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Biomedical subjects

G Powis

Publications and source records attributed to G Powis.

At least 19 recordsLinked to original sources
PubMed2003

Increased thioredoxin-1 inhibits SSAT expression in MCF-7 human breast cancer cells.

Spermidine/spermine N(1)-acetyltransferase (SSAT) regulates polyamine catabolism. Thioredoxin-1 (Trx-1) is a redox protein that is overexpressed in human cancer leading to increased cell proliferation, decreased apoptosis, and decreased patient survival. We report that SSAT mRNA expression is decreased in Trx-1 transfected MCF-7 human breast cancer cells. There is also a decrease in SSAT enzyme activity and lower putrescine levels but no change in spermine or spermidine levels. The expression of SSAT is regulated by the NF-E2-related factor 2 (Nrf-2) and polyamine modulated factor-1 (PMF-1) transcription factor complex. Trx-1 transfected MCF-7 cells showed decreased Nrf-2/PMF-1 DNA binding without a change in Nrf-2 or PMF-1 protein expression. The results suggest that Trx-1 may play a role in the redox regulation of SSAT expression and polyamine homeostasis that could contribute to the biological effects of Trx-1.

Acetyltransferases↗
PubMed2001

New inhibitors of the thioredoxin-thioredoxin reductase system based on a naphthoquinone spiroketal natural product lead.

Natural products of the naphthoquinone spiroketal structural type served as lead structures for the development of novel inhibitors of the thioredoxin-thioredoxin reductase redox system. The most potent compound in this series inhibited thioredoxin with an IC(50) of 350 nM, and many derivatives showed low micromolar activities for growth inhibition against two breast cancer cell lines.

Antineoplastic Agents↗
PubMed2001

Thioredoxin peroxidase-1 (peroxiredoxin-1) is increased in thioredoxin-1 transfected cells and results in enhanced protection against apoptosis caused by hydrogen peroxide but not by other agents including dexamethasone, etoposide, and doxorubicin.

Thioredoxin-1 (Trx-1) is a small redox oncoprotein whose expression is increased in a number of human primary cancers where it is associated with aggressive tumor growth, inhibition of apoptosis and decreased patient survival. We report that Trx-1-transfected MCF-7 human breast cancer cells have increased expression of thioredoxin peroxidase-1 (TrxP-1) a peroxiredoxin family member that scavenges H(2)O(2) using Trx-1 as a source of reducing equivalents. Our work shows that TrxP-1 is more effective than selenium-dependent glutathione peroxidase in protecting cells against H(2)O(2) damage. Transfection of mouse WEHI7.2 lymphoma cells with human TrxP-1 or TrxP-2, but not TrxP-4, protects the cells against H(2)O(2) induced apoptosis but does not protect against apoptosis induced by dexamethasone, etoposide, or doxorubicin. The results show that an increase in TrxP-1 expression contributes to the protection against H(2)O(2) induced apoptosis caused by Trx-1, but does not protect against apoptosis induced by other agents.

Animals↗
PubMed2001

Alternative splicing is associated with decreased expression of the redox proto-oncogene thioredoxin-1 in human cancers.

Human thioredoxin-1 (Trx-1) is a small redox protein that is overexpressed in a number of human primary tumors, where it is associated with rapid cell proliferation and inhibited apoptosis. Mutation scanning denaturing high-performance liquid chromatography of Trx-1 mRNA in 58 human tumor cell lines found no evidence for changes in the base sequence of human Trx-1 mRNA. An alternatively spliced form of Trx-1 mRNA lacking exons 2 and 3 was found in 7 of the cell lines but it was not translated. The cell lines having the alternatively spliced Trx-1 mRNA had 73% lower total Trx-1 mRNA than the other cell lines, suggesting that alternative splicing may control the level of Trx-1 mRNA in some cancer cells.

Alternative Splicing↗
PubMed2001

Molecular modeling studies of the Akt PH domain and its interaction with phosphoinositides.

