Background Early identification of patients susceptible to chemotherapy-induced cardiotoxicity could lead to targeted treatment to reduce cardiac dysfunction. Rats treated with doxorubicin (DOX), a chemotherapeutic agent, have increased cardiac expression of 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), a bioactive lipid implicated in hypertension and coronary artery disease. However, the utility of 14,15-DHET as plasma biomarkers was not defined. The aim of this study is to investigate if levels of 14,15-DHET are an early blood biomarker to predict the subsequent occurrence of cardiotoxicity in cancer patients after chemotherapy. Methods H9c2 rat cardiomyocytes were treated with DOX (1 μM) for 2 h and levels of 14,15-DHET in cell media was quantified at 2, 6 or 24 h in media after DOX treatment. Similarly, female Sprague–Dawley rats were treated with DOX for two weeks and levels of 14,15-DHET was assessed in plasma at 48 h and 2 weeks after DOX treatment. Changes in brain natriuretic peptide (BNP) mRNA, an early cardiac hypertrophy process, were determined in the H9c2 cells and rat cardiac tissue. Results were confirmed in human subjects by assessment of levels of 14,15-DHET in plasma of breast cancer patients before and after DOX treatment and left ventricular ejection fraction (LVEF), a clinical marker of cardiotoxicity. Results Levels of 14,15-DHET in cell media and rat plasma increased ~ 3-fold and was accompanied with increase in BNP mRNA in H9c2 cells and rat cardiac tissue after DOX treatment. In matched plasma samples from breast cancer patients, levels of 14,15-DHET were increased in patients that developed cardiotoxicity at 3 months before occurrence of LVEF decrease. Conclusions Together, these results indicate that levels of 14,15-DHET are elevated prior to major changes in cardiac structure and function after exposure to anthracyclines. Increased levels of 14,15-DHET in plasma may be an important clinical biomarker for early detection of anthracycline-induced cardiotoxicity in cancer patients.

Abstract 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 in 62% and 71% of Stage I and 88% and 100% of Stage II and 91 and 100% of Stage III lung AC patients. Ratios of miR-486/miR-16 and miR-29c/miR-16 were ~10-fold higher in A549 lung AC cell lysates and media compared with renal ACHN cell lysates and media and both miRNAs were higher in A549 cells when compared to other lung cancer cell lines. Conclusions: Upregulation of miR-486 and miR-29c in blood can be used as a biomarker for early AC lung cancer diagnosis.

Strategies to improve the early diagnosis of prostate cancer will provide opportunities for earlier intervention. The blood-based prostate-specific antigen (PSA) assay is widely used for prostate cancer diagnosis but specificity of the assay is not satisfactory. An algorithm based on serum levels of PSA combined with other serum biomarkers may significantly improve prostate cancer diagnosis. Plasma glycan-binding IgG/IgM studies suggested that glycan patterns differ between normal and tumor cells. We hypothesize that in prostate cancer glycoproteins or glycolipids are secreted from tumor tissues into the blood and induce auto-immunoglobulin (Ig) production. A 24-glycan microarray and a 5-glycan subarray were developed using plasma samples obtained from 35 prostate cancer patients and 54 healthy subjects to identify glycan-binding auto-IgGs. Neu5Acα2-8Neu5Acα2-8Neu5Acα (G81)-binding auto-IgG was higher in prostate cancer samples and, when levels of G81-binding auto-IgG and growth differentiation factor-15 (GDF-15 or NAG-1) were combined with levels of PSA, the prediction rate of prostate cancer increased from 78.2% to 86.2% than with PSA levels alone. The G81 glycan-binding auto-IgG fraction was isolated from plasma samples using G81 glycan-affinity chromatography and identified by N-terminal sequencing of the 50 kDa heavy chain variable region of the IgG. G81 glycan-binding 25 kDa fibroblast growth factor-1 (FGF1) fragment was also identified by N-terminal sequencing. Our results demonstrated that a multiplex diagnostic combining G81 glycan-binding auto-IgG, GDF-15/NAG-1 and PSA (≥ 2.1 ng PSA/ml for cancer) increased the specificity of prostate cancer diagnosis by 8%. The multiplex assessment could improve the early diagnosis of prostate cancer thereby allowing the prompt delivery of prostate cancer treatment.

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.

