Overexpression Of Glutathione S-transferases Research Articles

Rapid discovery of a novel “green” and natural GST inhibitor for sensitizing hepatocellular carcinoma to Cisplatin by visual screening strategy

Over-expression of glutathione S-transferase (GST) can promote Cisplatin resistance in hepatocellular carcinoma (HCC) treatment. Hence, inhibiting GST is an attractive strategy to improve Cisplatin sensitivity in HCC therapy. Although several synthesized GST inhibitors have been developed, the side effects and narrow spectrum for anticancer seriously limit their clinical application. Considering the abundance of natural compounds with anticancer activity, this study developed a rapid fluorescence technique to screen “green” natural GST inhibitors with high specificity. The fluorescence assay demonstrated that schisanlactone B (hereafter abbreviated as C1) isolated from Xue tong significantly down-regulated GST levels in Cisplatin-resistant HCC cells in vitro and in vivo. Importantly, C1 can selectively kill HCC cells from normal liver cells, effectively improving the therapeutic effect of Cisplatin on HCC mice by down-regulating GST expression. Considering the high GST levels in HCC patients, this compound demonstrated the high potential for sensitizing HCC therapy in clinical practice by down-regulating GST levels.

Overexpression of Glutathione S-Transferases in Human Diseases: Drug Targets and Therapeutic Implications.

Glutathione S-transferases (GSTs) are a major class of phase II metabolic enzymes. Besides their essential role in detoxification, GSTs also exert diverse biological activities in the occurrence and development of various diseases. In the past few decades, much research interest has been paid to exploring the mechanisms of GST overexpression in tumor drug resistance. Correspondingly, many GST inhibitors have been developed and applied, solely or in combination with chemotherapeutic drugs, for the treatment of multi-drug resistant tumors. Moreover, novel roles of GSTs in other diseases, such as pulmonary fibrosis and neurodegenerative diseases, have been recognized in recent years, although the exact regulatory mechanisms remain to be elucidated. This review, firstly summarizes the roles of GSTs and their overexpression in the above-mentioned diseases with emphasis on the modulation of cell signaling pathways and protein functions. Secondly, specific GST inhibitors currently in pre-clinical development and in clinical stages are inventoried. Lastly, applications of GST inhibitors in targeting cell signaling pathways and intracellular biological processes are discussed, and the potential for disease treatment is prospected. Taken together, this review is expected to provide new insights into the interconnection between GST overexpression and human diseases, which may assist future drug discovery targeting GSTs.

The mechanisms of metabolic resistance to pyrethroids and neonicotinoids fade away without selection pressure in the tarnished plant bug Lygus lineolaris.

Heavy selection pressure prompted the development of resistance in a serious cotton pest tarnished plant bug (TPB), Lygus Lineolaris in the mid-southern USA. Conversely, a laboratory resistant TPB strain lost its resistance to five pyrethroids and two neonicotinoids after 36 generations without exposure to any insecticide. It is worthwhile to examine why the resistance diminished in this population and determine whether the resistance fade away has practical value for insecticide resistance management in TPB populations. A field-collected resistant TPB population in July (Field-R1) exhibited 3.90-14.37-fold resistance to five pyrethroids and two neonicotinoids, while another field-collected TPB population in April (Field-R2) showed much lower levels of resistance (0.84-3.78-fold) due to the absence of selection pressure. Interestingly, after 36 generations without exposure to insecticide, the resistance levels in the same population (lab-R) significantly decreased to 0.80-2.09-fold. The use of detoxification enzyme inhibitors had synergistic effects on permethrin, bifenthrin and imidacloprid in resistant populations of L. lineolaris. The synergism was more pronounced in field-R2 than lab-S and lab-R TPB population. Moreover, esterase, GST, and P450 enzyme activities increased significantly by approximately 1.92-, 1.43-, and 1.44- fold in field-R1, respectively, and 1.38-fold increased P450 enzyme activities in field-R2 TPB population, compared to the lab susceptible (lab-S) TPB. In contrast, the three enzyme activities in the lab-R strain were not significantly elevated anymore relative to the lab-S population. Additionally, field-R1 TPB showed elevated expression levels of certain esterase, GST and P450 genes, respectively, while field-R2 TPB overexpressed only P450 genes. The elevation of these gene expression levels in lab-R expectedly diminished to levels close to those of the lab susceptible (lab-S) TPB populations. Our results indicated that the major mechanism of resistance in TPB populations was metabolic detoxification, and the resistance development was likely conferred by increased gene expressions of esterase, GST, and P450 genes, the fadeaway of the resistance may be caused by reversing the overexpression of esterase, GST and P450. Without pesticide selection, resistant gene (esterase, GST, P450s) frequencies declined, and detoxification enzyme activities returned to lab-S level, which resulted in the recovery of the susceptibility in the resistant TPB populations. Therefore, pest's self-purging of insecticide resistance becomes strategically desirable for managing resistance in pest populations. This article is protected by copyright. All rights reserved.

