RESUMEN
Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs have specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP-ribose) polymerase hyperactivation and cell death and reduced cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. Moreover, this study also shows that restoration of hCNT1 expression is able to reduce tumor growth in a mouse model of pancreatic adenocarcinoma. These data predict a novel role for a NT protein, hCNT1, which appears to be independent of its role as mediator of nucleoside uptake by cells. Thereby, hCNT1 fits the profile of a transceptor in a substrate translocation-independent manner and is likely to be relevant to tumor biology.
Asunto(s)
Adenocarcinoma/metabolismo , Proteínas de Transporte de Membrana/fisiología , Nucleósidos/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/patología , Adenoviridae/genética , Transporte Biológico , Ciclo Celular , Muerte Celular , Línea Celular Tumoral , Forma de la Célula , Supervivencia Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica , Vectores Genéticos , Humanos , Sistema de Señalización de MAP Quinasas , Trasplante de Neoplasias , Neoplasias Pancreáticas/patología , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Carga TumoralRESUMEN
BACKGROUND: Pancreatic cancer, the fifth leading cause of adult cancer death in Western countries, lacks early detection, and displays significant dissemination ability. Accumulating evidence shows that integrin-mediated cell attachment to the extracellular matrix induces phenotypes and signaling pathways that regulate tumor cell growth and migration. METHODS: In view of these findings, we examined the role of ß(3) in pancreatic cancer by generating two stable ß(3)-expressing pancreatic human cell lines and characterizing their behavior in vitro and in vivo. RESULTS: Transduction of ß(3) selectively augmented the functional membrane α(v)ß(3) integrin levels, as evident from the enhanced adhesion and migration abilities related to active Rho GTPases. No effects on in vitro anchorage-dependent growth, but higher anoikis were detected in ß(3)-overexpressing cells. Moreover, tumors expressing ß(3) displayed reduced growth. Interestingly, treatment of mice with an α(v)-blocking antibody inhibited the growth of ß(3)-expressing tumors to a higher extent. CONCLUSIONS: Our results collectively support the hypothesis that α(v)ß(3) integrin has dual actions depending on the cell environment, and provide additional evidence on the role of integrins in pancreatic cancer, which should eventually aid in improving prediction of the effects of therapies addressed to modulate integrin activities in these tumors.
Asunto(s)
Integrina beta3/metabolismo , Neoplasias Pancreáticas/metabolismo , Subunidades de Proteína/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Humanos , Ratones , Neoplasias Pancreáticas/patología , Proteínas de Unión al GTP rho/metabolismoRESUMEN
BACKGROUND: pancreatic cancer, the fifth leading cause of adult cancer death in Western countries, lacks early detection, and displays significant dissemination ability. Accumulating evidence shows that integrin-mediated cell attachment to the extracellular matrix induces phenotypes and signaling pathways that regulate tumor cell growth and migration. METHODS: in view of these findings, we examined the role of ß3 in pancreatic cancer by generating two stable ß3-expressing pancreatic human cell lines and characterizing their behavior in vitro and in vivo. RESULTS: transduction of ß3 selectively augmented the functional membrane αvß3 integrin levels, as evident from the enhanced adhesion and migration abilities related to active Rho GTPases. No effects on in vitro anchorage-dependent growth, but higher anoikis were detected in ß3-overexpressing cells. Moreover, tumors expressing ß3 displayed reduced growth. Interestingly, treatment of mice with an αv-blocking antibody inhibited the growth of ß3-expressing tumors to a higher extent. CONCLUSIONS: our results collectively support the hypothesis that αvß3 integrin has dual actions depending on the cell environment, and provide additional evidence on the role of integrins in pancreatic cancer, which should eventually aid in improving prediction of the effects of therapies addressed to modulate integrin activities in these tumors.
Asunto(s)
Integrina alfaVbeta3/metabolismo , Neoplasias Pancreáticas/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Neoplasias Pancreáticas/patología , Transducción Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We analyzed the differential gene expression in the pancreatic cancer cell line NP-18 upon induction of apoptosis caused by cyclin-dependent kinase inhibition triggered by either overexpression of the tumor suppressor gene p16(INK4A)using an adenoviral construction or incubation with the chemical inhibitors, roscovitine or olomoucine. Screening was performed using cDNA arrays from Clontech that allowed the determination of the expression of 1,176 genes specifically related with cancer. The analysis was carried out using the Atlas Image 2.01 (Clontech) and GeneSpring 4.2 (Silicon Genetics) softwares. Among the differentially expressed genes, we chose for further validation histone deacetylase 1 (HDAC1), von Hippel Lindau and decorin as upregulated genes, and Sp1, hypoxia-inducible factor-1 alpha and DNA primase as downregulated genes. The changes in the expression of these genes to mRNA were validated by quantitative RT-PCR and the final translation into protein by Western blot analysis. Inhibition of HDAC activity, Sp1 binding and DNA primase expression led to an increase in the level of apoptosis, both in parental cells and in doxorubicin-resistant cells. Therefore, these proteins could constitute possible targets to develop modulators in cancer chemotherapy that would increase or restore apoptosis.