RESUMEN
PURPOSE: The Bcl-2 mediated resistance is one of the most critical obstacle in cancer therapy. Conventional chemotherapeutics such as Paclitaxel, a commonly used in the treatment of metastatic breast cancer, is not sufficient to overcome Bcl-2 mediated drug resistance mechanism. Thus, combinational drug regimes are favored by researchers to overcome resistance phenotype against drugs. N1,N11-diethylnorspermine (DENSpm), a polyamine analogue, which is a promising drug candidate induced-cell cycle arrest and apoptosis in various cancer cells such as prostate, melanoma, colon and breast cancer cells via activated polyamine catabolism and reactive oxygen generation. Recent studies indicated the potential therapeutic role of DENSpm in phase I and II trials in breast cancer cases. Although the molecular targets of Paclitaxel in apoptotic cell death mechanism is well documented, the therapeutic effect of DENSpm and Paclitaxel in breast cancer cells has not been investigated yet. In this study, our aim was to determine the time dependent effect of DENSpm and Paclitaxel on apoptotic cell death via determination of polyamine metabolism related targets in wt and Bcl-2 overexpressing MCF-7 breast cancer cells. RESULTS: In our experimental study, Paclitaxel decreased cell viability in dose-dependent manner within 24h. Co-treatment of Paclitaxel (30nM) with DENSpm (20µM) further increased the cytoxicity of Paclitaxel (30nM) compared to alone Paclitaxel (30nM) treatment in MCF-7 Bcl-2+ breast cancer cells. In addition, we determined that resistance against Paclitaxel-induced apoptotic cell death in Bcl-2 overexpressed MCF-7 cells was overcome due to activation of polyamine catabolic pathway, which caused depletion of polyamines. CONCLUSIONS: DENSpm combinational treatment might increase the effect of low cytotoxic paclitaxel in drug-resistant breast cancer cases.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias de la Mama/metabolismo , Paclitaxel/administración & dosificación , Poliaminas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Espermina/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Femenino , Humanos , Células MCF-7 , Espermina/administración & dosificaciónRESUMEN
Ca2+ influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor triggers activation and postsynaptic accumulation of Ca2+/calmodulin-dependent kinase II (CaMKII). CaMKII, calmodulin, and alpha-actinin directly bind to the short membrane proximal C0 domain of the C-terminal region of the NMDA receptor NR1 subunit. In a negative feedback loop, calmodulin mediates Ca2+-dependent inactivation of the NMDA receptor by displacing alpha-actinin from NR1 C0 upon Ca2+ influx. We show that Ca2+-depleted calmodulin and alpha-actinin simultaneously bind to NR1 C0. Upon addition of Ca2+, calmodulin dislodges alpha-actinin. Either the N- or C-terminal half of calmodulin is sufficient for Ca2+-induced displacement of alpha-actinin. Whereas alpha-actinin directly antagonizes CaMKII binding to NR1 C0, the addition of Ca2+/calmodulin shifts binding of NR1 C0 toward CaMKII by displacing alpha-actinin. Displacement of alpha-actinin results in the simultaneous binding of calmodulin and CaMKII to NR1 C0. Our results reveal an intricate mechanism whereby Ca2+ functions to govern the complex interactions between the two most prevalent signaling molecules in synaptic plasticity, the NMDA receptor and CaMKII.
Asunto(s)
Actinina/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Sitios de Unión , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Humanos , Estructura Terciaria de Proteína , Ratas , Receptores de N-Metil-D-Aspartato/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
Calmodulin (CaM) is the major Ca2+ sensor in eukaryotic cells. It consists of four EF-hand Ca2+ binding motifs, two in its N-terminal domain and two in its C-terminal domain. Through a negative feedback loop, CaM inhibits Ca2+ influx through N-methyl-D-aspartate-type glutamate receptors in neurons by binding to the C0 region in the cytosolic tail of the NR1 subunit. Ca2+ -depleted (apo)CaM is pre-associated with a variety of ion channels for fast and effective regulation of channel activities upon Ca2+ influx. Using the NR1 C0 region for fluorescence and circular dichroism spectroscopy studies we found that not only Ca2+ -saturated CaM but also apoCaM bound to NR1 C0. In vitro interaction assays showed that apoCaM also binds specifically to full-length NR1 solubilized from rat brain and to the complete C terminus of the NR1 splice form that contains the C0 plus C2' domain. The Ca2+ -independent interaction of CaM was also observed with the isolated C-but not N-terminal fragment of calmodulin in the independent spectroscopic assays. Fluorescence polarization studies indicated that apoCaM associated via its C-terminal domain with NR1 C0 in an extended conformation and collapsed to adopt a more compact conformation of faster rotational mobility in its complex with NR1 C0 upon addition of Ca2+. Our results indicate that apoCaM is associated with NR1 and that the complex of CaM bound to NR1 C0 undergoes a dramatic conformational change when Ca2+ binds to CaM.