Genetic Polymorphisms of Cytochrome P450 and Risk on Prostate and Bladder Cancer
GUNES S 1, ALTAYLI E1, BAGCI H1, YILMAZ AF2, SARIKAYA S2
1Ondokuz Mayis University, Department of Medical Biology, Samsun, Turkey; 2Ondokuz Mayis University, Department of Urology, Samsun, Turkey.
Background: Human cytochrome P450 (CYPs) enzymes are oxidative enzymes responsible for the metabolic activation of many precarcinogens and participate in the activation and inactivation of anticancer drugs. Genetic differences of the detoxification system may cause an increase in susceptibility to environmentally induced bladder cancer. Genetic differences of the detoxification system may cause an increase in susceptibility to environmentally induced bladder cancer and prostate cancer. Aims: 1) To investigate the potential association between the cytochrome p450 1A2 (CYP1A2) 734 C>A and cytochrome p450 2D6 (CYP2D6) 1934 G>A gene polymorphisms and the risk of bladder cancer in a Turkish population. 2) To investigate the relationship of prostate cancer (PCa) with genetic polymorphism of 17-hydroxylase (CYP17) (-34 T/C) and CYP1A1 (T/C) genes in a Turkish population.
Methods: This study included 135 bladder cancer patients and 128 age and sex matched cancer-free controls; 148 prostate cancer patients, 136 benign prostatic hyperplasia patients, and 102 healthy individuals as controls. The polymorphisms were analyzed using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay. Genotype and allele frequencies and their associations with bladder and prostate cancer risk, demographic factors, smoking status, and tumor stage were investigated.
Results: 1) There was no association between studied polymorphisms of CYP1A2, and CYP2D6 genes and bladder cancer. 2) No statistically significant association was observed between smoking status of the patients and any of the polymorphisms studied. 3) We have observed that there is statistically significant association between the smokers with the CYP1A2 CC genotype and the bladder cancer risk but not with the non-smoker subjects with CC genotype (OR=2.55; %95CI, 1.030-6.316). 4) No association was observed between prostate cancer and 17 hydroxylase gene polymorphism. 5) There was also an association between 17 hydroxylase polymorphism and benign prostatic hyperplasia (P=0.004).
Conclusions: These data demonstrate that cytochrome P450 enzymes polymorphisms studied may not be associated with bladder cancer and CaP population studied. In addition, the results suggest that the genotypes of CYP1A2 polymorphism may be associated with increased risk of bladder cancer in smokers.
Keywords: CYP450 polymorphisms, bladder cancer, prostate cancer
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Cholinesterase Inhibitors and NMDA Receptor Antagonists in Alzheimer’s Disease and Pesticide Poisoning.
GUPTA RC
Murray State University, Hopkinsville, KY; Dejan Milatovic, Vanderbilt University, Nashville, TN, USA.
Both Alzheimer’s disease (AD) and anticholinesterase pesticide (organophosphate and carbamate) poisoning are characterized by alterations in the cholinergic and glutamatergic systems. In addition to neurodegeneration, common biochemical mechanisms in the discrete brain areas of concern, cortex and hippocampus, involve up-regulation of NMDA receptors, excess free radical generation, oxidative/nitrosative stress, depletion of enzyme-based antioxidant system and high- energy phosphates. While AD is characterized by a marked decline in acetylcholine (ACh) due to the loss of cholinergic neurons (as much as 90%), immediate reaction in the organophosphate (OP) and carbamate (CM) poisoning is acetylcholinesterase (AChE) inhibition, followed by ACh accumulation. Currently, donepezil (Aricept), rivastigmine (Exelon), and galantamine (Reminyl) are the three reversible AChE inhibitors most commonly indicated in AD patients to elevate the levels of ACh, which is required to sustain precious memory. However, these AChE inhibitors are indicated only in mild to moderate AD, and patients often become refractory to the beneficial effects after a year of treatment. Our findings revealed that a combination therapy with donepezil (0.75 or 1.5 mg/kg, ip) or rivastigmine (0.35 or 0.7 mg/kg, ip) and an NMDA receptor antagonist like memantine (10 mg/kg, ip) or neramexane (3.1, 6.2, or 12.4 mg/kg, ip), has no interaction at the AChE level and may provide the optimal therapeutic effect. Our study with cholinesterase inhibitors and NMDA receptor antagonist in the animal model revealed that pretreatment with memantine (18 mg/kg, sc) in combination with atropine sulfate (16 mg/kg, sc) provides remarkable protection against an OP compound DFP (1.5 mg/kg, sc) or a carbamate compound carbofuran (1.5 mg/kg, sc) induced behavioral toxicity, AChE inhibition and decline of high-energy phosphates. In addition, these antidotal pretreatments suppressed DFP or carbofuran induced increase in markers of oxidative (F2-isoprostane and F4-neuroprostane) and nitrosative (citrulline) stress, inflammation (PGE2) and morphological changes in the dendritic system of pyramidal neurons from CA1 hippocampal area. It can be concluded that the lack of interaction between AChE inhibitors (donepezil or rivastigmine) and NMDA receptor antagonists (memantine or neramexane) at the AChE level benefits in treatment of AD, while interaction benefits in prophylaxis and treatment of OP/CM poisoning.
