when the varied substrate was CDNB, the inhibition types were non-competitive for GST-π (Ki = 1.98 ± 0.31 mM) and competitive for GST-α (Ki = 1.57 ± 0.16 mM).Amitriptyline, in addition to its antidepressant effect, might also have a minor supportive role on the effectiveness of the anticancer drugs by decreasing their elimination through inhibiting GST-π and GST-α.

Influence of GST Gene Polymorphisms on the Clearance of Intravenous Busulfan in Adult Patients Undergoing Hematopoietic Cell Transplantation

Intravenous (i.v.) busulfan can produce a more consistent pharmacokinetic profile than oral formulations can, but nonetheless, significant interpatient variability is evident. We investigated the influence of polymorphisms of 3 GST isozyme genes (GSTA1, GSTM1, and GSTT1) on i.v. busulfan clearance. Fifty-eight adult patients who received 3.2 mg/kg/day of busulfan as conditioning for hematopoietic cell transplantation were included in this study. Stepwise multiple linear regression demonstrated that GSTA1 variant GSTA1∗B (P = .004), GSTM1/GSTT1 double-null genotype (P = .039), and actual body weight (P = .001) were significantly associated with lower clearance of i.v. busulfan. A trend test analyzing the overall effect of GST genotype on busulfan pharmacokinetics, combining GSTA1 gene polymorphism and the number of GSTM1- and GSTT-null genotypes, showed a significant correlation between GST genotype and busulfan clearance (P = .001). The clearance of i.v. busulfan was similar between patients with GSTA1∗A/∗A and GSTM1/GSTT1 double-null genotypes and those with GSTA1∗A/∗B and GSTM1/GSTT1 double-positive genotypes. In conclusion, a pharmacogenetic approach using GST gene polymorphisms may be valuable in optimizing the i.v. busulfan dosage scheme. Our results also highlight the importance of including polygenic analyses and addressing interactions among isozyme genes in pharmacogenetic studies.

Role of alpha class glutathione transferases (GSTs) in chemoprevention: GSTA1 and A4 overexpressing human leukemia (HL60) cells resist sulforaphane and curcumin induced toxicity

Alpha-class glutathione transferases (α-GSTs) have been shown to protect cells from the harmful effects of reactive oxygen species (ROS) induced lipid peroxidation (LPO) during oxidative stress caused by various physico-chemical agents. While GSTA1-1/A2-2 isozymes exhibit high activity towards lipid and fatty acid hydroperoxides through their selenium independent glutathione peroxidase (GPx) activity, the GSTA4-4 isozyme efficiently metabolizes the LPO product 4-hydroxynonenal (4-HNE) by conjugating it with glutathione (GSH). Because of the fact that ROS generated by the chemopreventive agents, sulforaphane (SFN) and curcumin (Cur), are implicated in the mechanisms of cancer cell killing, the present studies were designed to investigate the contribution of ROS induced LPO in the cytotoxic effects of these agents and the role of α-class GSTs in modulating their toxicity. Human erythroleukemic (HL60) cells were stably transfected with the cDNA encoding the hGSTA1-1 and mGsta4-4 isozymes. After analysing the expression and activities of the respective GST isozymes, the effects of SFN and Cur on the extent of LPO, cytotoxicity and apoptosis were compared in empty vector (VT), hGSTA1-1 and mGsta4-4 expressing HL60 cells. These studies demonstrate that when compared with SFN, Cur was relatively more cytotoxic to HL60 cells. The ectopic expression of hGSTA1-1 and mGsta4-4 isozymes provided resistance to SFN and Cur induced cytotoxicity and apoptosis through a significant suppression of LPO in these cells. Overall, the results suggest that the expression of α-class GSTs in cancer cells can modulate the therapeutic efficacy of chemopreventive agents.