The Multifaceted Role of Glutathione S-Transferases in Health and Disease.

In humans, the cytosolic glutathione S-transferase (GST) family of proteins is encoded by 16 genes presented in seven different classes. GSTs exhibit remarkable structural similarity with some overlapping functionalities. As a primary function, GSTs play a putative role in Phase II metabolism by protecting living cells against a wide variety of toxic molecules by conjugating them with the tripeptide glutathione. This conjugation reaction is extended to forming redox sensitive post-translational modifications on proteins: S-glutathionylation. Apart from these catalytic functions, specific GSTs are involved in the regulation of stress-induced signaling pathways that govern cell proliferation and apoptosis. Recently, studies on the effects of GST genetic polymorphisms on COVID-19 disease development revealed that the individuals with higher numbers of risk-associated genotypes showed higher risk of COVID-19 prevalence and severity. Furthermore, overexpression of GSTs in many tumors is frequently associated with drug resistance phenotypes. These functional properties make these proteins promising targets for therapeutics, and a number of GST inhibitors have progressed in clinical trials for the treatment of cancer and other diseases.

MRNA Sequencing Reveals Upregulation of Glutathione S-Transferase Genes during Acanthamoeba Encystation.

Some members of the genus Acanthamoeba are facultative pathogens typically with a biphasic lifestyle: trophozoites and cysts. Acanthamoeba is capable of infecting the cornea, resulting in Acanthamoeba keratitis. The cyst is one of the key components for the persistence of infection. Gene expression during Acanthamoeba encystation showed an upregulation of glutathione S-transferase (GST) genes and other closely related proteins. mRNA sequencing showed GST, and five genes with similar sequences were upregulated after 24 h of inducing encystation. GST overexpression was verified with qPCR using the HPRT and the cyst-specific protein 21 genes as controls. The GST inhibitor ethacrynic acid was found to decrease cell viability by 70%. These results indicate a role of GST in successful encystation, possibly by maintaining redox balance. GST and associated processes could be targets for potential treatments alongside regular therapies to reduce relapses of Acanthamoeba infection.

Alpha-class glutathione S-transferases involved in the detoxification of aflatoxin B1 in ducklings.

The objective of this study was to identify the key glutathione S-transferase (GST) isozymes involved in the detoxification of Aflatoxin B1 (AFB1) in ducks' primary hepatocytes. The full-length cDNA encoding the 10 GST isozymes (GST, GST3, GSTM3, MGST1, MGST2, MGST3, GSTK1, GSTT1, GSTO1 and GSTZ1) were isolated/synthesized from ducks' liver and cloned into the pcDNA3.1(+) vector. The results showed that pcDNA3.1(+)-GSTs plasmids were successfully transferred into the ducks' primary hepatocytes and the mRNA of the 10 GST isozymes were overexpressed by 1.9-3274.7 times. Compared to the control, 75μg/L (IC30) or 150μg/L (IC50) AFB1 treatment reduced the cell viability by 30.0-50.0% and increased the LDH activity by 19.8-58.2% in the ducks' primary hepatocytes. Notably, the AFB1-induced changes in cell viability and LDH activity were mitigated by overexpression of GST and GST3. Compared to the cells treated with AFB1, exo-AFB1-8,9-epoxide (AFBO)-GSH, as the major detoxified product of AFB1, was increased in the cells overexpression of GST and GST3. Moreover, the sequences, phylogenetic and domain analysis revealed that the GST and GST3 were orthologous to Meleagris gallopavo GSTA3 and GSTA4. In conclusion, this study found that the ducks' GST and GST3 were orthologous to Meleagris gallopavo GSTA3 and GSTA4, which were involved in the detoxification of AFB1 in ducks' primary hepatocytes.

