The Glutathione-S-transferases exist as cytosolic, mitochondrial, and microsomal which can participate in signal transduction by not phosphorylating any factor that is directly involved in which cell growth and death. This signal transduction is considered to be a new way of implication in cell metabolic pathways due to the influence of external, such as xenobiotics and UV radiation, and internal, such as oxidative stress, free radicals, etc., agents. The GST binding assay studies revealed that they participate in the inhibition of various proteins, for example, phosphoproteins, AP-1, JNK, etc., in the systems to regulate cell mechanisms during cell synthesis.
Biomembranes and Signal Transduction
A biomembrane is an enclosing or separating membrane that acts as a selective barrier, within or around a cell. It consists of a lipid bilayer with embedded proteins that may constitute close to 50% of membrane content (Mark Latash 2007). It has a selective permeable structure, which is essential for effective separation of a cell or organelle from its surroundings. Biomembrane has certain mechanical or elastic properties. These membranes contain receptors on their surface and receive signals from various influences. During this process an extracellular signaling molecule activates a membrane receptor that in turn alters intracellular molecules creating a response (Silverthorn 2006) called signal transduction. In signal transduction, the chemical signal binds to the outer portion of the transmembrane receptor, changing its shape and conveying another signal inside the cell. Some chemical messengers, like testosterone, can pass through the cell membrane, and bind directly to receptors in the cytoplasm or nucleus. Sometimes these chemicals can have a cascade of amplified signals, so a small signal can result in a large response (Reece and Campbell 2002). Eventually, the signal creates a change in the cell, either in the expression of the DNA in the nucleus or in the activity of enzymes in the cytoplasm. These processes can take milliseconds (for ion flux), minutes (for protein- and lipid-mediated kinase cascades), hours, or days (for gene expression). In 1980, Martin Rodbell examined the effects of glucagon on a rat’s hepatocyte membrane receptor and noted that guanosine triphosphate disassociated glucagon from this receptor, and stimulated the G-protein, which heavily influenced the cell’s metabolism. Thus, he deduced that the G-protein was a transducer to accept glucagon molecules and affect the cell (Rodbell 1980). For this, he shared the 1994 Nobel Prize in Physiology/Medicine with Alfred G. Gilman. The current understanding of signal transduction processes reflects contributions made by Rodbell and many other research groups throughout the years.
Signal transduction involves the binding of extracellular signaling molecules, and intracellular signaling cascades can be started through cell–substratum interactions (Beato et al. 1996). Most of the chemicals have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter region of their responsive genes (Hammes 2003). Examples of signaling molecules include the hormone melatonin (Sugden et al. 2004), the neurotransmitter acetylcholine (Kistler et al. 1982), the cytokine interferon γ (Schroder et al. 2004), environmental stimuli, and certain microbial molecules. All of these may be independent of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling. Single-celled organisms may respond to environmental stimuli through the activation of signal transduction pathways. For example, slime molds secrete cyclic adenosine monophosphate upon starvation, stimulating individual cells in the immediate environment to aggregate (Hanna et al. 1984), and yeast cells use mating factors to determine the mating types of other cells and participate in sexual reproduction (Sprague 1991).
Receptors can be roughly divided into two major classes: extracellular receptors and intracellular receptors.
Extracellular receptors are integral transmembrane proteins and make up most receptors. They span the plasma membrane of the cell, with one part of the receptor on the outside of the cell and the other on the inside. Signal transduction occurs as a result of a ligand binding to the outside; the molecule does not pass through the membrane. This binding stimulates a series of events inside the cell; different types of receptor stimulate different responses and receptors typically respond to only the binding of a specific ligand. Upon binding, the ligand induces a change in the chemical confirmation of the inside part of the receptor. These result in either the activation of an enzyme in the receptor or the exposure of a binding site for other intracellular signaling proteins within the cell, eventually propagating the signal through the cytoplasm.
Intracellular receptors include nuclear receptors and cytoplasmic receptors, and are soluble proteins localized within the nucleoplasm or the cytoplasm, respectively. The typical ligands for nuclear receptors are lipophilic hormones, with steroid hormones (e.g., testosterone, progesterone, and cortisol) and derivatives of vitamin A and D among them. To reach its receptor and initiate signal transduction, the hormone must pass through the plasma membrane, usually by passive diffusion. The nuclear receptors are ligand-activated transcription activators; on binding with the ligand (the hormone), the ligands will pass through the nuclear membrane into the nucleus and enable the transcription of a certain gene and, thus, the production of a protein. Intracellular signal transduction is by and large carried out by second messenger molecules.
