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Papers by David Putt

Background: The majority of lung cancer patients are diagnosed at an advanced or metastatic stage... more Background: The majority of lung cancer patients are diagnosed at an advanced or metastatic stage and their 5-year survival rate is < 5%. However, when patients are diagnosed at an early stage, Stage I, they have ~70% 5-year survival rate. The aim of the study was to identify Stage I lung cancer miRNA biomarkers for early lung cancer detection. Methods: RNA was isolated from serum of 11 healthy subjects and 45 non-small cell lung cancer (NSCLC) patients comprised of 21 Stage I, 8 Stage II and 11 Stage III adenocarcinoma (AC) and 5 squamous cell carcinoma (SCC) patients, and from NSCLC (A549, H23, H1437, H460 and H1299) and kidney (ACHN) cell lines. Eight miRNA lung cancer biomarker candidates were selected based on previously published studies, were analyzed by qRT-PCR/Taqman® analyses. Results: MiR486 levels were up-regulated in Stages I, II and III AC. Whereas MiR-122 and miR-29c were not detected in serum samples in most healthy subjects, these respective miRNAs were expressed...

Journal of Pharmacology and Experimental Therapeutics, 2003

Journal of Controlled Release, 2010

Redox-responsive polyplexes represent a promising class of non-viral gene delivery vectors. The r... more Redox-responsive polyplexes represent a promising class of non-viral gene delivery vectors. The reducible disulfide bonds in the polyplexes undergo intracellular reduction owing to the presence of high concentrations of reduced glutathione (GSH). Available evidence suggests improved transfection activity of redox-sensitive polyplexes upon artificial modulation of intracellular GSH. This study investigates the effect of innate differences in GSH concentration in a panel of human pancreatic cancer cell lines on activity of reducible polyplexes of the four major classes of nucleic acid therapeutics: plasmid DNA (pDNA), messenger RNA (mRNA), antisense oligodeoxynucleotides (AON) and siRNA. In general, reducible polyplexes of linear poly(amido amines) (PAA) show improved activity compared to non-reducible polyplexes of PAA. Results demonstrate that increased GSH levels are associated with improved transfection of mRNA polyplexes but no clear trend is observed for pDNA, AON and siRNA polyplexes.

Primary cultures of rat renal proximal tubular (PT) and distal tubular (DT) cells from control an... more Primary cultures of rat renal proximal tubular (PT) and distal tubular (DT) cells from control and uninephrectomized (NPX) Sprague-Dawley rats were established to study whether the altered toxicological responses identified in freshly isolated cells are maintained in culture. Previous work showed that primary cultures of PT cells from hypertrophied rat kidneys maintained their differentiated properties, as evidenced by their high respiratory rate, active transport function, transport and metabolism of glutathione, and their hypertrophic phenotype. In the present study, primary cultures of PT cells from NPX rat kidneys, but to a much lesser extent DT cells, were more susceptible to cellular injury induced by either mercuric chloride, KCN, or tert-butyl hydroperoxide (tBH), than corresponding cells from normal rat kidneys. Direct comparisons of cytotoxicity and lipid peroxidation induced by tBH in freshly isolated renal cells showed that the primary cultures of cells from NPX rat kidn...

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The FASEB Journal, 2014

More aggressive or invasive prostate cancer cells (PCCs) are often resistant to chemotherapy and ... more More aggressive or invasive prostate cancer cells (PCCs) are often resistant to chemotherapy and have poor prognosis. Two immortalized human PCC lines, PC-3 (more aggressive) and LNCaP (less aggres...

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is the penultimate nephrotoxic metabolite of the environm... more S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is the penultimate nephrotoxic metabolite of the environmental contaminant trichloroethylene. Although metabolism of DCVC by the cysteine conjugate β-lyase is the most studied bioactivation pathway, DCVC may also be metabolized by the flavin-containing monooxygenase (FMO) to yield DCVC sulfoxide (DCVCS). Renal cellular injury induced by DCVCS was investigated in primary cultures of human proximal tubular (hPT) cells by assessment of time-and concentration-dependent effects on cellular morphology, acute cellular necrosis, apoptosis, mitochondrial function, and cellular GSH status. Confluent hPT cells incubated with as little as 10 µM DCVCS for 24 hr exhibited morphological changes, although at least 100 µM DCVCS was required to produce marked changes. Acute cellular necrosis did not occur until 48 hr with at least 200 µM DCVCS, indicating that this is a high-dose, late response. The extent of necrosis was similar to that with DCVC. In contrast, apoptosis occurred as early as 1 hr with as little as 10 µM DCVCS and the extent of apoptosis was much less than that with DCVC. Mitochondrial function was maintained with DCVCS concentrations up to 100 µM, consistent with hPT cells only being competent to undergo apoptosis at early time points and relatively low concentrations. Marked depletion (> 50%) of cellular GSH content was only observed with 500 µM DCVCS. These results, combined with earlier studies showing protection from DCVC-induced necrosis and apoptosis by the FMO inhibitor methimazole, suggest that formation of DCVCS plays a significant role in trichloroethylene-induced renal cellular injury in hPT cells.

