CEdG – a glycated DNA adduct linking altered metabolism and genetic instability

This dissertation details original work focused on the DNA adduct N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG). This DNA adduct results from the spontaneous reaction of DNA with the endogenous and exogenous formed, carbohydrate-derived, reactive carbonyl species, methylglyoxal. Using in vitro steady state kinetics, we have shown that CEdG in template DNA leads to DNA miscoding effects when the model replicative polymerase, exonuclease-free Klenow fragment (KF-) is used. The development, validation and application of a novel stable isotope dilution, triple quadrupole mass spectrometric method for the quantitation of CEdG is also detailed. This method was used to quantitate CEdG in urine from diabetic rats, urine from human patients, human tumor and adjacent biopsy tissue, diabetic animal tissue and DNA treated with methylglyoxal. Finally, we detail the adaptation, validation and application of a novel, commercially-available microfluidic HPLC-chip for increased sensitivity in the quantitation of CEdG and also apply it to the quantitation of the RNA analogue, CEG. Combined, these studies establish CEdG as a potential biomarker for glycation and point to a viable avenue for connecting chronic glycolytic flux with genetic instability

Enhanced radiation response in radioresistant MCF-7 cells by targeting peroxiredoxin II.

In our previous study, we identified that a protein target, peroxiredoxin II (PrxII), is overexpressed in radioresistant MCF+FIR3 breast-cancer cells and found that its expression and function is associated with breast-cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast-cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by the siRNA that inhibits PrxII gene expression. Our results, presented here, show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca(2+) efflux from the cells, and perturbing the intracellular Ca(2+) homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to this technology's property of targeting to specific cancer-related genes

Stress hormones increase cell proliferation and regulates interleukin-6 secretion in human oral squamous cell carcinoma cells

Patients with oral cancer can have high psychological distress levels, but the effects of stress-related hormones on oral cancer cells and possible mechanisms underlying these relationships are unknown. In this study, we have investigated the effects of stress-related hormones on interleukin-6 (IL-6) secretion and proliferation of oral squamous cell carcinoma (OSCC) cells. The effects of norepinephrine (NE), and cortisol were studied in SCC9. SCC15, and SCC25 cells and effects of isoproterenol in SCC9 and SCC25 cells. Real-time PCR studies revealed constitutive beta 1- and beta 2-adrenergic receptors (beta-ARs) expression in the SCC9. SCC15, and SCC25 cells. The results showed that NE and isoproterenol significantly enhanced IL-6 mRNA expression and protein production in supernatants of SCC9 and SCC25 cells. Physiological stress levels of NE and isoproterenol (10 mu M) at 1 h elicited the most robust IL-6 increase. Regarding IL-6 secretion, 10 mu M NE induced a 5-fold increase at 1 h, 3.7-fold increase at 6 h, and 3.2-fold at 24 h in SCC9 cells. These effects were blocked by the beta-adrenergic antagonist propranolol, supporting a role for beta-ARs in IL-6 secretion. The effects of cortisol varied according to the hormone concentration. Pharmacological concentrations of cortisol (1000 nM) inhibited IL-6 production by SCC9 and SCC25 cells. Cortisol dose that simulates stress conditions (10 nM) tended to increase IL-6 expression in SCC9 cells. Hormonal doses that simulate stress conditions (10 mu M NE, at 6 h in SCC9 and SCC15 cells and 10 nM cortisol, at 48 h in SCC15 cells) stimulated increased cell proliferation. Treatment of SCC9 cells with IL-6 neutralizing ab (10 mu g/mL) partially inhibited NE-induced proliferation. Finally, 20 OSCC biopsies were shown to express beta 1- and beta 2-ARs. These findings suggest that stress hormones can affect oral cancer cells behavior. (C) 2010 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Vaccinations with Recombinant Variants of Aspergillus fumigatus Allergen Asp f 3 Protect Mice against Invasive Aspergillosis

A vaccine that effectively protects immunocompromised patients against invasive aspergillosis is a novel approach to a universally fatal disease. Here we present a rationale for selection and in vivo testing of potential protein vaccine candidates, based on the modification of an immunodominant fungal allergen for which we demonstrate immunoprotective properties. Pulmonary exposure to viable Aspergillus fumigatus conidia as well as vaccination with crude hyphal extracts protects corticosteroid-immunosuppressed mice against invasive aspergillosis (J. I. Ito and J. M. Lyons, J. Infect. Dis. 186:869-871, 2002). Sera from the latter animals contain antibodies with numerous and diverse antigen specificities, whereas sera from conidium-exposed mice contain antibodies predominantly against allergen Asp f 3 (and some against Asp f 1), as identified by mass spectrometry. Subcutaneous immunization with recombinant Asp f 3 (rAsp f 3) but not with Asp f 1 was protective. The lungs of Asp f 3-vaccinated survivors were free of hyphae and showed only a patchy low-density infiltrate of mononuclear cells. In contrast, the nonimmunized animals died with invasive hyphal elements and a compact peribronchial infiltrate of predominately polymorphonuclear leukocytes. Three truncated versions of rAsp f 3, spanning amino acid residues 15 to 168 [rAsp f 3(15-168)], 1 to 142, and 15 to 142 and lacking the known bipartite sequence required for IgE binding, were also shown to be protective. Remarkably, vaccination with either rAsp f 3(1-142) or rAsp f 3(15-168) drastically diminished the production of antigen-specific antibodies compared to vaccination with the full-length rAsp f 3(1-168) or the double-truncated rAsp f 3(15-142) version. Our findings point to a possible mechanism in which Asp f 3 vaccination induces a cellular immune response that upon infection results in the activation of lymphocytes that in turn enhances and/or restores the function of corticosteroid-suppressed macrophages to clear fungal elements in the lungs