Pathways regulated by receptor tyrosine kinases
Ligand- or current-regulated ion channels
A direct recognition of Ca2+ by the protein
Binding of Ca2+ in the active site of an enzyme
One of the best-studied interactions of Ca2+ with a protein is the regulation of calmodulin by Ca2+. Calmodulin itself can regulate other proteins, or be part of a larger protein (e.g., phosphorylase kinase). The Ca2+–calmodulin complex plays an important role in proliferation, mitosis, and neural signal transduction. The response of cells to extracellular stimuli is in part mediated by a number of intracellular kinase and phosphatase enzymes (Hunter 1995). The mitogen-activated protein (MAP) kinases are members of discrete signaling cascades, which are focal points for diverse extracellular stimuli, and function to regulate fundamental cellular processes. Four distinct subgroups within the MAP kinase family have been described. These include (1) extracellular signal-regulated kinases (ERKs), (2) c-jun N-terminal or stress-activated protein kinases (JNK/SAPK), (3) ERK5/big MAP kinase 1 (BMK1), and (4) the p38 group of protein kinases. The JNK group of protein kinases is activated in response to a number of cellular stresses, including high osmolarity and oxidation (Ip and Davis 1998). The ERK5/BMK1 MAP kinase signaling pathway regulates serum-induced early gene expression (Kato et al. 1997). The p38 group kinases have been found to be involved in inflammation, cell growth, cell differentiation, cell cycle, and cell death (New and Han 1998).
The JNK Signal Transduction Pathway
The c-Jun NH2-terminal kinase (JNK) is a member of an evolutionarily conserved subfamily of mitogen-activated protein (MAP) kinases. Recent studies have led to progress toward understanding the physiological function of the JNK signaling pathway. Mitogen-activated protein (MAP) kinase signaling pathways relay, amplify, and integrate signals from a diverse range of extracellular stimuli, thereby controlling the genomic and physiological response of a cell to changes in the environment. In mammalian systems, these responses include cellular proliferation, differentiation, development, inflammatory response, and apoptosis. The c-Jun NH2-terminal kinase (JNK) represents one subgroup of MAP kinases that is activated primarily by cytokines and exposure to environmental stress. A major target of the JNK signaling pathway is the activation of the AP-1 (Activator protein-1) transcription factor that is mediated, in part, by the phosphorylation of c-Jun and related molecules. The JNK proteins are encoded by three genes such as JNK1, JNK2, and JNK3. Recent mutation study on mice has revealed that the JNK proteins are required for cell viability; however, they are required for cellular physiology.
Two protein kinases that activate JNK have been identified (MKK4 and MKK7). During embryo development, the modification of MKK4 alone causes death to hepatic cells, but MKK7 is necessary for embryo viability (Davis 2000). Disruption of either MKK4 or MKK7 was found to cause partial defects in stress-stimulated JNK activation (e.g., ultraviolet light). In contrast, disruption of both genes prevented JNK activation by these stimuli (Tournier et al. 2001). Interestingly, MKK4 and MKK7 preferentially phosphorylate JNK on Tyr and Thr, respectively (Lawler et al. 1998), and both protein kinases are activated in response to environmental stress (Davis 2000). As dual phosphorylation of JNK on Tyr and Thr is required for full activation, it is likely that MKK4 and MKK7 cooperate to activate JNK in response to environmental stress. However, the roles of MKK4 and MKK7 are different in cells stimulated with cytokines. Treatment of cells with tumor necrosis factor (TNF) causes activation of MKK7, but not MKK4. Gene-knockout studies demonstrate that MKK7 is essential for TNF-stimulated JNK activation and that MKK4 deficiency causes some reduction in JNK activation (Tournier et al. 2001). Together, these data indicate that TNF stimulates MKK7 activity, that JNK activation is triggered by MKK7, and that the basal activity of MKK4 is required for full activation of JNK in response to TNF. In yeast, four groups of potential scaffold proteins that may coordinate JNK signaling modules have been reported: CrkII; filamin; β-arrestin; and JIP (JNK-interacting protein). This can occur due to binary complex formation of JNK proteins. In Drosophila, the JNK signaling pathway is required for the morphogenetic process of dorsal closure (Davis 2000) with the help of the TGF-β family member Decapentaplegic (Dpp)-initiated elongation and migration of dorsal epithelial cells during dorsal closure.
