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Biomedical subjects

Kenneth D Tew

Publications and source records attributed to Kenneth D Tew.

At least 19 recordsLinked to original sources
PubMed2006

PABA/NO as an anticancer lead: analogue synthesis, structure revision, solution chemistry, reactivity toward glutathione, and in vitro activity.

PABA/NO is a diazeniumdiolate of structure Me(2)NN(O)=NOAr (where Ar is a 5-substituted-2,4-dinitrophenyl ring whose 5-substituent is N-methyl-p-aminobenzoic acid). It has shown activity against human ovarian cancer xenografts in mice rivaling that of cisplatin, but it is poorly soluble and relatively unstable in water. Here we report structure-based optimization efforts resulting in three analogues with improved solubility and stability in aqueous solution. We sought to explain PABA/NO's physicochemical uniqueness among these four compounds, whose aminobenzoic acid precursors differ structurally only in the presence or absence of the N-methyl group and/or the position of the carboxyl moiety (meta or para). Studies revealed that PABA/NO's N-methyl-p-aminobenzoic acid substituent is bound to the dinitrobenzene ring via its carboxyl oxygen while the other three are linked through the aniline nitrogen. This constitutes a revision of the previously published PABA/NO structure. All four analogues reacted with GSH to produce bioactive nitric oxide (NO), but PABA/NO was the most reactive. Consistent with PABA/NO's potent suppression of A2780 human ovarian cancer xenograft growth in mice, it was the most potent of the four in the OVCAR-3 cell line.

4-Aminobenzoic Acid↗
PubMed2005

A glutathione S-transferase pi-activated prodrug causes kinase activation concurrent with S-glutathionylation of proteins.

Nitric oxide (NO) is an endogenous, diffusible, transcellular messenger shown to affect regulatory and signaling pathways with impact on cell survival. Exposure to NO can impart direct post-translational modifications on target proteins such as nitration and/or nitrosylation. As an alternative, after interaction with oxygen, superoxide, glutathione, or certain metals, NO can lead to S-glutathionylation, a post-translational modification potentially critical to signaling pathways. A novel glutathione S-transferase pi (GSTpi)-activated pro-drug, O(2)-[2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), liberates NO and elicits toxicity in vitro and in vivo. We now show that PABA/NO induces nitrosative stress, resulting in undetectable nitrosylation, limited nitration, and high levels of S-glutathionylation. After a single pharmacologically relevant dose of PABA/NO, S-glutathionylation occurs rapidly (<5 min) and is sustained for approximately 7 h, implying a half-life for the deglutathionylation process of approximately 3 h. Two-dimensional SDS-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to S-glutathionylated residues indicated that numerous proteins were S-glutathionylated. Subsequent matrix-assisted laser desorption ionization/time of flight analysis identified 10 proteins, including beta-lactate dehydrogenase, Rho GDP dissociation inhibitor beta, ATP synthase, elongation factor 2, protein disulfide isomerase, nucleophosmin-1, chaperonin, actin, protein tyrosine phosphatase 1B (PTP1B), and glucosidase II. In addition, we showed that sustained S-glutathionylation was temporally concurrent with drug-induced activation of the stress kinases, known to be linked with cell death pathways. This is consistent with the fact that PABA/NO induces S-glutathionylation and inactivation of PTP1B, one phosphatase that can participate in deactivation of kinases. These effects were consistent with the presence of intracellular PABA/NO or metabolites, because cells overexpressing MRP1 were less sensitive to the drug and had reduced levels of S-glutathionylated proteins.

4-Aminobenzoic Acid↗
PubMed2005

Evaluation of lipophilins as determinants of tumor cell response to estramustine.

Estramustine administered orally as estramustine phosphate (EMP) remains a major tool in hormone refractory prostate cancer chemotherapy. The presence of estramustine binding protein, prostatin, in prostate tissue may be a determinant of response to treatment. Lipophilins are secretory proteins with homology to prostatin. Reverse transcription-polymerase chain reaction was performed to estimate expression patterns of lipophilins A to C in human biopsies and cell lines resistant to estramustine. Although lipophilin A was not expressed in prostate tissue, both lipophilins B and C were expressed in normal and tumor prostate without significant differences. For lipophilin C, a somatic mutation (T to C transition at positions 409 and 412) was found in human tumor samples and absent in normal prostate tissue. No consistent response to EMP was observed in enhanced green fluorescent protein (EGFP)-tagged lipophilin C-transfected PC3 cells compared with parental controls. Among these EGFP-lipophilin C clones, no direct correlation between response to EMP treatment (IC50 values) and EGFP expression was observed (p = 0.73). Lipophilin C mRNA levels did not vary significantly between wild-type and estramustine-resistant cells in prostate (DU145 and PC3) and ovarian (SKOV3) cancer cell lines. Overall, these results suggest that lipophilins are not specific determinants of estramustine efficacy.

