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Biomedical subjects

G Powis

Publications and source records attributed to G Powis.

At least 55 records · Page 3Linked to original sources
PubMed1997

Thioredoxin, a gene found overexpressed in human cancer, inhibits apoptosis in vitro and in vivo.

The redox protein thioredoxin plays an important role in controlling cancer cell growth through regulation of DNA synthesis and transcription factor activity. Thioredoxin is overexpressed by a number of human primary cancers and its expression is decreased during dexamethasone-induced apoptosis of mouse WEHI7.2 thymoma cells. We examined the ability of WEHI7.2 cells stably transfected with human thioredoxin cDNA showing increased levels of cytoplasmic thioredoxin to undergo apoptosis in vitro and in vivo. The cells were protected from apoptosis induced by dexamethasone, staurosporine, etoposide, and thapsigargin, but not by N-acetyl-sphingosine. When inoculated into severe combined immunodeficient mice, the trx-transfected cells formed tumors that showed increased growth compared to wild-type, as well as bcl-2-transfected, WEHI7.2 cells. The trx- and bcl-2-transfected cell tumors both showed less spontaneous apoptosis than tumors formed by the wild-type cells. Unlike tumors formed by the wild-type and bcl-2-transfected WEHI7.2 cells, trx-transfected cell tumors did not show growth inhibition upon treatment with dexamethasone. This study suggests that increased thioredoxin expression in human cancers may result in an increased tumor growth through inhibition of spontaneous apoptosis and a decrease in the sensitivity of the tumor to drug-induced apoptosis.

Animals↗
PubMed1997

Mechanisms of the regulation of thioredoxin reductase activity in cancer cells by the chemopreventive agent selenium.

Selenium is an essential trace element, the deficiency of which is associated with an increased incidence of some human cancers. Dietary supplementation with selenium has been reported to produce a decrease in the incidence of some cancers in humans. Thioredoxin reductase (TR) is a newly discovered homodimeric selenocysteine (SeCys)-containing protein that catalyzes the NADPH-dependent reduction of the redox protein thioredoxin (Trx). Trx is overexpressed by a number of human tumors, and experimental studies have shown that Trx contributes to the growth and to the transformed phenotype of some human cancer cells. Thus, TR, by reducing Trx, could play a role in regulating the growth of normal and cancer cells. We have investigated mechanisms by which selenium, in the form of sodium selenite, added to serum-free growth medium regulates TR activity in cancer cell lines. Selenium caused a dose-dependent increase in cellular TR activity. The increase in TR activity produced by 1 microM Se compared to medium with no added selenium was: for MCF-7 breast cancer cells, 37-fold; for HT-29 colon cancer cells, 19-fold; and for A549 lung cancer cells, 8-fold. In contrast, Jurkat and HL-60 leukemia cells showed no increase in TR activity. The half-life of the time course of induction of TR in HT-29 cells after adding selenium was 10 h. The increase in TR activity was accompanied by an increase in TR protein levels up to 3-fold and an increase in the specific activity of the enzyme of 5-32-fold, depending on the cell line. Studies using 75Se showed that the amount of selenium incorporated into TR increased with increasing selenium concentration up to a ratio of 1 selenium per TR monomer. There was an increase in TR mRNA levels of 2-5-fold at 1 microM selenium and an increase in the stability of TR mRNA with a half-life for degradation of 21 h compared to 10 h in the absence of selenium. Trx mRNA and protein levels and Trx mRNA stability were not affected by selenium. The results of the study show that the increase in TR activity caused by selenium is specific and due to several effects, including an increase in the stability of TR mRNA leading to increased TR mRNA levels, an increase in TR protein, but predominantly to an increase in the specific activity of TR associated with increased incorporation of selenium into the enzyme.

Anticarcinogenic Agents↗
PubMed1997

The expression of the molecular chaperone calnexin is decreased in cancer cells grown as colonies compared to monolayer.

Differential display was used to identify genes which were differentially expressed when HT-29 human colon adenocarcinoma cells were grown as monolayers attached to plastic or as colonies in soft agarose. One of the gel bands differentially displayed corresponded to a 171 bp fragment that showed 99% identity with a sequence of mRNA for human calnexin. The decrease in calnexin gene expression by HT-29 cells growing as colonies in soft agarose was confirmed by reverse transcriptase-PCR (RT-PCR) using calnexin-specific primers. We also used RT-PCR to show that the expression of calnexin was decreased in HT-29 cells and MCF-7 human breast adenocarcinoma cells growing in suspension in poly(hydroxyethyl methacrylate)-coated wells compared to cells growing as monolayers. The results suggest that there is a signal for the up-regulation of calnexin expression when cells contact a substrate which allows cell adhesion.

