Class Of Glutathione S-transferase Research Articles

Inhibition of JNK by pi class of glutathione S-transferase through PKA/CREB pathway is associated with carnosic acid protection against 6-hydroxydopamine-induced apoptosis

Pi class of glutathione S-transferase (GST) is known to suppress c-Jun N-terminal kinase (JNK)-related apoptosis through protein-protein interactions. Moreover, signaling by PKA/cAMP response element binding protein (CREB) is necessary for GSTP up-regulation. This study explored whether carnosic acid (CA) from rosemary prevents 6-hydroxydopamine (6-OHDA)-induced neurotoxicity by inhibition of JNK through GSTP via PKA/CREB signaling. Results indicated that the GSTP protein was increased in SH-SY5Y cells treated with CA for 18 and 24 h. However, CA had no significant effect on alpha or mu class of GST. Treatment of CA increased the induction of p-PKAα, nuclear p-CREB, and CRE-DNA binding activity. These effects of CA were attenuated in cells pretreated with the PKA inhibitor H89. CA pretreatment suppressed 6-OHDA-induced apoptosis by inhibition of JNK phosphorylation, poly(ADP)-ribose polymerase cleavage, and nuclear condensation. Pretreatment with H89 and GSTP siRNA attenuated the ability of CA to reverse 6-OHDA-induced apoptosis. By use of immunoprecipitation with JNK antibody to examine the interaction of GSTP-JNK with CA, we showed that CA pretreatment increased the immunoprecipitation of GSTP after 6-OHDA treatment, which suggests that CA promoted the interaction between GSTP and JNK. Conclusion: CA prevents 6-OHDA-induced apoptosis via inhibition of JNK by GSTP through the PKA/CREB pathway.

Functional classification and biochemical characterization of a novel rho class glutathione S-transferase in Synechocystis PCC 6803

We report a novel class of glutathione S-transferase (GST) from the model cyanobacterium Synechocystis PCC 6803 (sll1545) which catalyzes the detoxification of the water pollutant dichloroacetate and also shows strong glutathione-dependent peroxidase activity representing the classical activities of zeta and theta/alpha class respectively. Interestingly, sll1545 has very low sequence and structural similarity with these classes. This is the first report of dichloroacetate degradation activity by any bacterial GST. Based on these results we classify sll1545 to a novel GST class, rho. The present data also indicate potential biotechnological and industrial applications of cyanobacterial GST in dichloroacetate-polluted areas.

Cloning and expression of a JHA-inducible glutathione S-transferase gene in the common cutworm Spodoptera litura (Lepidoptera: Noctuidae)

The juvenile hormone titer during the last instar is a crucial determining switch for metamorphosis in lepidopterans. We previously observed that the induction of glutathione S-transferase (GST) activity by a juvenile hormone analog (JHA) is reversely proportional to the occurrence of JHA-induced supernumerary instars following topical application of JHA to Spodoptera litura during the last instar period. In this paper, at least five JHA-induced GSTs were purified by glutathione-affinity chromatography, and one was further isolated for determination of the N-terminal sequence. The corresponding cDNA was cloned and named SlGST1 (GenBank accession no. AY506545). SlGST1 was classified into the epsilon class of GSTs by phylogenetic analysis. Northern blot analyses further showed that the SlGST1 in fat bodies can be induced by JHA, particularly in 0-day-old sixth-instar larvae, in which induction was much higher than that in 1- and 2-day-old sixth-instar larvae. To our knowledge, this is the first JHA-inducible GST to have been identified, and we believe that it will provide new insight into the role of GST in insect development, particularly during metamorphosis.

