RESUMO
Despite an initial positive response, breast cancer cells inevitably acquire resistance to doxorubicin (Dox). Alpha-naphthoflavone (ANF) is a well-known chemopreventive agent; however, its anti-cancer properties have not been established. We examined the therapeutic efficacy of ANF in doxorubicin-resistant MCF-7 (MCF-7/Dox) breast cancer cells and investigated its underlying molecular mechanisms of action. MCF-7/Dox cells expressed constitutively active forms of the tyrosine kinases: focal adhesion kinase (FAK-Y397) and protein tyrosine kinase 2 beta (Pyk2- Y579/580) compared with parental MCF-7 cells. ANF significantly enhanced the sensitivity of MCF-7/Dox cells to Dox cytotoxicity in vitro and when co-administered in vivo. This ANF-mediated chemosensitization has dual mechanisms of action: (a) intracellular Dox retention via suppression of P-glycoprotein pump activity, and (b) inhibition of clonogenic cell survival via de-phosphorylation of FAK, Pyk2, and EGF-induced Akt in MCF-7/Dox cells and tumor xenografts. Because of its strong chemosensitization action, broad safety profile, and health benefits, ANF is an attractive anti-cancer drug with therapeutic implications to circumvent drug resistance in breast cancer patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzoflavonas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/farmacologia , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 2 de Adesão Focal/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Benzoflavonas/administração & dosagem , Neoplasias da Mama/enzimologia , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Quinase 1 de Adesão Focal/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
DNA de Cadeia Simples/química , RNA Interferente Pequeno/biossíntese , Sequência de Bases , Linhagem Celular Tumoral , Inativação Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Dados de Sequência Molecular , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Neoplasias da Próstata/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Benzoquinonas/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Perfilação da Expressão Gênica , Humanos , Concentração Inibidora 50 , Masculino , Nigella sativa/química , Transdução de SinaisRESUMO
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.
Assuntos
Proteína Morfogenética Óssea 2/fisiologia , Calcificação Fisiológica , NF-kappa B/fisiologia , Osteoblastos/fisiologia , Neoplasias da Próstata/patologia , Fator de Necrose Tumoral alfa/fisiologia , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Masculino , Metástase Neoplásica , Osteoblastos/citologia , Ligante RANK/genéticaRESUMO
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.
Assuntos
Benzoquinonas/farmacologia , Glucose/farmacologia , Inibidores da Protease de HIV/toxicidade , Insulina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Sulfato de Atazanavir , Linhagem Celular , Citosol/efeitos dos fármacos , Citosol/enzimologia , Glutationa/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/metabolismo , Canais Iônicos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Nelfinavir/toxicidade , Nigella sativa/química , Oligopeptídeos/toxicidade , Óleos de Plantas/farmacologia , Piridinas/toxicidade , Ratos , Espécies Reativas de Oxigênio/metabolismo , Saquinavir/toxicidade , Sementes/química , Superóxido Dismutase/metabolismo , Proteína Desacopladora 2RESUMO
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Assuntos
Proteína Morfogenética Óssea 2/fisiologia , NF-kappa B/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Smad/fisiologia , Transcrição Gênica , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 4/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Masculino , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Proteínas de Neoplasias/metabolismo , Plasmídeos , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: These findings clearly demonstrate the feasibility of developing an effective oral formulation of WR-1065 as a radioprotective agent.
Assuntos
Mercaptoetilaminas/administração & dosagem , Mercaptoetilaminas/farmacologia , Nanopartículas/química , Poliglactina 910/química , Protetores contra Radiação/administração & dosagem , Protetores contra Radiação/farmacologia , Administração Oral , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Jejuno/citologia , Jejuno/efeitos dos fármacos , Jejuno/efeitos da radiação , Masculino , Mercaptoetilaminas/química , Camundongos , Protetores contra Radiação/química , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/efeitos da radiação , Taxa de SobrevidaRESUMO
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.
Assuntos
Benzimidazóis/farmacologia , Neoplasias da Mama/metabolismo , Canais de Cálcio Tipo T/metabolismo , Proliferação de Células/efeitos dos fármacos , Mama/efeitos dos fármacos , Mama/metabolismo , Neoplasias da Mama/patologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Ciclopropanos , Humanos , Naftalenos , RNA Interferente Pequeno/farmacologiaRESUMO
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.