The serine-threonine protein kinase Akt is a direct downstream target of phosphatidylinositol 3-kinase (PI3-K). The PI3-K-generated phospholipids regulate Akt activity via directly binding to the Akt PH domain. The binding of PI3-K-generated phospholipids is critical to the relocalization of Akt to the plasma membrane, which plays an important role in the process of Akt activation. Activation of the PI3-K/Akt signaling pathway promotes cell survival. To elucidate the structural basis of the interaction of PI3-K-generated phospholipids with the Akt PH domain with the objective of carrying out structure-based drug design, we modeled the three-dimensional structure of the Akt PH domain. Comparative modeling-based methods were employed, and the modeled Akt structure was used in turn to construct structural models of Akt in complex with selected PI3-K-generated phospholipids using the computational docking approach. The model of the Akt PH domain consists of seven beta-strands forming two antiparallel beta-sheets capped by a C-terminal alpha-helix. The beta1-beta2, beta3-beta4, and beta6-beta7 loops form a positively charged pocket that can accommodate the PI3-K-generated phospholipids in a complementary fashion through specific hydrogen-bonding interactions. The residues Lys14, Arg25, Tyr38, Arg48, and Arg86 form the bottom of the binding pocket and specifically interact with the 3- and 4-phophate groups of the phospholipids, while residues Thr21 and Arg23 are situated at the wall of the binding pocket and bind to the 1-phosphate group. The predicted binding mode is consistent with known site-directed mutagenesis data, which reveal that mutation of these crucial residues leads to the loss of Akt activity. Moreover, our model can be used to predict the binding affinity of PI3-K-generated phospholipids and rationalize the specificity of the Akt PH domain for PI(3,4)P2, as opposed to other phospholipids such as PI(3)P and PI(3,4,5)P3. Taken together, our modeling studies provide an improved understanding of the molecular interactions present between the Akt PH domain and the PI3-K-generated phospholipids, thereby providing a solid structural basis for the design of novel, high-affinity ligands useful in modulating the activity of Akt.

Amino Acid Sequence↗
PubMed2001

Catalase-overexpressing thymocytes are resistant to glucocorticoid-induced apoptosis and exhibit increased net tumor growth.

Glucocorticoids are used for the treatment of lymphoid neoplasms, taking advantage of the well-known ability of these compounds to cause apoptosis in lymphoid tissues. Previously, we have shown that dexamethasone, a synthetic glucocorticoid, causes a down-regulation of several antioxidant defense enzymes and proteins, including catalase and thioredoxin, concomitant with the induction of apoptosis in WEHI7.2 mouse thymoma cells. To test whether this down-regulation plays a critical role in the mechanism of steroid-induced apoptosis, WEHI7.2 cells were transfected with rat catalase. Two clones, expressing 1.4-fold and 2.0-fold higher catalase specific activity, respectively, when compared with vectoronly transfectants were selected for further study. An increase to 1.4-fold parental cell catalase activity delayed cell loss after dexamethasone treatment, whereas a 2.0-fold parental catalase activity prevented dexamethasone-induced cell loss for 48 h after treatment. Dexamethasone treatment of the WEHI7.2 cells stimulated a release of cytochrome c into the cytosol. Catalase-overexpressing cells showed a delay or lack of cytochrome c release from the mitochondria, which correlated temporally with the delay or prevention of cell loss in the culture after dexamethasone treatment. A decreased amount of cell death from WEHI7.2 cells overexpressing catalase was also seen in tumor xenografts in severe combined immunodeficient mice when compared with tumors from vector-only transfected cells. Similarly, thioredoxin-overexpressing WEHI7.2 cells, shown previously to be apoptosis resistant, showed decreased cell death in tumor xenografts. This resulted in larger tumors from cells overexpressing these proteins. Cell death in control transfectant tumor xenografts was primarily attributable to apoptosis. In contrast, the cell death we observed in tumors from thioredoxin- or catalase-overexpressing cells had a higher frequency of a nonapoptotic, nonnecrotic type of cell death termed para-apoptosis. These data suggest that: (a) oxidative stress plays a critical role in steroid-induced apoptosis prior to the commitment of the cells to undergo apoptosis; and (b) resistance to oxidative stress can contribute to tumor growth.

