Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (gamma-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1mM CHB with 5 mM GSH at 37 degrees C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl, N-2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[p-[2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivative, 4-[p-[N-2-S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH2), 4-[p-[N-2-chloroethyl, N-2-S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG2, CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR.     

Purification and characterization of acidic form of glutathione S-transferase in human fetal livers: high similarity to placental form.

An acidic form of glutathione S-transferase (GST) was purified from human fetal livers by means of affinity chromatography and chromatofocusing. The major peak of the acidic form of GST was focused between pH 4.8 and 4.9. Judging by SDS-PAGE, the purified acidic GST was apparently homogeneous; the subunit molecular weight was estimated to be 23,000. The acidic GST catalyzed the conjugations of glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA). The immunochemical properties of the purified acidic GST were indistinguishable from those of human placental GST-pi. The N-terminal amino acid sequence of the acidic GST was identical with that of GST-pi from human placenta. The level of expression of the acidic form of GST was clearly different between human adult and fetal livers as examined on the levels of mRNA and protein.     

The influence of diet on the regional distribution of glutathione S-transferase activity in channel catfish intestine

There is evidence that glutathione conjugates are the major metabolites formed following systemic uptake of carcinogenic contaminants from the intestine. The effect of commercial diet versus a semi-purified diet on the distribution of glutathione S-transferase (GST) activity was examined in proximal, medial, and distal sections of catfish intestine. The bulk of GST activity with 1-chloro-2,4-dinitrobenzene, ethacrynic acid, and 3H-benzo[a]pyrene-4,5-oxide, and the percent cytosolic protein cross-reacting with anti-catfish GST-pi were in the more proximal segments and dropped off distally in the two diet groups. However, the total GST-pi cross-reacting protein in the proximal section was significantly higher in fish fed a chow diet. Western blot analysis revealed pi-class GST to be expressed principally in the proximal intestine. Cytosol samples cross-reacted with antibodies to human GST-alpha, -mu, and -pi, but not -theta, classes. Alpha-like GST isoforms of MW 26,200 and 24,600, absent in sections from fish fed a purified diet, were differentially expressed only in the distal section of chow-fed fish. These results indicate that diet significantly elicits regional differences in GST protein levels, that components of the commercial chow affect GST protein expression in the distal intestine, and that maintenance diet should be taken into consideration during dietary exposure studies.     

Mapping genes encoding drug-metabolizing enzymes in recombinant inbred mice.

Probes for cytochrome P450IVA (P450IVA), alpha- and pi-class glutathione S-transferases (GST), and phenol-metabolizing UDP-glucuronyltransferase (UDPGT-K39) detected restriction fragment length variants (RFLVs) between C57BL/6J and DBA/2J mice. These variants were used to map the P450IVA genes (Cyp4 alpha) to chromosome 4, close to Mtv-13 and Pmv-19, midway between brown (b) and Gpd-1; GST alpha genes were mapped to chromosome 9, with a cross-hybridizing sequence mapping to another chromosome; the GST pi genes were mapped to the distal end of chromosome 1 near Pmv-21; one UDPGT-K39 variant to chromosome 1, between Acrg and Emv-17, and another showed linkage to Odc-10 on an unidentified chromosome. No RFLVs were detected with probes for P450IID, P450 reductase, androsterone-metabolizing UDPGT, GST mu, or microsomal GST.     

MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents

O(6)-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in the defense against alkylating agents that generate, among other lesions, O(6)-alkylguanine in DNA (collectively termed O(6)-alkylating agents [O(6)AA]). The defense is highly important, since O(6)AA are common environmental carcinogens, are formed endogenously during normal cellular metabolism and possibly inflammation, and are being used in cancer therapy. O(6)AA induced DNA damage is subject to repair, which is executed by MGMT, AlkB homologous proteins (ABH) and base excision repair (BER). Although this review focuses on MGMT, the mechanism of repair by ABH and BER will also be discussed. Experimental systems, in which MGMT has been modulated, revealed that O(6)-methylguanine (O(6)MeG) and O(6)-chloroethylguanine are major mutagenic, carcinogenic, recombinogenic, clastogenic and killing lesions. O(6)MeG-induced clastogenicity and cell death require MutS alpha-dependent mismatch repair (MMR), whereas O(6)-chloroethylguanine-induced killing occurs independently of MMR. Extensive DNA replication is required for O(6)MeG to provoke cytotoxicity. In MGMT depleted cells, O(6)MeG induces apoptosis almost exclusively, barely any necrosis, which is presumably due to the remarkable ability of secondarily formed DNA double-strand breaks (DSBs) to trigger apoptosis via ATM/ATR, Chk1, Chk2, p53 and p73. Depending on the cellular background, O(6)MeG activates both the death receptor and the mitochondrial apoptotic pathway. The inter-individual expression of MGMT in human lymphocytes is highly variable. Given the key role of MGMT in cellular defense, determination of MGMT activity could be useful for assessing a patient's drug sensitivity. MGMT is expressed at highly variable amounts in human tumors. In gliomas, a correlation was found between MGMT activity, MGMT promoter methylation and response to O(6)AA. Although the human MGMT gene is inducible by glucocorticoids and genotoxins such as radiation and alkylating agents, the role of this induction in the protection against carcinogens and the development of chemotherapeutic alkylating drug resistance are still unclear. Modulation of MGMT expression in tumors and normal tissue is currently being investigated as a possible strategy for improving cancer therapy.     