Adenocarcinoma↗
PubMed1997

Pharmacokinetic studies of the herbicide and antitumor compound oryzalin in mice.

Oryzalin [3,5-dinitro-N,N-di(n-propyl)benzensulfanilamide] is a widely used sulfonamide herbicide that selectively inhibits microtubule formation in algae and higher plants. Oryzalin has also been found to be an inhibitor of intracellular free Ca2+ signaling in mammalian cells and to have antitumor activity in animals. Despite its widespread use there have been no reports of the pharmacokinetics of oryzalin in animals or man. A reversed-phase high-performance liquid chromatographic (HPLC) method was developed to measure oryzalin in biological fluids. Following repeated daily administration of oryzalin to mice by the i.p. route at 200 mg/kg, or the p.o. route at 300 mg/kg, peak plasma concentrations of up to 25 micrograms/ml were achieved. The half life for oryzalin in plasma of mice given i.p. oryzalin was 14.3 h with a clearance of 0.07 l/h. A major metabolite of oryzalin, N-depropyloryzalin, was identified in plasma and its structure confirmed by mass spectral analysis (M+H+ = 305). This metabolite was cleared more rapidly than oryzalin with a half life of 1.15 h and a clearance of 0.17 l/h. N-Depropyloryzalin caused similar inhibition of colony formation by HT-29 colon cancer cells as oryzalin with IC50 = 8 micrograms/ml. The results suggest that oryzalin and its N-depropyl metabolite can inhibit tumor colony formation at pharmacologically achievable levels.

Animals↗
PubMed1997

Inhibition of intracellular Ca2+ signalling, cytotoxicity and antitumor activity of the herbicide oryzalin and its analogues.

PURPOSE: Studies were conducted on oryzalin (3,5-dinitro-N,N-di(n-propyl)sulfanilamide), a widely used dinitroaniline sulfonamide herbicide, which was identified from plant extracts as an inhibitor of mitogen- and growth factor-mediated intracellular free Ca2+ ([Ca2+]i) signalling in mammalian cells. METHODS AND RESULTS: Oryzalin inhibited vasopressin, bradykinin and platelet-derived growth factor [Ca2+]i signalling in Swiss 3T3 fibroblasts with IC50 values of 14, 16 and 18 microM, respectively. 45Ca2+ uptake into nonmitochondrial stores of saponin-permeabilized Swiss 3T3 cells was inhibited by oryzalin with an IC50 of 34 microM. Oryzalin inhibited colony formation of HT-29 colon carcinoma cells with an IC50 of 8 microM and inhibited the growth of a number of other cancer cell lines and primary human tumors in vitro with IC50 values in the range 3 to 22 microM. A number of oryzalin analogues were studied and an association was found between the ability to inhibit [Ca2+]i signalling and inhibition of the growth of HT-29 human colon cancer cells (P = 0.001) and of CCRF-CEM human leukemia cells (P = 0.016). Oryzalin at doses up to 600 mg/kg administered orally or subcutaneously daily to mice for 3 to 10 days beginning a day after tumor inoculation inhibited the growth of murine B16 melanoma by 63% but showed no appreciable activity when administered subcutaneously or intraperitoneally to mice beginning a number of days after tumor inoculation against a variety of human tumor xenografts. The peak plasma concentration of oryzalin following repeated subcutaneous administration of oryzalin at 600 mg/kg per day to mice was 37 microM and of its major metabolite N-depropyl oryzalin was 53 microM. CONCLUSION: It is unlikely that the absence of significant antitumor activity of oryzalin is a result of the inability to achieve adequate plasma concentrations.

3T3 Cells↗
PubMed1997

Selenium and the thioredoxin redox system: effects on cell growth and death.

Thioredoxin is a redox protein found overexpressed in some human tumors. Thioredoxin is secreted by tumor cells and enhances the sensitivity of the cancer cells to other growth factors. Redox activity is essential for stimulation of cell growth by thioredoxin. Cells transfected with thioredoxin cDNA show increased tumor growth and decreased apoptosis in vivo and decreased sensitivity to apoptosis induced by a variety of agents both in vitro and in vivo. Cells transfected with a redox-inactive mutant thioredoxin show inhibited tumor growth in vivo. Dietary selenium has been shown to prevent some forms of human cancer. Selenocysteine is an essential component of thioredoxin reductase, the flavoenzyme that is responsible for the reduction of thioredoxin. Selenium added to the culture medium increases thioredoxin reductase activity due to an increase in thioredoxin reductase protein but mostly due to an increase in the specific activity of the enzyme. Some diaryl chalcogenide (selenium and tellurium) compounds have been studied as inhibitors of thioredoxin reductase. The most active were diaryl tellurium compounds, which were noncompetitive inhibitors of thioredoxin reductase with Ki values of 2-10 microM. Several of the compounds inhibited cancer cell colony formation in vitro with IC50s as low as 2 microM.