Structural and Functional Evolution of Positively Selected Sites in Pine Glutathione S-Transferase Enzyme Family*

Phylogenetic analyses have identified positive selection as an important driver of protein evolution, both structural and functional. However, the lack of appropriate combined functional and structural assays has generally hindered attempts to elucidate patterns of positively selected sites and their effects on enzyme activity and substrate specificity. In this study we investigated the evolutionary divergence of the glutathione S-transferase (GST) family in Pinus tabuliformis, a pine that is widely distributed from northern to central China, including cold temperate and drought-stressed regions. GSTs play important roles in plant stress tolerance and detoxification. We cloned 44 GST genes from P. tabuliformis and found that 26 of the 44 belong to the largest (Tau) class of GSTs and are differentially expressed across tissues and developmental stages. Substitution models identified five positively selected sites in the Tau GSTs. To examine the functional significance of these positively selected sites, we applied protein structural modeling and site-directed mutagenesis. We found that four of the five positively selected sites significantly affect the enzyme activity and specificity; thus their variation broadens the GST family substrate spectrum. In addition, positive selection has mainly acted on secondary substrate binding sites or sites close to (but not directly at) the primary substrate binding site; thus their variation enables the acquisition of new catalytic functions without compromising the protein primary biochemical properties. Our study sheds light on selective aspects of the functional and structural divergence of the GST family in pine and other organisms.

Indigofera suffruticosa Mill extracts up-regulate the expression of the π class of glutathione S-transferase and NAD(P)H: quinone oxidoreductase 1 in rat Clone 9 liver cells

Because induction of phase II detoxification enzyme is important for chemoprevention, we study the effects of Indigofera suffruticosa Mill, a medicinal herb, on the expression of π class of glutathione S-transferase (GSTP) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in rat Clone 9 liver cells. Both water and ethanolic extracts of I. suffruticosa significantly increased the expression and enzyme activities of GSTP and NQO1. I. suffruticosa extracts up-regulated GSTP promoter activity and the binding affinity of nuclear factor erythroid 2-related factor 2 (Nrf2) with the GSTP enhancer I oligonucleotide. Moreover, I. suffruticosa extracts increased nuclear Nrf2 accumulation as well as ARE transcriptional activity. The level of phospho-ERK was augmented by I. suffruticosa extracts, and the ERK inhibitor PD98059 abolished the I. suffruticosa extract-induced ERK activation and GSTP and NQO-1 expression. Moreover, I. suffruticosa extracts, especially the ethanolic extract increased the glutathione level in mouse liver and red blood cells as well as Clone 9 liver cells. The efficacy of I. suffruticosa extracts in induction of phase II detoxification enzymes and glutathione content implies that I. suffruticosa could be considered as a potential chemopreventive agent.

Expression of a rice Lambda class of glutathione S-transferase, OsGSTL2, in Arabidopsis provides tolerance to heavy metal and other abiotic stresses

Global industrial growth has contaminated the soil and water with many hazardous compounds, including heavy metals. These heavy metals are not only toxic to plants but also cause severe human health hazards when leach out into food chain. One of the approaches employed for the decontamination of environment includes identification and overexpression of genes involved in the detoxification mechanism of plants. Glutathione S-transferases (GSTs) are a superfamily of enzymes, principally known for their role in detoxification reactions. Different classes of GSTs have been used to develop plants with improved detoxification mechanism, but not much information is available for Lambda class of GSTs. Here, we studied expression of OsGSTLs in different rice genotypes under arsenic stress. The study suggests differential expression of these genes in arsenic sensitive and tolerant genotypes. Further, the role of one member of Lambda class OsGSTL2 was studied by expressing in heterologous system, Arabidopsis. Transgenic lines developed were analysed for their response to different abiotic stresses including heavy metals. Analysis suggests that OsGSTL2 provides tolerance for heavy metals and other abiotic stresses like cold, osmotic stress and salt. We conclude that OsGSTLs can be utilized for developing plant varieties tolerant to different abiotic stresses including heavy metals.

Bivalent Enzyme Inhibitors Discovered Using Dynamic Covalent Chemistry

A bivalent dynamic covalent chemistry (DCC) system has been designed to selectively target members of the homodimeric glutathione-S-transferase (GST) enzyme family. The dynamic covalent libraries (DCLs) use aniline-catalysed acylhydrazone exchange between bivalent hydrazides and glutathione-conjugated aldehydes and the bis-hydrazides act as linkers to bridge between each glutathione binding site. The resultant DCLs were found to be compatible and highly responsive to templating with different GST isozymes, with the best results coming from the M and Schistosoma japonicum (Sj) class of GSTs, targets in cancer and tropical disease, respectively. The approach yielded compounds with selective, nanomolar affinity (K(i) =61 nM for mGSTM1-1) and demonstrates that DCC can be used to simultaneously interrogate binding sites on different subunits of a dimeric protein.