KDM6B promotes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key regulator of PARthanatos. Loss of KDM6B protein or its demethylase activity conferred cancer cell resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O⁶-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second XRCC1-dependent repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.     

Preparation of monoclonal antibodies to glutathione S-transferase-pi and application to immunohistochemical study

Glutathione S-transferase (GST) EC 2.5.2.18) catalyzes conjugation of reduced glutathione with hydrophobic substrates, such as S-epoxide active molecules. It participates in glutathione metabolism and the gamma-glutamyl cycle, playing an important role in detoxification and biosynthesis of many compounds. It is also known as a marker of pre-neoplasia in chemical hepatocarcinogenesis. Isoelectric focusing studies have revealed that this enzyme is composed of several isozymes, one of which, an acidic form of GST called GST-pi, has been extracted from human placenta. In this study, we prepared monoclonal antibodies (MAb) against human GST-pi from placenta. Specificity was confirmed by immunoblots of GST-pi after polyacrylamide gel electrophoresis and inhibition testing of enzyme activity by the antibody. The subclass of the antibody was IgG1 and the light chain was kappa. In light microscopic immunohistochemical studies of human placenta using the MAb, GST-pi was localized diffusely in the cytoplasm and along the apical cell membranes of syncytial cells in villi and in the cytoplasm of cytotrophoblastic cells in the basal plate. The MAb we prepared may also be useful for analyzing the enzyme's function in detoxification and biosynthesis of many compounds, as well as for oncological studies, such as diagnosis of malignant disease and localization of oncofetal proteins in malignant tissues.     

Carcinogen mmetabolism and DNA adducts in human lung tissues as affected by tobacco smoking or metabolic phenotype: a case-control study on lung cancer patients

Individual variations in activity of pulmonary enzymes that metabolize tobacco-derived carcinogens may affect an individual's cancer risk from cigarette smoking. To investigate whether some of these enzymes (e.g., cytochrome P450IA-related) can serve as markers for carcinogen-induced DNA damage accumulating in the lungs of smokers, non-tumorous lung tissue specimens were taken during surgery from middle-aged men with either lung cancer (n = 54) or non-neoplastic lung disease (n = 20). Phase I (AHH, ECDE) and phase II (EH, UDPGT, GST) enzyme activities, glutathione and malondialdehyde contents were determined in lung parenchyma and/or bronchial tissues; some samples were analyzed for DNA adducts, using ³²P-postlabeling.

Data analysis of subsets or the whole group of patients yielded the following results. (1) Phase I and II drug-metabolizing enzyme (AHH, EH, UDPGT, GST) activities in histologically normal surgical specimens of lung parenchyma were correlated with the respective enzyme activities in bronchial tissues of the same subject. (2) In lung parenchyma, enzyme (AHH, ECDE, EH, UDPGT) activities were significantly and positively related to each other, implying a similar regulatory control of their expression. (3) Mean activities of pulmonary enzymes (AHH, ECDE) were significantly (2- and 7-fold, respectively) higher in lung cancer patients who had smoked within 30 days before surgery (except GST, which was depressed) than in cancer-free subjects with a similar smoking history. (4) In the cancer patients, the time required for AHH, EH and UDPGT activities to return to the level found in non-smoking subjects was several weeks. (5) Bronchial tree and peripheral lung parenchyma preparations exhibited a poor efficiency in activating promutagens to bacterial mutagens in Salmonella. However, they decreased the mutagenicity of several direct-acting mutagens, an effect which was more pronounced in tissue from recent smokers. GSH concentration and GST activity were positively correlated with mutagen inactivation in the same sample. (6) In recent smokers, AHH activity in lung parenchyma was positively correlated with the level of tobacco smoke-derived DNA adducts. (7) Pulmonary AHH and EH activity had prognostic value in tobacco-related lung cancer patients. (8) An enhanced level of pro-oxidant state in the lungs was associated with recent cigarette smoking. Malondialdehyde level in lung parenchyma was associated with the degree of small airway obstruction, suggesting a common free radical-mediated pathway for both lung cancer induction and small airway obstruction.