The JNK signaling pathway has been implicated in many pathological conditions, including cancer, stroke, heart disease, and inflammatory diseases (Davis 2000). Drugs that inhibit JNK signaling may, therefore, be therapeutically beneficial. Furthermore, such drugs will facilitate research on JNK function. In addition, the JNK pathway participates in activating transcription factor 2, ELK-1, and the SAP-1a transcription factor, and JNK may influence p53 and NF-κB pathways (Beuckmann et al. 2000). Extracellular signals such as growth factors, transforming oncoproteins, and UV irradiation stimulate phosphorylation of c-Jun at ser-63/73 and activate c-Jun-dependent transcription. The binding of JNK to the N-terminal region of c-Jun permits substrate phosphorylation. This pathway has also been shown to be important in the control of cell survival and death pathways by the induction of apoptosis.
The p38 Signal Transduction Pathway
The p38 signaling transduction pathway, a mitogen-activated protein (MAP) kinase pathway, plays an essential role in regulating many cellular processes including inflammation, cell differentiation, and cell growth and death. Activation of p38 often through extracellular stimuli, such as bacterial pathogens and cytokines, mediates signal transduction into the nucleus to turn on the responsive genes. p38 also transduces signals to other cellular components to execute different cellular responses.
Protein p38α (or simply p38) was first isolated as 38 kDa protein, which has specificity to phosphorylate tyrosine in response to LPS stimulation (Han et al. 1994). Three p38 homologues of alpha, p38β, p38γ (or ERK6, SAPK3), and p38 δ (or SAPK4) are cloned in mammals. The p38α and p38β genes are ubiquitously expressed. However, p38γ and δ are differentially expressed in different tissues. p38γ is predominantly expressed in skeletal muscle (Lechner et al. 1996), and p38δ is enriched in lung, kidney, testis, pancreas, and small intestine. p38γ expression was reported to be induced during muscle differentiation and p38β expression was shown to be developmentally regulated (Lechner et al. 1996). An upregulation of the expression of p38 isoforms was observed in the inflammatory cell lineages. Sequence comparisons revealed that each p38 isoform has more than 60% identity within this group, but only 40–45% to the other MAP kinase family members.
p38 homologues have been identified and cloned in both low and high eukaryotic species, including fly, frog, and yeast. Their role has been implicated in osmoregulation, responses to extracellular stress stimuli, and cell cycle events. Mammalian p38δ are also activated by environmental stresses. Since mammalian p38 was identified in studies designed to understand signaling pathways during inflammation (Han et al. 1994), extensive data of p38 regulation have been developed in immune systems. p38 activation has been observed in inflammatory responses, as in LPS-treated macrophages (Han et al. 1994), TNF-stimulated endothelial cells, IL-17-stimulated chondrocytes, IL-18-stimulated U1 monocytic cell line, human platelets stimulated with thrombin, and chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP)-treated or phorbol myristate acetate (PMA)-treated human neutrophils.
MAP kinase-activated protein kinase 2 (MAPKAPK2 or M2) was the first identified p38α substrate. Subsequently, a closely related protein kinase, M3 (or 3pk), was also found to be a substrate of p38α. Moreover, activated M2 and M3 phosphorylate various substrates including small heat shock protein 27 (HSP27), lymphocyte-specific protein 1 (LSP1), cAMP response element-binding protein (CREB), ATF1, and tyrosine hydroxylase. The expression of many cytokines, transcription factors, and cell surface receptors was found to be coordinated by p38. The activation of the p38 pathway plays an essential role in: (1) production of proinflammatory cytokines such as IL-1b, TNF-a, and IL-6; (2) induction of enzymes such as COX-2, which controls connective tissue remodeling in pathological condition; (3) expression of an intracellular enzyme such as iNOS, which regulates oxidation; and (4) induction of adherent proteins such as VCAM-1 and many other inflammatory-related molecules. In addition to these, the p38 pathway plays a regulatory role in the proliferation and differentiation of cells of the immune system.