Enhanced soluble expression of glutathione S-transferase Mu from Rutilus kutum by co-expression with Hsp70 and introducing a novel inhibitor for its activity

One of the most critical challenges in recombinant protein production is the increment of protein solubility. The overexpression of glutathione S-transferases (GSTs) in cancers leads to the fast detoxification of drug substrates that is subsequently followed by increased drug resistance. In this study, the co-expression of Rutilus kutum Hsp70 (RkHsp70) with the mu-class GST (RkGST-μ) from the same source was analyzed to increase the solubility of aggregate-prone RkGST-μ. Based on the results, the co-expressed RkGST-μ cells showed substantially higher solubility and specific activity compared to control. The kinetic properties of the enzyme were measured and its optimum pH and temperature were observed at pH 8 and 35 °C. Furthermore, this study introduces ZM-093, a sulfamethoxazole-based azo with IC50 values of 24.45 μM (p < 0.0001), as a novel RkGST-μ inhibitor. Overall, the present study provides clear findings in support of using molecular chaperones to enhance the solubility of recombinant proteins.

Genome Mining and Expression Analysis of Carboxylesterase and Glutathione S-Transferase Genes Involved in Insecticide Resistance in Eggplant Shoot and Fruit Borer, Leucinodes orbonalis (Lepidoptera: Crambidae).

The shoot and fruit borer, Leucinodes orbonalis (Lepidoptera: Crambidae) is the major cause of low productivity in eggplant and insecticides being the mainstay of management of L. orbonalis. However, field control failures are widespread due to the evolution of insecticide resistance. Taking advantage of the whole genome sequence information, the present study investigated the level of insecticide resistance and the expression pattern of individual carboxylesterase (CE) and glutathione S-transferases (GSTs) genes in various field collected populations of L. orbonalis. Dose-mortality bioassays revealed a very high level of resistance development against fenvalerate (48.2–160-fold), phosalone (94-534.6-fold), emamectin benzoate (7.2–55-fold), thiodicarb (9.64–22.7-fold), flubendiamide (187.4–303.0-fold), and chlorantraniliprole (1.6–8.6-fold) in field populations as compared to laboratory-reared susceptible iso-female colony (Lo-S). Over-production of detoxification enzymes viz., CE and GST were evident upon enzyme assays. Mining of the draft genome of L. orbonalis yielded large number of genes potentially belonging to the CE and GST gene families with known history of insecticide resistance in other insects. Subsequent RT-qPCR studies on relative contribution of individual genes revealed over-expression of numerous GSTs and few CEs in field populations, indicating their possible involvement of metabolic enzymes in insecticide resistance. The genomic information will facilitate the development of novel resistance management strategies against this pest.

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases.

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)-two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 ± 3.6 µM and a Ki of 23.2 ± 3.2 µM. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

Caffeic Acid Phenethyl Ester Assisted by Reversible Electroporation-In Vitro Study on Human Melanoma Cells.

Melanoma is one of the most serious skin cancers. The incidence of this malignant skin lesion is continuing to increase worldwide. Melanoma is resistant to chemotherapeutic drugs and highly metastatic. Surgical resection can only be used to treat melanoma in the early stages, while chemotherapy is limited due to melanoma multi-drug resistance. The overexpression of glutathione S-transferase (GST) may have a critical role in this resistance. Caffeic acid phenethyl ester (CAPE) is a natural phenolic compound, which occurs in many plants. Previous studies demonstrated that CAPE suppresses the growth of melanoma cells and induces reactive oxygen species generation. It is also known that bioactivation of CAPE to its corresponding quinone metabolite by tyrosinase would lead to GST inhibition and selective melanoma cell death. We investigated the biochemical toxicity of CAPE in combination with microsecond electropermeabilization in two human melanoma cell lines. Our results indicate that electroporation of melanoma cells in the presence of CAPE induced high oxidative stress, which correlates with high cytotoxicity. Moreover, it can disrupt the metabolism of cancer cells by inducing apoptotic cell death. Electroporation of melanoma cells may be an efficient CAPE delivery system, enabling the application of this compound, while reducing its dose and exposure time.