Abstract The blood prostate-specific antigen (PSA) assay is used for prostate cancer diagnosis but specificity of the assay is not satisfactory. Previously a combined PSA and GDF-15 (NAG-1) score was shown to improve specificity of prostate cancer detection. Our hypothesis was that, in prostate cancer, glycoproteins were secreted from tumor tissues into blood and induce autoimmune immunoglobulin G (auto-IgG) production and, thus, measurement of the glycan-auto-IgG in blood would improve prostate cancer diagnosis. The 24 glycan-containing microarray analyses have been carried out with plasma samples obtained from 35 prostate cancer patients and 46 healthy subjects to identify glycan-binding auto-IgG biomarker candidates by incubating the auto-IgG captured by glycans spotted on the slide with biotinylated anti-human secondary IgG/streptavidin-Alexa647. Among the 24 glycans, GlcNAc-polyacrylamide (PAA) (G09) and Fucα1-3GlcNAcβ-PAA (G24) glycans showed lower signals of the auto-IgG in the prostate cancer after quantile normalization. β-D-Galactose-PAA (G04) and L-rhamnose-PAA (G08) glycans showed higher signals of the auto-IgG in prostate cancer. No auto-IgM signal was detected. Subsequently, a 5-glycan subarray analysis was developed and lower signals of G09 and G24 glycan-binding auto-IgG were verified by the subarray analysis using 35 prostate cancer plasma samples compared with 54 controls. A higher signal of Neu5Acα2-8Neu5Acα2-8Neu5Acα-sp-PAA (G81)-binding auto-IgG in prostate cancer was detected by the subarray analysis. When the result obtained with the G81-binding auto-IgG was combined with levels of PSA and NAG-1, the prediction rate of prostate cancer increased to 86.2% from 78.2% with PSA levels alone, improving diagnostic accuracy of prostate cancer. The G81 glycan-binding auto-IgG was isolated from prostate cancer and control plasma samples using G81 glycan-affinity chromatography. Western blot analysis of the auto-IgG eluate with a secondary IgG antibody revealed that the level of the 50 kDa heavy chain of the auto-IgG obtained from the prostate cancer patient was ~3-fold higher than the control sample. The 50 kDa fragment was identified as an IgG heavy chain variable region by N-terminal sequencing (Edman's degradation). Our result demonstrated that a multiplex biomarker diagnostic consisting of glycan-binding auto-IgG, PSA and NAG-1 increased specificity and sensitivity of prostate cancer diagnosis. Supported by NCI SBIR Phase I, CA159721. Citation Format: Hyesook Kim, Julia Matzenbacher Santos, Aby Joiakim, David Kaplan, Ben Rybicki, Alan Dombkowski, David A. Putt. Improvement of prostate cancer diagnosis using a multiplex test of PSA, GDF-15 (NAG-1) and glycan-binding auto-IgG in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 690.

Abstract Whereas over 75% of lung cancer patients are diagnosed at an advanced or metastatic stage and their 5-year survival rate is <5%, patients diagnosed at an early stage, Stage I, have ~70% 5-year survival rate. In the present study, Stage I lung cancer miRNA biomarkers were identified using qRT-PCR and novel label-free facile nanoarray technology for early lung cancer detection. Total RNA from human A549 lung and ACHN kidney cancer cell lysates and media were isolated and qRT-PCR/Taqman® analyses (two-step RT-PCR) were carried out for 8 miRNA lung cancer biomarker candidates. The 8 miRNA levels were normalized by the miR16 level (no change reported in lung cancer). Ratios of miR486/miR16 and miR29c/miR16 increased ~10-fold in A549 lung cancer cell lysates and media compared with those in ACHN kidney cells. Total RNA fractions were isolated from 11 healthy subjects, 21 Stage I and 19 Stage II/III adenocarcinoma and 5 squamous lung cancer patients and levels of the 8 miRNAs were measured by qRT-PCR/Taqman® analysis (in total, 1,026 analyses) and normalized by the miR16 level. miR486 levels were up-regulated (p=0.02) and miR203 (p=0.0005) and miR205 (p=0.041) levels were down-regulated in Stages I and II/III adenocarcinoma. Whereas miR122 and miR29c were not detected in serum samples from the majority of healthy subjects, the miRNAs were expressed in 82% and 71%, respectively, of Stage I and 90% and 100%, respectively, of Stages II/III lung cancer patients. A novel label-free facile 90 nm (diameter) miRNA nanowell technology was developed. Electrochemical analyses of the early lung cancer biomarker candidates, miR486 and miR29c, and, an internal control, miR16, were carried out using various concentrations of miRNA standards (0 to 100 fM) with biotinylated cDNA captured by streptavidin coated on the nanogold surface. The Nyquist plots showed a dose-dependent increase in impedance (-Z” kohm) and sensitivity of the miRNA nanowell electrochemical technology was ≤1 fM. The impedance level obtained with miR29c in A549 lung cancer cell media was ~2.8-fold higher (mean value ± SD, 163.9 ± 32.8 kohm) compared with ACHN kidney cancer cell media (60.8 ± 33.1 kohm) by 90 nm nanowell analysis (p=0.000019). Electrochemical analyses of a serum sample obtained from a lung cancer patient revealed that miR486 and miR29c levels were ~2-fold (p=0.0031) and 9-fold (p=0.0006) higher, respectively, compared to the pooled human control sera. This result agreed with the result obtained by qRT-PCR/Taqman® analysis of human serum samples. Our results suggest that miR486 and miR203 levels up- and down-regulated, respectively, in lung cancer serum samples are biomarkers for early (Stage I) lung cancer diagnosis. Supported by NCI SBIR Phase I contract, N261201500040C (Topic 337). Citation Format: Julia M. Santos, Aby Joiakim, David A. Putt, Pilar Herrera-Fierro, Vishva Ray, Alan Dombokowski, Guoan Chen, David G. Beer, Hyesook Kim. Identification of early lung cancer miRNA biomarkers using qRT-PCR and novel label-free nanoarray technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5436. doi:10.1158/1538-7445.AM2017-5436