Animals↗
PubMed2001

Syntheses and biological activities of a novel group of steroidal derived inhibitors for human Cdc25A protein phosphatase.

Silica gel supported pyrolysis of an azido-homo-oxa steroid led to rearrangement, presumably by a mechanism similar to that of solution phase Schmidt fragmentation, to produce a group of novel inhibitors for the oncogenic cell cycle regulator Cdc25A phosphatase. Cyano-containing acid 17, one of the best inhibitors in this group, inhibited the activity of Cdc25A protein phosphatase reversibly and noncompetitively with an IC(50) value of 2.2 microM. Structure-activity relationships revealed that a phosphate surrogate such as a carboxyl or a xanthate group is required for inhibitory activity, and a hydrophobic alkyl chain, such as the cholesteryl side chain, contributes greatly to the potency. Without the cyano group, acid 26 and xanthate 27 were found to be more selective over Cdc25A (IC(50) = 5.1 microM and 1.1 microM, respectively) than toward CD45 (IC(50) > 100 microM, in each case), a receptor protein tyrosine phosphatase. Several of these inhibitors showed antiproliferative activities in the NCI 60-human tumor cell line screen. These steroidal derived Cdc25 inhibitors provide unique leads for the development of dual-specificity protein phosphatase inhibitors.

Antineoplastic Agents↗
PubMed2001

DNA microarray reveals increased expression of thioredoxin peroxidase in thioredoxin-1 transfected cells and its functional consequences.

The mammalian thioredoxins are a family of small redox proteins that undergo NADPH dependent reduction by thioredoxin reductase. Reduced thioredoxins reduce oxidized cysteine groups on proteins including transcription factors to increase their binding to DNA, and is a source of reducing equivalents for enzymes such as thioredoxin peroxidase which removes H2O2 and alkyl peroxides. Thioredoxin-1 is over expressed in many human tumors where it is associated with aggressive tumor growth, inhibited apoptosis and decreased patient survival. Transfection of cells with thioredoxin-1 has been shown to increase cell growth and inhibit apoptosis. We have used DNA micro array to investigate the effects of thioredoxin-1 transfection on the expression of a panel of 520 redox, apoptosis and cell growth related genes in MCF-7 human breast cancer cells. One of the genes whose expression was increased as a result of thioredoxin-1 over expression was thioredoxin peroxidase-2. This increase was confirmed by Northern blotting. Transfection of mouse WEHI7.2 thymoma cells with human thioredoxin peroxidase-2 was found to protect the cells from apoptosis induced by H2O2 but not from apoptosis induced by dexamethasone, doxorubicin or etoposide. Thus, increased thioredoxin peroxidase-2 expression does not explain the widespread antiapoptotic effects of thioredoxin-1.

Animals↗
PubMed2001

Structure-activity relationship of aza-steroids as PI-PLC inhibitors.

A number of aza-steroids were synthesized as potent phosphatidylinositol phospholipase C (PI-PLC) inhibitors. The epimeric mixtures 22,25-diazacholesterol (8a) and 3beta-hydroxy-22,25-diazacholestane (8b) were among the most active of these inhibitors, with IC(50) values of 7.4 and 7.5 microM, respectively. The 20alpha epimer, 8a2 (IC(50)=0.64 microM), whose stereochemistry at C-20 coincides with that of cholesterol, was found 50 times more potent than the 20beta epimer, 8a1 (IC(50)=32.2 microM). In diaza-estrone derivatives, the 3-methoxy group on the aromatic A-ring of 23 exhibited moderate PI-PLC inhibitory activity (IC(50)=19.7 microM), while compound with a free hydroxyl group (21) was inactive. However, in diaza-pregnane derivatives, epimers with a 3-hydroxyl group (8a, IC(50)=7.4 microM) exhibited more potent PI-PLC inhibitory activity than their counterparts with 3-methoxyl group on the non-aromatic A-ring (26, IC(50)=17.4 microM). We have illustrated in our previous publication that 3-hydroxyl-6-aza steroids are potent PI-PLC inhibitors.(3) However, simultaneous presence of the 6-aza and 22,25-diaza moieties in one molecule as in 13, led to loss of activity. Epimeric mixture 8a showed selective growth inhibition effects in the NCI in vitro tumor cell screen with a mean GI(50) value (MG-MID) of 5.75 microM for 54 tumors.