Antineoplastic Agents, Hormonal↗
PubMed2005

Influence of glutathione S-transferase pi and p53 expression on tumor frequency and spectrum in mice.

The role of glutathione S-transferase pi (GSTpi) in tumor development has been previously suggested; however the exact function of this enzyme in carcinogenesis remains unclear. GSTpi has been identified as a modulator of cell signaling by interacting with and inhibiting c-Jun N-terminal kinase (JNK). This kinase has been in turn described as a regulator of p53 stability and transcriptional activity. To study the possible interaction between GSTpi and p53, we crossed GSTpi-deficient animals with p53(-/-) mice. Double knock out animals were viable but developed tumors within 6 months of age; the life span of these animals was however similar to that of GSTpi(+/-)/p53(-/-) and GSTpi(+/+)/p53(-/-). Mice heterozygous for p53 lived significantly longer than the p53(-/-) animals and developed tumors much later, and the expression of GSTpi did not significantly modify the life span of the animals. In contrast, in a wild-type p53 background, GSTpi(-/-) mice developed tumors with a significantly higher frequency than heterozygous and wild-type animals with a median tumor free life span 20 weeks shorter. In addition, in p53(+/+) background, one third of the GSTpi(-/-) animals developed lung adenomas, while less than 10% of GSTpi(+/-) and GSTpi(+/+) presented such tumors. GSTpi expression did not alter the expression of tumorigenesis markers such as COX-2 or ornithine decarboxylase in response to phorbol ester. Furthermore, GSTpi-deficient mouse embryo fibroblasts were more sensitive to H(2)O(2)-induced apoptosis. P53(-/-) cells, independent of GSTpi status, were more sensitive to UV and other DNA damaging agents than their wild-type counterparts. These results suggest that GSTpi may play a protective role in the development of spontaneous tumors.

Animals↗
PubMed2005

Glutathione S-transferases as regulators of kinase pathways and anticancer drug targets.

Anticancer drug development using the platform of glutathione (GSH), glutathione S-transferases (GST) and pathways that maintain thiol homeostasis has recently produced a number of lead compounds. GSTpi is a prevalent protein in many solid tumors and is overexpressed in cancers resistant to drugs. It has proved to be a viable target for pro-drug activation with at least one candidate in late-stage clinical development. In addition, GSTpi possesses noncatalytic ligand-binding properties important in the direct regulation of kinase pathways. This has led to the development and testing of agents that bind to GSTpi and interfere with protein-protein interactions, with the phase II clinical testing of one such drug. Attachment of glutathione to acceptor cysteine residues (glutathionylation) is a posttranslational modification that can alter the structure and function of proteins. Two agents in preclinical development (PABA/NO, releasing nitric oxide on GST activation, and NOV-002, a pharmacologically stabilized pharmaceutical form of GSSG) can lead to glutathionylation of a number of cellular proteins. The biological significance of these modifications is linked with the mechanism of action of these drugs. In the short term, glutathione-based systems should continue to provide viable targets and a platform for the development of novel cancer drugs.

4-Aminobenzoic Acid↗
PubMed2005

TLK-286: a novel glutathione S-transferase-activated prodrug.

Telik, Inc. (Palo Alto, CA, USA) is currently developing TLK-286, a novel prodrug that is preferentially activated by glutathione S-transferase P1-1 (GST-pi). TLK-286 is the lead clinical candidate from a group of rationally designed glutathione analogues designed to exploit high GST-pi levels in solid tumours and drug-resistant cell populations. This concept was based on extensive literature showing that the overexpression of GST-pi in human tumours is associated with malignancy, poor prognosis and the development of drug resistance. Thus, the selective targeting of susceptible tumour phenotypes is a strategy that should result in the release of more active drug in malignant cells compared with normal tissue, thereby achieving an improved therapeutic index. A number of published preclinical studies have confirmed the mechanism of action of this drug. In a series of Phase II clinical trials, TLK-286 was initially shown to have clinical activity and a favorable toxicity profile as a single agent in the salvage setting in ovarian, non-small cell lung, breast and colorectal cancers. Recently, Phase II trials have been reported that demonstrated TLK-286 is active and did not increase the toxicity in combination treatment regimens with standard chemotherapeutic agents, including platinums, taxanes and anthracyclines in previously treated patients with ovarian and non-small cell lung cancers, and in the first-line treatment setting in non-small cell lung cancer patients. TLK-286 is also presently under active testing in Phase III settings for non-small cell lung and ovarian cancers.