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Antileishmanial Efficacy of Amphotericin B bearing Emulsomes against Experimental Visceral Leishmaniasis
GUPTA S1, DUBE A2, VYAS SP1,3
1Indo Soviet Friendship College of Pharmacy, Moga (PB), India; 2Central Drug Research Institute, Lucknow (UP), India; 3Dr. Hari Singh Gour Vishwavidyalaya, Sagar (MP), India.
Background: Because lipid particles are phagocytized by the reticuloendothelial system, lipid associated amphotericin B (AmB) should be concentrated in infected macrophages (MQs) of liver and spleen and be very effective against visceral leishmaniasis (VL). Aims: 1) To develop emulsomes for effective and site specific localization of AmB. 2) Passive and active targeting of AmB inside the MQs. 3) To treat the VL.
Methods: AmB loaded plain emulsomes (PE) and O-palmitoyl mannan coated emulsomes (CE) were prepared by cast film method followed by homogenization. Organ distribution study included 72 male albino rats (weight 150-200g, 18 rats per treatment, one control group). AmB-deoxycholate (AD), PE and CE containing doses equivalent to 1mg/kg of AmB were administered i.v. to different groups. The antileishmanial activity of AD, PE and CE was tested in vitro at different drug doses (0.03, 0.08, 0.13 and 0.2 μg/ml) in Leishmania donovani infected MQ-amastigote system (J774A.1 cells). L. donovani infected hamsters (weight 80-100 g) harboring 38-40 amastigotes/100 MQ nuclei were distributed (6 in each group, total 36, 3 control groups) for drug (equivalent to 0.5 mg/kg) treatment intracardially. Each experiment was run for 3 times.
Results: The in vitro antileishmanial activity showed higher efficacy of CE (92.275.7% parasite inhibition, PI) over PE (82.575.4% PI) and AD (65.715.1% PI) (p<0.05 for AD vs PE; p<0.01 for AD vs CE) [data at dose of 0.08 μg/ml]. The extent of accumulation of AmB in MQs rich organs, liver and spleen was significantly high from developed systems (54.47±3.4 and 13.48±0.91% from PE; 59.674.2 and 14.120.9% from CE after 24 h) when compared against AD (15.741.1 and 1.140.08% after 24 h). Formulation CE eliminated L. donovani amastigotes within splenic MQs more efficiently (73.76.7% PI) than PE (51.75.4% PI) (p<0.01) or AD (30.44.8% PI) (p<0.001).
Conclusions: 1) The proposed systems showed excellent potential for MQ targeting as shown by drug levels in liver and spleen. 2) The formulations could significantly modify the pharmacokinetics of AmB as compared to AD, providing prolonged action at comparatively low drug doses thereby reducing the toxicity problems like nephrotoxicity, cardiac arrhythmia etc.
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Ciprofloxacin Induces Oxidative Stress and Exerts Biphasic Cytotoxicity in Primary Culture of Rat Astrocytes and Human Fibroblast Cells.
GÜRBAY A1,2, GONTHIER B2, FAVIER A3, HINCAL F1
1Univ. of Hacettepe, Ankara, Turkey; 2Univ. Joseph Fourier, La Tronche, France; 3CEA/Grenoble, Grenoble, France
Background: Mechanism underlying adverse effects of ciprofloxacin (CPFX) is still not well known. Aims: The possible cytotoxic and oxidative stress inducing effects of CPFX were investigated on primary cultures of rat astrocytes and human fibroblast cells.
