[Show abstract][Hide abstract] ABSTRACT: In human immunodeficiency virus 1 (HIV-1)-infected individuals, exposure to a protease inhibitor (PI)-based highly active antiretroviral therapy (HAART) regimen increases cardiovascular disease and endothelial dysfunction. However, the mechanisms of PI-induced effects on endothelial cells (ECs) are not known. Furthermore, strategies to suppress these deleterious outcomes of PIs need to be developed. Insulin-induced PI3K/Akt signaling and endothelial nitric oxide (NO)-synthase (eNOS) phosphorylation regulates NO production by ECs that maintain vascular homeostasis. We evaluated whether nelfinavir (NEL), a potent HIV-1 PI that suppresses Akt phosphorylation, can alter insulin-induced NO production in human aortic endothelial cells (HAECs) and whether insulin sensitization of HAECs via the peroxisome proliferator-activated receptor-gamma agonists, thiazolidinediones, can ameliorate these side effects.
Real-time NO production in HAECs was monitored by fluorimetric dyes DAF-FM DA and DAF-2 DA. Immunodetection studies were used to determine the phosphorylation of Akt, eNOS, insulin receptor-β (IR-β), insulin receptor substrate-1 (IRS-1), and PI3K/p85α. Expression of eNOS messenger RNA was measured by reverse transcription polymerase chain reaction.
In vitro exposure (72 hours) of HAECs to NEL (0.25-2 μg/mL) decreased both basal (2.5-fold) and insulin-induced NO production (4- to 5-fold). NEL suppressed insulin-induced phosphorylation of both Akt and eNOS at serine residues 473 and 1177, respectively. NEL decreased tyrosine phosphorylation of IR-β, IRS-1, and PI3K. Coexposure to troglitazone (TRO; 250 nM) ameliorated the suppressive effects of NEL on insulin signaling and NO production. Coexposure to TRO also increased eNOS expression in NEL-treated HAECs.
Our findings indicate that treatment with potent insulin sensitizers may protect against PI-mediated endothelial dysfunction during long-term HAART.
[Show abstract][Hide abstract] ABSTRACT: SOX9, a high mobility group (HMG) box transcription factor, is required for development, differentiation and lineage commitment. It is known to exert its effects through nuclear translocation, such as cell cycle changes in response to retinoic acid treatment in breast cancer cells. However, it is not known whether SOX9 has prognostic significance in human breast cancer. Over-expression and cytoplasmic sequestration of nuclear proteins are implicated in tumor progression. To determine whether SOX9 has any prognostic significance in human breast cancer, its expression and subcellular localization were analyzed in more than 200 human breast carcinomas (BCs). SOX9 mRNA expression data for human BCs were computed from microarray studies available in public databases and correlated with known poor prognostic parameters of BCs. SOX9 protein expression and its correlation with Ki-67 staining in human BCs were assessed using immunohistochemistry. Higher SOX9 mRNA levels were significantly associated with estrogen receptor negative (P ≤ 0.001) and higher grade (P ≤ 0.01) human breast tumors. Patients with higher SOX9 mRNA level had significantly shorter overall survival (P ≤ 0.0001). SOX9 protein, which is normally nuclear, was instead localized in the cytoplasm of 25-30% invasive ductal carcinomas (IDCs) and lymph node metastases. Its cytoplasmic accumulation significantly correlated with enhanced proliferation in breast tumors (Kendall's tau = 0.337 with a P value < 0.0001). Cytoplasmic SOX9 can serve as a valuable prognostic marker for IDCs and metastatic breast cancer. Its significant correlation with breast tumor cell proliferation implies that SOX9 directly contributes to the poor clinical outcomes associated with invasive breast cancer.