Animals↗
PubMed2004

Human ATP-binding cassette transporter-2 (ABCA2) positively regulates low-density lipoprotein receptor expression and negatively regulates cholesterol esterification in Chinese hamster ovary cells.

We present evidence that the ATP binding-cassette transporter-2 (ABCA2) is a sterol-responsive gene that has a role in the trafficking of low-density lipoprotein-derived free cholesterol (LDL-FC). In HepG2 cells ABCA2 was coordinately expressed with other sterol-responsive genes. Stable constitutive expression of ABCA2 in Chinese hamster ovary cells (CHOA2) was accompanied by an increase the expression of the low-density lipoprotein receptor (LDLR) and other genes involved in the regulation of cholesterol homeostasis. LDLR mRNA was elevated greater than ninefold and 3-hydroxy-3-methylglutaryl CoA synthase (HMGCoA S) expression was elevated sevenfold in CHOA2 cells. The increase in LDLR expression was regulated at the level of transcription; however, culture of CHO and CHOA2 cells in medium containing lipoprotein-deficient serum (LPDS) results in similar levels of LDLR promoter expression. No differences were measured in the dose-dependent uptake of fluorescently labeled 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchorate-LDL (DiI-LDL) between CHO and CHOA2 cells cultured in medium containing LPDS. Ultraviolet microscopy revealed a similar distribution of the DiI-LDL label in cytoplasmic vesicles. We measured an LDL dose-dependent reduction in esterification of LDL-FC in intact CHOA2 cells cultured in medium containing LPDS, however, no significant difference was measured in acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in cell-free extracts of CHO and CHOA2 cells. CHO cells or CHOA2 cells treated with the hydrophobic amine, U18666A, showed similar filipin staining of unesterified cholesterol in cytoplasmic vesicles. Addition of progesterone or U18666A to CHO cells elevated ABCA2 expression. Finally, we found that ABCA2 expression was elevated in Niemann-Pick type C1 (NPC1) fibroblasts and in Familial Hypercholesterolemia (FHC) fibroblasts.

ATP-Binding Cassette Transporters↗
PubMed2004

Association of ABCA2 expression with determinants of Alzheimer's disease.

With the use of a novel method for detecting differential gene expression, alterations in functional gene clusters related to transport or oxidative stress response and beta-amyloid (Abeta) peptide metabolism were identified in a HEK293 cell line engineered to overexpress the human ATP binding cassette transporter ABCA2. These included fatty acid binding protein, phospholipid binding protein, phospholipid synthesis protein, transporter cofactors, seladin-1, Abeta precursor protein (APP), vimentin, and low-density lipoprotein receptor-related protein. ABCA2 was highly expressed in neuroblastoma cells and colocalized with Abeta and APP. Additionally, increased APP protein levels were detected within ABCA2/APP double-transfected cells, and increased Abeta was detected in the media of ABCA2-transfected cells relative to controls. The transporter was abundant in the temporal and frontal regions of both normal and Alzheimer's disease (AD) brain but was detected at lower concentrations in the parietal, occipital, and cerebellar regions. The ABCA2 transfected cell line expressed resistance to a free radical initiator, confirming involvement in protection against reactive oxygen species and suggesting a further possible link to AD.

ATP-Binding Cassette Transporters↗
PubMed2004

Identification of a novel first exon of the human ABCA2 transporter gene encoding a unique N-terminus.

The human ABCA2 transporter is a member of a large family of ATP-binding proteins that transport a variety of molecules across biological membranes. Using RNA ligation-mediated PCR (RLM-PCR), we have identified a novel first exon, which we designate 1B that is located 699 bp upstream of the previously characterized first exon, which we designate 1A. These first exons are alternatively spliced to the second exon of the ABCA2 transcript resulting in a protein that has a unique amino terminus. For exon 1B, the new amino terminus encoded by the first exon is 52 amino acids and for exon 1A, 22 amino acids. We observed that among adult tissues examined, the highest expression of the 1B isoform was in peripheral blood leukocytes (PBL). Laser scanning confocal microscopy revealed that the 1A isoform and the 1B isoform co-localize with lysosome-associated membrane proteins-1 and -2 (LAMP-1 and -2). Cytotoxicity assays suggested a role for ABCA2 in estramustine and estradiol resistance, and overexpression of ABCA2 is seen in an estramustine-resistant prostate carcinoma line. Since both isoforms of the ABCA2 transporter have identical subcellular localization and both are overexpressed in a resistant cell line, we propose that they are also functionally redundant. It is likely that expression of ABCA2 by two independent promoters constitutes locus of regulation controlling expression of the protein to meet requirements in different tissues.

