The influence of temperature on the metabolic activity of CYP2C9, CYP2C19, and CYP3A4 genetic variants in vitro.

1. Temperature is considered to affect the activity of drug-metabolizing enzymes; however, no previous studies have compared temperature dependency among cytochrome P450 genetic variants. This study aimed to analyse warfarin 7-hydroxylation by CYP2C9 variants; omeprazole 5-hydroxylation by CYP2C19 variants; and midazolam 1-hydroxylation by CYP3A4 variants at 34 °C, 37 °C, and 40 °C.2. Compared with that seen at 37 °C, the intrinsic clearance rates (Vmax/Km) of CYP2C9.1 and .2 were decreased (76 ∼ 82%), while that of CYP2C9.3 was unchanged at 34 °C. At 40 °C, CYP2C9.1, .2, and .3 exhibited increased (121%), unchanged and decreased (87%) intrinsic clearance rates, respectively. At 34 °C, the clearance rates of CYP2C19.1A and .10 were decreased (71 ∼ 86%), that of CYP2C19.1B was unchanged, and those of CYP2C19.8 and .23 were increased (130 ∼ 134%). At 40 °C, the clearance rates of CYP2C19.1A, .1B, .10, and .23 remained unaffected, while that of CYP2C19.8 was decreased (74%). At 34 °C, the clearance rates of CYP3A4.1 and .16 were decreased (79 ∼ 84%), those of CYP3A4.2 and .7 were unchanged, and that of CYP3A4.18 was slightly increased (112%). At 40 °C, the clearance rate of CYP3A4.1 remained unaffected, while those of CYP3A4.2, .7, .16, and .18 were decreased (58 ∼ 82%).3. These findings may be clinically useful for dose optimisation in patients with hypothermia or hyperthermia.

Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 1.

Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress.

CD44-positive Cancer Stem-like Cells as a Potential Source of Peritoneal Metastasis After Surgery.

BACKGROUND/AIM: The role of CD44 in gastric cancer-derived peritoneal metastasis is currently unknown. It was previously shown that viable, tumorigenic cancer cells are spilled into the peritoneal cavity during surgery, providing a potential cause of peritoneal recurrence after surgery. The purpose of this study was to elucidate the mechanism of peritoneal metastasis of gastric cancer through the expression of CD44 and to propose a method for preventing peritoneal recurrence. MATERIALS AND METHODS: Gastric cancer cell line MKN-45 was sorted into CD44+ and CD44- cells and then injected intraperitoneally into NOD/ShiJic-scidJcl mice. Differences in tumor-initiating capacity between the two groups were assessed using in vivo limiting dilution assays. Tumors harvested from both groups were examined for CD44 and ALDH1A1 expression using immunohistochemistry. The effects of CD44 blockade with anti-CD44 antibody on cell invasion and peritoneal metastasis formation in vivo were assessed. RESULTS: CD44+ cells showed significantly higher efficiency in initiating peritoneal tumor than CD44- cells. Blockade of CD44 significantly reduced peritoneal dissemination of CD44+ cells in vivo, indicating that the CD44 function of intraperitoneally disseminated cancer cells helped promote the formation of peritoneal metastasis. The margin of established tumors showed clusters of cells co-expressing CD44 and ALDH1A1. Peritoneally administered CD44- cells resulted in peritoneal metastases consisting of CD44+ and CD44- cancer cells. CONCLUSION: CD44 expressing cells are a potential source of peritoneal metastasis after surgery and could be a promising target for preventing peritoneal recurrence.

Enhancement of antitumor immune response by radiation therapy combined with dual immune checkpoint inhibitor in a metastatic model of HER2-positive murine tumor.