Assuntos
Proteína de Ligação a CREB/metabolismo , Diferenciação Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Produtos do Gene tat/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Megacariócitos/metabolismo , Carcinógenos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Produtos do Gene tat/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Integrina beta3/biossíntese , Células K562 , Fosforilação , Elementos de Resposta/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
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.
Assuntos
Quimiocinas CXC/farmacologia , Células Endoteliais/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , NF-kappa B/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores CXCR4/biossíntese , Neoplasias Ósseas/secundário , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Células Endoteliais/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , NF-kappa B/antagonistas & inibidores , Fosforilação , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Receptores CXCR4/genética , Proteínas Recombinantes/farmacologia , Fator de Transcrição RelA/biossíntese , Fator de Transcrição RelA/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
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.
Assuntos
Produtos do Gene tat/fisiologia , HIV-1/fisiologia , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Sequência de Bases , Ácido Butírico/farmacologia , Diferenciação Celular/fisiologia , Primers do DNA , Citometria de Fluxo , Glicoforinas/metabolismo , Humanos , Células K562 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serotonina/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Homocysteine (Hcy) is a metabolite of the essential amino acid methionine. Hyperhomocysteinemia is associated with vascular disease, particularly carotid stenosis. Rosiglitazone, a ligand of the peroxisome proliferator-activated receptor gamma , attenuates balloon catheter-induced carotid intimal hyperplasia in type 2 diabetic rats. We studied 4 groups (n = 7 per group) of adult female Sprague-Dawley rats fed (a) powdered laboratory chow (control), (b) control diet with rosiglitazone (3.0 mg/kg/d), (c) diet containing 1.0% l -methionine, and (d) diet containing methionine and rosiglitazone. After 1 week on high methionine diet, the rats were administered an aqueous preparation of rosiglitazone by oral gavage. One week after initiation of rosiglitazone, balloon catheter injury of the carotid artery was carried out using established methods, and the animals continued on their respective dietary and drug regimens for another 21 days. At the end of the experimental period, blood samples were collected, and carotid arteries and liver were harvested. Serum Hcy increased significantly on methionine diet compared with controls (28.9 +/- 3.2 vs 6.3 +/- 0.04 micromol/L). Development of intimal hyperplasia was 4-fold higher in methionine-fed rats; this augmentation was significantly reduced ( P < .018) in rosiglitazone-treated animals. Rosiglitazone treatment significantly ( P < .001) suppressed Hcy levels and increased the activity of the Hcy metabolizing enzyme, cystathionine-beta-synthase in the liver samples. Hcy (100 micromol/L) produced a 3-fold increase in proliferation of rat aortic vascular smooth muscle cells; this augmentation was inhibited by incorporating rosiglitazone (10 micromol/L). After balloon catheter injury to the carotid artery of animals on a high methionine diet, there was an increase in the rate of development of intimal hyperplasia consistent with the known effects of Hcy. It is demonstrated for the first time that the peroxisome proliferator-activated receptor gamma agonist rosiglitazone can attenuate the Hcy-stimulated increase in the rate of development of intimal hyperplasia indirectly by increasing the rate of catabolism of Hcy by cystathionine-beta-synthase and directly by inhibiting vascular smooth muscle cell proliferation. These findings may have important implications for the prevention of cardiovascular disease and events in patients with hyperhomocysteinemia (HHcy).