Apoptosis↗
PubMed1997

Redox active disulfides: the thioredoxin system as a drug target.

Thioredoxin, and particularly extracellular thioredoxin, presents an attractive target for developing novel agents to treat cancer. Our studies have involved the examination of a series of alkyl 2-imidazolyl disulfides as inhibitors of the growth-stimulatory activity of the thioredoxin system. We originally determined the disulfides to be weak reversible inhibitors of thioredoxin reductase. Subsequently, we have shown that alkyl 2-imidazolyl disulfides interact directly with thioredoxin, thioalkylating critical cysteine residues or causing dimerization of the protein leading to its loss of biological activity. One of the analogues that binds to thioredoxin, 1-methylpropyl 2-imidazolyl disulfide (IV-2), selectively inhibits the thioredoxin-dependent growth of tumor cells in culture and has antitumor activity against MCF-7 and HL-60 tumors in vivo. Our work involves the development of a parallel combinatorial synthetic method to produce a large number of disulfide analogues at one time. These analogues, which differ sterically, electronically, and physically, were produced in a 96-well plate. The biological activity of these analogues was evaluated, also in the 96-well plate format. This rapid method of evaluating biological activity is a means to identify agents with specificity for inhibition of the thioredoxin system, and may provide novel antitumor agents with activity against solid tumor cancers.

Animals↗
PubMed1997

Cell line-directed screening assay for inhibitors of thioredoxin reductase signaling as potential anti-cancer drugs.

We have used a cell line-directed screening approach (CDSA) to identify novel inhibitors of the thioredoxin reductase signaling pathway which contributes to the transformed phenotype of some human tumors. Two 2-imidazolyl disulfide compounds, previously identified as inhibitors of thioredoxin reductase, were screened for growth inhibitory activity in the National Cancer Institute (NCI) human cancer cell line panel. The COMPARE pattern recognition algorithm was used to identify similar compounds from > 60,000 compounds in the NCI investigational drug database. Of 47 nondiscreet compounds tested in a thioredoxin reductase/thioredoxin insulin reduction assay, 37 (77%) were inhibitors with IC50s < or = 10 micrograms/ml and 15 of those (32%) had IC50s < or = 1 microgram/ml. These compounds were all as selective or more selective for thioredoxin reductase than for glutathione reductase, while three compounds were inhibitors of thioredoxin. In comparison to CDSA, the number of compounds with IC50s < or = 1 microgram/ml identified by screening of 52 compounds from the database whose growth inhibiting activity was unrelated to the activity of the disulfide compounds was only 2%. Screening of 221 randomly selected natural products gave only 3% of compounds with IC50s < or = 1 microgram/ml. Thus, the CDSA using data from the NCI cancer cell panel and known inhibitors of the selected target as seed compounds can greatly increase hit rates, compared with random screening, for identifying novel inhibitors of a target, in this case thioredoxin signaling.

Antineoplastic Agents↗
PubMed1996

Transfection with human thioredoxin increases cell proliferation and a dominant-negative mutant thioredoxin reverses the transformed phenotype of human breast cancer cells.

Thioredoxin, a redox protein with growth factor activity that modulates the activity of several proteins important for cell growth, has been reported to be overexpressed in a number of human primary cancers. In the present study, the effects of stably transfecting mouse NIH 3T3 cells and MCF-7 human breast cancer cells with cDNA for wild-type human thioredoxin or a redox-inactive mutant thioredoxin, Cys32-->Ser32/Cys35-->Ser35 (C32S/C35S), on cell proliferation and transformed phenotype have been investigated. NIH 3T3 cells transfected with thioredoxin achieved increased saturation densities compared with vector alone-transfected cells, but were not transformed as assessed by tumor formation in immunodeficient mice. Thioredoxin-transfected MCF-7 cells showed unaltered monolayer growth on plastic surfaces compared with vector alone-transfected cells, but exhibited severalfold increased colony formation in soft agarose. Stable transfection of NIH 3T3 and MCF-7 cells with C32S/C35S resulted in inhibition of monolayer growth on plastic surfaces, and up to 73% inhibition of colony formation by MCF-7 cells in soft agarose. When inoculated into immunodeficient mice, thioredoxin-transfected MCF-7 cells formed tumors, although with a 38-57% growth rate compared with vector alone-transfected cells, whereas tumor formation by C32S/C35S-transfected MCF-7 cells was almost completely inhibited. The results of the study suggest that thioredoxin plays an important role in the growth and transformed phenotype of some human cancers. The inhibition of tumor cell growth by the dominant-negative redox-inactive mutant thioredoxin suggests that thioredoxin could be a novel target for the development of drugs to treat human cancer.