Increased plasma and salivary cortisol levels in patients with oral cancer and their association with clinical stage

Objectives Dysregulation of the hypothalamus-pituitary-adrenal axis has been observed in patients with cancer. This cross-sectional study investigated whether patients with oral and oropharyngeal squamous cell carcinoma (SCC) show changes in cortisol levels in saliva and plasma compared with three control groups, and evaluated its correlation with clinicopathological data.Methods Salivary and plasma cortisol levels of 34 patients with oral SCC were compared with hormonal levels of 17 oropharyngeal SCC patients, 17 oral leukoplakia patients, 27 smokers and/or drinkers and 25 healthy volunteers. Multivariate analysis was used to evaluate the impact of clinical variables on the cortisol levels.Results The plasma (p<0.05) and salivary (p<0.01) cortisol levels were significantly higher in patients with oral SCC compared with all groups. Patients with oropharyngeal SCC had higher levels of salivary cortisol compared with smokers and/or drinkers (p<0.05) and patients with leukoplakia (p<0.01). Patients with advanced-stage oral SCC showed significantly higher levels of cortisol than those in an initial clinical stage. Men with oral SCC had higher salivary cortisol levels than women (p<0.05). Age, smoking, alcohol consumption, presence of teeth and awareness of cancer diagnosis had no significant effect on cortisol levels.Conclusions These results indicate a dysregulation of cortisol secretion in patients with oral cancer and suggest that this hormone can be a biomarker associated with the disease's clinical status.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

DNA Advanced Glycation End Products (DNA-AGEs) Are Elevated in Urine and Tissue in an Animal Model of Type 2 Diabetes.

More precise identification and treatment monitoring of prediabetic/diabetic individuals will require additional biomarkers to complement existing diagnostic tests. Candidates include hyperglycemia-induced adducts such as advanced glycation end products (AGEs) of proteins, lipids, and DNA. The potential for DNA-AGEs as diabetic biomarkers was examined in a longitudinal study using the Leprdb/db animal model of metabolic syndrome. The DNA-AGE, N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) was quantified by mass spectrometry using isotope dilution from the urine and tissue of hyperglycemic and normoglycemic mice. Hyperglycemic mice (fasting plasma glucose, FPG, ≥ 200 mg/dL) displayed a higher median urinary CEdG value (238.4 ± 112.8 pmol/24 h) than normoglycemic mice (16.1 ± 11.8 pmol/24 h). Logistic regression analysis revealed urinary CEdG to be an independent predictor of hyperglycemia. Urinary CEdG was positively correlated with FPG in hyperglycemic animals and with HbA1c for all mice. Average tissue-derived CEdG was also higher in hyperglycemic mice (18.4 CEdG/106 dG) than normoglycemic mice (4.4 CEdG/106 dG). Urinary CEdG was significantly elevated in Leprdb/db mice relative to Leprwt/wt, and tissue CEdG values increased in the order Leprwt/wt &lt; Leprwt/db &lt; Leprdb/db. These data suggest that urinary CEdG measurement may provide a noninvasive quantitative index of glycemic status and augment existing biomarkers for the diagnosis and monitoring of diabetes

Discovery of (<i>R</i>)‑2-(6-Methoxynaphthalen-2-yl)butanoic Acid as a Potent and Selective Aldo-keto Reductase 1C3 Inhibitor

Type 5 17β-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ⁴-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively, in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogues and find that (<i>R</i>)-2-(6-methoxynaphthalen-2-yl)­butanoic acid (in which the methyl group of <i>R</i>-naproxen was replaced by an ethyl group) acts as a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. <i>R</i>-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids and in the context of advanced prostate cancer <i>R</i>-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease

Discovery of (<i>R</i>)‑2-(6-Methoxynaphthalen-2-yl)butanoic Acid as a Potent and Selective Aldo-keto Reductase 1C3 Inhibitor

Type 5 17β-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ⁴-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively, in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogues and find that (<i>R</i>)-2-(6-methoxynaphthalen-2-yl)­butanoic acid (in which the methyl group of <i>R</i>-naproxen was replaced by an ethyl group) acts as a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. <i>R</i>-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids and in the context of advanced prostate cancer <i>R</i>-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease