RESUMEN
Folate, an important nutrient in the human diet, has been implicated in cancer, but its role in metastasis is not established. We have shown previously that the withdrawal of medium folate leads to the inhibition of migration and invasion of A549 lung carcinoma cells. Here we have demonstrated that medium folate regulates the function of Rho GTPases by enabling their carboxyl methylation and translocation to plasma membrane. Conversely, the lack of folate leads to the retention of these proteins in endoplasmic reticulum. Folate also promoted the switch from inactive (GDP-bound) to active (GTP-bound) GTPases, resulting in the activation of downstream kinases p21-activated kinase and LIM kinase and phosphorylation of the actin-depolymerizing factor cofilin. We have further demonstrated that in A549 cells two GTPases, RhoA and Rac1, but not Cdc42, are immediate sensors of folate status: the siRNA silencing of RhoA or Rac1 blocked effects of folate on cofilin phosphorylation and cellular migration and invasion. The finding that folate modulates metastatic potential of cancer cells was confirmed in an animal model of lung cancer using tail vein injection of A549 cells in SCID mice. A folate-rich diet enhanced lung colonization and distant metastasis to lymph nodes and decreased overall survival (35 versus 63 days for mice on a folate-restricted diet). High folate also promoted epithelial-mesenchymal transition in cancer cells and experimental mouse tumors. Our study provides experimental evidence for a mechanism of metastasis promotion by dietary folate and highlights the interaction between nutrients and metastasis-related signaling.
Asunto(s)
Adenocarcinoma/enzimología , Cofilina 1/metabolismo , Ácido Fólico/administración & dosificación , Neoplasias Pulmonares/enzimología , Proteína de Unión al GTP rac1/fisiología , Proteína de Unión al GTP rhoA/fisiología , Adenocarcinoma/secundario , Administración Oral , Animales , Línea Celular Tumoral , Membrana Celular/enzimología , Movimiento Celular/efectos de los fármacos , Supervivencia Celular , Suplementos Dietéticos , Retículo Endoplásmico/enzimología , Transición Epitelial-Mesenquimal , Ácido Fólico/farmacología , Humanos , Neoplasias Pulmonares/patología , Metástasis Linfática , Masculino , Metilación , Ratones SCID , Trasplante de Neoplasias , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Transducción de Señal , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/química , Proteína de Unión al GTP rac1/químicaRESUMEN
Glycine N-methyltransferase (GNMT), an abundant cytosolic enzyme, catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine generating S-adenosylhomocysteine and sarcosine (N-methylglycine). This reaction is regulated by 5-methyltetrahydrofolate, which inhibits the enzyme catalysis. In the present study, we observed that GNMT is strongly down regulated in human cancers and is undetectable in cancer cell lines while the transient expression of the protein in cancer cells induces apoptosis and results in the activation of ERK1/2 as an early pro-survival response. The antiproliferative effect of GNMT can be partially reversed by treatment with the pan-caspase inhibitor zVAD-fmk but not by supplementation with high folate or SAM. GNMT exerts the suppressor effect primarily in cells originated from malignant tumors: transformed cell line of non-cancer origin, HEK293, was insensitive to GNMT. Of note, high levels of GNMT, detected in regenerating liver and in NIH3T3 mouse fibroblasts, do not produce cytotoxic effects. Importantly, GNMT, a predominantly cytoplasmic protein, was translocated into nuclei upon transfection of cancer cells. The presence of GNMT in the nuclei was also observed in normal human tissues by immunohistochemical staining. We further demonstrated that the induction of apoptosis is associated with the GNMT nuclear localization but is independent of its catalytic activity or folate binding. GNMT targeted to nuclei, through the fusion with nuclear localization signal, still exerts strong antiproliferative effects while its restriction to cytoplasm, through the fusion with nuclear export signal, prevents these effects (in each case the protein was excluded from cytosol or nuclei, respectively). Overall, our study indicates that GNMT has a secondary function, as a regulator of cellular proliferation, which is independent of its catalytic role.
Asunto(s)
Núcleo Celular/metabolismo , Glicina N-Metiltransferasa/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Catálisis , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Daño del ADN , Activación Enzimática , Ácido Fólico/química , Ácido Fólico/metabolismo , Expresión Génica , Glicina N-Metiltransferasa/química , Glicina N-Metiltransferasa/genética , Glicina N-Metiltransferasa/farmacología , Humanos , Ratones , Modelos Moleculares , Conformación Molecular , Mutación , Unión Proteica , Transporte de Proteínas , Proteínas Recombinantes/farmacología , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/farmacologíaRESUMEN
10-Formyltetrahydrofolate dehydrogenase (FDH) converts 10-formyltetrahydrofolate to tetrahydrofolate (THF). Expression of the enzyme in FDH-deficient cancer cells induces cytotoxicity that can be reversed by supplementation with high concentrations of a reduced folate, 5-formyl-THF (leucovorin). In contrast, non-tumor cells are resistant to FDH. The present study was undertaken to investigate mechanisms that could protect cells against FDH suppressor effects. Using 10 microM leucovorin supplementation of FDH-sensitive A549 cells transfected for FDH expression, we selected clones that have acquired resistance against FDH. Resistant cells expressed high levels of FDH and were capable of growing after withdrawal of leucovorin. These cells, however, have increased doubling time due to prolonged S phase. They also have significantly increased levels of total folate pool and THF/5,10-methylene-THF pool while the level of 10-formyl-THF was two-fold lower than in parental FDH-sensitive cells. We have shown that the FDH-catalyzed reaction proceeds at about a three-fold slower rate at the ratio of 10-formyl-THF/THF corresponding to the resistant cells than at the ratio corresponding to parental sensitive cells, due to product inhibition (KI is 2.35 microM). FDH-resistant cells have strongly up-regulated dihydrofolate reductase (DHFR) that is proposed to be a mechanism for the alteration of folate pools and a key component of the acquired resistance. Elevation of DHFR in A549 cells by transient transfection decreased sensitivity to FDH toxicity and allowed selection of FDH-resistant clones. DHFR-induced repression of FDH catalysis could be an S phase-related metabolic adjustment that provides protection against FDH suppressor effects.
Asunto(s)
Leucovorina/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Tetrahidrofolato Deshidrogenasa/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Secuencia de Bases , Catálisis , Línea Celular , Cartilla de ADN , Resistencia a Medicamentos , Ácido Fólico/metabolismo , HumanosRESUMEN
Our studies showed that an abundant folate enzyme, 10-formyltetrahydrofolatedehydrogenase (FDH), is strongly down-regulated in several types of cancer on both the mRNA and the protein level. Transient expression of FDH in several human prostate cancer cell lines, a hepatocarcinoma cell line, HepG2, and a lung cancer cell line, A549, suppressed proliferation and resulted in cytotoxicity. In contrast, overexpression of a catalytically inactive FDH mutant did not inhibit proliferation, which suggests that the suppressor effect of FDH is a result of its enzymatic function. Because the FDH substrate, 10-formyltetrahydrofolate, is required for de novo purine biosynthesis, we hypothesized that the inhibitory effects of FDH occur through the depletion of intracellular 10-formyltetrahydrofolate followed by the loss of de novo purine biosynthesis. The ultimate impact is diminished DNA/RNA biosynthesis. Indeed, supplementation of FDH-overexpressing cells with 5-formyltetrahydrofolate or hypoxanthine reversed the FDH growth-inhibitory effects. Hence, down-regulation of FDH in tumors is proposed to be one of the cellular mechanisms that enhance proliferation.