Stimulatory effect of copper and zinc sulphate on plant regeneration, glutathione-S-transferase analysis and assessment of antioxidant activities in Mucuna pruriens L. (DC)

The effect of heavy metals, CuSO4 and ZnSO4 on morphogenic response of cotyledonary node explants of Mucuna pruriens (L.), a fast growing tropical medicinal legume was evaluated. The climber holds economic importance in traditional as well as modern pharmaceutical areas. Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with various strength (0.5, 2.5, 4.5, 6.5 or 8.5 µM) of BA (6-benzyladenine), Kn (Kinetin) and 2-iP (2-isopentenyl adenine) were screened to establish optimum culture conditions for in vitro regeneration using cotyledonary node explant. Optimized medium (MS + BA 2.5 µM) was augmented with different concentration of CuSO4 (0.5–25.0 µM) and ZnSO4 (5–150 µM) to evaluate the effect of heavy metals on regeneration potential of the explants. Among various concentrations tested, MS medium with optimized BA (2.5 µM) along with 5.0 µM CuSO4 or 80.0 µM ZnSO4 proved beneficial, inducing 11.20 and 13.40 shoots per explant, respectively. The results demonstrated that glutathione-S-transferase (GSTs) was involved in fighting heavy metal stress, and might also have a role in in vitro regeneration. Elongated healthy microshoots on transfer to half strength MS medium produced 3.40 roots per shoot after 28 days of incubation without the necessity of exogenous auxin. The in vitro grown regenerants were hardened in Soilrite, and then established in garden and sandy soil (1:1) under natural light where they showed normal growth and development. The work highlights the enhanced multiplication and better growth of the regenerants using elevated concentration of CuSO4 and ZnSO4. GSTs overexpression activity not only confers to overcome the stress but enhanced the in vitro regeneration of M. pruriens also.

Genetic Clearness Novel Strategy of Group I Bacillus Species Isolated from Fermented Food and Beverages by Using Fibrinolytic Enzyme Gene Encoding a Serine-Like Enzyme.

Fibrinolytic enzyme gene (fibE) is widely conserved among Bacillus spp. belonging to group I species. This is encoding a serine-like enzyme (FibE) secreted in extracellular medium. This present work aims to assess the molecular usefulness of this novel conserved housekeeping gene among group I Bacillus spp. to identify and discriminate some related strains in traditional fermented food and beverages in Republic of Congo. First of all 155 isolates have been screened for enzymatic activities using caseinolytic assays. PCR techniques and nested PCR method using specific primers and correlated with 16S RNA sequencing were used. Blotting techniques have been performed for deep comparison with molecular methods. As a result B. amyloliquefaciens (1), B. licheniformis (1), B. subtilis (1), B. pumilus (3), B. altitudinis (2), B. atrophaeus (1), and B. safensis (3) have been specifically identified among 155 isolates found in fermented food and beverages. Genetic analysis and overexpression of glutathione S-transferases (GSTs) fused to mature protein of FibE in Escherichia coli BL21 and TOP10 showed 2-fold higher enzymatic activities by comparison with FibE wild type one. Immunodetection should be associated but this does not clearly discriminate Bacillus belonging to group I.

Multifunctional Pt(iv) complexes containing a glutathione S-transferase inhibitor lead to enhancing anticancer activity and preventing metastasis of osteosarcoma cells.

Cisplatin has been clinically applied in the treatment of osteosarcoma (OS), but its efficacy is severely limited due to drug resistance and metastasis. One of the chief culprits is the overexpression of glutathione S-transferases (GSTs) in cancer cells, which can accelerate the interaction of glutathione (GSH) with cisplatin, reducing its biological effects. In this study, three Pt(iv) complexes derived from cisplatin conjugated with a GST inhibitor (NBDHEX) were designed and synthesized. The stabilities and releasing capabilities of these complexes, as well as their abilities to inhibit GSTs, were investigated together with their in vitro anticancer activities toward osteosarcoma cells. Among them, complex 2, bearing one NBDHEX derivative and a hydroxyl group at the axial positions, could markedly kill human OS cells due to its suitable stability and prominent ability to inhibit GSTs. Meanwhile, it can prevent the metastasis of OS via down-regulating Akt. Thus, complex 2 has the potential for further research for the treatment of OS.

Glutathione S-Transferase Enzymes in Plant-Pathogen Interactions.

Plant glutathione S-transferases (GSTs) are ubiquitous and multifunctional enzymes encoded by large gene families. A characteristic feature of GST genes is their high inducibility by a wide range of stress conditions including biotic stress. Early studies on the role of GSTs in plant biotic stress showed that certain GST genes are specifically up-regulated by microbial infections. Later numerous transcriptome-wide investigations proved that distinct groups of GSTs are markedly induced in the early phase of bacterial, fungal and viral infections. Proteomic investigations also confirmed the accumulation of multiple GST proteins in infected plants. Furthermore, functional studies revealed that overexpression or silencing of specific GSTs can markedly modify disease symptoms and also pathogen multiplication rates. However, very limited information is available about the exact metabolic functions of disease-induced GST isoenzymes and about their endogenous substrates. The already recognized roles of GSTs are the detoxification of toxic substances by their conjugation with glutathione, the attenuation of oxidative stress and the participation in hormone transport. Some GSTs display glutathione peroxidase activity and these GSTs can detoxify toxic lipid hydroperoxides that accumulate during infections. GSTs can also possess ligandin functions and participate in the intracellular transport of auxins. Notably, the expression of multiple GSTs is massively activated by salicylic acid and some GST enzymes were demonstrated to be receptor proteins of salicylic acid. Furthermore, induction of GST genes or elevated GST activities have often been observed in plants treated with beneficial microbes (bacteria and fungi) that induce a systemic resistance response (ISR) to subsequent pathogen infections. Further research is needed to reveal the exact metabolic functions of GST isoenzymes in infected plants and to understand their contribution to disease resistance.

Sulfur mustard triggers oxidative stress through glutathione depletion and altered expression of glutathione-related enzymes in human airways

Context: Sulfur mustard (SM) is a lipophilic and reactive chemical compound that targets human airway system.Objective: Glutathione (GSH) depletion, oxidative stress (OS) status, and changes in expression of GSH-dependent antioxidant enzymes were considered in human mustard lungs.Materials and methods: Lung biopsies and bronchoalveolar lavage (BAL) were collected from non-exposed (n = 10) individuals and SM-exposed patients (n = 12). Alterations in expression of GSH-dependent enzymes were studied using RT2 Profiler™ PCR array. OS was evaluated by determining BAL fluid levels of total antioxidant capacity (TAC), malondialdehyde (MDA), and GSH.Results: Mean TAC (0.142 ± 0.027 µmol/l) and GSH (4.98 ± 1.02 nmol/l) in BAL fluids of control group was significantly higher (p < .05) than those in SM-exposed patients (TAC = 0.095 ± 0.018 µmol/l and GSH= 3.09 ± 1.02 nmol/l), while MDA level in BAL fluids of these patients (0.71 ± 0.06 nmol/l) was significantly (p = .001) higher than that in controls (0.49 ± 0.048 nmol/l). Glutathione peroxidases (GPXs), glutathione-s-transferases (GSTs), and glutathione synthetase (GSS) enzymes were overexpressed in mustard lung biopsies, while glutathione reductase (GSR) was significantly downregulated (14.95-fold).Conclusions: GSH depletion induced by GSR downregulation may be a major mechanism of SM toxicity on human lung. Despite overexpression of GSTs and GPXs genes, GSH depletion may decline the productivity of these enzymes and total antioxidants capacity, which is associated with OS.