The FASEB Journal

GSH transport across the basolateral plasma membrane (BLM) into the renal proximal tubule occurs ... more GSH transport across the basolateral plasma membrane (BLM) into the renal proximal tubule occurs by both sodium-coupled and sodium-independent pathways and may be mediated by one or more of several...

Environmental Disease

Bisphenol A (BPA) is a phenolic environmental estrogen that disrupts endocrine activity thereby i... more Bisphenol A (BPA) is a phenolic environmental estrogen that disrupts endocrine activity thereby increasing the risk of hormone-related health problems. The human population is highly exposed to BPA and food is believed to be a primary source of BPA exposure. The aim of this study was to test the sensitivity and specificity of a BPA enzyme-linked immunosorbent assay (ELISA) and to measure levels of BPA in supernatants obtained from various canned foods from different countries. The concentration of BPA was measured in supernatant from different types of canned soup and vegetable mixes produced by US companies and two companies each from three different Asian countries (Korea, Japan and China), which are available at markets in the USA. ELISA results were confirmed by LC/MS/MS and shown to be in agreement. Cross-reactivity tests demonstrated that BPA ELISA kit does not cross-react with other tested phenolic compounds. There was no significant difference of BPA levels in different types of soups from different US companies. However, levels of BPA in supernatants of canned vegetable mixes of a company in the USA were 200-fold lower than the levels in canned vegetable soups of the US companies. BPA levels varied greatly among canned foods among companies in various countries. Thus, this study validated the use of a simple ELISA assay to measure levels of BPA in supernatants of canned food, which would facilitate the routine monitoring of dietary exposure to BPA. Decreasing the consumption of BPA will lead to a reduction in the risk of adverse health effects.

Medical Hypotheses

Preeclampsia is a serious complication of pregnancy characterized by the development of vasospasm... more Preeclampsia is a serious complication of pregnancy characterized by the development of vasospasm, hypertension and often associated with proteinuria after the 20th week of gestation. Because termination of pregnancy results in the most efficacious resolution of preeclampsia, it is a leading cause of premature delivery worldwide. In pregnancy, 14,15-epoxyeicosatrienoic acids (EETs) have been shown to facilitate uterine blood flow during preeclampsia, in which the classic vasodilator agents such as nitric oxide and prostacyclin are reduced. EETs are converted to dihydroxyeicosatrienoic acids (DHETs) by the activity of soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH activity is increased in preeclampsia by measuring urinary 14,15-DHET in healthy and preeclamptic pregnant women. Urine samples were collected and incubated with or without β-glucuronidase to enable the measurement of both the glucuronidated and free forms of 14,15-DHET, which were quantified using a 14,15-DHET ELISA. Levels of total (free+glucuronidated) 14,15-DHET, which is a measurement of EET-dependent sEH activity, were higher in urine samples obtained from preeclamptic women compared to healthy pregnant women. Considering the fact that free+glucuronidated 14,15-DHET levels are increased in urine of preeclamptic women, we hypothesize that sEH expression or activity is augmented in these patients, reducing EET and increasing blood pressure. Moreover we suggest that novel anti-hypertensive agents that target sEH might be developed as therapeutics to control high blood pressure in women with preeclampsia.