These results demonstrate the pronounced effect of recent cigarette smoke exposure on pulmonary xenobiotic metabolism and lipid peroxidation and lend further support to the hypothesis that the inducibility of pulmonary AHH activity (cytochrome P450IA1 levels) in tobacco smokers is associated with lung cancer risk. Results on DNA adducts in smokers' lung tissue may help to explain why a certain metabolic phenotype accumulates more DNA damage in lung cells.     

Glutathione S-transferase and UDP-glucuronyltransferase activity in primary cultures of rainbow trout gill epithelial cells

The objective of this study was to characterize rainbow trout (Oncorhynchus mykiss) gill epithelial cells in primary culture by evaluating their ability to maintain glutathione and glucuronide conjugating enzymes. The activity and inducibility of the phase II enzymes was investigated as a function of culture time. Glutathione S-transferase (GST) and UDP-glucuronyltransferase (UDPGT) enzyme activities were measured in freshly isolated cells and in cells cultured for 7 and 12 days. GST activity, determined with 1-chloro-2,4-dinitrobenzene, decreased gradually to 72% after 7 days and to 38% after 12 days in culture compared with freshly isolated cells. There was no significant difference between UDPGT activities in freshly isolated cells compared with cells cultured up to 12 days although a transient decrease in activity was observed at day 7. In vitro induction of the enzymes was studied using beta-naphtoflavone (BNF) and 3-methylcholanthrene (3-MC) as inducers. GST activity increased 2-fold after exposure to BNF and 1.5-fold after 3-MC exposure for 48 h in 7 days old cultures. No induction was observed in 12 days old cultures. UDPGT activity was not induced either at day 7 or 12.     

Structures of thermolabile mutants of human glutathione transferase P1-1

An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix alpha6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the alpha-helical propensity of the isolated alpha6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37 degrees C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue.     

O6-methylguanine-DNA methyltransferase deficiency in developing brain: implications for brain tumorigenesis

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a cardinal defense against the mutagenic and carcinogenic effects of alkylating agents. We have reported evidence that absence of detectable MGMT activity (MGMT(-) phenotype) in human brain is a predisposing factor for primary brain tumors that affects ca. 12% of individuals [J.R. Silber, A. Blank, M.S. Bobola, B.A. Mueller, D.D. Kolstoe, G.A. Ojemann, M.S. Berger, Lack of the DNA repair protein O(6)-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary brain tumors, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 6941-6946]. We report here that MGMT(-) phenotype in the brain of children and adults, and the apparent increase in risk of neurocarcinogenesis, may arise during gestation. We found that MGMT activity in 71 brain specimens at 6-19 weeks post-conception was positively correlated with gestational age (P相似文献   

Eukaryotic translation elongation factor 1γ contains a glutathione transferase domain—Study of a diverse,ancient protein super family using motif search and structural modeling

Using computer methods for multiple alignment, sequence motif search, and tertiary structure modeling, we show that eukaryotic translation elongation factor 1γ (EF1γ) contains an N-terminal domain related to class θ glutathione S-transferases (GST). GST-like proteins related to class θ comprise a large group including, in addition to typical GSTs and EF1γ, stress-induced proteins from bacteria and plants, bacterial reductive dehalogenases and β-etherases, and several uncharacterized proteins. These proteins share 2 conserved sequence motifs with GSTs of other classes (α, μ, and π). Tertiary structure modeling showed that in spite of the relatively low sequence similarity, the GST-related domain of EF1γ is likely to form a fold very similar to that in the known structures of class α, μ, and π GSTs. One of the conserved motifs is implicated in glutathione binding, whereas the other motif probably is involved in maintaining the proper conformation of the GST domain. We predict that the GST-like domain in EF1γ is enzymatically active and that to exhibit GST activity, EF1γ has to form homodimers. The GST activity may be involved in the regulation of the assembly of multisubunit complexes containing EF1 and aminoacyl-tRNA synthetases by shifting the balance between glutathione, disulfide glutathione, thiol groups of cysteines, and protein disulfide bonds. The GST domain is a widespread, conserved enzymatic module that may be covalently or noncovalently complexed with other proteins. Regulation of protein assembly and folding may be 1 of the functions of GST.     