Overcoming resistance to cisplatin by inhibition of glutathione S-transferases (GSTs) with ethacraplatin micelles in vitro and in vivo

Platinum-based DNA-adducting agents are used extensively in the clinic for cancer chemotherapy. However, the anti-tumor efficacy of these drugs is severely limited by cisplatin resistance, and this can lead to the failure of chemotherapy. One of cisplatin resistance mechanisms is associated with overexpression of glutathione S-transferases (GSTs), which would accelerate the deactivation of cisplatin and decrease its antitumor efficiency. Nanoscale micelles encapsulating ethacraplatin, a conjugate of cisplatin and ethacrynic acid (an effective GSTs inhibitor), can enhance the accumulation of active cisplatin in cancer cells by inhibiting the activity of GSTs and circumventing deactivation of cisplatin. Invitro and invivo results provide strong evidence that GSTs inhibitor-modified cisplatin prodrug combined with nanoparticle encapsulation favor high effective platinum accumulation, significantly enhanced antitumor efficacy against cisplatin-resistant cancer and decreased system toxicity. It is believed that these ethacraplatin-loaded micelles have the ability of overcoming resistance of cancers toward cisplatin and will improve the prospects for chemotherapy of cisplatin-resistant cancers in the near future.

Abstract 5634: Characterization of molecular mechanisms of cytoplasmic trafficking and nuclear translocation of AR splice variants ARv7

Abstract Androgen receptor (AR) signaling is critical to not only hormone-sensitive but also advanced castration-resistant prostate cancer. AR inhibitors (abiraterone and enzalutamide), the next generation of androgen deprivation therapy (ADT) have been used for metastatic castration-resistant PC (mCRPC) treatment, however, the majority of patients progress due to the development of drug resistance. AR variants has emerged as one of the mechanisms of resistance to these drugs. ARv7 and Arv567 splice variants that found lacking the ligand -binding domain are constitutively active in the nucleus and thus restore AR function despite AR inhibitors treatment. We have reported that microtubules and dynein motor protein is required as transportation system for AR for its nuclear translocation and activity and that taxanes inhibit AR signaling downstream of microtubule inhibitors. In addition, we identified that the AR hinge region mediates binding to microtubules is present in ARv567 but missing from ARv7. ARv7 is expressed in about 60% of CRPC patients and has been shown to confer clinical resistance to next generation AR signaling inhibitors. Currently, there is no therapeutic modality targeting specifically ARv7 expression or function. Using fluorescent recovery after photobleaching experiments (FRAP) we have shown that ARv7 translocation to the nucleus occurs much faster than AR-FL (t1/2 11s and 23 s respectively) and that its nuclear import is independent of the importin a/b pathway utilized by AR-FL and proteins with canonical NLS. Furthermore, transient expression of the Q69L RanGTP mutant protein, which acts as dominant negative by occluding the nucleoporins, had minimal effect on ARv7 nuclear import, while it completely blocked AR-FL and AR-v567. Collectively, these data suggest that the nuclear import pathway for ARv7 is distinct from that utilized by AR-FL. We believe that ARv7 undoubtedly uses facilitated transport because of its size (75 kDa), therefore we are tackling three possibilities of nuclear translocation of ARv7. First, ARv7 could interact directly with FG repeat nucleoporins (without receptor) as reported for beta catenin. We are investigating this by the use of importin β-(71-876) mutant cell line for in vitro studies which blocks all members of the importin family. In addition, we are investigating the role of karyopherin transport receptors by individual knockdown experiments coupled with live cell imaging and kinetic quantitation of ARv7 nucleocytoplasmic trafficking. Further, ARv7could translocate to nucleus by utilizes a piggyback on another protein with a classical nuclear localization signals. Overexpression of glutathione S-transferase- importin β binding domain fragment which will block all importin β pathways will exclude this mechanism. Elucidation of this mechanism will be vital to facilitate design an alternative therapeutic modality to halt AR signaling for CRPC treatment. Citation Format: Eiman Mukhtar, Seaho Kim, Paraskevi Giannakakou. Characterization of molecular mechanisms of cytoplasmic trafficking and nuclear translocation of AR splice variants ARv7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5634. doi:10.1158/1538-7445.AM2017-5634

Synchronous overexpression of glutathione-S-transferase and cyanidase maintains the redox homeostasis ‎and improves cyanide remediation capacity in tobacco