Experimental Biology and Medicine 02/2011; 236(2):145-55. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute neurotoxic effects of high-dose methylmercury (MeHg) in humans have been well documented in the scientific literature. However, low-dose effects are less well described. This study was designed to evaluate the effects of low-dose MeHg (<100 nM) on human brain cells in a tissue culture model. Neuroblastoma (NB) cells (SH-SY5Y) were used in the cell culture model to study low-dose effects of MeHg on cell growth, cell survival, reactive oxygen species (ROS), and the phosphorylation of tau protein, as a measure of potential markers of cellular events associated with tauopathies. When cells were incubated in culture with MeHg (50 and 100 nM), there were significant decreases in cell viability as well as significant increase in ROS generation as determined by fluorescent dye analysis (H(2)DCFDA). Furthermore, a concomitant decrease in glutathione levels to 25% of control was observed at both 50 and 100 nM MeHg. In addition, the level of phosphorylated tau was significantly increased after treatment at both 50 and 100 nM MeHg, compared with controls. Pretreatment of NB cells with the antioxidant, N-acetylcysteine (1.25 mM) and the calpain inhibitor, MDL-28170 (10 μM), significantly attenuated the effects of MeHg (50 and 100 nM) on cell viability as well as on tau phosphorylation. These results indicate that low-dose MeHg toxicity may be related to an induction of tau phosphorylation through an oxidative stress-dependent mechanism and that blockade of this pathway may attenuate the toxic effects of MeHg.
[Show abstract][Hide abstract] ABSTRACT: RNA interference is a post-transcriptional silencing mechanism triggered by the bioavailability and/or exogenous introduction of double-stranded RNA (dsRNA) into cells. Here we describe a novel method for the synthesis of siRNA in a single vessel. The method employs in vitro transcription and a single-stranded DNA (ssDNA) template and design, which incorporates upon self-annealing, two promoters, two templates, and three loop regions. Using this method of synthesis we generated efficacious siRNAs designed to silence both exogenous and endogenous genes in mammalian cells. Due to its unique design the single-stranded template is easily amenable to adaptation for attachment to surface platforms for synthesis of siRNAs. A siRNA synthesis platform was generated using a 3' end-biotinylated ssDNA template tethered to a streptavidin coated surface that generates stable siRNAs under multiple cycles of production. Together these data demonstrate a unique and robust method for scalable siRNA synthesis with potential application in RNAi-based array systems.
[Show abstract][Hide abstract] ABSTRACT: Thymoquinone (TQ), an active ingredient of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against a variety of experimental tumors. However, the precise mechanism of action of TQ is not known. We investigated the mechanism of action of TQ in androgen receptor (AR)-independent (C4-2B) and AR naïve (PC-3) prostate cancer cells, as models of aggressive prostate cancers. Exposure (24-48 h) to TQ (25-150 micromol/L) inhibited the growth of both C4-2B and PC-3 cells, with IC(50) values of approximately 50 and 80 micromol/L, respectively. Within one hour, TQ increased reactive oxygen species (ROS) levels (3-fold) and decreased glutathione (GSH) levels (60%) in both cell types. Pretreatment with N-acetylcysteine (NAC) inhibited both TQ-induced ROS generation and growth inhibition. TQ did not increase the activity of caspases and the caspase inhibitor, z-VAD-FMK did not decrease TQ-induced apoptosis. Furthermore, although TQ treatment resulted in the activation of Jun kinase (JNK), pretreatment with the JNK inhibitor, SP600125, did not protect cells from TQ. However, TQ significantly up-regulated the expressions of growth arrest and DNA damage inducible gene (GADD45alpha) and apoptosis-inducing factor-1 and down-regulated the expressions of several Bc12-related proteins, such as BAG-1, Bcl2, Bcl2A1, Bcl2L1 and BID. In C4-2B cells, TQ dose dependently inhibited both total and nuclear AR levels (4-5 fold) and AR-directed transcriptional activity (10-12 fold). Interestingly, this suppressive effect on AR was not prevented by NAC, which clearly suggested that TQ-induced cytotoxicity is not due to changes in AR regulation. These data suggest that TQ-induced cell death is primarily due to increased ROS generation and decreased GSH levels, and is independent of AR activity.
