RESUMEN
Arsenic trioxide appears to be effective in the treatment of pro-myelocytic leukaemia. The substituted phenylarsen(III)oxides are highly polar, they have a high tendency to undergo oxidation to As (V) and to form oligomers, to prevent this we protected the As-(OH)(2) group as cyclic dithiaarsanes. To increase the compound's biological stability and passive diffusion we conjugated the compound of interest with lipoamino acids (Laas). Alternatively, we further conjugated the dithiaarsane derivative with a carbohydrate to utilize active transport systems and to target compound. We investigated two novel glyco-lipid arsenicals (III) (compounds 9 and 11) for their ability to initiate MCF-7 breast cancer cell death and characterized the mechanism by which death was initiated. A significant decrease in MCF-7 cell proliferation was observed using 1 microM and 10 microM compound (11) and 10 microM of compound (9). Treatment with compound (11) triggered apoptosis of MFC-7 cells while compound (9) induced inhibition of cellular proliferation was not via rapid induction of apoptosis and more likely reflected necrosis and/or alterations in the cell cycle. Differences in the anti-proliferative potency of the two compounds indicate that structural modifications influence effectiveness.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias de la Mama/patología , Cationes , Muerte Celular/efectos de los fármacos , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Glucolípidos/química , Glucolípidos/farmacología , Humanos , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The phthalates di(2-ethylhexyl)phthalate (DEHP) and di-n-butyl phthalate (DBP) are environmental contaminants with significant human exposures. Both compounds are known reproductive toxins in rodents and DEHP also induces rodent hepatocarcinogenesis in a process believed to be mediated via the peroxisome proliferator-activated receptor alpha (PPARalpha). DEHP and DBP are metabolised to their respective monoesters, mono-(2-ethylhexyl)phthalate (MEHP) and mono-n-butyl phthalate (MBP), which are the active metabolites. MEHP also activates another member of the PPAR subfamily, PPARgamma. The effects of PPARalpha and PPARgamma activation in human breast cells appears to be opposing; PPARalpha activators in breast cells cause an increase in proliferation, while PPARgamma activation in breast cells is associated with differentiation and an inhibition of cell proliferation. Further to this the activation of the PPARs is cell and ligand specific, suggesting the importance of examining the effect of MEHP and MBP on the activation of PPARalpha, PPARbeta and PPARgamma in human breast. We used the common model of human breast cancer MCF-7 and examined the ability of MEHP and MBP to activate human PPARs in this system. The ability of MBP and MEHP to block PPAR responses was also assessed. We found that both human PPARalpha and PPARgamma were activated by MEHP whereas MEHP could not activate PPARbeta. MBP was unable to activate any PPAR isoforms in this breast model, despite being a weak peroxisome proliferator in liver, although MBP was an antagonist for both PPARgamma and PPARbeta. Our results suggest that the toxicological consequences of MEHP in the breast could be complex given the opposing effects of PPARalpha and PPARgamma in human breast cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Dietilhexil Ftalato/análogos & derivados , PPAR alfa/metabolismo , PPAR gamma/metabolismo , Ácidos Ftálicos/farmacología , Plastificantes/farmacología , Neoplasias de la Mama/genética , Butiratos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dietilhexil Ftalato/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , PPAR alfa/genética , PPAR gamma/genética , PPAR-beta/genética , PPAR-beta/metabolismo , Compuestos de Fenilurea/farmacología , Estadísticas no Paramétricas , Tiazoles/farmacología , Tiazolidinedionas/farmacología , TransfecciónRESUMEN
Peroxisome proliferator-activated receptors are ligand-activated transcription factors with a potential role in cancer. We investigated peroxisome proliferator-activated receptor alpha expression in breast cancer cell lines and showed a relationship between mean peroxisome proliferator-activated receptor alpha and estrogen receptor alpha mRNA levels in estrogen receptor alpha positive breast cancer cells. Transfection of estrogen receptor alpha into the estrogen receptor alpha negative cell line, MDA-MB-231 decreased peroxisome proliferator-activated receptor alpha mRNA and conversely inhibition of estrogen receptor alpha by ICI-182 780 in estrogen receptor alpha positive, MCF-7 cells increased peroxisome proliferator-activated receptor alpha mRNA levels. Estrogen receptor alpha levels can be modulated by histone deacetylase inhibitors and such agents are in clinical trials for cancer treatment. We found the histone deacetylase inhibitor, sodium butyrate, increased peroxisome proliferator-activated receptor alpha mRNA levels within 4h of treatment. Peroxisome proliferator-activated receptor alpha modulation was independent of estrogen receptor alpha, as a similar increase was observed in the estrogen receptor alpha negative MDA-MB-231 cells. To further investigate the relationship between sodium butyrate and peroxisome proliferator-activated receptor alpha expression, we created an MCF-7 cell line that conditionally over-expresses human peroxisome proliferator-activated receptor alpha. Over-expression of the peroxisome proliferator-activated receptor protected MCF-7 cells from sodium butyrate-mediated inhibition of proliferation and attenuated sodium butyrate-mediated induction of histone deacetylase 3 mRNA, indicating that elevated levels of peroxisome proliferator-activated receptor alpha may reduce the sensitivity of cells to histone deacetylase inhibitors. The estrogen receptor alpha dependence of peroxisome proliferator-activated receptor alpha levels may be significant since estrogen receptor alpha negative breast cancer cells are associated with a more aggressive phenotype. Our studies also suggest that peroxisome proliferator-activated receptor alpha levels may be a marker of breast cancer cell sensitivity to histone deacetylase inhibitors.
