RESUMO
The effects of radiation are known to be potentiated by N-3 polyunsaturated fatty acids, which modulate several signaling pathways, but the molecular mechanisms through which these fatty acids enhance the anticancer effects of irradiation in colorectal cancer (CRC) treatment remain poorly elucidated. Here, we aimed to ascertain whether the fatty acid docosahexaenoic acid (DHA) exerts a modulating effect on the response elicited by radiation treatment (RT). Two CRC cell lines, Caco-2 and HT-29, were exposed to RT, DHA, or both (DHA + RT) for various times, and then cell viability, proliferation, and clonogenicity were assessed. Moreover, cell cycle, apoptosis, and necrosis were analyzed using flow cytometry, and the involvement of WNT/ß-catenin signaling was investigated by immunofluorescence to determine nuclear ß-catenin, GSK3ß phosphorylation status, and TCF/LEF-activity reporter. DHA and RT applied separately diminished the viability of both HT-29 and Caco-2 cells, and DHA + RT caused a further reduction in proliferation mainly in HT-29 cells, particularly in terms of colony formation. Concomitantly, our results verified cell cycle arrest in G0/G1 phase, a reduction of cyclin D1 expression, and a decrease in GSK3ß phosphorylation after the combined treatment. Furthermore, immunofluorescence quantification revealed that nuclear ß-catenin was increased in RT-exposed cells, but this effect was abrogated in cells exposed to DHA + RT, and the results of TCF/LEF-activity assays confirmed that DHA attenuated the increase in nuclear ß-catenin activity induced by irradiation. Our finding shows that DHA applied in combination with RT enhanced the antitumor effects of irradiation on CRC cells, and that the underlying mechanism involved the WNT/ß-catenin pathway. © 2018 BioFactors, 45(1):24-34, 2019.
Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Raios gama , Regulação Neoplásica da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , beta Catenina/genética , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Células CACO-2 , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Ciclina D1/genética , Ciclina D1/metabolismo , Relação Dose-Resposta a Droga , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HT29 , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Fator 1 de Transcrição de Linfócitos T/genética , Fator 1 de Transcrição de Linfócitos T/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
BACKGROUND: To date, there is no effective therapy for patients with advanced/metastatic adrenocortical cancer (ACC). The activation of the Wnt/beta-catenin signaling is frequent in ACC and this pathway is a promising therapeutic target. AIM: To investigate the effects of the inhibition of the Wnt/beta-catenin in ACC cells. METHODS: Adrenal (NCI-H295 and Y1) and non-adrenal (HeLa) cell lines were treated with PNU-74654 (5-200 µM) for 24-96 h to assess cell viability (MTS-based assay), apoptosis (Annexin V), expression/localization of beta-catenin (qPCR, immunofluorescence, immunocytochemistry and western blot), expression of beta-catenin target genes (qPCR and western blot), and adrenal steroidogenesis (radioimmunoassay, qPCR and western blot). RESULTS: In NCI-H295 cells, PNU-74654 significantly decreased cell proliferation 96 h after treatment, increased early and late apoptosis, decreased nuclear beta-catenin accumulation, impaired CTNNB1/beta-catenin expression and increased beta-catenin target genes 48 h after treatment. No effects were observed on HeLa cells. In NCI-H295 cells, PNU-74654 decreased cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. Additionally, in NCI-H295 cells, PNU-74654 decreased SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase 48 h after treatment. In Y1 cells, PNU-74654 impaired corticosterone secretion 24 h after treatment but did not decrease cell viability. CONCLUSIONS: Blocking the Tcf/beta-catenin complex inhibits the Wnt/beta-catenin signaling in adrenocortical tumor cells triggering increased apoptosis, decreased cell viability and impairment of adrenal steroidogenesis. These promising findings pave the way for further experiments inhibiting the Wnt/beta-catenin pathway in pre-clinical models of ACC. The inhibition of this pathway may become a promising adjuvant therapy for patients with ACC.
Assuntos
Neoplasias do Córtex Suprarrenal/patologia , Antineoplásicos/farmacologia , Fator 1 de Transcrição de Linfócitos T/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Radioimunoensaio , Reação em Cadeia da Polimerase em Tempo Real , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
CONTEXT: CTNNB1/ß-catenin mutations and activation of Wnt/ß-catenin pathway are frequent in adult adrenocortical tumors (ACT), but data on childhood ACT are lacking. OBJECTIVE: The aim of the study was to investigate the presence of Wnt/ß-catenin pathway abnormalities in childhood ACT. PATIENTS AND METHODS: Clinicopathological findings and outcome of 62 childhood ACT patients were analyzed regarding CTNNB1 mutations and the expression of Wnt-related genes (CTNNB1; WNT4, a Wnt ligand; SFRP1, DKK3, and AXIN1, Wnt inhibitors; TCF7, a transcription factor; and MYC and WISP2, target genes) by quantitative PCR and immunohistochemistry. RESULTS: CTNNB1-activating mutations were found in only four of 62 ACT (6%), all of them harboring TP53 mutation. There was association between the presence of CTNNB1 mutations and death (P = 0.02). Diffuse ß-catenin accumulation was found in 71% of ACT, even in ACT without CTNNB1 mutations. Compared to normal adrenals, ACT presented increased expression of CTNNB1 (P = 0.008) and underexpression of Wnt inhibitor genes: DKK3 (P < 0.0001), SFRP1 (P = 0.05), and AXIN1 (P = 0.04). With regard to Wnt/ß-catenin target genes, ACT presented increased expression of WISP2 but lower expression of MYC. Higher overall survival was associated with underexpression of SFRP1 (P = 0.01), WNT4 (P = 0.004), and TCF7 (P < 0.01). CONCLUSIONS: CTNNB1 mutations are not common in childhood ACT but appear to associate with poor prognosis. Nevertheless, most ACT exhibit increased expression of ß-catenin and WISP2 and reduced expression of Wnt inhibitor genes (DKK3, SFRP1, and AXIN1). Thus, in addition to CTNNB1 mutations, other genetic events affecting the Wnt/ß-catenin pathway may be involved in childhood adrenocortical tumorigenesis.
Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Adolescente , Neoplasias do Córtex Suprarrenal/genética , Proteína Axina/fisiologia , Proteínas de Sinalização Intercelular CCN , Criança , Pré-Escolar , Estudos de Coortes , DNA/genética , DNA/isolamento & purificação , Feminino , Humanos , Imuno-Histoquímica , Lactente , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Masculino , Mutação/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Análise de Sobrevida , Fator 1 de Transcrição de Linfócitos T/fisiologia , Fatores de Transcrição/fisiologia , Resultado do Tratamento , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/genética , Proteína Wnt4/fisiologia , beta Catenina/genéticaRESUMO
Comparative studies of the tetrapod raldh2 (aldh1a2) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1) interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox and ventral Lim-homeodomain binding sites. Raldh2 and Math1/Cath1 expression in mouse and chicken highlights a novel, transient, endogenous Raldh2 expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits. Consistent with expression of raldh2 in the dorsal interneurons of tetrapods, we also found that raldh2 is expressed in dorsal interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal proprioception.