Experimental Biology and Medicine 06/2010; 235(6):751-60. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Complexes of Zn(II) with radioprotective thiol ligands such as cysteamine, L-cysteine and N-(2-mercaptopropionyl)glycine have been synthesized, characterized, and evaluated for cytotoxicity and radioprotective effect. Zn2(N-(2-mercaptopropionyl)glycine)2 when given intraperitoneally at a dose of 14.1 mgkg−1, 30 min before whole-body γ-irradiation (9.0 Gy, 1 Gy min−1), resulted in 68% 30-day survival of CD2F1 mice. This radioprotection was significantly better than that afforded by equimolar doses of ZnCl2 (7%), by N-(2-mercaptopropionyl)glycine (0%), or by a mixture of ZnCl2 and N-(2-mercaptopropionyl)-glycine (0%). Zn(cysteamine)2 and Zn(L-cysteine)2 afforded 14% and 7% survival of CD2F1 mice. These data show that the radioprotective effect of thiols such as N-(2-mercaptopropionyl)glycine can be enhanced by complexation with Zn(II).
Pharmacy and Pharmacology Communications. 02/2010; 6(7):299 - 302.
[Show abstract][Hide abstract] ABSTRACT: Aetiology (case control) Level of Evidence 3b OBJECTIVE To evaluate the effect of N(G)-nitro-L-arginine methyl ester (L-NAME)-induced hypertension (HT) on erectile function in the rat and determine if the phosphodiesterase (PDE)-5 inhibitor, sildenafil, can reverse the effects of nitric oxide (NO) deficiency, as HT is a risk factor for erectile dysfunction (ED) and the NO synthase (NOS) inhibitor L-NAME induces NO-deficient HT.
Thirty-six adult Sprague-Dawley male rats were divided into three groups, i.e. a control, L-NAME-HT (40 mg/rat/day in the drinking water for 4 weeks), and sildenafil-treated L-NAME-HT (1.5 mg/rat/day sildenafil, by oral gavage concomitantly with L-NAME). The erectile response expressed as a ratio of intracavernosal pressure (ICP)/mean arterial pressure (MAP), evaluated after electrical stimulation of the right cavernous nerve. The isometric tension of corpus cavernosum smooth muscle (CCSM) was measured in organ-bath experiments. NOS expression was determined immunohistochemically for neuronal (n)NOS and by Western blot analysis for endothelial (e) and inducible (i) NOS protein. cGMP levels were evaluated by enzyme-linked immunosorbent assay.
The erectile response was diminished in the HT group. Nitrergic and endothelium-dependent relaxation was reduced, while the relaxation response to sodium nitroprusside and contractile response to phenylephrine were not altered in CCSM from L-NAME-treated rats. HT rats showed decreased expression of nNOS, whereas eNOS and iNOS protein expression was increased. Sildenafil partly restored endothelial and molecular changes in CCSM from HT rats, but did not reverse the decreased erectile response, even as cGMP levels returned to normal levels.
Sildenafil treatment did not correct the ED in L-NAME-treated HT rats. Under sustained high blood pressure, up-regulation of PDE5 expression failed to reverse the depletion of neuronal NO and/or impaired nNOS activity. However, endothelium-dependent relaxation was restored. Drug targeting of neuronal dysfunction might delay the onset of ED in HT.
BJU International 12/2009; 106(1):78-83. · 3.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The molecular mechanisms involved in prostate cancer (PC) metastasis and bone remodeling are poorly understood. We recently reported that phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) mediates transcriptional regulation and activation of bone morphogenetic protein (BMP)-2 signaling by nuclear factor (NF)-kappaB in bone metastatic prostate cancer cells. In the present study, we demonstrate that NF-kappaB, whether activated by recombinant human tumor necrosis factor (TNF)-alpha or by ectopic expression of the p65 subunit, is involved in extracellular matrix adhesion and invasion of osteotropic PC-3 and C4-2B, but not LNCaP, cells. The enhanced metastatic potential was associated with transcriptional upregulation of osteopontin, osteocalcin, and collagen IA1 in osteotropic PC cells, suggesting their role in osteomimicry of PC cells. Unlike BMP-4, BMP-2 protein enhanced the invasive properties of C4-2B cells, but not in LNCaP cells. Also, this effect was nullified by Noggin. In addition, BMP-2 mediates TNF-alpha-induced invasion of C4-2B cells in a NF-kappaB-dependent fashion. TNF-alpha or conditioned media (CM) of TNF-alpha-stimulated C4-2B cells upregulated BMP-2 and BMP-dependent Smad transcripts and inhibited receptor activator of NF-kappaB ligand transcripts in RAW 264.7 preosteoclast cells, respectively, implying that this factor may contribute to suppression of osteoclastogenesis via direct and paracrine mechanisms. In contrast, CM of TNF-alpha-stimulate or BMP2-stimulated C4-2B cells induced in vitro mineralization of MC3T3-E1 osteoblast cells in a BMP-2-dependent and NF-kappaB-dependent manner, respectively. Taken together, the results suggest that mutual interactions between these factors may be pivotal not only in enhancing the osteomimicry and metastatic potential of PC cells, but also in bone remodeling and in shifting the balance from osteoclastogenesis towards osteoblastogenesis.
Cancer Science 09/2009; 101(1):103-11. · 3.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prolonged use of highly active antiretroviral therapy (HAART) is associated with insulin resistance in HIV-1-positive patients. Small animal models that recapitulate the long-term effects of HAART may facilitate the identification of therapeutic agents to suppress these side effects. We investigated the protective effects of black seed oil (BSO) from Nigella sativa in Sprague-Dawley rats treated with a daily HAART regimen for 7 months. The antiretroviral drugs, consisting of nelfinavir (200 mg/kg), zidovudine (50 mg/kg), and efavirenz (20 mg/kg), were mixed with diet with or without BSO (400 microL/kg) supplementation. Significant increases in insulin and C-peptide levels were observed in HAART-treated groups, and concomitant BSO treatment reduced this hyperinsulinemia. Interestingly, HAART-treated rats showed reduced size of pancreatic islets that was not seen in BSO-exposed rats. In vitro studies showed that nelfinavir, alone and in combination with HAART, induced oxidative stress and decreased glucose-induced insulin production in INS-1 cells. Suppressed insulin production was restored in cells coexposed to either BSO or thymoquinone. Our findings demonstrated that chronic HAART may increase serum insulin levels by dysregulating both insulin production by beta cells and insulin action at the periphery. These deleterious effects may be prevented by dietary supplementation with BSO.
Canadian Journal of Physiology and Pharmacology 05/2009; 87(4):300-9. · 1.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The highly active anti-retroviral therapy (HAART) regimen has considerably reduced the mortality rate in HIV-1 positive patients. However, long-term exposure to HAART is associated with a metabolic syndrome manifesting cardiovascular dysfunction, lipodystrophy, and insulin resistance syndrome (IRS). The inclusion of HIV-1 protease inhibitors (PIs) in HAART has been linked to the induction of IRS. Although several molecular mechanisms of PI-induced effects on insulin action have been postulated, the deleterious effects of PIs on insulin production by pancreatic beta-cells have not been fully investigated and therapeutic strategies to ameliorate insulin dysregulation at this level have not been targeted. The present study showed that exposure to several different PIs, nelfinavir (5-10 microM), saquinavir (5-10 microM) and atazanavir (8-20 microM), decreases glucose stimulated insulin secretion from rat pancreatic beta-cells (INS-1). Nelfinavir significantly increased reactive oxygen species (ROS) generation and suppressed cytosolic, but not mitochondrial superoxide dismutase (SOD) levels. Nelfinvair also decreased both glutathione and ATP and increased UCP2 levels in these cells. Simultaneous treatment with thymoquinone (TQ) (2.5 microM), an active ingredient of black seed oil, significantly inhibited the effect of nelfinavir on augmented ROS production and suppressed SOD levels. Both TQ and black seed oil exposure increased glucose stimulated insulin secretion and ameliorated the suppressive effect of nelfinavir. The present findings imply a direct role of ROS in PI induced deleterious effects on pancreatic beta-cells. Our findings also suggest that TQ may be used as a potential therapeutic agent to normalize the dysregulated insulin production observed in HAART treated patients.
Experimental Biology and Medicine 03/2009; 234(4):442-53. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: N-(2-mercaptoethyl)1,3-diaminopropane (WR-1065), is the active metabolite of amifostine, a broad spectrum cytoprotective agent used in conjunction with both chemo- and radiotherapy of certain cancers. This report describes for the first time an oral formulation of WR-1065 and follows on from our earlier report of a similar oral formulation of amifostine.
The nanoparticles of WR-1065 were prepared by spray drying technique using poly lactide-co-glycolide (PLGA) as the polymer matrix. Radioprotection was determined by measuring reductions in radiation-induced: (i) 30-day survival; (ii) bone marrow suppression; and (iii) intestinal injury following 9 Gray (Gy) whole body gamma irradiation in mice. All treatments were given 1 hour pre-irradiation and WR-1065 was tested at the dose of 500 mg/kg.
The WR-1065/PLGA nanoparticles were smooth and spherical with the average diameter of 206 nm and contained 21.7% (w/w) WR-1065. While irradiation markedly reduced 30-day survival in non-treated control mice, and caused significant bone marrow suppression and intestinal injury in surviving mice, oral administration of WR-1065/PLGA nanoparticles resulted in significant radioprotection as evidenced by a marked reduction in all three of the above mentioned parameters of radiation injury.
These findings clearly demonstrate the feasibility of developing an effective oral formulation of WR-1065 as a radioprotective agent.
International Journal of Radiation Biology 12/2008; 84(11):900-8. · 1.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine how partial bladder outlet obstruction (PBOO) in a rat model affects erectile function, and whether an uroselective alpha1-adrenoceptor antagonist, alfuzosin (Sanofi-Aventis, Paris, France) attenuates any erectile dysfunction (ED).
Adult male Sprague-Dawley rats (120) were randomized into four groups: 1, sham-operated; 2, alfuzosin-treated; 3, PBOO; and 4, alfuzosin-treated with PBOO. Groups 3 and 4 were subjected to PBOO for 6 weeks by ligation of the urethra, while groups 2 and 4 rats received daily oral alfuzosin (10 mg/day) for 6 weeks. In vivo erectile responses were monitored by evaluating ratios of intracavernosal pressure (ICP)/mean arterial pressure, and total ICP (area under the curve). Organ-bath studies were performed on corpus cavernosum smooth muscle (CCSM) strips. Nitric oxide synthase (NOS) expression was determined immunohistochemically (IHC) for neuronal (n)NOS and by Western blot analysis for endothelial (e) and inducible (i) NOS protein.
Rats with PBOO showed lower erectile responses than controls. Maximum electrical field stimulation-mediated and endothelium-dependent acetylcholine-induced relaxations and contractile responses to phenylephrine were significantly reduced in CCSM strips from the PBOO group. The NO donor sodium nitroprusside completely relaxed CCSM from rats in all groups. IHC analyses showed decreased expression of nNOS in PBOO groups compared with controls; by contrast, protein expression of eNOS and iNOS was increased. Alfuzosin-treatment partially attenuated functional and molecular changes in penises of PBOO rats.
Rats with PBOO show ED, most likely due to altered NOS expression and NO bioavailability. The alpha-adrenoreceptor antagonist alfuzosin reversed this ED by altering sympathetic tone, increasing NO-induced relaxation and augmenting blood flow in the penis. This study suggests a rationale for further clinical trials using combinations of alpha-adrenoceptor antagonists and phosphodiesterase-5 inhibitors in patients with ED and lower urinary tract symptoms.
BJU International 11/2008; 102(11):1651-7. · 3.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bone morphogenetic proteins (BMPs) exert osteoinductive effects in prostate cancer (PC) via uncharacterized mechanisms. In this study, we investigated whether the nuclear transcription factor NF-kappaB, implicated in PC metastasis, is involved in transcriptional regulation and activation of BMP-2 or BMP-4/Smad signaling in PC cells.
NF-kappaB inhibition was achieved by IkappaBalpha super-repressor adenoviral vector and activation was monitored by EMSA and reporter assays. BMP expression and activation was measured by PCR and reporter assays. Promoter binding assay was performed by chromatin immunoprecipitation (ChIP) assay. Smad1/5/8 phosphorylation was measured by Western blot analysis.