Protein Kinase C (PKC)
Protein kinase C was originally identified as a serine/threonine kinase that was maximally active in the presence of diacylglycerols (DAG) and calcium ion. It is now known that there are at least ten proteins of the PKC family. Each of these enzymes exhibits specific patterns of tissue expression and activation by lipid and calcium. PKCs are involved in the signal transduction pathways initiated by certain hormones, growth factors, and neurotransmitters. The phosphorylation of various proteins, by PKC, can lead to either increased or decreased activity. Of particular importance is the phosphorylation of the EGF receptor by PKC, which downregulates the tyrosine kinase activity of the receptor. This effectively limits the length of the cellular responses initiated through the EGF receptor.
Glutathione transferases (EC 18.104.22.168; GSTs) are members of a multigene family of isoenzymes ubiquitously expressed in most living organisms. GSTs are a family of Phase II detoxification enzymes that catalyze the conjugation of glutathione (GSH) to a wide variety of xenobiotics. This detoxification ability plays a role in cellular protection from environmental and oxidative stress, yet is also implicated in cellular resistance to drugs. Advances in the molecular biology of the GSTs over the past several years have revealed a broader role for these enzymes. Indeed, GSTs have been found to be involved in the biosynthesis and metabolism of prostaglandins (Beuckmann et al. 2000), steroids, and leukotrienes; in the management of toxic products of lipid oxidation and S-glutathionated proteins generated by oxidative stress (Ruxana Begum and Kedam 2010); and in the acquisition of resistance to chemotherapeutic agents (Tew 1994).
The GST proteins are also extensively involved in the control of ROS, which can cause oxidative stress. Excess free radicals are formed in many ways, for example, trauma, medications, metabolism of lipids, etc., and can create a potentially dangerous and unstable cellular environment, linked to pathology of tissue damage, degenerative diseases such as Parkinson’s, and accelerated aging (Divya et al. 2014). Steroid hormone synthesis has also been linked to GST activity, where GST catalysis is in part responsible for progesterone and testosterone synthesis. Membrane-bound GSH transferases (mitochondrial) are more active and specific for leukotriene synthesis.
Types of GSTs
Soluble, predominantly found in the cytosol of hepatocytes. Highly polymorphic
Come to be known as “MAPEG” – membrane-associated proteins in eicosanoid and glutathione metabolism. GST1–3. These enzymes form part of a larger super family of small membrane proteins, additional members being leukotriene C4 synthase and 5-lipoxygenase-activating protein. Largely involved in eicosanoid synthesis. Six identified to date
Cytosolic and mitochondrial forms are soluble enzymes sharing similarities in 3D structure, but share no resemblance with microsomal forms. Cytosolic GSTs, the most studied of the GSTs, were originally defined based on their substrate/inhibitor specificity.
Most GST classes show a high degree of polymorphism and include several subunits. Each subunit with 199–244 amino acids in length, and 22–29 kDa mass, contains a catalytically independent active site that consists of a GSH-binding site (“G-site”) in the amino-terminal domain and a site that binds the hydrophobic substrate (“H-site”) in the carboxy-terminal domain. More than a dozen cytosolic GST subunits have been identified in humans. As the functional enzymes are dimeric, and those of α and μ classes, in addition to homodimers, can also form heterodimers, the number of isoenzymes that can be generated from these subunits is significantly larger. The isoenzymes are named according to their class and subunit composition, with each subunit designated by an Arabic numeral (e.g., GSTA1–2 denotes the enzyme composed of subunits 1 and 2 of α class). Expression of the different classes of GSTs varies among tissues and with developmental stage. For example, α-class GSTs are predominantly expressed in liver, testis, and kidney and their expression levels are similar in both adult and fetal tissues. In contrast, GSTπ (GSTP1–1), originally isolated from placenta, is found mainly in brain, lung, and heart; its expression in liver decreases during embryonic development, becoming very low in adult tissue and expressed on phenobarbitol treatment in hepatocyte of rat.
