RESUMO
Pleiotrophin (PTN) stimulates endothelial cell migration through binding to receptor protein tyrosine phosphatase beta/zeta (RPTPß/ζ) and ανß3 integrin. Screening for proteins that interact with RPTPß/ζ and potentially regulate PTN signaling, through mass spectrometry analysis, identified cyclin-dependent kinase 5 (CDK5) activator p35 among the proteins displaying high sequence coverage. Interaction of p35 with the serine/threonine kinase CDK5 leads to CDK5 activation, known to be implicated in cell migration. Protein immunoprecipitation and proximity ligation assays verified p35-RPTPß/ζ interaction and revealed the molecular association of CDK5 and RPTPß/ζ. In endothelial cells, PTN activates CDK5 in an RPTPß/ζ- and phosphoinositide 3-kinase (PI3K)-dependent manner. On the other hand, c-Src, ανß3 and ERK1/2 do not mediate the PTN-induced CDK5 activation. Pharmacological and genetic inhibition of CDK5 abolished PTN-induced endothelial cell migration, suggesting that CDK5 mediates PTN stimulatory effect. A new pyrrolo[2,3-α]carbazole derivative previously identified as a CDK1 inhibitor, was found to suppress CDK5 activity and eliminate PTN stimulatory effect on cell migration, warranting its further evaluation as a new CDK5 inhibitor. Collectively, our data reveal that CDK5 is activated by PTN, in an RPTPß/ζ-dependent manner, regulates PTN-induced cell migration and is an attractive target for the inhibition of PTN pro-angiogenic properties.
Assuntos
Proteínas de Transporte/farmacologia , Movimento Celular/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/genética , Citocinas/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Carbazóis/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Quinase 5 Dependente de Ciclina/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Guanina/análogos & derivados , Guanina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/farmacologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ratos , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/metabolismo , Roscovitina/farmacologia , Transdução de SinaisRESUMO
We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage (Alakhras et al. Cancer Lett 2011; 306: 15-26). To further study retinoic acid clastogenicity and evaluate DNA damaging potential we investigated the ability of (a) all-trans retinoic acid and its steroidal analogue EA-4 to induce DNA fragmentation by using Comet assay under alkaline unwinding and neutral condition electrophoresis, and (b) the retinoids under study to induce small (0-1 kb) DNA fragments. Two cell lines, C2C12 mouse cells and HL-60 human leukemic cells were used in this study. We found that all-trans retinoic acid and its steroidal analogue EA-4 (a) provoke DNA migration due to DNA fragmentation as it is shown by the increased values of Comet parameters, and (b) induce significantly small-size fragmented genomic DNA as indicated by the quantification of necrotic/apoptotic small DNA segments in both cell systems. A different response between the two cell lines was observed in relation to retinoid ability to increase the percentage of DNA in the tail as well as break DNA in to small fragments. Our findings confirm the ability of retinoic acid to provoke micronucleation by disrupting DNA into fragments, among which small pieces of double-stranded DNA up to 1 kb are identified.
Assuntos
Antineoplásicos/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Tretinoína/análogos & derivados , Tretinoína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaio Cometa , Células HL-60 , Humanos , CamundongosRESUMO
Topoisomerase I is one of the most significant molecular targets through which indolocarbazoles inhibit tumour growth. In the present work, we studied the effect of new pyrrolo[2,3-α]carbazole derivatives on topoisomerase I activity in vitro, as well as on the viability of glioma and endothelial cells in vitro and on angiogenesis in vivo. All the tested compounds significantly decreased topoisomerase I activity in a concentration dependent manner, with the most effective being 1c, 1d(1), 1d(2) and 1f. The number of viable glioma and endothelial cells in vitro was also decreased in a concentration-dependent manner by all the tested compounds, although efficacy and potency differed in endothelial compared with glioma cells. Compounds 1c, 1d(1), 1e and 1f were the most effective in glioma cells, while compounds 1d(2) and 1e were the most effective in decreasing the number of viable endothelial cells. Finally, all the tested compounds inhibited angiogenesis in the chicken embryo chorioallantoic membrane in a significant and dose-dependent manner, with the most effective inhibitor being compound 1d(2). These data suggest that the tested pyrrolo[2,3-α]carbazole derivatives inhibit topoisomerase I activity and may be potentially useful for inhibition of glioma cell growth and angiogenesis.