Genetically engineered plants could provide feasible and environmentally safe approach for phytoremediation of CN compounds. However, the survival capacity of transgenic plants under high doses of CN is disappointing. One of the possible causes of such phytotoxicity is the CN-induced inhibition of antioxidant systems that lead to accumulation of reactive oxygen species (ROS). Therefore, simultaneous overexpression of a CN degrading enzyme along with an ‎oxidative stress-relieving protein could maintain the internal redox homeostasis and enhance CN-phytoremediation capacity under high doses of CN. In the present study, transgenic tobacco plants overexpressing the ‎bacterial cyanidase (CYND) and the cyanobacterial glutathione-S-transferase (GST) separately ‎and ‎in combination have been generated. Significant growth recovery was observed for all transgenic plants under 10mM CN compared to wild type plants. However, GST+CYND overexpressors showed the highest biomass accumulation under CN stress. Similarly, reduction in photosynthetic pigments and total carbohydrate levels were significantly recovered in transgenic plants especially at higher doses of CN (7.5 and 10.0mM). Moreover, both GST and GST+CYND transgenic plants showed elevated activities of SOD and CAT compared to the wild type upon exposure to CN. MDA content, as an indicator of lipid peroxidation, was significantly decreased in all transgenic plants. However, the lowest level of MDA was observed in GST+CYND transgenic lines. These results suggest that synchronous overexpression of GST with CYND genes improves CN remediation capacity of tobacco by improvement of the antioxidant scavenging systems and keeping ROS under control.

Glutathione-Associated Enzymes In Anticancer Drug Resistance.

The importance of thiol-mediated detoxification of anticancer drugs that produce toxic electrophiles has been of considerable interest to many investigators. Glutathione and glutathione S-transferases (GST) are the focus of much attention in characterizing drug resistant cells. However, ambiguous and sometimes conflicting data have complicated the field. This article attempts to clarify some of the confusion. The following observations are well established: (a) tumors express high levels of GST, especially GST psi, although the isozyme components vary quite markedly between tissues and the isozymes are inducible; (b) nitrogen mustards are good substrates for the GST alpha family of isozymes which are frequently overexpressed in cells with acquired resistance to these drugs; (c) most drugs of the multidrug-resistant phenotype have not been shown to be GST substrates and although GST psi is frequently overexpressed in multidrug-resistant cells, most indications are that this is an accompaniment to, rather than a cause of, the resistant phenotype; (d) transfection of GST complementary DNAs has produced some lines with increased resistance to alkylating agents. Most studies of the relationships between GST and resistance have overlooked the potential importance of other enzymes involved in the maintenance of cellular glutathione homeostasis, and this has complicated data interpretation. Translational research aimed at applying our knowledge of glutathione pathways has produced preclinical and clinical testing of some glutathione and GST inhibitors, with some encouraging preliminary results. In brief, GSTs are important determinants of drug response for some, not all, anticancer drugs. Caution should be encouraged in assessing cause/effect relationships between GST overexpression and resistance mechanisms.

A novel anti-chemoresistance agent designed from natural products by targeting GSTO

The biological function of glutathione s-transferases (GSTs), a key member of phase II drug-metabolizing enzymes, plays an essential role in both cell detoxification and oxidative stress regulation through redox-mediated mechanism. Overexpression of GSTs in tumors is frequently associated with high capacity of detoxification, thereby endowing cancer cells with an increased capacity of drug detoxification even in the presence of chemotherapeutic agents. The correlation between GSTs and malignancy has been widely discussed, but the role and function of GSTs in tumorigenesis, metastasis, and drug resistance are not completely understood. Recently, we found that overexpressing GSTO1 is highly associated with tumor malignance. Protoapigenone (WYC02), first isolated from Thelypteris torresiana, show the anticancer activity. However, moderate effects of WYC02 impinged drug development. Recently, we have successfully synthesized the new generation of anticancer agents based on natural product protoapigenone for targeting GSTO in cancer. Two straightforward strategies including structure-based and ligand-based drug design were employed for best optimization. We conducted a structure-based designing strategy to modify natural product by introducing functional side chains mimicking GSH, a substrate of GST. Our team has also constructed a well-established pharmacophore for GSTO inhibitor. The newly design GSTO inhibitor shows the promising anticancer activity against chemoresistance. Furthermore, at least three classes of chemotherapeutic agents exhibit strong synergistic effects with the GSTO inhibitor. Altogether, we provide a potential therapeutic way for GSTO via naturally occurring compounds. Most importantly, this finding hopes to inspire further drug development.