PCR and chimeric BMP-2 and BMP-4 luciferase assays demonstrate that NF-kappaB confers robust and selective activation of BMP-2 in p65 overexpressing or rhTNF-alpha-stimulated PC cells. Inhibition of NF-kappaB significantly reduced transcript levels and autocrine production of BMP-2 by rhTNF-alpha stimulated C4-2B cells and to a lesser extent by the parental LNCaP cells. Selective inhibition of PI3K/Akt suppressed the NF-kappaB-induced BMP-2 promoter activity. Furthermore, suppression of NF-kappaB activation decreased the transcript levels and BMP-2-induced phosphorylation of Smad1/5/8, critical downstream targets of BMP-2 signaling in PC cells. Notably, the activation of BMPRII by BMP-2 is required for modulation of Smad activation by NF-kappaB in PC cells. Based on ChIP analysis, the transcriptional regulation of BMP-2 gene by NF-kappaB may be partially attributed to binding to kappab site on the BMP-2 promoter.
The data suggest that PI3K/Akt-NF-kappaB axis may promote PC bone metastasis in part by regulating transcription and activation of the BMP-2-Smad signaling cascade in osteotropic PC cells.
The Prostate 11/2008; 69(2):168-80. · 3.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cocaine remains the most frequently used illicit substance. Although cocaine-induced atherosclerosis is well documented, its mechanism of action on human vascular endothelial cells has not been determined. Nitric oxide (NO) and endothelin-1 (ET-1) are involved in endothelial cell activation and leukocyte recruitment. The present study monitored the effects of cocaine on NO and ET-1 production in human aortic endothelial cells (HAECs) and the effects of sodium nitroprusside (SNP) and BQ-123 on leukocyte adhesion to HAECs. Acute exposure to cocaine (1 and 3 muM) significantly increased ET-1 production (2-fold) and ET-1 receptor type-A (ET(A)R) protein expression, within 6-12 h. Cocaine exposure for a longer duration (24-72 h) showed a temporal decrease in both NO production and endothelial NO-synthase (eNOS) expression. The cocaine-mediated suppression of NO was ameliorated by co-treatment of cells with the ET(A)R blocker, BQ-123 (5 muM). Furthermore, both short-term (24 h) and long-term (72 h) exposure to cocaine increased endothelial adhesion of monocytes (U937 cells) by 20% and 40%, respectively, which were also suppressed by BQ-123 and SNP co-treatment. These data suggest that a concomitant increase in both ET-1 and ET(A)R expression in cocaine exposed HAECs may enhance signaling via the ET(A)R which decreases eNOS expression and NO production, and ultimately results in endothelial activation and leukocyte adhesion. Our findings implicate a molecular mechanism of action of cocaine and a therapeutic effect of ET(A)R-specific inhibitor in suppressing the cocaine-induced endothelial dysfunction.
[Show abstract][Hide abstract] ABSTRACT: We have measured the expression of T-type Ca2+ channel mRNA in breast cancer cell lines (MCF-7 (ERalpha+) using Western blot and quantitative real-time PCR (Q-RT-PCR). These results revealed that the MCF-7 cells express both alpha1G and alpha1H isoforms of T-type Ca2+ channels. In order to further clarify the role of T-type Ca2+ channels in proliferation, we tested the effects of a selective T-type Ca2+ channel inhibitor NNC-55-0396 on cellular proliferation. MCF-7 (ERalpha+) cellular proliferation was inhibited by the compound. In contrast, NNC-55-0396 at same concentration had no effect on the proliferation of MCF-10A cells, a non-cancer breast epithelial cell line. We also found that message expression of the T-type Ca2+ channels were only expressed in rapidly growing non-confluent cells but not in the cytostatic confluent cells. Knocking down the expression of T-type Ca2+ channels with siRNA targeting both alpha1G and alpha1H resulted in growth inhibition as much as 45%+/-5.0 in MCF-7 cells as compared to controls. In conclusion, our results suggest that T-type Ca2+ channel antagonism/silencing may reduce cellular proliferation in mitogenic breast cells.