PURPOSE: Treatments for metastatic human epidermal growth factor receptor 2 (HER2)-positive tumors are improving but remain inadequate. We investigated activating antitumor immune response by combining radiation therapy with immune checkpoint inhibitors using mouse tumors overexpressing HER2, a pivotal driver oncogenic antigen, to develop new immunotherapies for metastatic HER2-positive tumors. MATERIALS AND METHODS: NT2.5 cells were inoculated into the two mammary fat pads of FVB/N mice, which were divided into four groups: no treatment (Non), anti-PD-1 and anti-CTLA4 antibodies (P1C4), irradiation of the large tumor (Rad), and combination (R + P1C4) groups. Tumor growth, immunostaining of tumor-infiltrating lymphocytes, and the proportion of HER2-tumor antigen-specific CD8-positive T cells in the spleen and tumor-infiltrating lymphocytes were analyzed. RESULTS: In the Rad group, unirradiated and irradiated tumors shrank after treatment. Besides the directly irradiated tumors, the unirradiated tumors in the R + P1C4 group shrank the most. In the unirradiated tumors, CD8-positive T cells and FOXP3-positive T cells accumulated significantly more in the R + P1C4 group than in the P1C4 and the Rad groups (all p < 0.001). CD4-positive helper T cells accumulated significantly more in the R + P1C4 group than in the Rad group (p < 0.05), but this was not significantly different from the P1C4 group. HER2-specific CD8-positive T cells in the spleen and tumor-infiltrating lymphocytes were significantly increased in the R + P1C4 group compared to the P1C4 and Rad groups (all p < 0.0001). CONCLUSION: Irradiation of HER2-positive tumors induced an antitumor immune effect against the unirradiated tumor, which was enhanced by the combined use of immune checkpoint inhibitors and was mediated by enhanced recruitment of HER2-tumor antigen-specific cytotoxic T lymphocytes at the tumor site in an HER2-positive mouse tumor model. Harnessing the distant antitumor immune response induced by the combination of radiation therapy and immune checkpoint inhibitors could be a promising treatment strategy for metastatic HER2-positive tumors.

Long-term prognosis of patients with cancer-related genes detected in postoperative peritoneal washings obtained during curative gastrectomy.

BACKGROUND: Cancer cells in intraoperative peritoneal washings (PW) indicate increased peritoneal recurrence. Detection of CEA or CK20 genes indicates poor prognosis. We assessed long-term prognosis of patients with amplification of cancer-related genes in PW obtained intraoperatively during curative gastric cancer surgery. METHODS: PW was collected before and immediately after curative gastrectomy. CEA, CK20, TFF1, MUC2, and FABP1-mRNA were selected as marker genes for reverse transcription polymerase chain reaction. Peritoneal recurrence-free survival (PRFS) and overall survival (OS) after >7-year follow-up were examined using the Kaplan-Meier method. RESULTS: Of 138 patients who underwent gastrectomy with negative cytological findings at laparotomy, 80 patients showed negative cancer-related gene amplification in preoperative PW. Fifty-eight patients were excluded due to positive gene amplification, which suggested presence of preoperative peritoneal cancer cells. The 80 patients had mRNA amplification in PW after surgery. Amplification of multiple and single cancer-related marker genes was observed in 38 and 21 patients; 21 cases had marker-negative results. Five-year PRFS was 69.1%, 95.2%, and 100% in multi-marker-positive, single marker-positive, and marker-negative cases, respectively. Multi-marker-positive patients had significantly worse PRFS than the other groups (p < 0.05). Multivariate analysis in the Cox proportional hazards model identified multi-marker-positivity as an independent prognostic factor for PRFS (hazard ratio, 7.6; 95% confidence interval, 1.07-62.63; p = 0.046), and multi-marker-positive patients had significantly worse OS than other groups (p < 0.01). CONCLUSION: Multi-marker cancer-related gene amplification in PW is associated with worse prognosis in PRFS and OS even after a long follow-up; PRFS can be stratified by the number of genes amplified.

Variably protease-sensitive prionopathy: a new sporadic disease of the prion protein.

