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.
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.
This study investigates the effect of innate differences in GSH concentration in a panel of human pancreatic cancer cell lines on activity of redox polyplexes. Increased GSH levels are associated with improved transfection of mRNA polyplexes.
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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.
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.
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.
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.
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 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.
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.
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.
Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxoglutarate carriers (OGC; Slc25a11). To determine whether these carriers function similarly in ...liver mitochondria, we assessed the effect of competition with specific substrates or inhibitors on GSH uptake in isolated rat liver mitochondria. GSH uptake was uniphasic, independent of ATP hydrolysis, and exhibited Km and Vmax values of 4.08mM and 3.06nmol/min per mg protein, respectively. Incubation with butylmalonate and phenylsuccinate inhibited GSH uptake by 45–50%, although the individual inhibitors had no effect, suggesting in rat liver mitochondria, the DIC and OGC are only partially responsible for GSH uptake. H4IIE cells, a rat hepatoma cell line, were stably transfected with the cDNA for the OGC, and exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by H2O2, methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver.
To further develop primary cultures of human proximal tubular (hPT) cells for study of drug disposition, we determined kinetics and protein expression of several key transporters for organic anions ...and cations, peptides, and neutral amino acids.
p-Aminohippurate uptake exhibited similar kinetics as published values, was inhibited by cephaloridine, cimetidine, methotrexate, and urate, consistent with function of both organic anion transporter 1 (OAT1) and OAT3. Transport rates by organic cation transporters (OCTs) were up to three-fold higher than those of OATs. Of the OCT substrates tested, triethanolamine exhibited the highest transport rates across the basolateral membrane (BLM). OCTN1 exhibited high-affinity, low-capacity BLM transport of
l-carnitine. Glycylsarcosine transport by PepT2 was rapid and comparable to that of OCTs. Amino acid System L on the BLM exhibited comparable kinetic parameters for transport of
l-leucine as the OATs. Efflux of verapamil across the brush-border membrane by P-glycoprotein was very rapid. Expression of carriers was generally maintained throughout 5 days of culture. Of the four OAT proteins studied (OAT1-4), expression of OAT1 and OAT3 was the most readily detected and exhibited interindividual variation. OCTN2 was the major OCT in hPT cells. Expression was also quantified for multidrug resistance-associated proteins 2 and 5 and P-glycoprotein. These results show that primary cultures of hPT cells express a diverse array of transporters for major classes of important drugs and are suitable for study of drug transport and disposition and assessment of potential drug–drug interactions in human kidney.
Abstract We previously catalogued expression and activity of organic anion and cation, amino acid, and peptide transporters in primary cultures of human proximal tubular (hPT) cells to establish them ...as a cellular model to study drug transport in the human kidney Lash, L.H., Putt, D.A., Cai, H., 2006. Membrane transport function in primary cultures of human proximal tubular cells. Toxicology 228, 200–218. Here, we extend our analysis to drug metabolism enzymes. Expression of 11 cytochrome P450 (CYP) enzymes was determined with specific antibodies. CYP1B1, CYP3A4, and CYP4A11 were the only CYP enzymes readily detected in total cell extracts. These same CYP enzymes, as well as CYP3A5 and possibly CYP2D6, were detected in microsomes from confluent hPT cells, although expression levels varied among kidney samples. In agreement with Western blot data, only activity of CYP3A4/5 was detected among the enzyme activities measured. Expression of all three glutathione S -transferases (GSTs) known to be found in hPT cells, GSTA, GSTP, and GSTT, was readily detected. Variable expression of three sulfotransferases (SULTs), SULT1A3, SULT1E, and SULT2A1, and three UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A6, and UGT2B7, was also detected. When examined over the course of cell growth to confluence, expression of all enzymes was generally maintained at readily measurable levels, although they were often lower than in fresh tissue. These results indicate that primary cultures of hPT cells possess significant capacity to metabolize many classes of drugs, and can be used as an effective model to study drug metabolism.
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