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.

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 index. 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 protein expression in different cells. Therefore, this review discusses the recent reports that link epigenetic modifications in the hypothalamus to changes on long-term feeding control and its role in the onset of obesity. (C) 2016 Elsevier Ltd. All rights reserved.

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 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&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.

Protein O-GlcNAcylation (N-acetylglucosamine modification) plays a critical role in cell-cycle regulation, apoptosis and signal transduction. Most serine (Ser) and threonine (Thr) sites for O-GlcNAcylation are on or near to phosphorylation sites, which creates a system of mutual elimination. O-GlcNAcylation of p53, a tumor suppressor protein, at Ser-149 prevents p53 degradation and stabilizes p53 proteins expressed in MCF-7 human breast cancer cells whereas phosphorylation of the p53 at Thr-155 induces ubiquitin-dependent degradation, which decreases p53 levels. Accumulation of the p53 protein in cancer cells induces arrest of cell growth and apoptosis in response to DNA damage. Ser-149 site-specific anti-peptide antibodies have been produced using KLH conjugated with p53 peptides with and without O-GlcNAcylation at Ser-149 by immunization of rabbits for polyclonal and mice for monoclonal antibodies. Whereas polyclonal antibodies and hybridoma media for O-GlcNAcylated p53 cross-reacted with BSA conjugated with Ser-149-O-GlcNAcylated p53 peptides in ELISA or Western blot analysis, the antibodies did not cross-react with BSA conjugated with p53 peptides without O-GlcNAcylation. Anti-peptide polyclonal antibodies produced for the unmodified p53 peptides did not cross-react with BSA conjugated with Ser-149 O-GlcNAcylated p53 peptides. Using the hybridoma media for Ser-149-O-GlcNAcylated p53, Western blot analysis was carried out with nuclear lysates and cytosol-containing supernatant obtained from MCF10A (normal breast cancer cell line) and MCF-7 cells. It was found that, in MCF-10A cells, Ser-149-O-GlcNAcylated p53 proteins (∼55 kDa) were minimally expressed in the nuclear fraction but highly expressed in the cytosolic fraction whereas, in MCF-7 cells, the level of cytosolic Ser-149-O-GlcNAcylated p53 protein was low compared with the level in MCF-10A cells. MCF-7 cells were treated for 24 hr with 1% DMSO or 2 mM β-N-acetylglucosaminidase (O-GlcNAcase) inhibitor, acetazolamide, dissolved in DMSO and levels of Ser-149-O-GlcNAcylated p53 were determined by Western blot analysis using antibodies for the Ser-149-O-GlcNAcylated p53. Whereas the level of O-GlcNAcylated p53 did not increase after 1% DMSO treatment, the level dramatically increased after treatment of acetazolamide in 1% DMSO and induced cell death. Our results showed that acetazolamide treatment increased Ser-149-O-GlcNAcylated p53 level and induced cell death in MCF-7 cells, which may decrease invasiveness of the breast cancer cells. Supported by NCI SBIR Phases I and II Contracts No. N261201100073C and N261201300058C. Citation Format: Hyesook Kim, So Hee KIm, Aby Joiakim, David Kaplan, Sung Hun Bae, David Putt. Detection of increased O-GlcNAcylated p53 levels in MCF-7 cells after O-GlcNAcase inhibitor treatment using antibodies specific for Ser-149-O-GlcNAcylated p53 proteins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1230. doi:10.1158/1538-7445.AM2015-1230

More aggressive prostate cancer cells (PCCs) are often resistant to chemotherapy. Differences exist in redox status and mitochondrial metabolism that may help explain this phenomenon. Two human PCC lines, PC-3 cells (more aggressive) and LNCaP cells (less aggressive), were compared with regard to cellular glutathione (GSH) levels, susceptibility to either oxidants or GSH depletors, and expression of several proteins involved in apoptosis and stress response to test the hypothesis that more aggressive PCCs exhibit higher GSH concentrations and are relatively resistant to cytotoxicity. PC-3 cells exhibited 4.2-fold higher GSH concentration than LNCaP cells but only modest differences in acute cytotoxicity were observed at certain time points. However, only LNCaP cells underwent diamide-induced apoptosis. PC-3 cells exhibited higher levels of Bax and caspase-8 cleavage product but lower levels of Bcl-2 than LNCaP cells. However, LNCaP cells exhibited higher expression of Fas receptor (FasR) but also higher levels of several stress response and antioxidant proteins than PC-3 cells. LNCaP cells also exhibited higher levels of several mitochondrial antioxidant systems, suggesting a compensatory response. Thus, significant differences in redox status and expression of proteins involved in apoptosis and stress response may contribute to PCC aggressiveness.

Abstract 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 5'-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 5'-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 < 0.05). 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.

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. Whereas Thr-58 O-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. Using form-specific antibodies for Thr-58-O-GlcNAcylated c-Myc, Western blot analysis was carried out and found that Thr-58-O-GlcNAcylated c-Myc proteins were detected primarily in the nuclear fraction as 68 kDa proteins. MCF-7 cells were treated for 4 hr with 1% DMSO or 2 mM ketoconazole [β-N-acetylglucosaminidase (O-GlcNAcase) inhibitor, dissolved in DMSO]. Western blot analysis of nuclear lysates of MCF-7 cells revealed that the levels of Thr-58-O-GlcNAcylated c-Myc dramatically increased after ketoconazole treatment. Thr-58-O-GlcNAcylated c-Myc levels also increased in the nuclear lysates of Mia Paca-2 and SW480 cells treated with 50 µg/ml for 48 hours. The ketoconazole treatment increased cell death in MCF-7 cells 84% higher than in MCF10A non-cancerous cells by lactate dehydrogenase (LDH) cytotoxicity assays. Treatment with 50 µg/ml ketoconazole for 72 hr inhibited cell proliferation of colon cancer cells, SW480 and HT29, by 63.3% and 67.4%, respectively, lung cancer cells, H1437 and A549, by 44.9% and 51.3%, respectively, and a pancreatic cancer cell, Mia Paca-2, by 60.2% in a dose-dependent manner. These results demonstrated that treatment of ketoconazole inhibited the proliferation of MCF-7 breast cancer cells as well as colon, lung and pancreatic cancer cells. Wound healing assays were carried out to verify the inhibition of cell migration. Whereas 55.3% of Mia Paca-2 cells migrated toward a scratched field after 24-hr treatment, only 11.6% of the cells migrated after treatment with 100 µg/ml ketoconazole. PANC-1 cells, which failed to increase Thr-58-O-GlcNAcylated c-Myc expression and cell proliferation after ketoconazole treatment, showed no significant difference of cell migration after 24 hr with 100 µg/ml ketoconazole treatment. Surprisingly, colon cancer cells, SW480 and HT29, showed an inhibition of invasiveness as evidenced by the inhibition of cell proliferation and cell migration after treatment with 100 µg/ml ketoconazole. Collectively, our results demonstrated that treatment of ketoconazole, an O-GlcNAcase inhibitor, increased Thr-58-O-GlcNAcylated c-Myc level and inhibited cell proliferation in MCF-7, SW480, HT29, H1437, A549 and Mia Paca-2 cells, which may decrease invasiveness and migration of the breast, colon, lung and pancreatic cancer cells. Supported by NCI SBIR Phase II Contract No. HHSN261201300058C. Citation Format: So Hee Kim, Eun-Sin Du, Aby Joiakim, Sung-Su Park, David Kaplan, David Putt, Hyesook Kim. Ketoconazole-induced O-GlcNAcylated c-Myc expression and inhibition of cell proliferation in cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3513. doi:10.1158/1538-7445.AM2014-3513