Effect of excessive intake of ascorbic acid on hepatic and extra-hepatic phase I and phase II drug metabolism in rat

Guinea pig is the animal model of choice for studies on effects of ascorbic acid (AA). However, rat is one of the largely used animals for investigations related to chemical carcinogenesis. Therefore, the present study was designed to evaluate the changes induced by high intake of the vitamin in xenobiotic and carcinogen metabolizing status of the organs. Male Wistar rats, dosed daily with 50 mg AA/100 g body weight for 10 weeks, demonstrated a small non-significant increase in hepatic, pulmonary and colon cytochrome P-450 (Cyt. P-450) contents, which was accompanied with a significant increase in hepatic and pulmonary arylhydrocarbon hydroxylase (AHH) activities. Phase II enzymes of drug metabolism responded in different ways to increased intake of AA. UDP-glucuronyltransferase (UDPGT) activity was unaffected in liver and colon, but it was increased (p less than 0.005) in lung. Activities of glutathione S-transferase (GST) were decreased in the three organs. Inducibility of AHH by 3-methylcholanthrene (MCA) or phenobarbital (PB) was largely reduced due to AA feeding. Besides this, MCA and PB had differential effects on enzymatic levels in AA fed rats. When compared with our earlier observations in guinea pig, it was found that rat responded similarly to guinea pig to increased intake of AA with regard to hepatic AHH, Cyt. P-450, UDPGT and GST, pulmonary AHH, Cyt. P-450 and Cyt. b5, and all studied colon enzymes, except GST.     

Purification and subunit-structural and immunological characterization of five glutathione S-transferases in human liver, and the acidic form as a hepatic tumor marker

Five glutathione S-transferase (GST, EC 2.5.1.18) forms were purified from human liver by S-hexylglutathione affinity chromatography followed by chromatofocusing, and their subunit structures and immunological relationships to rat liver glutathione S-transferase forms were investigated. They were tentatively named GSTs I, II, III, IV and V in order of decreasing apparent isoelectric points (pI) on chromatofocusing. Their subunit molecular weights assessed on SDS-polyacrylamide gel electrophoresis were 27 (Mr X 10(-3)), 27, 27.7,27 and 26, respectively, (26, 26, 27, 26, and 24.5 on the assumption of rat GST subunit Ya, Yb and Yc as 25, 26.5 and 28, respectively), indicating that all forms are composed of two subunits identical in size. However, it was suggested by gel-isoelectric focusing in the presence of urea that GSTs I and IV are different homodimers, consisting of Y1 and Y4 subunits, respectively, which are of identical Mr but different pI, while GST II is a heterodimer composed of Y1 and Y4 subunits. This was confirmed by subunit recombination after guanidine hydrochloride treatment. GST III seemed to be identical with GST-mu with regard to Mr and pI. GST V was immunologically identical with the placental GST-pi. On double immunodiffusion or Western blotting using specific antibodies to rat glutathione S-transferases, GST I, II and IV were related to rat GST 1-1 (ligandin), GST III(mu) to rat GST 4-4 (D), and GST V (pi) to rat GST 7-7 (P), respectively. GST V (pi) was increased in hepatic tumors.     

The nuclear targeting and nuclear retention properties of a human DNA repair protein O6-methylguanine-DNA methyltransferase are both required for its nuclear localization: the possible implications.