The GSTα isoform is mainly expressed in the liver and is encoded by a gene cluster localized on chromosome 6p12. This cluster contains five genes encoding proteins belonging to GSTA1–A5. Human tissues widely express transcripts for GSTA1, A2 and A4, whereas expression of GSTA3 is rare and GSTA5 has yet to be detected in human tissues. Epidemiological results show that aberrant expression of GSTα has been linked to an increased risk in colorectal cancer, ovarian cancer, and clear cell renal cell carcinoma. The GSTA1 gene contains seven exons and is ∼12 kb in length. A genetic polymorphism of GSTA1 is characterized by two alleles, GSTA1*A and GSTA1*B. These alleles differ in promoter regions based on three linked single nucleotide substitutions at positions −567, −69, and −52. Specifically, the −52 substitution has been shown to increase promoter activity in GSTA1*A, thus making it predominantly expressed. Coles et al. (2001) previously showed a correlation between GSTA1*B expression and an increased risk of colorectal cancer. However, as with many such published accounts, other groups have recently reported conflicting evidence finding no association between GST expression and susceptibility to colorectal cancer. GSTA2 has not been extensively studied, but it is known to have several variants (GSTA2*A–E). The catalytic properties of GSTA2 variants A–D do not seem to differ; however, the novel variant GSTA2E shows reduced rates of catalysis when compared to A–D. This may be due to substitution of the highly conserved Pro residue. GSTA3 is selectively expressed in steroidogenic tissues and plays a role in steroid hormone biosynthesis. Three GSTA3 transcripts have been identified and found exclusively in African populations. Polymorphisms in GSTA3 may affect steroidogenesis through altered protein levels or function, and it has recently been hypothesized that alterations in genes involved in steroidogenesis and sex steroid metabolism could potentiate risk factors for the development of ovarian cancer.
The Omega class of GSTs contains two functional members, GSTO1 and GSTO2, and a pseudogene (GSTO3p). Based on two defining features, the Omega class is both structurally and functionally distinct from other eukaryotic GSTs (Townsend and Tew 2003). This protein on X-ray crystallography shows a unique 19-residue N-terminus extension that forms a structural unit quite unlike any found in other classes. At present, its function remains undefined (Board et al. 2000) and known substrates of other GSTs are not catalyzed by GSTO.
GSTO1 is a single gene located on chromosome 10 that codes for GSTO1 proteins expressed abundantly in the liver, macrophages, glial, and endocrine cells. To date, four polymorphisms have been identified, GSTO1*A–D. Among the Australian, African, and Chinese populations, GSTO1*A was the most prevalent haplotype with a frequency ranging from 0.6 to 0.9, whereas GSTO1*B*A was the least common, with a frequency of 0.01–0.05. GSTO1*A demonstrated a GSH-dependent reduction of dehydroascorbate, a function characteristic of glutaredoxins rather than GSTs (Board et al. 2000). This allele was first described as the human monomethylarsenic acid reductase, (MMA [V]), and is the rate-limiting enzyme of inorganic arsenic metabolism. GSTO2, while separated from GSTO1 by 7.5 kb on chromosome 10, shares 64% amino acid identity. GSTO2, like GSTO1, is ubiquitously expressed and shares GSH-dependent dehydroascorbate reductase activity. However, GSTO2 has a high catalytic activity toward CDNB, and its overexpression induced apoptosis, suggesting a possible role in cell signaling (Wang et al. 2005). This is the first report made on cell signaling by GSTs.
The zeta class (GSTZ1) is a single 10.9 kb gene located on chromosome 14 that codes for a 29-kDa protein. GSTZ1 was independently characterized as maleylacetoacetate isomerase (MAAI) and plays a putative isomerase role in the catabolic pathway of phenylalanine and tyrosine in addition to the GSH-dependent transformation of α-halogenated acids. GSTZ1 is preferentially expressed in hepatocytes and renal proximal tubule cells where phenylalanine and tyrosine are catabolized.
GSTZ1-deficient mice have an elevated urinary excretion of FAA (free amino acid) and were subject to renal injury following phenylalanine and tyrosine overload. Four families have been identified that lack GSTZ1 members that were died within the first year of life. While the clinical data for GSTZ1 are insufficient to deduce a role for GSTZ1 in inherited genetic disease, it is plausible that a perturbation in GSTZ1-mediated tyrosine metabolism is contributory to the described pathology. Whether this tyrosine undergoes signal mechanism is not known.
Five GST isoforms belonging to the mu class (GSTM1–5) have been described. A gene cluster located on chromosome 1 encodes for GSTM1–5. The GSTM1 gene contains four different alleles allowing for several M1 class polymorphisms. GSTM1*A and M1*B are functionally identical and differ at K173 N amino acid substitution (McLellan et al. 1997). The presence of the GSTM1*A allele has been associated with a decreased risk of bladder cancer with the implication that detoxification of possible bladder-specific carcinogens may occur in these individuals. In addition, McLellan et al. (1997) described a rapid enzyme activity phenotype in Saudi Arabian individuals attributed to a tandem M1 gene duplication resulting in two functional M1 genes. It was inferred that this rapid detoxification phenotype could have an increased protective effect against carcinogens.
Loss of GSTM enzyme function is ascribed to a homozygous deletion of this gene resulting in the GSTM1*0 allele. It has been suggested that the mutation is a result of an unequal crossing-over of the M1 and M2 loci which are in close physical proximity and share 99% nucleotide sequence identity. The frequency of GTSM1*0 individuals is approximately 67% in Australians, 50% in Caucasians, and 22% in Nigerians. The GSTM null phenotype is associated with an increased risk of the lung, colon, and bladder cancer and has also been associated with response rates to some chemotherapy. Cytosolic prostaglandin E was purified from the human brain and characterized as GSTM2 (Beuckmann et al. 2000). The GSTM2*B allele has been shown to catalyze the conjugation of GSH to aminochrome, a redox cycling product of dopamine. Products formed during the redox cycling of catecholamines (such as dopamine) contribute to the processes involved in neurodegenerative diseases such as Parkinson’s disease and schizophrenia; therefore, GSTM2*B may play a cytoprotective role in neurodegeneration.
The GSTM3 locus contains two alleles, A and B. The GSTM3*B allele has a three-base pair deletion in intron 6 that introduces a recognition motif for the transcription factor YY1. GSTM3*A and *B are expressed in the brain; yet, there appears to be no direct relationship between GSTM3 expression and the incidence of astrocytomas. The GSTM3*AA genotype is associated with an increased risk for laryngeal squamous cell carcinoma, whereas GSTM3*BB was putatively protective. In addition, GSTM3*AA was shown to occur more frequently in patients with multiple cutaneous basal cell carcinoma than GSTM3*BB. The roles of GST M-4 and GST M-5 were not known.
The theta class of GSTs consists of two different subfamilies: GSTT1 and GSTT2. Genes encoding both proteins are colocalized on chromosome 22 and are separated by 50 kb. Polymorphisms exist between these two and within both genes including a null phenotype (GSTT1*0) that exhibits decreased catalytic activity and has been associated with an increased risk of cancers of the head, neck, and oral cavity (Strange and Fryer 1999).
The GST Pi class is encoded by a single gene spanning approximately 3 kb and located on chromosome 11. In this group, four active, functionally different polymorphisms (GSTP1*A–D) have been identified. The GSTP1 genotype has been associated with differences in chemotherapeutic response and cancer susceptibility and is overexpressed in a wide variety of tumors including ovarian, NSCLC, breast, colon, pancreas, and lymphoma (Tew 1994). Although it is well established that GSTπ is overexpressed in a wide variety of solid tumors, prostate cancer is the only example in which the absence or reduced expression of GSTπ is associated with tumor incidence. GSTπ is widely expressed in normal prostate tissue; however, its presence is undetectable in malignant cells. Studies examining the absence of GSTπ in human prostate cancer show that hypermethylation of the GSTπ regulatory region is the most common somatic alteration identified. This alteration results in the loss of GSTπ expression and is proposed to occur during pathogenesis of the disease. Recently, a methyl-CpG-binding domain protein has been identified that mediates hypermethylation of the GSTπ regulatory region. These findings provide a possible target for restoration of GSTπ activity. Therefore, the expression of GST proteins is independent on the concentration of each class of GST protein in any tissue. GST expression (and/or activity) of specific isoforms is lost in some individuals with allelic variation.
Recently, Dang et al. (2005) examined the effect of GSTP1 on the survival and proliferation of human colon cancer cells. GSTP1 wild-type and deficient colon cancer cells were grown under serum deprivation and low-density seeding conditions, and cellular apoptosis, oxidative stress, and kinase signaling were examined. Lack of GSTP1 expression resulted in an increase in cellular oxidative stress and resulted in apoptosis when cells were cultured under growth-limiting conditions. In addition, the presence of GSTP1 was essential for the mediation of MAPK kinase and ERK kinase signaling. GSTP1 plays a critical role in protection from cell cycle arrest and oxidative stress under growth-limiting conditions.