3T3 Cells↗
PubMed1996

Oxidative inactivation of thioredoxin as a cellular growth factor and protection by a Cys73-->Ser mutation.

Thioredoxin (Trx) is a widely distributed redox protein that regulates several intracellular redox-dependent processes and stimulates the proliferation of both normal and tumor cells. We have found that when stored in the absence of reducing agents, human recombinant Trx undergoes spontaneous oxidation, losing its ability to stimulate cell growth, but is still a substrate for NADPH-dependent reduction by human thioredoxin reductase. There is a slower spontaneous conversion of Trx to a homodimer that is not a substrate for reduction by thioredoxin reductase and that does not stimulate cell proliferation. Both conversions can be induced by chemical oxidants and are reversible by treatment with the thiol reducing agent dithiothreitol. SDS-PAGE suggests that Trx undergoes oxidation to monomeric form(s) preceding dimer formation. We have recently shown by X-ray crystallography that Trx forms a dimer that is stabilized by an intermolecular Cys73-Cys73 disulfide bond. A Cys73-->Ser mutant Trx (C73S) was prepared to determine the role of Cys73 in oxidative stability and growth stimulation. C73S was as effective as Trx in stimulating cell growth and was a comparable substrate for thioredoxin reductase. C73S did not show spontaneous or oxidant-induced loss of activity and did not form a dimer. The results suggest that Trx can exist in monomeric forms, some of which are mediated by Cys73 that do not stimulate cell proliferation but can be reduced by thioredoxin reductase. Cys73 is also involved in formation of an enzymatically inactive homodimer, which occurs on long term storage or by chemical oxidation. Thus, although clearly involved in protein inactivation, Cys73 is not necessary for the growth stimulating activity of Trx.

Growth Substances↗
PubMed1996

Human thioredoxin reductase gene localization to chromosomal position 12q23-q24.1 and mRNA distribution in human tissue.

Thioredoxin reductase is a member of the pyridine nucleotide-disulfide oxidoreductase family of enzymes. By delivering reducing equivalents to thioredoxin, thioredoxin reductase exerts control over a number of redox-sensitive factors in the cell, including ribonucleotide reductase and several transcription factors. We have localized the human thioredoxin gene to chromosomal position 12q23-q24.1 by in situ hybridization. We have also determined the relative tissue distribution of thioredoxin reductase mRNA as well as thioredoxin mRNA by probing a Northern blot of several human normal tissues.

Chromosome Banding↗
PubMed1996

Crystal structures of reduced, oxidized, and mutated human thioredoxins: evidence for a regulatory homodimer.

BACKGROUND: Human thioredoxin reduces the disulfide bonds of numerous proteins in vitro, and can activate transcription factors such as NFkB in vivo. Thioredoxin can also act as a growth factor, and is overexpressed and secreted in certain tumor cells. RESULTS: Crystal structures were determined for reduced and oxidized wild type human thioredoxin (at 1.7 and 2.1 A nominal resolution, respectively), and for reduced mutant proteins Cys73-->Ser and Cys32-->Ser/Cys35-->Ser (at 1.65 and 1.8 A, respectively). Surprisingly, thioredoxin is dimeric in all four structures; the dimer is linked through a disulfide bond between Cys73 of each monomer, except in Cys73-->Ser where a hydrogen bond occurs. The thioredoxin active site is blocked by dimer formation. Conformational changes in the active site and dimer interface accompany oxidation of the active-site cysteines, Cys32 and Cys35. CONCLUSIONS: It has been suggested that a reduced pKa in the first cysteine (Cys32 in human thioredoxin) of the active-site sequence is important for modulation of the redox potential in thioredoxin. A hydrogen bond between the sulfhydryls of Cys32 and Cys35 may reduce the pKa of Cys32 and this pKa depression probably results in increased nucleophilicity of the Cys32 thiolate group. This nucleophilicity, in tum, is thought to be necessary for the role of thioredoxin in disulfide-bond reduction. The physiological role, if any, of thioredoxin dimer formation remains unknown. It is possible that dimerization may provide a mechanism for regulation of the protein, or a means of sensing oxidative stress.

Amino Acid Sequence↗
PubMed1996

Decreased antioxidant defence and increased oxidant stress during dexamethasone-induced apoptosis: bcl-2 prevents the loss of antioxidant enzyme activity.