Human O6-methylguanine-DNA methyltransferase (MGMT) protects human cells from the mutagenic effects of alkylating agents by repairing the O6-alkylguanine residues formed by these agents in the nuclear DNA. We report here a study showing a possible two-step model for the nuclear localization of the 21 kDa human protein. The first step is the translocation of the protein from the cytosol to the nucleus. This appears to require the nuclear targeting property associated with the holoprotein in combination with a cellular factor(s) to effect the nuclear translocation of MGMT. The second step involves the nuclear retention of MGMT (to prevent its export from the nucleus). This requires a basic region (PKAAR, codons 124-128) that can bind to the non-diffusible DNA elements in the nucleus. Supporting data for such mechanisms are: (i) the holoprotein can target the cytosolic 110 kDa beta-galactosidase into the nucleus; (ii) purified recombinant MGMT requires a cellular factor for transport across the nuclear membrane; (iii) nuclear MGMT can be removed selectively by DNase I; (iv) the repair-positive K125L mutant, which alters the PKAAR motif, remains in the cytosol and fails to bind DNA in vitro; and (v) polypeptide containing the PKAAR motif has no nuclear targeting property. Interestingly, mutants in another basic region, KLLKVVK (codons 101-107) are DNA binding and repair deficient but entirely nuclear. As these substitutions affect the functional properties of human MGMT, they are potential targets for genetic screening of individuals for risk assessment to alkylating agents.     

CYP2E1-mediated oxidative stress regulates HO-1 and GST expression in maneb- and paraquat-treated rat polymorphonuclear leukocytes

Cytochrome P4502E1 (CYP2E1), glutathione-S-transferase A4-4 (GSTA4-4), and inducible nitric oxide synthase (iNOS) are implicated in maneb- and paraquat-induced toxicity leading to various pathological conditions. The study aimed to investigate the role of CYP2E1 in maneb- and paraquat-induced oxidative stress in rat polymorphonuclear leukocytes (PMNs) and its crosstalk with iNOS-mediated nitrosative stress and GSTA4-4-linked protective effect, if any and their consequent links with the nuclear factor erythoid 2-related factor 2 (Nrf2) activation and heme oxygenase-1 (HO-1) expression. Rats were treated with/without maneb and/or paraquat for 1, 2, and 3 weeks along with vehicle controls. Subsets of rats were also treated with diallyl sulfide (DAS) or aminoguanidine (AG) along with the respective controls. Maneb and paraquat augmented the reactive oxygen species (ROS), lipid peroxidation (LPO) and 4-hydroxy nonenal (4-HNE) contents, and superoxide dismutase (SOD) activity in the PMNs. However, maneb and paraquat attenuated the reduced glutathione (GSH) level and the expression/activity of total GST and GST-pi. Maneb and paraquat increased the expression/activity of CYP2E1, GSTA4-4, iNOS, Nrf2 and HO-1, and nitrite content. CYP2E1 inhibitor, DAS noticeably alleviated maneb- and paraquat-induced ROS, LPO, 4-HNE, SOD, Nrf2 and HO-1, GST, GSH, and GST-pi while iNOS, nitrite content and GSTA4-4 levels were unchanged. Conversely, AG, an iNOS inhibitor, attenuated maneb- and paraquat-directed changes in nitrite, LPO, iNOS but it did not alter ROS, GSH, SOD, GST, GST-pi, Nrf2, HO-1, CYP2E1, and GSTA4-4. The results demonstrate that CYP2E1 induces iNOS-independent free radical generation and subsequently modulates the Nrf2-dependent HO-1 and 4-HNE-mediated GST expression in maneb- and paraquat-treated PMNs.     

溴化1-己基-3-甲基咪唑对斜生栅藻谷胱甘肽及其代谢酶的影响

研究了浓度为0、1、5、10、15、20 mg/L的新兴离子液体溴化1-己基-3-甲基咪唑([C6mim]Br)在24h、48h、72h和96h对斜生栅藻还原型谷胱甘肽（GSH）及其代谢酶-谷胱甘肽过氧化物酶（GPX）、谷胱甘肽转硫酶（GST）和谷胱甘肽还原酶（GR）的影响。结果表明：GSH含量在24h、48h和72h时,在最低处理浓度下不变,其他处理浓度下随胁迫浓度增加而降低,96h时则与对照无差异或较小;GPX和GST的活性在72h之前明显升高（最高浓度组的GST活性有波动）,96h时均降低至对照水平;GR活性在24h时,[C6mim]Br=1 mg/L时升高,之后降低,在48h增高至对照水平,72h时,[C6mim]Br≥10 mg/L的处理组高于对照水平,96h时,除最低处理组外,均降至对照水平以下。GR是GSH系统中的限速酶,GST则是该系统中活性和灵敏性最高的酶,可作为[C6mim]Br胁迫时的敏感的生物标志物。1 mg/L的[C6mim]Br可引起藻细胞的氧化胁迫,具有环境毒性。     

Zebularine partially reverses GST methylation in prostate cancer cells and restores sensitivity to the DNA minor groove binder brostallicin

Brostallicin is a DNA minor groove binder that shows enhanced antitumor activity in cells with high glutathione S-transferase (GST)/glutathione content. Prostate cancer cells present, almost invariably, methylation of the GSTP1 gene promoter and, as a consequence, low levels of GST-pi expression and activity. In these cells, brostallicin shows very little activity. We tested whether pretreatment of heavily GST-methylated prostate cancer cells with demethylating agents could enhance the activity of brostallicin. Human prostate cancer cells LNCaP and DU145 were used for these studies both in vitro and in vivo. The demethylating agent zebularine was used in combination with brostallicin. Methylation specific PCR and pyrosequencing were used to determine the level of GST methylation. Pretreatment with demethylating agents enhanced the in vitro activity of brostallicin in LNCaP cells. Zebularine, in particular, induced an enhancement of activity in vivo comparable to that obtained by transfecting the human GSTP1 gene in LNCaP cells in vitro. Molecular analysis performed on tumor xenografts in mice pretreated with zebularine failed to detect re-expression of GST-pi and demethylation of GSTP1. However, we found demethylation in the GSTM1 gene, with consequent re-expression of GST-mu at the mRNA level. These results indicate that zebularine, both in vitro and in vivo, enhances the activity of brostallicin and that this enhancement correlates with re-expression of GST-pi and GST-mu. These findings highlight the potential therapeutic value of combining demethylating agents and brostallicin in tumors with GST methylation that poorly respond to brostallicin.     

O(6)-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: Enzyme activity, promoter methylation and immunohistochemistry

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O(6)-methylguanine. It also repairs larger adducts on the O(6)-position of guanine, such as O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine and O(6)-chloroethylguanine. These adducts are formed in response to alkylating environmental pollutants, tobacco-specific carcinogens and methylating (procarbazine, dacarbazine, streptozotocine, and temozolomide) as well as chloroethylating (lomustine, nimustine, carmustine, and fotemustine) anticancer drugs. MGMT is therefore a key node in the defense against commonly found carcinogens, and a marker of resistance of normal and cancer cells exposed to alkylating therapeutics. MGMT also likely protects against therapy-related tumor formation caused by these highly mutagenic drugs. Since the amount of MGMT determines the level of repair of toxic DNA alkylation adducts, the MGMT expression level provides important information as to cancer susceptibility and the success of therapy. In this article, we describe the methods employed for detecting MGMT and review the literature with special focus on MGMT activity in normal and neoplastic tissues. The available data show that the expression of MGMT varies greatly in normal tissues and in some cases this has been related to cancer predisposition. MGMT silencing in tumors is mainly regulated epigenetically and in brain tumors this correlates with a better therapeutic response. Conversely, up-regulation of MGMT during cancer treatment limits the therapeutic response. In malignant melanoma, MGMT is not related to the therapeutic response, which is due to other mechanisms of inherent drug resistance. For most cancers, studies that relate MGMT activity to therapeutic outcome following O(6)-alkylating drugs are still lacking.     

Cafestol, a coffee-specific diterpene, induces apoptosis in renal carcinoma Caki cells through down-regulation of anti-apoptotic proteins and Akt phosphorylation

Cafestol, one of the major compounds in coffee beans, has been reported for its tumor cell growth inhibitory activity and anti-carcinogenic activity, although the mechanism of action is poorly understood. In the present study, we investigated the effect of cafestol on the apoptotic pathway in human renal Caki cells and other cancer cell lines. Cafestol treatment inhibited Caki cells viability a dose-dependent manner by inducing apoptosis, as evidenced by DNA fragmentation and the accumulation of sub-G1 phase. Cafestol-induced apoptosis is associated with the reduction of mitochondrial membrane potential (MMP), activation of caspase 3, cytochrome c release, and down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1 and cFLIP). Cafestol-induced apoptosis was blocked by pretreatment with broad caspase inhibitor z-VAD-fmk, showing its dependence on caspases. Ectopic expression of Bcl-2 or Mcl-1 in Caki cells attenuates cafestol-induced apoptosis. In addition, we have also shown that cafestol inhibits phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, and PI3K inhibitor LY29004 significantly increases cafestol-induced apoptosis in Caki cells. Taken together, our results show the activity of cafestol to modulate multiple components in apoptotic response of human renal Caki cells and a potential as a therapeutic agent for preventing cancers such as renal carcinoma.