GSTs as Signaling Molecule
GSTs comprise a multigene family, of which GSTp is the most prevalent and ubiquitous nonhepatic isozyme. Recently, GSTs have also been shown to act as modulators of signal transduction pathways that control cell proliferation and cell death. Because of their cytoprotective role and their involvement in the development of resistance to anticancer agents, GSTs have become attractive drug targets. Among cellular functions attributed to GSTs is ligand binding and xenobiotic detoxification (Tew 1994). Reduced glutathione (GSH) binds to the “G” site of GSTp (and other GST isozymes) and plays an important role in detoxification of reactive oxygen species (ROS) and the maintenance of the cellular redox state. Among factors implicated in regulating JNK activity are ROS and altered redox potential. ROS have also been associated with regulation of other signaling cascades, for example, certain isozymes of protein kinase C and mitogen-activated protein kinase. The addition of exogenous oxidants or antioxidants has been found to influence the activation of MAPK/JNK.
It has been speculated that the absence or decreased expression of GSTπ results in a reduced detoxification of possible carcinogens that may be causal to malignant transformation and disease progression. In addition, the GST-mediated conjugation of GSH to a number of anticancer drug substrates has long been linked to anticancer drug resistance in a variety of tumors. A disparity of this is that GSTπ has a weak affinity for the majority of anticancer drugs, although its increased expression is highly correlated with multidrug resistance. From this, it can be inferred that the capacity of GSTs to regulate kinase-dependent proliferation pathways, especially in the case of GSTπ, may be of more consequence than its catalytic properties alone.
Under nonstressed conditions, GSTπ inhibits JNK phosphorylation by sequestering JNK/c-Jun. Exposure to anticancer drugs or oxidative stress can alter the redox potential of the cell resulting in the oligomerization of GSTπ and the dissociation of the GSTπ–JNK complex. JNK can then become phosphorylated and subsequently activate downstream kinases and transcription factors. In some cases, transient or low exposure to stress can induce cell proliferation. During prolonged or high exposure, apoptosis can be induced.
This mechanism is similar to the one proposed for GSTπ–JNK. In conditions, such as oxidative stress or heat shock, GSTM1 oligomerizes allowing for the release of ASK1 and subsequent induction of apoptosis (Dorion et al. 2002). GSTM1 plays a regulatory role in the heat shock–sensing pathway, while thioredoxin plays a regulatory role in the oxidative stress–sensing pathway that leads to p38 activation. Impaired clinical response to therapy in a variety of tumor types has been associated with an altered expression of GSTM1. Thus, any enzymatic influence GSTM1 plays in anticancer drug resistance is only further augmented by its role in kinase regulation.
GSTμ and thioredoxin (Trx) can act as inhibitors of ASK1. Stresses such as heat shock or reactive oxygen species can result in the release of ASK1 from the GSTμ–ASK1 or TRX–ASK1 complex (respectively). ASK1 oligomerizes and is activated through autophosphorylation, which in turn activates downstream kinases such as MKK4/MKK7, MKK3/MKK6, JNK, and p38. The fate of the cell (either proliferation or apoptosis) is dependent upon the time/concentration exposure to the stress.
Forced expression of GSTM1 blocked ASK1 oligomerization and repressed ASK1-dependent apoptotic cell death. GST-mediated regulation of the kinase pathways adds a new dimension to their known role in metabolism and cellular homeostasis.
In addition to acting as a negative regulator of kinase activation, GSTπ has also been shown to play a necessary role in the glutathionylation of 1-cys peroxiredoxin (1-cysPrx). Oxidation of the catalytic cysteine of 1-cysPrx has been associated with its loss of peroxidase activity. Recently, it was shown that heterodimerization of 1-cysPrx with GSTπ mediates the glutathionylation of the previously oxidized cysteine, thus restoring its peroxidase activity. From this study, it was concluded that the glutathionylation and subsequent GSH-mediated reduction of 1-cysPrx requires heterodimerization with GSTπ. This provides the first example in which GSTπ functions in the glutathionylation of oxidized cysteine residues. In addition, Townsend and Tew (2003) have found that cells deficient in GSTP1–1 and/or GSTP2–2 have a reduced capacity to respond to oxidative or nitrosative stress by enacting glutathionylation of a select group of target proteins. These findings imply that GSTπ may play a direct role in control of posttranslational glutathionylation reactions.
To determine whether GSTp affected the degree of JNK or Jun phosphorylation, increasing concentrations of GSTp (purified form) were added to preformed Jun–JNK complex, which contained the phosphorylated form of JNK, obtained from UV-treated cells. GSTp decreased JNK phosphorylation of c-Jun in a dose-dependent manner (within a range of 0.05–1 mg), but it did not decrease the number of phosphate groups on JNK, as revealed by immunoblots with phospho-JNK antibodies. Dual activity protein phosphatase (Ishibashi et al. 1992) was used as a positive control in these reactions.