Assuntos
Cateterismo/efeitos adversos , Homocisteína/sangue , Metionina/administração & dosagem , Músculo Liso Vascular/patologia , Tiazolidinedionas/farmacologia , Túnica Íntima/patologia , Animais , Artérias Carótidas , Divisão Celular/efeitos dos fármacos , Cistationina beta-Sintase/metabolismo , DNA/biossíntese , Dieta , Feminino , Homocisteína/antagonistas & inibidores , Hiperplasia , Ligantes , Fígado/enzimologia , Metionina/farmacologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , PPAR gama/metabolismo , Ratos , Ratos Sprague-Dawley , Rosiglitazona , Tiazolidinedionas/administração & dosagem , Tiazolidinedionas/metabolismo , Túnica Íntima/efeitos dos fármacosRESUMO
Highly active antiretroviral therapy (HAART) has significantly improved the prognosis of HIV-1-infected patients but is associated with significant side effects such as diabetes, atherosclerosis, and cardiovascular complications. Oxidative stress can disrupt endothelial homeostasis by dysregulating the balance between pro- and antiatherogenic factors. We hypothesized that chronic exposure to HAART results in endothelial oxidative stress and activation of mononuclear cell recruitment, an early event in atherosclerosis. We studied the effects of HAART drug combinations, consisting of zidovudine, a nucleoside reverse transcriptase inhibitor; efavirenz, a nonnucleoside reverse transcriptase inhibitor; and either of the two protease inhibitors (PIs), indinavir or nelfinavir, on human aortic endothelial cells (HAECs) by monitoring the following parameters: (1) generation of reactive oxygen species (ROS), (2) mono-nuclear cell (Jurkat or U-937) adhesion, and (3) expression of cell adhesion molecules (CAMs). HAART exposure increased ROS formation in HAECs. Exposure to PIs alone and in HAART combinations increased mononuclear cell adhesion to HAECs in a concentration-dependent manner. Mononuclear cell adhesion to HAART-exposed HAECs was significantly enhanced following acute (24-h) exposure to the inflammatory cytokines, tumor necrosis factor (TNF)-alpha or interleukin (IL)-1beta and was suppressed by the antioxidants N-ace-tylcysteine and glutathione. Exposure to HAART increased intercellular adhesion molecule-1 (ICAM-1) gene expression and concomitant exposure to TNF-alpha further increased ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and endothelial-leukocyte adhesion molecule cell surface protein levels. These studies indicate that chronic HAART exposure increases oxidative stress in endothelial cells and induces mononuclear cell recruitment, which may eventually precipitate the cardiovascular diseases observed in HIV-1+ individuals on antiretroviral therapy.
Assuntos
Antioxidantes/farmacologia , Terapia Antirretroviral de Alta Atividade/efeitos adversos , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Estresse Oxidativo , Acetilcisteína/farmacologia , Alcinos , Benzoxazinas , Adesão Celular , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Células Cultivadas , Ciclopropanos , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Endotélio Vascular/metabolismo , Expressão Gênica/efeitos dos fármacos , Glutationa/farmacologia , Humanos , Indinavir/administração & dosagem , Indinavir/efeitos adversos , Interleucina-1/farmacologia , Leucócitos Mononucleares/fisiologia , Nelfinavir/administração & dosagem , Nelfinavir/efeitos adversos , Oxazinas/administração & dosagem , Oxazinas/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Zidovudina/administração & dosagem , Zidovudina/efeitos adversosRESUMO
Both erythromycin and clarithromycin have been reported to cause QT prolongation and the cardiac arrhythmia torsade de pointes in humans, however direct evidence documenting that these drugs produce this effect by blocking human cardiac ion channels is lacking. The goal of this study was to test the hypothesis that these macrolide antibiotics significantly block the delayed rectifier current (IKr) encoded by HERG (the human ether-a-go-go-related gene) at drug concentrations, temperature and ionic conditions mimicking those occurring in human subjects. Potassium currents in HEK 293 cells stably transfected with HERG were recorded using a whole cell voltage clamp method. Exposure of cells to erythromycin reduced the HERG encoded potassium current in a concentration dependent manner with an IC50 of 38.9 +/- 1.2 microM and Hill Slope factor of 0.4 +/- 0.1. Clarithromycin produced a similar concentration-dependent block with an IC50 of 45.7 +/- 1.1 microM and Hill Slope factor of 1.0 +/- 0.1. Erythromycin (25-250 microM) and clarithromycin (5 or 25 microM) also produced a significant decrease in the integral of the current evoked by an action potential shaped voltage clamp protocol. The results of this study document that both erythromycin and clarithromycin significantly inhibit the HERG potassium current at clinically relevant concentrations.