Primary cultures of human proximal tubular (hPT) cells are a useful experimental model to study transport, metabolism, cytotoxicity, and effects on gene expression of a diverse array of drugs and environmental chemicals because they are derived directly from the in vivo human kidney. To extend the model to investigate longer-term processes, primary cultures (P0) were passaged for up to four generations (P1–P4). hPT cells retained epithelial morphology and stained positively for cytokeratins through P4, although cell growth and proliferation successively slowed with each passage. Necrotic cell death due to the model oxidants tert-butyl hydroperoxide (tBH) and methyl vinyl ketone (MVK) increased with increasing passage number, whereas that due to the selective nephrotoxicant S-(1,2-dichlorovinyl)-l-cysteine (DCVC) was modest and did not change with passage number. Mitochondrial activity was lower in P2–P4 cells than in either P0 or P1 cells. P1 and P2 cells were most sensitive to DCVC-induced apoptosis. DCVC also increased cell proliferation most prominently in P1 and P2 cells. Modest differences with respect to passage number and response to DCVC exposure were observed in expression of three key proteins (Hsp27, GADD153, p53) involved in stress response. Hence, although there are some modest differences in function with passage, these results support the use of multiple generations of hPT cells as an experimental model.

Approximately 5–8% of pregnant women have preeclampsia. Epoxyeicosatrienoic acids (EETs) generated by cytochromes (CYP) 2C and 2J have anti‐hypertensive activity. Urinary 14,15‐dihydroxyeicosatrienoic acid (DHET) levels in spontaneously hypertensive rats (SHR) were found to be ~35‐fold higher than levels in normotensive Wistar Kyoto (WKY) rats, suggesting that soluble epoxide hydrolase (sEH) induced developmental hypertension. Urinary levels of 14,15‐DHET were measured by ELISA in 7 preeclamptic and 9 healthy pregnant women. Mean systolic/diastolic pressures of the preeclamptic and healthy women were 152/95 and 120/74 mm Hg, respectively (p<0.01 for both). Contrary to the rat study, urinary free 14,15‐DHET levels did not significantly differ among the pregnant women. However, glucuronidated 14,15‐DHET levels in preeclamptic women was significantly higher (p<0.03) than controls. Glucuronidation of 14,15‐DHET (74 μM) by UDP‐glucuronosyltransferase (UGT) in pooled human liver microsomes was inhibited by 87% after treatment with 1.3 μM 12‐(3‐adamantan‐1‐yl‐ureido)dodecanoic acid (AUDA). The results demonstrated that increased urinary glucuronidated 14,15‐DHET is a biomarker for sEH‐ and UGT‐dependent preeclampsia and that AUDA inhibits both sEH and UGT, putative target enzymes for medical intervention. Supported in part by NIEHS SBIR Phase I (N43ES85430) and Phase II (N44ES05459).

Abstract Protein O-GlcNAcylation (N-acetylglucosamine modification) plays a critical role in cell-cycle regulation, apoptosis and signal transduction. Most serine (Ser) and threonine (Thr) sites for O-GlcNAcylation are on or near to phosphorylation sites, which creates a system of mutual elimination. 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. Whereas Thr-58 O-GlcNAcylation induces ubiquitin-dependent c-myc degradation, Thr-58/Ser-62 phosphorylation increases c-myc stability and invasiveness and tumorigenesis of MCF-7 human breast cancer cells. Thr-58 site-specific anti-peptide antibodies have been produced by immunization of rabbits with KLH conjugated with c-myc peptides with and without Thr-58 O-GlcNAcylation. Whereas the antibodies for O-GlcNAcylated c-myc cross-reacted with BSA conjugated with Thr-58-O-GlcNAcylated c-myc peptides in ELISA and Western blot analysis, the antibodies did not cross-react with BSA conjugated with c-myc peptides without O-GlcNAcylation. Anti-peptide antibodies produced for the unmodified c-myc peptides did not cross-react with BSA conjugated with Thr-58-O-GlcNAcylated c-myc peptides. Using the antibodies for Thr-58-O-GlcNAcylated c-myc, Western blot analysis was carried out with nuclear lysates and cytosol-containing supernatant obtained from MCF-7 cells and it was found that Thr-58-O-GlcNAcylated c-myc proteins were minimally expressed in the cytosolic fraction but highly expressed in the nuclear fraction. Whereas c-myc proteins without Thr-58-O-GlcNAcylation in the nuclear fraction were detected primarily as 65 kDa proteins in Western blot analyses, Thr-58-O-GlcNAcylated c-myc proteins in the nuclear fraction were detected primarily as 68 kDa proteins. MCF-7 cells were treated for 4 hr with 1% DMSO and 2 mM β-N-acetylglucosaminidase (O-GlcNAcase) inhibitors (buspirone or ketoconazole) dissolved in DMSO and levels of Thr-58-O-GlcNAcylated and unmodified c-myc were determined by Western blot analysis using antibodies for the Thr-58-O-GlcNAcylated or unmodified c-myc. Whereas the O-GlcNAcylated c-myc level did not increase after buspirone treatment, the level dramatically increased after ketoconazole treatment. The Thr-58-unmodified c-myc protein level failed to increase after 2 mM ketoconazole treatment. The ketoconazole treatment increased cell death in MCF-7 cells 84% higher than in MCF10A non-cancerous cells as evidenced by lactate dehydrogenase (LDH) cytotoxicity assays. Our results showed that ketoconazole treatment increased Thr-58-O-GlcNAcylated c-myc level and induced cell death in MCF-7 cells, which may decrease invasiveness of the breast cancer cells. Supported by NCI SBIR Phase I Contract No. HHSN261201100073C. Citation Format: Hyesook Kim, Sohee Kim, Aby Joiakim, David Kaplan, David Putt. Production of Thr-58-O-GlcNAcylated c-myc antibodies and detection of increased O-GlcNAcylated c-myc levels in MCF-7 cells after ketoconazole treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-58. doi:10.1158/1538-7445.AM2013-LB-58 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

We hypothesize that overexpression of mitochondrial glutathione (mtGSH) transporters in renal proximal tubular (PT) cells from diabetic rats will reverse the oxidative stress and diminish susceptibility to chemically induced cytotoxicity by producing sustained increases in mtGSH content. cDNAs encoding either the dicarboxylate (DIC; Slc25a10) or 2‐oxoglutarate (OGC; Slc25a11) carrier were expressed in renal PT cells from either control or streptozotocin‐induced diabetic Sprague‐Dawley rats using Lipofectamine. Transfection with either DIC or OGC cDNA produced 7,800‐ or 5,000‐fold increases in DIC or OGC mRNA, respectively, in cells from control rats and 600‐ and 1900‐fold increases in DIC or OGC mRNA, respectively, in cells from diabetic rats. Carrier overexpression provided significant protection, as judged by LDH release and MTT fluorescence, from injury due to the mitochondrial toxicant antimycin A. These results suggest that mtGSH carrier overexpression may be a viable approach to improving redox status of renal cells in chronic disease states.(Supported by DOD Grant PR64340)

Nephropathy is a serious and common complication of diabetes. In the streptozotocin (STZ)-treated rat model of diabetes, nephropathy does not typically develop until 30 to 45days post-injection, although hyperglycemia occurs within 24h. We tested the hypothesis that chronic hyperglycemia results in a modest degree of oxidative stress that is accompanied by compensatory changes in certain antioxidants and mitochondrial redox status. We propose that as kidneys progress to a state of diabetic nephropathy, further adaptations occur in mitochondrial redox status. Basic parameters of renal function in vivo and several parameters of mitochondrial function and glutathione (GSH) and redox status in isolated renal cortical mitochondria from STZ-treated and age-matched control rats were examined at 30days and 90days post-injection. While there was no effect of diabetes on blood urea nitrogen, measurement of other, more sensitive parameters, such as urinary albumin and protein, and histopathology showed significant and progressive worsening in diabetic rats. Thus, renal function is compromised even prior to the onset of frank nephropathy. Changes in mitochondrial respiration and enzyme activities indicated existence of a hypermetabolic state. Higher mitochondrial GSH content and rates of GSH transport into mitochondria in kidneys from diabetic rats were only partially due to changes in expression of mitochondrial GSH carriers and were mostly due to higher substrate supply. Although there are few clear indicators of oxidative stress, there are several redox changes that occur early and change further as nephropathy progresses, highlighting the complexity of the disease.