OBJECTIVE: The objective of the study is to report 2 new genotypic forms of protease-sensitive prionopathy (PSPr), a novel prion disease described in 2008, in 11 subjects all homozygous for valine at codon 129 of the prion protein (PrP) gene (129VV). The 2 new PSPr forms affect individuals who are either homozygous for methionine (129MM) or heterozygous for methionine/valine (129MV). METHODS: Fifteen affected subjects with 129MM, 129MV, and 129VV underwent comparative evaluation at the National Prion Disease Pathology Surveillance Center for clinical, histopathologic, immunohistochemical, genotypical, and PrP characteristics. RESULTS: Disease duration (between 22 and 45 months) was significantly different in the 129VV and 129MV subjects. Most other phenotypic features along with the PrP electrophoretic profile were similar but distinguishable in the 3 129 genotypes. A major difference laid in the sensitivity to protease digestion of the disease-associated PrP, which was high in 129VV but much lower, or altogether lacking, in 129MV and 129MM. This difference prompted the substitution of the original designation with "variably protease-sensitive prionopathy" (VPSPr). None of the subjects had mutations in the PrP gene coding region. INTERPRETATION: Because all 3 129 genotypes are involved, and are associated with distinguishable phenotypes, VPSPr becomes the second sporadic prion protein disease with this feature after Creutzfeldt-Jakob disease, originally reported in 1920. However, the characteristics of the abnormal prion protein suggest that VPSPr is different from typical prion diseases, and perhaps more akin to subtypes of Gerstmann-Sträussler-Scheinker disease.

Characterization of new potential anticancer drugs designed to overcome glutathione transferase mediated resistance.

Resistance against anticancer drugs remains a serious obstacle in cancer treatment. Here we used novel strategies to target microsomal glutathione transferase 1 (MGST1) and glutathione transferase pi (GSTP) that are often overexpressed in tumors and confer resistance against a number of cytostatic drugs, including cisplatin and doxorubicin (DOX). By synthetically combining cisplatin with a GST inhibitor, ethacrynic acid, to form ethacraplatin, it was previously shown that cytosolic GST inhibition was improved and that cells became more sensitive to cisplatin. Here we show that ethacraplatin is easily taken up by the cells and can reverse cisplatin resistance in MGST1 overexpressing MCF7 cells. A second and novel strategy to overcome GST mediated resistance involves using GST releasable cytostatic drugs. Here we synthesized two derivatives of DOX, 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) and 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) and showed that they are substrates for MGST1 and GSTP (releasing DOX). MGST1 overexpressing cells are resistant to DOX. The resistance is partially reversed by DNS-DOX. Interestingly, the less reactive MNS-DOX was more cytotoxic to cells overexpressing MGST1 than control cells. It would appear that, by controlling the reactivity of the prodrug, and thereby the DOX release rate, selective toxicity to MGST1 overexpressing cells can be achieved. In the case of V79 cells, DOX resistance proportional to GSTP expression levels was noted. In this case, not only was drug resistance eliminated by DNS-DOX but a striking GSTP-dependent increase in toxicity was observed in the clonogenic assay. In summary, MGST1 and GSTP resistance to cytostatic drugs can be overcome and cytotoxicity can be enhanced in GST overexpressing cells.

Alkylating antitumor drug mechlorethamine conceals a structured PrP domain and inhibits in vitro prion amplification.

Prion diseases are a group of incurable transmissible neurodegenerative disorders. The key molecular event in the pathogenesis of prion diseases is the conversion of the cellular prion protein (PrP(C)) into its pathological isoform (PrP(Sc)), accompanied by a conformational transition of α-helix into ß-sheet structure involving the structured α-helix 1 domain from residues 144-154 of the protein (PrP144-154). Blocking the accessibility of PrP144-152 with anti-PrP antibody 6H4 was found to prevent PrP conversion and even to cure prion infection in cell models ( Enari et al. 2001 ). Previously, Yuan et al. (2005 ) demonstrated that the reduction and alkylation of PrP induced concealment of the 6H4 epitope. This study examined the ability of mechlorethamine (MCT), an alkylating antitumor drug, to conceal the 6H4 epitope and block PrP conversion in the presence of a reducing reagent. Mechlorethamine treatment significantly decreased in vitro amplification of PrP(Sc) in the highly efficient protein misfolding cyclic amplification system. Our findings suggest that MCT may serve as a potential therapeutic agent for prion diseases.

Bioinformatic and enzymatic characterization of the MAPEG superfamily.

The membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily includes structurally related membrane proteins with diverse functions of widespread origin. A total of 136 proteins belonging to the MAPEG superfamily were found in database and genome screenings. The members were found in prokaryotes and eukaryotes, but not in any archaeal organism. Multiple sequence alignments and calculations of evolutionary trees revealed a clear subdivision of the eukaryotic MAPEG members, corresponding to the six families of microsomal glutathione transferases (MGST) 1, 2 and 3, leukotriene C4 synthase (LTC4), 5-lipoxygenase activating protein (FLAP), and prostaglandin E synthase. Prokaryotes contain at least two distinct potential ancestral subfamilies, of which one is unique, whereas the other most closely resembles enzymes that belong to the MGST2/FLAP/LTC4 synthase families. The insect members are most similar to MGST1/prostaglandin E synthase. With the new data available, we observe that fish enzymes are present in all six families, showing an early origin for MAPEG family differentiation. Thus, the evolutionary origins and relationships of the MAPEG superfamily can be defined, including distinct sequence patterns characteristic for each of the subfamilies. We have further investigated and functionally characterized representative gene products from Escherichia coli, Synechocystis sp., Arabidopsis thaliana and Drosophila melanogaster, and the fish liver enzyme, purified from pike (Esox lucius). Protein overexpression and enzyme activity analysis demonstrated that all proteins catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with reduced glutathione. The E. coli protein displayed glutathione transferase activity of 0.11 micromol.min(-1).mg(-1) in the membrane fraction from bacteria overexpressing the protein. Partial purification of the Synechocystis sp. protein yielded an enzyme of the expected molecular mass and an N-terminal amino acid sequence that was at least 50% pure, with a specific activity towards 1-chloro-2,4-dinitrobenzene of 11 micromol.min(-1).mg(-1). Yeast microsomes expressing the Arabidopsis enzyme showed an activity of 0.02 micromol.min(-1).mg(-1), whereas the Drosophila enzyme expressed in E. coli was highly active at 3.6 micromol.min(-1).mg(-1). The purified pike enzyme is the most active MGST described so far with a specific activity of 285 micromol.min(-1).mg(-1). Drosophila and pike enzymes also displayed glutathione peroxidase activity towards cumene hydroperoxide (0.4 and 2.2 micromol.min(-1).mg(-1), respectively). Glutathione transferase activity can thus be regarded as a common denominator for a majority of MAPEG members throughout the kingdoms of life whereas glutathione peroxidase activity occurs in representatives from the MGST1, 2 and 3 and PGES subfamilies.

Activation of rat liver microsomal glutathione S-transferase by gallic acid.

The effect of phenolic antioxidants on the rat liver microsomal glutathione S-transferase (MGST1) was investigated in vitro. When microsomes were incubated with various polyphenolic antioxidants, gallic acid (3,4,5-trihydroxybenzoic acid) markedly increased MGST1 activity and the increase was prevented in the presence of superoxide dismutase (SOD) or catalase. The MGST1 activity increased by gallic acid was decreased by further incubation with sodium arsenite, a sulfenic acid reducing agent, but was not with dithiothreitol, a disulfide bond reducing agent. The incubation of microsomes with gallic acid in the presence of the NADPH generating system which generates reactive oxygen species (ROS) through cytochrome P-450 system increased the MGST1activity in spite of scavenging the ROS and the increase was also depressed by SOD/catalase. The increase of MGST1 activity by gallic acid was prevented by co-incubation with a stable radical, 1,1-diphenyl-2-picrylhydrazyl or ferric chloride. These results suggest that the gallic acid acts as a pro-oxidant and activates MGST1 through oxidative modification of the enzyme.

NADPH dependent activation of microsomal glutathione transferase 1.

Microsomal glutathione transferase 1 (MGST1) can become activated up to 30-fold by several mechanisms in vitro (e.g. covalent modification by reactive electrophiles such as N-ethylmaleimide (NEM)). Activation has also been observed in vivo during oxidative stress. It has been noted that an NADPH generating system (g.s.) can activate MGST1 (up to 2-fold) in microsomal incubations, but the mechanism was unclear. We show here that NADPH g.s treatment impaired N-ethylmaleimide activation, indicating a shared target (identified as cysteine-49 in the latter case). Furthermore, NADPH activation was prevented by sulfhydryl compounds (glutathione and dithiothreitol). A well established candidate for activation would be oxidative stress, however we could exclude that oxidation mediated by cytochrome P450 2E1 (or flavine monooxygenase) was responsible for activation under a defined set of experimental conditions since superoxide or hydrogen peroxide alone did not activate the enzyme (in microsomes prepared by our routine procedure). Actually, the ability of MGST1 to become activated by hydrogen peroxide is critically dependent on the microsome preparation method (which influences hydrogen peroxide decomposition rate as shown here), explaining variable results in the literature. NADPH g.s. dependent activation of MGST1 could instead be explained, at least partly, by a direct effect observed also with purified enzyme (up to 1.4-fold activation). This activation was inhibited by sulfhydryl compounds and thus displays the same characteristics as that of the microsomal system. Whereas NADPH, and also ATP, activated purified MGST1, several nucleotide analogues did not, demonstrating specificity. It is thus an intriguing possibility that MGST1 function could be modulated by ligands (as well as reactive oxygen species) during oxidative stress when sulfhydryls are depleted.