Environmental Monitoring and Assessment, 2016

The endocrine disruptor Bisphenol A (BPA) is ubiquitous in both aquatic and surface sediment envi... more The endocrine disruptor Bisphenol A (BPA) is ubiquitous in both aquatic and surface sediment environments because it is continuously released into sewage wastewater effluent. The measurement of BPA at wastewater treatment plants is rarely performed even though the United States Environmental Protection Agency (EPA) states that current levels of environmental BPA could be a threat to aquatic organisms. Therefore, the aims of this study were to measure BPA levels in sewage wastewater at different collection points over a 1-year period and to compare the levels of BPA to 8-isoprostane, a human derived fatty acid, found in sewage wastewater. We analyzed pre-treated sewage samples collected from three source points located in different communities in the metropolitan Detroit area provided by the Detroit Water and Sewerage Department. Human urine samples were also used in the study. BPA and 8-isoprostane were measured using ELISA kits from Detroit R&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;D, Inc. BPA levels from the same collection point oscillated more than 10-fold over 1 year. Also, BPA levels fluctuated differentially at each collection point. Highly fluctuating BPA values were confirmed by LC/MS/MS. The concentration of BPA in sewage wastewater was ~100-fold higher than the concentration of 8-isoprostane, while urinary concentration was ~20-fold higher. Thus, BPA levels discharged into the sewage network vary among communities, and differences are also observed within communities over time. The difference in BPA and 8-isoprostane levels suggest that most of the BPA discharged to sewage wastewater might be derived from industries rather than from human urine. Therefore, the continuous monitoring of BPA could account for a better regulation of BPA release into a sewage network.

Appetite, Dec 23, 2016

The incidence of obesity, one of the main risks for type 2 diabetes and cardiovascular disease, h... more The incidence of obesity, one of the main risks for type 2 diabetes and cardiovascular disease, has been rising, and changes in eating behavior are associated with this increasing rate. Body weight is maintained via a complex integration of endocrine and neuronal inputs that regulate the control of orexigenic and anorexigenic neuropeptides in the arcuate nucleus of the hypothalamus. Overfeeding may disrupt the mechanisms of feeding control, increasing orexigenic peptides such as neuropeptide Y (NPY), and/or decreasing the anorexigenic peptide proopiomelanocortin (POMC) leading to a change in energy balance and body-weight instrumentation. Despite of the great interest in this field, the mechanism by which expression of POMC and NPY is modified is not entirely clear. Over the past decades, studies have demonstrated that epigenetic modifications such as DNA methylation, histone modification and changes in miRNA dynamics, could be modulated by external stimuli and these could affect pr...

The Faseb Journal, Mar 1, 2006

Medical Hypotheses, 2016

Over the past decades, life-styles changing have led to exacerbated food and caloric intake and a... more Over the past decades, life-styles changing have led to exacerbated food and caloric intake and a reduction in energy expenditure. Obesity, main outcome of these changes, increases the risk for developing type 2 diabetes, cardiovascular disease and metabolic syndrome, the leading cause of death in adult and middle age population. Body weight and energy homeostasis are maintained via complex interactions between orexigenic and anorexigenic neuropeptides that take place predominantly in the hypothalamus. Overeating may disrupt the mechanisms of feeding control, by decreasing the expression of proopiomelanocortin (POMC) and α-melanocyte stimulating hormone (α-MSH) and increasing orexigenic neuropeptide Y (NPY) and agouti-related peptide (AgRP), which leads to a disturbance in appetite control and energy balance. Studies have shown that regular physical exercise might decrease body-weight, food intake and improve the metabolic profile, however until the currently there is no consensus about its effects on the expression of orexigenic/anorexigenic neuropeptides expression. Therefore, we propose that the type and length of physical exercise affect POMC/αMSH and NPY/AgRP systems differently and plays an important role in feeding behavior. Moreover, based on the present reports, we hypothesize that increased POMC/αMSH overcome NPY/AgRP expression decreasing food intake in long term physical exercise and that results in amelioration of several conditions related to overweight and obesity.