Asunto(s)
Butiratos/farmacología , Receptor alfa de Estrógeno/metabolismo , PPAR alfa/metabolismo , Línea Celular Tumoral , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , PPAR alfa/genéticaRESUMEN
We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-1R rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK1/2 phosphorylation. Using toll-like receptor (tlr)9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA microarrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B (itm2b), receptor activity-modifying protein 2 (ramp2) and macrophage-specific gene 1 (mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Animales , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/inmunología , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Activación de Macrófagos/inmunología , Factor Estimulante de Colonias de Macrófagos/inmunología , Macrófagos/inmunología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/análisis , Receptor de Factor Estimulante de Colonias de Macrófagos/efectos de los fármacos , Receptor de Factor Estimulante de Colonias de Macrófagos/inmunología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 9RESUMEN
Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anti-cancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.
Asunto(s)
Neoplasias de la Mama/patología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/fisiología , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/fisiología , ADN sin Sentido/farmacología , Señalización del Calcio , ATPasas Transportadoras de Calcio/análisis , Proteínas de Transporte de Catión/análisis , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , ATPasas Transportadoras de Calcio de la Membrana Plasmática , ATPasas Transportadoras de Calcio del Retículo SarcoplásmicoRESUMEN
The ligand-activated transcription factor peroxisome proliferator-activated receptor beta (PPARbeta) is present in the brain and is implicated in the regulation of genes with potential roles in neurotoxicity. We sought to examine the role of PPARbeta in neuronal cell death by using the PPARbeta ligand GW0742. Primary cultures of rat cerebellar granule neurons were prepared from 7-day-old pups. Reverse transcriptase-polymerase chain reaction and in situ hybridization were used to verify that PPARbeta mRNA was present in neurons. After 10-12 days in culture, the neuronal cells were incubated in the presence of GW0742, and cell death was measured with a lactate dehydrogenase release (LDH) assay. After 24 hr of exposure, PPARbeta activation by GW0742 was not inherently toxic to cerebellar granule neurons. However, toxicity was observed after 48 hr, with cell death mediated via an apoptotic mechanism. In an effect opposite to that observed with PPARalpha-activating ligands, PPARbeta activation exhibited neuroprotective properties. Treatment with GW0742 significantly reduced cell death during a 12-hr exposure to low-KCl media. These results clearly reinforce very specific roles for the PPAR isoforms in neurons and suggest that PPARbeta is worthy of further investigation regarding its potential role as a therapeutic target in neurodegenerative states.
Asunto(s)
Muerte Celular/fisiología , Degeneración Nerviosa/metabolismo , Neuronas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Tiazoles/farmacología , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , L-Lactato Deshidrogenasa/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Degeneración Nerviosa/inducido químicamente , Neuronas/efectos de los fármacos , Neurotoxinas/antagonistas & inhibidores , Neurotoxinas/metabolismo , Deficiencia de Potasio/metabolismo , Proteínas Proto-Oncogénicas c-jun/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Pirimidinas/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/agonistas , Factores de Transcripción/genéticaRESUMEN
Transporters of Ca2+ are potential drug targets and Ca2+ is a useful signal in the assessment of G-protein-coupled receptor activation. Assays involving the assessment of intracellular Ca2+ using microplate readers most often use Ca2+ indicators which do not exhibit a spectra shift on Ca2+ binding (e.g. fluo-3). Indicators that do exhibit a spectral shift upon Ca2+ binding (e.g. fura-2) offer potential advantages for the calibration of intracellular Ca2+ levels. However, experimental limitations may limit the use of ratiometric dyes in microplate readers capable of screening. In this study, we compared the assessment of intracellular Ca2+ in adherent breast cancer cells using ratiometric and nonratiometric Ca2+ indicators. Our results demonstrate that both fluo-3 and fura-2 detect ATP dose-dependent increases in intracellular Ca2+ in the MCF-7 breast cancer cell line and that some of the limitations in the use of fura-2 appear to be overcome by the use of glass bottom microplates. The calibrated intracellular Ca2+ levels derived using fura-2 are consistent with those from microscopy and cuvette-based studies. Fura-2 may be useful in microplate studies, where cell lines with different properties are compared or where screening treatments lead to differences in the number of cells or dye loading.