ATP-Binding Cassette Transporters↗
PubMed2004

Sulfur containing amino acids and human disease.

Sulfur containing amino acids contribute substantially to the maintenance and integrity of cellular systems by influencing cellular redox state and cellular capacity to detoxify toxic compounds, free radicals and reactive oxygen species. Methionine and cysteine are the two primary sulfur-containing amino acids in mammals. Methionine is an essential amino acid, obtained by dietary intake while cysteine is non-essential and a metabolite of methionine metabolism. Each of these amino acids contributes significantly to the cellular pool of organic sulfur and generally to sulfur homeostasis as well as playing a significant role in regulation of one carbon metabolism. Genetic defects in the enzymes regulating sulfur pools produce a variety of human pathologies, including homo- and cystinuria, homo- and cysteinemia, and neural tube defects. In addition, thiol imbalance has been associated with multiple disorders, including vascular disease, Alzheimer's, HIV and cancer. Possible treatments to restore the thiol balance are also discussed.

Animals↗
PubMed2004

Organosulfur compounds from alliaceae in the prevention of human pathologies.

A strong association between elevated plasma low-density-lipoprotein (LDL) and the development of cardiovascular diseases (CVD) has been established. Oxidation of LDL (Ox-LDL) promotes vascular dysfunction, enhances the production and release of inflammatory mediators such as reactive oxygen species and contribute to the initiation and progression of atherosclerosis. In addition, Ox-LDL enhances the production and release of tumor necrosis factor (TNF-alpha), interleukin (IL)-6, arachidonic acid metabolites and nitric oxide (NO) that are responsible for various human pathologies including cancer. Organosulfur compounds (OSC) from alliaceae modulate the glutathione (GSH) redox cycle and inhibits NFkappa-B activation in human T cells. Furthermore, OSC bioactivities include antioxidant, antibacterial, anticarcinogenic, antiatherogenic, immunostimulatory, and liver protection potential.

Allyl Compounds↗
PubMed2004

Tumor cell responses to a novel glutathione S-transferase-activated nitric oxide-releasing prodrug.

We have used structure-based design techniques to introduce the drug O(2)-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino) phenyl] 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), which is efficiently metabolized to potentially cytolytic nitric oxide by the pi isoform of glutathione S-transferase, an enzyme expressed at high levels in many tumors. We have used mouse embryo fibroblasts (MEFs) null for GSTpi (GSTpi(-/-)) to show that the absence of GSTpi results in a decreased sensitivity to PABA/NO. Cytotoxicity of PABA/NO was also examined in a mouse skin fibroblast (NIH3T3) cell line that was stably transfected with GSTpi and/or various combinations of gamma-glutamyl cysteine synthetase and the ATP-binding cassette transporter MRP1. Overexpression of MRP1 conferred the most significant degree of resistance, and in vitro transport studies confirmed that a GSTpi-activated metabolite of PABA/NO was effluxed by MRP1 in a GSH-dependent manner. Additional studies showed that in the absence of MRP1, PABA/NO activated the extracellular-regulated and stress-activated protein kinases ERK, c-Jun NH(2)-terminal kinase (JNK), and p38. Selective inhibition studies showed that the activation of JNK and p38 were critical to the cytotoxic effects of PABA/NO. Finally, PABA/NO produced antitumor effects in a human ovarian cancer model grown in SCID mice.

3T3 Cells↗
PubMed2004

Amplified differential gene expression microarray.

Amplified Differential Gene Expression (ADGE) and DNA microarray provides a new concept that the ratios of differentially expressed genes are magnified prior to detecting them. The ratio magnification is achieved with the integration of DNA reassociation and polymerase chain reaction (PCR) amplification and ensured with the design of the adapters and primers. The ADGE technique can be used either as a stand-alone method or in series with DNA microarray. ADGE is used in sample preprocessing and DNA microarray is used as a displaying system in the series combination. The combination of ADGE and DNA microarray provides a mutual complement of their strengths: the magnification of ratios of differential gene expression improves the detection sensitivity; the PCR amplification and efficient labeling enhance the signal intensity and reduce the requirement for large amounts of starting material; and the high throughput for DNA microarray is maintained.

DNA Primers↗
PubMed2003

Increased myeloproliferation in glutathione S-transferase pi-deficient mice is associated with a deregulation of JNK and Janus kinase/STAT pathways.