Ciaccio et al.’s study, as conducted above, has revealed that GSTp in different forms can associate with JNK proteins, and in the presence of inhibitor α, μ, and π, GSTs are associated more as indicated by antibody interaction studies (Fig. 4a).
Changes in ROS affect GST oligomerization, and its association with, and inhibition of, JNK was next assessed whether modulation of the cellular redox potential would affect JNK inhibition by GSTp. Further they monitored the GST–JNK complex in vivo by means of immunoprecipitation followed by immunoblot analysis. Exposure of mouse fibroblast cells to either UV or H2O2 reduced the amount of the JNK–GSTp complex and increased JNK activity, whereas pretreating cells with the free radical scavengers N-acetylcysteine (NAC) or the ethyl-ester of glutathione (eeGSH) prevented JNK dissociation from GSTp and maintained GSTp inhibitory activity. GST–JNK–Jun association is inversely correlated with JNK activity. Only the monomeric form of GSTp was capable of mediating efficient inhibition of JNK phosphorylation of c-Jun. GSTp inhibition of JNK is due primarily to their association, which is released upon the conversion of GSTp from a monomer to a dimer form.
Since JNK efficiently targets the ubiquitination of its nonphosphorylated associated proteins c-Jun, ATF2, and p53, we determined the possible effects of GSTp on ubiquitination of JNK substrates in this reaction. Under nonstressed growth conditions, c-Jun exhibits a short half-life, which is prolonged upon phosphorylation by JNK (Musti et al. 1997). Transfection of GSTp cDNA into 3T3 mouse fibroblasts increased the level of c-Jun ubiquitination in vivo. Since the level of ubiquitinated Jun is inversely correlated with its degree of phosphorylation (Musti et al. 1997), the increase in ubiquitinated c-Jun is an expected result of the GSTp inhibition of basal JNK activity, which reduces the number of c-Jun molecules that undergo phosphorylation. The noticeable increase in ubiquitinated c-Jun molecules provides an example of the physiological significance of JNK inhibition under normal growth conditions.
The link between GSTs and the MAP kinase pathway provides a rationale as to why many of the selecting drugs are neither subject to conjugation with GSH, nor substrates for GSTs (Tew 1994). Many anticancer agents induce apoptosis via activation of the MAP kinase pathway, specifically via JNK and p38 (Davis 2000). Elevated levels of GST are associated with increased resistance to apoptosis initiated by a variety of stimuli (Cumming et al. 2001).
Aberrant cellular signaling is also a hallmark of the malignant phenotype, and thus high levels of GSTπ in many tumors may be either a cause or effect of the transformation process. The pathology of prostate cancer strongly supports these conclusions. Hypermethylation of the GSTπ regulatory region is the most common somatic alteration identified in human prostate cancer (Lin et al. 2001). This alteration results in the loss of GSTπ expression, and is proposed to occur during pathogenesis of the disease. Recently, a methyl-CpG-binding domain (MBD) protein that mediates hypermethylation of the GSTπ regulatory region has been identified. These findings provide a possible target for restoration of GSTπ activity. GST expression and/or activity of specific isoforms are lost in some individuals with allelic variation. Although it has been speculated that reduced detoxification of possible carcinogens may be causal to malignant transformation and disease progression, a more plausible link may be through an altered capacity to regulate kinase-dependent proliferation pathways.
As GST isozymes (in particular GSTπ) are frequently upregulated in many solid tumors and lymphomas, prodrugs activated by GST-mediated catalysis have become a viable drug design concept. Additionally, the regulatory properties of GSTπ in kinase cascades have provided a translational opportunity to target GSTs in myeloproliferative pathways, with the consequent clinical testing of new agents in myelodysplastic syndrome.
The first GST described was originally identified as “ligandin” due to its ability to interact covalently and noncovalently with various compounds that are not substrates for enzymatic activity, including steroids, thyroid hormones, bile acid, bilirubin, and heme (Danielson and Mannervik 1985). While the ligand-binding function remains unclear, sequestering molecules may serve a regulatory role, preventing cytotoxic ligands from interacting with their targets. Supporting this conclusion, recent studies have demonstrated a regulatory role for the π and μ classes of GSTs in the mitogen-activated protein (MAP) kinase pathway that participates in cellular survival and death signaling.
Our department is funded by DST FIST and UGC BSR, New Delhi Grants.
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