Cancer Letters 09/2008; 267(1):116-24. · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the peripheral mechanisms of erectile dysfunction (ED) in a rat model of triple-binge cocaine administration.
Adult male Sprague-Dawley rats (n=24) were divided into two groups: group 1, control rats receiving vehicle (saline); group 2, rats receiving binge cocaine injections. After completion of triple-binge cocaine or saline injections, both groups underwent an in vivo, neurogenic-mediated erectile response protocol to assess intracavernosal pressure (ICP). Penile endothelin-A and -B receptors (ET(A)R and ET(B)R), plasma levels of big endothelin-1 (big-ET-1), and endothelial nitric oxide synthase (eNOS) protein expression were assessed. To analyze nitric oxide (NO) production, we measured plasma nitrate-nitrite levels and quantitated myeloperoxidase (MPO) activity in cavernosal tissues to determine reactive oxygen species generation. Endothelium-dependent and -independent relaxation responses were evaluated in vitro. Data were analyzed with Student t test.
Triple-binge cocaine administration caused significantly decreased erectile responses as measured by ICP in vivo. Plasma big-ET-1 levels were significantly increased in the triple-binge cocaine treatment group compared with control animals. In the penis, triple-binge cocaine administration significantly increased ET(A)R expression compared with saline controls, while ET(B)R expression was not altered. Cocaine-treated rats had significantly decreased eNOS expression and NO production. The activity of tissue MPO was significantly increased in the cocaine group compared with control rats. Organ bath studies demonstrated that triple-binge cocaine resulted in a 64% reduction in maximal relaxation compared with the control group.
This study demonstrates that triple-binge cocaine administration significantly reduces erectile function in rats. The pathophysiologic mechanisms that are likely involved include increased plasma big-ET-1 levels, increased penile ET(A)R expression, increased penile MPO activity, and reduced penile eNOS expression.
European Urology 09/2007; 52(2):555-63. · 10.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) Tat protein regulates transcription factor functions and alters cellular gene expression. Because hematopoietic progenitor cell (HPC) differentiation requires activation of lineage-specific transcription factors, Tat may affect hematopoiesis in HIV-1-infected micro-environments. We have monitored the molecular effects of Tat on megakaryocytic differentiation in the HPC line, K562. Flow cytometry analysis of CD61 indicated that phorbol myristate acetate (PMA) (16 nM) stimulated megakaryocytic commitment of K562 cells was increased (3- to 4-fold) following exposure to Tat (1-100 ng/ml). Activation of the megakaryocytic transcription factor cAMP regulatory element binding protein (CREB) and its coactivation by the CREB binding protein (CBP) was subsequently monitored. CREB phosphorylation and DNA binding were measured by Western immunodetection and electrophoretic mobility shift assays (EMSA), respectively. Within 2 hrs after stimulation, Tat increased both CREB phosphorylation and DNA binding by 7- to 10-fold. Transient cotransfection with CREB reporter and CBP expression plasmids demonstrated that Tat treatment increases (3- to 4-fold) both PMA-stimulated and CBP-mediated transcription via the cAMP regulatory element. Histone acetyl transferase (HAT) activity was increased (8- to 10-fold) in Tat-stimulated cells, which suggested increased chromosomal accessibility of transcription factors. Two-hybrid cotransfection assays using reporter plasmid containing the GAL4 DNA-binding domain and expression plasmid coding for the GAL4-CBP fusion protein, showed that Tat increases (2-fold) CBP-mediated coactivation of CREB. Both reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis showed that Tat treatment increases CBP gene expression (7- to 9-fold) and protein levels (5- to 7-fold) within 6-12 hrs after stimulation. Our findings indicated that Tat treatment increases both CREB function and CREB coactivation by CBP, which may facilitate megakaryocytic commitment of K562 cells. Induction of this molecular signaling by HIV-1 Tat protein may have relevance in understanding the HIV-induced hematologic manifestations and possibly in regulation of viral infectivity parameters in progenitor cell reservoirs.