It has been shown that glutathione S-transferase pi (GSTpi) interacts with and suppresses the activity of c-Jun NH(2)-terminal kinase (JNK). GST-deficient mice (GSTpi(-/-)) have higher levels of circulating white blood cells, with similar proportions of lymphocytes, monocytes, and granulocytes. Interestingly, a selective expansion of splenic B lymphocytes was observed in GSTpi(-/-) animals but no change in T lymphocytes or natural killer cells. A peptidomimetic inhibitor of GSTpi that disrupts the interaction between GSTpi and JNK mimics in wild type mice the increased myeloproliferation observed in GSTpi(-/-) animals. Until now, the molecular basis for this effect has not been defined. In an in vitro hematopoiesis assay, interleukin-3, granulocyte colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor were more effective at stimulating proliferation of hematopoietic cells in GSTpi(-/-) mice than in wild type. The JNK inhibitor SP600125 which caused little inhibition of cytokine-induced myeloproliferation in wild type mice, decreased the number of colonies in GSTpi(-/-) animals. A more sustained phosphorylation of the STAT family of proteins was also observed in GSTpi(-/-) bone marrow-derived mast cells exposed to interleukin-3. This was associated with an increased proliferation and a down-regulation of expression of negative regulators of the Janus kinase-STAT pathway SHP, Src homology 2 domain-containing tyrosine phosphatase-1 and -2. The increased activation of JNK and STATs in GSTpi-deficient mice provides a viable mechanism for the increased myeloproliferation in these animals. These data also confirm the important role that GSTpi plays in the regulation of cell signaling pathways in a myeloproliferative setting.

Animals↗
PubMed2003

The role of glutathione-S-transferase in anti-cancer drug resistance.

Glutathione-S-transferases (GSTs) are a family of Phase II detoxification enzymes that catalyse the conjugation of glutathione (GSH) to a wide variety of endogenous and exogenous electrophilic compounds. GSTs are divided into two distinct super-family members: the membrane-bound microsomal and cytosolic family members. Microsomal GSTs are structurally distinct from the cytosolic in that they homo- and heterotrimerize rather than dimerize to form a single active site. Microsomal GSTs play a key role in the endogenous metabolism of leukotrienes and prostaglandins. Human cytosolic GSTs are highly polymorphic and can be divided into six classes: alpha, mu, omega, pi, theta, and zeta. The pi and mu classes of GSTs play a regulatory role in the mitogen-activated protein (MAP) kinase pathway that participates in cellular survival and death signals via protein : protein interactions with c-Jun N-terminal kinase 1 (JNK1) and ASK1 (apoptosis signal-regulating kinase). JNK and ASK1 are activated in response to cellular stress. GSTs have been implicated in the development of resistance toward chemotherapy agents. It is plausible that GSTs serve two distinct roles in the development of drug resistance via direct detoxification as well as acting as an inhibitor of the MAP kinase pathway. The link between GSTs and the MAP kinase pathway provides a rationale as to why in many cases the drugs used to select for resistance are neither subject to conjugation with GSH, nor substrates for GSTs. GSTs have emerged as a promising therapeutic target because specific isozymes are overexpressed in a wide variety of tumors and may play a role in the etiology of other diseases, including neurodegenerative diseases, multiple sclerosis, and asthma. Some of the therapeutic strategies so far employed are described in this review.

Antineoplastic Agents↗
PubMed2003

Sensitivity and fidelity of DNA microarray improved with integration of Amplified Differential Gene Expression (ADGE).

BACKGROUND: The ADGE technique is a method designed to magnify the ratios of gene expression before detection. It improves the detection sensitivity to small change of gene expression and requires small amount of starting material. However, the throughput of ADGE is low. We integrated ADGE with DNA microarray (ADGE microarray) and compared it with regular microarray. RESULTS: When ADGE was integrated with DNA microarray, a quantitative relationship of a power function between detected and input ratios was found. Because of ratio magnification, ADGE microarray was better able to detect small changes in gene expression in a drug resistant model cell line system. The PCR amplification of templates and efficient labeling reduced the requirement of starting material to as little as 125 ng of total RNA for one slide hybridization and enhanced the signal intensity. Integration of ratio magnification, template amplification and efficient labeling in ADGE microarray reduced artifacts in microarray data and improved detection fidelity. The results of ADGE microarray were less variable and more reproducible than those of regular microarray. A gene expression profile generated with ADGE microarray characterized the drug resistant phenotype, particularly with reference to glutathione, proliferation and kinase pathways. CONCLUSION: ADGE microarray magnified the ratios of differential gene expression in a power function, improved the detection sensitivity and fidelity and reduced the requirement for starting material while maintaining high throughput. ADGE microarray generated a more informative expression pattern than regular microarray.

Cell Division↗