When the WEHI7.2 mouse lymphoid cell line was treated with dexamethasone to induce apoptosis the activities and transcript levels of the antioxidant defence enzymes catalase, superoxide dismutase (SOD) and DT-diaphorase exhibited a progressive decrease over 48 hours. Catalase activity was maintained and total SOD and DT-diaphorase activity showed smaller decreases following dexamethasone treatment of WEHI7.2 cells transfected with the bcl-2 oncogene, which protects the cells against apoptosis. Treatment of wild-type WEHI7.2 and bcl-2 transfected cells with a catalase inhibitor, aminotriazole, was not sufficient to induce apoptosis. Antioxidants, including bovine liver catalase, bovine erythrocyte CuZn-SOD, sodium selenite and Trolox, a water soluble vitamin E analogue, as well as hypoxia, inhibited dexamethasone-induced apoptosis. These results suggest that oxidant stress due to the decreased activity of antioxidant defence enzymes may play a role in dexamethasone-mediated lymphoid cell apoptosis and that bcl-2 may prevent apoptosis by maintaining the level of critical antioxidant defence mechanisms, which include catalase.

Journal Article↗
PubMed1996

Response of human multiple myeloma-derived cell lines to alkyl-lysophospholipid.

Clinical studies using high-dose chemotherapy and autologous bone marrow transplantation in multiple myeloma report improvement in tumor regression. The potential success of this approach may be compromised because of the myeloma cell burden. A promising approach is chemopurging of bone marrow with alkyl-lysophospholipids (ALP), a new class of antitumor agents. Using a limiting dilution assay, we tested ALP against several drug-resistant human myeloma cell lines: RPMI 8226/S, 8226/DOX40, 8226/LR-5, 8226/MR-20, 8226/DOX1V, and 8226/MDR10V. IC50 values were approximately 6 microg/mL, for all cell lines evaluated. ALP was effective against all cells, with mean log kills ranging from 2.48 to 4.95. Cytotoxicity was temperature-dependent. Survival curves of cell lines exposed to ALP showed a steep slope during the first 4 hours of exposure, with little additional cell kill after 24 hours. Dose-response curves after drug exposure for 4 and 24 hours reached plateaus at 75 and 25 microg/mL, respectively. Increasing concentrations of ALP caused a sustained increase in resting [Ca2+]i of sufficient magnitude to induce cell death. These results suggest that ALP is an effective cytotoxic agent even in cells known to be resistant to alkylating agents and other classes of cytotoxic drugs.

Antineoplastic Agents, Alkylating↗
PubMed1995

Cloning and sequencing of a human thioredoxin reductase.

The DNA sequence encoding human placental thioredoxin reductase has been determined. Of the 3826 base pairs sequenced, 1650 base pairs were in an open reading frame encoding a mature protein with 495 amino acids and a calculated molecular mass of 54,171. Sequence analysis showed strong similarity to glutathione reductases and other NADPH-dependent reductases. Human thioredoxin reductase contains the redox-active cysteines in the putative FAD binding domain and has a dimer interface domain not previously seen with prokaryote and lower eukaryote thioredoxin reductases.

Amino Acid Sequence↗
PubMed1995

An alternatively spliced form of NQO1 (DT-diaphorase) messenger RNA lacking the putative quinone substrate binding site is present in human normal and tumor tissues.

DT-diaphorase is a ubiquitously expressed flavoenzyme responsible for the two-electron reduction of a number of quinone and other anticancer drugs. The majority of DT-diaphorase enzyme activity in human tissues is the product of the NQO1 gene. We have now identified a novel alternatively spliced form of human NQO1 mRNA lacking exon 4 at levels equal to or exceeding those of wild-type NQO1 mRNA. Exon 4 codes for the putative quinone substrate binding site of DT-diaphorase derived from NQO1 and the recombinant protein from alternatively spliced NQO1 mRNA lacking exon 4 has minimal enzyme activity with quinoid and other known substrates of DT-diaphorase. The physiological substrate of DT-diaphorase is unknown, and it is possible that the protein derived from the alternatively spliced NQO1 mRNA could have enzyme activity with an appropriate substrate. We found full-length DT-diaphorase protein but could not detect expression of an appropriately smaller form of DT-diaphorase in human tissues using polyclonal antibody to DT-diaphorase, suggesting that alternatively spliced NQO1 mRNA lacking exon 4 may not be translated or that the protein product is rapidly degraded. Alternative splicing of NQO1 RNA could provide an important mechanism for regulating NQO1 gene expression.

Adenocarcinoma↗