Methods: The cultured cells were incubated with various concentrations of CPFX (0.5-300 mg/l for astrocytes, n= 4; 5-150 mg/l for fibroblast cells, n= 3), and cytotoxicity was determined by neutral red (NR) and/or MTT assays. Oxidative stress induction possibility by the drug were assayed measuring lipid peroxidation (LP) and total glutathione (GSH) levels, and activity of catalase (Cat) in both cell types. Superoxide dismutases and glutathione peroxidase activities were determined only in fibroblast cells.
Results: 1) Survival profile of astrocyte cells was biphasic in NR assay: While CPFX did not cause any alteration at any concentration for 7 h, ¡Ü50 mg/l concentrations induced significant (p< 0.05) cell proliferation in 24, 48, 72, and 96 h. However, cell proliferation gradually decreased at higher concentrations, and 200 and 300 mg/l of CPFX was found to be significantly (p< 0.05) cytotoxic at all time periods. With MTT assay, no alteration was noted for 7 h. But, viability decreased with ¡Ý 50 mg/l CPFX exposure in all other time periods. Cell proliferation was only seen in 24 h with 0.5 and 5 mg/l CPFX. A significant enhancement of LP was observed with the 300 mg/l of the drug, but GSH, and Cat content of cells did not change. 2) Cytotoxicity was not observed with 5-150 mg/l CPFX when the human fibroblast cells were incubated for 24 h. 5-12.5 mg/l of CPFX increased the cell growth in all incubation periods tested. Marked decreases in the viability of fibroblasts were observed at 50 and 75 mg/l, and ¡Ý 50 mg/l, following 48 and 72 h exposure, respectively (p< 0.05). An induction of LP and a marked decrease in intracellular GSH were observed following incubation of cells with 75 mg/l CPFX for 48 h. Vitamin E pretreatment provided protection in both cell types.
Conclusions: 1) CPFX-induced cytotoxicity is related to oxidative stress. 2) The hormetic-like biphasic effects of CPFX possibly resulted from the complex dosedependent relationships between reactive oxygen species, cell proliferation, and cell viability.
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Transient Activation of the Small GTPase Rap1 is Functionally Required for the Regulation of Breast Cancer Cell Motile Responses to IGF-I
GUVAKOVA MA, LEE WSY, FURSTENAU DK
Univ. of Pennsylvania, Philadelphia, PA, USA
Background: The Ras family GTPase Rap1 is an essential regulator for adhesion receptors, actin dynamics, and cell migration. The mechanisms by which Rap1 controls motile responses in the epithelial cells remain unknown. In fact, current view is that activation of Rap1 tightens cell-cell junctions and thus may have anti-metastatic effects in cancer. Normal and cancer cells share common, and perhaps evolutionarily conserved, motility machinery activated by insulin-like growth factor I (IGF-I).
Methods: We used an in vivo tool, a fusion of enhanced green fluorescent protein (EGFP) to the Rap1 binding domain (RBD) of RalGDS that is recruited specifically to the sites of increased Rap1 activity. By confocal laser-scanning microscopy, we monitored dynamics of EGFP-RBD-RalGDS; by time-lapse video microscopy we tracked localization of the EGFP-Rap1 molecules in live cancer cells. By image-based quantitative immunohistochemistry, we compared 23 normal/benign (N/B) and 32 invasive breast cancer (InvBC) surgical specimens.
Results: We report that in human breast cancer cells a rapid enhancement and a subsequent gradual decrease of Rap1 activity induced by IGF-I promote cell motile responses: breakdown of cell-cell contacts and formation of lamellipodia. This transient activation of Rap1 requires the kinase activity of the IGF-I receptor (IGF-IR) and receptor internalization. Time-lapse video microscopy in live cells confirmed a disappearance of EGFP-tagged WTRap1 and constitutively active V12Rap1 from cell-cell contacts. We also found accumulation of the active endogenous Rap1 in lamellipodia of IGF-I-stimulated cells, whereas selective blocking of IGF-I-induced Rap1 activation by over-expressed RapGAP restrained lamellipodia formation. Quantitative analysis of surgical specimens revealed significantly higher protein levels of Rap1 (p=6.23E-04) and IGF-IR (p=4.32E-07) in InvBC compared with N/B breast tissue.
Conclusions: 1) Presented data provide experimental evidence that transient activation of Rap1 by IGF-IR-mediated mechanism promotes cancer cell motile responses and that active Rap1 does not prevent cell-cell separation induced by IGF-I. 2) Screens of surgical specimens show significant over-expression of IGF-IR and its target Rap1 in infiltrating carcinoma of the breast.
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