Activation of Nrf2 Is Required for Up-Regulation of the π Class of Glutathione S-Transferase in Rat Primary Hepatocytes with l-Methionine Starvation

Numerous genes expression is regulated in response to amino acid shortage, which helps organisms adapt to amino acid limitation. The expression of the π class of glutathione (GSH) S-transferase (GSTP), a highly inducible phase II detoxification enzyme, is regulated mainly by activates activating protein 1 (AP-1) binding to the enhancer I of GSTP (GPEI). Here we show the critical role of nuclear factor erythroid-2-related factor 2 (Nrf2) in up-regulating GSTP gene transcription. Primary rat hepatocytes were cultured in a methionine-restricted medium, and immunoblotting and RT-PCR analyses showed that methionine restriction time-dependently increased GSTP protein and mRNA expression over a 48 h period. Nrf2 translocation to the nucleus, nuclear proteins binding to GPEI, and antioxidant response element (ARE) luciferase reporter activity were increased by methionine restriction as well as by l-buthionine sulfoximine (BSO), a GSH synthesis inhibitor. Transfection with Nrf2 siRNA knocked down Nrf2 expression and reversed the methionine-induced GSTP expression and GPEI binding activity. Chromatin immunoprecipitation assay confirmed the binding of Nrf2 to the GPEI. Phosphorylation of extracellular signal-regulated kinase 2 (ERK2) was increased in methionine-restricted and BSO-treated cells. ERK2 siRNA abolished methionine restriction-induced Nrf2 nuclear translocation, GPEI binding activity, ARE-luciferase reporter activity, and GSTP expression. Our results suggest that the up-regulation of GSTP gene transcription in response to methionine restriction likely occurs via the ERK-Nrf2-GPEI signaling pathway.

Cloning and characterization of two glutathione S‐transferases from pyrethroid‐resistant Culex pipiens

The Marin strain of Culex pipiens Say is a pyrethroid-resistant population that was collected in Marin County, California, in 2001 and subsequently maintained in the laboratory under regular permethrin exposure. In this study, two cDNAs, CpGSTd1 and CpGSTd2, encoding glutathione S-transferase (GST) were cloned from Cx. pipiens Marin. Phylogenetic analysis of the deduced amino acid sequences, CpGSTD1 and CpGSTD2, of these genes indicated that they belong to the Delta class of insect GSTs. The nucleotide and deduced amino acid sequences of CpGSTd1 and CpGSTd2 were 59 and 48% identical respectively. CpGSTD1 and CpGSTD2 were expressed in Escherichia coli and purified by affinity chromatography. The recombinant GSTs exhibited unique selectivity towards the general GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB), and also differed in their sensitivity to known inhibitors of GSTs. CpGSTD1 exhibited peroxidase activity with cumene hydroperoxide, while CpGSTD2 appeared to lack this activity. CpGSTD1 was able to metabolize 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), while DDT metabolism by CpGSTD2 was not detectable. CpGSTD1 and CpGSTD2 showed no detectable metabolism of permethrin. Gene expression of CpGSTd1 and CpGSTd2 in Marin mosquitoes was elevated about twofold in comparison with that found in a pyrethroid-sensitive mosquito strain. The results indicate that CpGSTD1 and CpGSTD2 have unique biochemical characteristics, but they do not appear to play major roles in permethrin resistance in Marin mosquitoes.

Identification of the <i>glutathione</i> S-<i>transferase</i> gene responsible for flower color intensity in carnations

Two cDNAs with homology to glutathione S-transferase (GST) were isolated from the carnation (Dianthus caryophyllus); these cDNAs are termed here DcGSTF1 and DcGSTF2. Phylogenetic analysis suggested that both DcGSTF1 and DcGSTF2 belonged to the Phi class of GSTs. DcGSTF2 showed high levels of transcription at late stages of petal d evelopment when anthocyanin biosynthesis is most active. Sequencing of DcGSTF2 indicated that it consisted of three exons and two introns. A truncated DcGSTF2 gene, resulting from the insertion of a CACTA-type transposable element, was found in the genome of a mutableower line bearing deep pink sectors on pale pink petals. A full length DcGSTF2 gene driven by a continuous expression promoter was introduced into the epidermal cells of carnations with pale pink petals. �e t ransformed cells were deep pink. �ese results suggest that the DcGSTF2 gene is responsible forower color intensity in carnations.

Structure and Function Relationship Study of Allium Organosulfur Compounds on Upregulating the Pi Class of Glutathione S-Transferase Expression

Allium organosulfides are potential chemopreventive compounds due to their effectiveness on the induction of phase II detoxification enzyme expression. In this study, we examined the structure and function relationship among various alk(en)yl sulfides on the expression of the pi class of glutathione S-transferase (GSTP) in rat Clone 9 cells, and what mechanism is involved. Cells were treated with 300 μM dipropyl sulfide (DPS), dipropyl disulfide (DPDS), propyl methyl sulfide (PMS), and propyl methyl disulfide (PMDS) for 48 h. DPDS and PMDS displayed more potency on GSTP protein and mRNA induction than that of DPS and PMS. Next, we compared the effectiveness of DPDS, PMDS, and diallyl disulfide (DADS), which have the same number of sulfur atoms but differ in the side alk(en)yl groups. The maximum increases on protein expression, mRNA level, and enzyme activity were noted in cells treated with DADS, followed by DPDS and PMDS. A reporter assay showed that three disulfides increased GSTP enhancer I (GPE I) activity (P < 0.05) in the order DADS > DPDS ≥ PMDS. Electromobility gel shift assays showed that the DNA binding of GPE I to nuclear proteins reached a maximum at 1 to 3 h after alk(en)yl disulfide treatment. Supershift assay revealed that c-jun bound to GPE I. Silencing of extracellular signal-regulated kinase (ERK) 2 expression inhibited c-jun activation and GSTP induction. Results suggest that both the type of alk(en)yl groups and number of sulfur atoms are determining factors of allium organosulfides on inducing GSTP expression, and it is likely related to the ERK-c-Jun-GPE I pathway.

Andrographolide-induced pi class of glutathione S-transferase gene expression via PI3K/Akt pathway in rat primary hepatocytes

Andrographis paniculata is an herb widely used in China, Korea, and India for its anti-hepatotoxic, anti-viral, and anti-inflammatory effects. Andrographolide is the major bioactive diterpene lactone in A. paniculata. The pi class of glutathione S-transferase (GSTP) is one of the phase II biotransformation enzymes. Our previous study indicated that andrographolide upregulates the expression of GSTP. The aim of this study was to investigate the mechanism by which andrographolide induces GSTP gene expression in rat primary hepatocytes. In hepatocytes treated with 40μM andrographolide, immunoblots showed maximal Akt phosphorylation at 0.5h and maximal c-jun phosphorylation at 3h. However, pretreatment with PI3K inhibitors, wortmannin and LY294002, or siPI3K inhibited the andrographolide-induced phosphorylation of c-jun and GSTP protein expression. EMSA showed that pretreatment with wortmannin, LY294002, or siPI3K attenuated the AP-1-DNA-binding activity caused by andrographolide. Results of immunoprecipitation indicated that nuclear c-fos/c-jun heterodimer increases with andrographolide treatment. Addition of antibodies against c-jun and c-fos decreased nuclear protein bound to the AP-1 consensus DNA sequence. In summary, andrographolide induces GSTP gene expression in rat primary hepatocytes through activation of the PI3K/Akt, phosphorylation of c-jun, nuclear accumulation of AP-1, and subsequent binding to the response element in the gene promoter region.

Butein Up-Regulates the Expression of the π Class of GlutathioneS-Transferase in Rat Primary Hepatocytes through the ERK/AP-1 Pathway

Induction of phase II enzymes is an important mechanism of chemoprevention. Here we compared the effects of chalcones on the expression of the π class of glutathione S-transferase (GSTP) in rat primary hepatocytes. Hepatocytes were treated with 10 or 25 μM of phloretin or butein for 24 h. Both butein and phloretin dose-dependently increased GSTP protein expression, and the induction potency of butein was stronger than that of phloretin. The increase in GSTP mRNA in cells treated with 25 μM of phloretin and butein was 107% and 211%, respectively (P < 0.05). Butein increased GST enzyme activity by 27% compared with that in the control cells (P < 0.05). In contrast, phloretin had no significant effect on GST enzyme activity. The pTA-luciferase reporter construct with the rat -2.7 kb GSTP promoter region was transiently transfected into Clone 9 liver cells, and the luciferase activity in butein-treated cells was 1.1-fold higher than that in control cells (P < 0.05). GSTP enhancer 1 (GPE1) deletion abolished the induction of reporter activity by butein. The phosphorylation of extracellular signal-regulated kinase (ERK), but not of c-Jun NH2-terminal kinase (JNK) and p38, was stimulated in the presence of butein. Pretreatment with PD98059, an ERK inhibitor, alleviated the increase in activator protein-1 (AP-1)-DNA binding activity and also the activation of GSTP protein expression by butein. Moreover, c-Jun is likely to bind to the GPE1. Silencing of ERK2 by siRNA gene knockdown reduced the butein-induced expression of GSTP. In conclusion, the increased GSTP expression by butein is likely related to the ERK-AP-1 pathway.

Methionine restriction up‐regulates the expression of the pi class of glutathione S‐transferase partially via the extracellular signal‐regulated kinase‐activator protein‐1 signaling pathway initiated by glutathione depletion

Understanding the molecular events underlying gene regulation by amino acids has attracted increasing attention. Here, we explored whether the mechanism by which methionine restriction affects the expression of the pi class of glutathione S-transferase (GSTP) is related to oxidative stress initiated by glutathione (GSH) depletion. Rat primary hepatocytes were cultured in an L-15-based medium in the absence or presence of 200 muM L-buthionine sulfoximine (BSO) or in a methionine-restricted L-15 medium supplemented with 20 muM L-methionine up to 72 h. BSO and methionine restriction time-dependently induced GSTP mRNA and protein expression in a similar pattern accompanied by a decrease in the cellular GSH level. The phosphorylation of extracellular signal-regulated kinase (ERK), but not of c-Jun NH(2)-terminal kinase and p38, was stimulated by methionine restriction and BSO. Electromobility gel shift assay showed that the DNA-binding activity of nuclear activator protein-1 (AP-1) increased in cells exposed to methionine restriction or BSO. With the ERK inhibitor FR180204, AP-1 activation and GSTP expression were abolished. Moreover, the induction of GSTP by methionine restriction and BSO was reversed by GSH monoethyl ester and N-acetylcysteine. Our results suggest that methionine restriction up-regulates GSTP gene expression, which appears to be initiated by the ERK-AP-1 signaling pathway through GSH depletion in rat hepatocytes.

Sulforaphane and α-Lipoic Acid Upregulate the Expression of the π Class of Glutathione S-Transferase through c-Jun and Nrf2 Activation