Characterization of spontaneously generated prion-like conformers in cultured cells.

A distinct conformational transition from the α-helix-rich cellular prion protein (PrPC) into its ß-sheet-rich pathological isoform (PrPSc) is the hallmark of prion diseases, a group of fatal transmissible encephalopathies that includes spontaneous and acquired forms. Recently, a PrPSc-like intermediate form characterized by the formation of insoluble aggregates and protease-resistant PrP species termed insoluble PrPC (iPrPC) has been identified in uninfected mammalian brains and cultured neuronal cells, providing new insights into the molecular mechanism(s) of these diseases. Here, we explore the molecular characteristics of the spontaneously formed iPrPC in cultured neuroblastoma cells expressing wild-type or mutant human PrP linked to two familial prion diseases. We observed that although PrP mutation at either residue 183 from Thr to Ala (PrPT183A) or at residue 198 from Phe to Ser (PrPF198S) affects glycosylation at both N-linked glycosylation sites, the T183A mutation that results in intracellular retention significantly increased the formation of iPrPC. Moreover, while autophagy is increased in F198S cells, it was significantly decreased in T183A cells. Our results indicate that iPrPC may be formed more readily in an intracellular compartment and that a significant increase in PrPT183A aggregation may be attributable to the inhibition of autophagy.

Purification of liver serine protease which activates microsomal glutathione S-transferase: possible involvement of hepsin.

Rat liver microsomal glutathione S-transferase (MGST1) is known to be activated by trypsin, however, it has not been clarified whether MGST1 is activated by a protease present in liver. In the present study we purified the MGST1 activating protease from liver microsomes and finally identified that the protease is hepsin, a type II transmembrane serine protease. When the protease was incubated with the purified MGST1 or liposomal MGST1 at 4 degrees C, MGST1 activity was increased 3-4.5 fold after 3-6 d. In electrophoretic and immunoblot analyses after the incubation of MGST1 with the protease MGST1 dimer and its degraded fragment were detected. These results suggest that the rat liver microsomal hepsin functions as MGST1 activating/degrading enzyme.

Nonenzymatic synthesis of glycerolipids catalyzed by imidazole.

Imidazole catalyzed acylations of lysolipids by acyl-CoAs in water at room temperature and at a pH close to neutrality. In the presence of oleoyl-CoA and either lysophosphatidylcholine, 1-palmitoyl-sn-glycero-3-phosphocholine (LPC); lysophosphatidylglycerol, monoacyl-sn-glycero-3-phosphoglycerol; lysophosphatidyl acid, 1-oleoyl-sn-glycero-3-phosphate; lysophosphatidylserine, monoacyl-sn-glycero-3-phosphoserin; or lysophosphatidylethanolamine, monoacyl-sn-glycero-3-phosphoethanolamine, the corresponding phospholipids were synthesized. Similarly, the use of lyso-platelet activating factor, an ether analog of LPC, yielded the formation of 1-O-alkyl-2-oleoyl-sn-glycero-3-phosphocholine. In the presence of LPC, an imidazole-catalyzed synthesis of phosphatidylcholine (PC) occurred when medium, long, and very long chain acyl-CoAs were added. With hydroxyacyl-CoA, a similar PC synthesis was obtained. The process described in the present paper appears to offer several potential applications of interest for the synthesis of glycerophospholipids and triglycerides with labeled and/or an unusual or fragile fatty acid, or when suitable acyltransferases have not yet been described in the literature and/or are not commercially available. The method described is very safe and simple since lipids can be synthesized in tubes containing 0.7% imidazole in water, and left for a few hours at room temperature on the bench.