Experimental Biology and Medicine 01/2006; 230(11):872-84. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The chemokine stromal-derived factor-1alpha (SDF-1alpha/CXCL-12) and its receptor, CXCR4, play a crucial role in adhesion and transendothelium migration (TEM) of prostate cancer cells. We tested the hypothesis that enhanced expression of CXCR4 in prostate cancer cells is dependent upon SDF-1alpha-mediated activation of nuclear factor-kappaB (NF-kappaB). SDF-1alpha increased the CXCR4 mRNA and protein expression in PC-3 cells but not in LNCaP cells. Similarly, SDF-1alpha enhanced the NF-kappaB-dependent transcriptional activity in PC-3 cells but not in LNCaP cells. SDF-1alpha increased PC-3 cell adhesion to the human umbilical vein endothelial cell monolayer and enhanced TEM, which was abrogated with anti-CXCR4 monoclonal antibody (mAb). Suppression of NF-kappaB activity in PC-3 cells by a mutant IkappaBalpha super-repressor adenoviral vector decreased the CXCR4 mRNA expression and inhibited adhesion and TEM. Transient overexpression of p65 subunit of NF-kappaB in PC-3 cells up-regulated CXCR4 receptor expression and increased the adhesion and TEM of these cells in response to SDF-1alpha gradient. Treatment of PC-3 cells with SDF-1alpha leads to nuclear translocation of NF-kappaB protein within 15 to 30 minutes, which correlated with IkappaBalpha phosphorylation. A p42/44 mitogen-activated protein kinase [MAPK, extracellular signal regulated kinase-1/2 (ERK-1/2)] biphasic activation pattern was observed in these cells at 15 minutes and 3 hours after SDF-1alpha treatment. Phosphorylation of IkappaB kinase alpha was observed within 30 minutes, which was blocked by PD98059 [MAPK kinase (MEK) inhibitor]. PD98059 cotreatment significantly inhibited SDF-1alpha-induced NF-kappaB reporter activity and CXCR4 receptor expression as shown by flow cytometry. These data suggest that SDF-1alpha-induced expression of CXCR4 in PC-3 cells is dependent on MEK/ERK signaling cascade and NF-kappaB activation.
Cancer Research 12/2005; 65(21):9891-8. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The hematopoietic compartments act as long-term reservoirs for human immunodeficiency virus type-1 (HIV-1). Although hematopoietic progenitor cells (HPCs) are rarely infectable, HPCs committed to the megakaryocytic lineage can be infected and support a productive infection by both the X4 and R5 strains of HIV-1. Indeed, in contrast to the CD34+ progenitors, the lineage-committed HPCs express high levels of the HIV-1 co-receptors, CXCR4 and CCR5. The HIV-1 transactivator (Tat) protein has been shown to alter co-receptor expression in T lymphocytes and macrophages. We hypothesized that Tat may regulate co-receptor expression in lineage-specific HPCs as well. We have monitored the effects of Tat protein on co-receptor expression and on lineage-specific differentiation, using the HPC cell line, K562. Butyric acid (BA)-induced erythroid differentiation in K562 cells was suppressed by 1-100 ng/ml of Tat, as evident from a 70-80% decrease in hemoglobin (Hb) production and a 10-30-fold decrease in glycophorin-A expression. However, Tat treatment enhanced phorbol myristate acetate (PMA)-induced megakaryocytic differentiation, as evident from a 180-210% increase in 3H-serotonin uptake and a 5-12-fold increase in CD61 expression. Tat did not significantly alter co-receptor expression in erythroid cells. However, Tat co-treatment profoundly effected both CXCR4 and CCR5 gene expression and protein levels in megakaryocytic cells. In PMA-stimulated cells, Tat increased CXCR4 and decreased in CCR5 expression, this was potentiated in cells chronically exposed to Tat. In conclusion, Tat protein suppresses erythroid and facilitates megakaryocytic differentiation of K562 cells. In megakaryocytic cells, Tat differentially effected CXCR4 and CCR5 expression. Because megakaryocytes may play a crucial role in HIV-1 infectivity in viral reservoirs, our findings implicate a role for Tat protein in dictating co-receptor usage in lineage-committed HPCs.
Experimental Biology and Medicine 11/2005; 230(9):631-44. · 2.80 Impact Factor