The anticarcinogenic effect of dietary organosulfur compounds has been partly attributed to their modulation of the activity and expression of phase II detoxification enzymes. Our previous studies indicated that garlic allyl sulfides upregulate the expression of the π class of glutathione S-transferase (GSTP) through the activator protein-1 pathway. Here, we examined the modulatory effect of sulforaphane (SFN) and α-lipoic acid (LA) or dihydrolipoic acid (DHLA) on GSTP expression in rat Clone 9 liver cells. Cells were treated with LA or DHLA (50–600 μmol/L) or SFN (0.2–5 μmol/L) for 24 h. Immunoblots and real-time PCR showed that SFN, LA, and DHLA dose dependently induced GSTP protein and mRNA expression. Compared with the induction by the garlic organosulfur compound diallyl trisulfide (DATS), the effectiveness was in the order of SFN > DATS > LA = DHLA. The increase in GSTP enzyme activity in cells treated with 5 μmol/L SFN, 50 μmol/L DATS, and 600 μmol/L LA and DHLA was 172, 75, 122, and 117%, respectively (P < 0.05). A reporter assay showed that the GSTP enhancer I (GPEI) was required for GSTP induction by the organosulfur compounds. Electromobility gel shift assays showed that the DNA binding of GPEI to nuclear proteins reached a maximum at 0.5–1 h after SFN, LA, and DHLA treatment. Super-shift assay revealed that the transcription factors c-jun and nuclear factor erythroid-2 related factor 2 (Nrf2) were bound to GPEI. These results suggest that SFN and LA in either its oxidized or reduced form upregulate the transcription of the GSTP gene by activating c-jun and Nrf2 binding to the enhancer element GPEI.

Activation of the cAMP/CREB/Inducible cAMP Early Repressor Pathway Suppresses Andrographolide-Induced Gene Expression of the π Class of Glutathione S-Transferase in Rat Primary Hepatocytes

Andrographolide (Ap) is a bioactive compound in Andrographis paniculata that is a Chinese herb. The pi class of glutathione S-transferase (GSTP) is one kind of phase II detoxification enzyme. Here we show that induction of GSTP protein and mRNA expression in rat primary hepatocytes by Ap was inhibited by forskolin and a variety of cAMP analogues. The inhibitory effect of the cAMP analogues was partially blocked by pretreatment with H89. In the presence of Ap, forskolin, or both, the expression of phospho-cAMP response element-binding protein (CREB) was increased. Ap alone had no effect on inducible cAMP early repressor (ICER) mRNA expression; however, Ap played a potentiating role in forskolin-induced ICER mRNA expression. An EMSA and immunoprecipitation assay showed that ICER binding to cAMP-response element (CRE) was increased in cells cotreated with Ap and forskolin for 3 and 8 h. Taken together, these results suggest that ICER is likely to be involved in the suppression of Ap-induced GSTP expression caused by the increase of cAMP in rat primary hepatocytes.

Modulation of the expression of the π class of glutathione S-transferase by Andrographis paniculata extracts and andrographolide

Andrographis paniculata (Ap) is a commonly used herb for traditional medicine in many Southeast Asian countries. In the present study, we investigated the effect of Ap on the expression of the pi class of glutathione S-transferase (GSTP) in rat primary hepatocytes. Hepatocytes were treated with 25 or 50 μg/mL of ethanol or ethyl acetate extracts of Ap (ApEE or ApEAE) or 10 or 20 μM andrographolide, which is the major active diterpene lactone of Ap, for 48 h. ApEE, ApEAE, and andrographolide dose-dependently induced GSTP protein and mRNA expression. In a GST activity assay, GST activity was significantly higher in cells treated with the maximum concentrations of ApEE, ApEAE, and andrographolide than in control cells ( P < 0.05). The pTA-2713 luciferase reporter construct containing rat GSTP enhancer 1 (GPE1) was transiently transfected into Clone 9 liver cells. Cells treated with ApEE, ApEAE, and andrographolide showed a dose-dependent increase in luciferase activity. GPE1 deletion abolished the induction efficiency of Ap. Also, the induction of GSTP expression by Ap was inhibited by wortmannin, which is an inhibitor of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. These results indicate that ApEE, ApEAE, and andrographolide induce GSTP expression. This induction is likely related to the PI3K/Akt pathway, and GPE1, an enhancer element in GSTP promoter, is essential for the induction.

Diallyl Disulfide and Diallyl Trisulfide Up-Regulate the Expression of the π Class of Glutathione S-Transferase via an AP-1-Dependent Pathway

Garlic organosulfur compounds are recognized as potential chemopreventive compounds. This protection is related to the induction of phase II detoxification enzymes. We previously reported that diallyl disulfide (DADS) and diallyl trisulfide (DATS) up-regulate the gene expression of the pi class of glutathione S-transferase (GSTP) and that an enhancer element named GPE I is required for this induction. In the present study, we further investigated the signal pathway involved in DADS and DATS up-regulation of this detoxification enzyme in Clone 9 cells. Cells were cultured with 25-200 micromol/L of DADS or DATS for 24 h. Western and Northern blots showed that both garlic allyl sulfides concentration dependently induced GSTP protein and mRNA expression, respectively. Changes in GST activity toward ethacrynic acid were consistent with the increase in GSTP expression (P < 0.05). Electromobility gel shift assay showed that the DNA binding activity of nuclear activator protein-1 (AP-1) is concentration-dependently increased in the presence of DADS and DATS as compared with that of the control cells. The phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not of p38, was stimulated in the presence of both garlic allyl sulfides. Pretreatment with SP600125 and PD98059, which are JNK and ERK inhibitors, respectively, abolished the increase in AP-1-DNA binding activity and also the induction of GSTP protein by either allyl sulfide. Our results indicate that the effectiveness of DADS and DATS on GSTP expression is likely related to the JNK-AP-1 and ERK-AP-1 signaling pathways and, thus, that DADS and DATS enhance the binding of AP-1 to GPE I.

Increased sensitivity of Hep G2 cells toward the cytotoxicity of cisplatin by the treatment of piper betel leaf extract

Piper betel leaves (PBL) are used in Chinese folk medicine for the treatment of various disorders. PBL has the biological capabilities of de-toxication, anti-oxidation and anti-mutation. In this study we first examined the effect of PBL extract on the activity of Glutathione S-transferase (GST) isoforms, and found that it inhibited total GST and the alpha class of GST (GSTA), but not the pi class of GST (GSTP), and the mu class of GST (GSTM), activity in Hep G2 cells. RT-PCR results verified a reduction in the expression of GSTA1. Next, we examined whether PBL extract could increase the sensitivity of Hep G2 cells to anti-cancer drugs. The data showed that the cytotoxicity of cisplatin was significantly enhanced by the presence of PBL extract, accompanied by a reduction in the expression of multidrug resistance protein 2 (MRP2). These effects of PBL extract were compared to its major constitute, eugenol. Although eugenol decreased MRP2 level more effectively than PBL extract, it exhibited less sensitizing effect. In conclusion, we demonstrated that PBL extract was able to increase the sensitivity of Hep G2 cells to cisplatin via at least two mechanisms, reducing the expression of MRP2 and inhibiting the activity of total GST and the expression of GSTA. The data of this study support an application of PBL as an additive to reduce drug resistance.

Garlic Organosulfur Compounds Upregulate the Expression of the π Class of Glutathione S-Transferase in Rat Primary Hepatocytes

The chemopreventive property of garlic is related in part to its induction of phase II detoxification enzymes. In the present study, we investigated the modulatory effect of 3 garlic organosulfur compounds, i.e., diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), which differ in their number of sulfur atoms, on the gene expression of the pi class of glutathione S-transferase (GSTP). Hepatocytes isolated from male Sprague-Dawley rats were cultured with 50-200 micromol/L of DAS, DADS, or DATS for 24 h. DADS and DATS increased GST activity toward ethacrynic acid by 40 and 66%, respectively (P < 0.05). Moreover, both garlic allyl sulfides dose dependently induced GSTP mRNA and protein expression. DATS increased the protein level more than DADS (P < 0.05). In contrast, DAS did not affect the activity or the protein or mRNA levels of this phase II drug-metabolizing enzyme. In Clone 9 liver cells, the pTA-luciferase reporter assay showed that luciferase activity in DADS- and DATS-treated cells was 2.8- and 3.9-fold higher than that in control cells, respectively (P < 0.05). Again, luciferase activity was not affected by treatment with DAS. Deletion of -2.7 to -2.6 kb in the GSTP promoter region, which contains the GSTP enhancer (GPE) I element, abolished the upregulation of GSTP transcription by DADS and DATS. Deletion of GPE II, however, did not affect the induction of reporter activity. In conclusion, the effectiveness of 3 garlic allyl sulfides on GSTP expression was related to the number of sulfur atoms in the molecules, and GPE I was responsible for this upregulation.