Azacosterol↗
PubMed2001

Properties and biological activities of thioredoxins.

The mammalian thioredoxins are a family of small (approximately 12 kDa) redox proteins that undergo NADPH-dependent reduction by thioredoxin reductase and in turn reduce oxidized cysteine groups on proteins. The two main thioredoxins are thioredoxin- 1, a cytosolic and nuclear form, and thioredoxin-2, a mitochondrial form. Thioredoxin-1 has been studied more. It performs many biological actions including the supply of reducing equivalents to thioredoxin peroxidases and ribonucleotide reductase, the regulation of transcription factor activity, and the regulation of enzyme activity by heterodimer formation. Thioredoxin-1 stimulates cell growth and is an inhibitor of apoptosis. Thioredoxins may play a role in a variety of human diseases including cancer. An increased level of thioredoxin-1 is found in many human tumors, where it is associated with aggressive tumor growth. Drugs are being developed that inhibit thioredoxin and that have antitumor activity.

Animals↗
PubMed2001

Properties and biological activities of thioredoxins.

The mammalian thioredoxins are a family of small (approximately 12 kDa) redox proteins that undergo NADPH-dependent reduction by thioredoxin reductase and in turn reduce oxidized cysteine groups on proteins. The two main thioredoxins are thioredoxin-1, a cytosolic and nuclear form, and thioredoxin-2, a mitochondrial form. Thioredoxin-1 has been studied more. It performs many biological actions including the supply of reducing equivalents to thioredoxin peroxidases and ribonucleotide reductase, the regulation of transcription factor activity, and the regulation of enzyme activity by heterodimer formation. Thioredoxin-1 stimulates cell growth and is an inhibitor of apoptosis. Thioredoxins may play a role in a variety of human diseases including cancer. An increased level of thioredoxin-1 is found in many human tumors, where it is associated with aggressive tumor growth. Drugs are being developed that inhibit thioredoxin and that have antitumor activity.

Animals↗
PubMed2001

Thioredoxin expression is associated with lymph node status and prognosis in early operable non-small cell lung cancer.

PURPOSE: Thioredoxin (TRX), a low molecular weight protein, exerts reduction-oxidation control over a number of transcription factors involved in cell activation and proliferation. High TRX mRNA levels have been found in lung carcinomas, a trait associated with a growth and survival advantage. EXPERIMENTAL DESIGN: In this study, we examined the immunohistochemical expression of human TRX in normal lung and in 102 primary non-small cell lung carcinomas. RESULTS: In normal lung, the staining for TRX was cytoplasmic in the respiratory bronchial epithelium, alveolar epithelium, and alveolar macrophages. Bronchial glandular cells demonstrated a mixed nuclear and cytoplasmic staining. In lung carcinomas, the pattern of expression for TRX was predominantly cytoplasmic and only occasionally nuclear. A strong association between absence of TRX expression and regional lymph node negativity was observed (P = 0.004). High proliferation index, as detected with Ki-67 antibody, was associated with high TRX expression (P = 0.02). A significant correlation between high cytoplasmic p53 reactivity and low TRX expression was observed (P = 0.04). No association with grade, tumor stage, histology, or bcl-2 was noted. A significant coexpression of TRX with human activator protein endonuclease 1 was recorded (P = 0.04). Absence of TRX expression was associated with a better outcome (P < 0.05). CONCLUSIONS: We conclude that overexpression of TRX in non-small cell lung carcinomas is indicative of a more aggressive tumor phenotype and is associated with bad prognostic features and possibly with a poorer outcome.

Aged↗
PubMed2000

3-(Hydroxymethyl)-bearing phosphatidylinositol ether lipid analogues and carbonate surrogates block PI3-K, Akt, and cancer cell growth.

Phosphatidylinositol 3-kinase (PI3-K) phosphorylates the 3-position of phosphatidylinositol to give rise to three signaling phospholipids. Binding of the pleckstrin homology (PH) domain of Akt to membrane PI(3)P's causes the translocation of Akt to the plasma membrane bringing it into contact with membrane-bound Akt kinase (PDK1 and 2), which phosphorylates and activates Akt. Akt inhibits apoptosis by phosphorylating Bad, thus promoting its binding to and blockade of the activity of the cell survival factor Bcl-x. Herein we present the synthesis and biological activity of several novel phosphatidylinositol analogues and demonstrate the ability of the carbonate group to function as a surrogate for the phosphate moiety. Due to a combination of their PI3-K and Akt inhibitory activities, the PI analogues 2, 3, and 5 proved to be good inhibitors of the growth of various cancer cell lines with IC(50) values in the 1-10 microM range. The enhanced Akt inhibitory activity of the axial hydroxymethyl-bearing analogue 5 compared to its equatorial counterpart 6 is rationalized based upon postulated differences in the H-bonding patterns of these compounds in complex with a homology modeling generated structure of the PH domain of Akt. This work represents the first attempt to examine the effects of 3-modified PI analogues on these two crucial cell signaling proteins, PI3-K and Akt, in an effort to better understand their cell growth inhibitory properties.

Antineoplastic Agents↗
PubMed2000

A redox-inactive thioredoxin reduces growth and enhances apoptosis in WEHI7.2 cells.

Cancer cell lines transfected with thioredoxin show increased anchorage-independent growth and decreased sensitivity to induction of apoptosis by a number of anticancer drugs. The present studies were undertaken to evaluate further the role of thioredoxin in cell growth and drug-induced apoptosis. A redox-inactive mutant thioredoxin was stably transfected into WEHI7.2 mouse lymphocytic leukemia cells and two clones were examined for growth characteristics and the induction of apoptosis by dexamethasone, etoposide, doxorubicin, and staurosporine. These clones each exhibited a 71% increase in doubling time in solution and a 20 and 75% reduction in colony formation in soft agarose. The transfected cells also showed increased susceptibility to apoptosis induced by dexamethasone, etoposide, doxorubicin, and staurosporine compared with controls. The results of this study suggest that thioredoxin can regulate the growth rate of cells and that thioredoxin is a critical component in the pathway leading to drug-induced apoptosis in WEHI7.2 cells.

Animals↗
PubMed2000

Thioredoxin reductase.

The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases.

Animals↗
PubMed2000

Redox control of caspase-3 activity by thioredoxin and other reduced proteins.

Caspases are cysteine proteinases that play a critical role in the execution phase of apoptosis. The active site cysteine residue must be reduced for caspase activity. Thioredoxins are redox proteins that catalyze the reduction of cysteine residues. We have examined the ability of various recombinant human thioredoxins to activate caspase-3. The EC(50) for caspase-3 activation by reduced thioredoxin-1 was 2.5 microM, by reduced glutathione 1.0 mM and by dithiothreitol 3.5 mM. A catalytic site redox-inactive mutant thioredoxin-1 was almost as active as thioredoxin-1 in activating caspase-3. Caspase activation was shown to correlate with the number of reduced cysteine residues in the thioredoxins. Reduced insulin and serum albumin were as effective on a molar basis as thioredoxin-1 in activating caspase-3. Thus, caspase-3 activation is not a specific effect of thioredoxins but is a property shared by other reduced proteins.

Apoptosis↗