Assuntos
Antibacterianos/farmacologia , Claritromicina/farmacologia , Eritromicina/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Potenciais de Ação , Linhagem Celular , Relação Dose-Resposta a Droga , Canal de Potássio ERG1 , Eletrofisiologia , Canais de Potássio Éter-A-Go-Go , Humanos , Concentração Inibidora 50 , Macrolídeos/farmacologia , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Temperatura , TransfecçãoRESUMO
Recent evidence has demonstrated an appreciable expression of metallothionein (MT) in erythrocytes. However, the induction of the MT protein by hematopoietic growth factors and its subsequent functional significance on clonal expansion or differentiation of erythroid progenitor cells remain elusive. We therefore examined the effects of growth factors erythropoietin (EPO), granulocyte-monocyte colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) on MT gene expression in erythroid progenitor K562 cell line. EPO, but not IL-3 or GM-CSF, induced a 3-fold increase in MT transcripts in K562 cells. MT induction was associated with EPO-induced cellular proliferation, suggesting a specific role for MT induction by EPO in erythroid progenitor cells. However, EPO- or sodium butyrate-induced differentiation as monitored by hemoglobin formation was inhibited in K562 cells stably transfected with an expression vector containing human MT-IIA gene. This inhibition of differentiation was partially reversed in these cells by an MT antisense phosphorothioate oligonucleotide. Furthermore, the MT-induced inhibition of differentiation was associated with downregulation of EPO receptor transcripts in K562 cells. These data suggest that, among growth factors required for erythropoiesis, EPO is a potent inducer of MT, and that MT may plays a significant role in EPO-induced proliferation, but not in the erythroid-specific differentiation of the progenitor cells.
Assuntos
Eritropoetina/farmacologia , Regulação da Expressão Gênica , Metalotioneína/biossíntese , Metalotioneína/genética , Butiratos/farmacologia , Diferenciação Celular , Divisão Celular , Relação Dose-Resposta a Droga , Regulação para Baixo , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Substâncias de Crescimento/metabolismo , Humanos , Imuno-Histoquímica , Interleucina-3/metabolismo , Células K562 , Oligonucleotídeos Antissenso/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células-Tronco/citologia , TransfecçãoRESUMO
Therapeutic resistance remains an unresolved problem in the clinical management of human prostate cancer (PC). Despite initial positive response to androgen ablation therapy (AAT), virtually all PC patients will relapse due to acquisition of hormone refractory disease and selective outgrowth of tumor cells with multidrug resistance phenotype. We here provide the first experimental evidence that restoring a functional androgen receptor (AR) in the androgen-independent prostate cancer PC3 cells enhances their sensitivity to growth arrest and suppresses their colony-forming ability in response to paclitaxel and gamma-irradiation. Furthermore, functional AR increases the susceptibility of these cells to the apoptotic potentials of therapeutic agents, as evidenced by an increase in caspase activity, annexin V binding, and internucleosomal DNA fragmentation, by inducing caspase activation. The abrogation of the cytotoxic effects by 4-hydroxyflutamide suggests a crucial role for AR activation in enhancing the therapeutic sensitivity of these cells in a ligand-independent fashion. Our data thus demonstrate that a functional AR is a prerequisite for effective therapeutic response and that aberrant expression or blockade by AAT may trigger pathways leading to emergence of PC cells with therapeutic resistance phenotype. Since the mainstay of primary therapy for PC has been AAT by pharmaco-therapeutic or surgical means, this study thus provides a new frontier for revising the AAT therapeutic strategy in conjunction with radiation and/or chemotherapeutic agents.
Assuntos
Androgênios/fisiologia , Antineoplásicos/farmacologia , Caspases/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/fisiologia , Antineoplásicos/uso terapêutico , Sequência de Bases , Linhagem Celular Tumoral , Fragmentação do DNA , Primers do DNA , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológicoRESUMO
Acquirement of multi-drug resistance by tumor cells represents a major obstacle in the management of prostate cancer. Such resistance was demonstrated in the androgen-independent DU-145 cells in response to paclitaxel and the mechanisms by which these cell develops resistance was not understood. The objective of this study was to examine whether abrogation of the constitutively active NF-kappaB in the chemoresistant, androgen independent DU-145 prostate cancer cells will enhance their sensitivity to cytototoxic agents. Inhibition of NF-kappaB by a dominant negative super-repressor IkappaB mutant adenoviral construct enhanced the apoptotic potentials of paclitaxel and rhTNF-alpha in these cells. Using reporter assays and RT-PCR analysis, we demonstrate that paclitaxel-induced cell death was associated with an increase in NF-kappaB activation and MDR-1 gene expression. Abrogation of these effects by the dominant negative IkappaB adenoviral construct suggests that induction and/or constitutive activation of NF-kappaB can block the paclitaxel-induced apoptotic signaling pathways in this cell line, possibly by increasing the expression of anti-apoptotic and MDR-1 gene products, leading to development of chemoresistance in these cells. We conclude that inhibition of NF-kappaB activation may have therapeutic implications for prostate cancer.
Assuntos
Adenoviridae/genética , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Resistencia a Medicamentos Antineoplásicos , NF-kappa B/metabolismo , Paclitaxel/farmacologia , Neoplasias da Próstata/patologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Divisão Celular , Cloranfenicol O-Acetiltransferase/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Neoplásica da Expressão Gênica , Genes Dominantes , Humanos , Proteínas I-kappa B/farmacologia , Masculino , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Bone marrow abnormalities are frequently observed in HIV-1-infected individuals. Infection of marrow mesenchymal stem cells (MSCs) may abrogate their growth properties and hematopoietic supportive functions. To delineate the cell type infected, and factors responsible for the deleterious effects, human bone marrow cells were exposed to HIV-1 in vitro. By week 4, the ability of MSCs to form colonies of purely fibroblasts (CFU-F) and mixed colonies of fibroblasts and adipocytes (CFU-FA) was suppressed by 23 +/- 5 and 55 +/- 7%, respectively. The p24 concentration in culture supernatants steadily declined from 170 ng/ml in the inoculum to 134 +/- 30, 35 +/- 15, 2.3 +/- 3, and <0.02 ng/ml at the end of week 1, 2, 3, and 4, respectively. However, even at week 4, coculturing with MT-4 lymphocytes for 1 week dramatically increased p24 levels. Polymerase chain reaction (PCR) amplification, using HIV-1-specific primers, and in situ hybridization with an HIV-1 cDNA probe demonstrated the presence of virus-specific nucleic acids within stromal colonies. Coimmunostaining with antibody to CD83 implicated the presence of HIV-1 within dendritic progenitor cells. Immunostaining with HIV-1 Tat antibody demonstrated the presence of Tat protein and reverse transcriptase (RT)-PCR assays showed increased (160-220%) mRNA levels for inflammatory cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 1beta [IL-1beta], IL-6, and macrophage inflammatory protein 1alpha [MIP-1alpha]). A concentration-dependent decrease in CFU-STROs was observed on incubation with either Tat protein (1-100 ng/ml) or with TNF-alpha or IL-1beta (0.025-25 ng/ml). These results suggest that HIV-1 infection of stromal cells may produce inhibitory factors that suppress the clonogenic potential of MSCs.
Assuntos
Células da Medula Óssea/patologia , Citocinas/metabolismo , Produtos do Gene tat/metabolismo , HIV-1/patogenicidade , Células-Tronco Hematopoéticas/patologia , Células da Medula Óssea/fisiologia , Células da Medula Óssea/virologia , Diferenciação Celular , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Hematopoéticas/virologia , Humanos , Inflamação , Células Estromais/patologia , Células Estromais/virologia , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
In vitro exposure of murine bone marrow cells to increasing concentrations of zidovudine (AZT, 0.1-50 microM) had a concentration dependent suppressive effect on the growth of granulocyte-monocyte colony forming unit (CFU-GM) derived colonies. In our previous published study, the mechanism of AZT-induced suppression of erythroid colony forming unit (CFU-E) derived colonies was linked to a decrease in erythropoitin receptor (Epo-R) gene expression. In this study, we have observed that AZT exposure also induced a concentration dependent suppressive effect (35-90%) on GM-CSF receptor type alpha (GM-CSFR alpha) gene expression. The suppression of GM-CSFR alpha mRNA expression was specific, since AZT caused a much lower decrease (15-22%) on the IL-3 receptor type alpha (IL-3R alpha) message level, and had an insignificant effect on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and c-myc message levels. Erythropoietin (Epo) therapy has been used for reversal of AZT induced erythroid toxicity. Exposure to increasing concentrations (10-500 U/ml) of GM-CSF was unable to override the suppressive effect of AZT on CFU-GM derived colonies, however, treatment in combination with IL-3 (10-250 U/ml) ameliorated the suppressive effects of AZT on CFU-GM and on GM-CSFR alpha and IL-3R alpha gene expression. These findings suggest a mechanism via which AZT may suppress granulocyte-monocyte specific differentiation in murine bone marrow cells. These data also suggest that a combination of GM-CSF and IL-3 may be a superior therapeutic intervention for AZT-induced neutropenia.