The Faseb Journal, Mar 1, 2008

Molecular Cancer Research, 2015

Protein N-acetylglucosamine modification (O-GlcNAcylation) plays a critical role in cell-cycle re... more Protein N-acetylglucosamine modification (O-GlcNAcylation) plays a critical role in cell-cycle regulation, apoptosis and signal transduction. Thr-58 of c-myc, a mutational hot spot in lymphomas, is a site for both phosphorylation (primed by Ser-62 phosphorylation) and O-GlcNAcylation, which are conserved among human rat and mouse. Whereas Thr-58O-GlcNAcylation induces ubiquitin-dependent c-myc degradation, Thr-58/Ser-62 phosphorylation increases c-myc stability and thus induces invasiveness and tumorigenesis of MCF-7 human breast cancer cells. c-Myc antibodies specific for (a) Thr-58-O-GlcNAcylation, (b) Thr-58-unmodification and (c) Thr-58/Ser-62 phosphorylation were produced and specificities of the antibodies have been characterized by Western blot analyses using BSA conjugated with synthetic peptides containing the O-GlcNAcylated, unmodified and phosphorylated sites of c-myc. Western blot analysis of MCF-7 cells revealed that, whereas ~68 kDa Thr-58-O-GlcNAcylated c-myc proteins were primarily detected in the nuclear fraction, the 65-68 kDa Thr-58/Ser-62 phosphorylated and 65 kDa and 40 kDa Thr-58 unmodified c-myc proteins were expressed in both nuclear and cytosolic fractions. When MCF-7 cells were treated with 1% DMSO, 2 mM ketoconazole, a medicine-like human and bacterial β-N-acetylglucosaminidase (O-GlcNAcase) inhibitor, or 2 mM streptozotocin (STZ), an irreversible inhibitor of O-GlcNAcase, dissolved in DMSO (final concentration, 1%) for 4 hr, Thr-58-O-GlcNAcylated c-myc protein levels in nuclear and total cell lysates increased after 2 mM ketoconazole treatment but not with STZ treatment. Thr-58/Ser-62 phosphorylated protein levels did not change after either ketoconazole or STZ treatments. Treatment of the cells with 1% DMSO, 2 mM ketoconazole, buspirone or acetazolamide dissolved in DMSO (final concentration, 1%) or N6-methyladenosine 59-monophosphate (dissolved in media), a medicine-like O-GlcNAcase inhibitor, for 4 hr revealed that Thr-58-O-GlcNAcylated c-myc protein levels in nuclear lysates increased only when the cells were treated with 2 mM ketoconazole. None of the O-GlcNAcase inhibitors including ketoconazole treatments changed levels of c-myc proteins unmodified at the Thr-58 site or phosphorylated at the Thr-58/Ser-62 sites. Whereas treatment of the MCF-7 cells with 2 mM STZ and N6-methyladenosine 59-monophosphate failed to induce cell death and treatment with 2 mM buspirone and acetazolamide induced minimal cell death, 2 mM ketoconazole treatment, which dramatically increased Thr-58-O-GlcNAcylated c-myc protein levels, induced severe cell death. Subsequent lactate dehydrogenase (LDH) cytotoxicity assays demonstrated that the ketoconazole treatment increased cell death in MCF-7 cells 84% higher than in MCF10A non-cancerous cells by lactate dehydrogenase (LDH) cytotoxicity assays. Dose-dependent cell proliferation assays were carried out by treatment of the cells with and without 10, 20, 50 or 100 µM ketoconazole dissolved in DMSO for 72 hr, staining the cells with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). The ketoconazole treatment inhibited cell proliferation of the MCF-7 cells by 60% and 90% at 50 and 100 µM, respectively (p These results suggest that, in MCF-7 cells, ketoconazole, a medicine-like O-GlcNAcase inhibitor, dramatically increased Thr-58-O-GlcNAcylated c-myc proteins, which coincided with increased cell death and inhibited cell proliferation. Supported by NCI SBIR Phases I and II Contracts N261201100073C and N261201300058C. Citation Format: Hyesook Kim, So Hee Kim, Aby Joiakim, David Kaplan, David Putt. Effects of O-GlcNAcase inhibitors on O-GlcNAcylated c-myc expression in MCF-7 cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A26.

Journal of Pharmacology and Experimental Therapeutics

Expression of the cytochrome P450 (CYP) 2B subfamily in rat and rabbit hepatic tissues after pyri... more Expression of the cytochrome P450 (CYP) 2B subfamily in rat and rabbit hepatic tissues after pyridine (PY) treatment has been examined, and the molecular basis for enhanced 2B1/2B2 expression has been determined. P450 expression was monitored using metabolic activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses, and the identity of the proteins was confirmed through N-terminus microsequence analysis. PY caused a dose-dependent elevation of hepatic CYP2B1/B2B levels in rats, which ranged from 4- to 22-fold over the dosing regimen of 100 to 400 mg PY/kg/day, for 3 days, respectively. PY at low dose failed to induce CYP2B in rabbit hepatic tissue, suggesting a species-dependent response in 2B expression. Anti-2B1 IgG addition to PY-induced microsomes inhibited benzphetamine N-demethylase activity by only approximately 15%, in sharp contrast to the approximately 73% inhibition observed for phenobarbital-induced microsomes, suggesting the induction of other form(s) of P450 having benzphetamine N-demethylase activity. Northern blot analysis revealed that PY treatment increased 2B1 and 2B2 poly(A)+ RNA levels approximately 69- and approximately 34-fold, respectively, whereas the 2E1 poly(A)+ RNA levels failed to increase. The results of this study show that PY induces CYP2B1/2B2 and that induction is species-dependent and kinetically distinguishable from 2E1 induction. Moreover, 2B1/2B2 induction occurs as a result of elevated mRNA levels associated with either transcriptional activation or mRNA stabilization, and it differs from the mechanism of hepatic 2E1 induction by PY.