RESUMO
BACKGROUND AND AIMS: KITENIN was previously reported to promote metastasis in mouse colon tumour models; however, the signalling mechanism of KITENIN at the cellular level was unknown. Here the functional role of KITENIN with respect to colorectal cancer (CRC) cell invasion and its expression in CRC tissues were investigated. METHODS: The effect of KITENIN on cell motility was analysed in a migration and invasion assay upon its overexpression and knockdown. Immunoprecipitation was used to elucidate binding partners, and immunohistochemistry was used to study expression levels. RESULTS: KITENIN overexpression enhanced the migration of rat intestinal epithelial cells, whereas a loss of invasiveness was observed in CRC cells after KITENIN knockdown. Mechanically, KITENIN served as a scaffolding molecule that simultaneously recruited both Dishevelled (Dvl) and protein kinase C delta (PKC delta) through the membrane-spanning C-terminal region to form a complex that stimulated extracellular signal-regulated kinase (ERK)/activating protein-1 (AP-1) via a PKC delta component but also organised the actin filament via a Dvl component. The KITENIN complex controlled the invasiveness of CRC cells aetiologically harbouring various mutations in APC, beta-catenin or K-ras, in which AP-1 activation is redundant but the organisation of the actin filament is indispensable for cell motility. Clinically, KITENIN expression was significantly higher in colon cancer tissues from advanced stage (III, IV) than that of stage I CRC and also in corresponding metastatic tissues. CONCLUSIONS: The functional KITENIN complex acts as an executor with regard to cell motility and thereby controls CRC cell invasion, which may contribute to promoting metastasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Transporte/fisiologia , Neoplasias Colorretais/patologia , Proteínas de Membrana/fisiologia , Fosfoproteínas/fisiologia , Proteína Quinase C-delta/fisiologia , Citoesqueleto de Actina/fisiologia , Animais , Proteínas de Transporte/metabolismo , Movimento Celular/fisiologia , Células Cultivadas , Neoplasias Colorretais/genética , Neoplasias Colorretais/fisiopatologia , Proteínas Desgrenhadas , Genes ras/genética , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Membrana/metabolismo , Mutação , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/fisiologia , Ratos , Fator de Transcrição AP-1/fisiologia , Células Tumorais CultivadasRESUMO
Understanding the mechanisms of platinum compound resistance, including cisplatin resistance, has important implications for improving cancer treatments. Previous studies identified a potential role for mitogen-activated protein kinase phosphatase-1 (MKP-1) in cisplatin resistance. This work focuses on the regulation of poly(ADP-ribose) polymerase-1 (PARP-1) expression by MKP-1. We found that MKP-1 overexpression stimulates PARP-1 and poly(ADP-ribose) (PAR) protein expression and cisplatin resistance while its downregulation suppresses PARP-1 and PAR protein expression and cisplatin resistance. Silencing MKP-1 promoted PARP-1 ubiquitination, which decreased PARP-1 protein levels. We also found that silencing c-Jun N-terminal kinase 1/2 (JNK1/2) decreased PARP-1 ubiquitination while increasing total PARP-1 protein levels. Furthermore, we showed that acquired cisplatin-resistant ovarian cancer cells expressed high levels of MKP-1 and PARP-1 proteins, and that silencing MKP-1 or PARP-1 increased cisplatin sensitivity in resistant cells. Notably, the pharmacologic inhibition of PARP activity restored cisplatin sensitivity in MKP-1 overexpressing cells. Thus, this work indicates that suppression of JNK1/2 activity by MKP-1 maintains PARP-1 levels and suggests that MKP-1-mediated cisplatin resistance can be bypassed by PARP-1 inhibition.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Fosfatase 1 de Especificidade Dupla/genética , Neoplasias Ovarianas/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/genética , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Fosfatase 1 de Especificidade Dupla/antagonistas & inibidores , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Proteólise/efeitos dos fármacosRESUMO
Nitrogenase-mediated H(2) accumulation of Rhodobacter sphaeroides under photoheterotrophic conditions is reduced directly by the hydrogenase activity catalyzing H(2) uptake and indirectly by energy-demanding metabolic processes such as poly-beta-hydroxybutyrate (PHB) formation. H(2) accumulation of R. sphaeroides was examined during cell growth under illumination of 15, 7, and 3 W/m(2). Mutations in either hupSL (H(2)-uptake hydrogenase) or phbC (PHB synthase) had no effect on nitrogenase activity. The nitrogenase activity of R. sphaeroides grown at 15 W/m(2), however, was 70% higher than that of cells grown at 3 W/m(2), while the H(2)-uptake hydrogenase activity was approximately 3-fold higher in the same comparison. Accordingly, H(2) uptake by hydrogenase, monitored by measuring the difference in H(2) accumulation between a hupSL-deletion mutant and the corresponding parental strain, appeared to reach a maximum level as illumination was increased to 15 W/m(2). On the other hand, the surplus energy due to lack of PHB formation led to a fixed increase in H(2) accumulation independent of light intensity, reflecting the fact that the cellular PHB content was not changed significantly depending on light intensity. Therefore, H(2) uptake by hydrogenase should be suppressed to achieve higher H(2) accumulation of R. sphaeroides, especially at 15 W/m(2).