Asunto(s)
Neoplasias de la Mama/química , Calcio/análisis , Adenosina Trifosfato/metabolismo , Compuestos de Anilina , Neoplasias de la Mama/metabolismo , Calcio/metabolismo , Línea Celular , Femenino , Colorantes Fluorescentes , Fura-2 , Humanos , Líquido Intracelular/química , XantenosRESUMEN
Peroxisome proliferator-activated receptor gamma (PPARgamma) has been the focus of studies assessing its potential neuroprotective role. These studies have shown either neuroprotection or neurotoxicity by PPARgamma ligands. Comparison of these studies is complicated by the use of different PPARgamma ligands, mechanisms of neurotoxicity induction, and neuronal cell type. In this study, we compared the effects of the synthetic PPARgamma ligand ciglitazone with an endogenous PPARgamma ligand, 15-deoxy-delta(12,14)-prostaglandin J(2) (15-deoxy PGJ(2)), on inherent neurotoxicity and neuroprotection using a reduction in extracellular KCl in rat cultured cerebellar granule neurons (CGN). We also assessed the effects of these ligands on c-Jun protein expression, which is up-regulated on induction of low-KCl-mediated neuronal apoptosis as well as being associated with PPAR in other cell types. We showed that PPARgamma mRNA is expressed in CGN cultures and observed ciglitazone- and 15-deoxy PGJ(2)-mediated inherent neurotoxicity that was concentration and time dependent. c-Jun was only modestly increased in the presence of ciglitazone but was markedly up-regulated by 15-deoxy PGJ(2) after 12 hr. Treatment of CGN cultures with ciglitazone simultaneous with KCl withdrawal resulted in a modest, time-dependent neuroprotection. Such neuroprotection after KCl withdrawal was not observed with 15-deoxy PGJ(2). Despite the absence of neuroprotection, 15-deoxy PGJ(2) markedly inhibited the early up-regulation of c-Jun during KCl withdrawal. These studies suggest that ciglitazone and 15-deoxy PGJ(2) have markedly different effects on inherent and low-KCl-induced toxicity and c-Jun expression in CGN, indicating potential non-PPARgamma mechanisms.
Asunto(s)
Cerebelo/efectos de los fármacos , Neuronas/efectos de los fármacos , Prostaglandina D2/farmacología , Receptores Citoplasmáticos y Nucleares/biosíntesis , Tiazoles/farmacología , Tiazolidinedionas , Factores de Transcripción/biosíntesis , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Cerebelo/citología , Cerebelo/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Ligandos , Neuronas/citología , Neuronas/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina D2/toxicidad , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/agonistas , Tiazoles/toxicidad , Factores de Transcripción/agonistasRESUMEN
Peroxisome proliferator-activated receptor (PPAR) alpha is a ligand-activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARalpha mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARalpha mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARalpha is expressed and dynamically regulated in human breast cancer MCF-7 and MDA-MB-231 cells. Having established the presence of PPARalpha in both cell types, we then examined the consequence of PPARalpha activation, by its ligands Wy-14,643 and clofibrate, on proliferation. With real-time reverse transcriptase-polymerase chain reaction, we showed that PPARalpha mRNA was dynamically regulated in MDA-MB-231 cells and that PPARalpha activation significantly increased proliferation of the cell line. In contrast, PPARalpha expression in MCF-7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA-MB-231 cells. However, PPARalpha ligand activation still significantly increased the proliferation of MCF-7 cells. The promotion of proliferation in breast cancer cell lines following PPARalpha activation was in stark contrast to the effects of PPARgamma-activating ligands that decrease proliferation in human breast cancer cells. Our results established the presence of PPARalpha in human breast cancer cell lines and showed for the first time that activation of PPARalpha in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis.