RESUMO
Mutation of galectin-3 at position 191 (rs4644) substituting proline to histidine (gal-3H(64)) resulted in the acquisition of resistance to drug-induced apoptosis by breast cancer cells. This study employed gastric cancer cells and patient tissues in attempts to elucidate how and why this mutation in galectin-3 (gal-3H(64)) enhances cancer progression, compared to wild type galectin-3 (gal-3P(64)). First, we prepared lenti-virus constructs containing gal-3P(64), gal-3H(64) and LacZ, and used them to infect galectin-3 null SNU-638 cells. We found that gal-3H(64) over-expression increases gastric cancer cell growth more than gal-3P(64) or LacZ over-expression. Also, gal-3H(64) over-expression conferred more resistance to cisplatin or 5-FU induced cytotoxicity than gal-3P(64). Gal-3H(64) also enhanced nuclear accumulation of ß-catenin as well as increased expression of TCF-4 target genes, such as fascin-1 and c-Myc through the augmented promoter binding activity of TCF-4, than gal-3P(64). We also demonstrated stronger staining of ß-catenin and galectin-3 in malignant tissues from gastric cancer patients with mutated galectin-3 at position 191 (gal-3 191) (A/A) (H(64)) and greater localization in the nucleus than in gal-3 191 A/C (P(64)) cancer patients. Taken together, we elucidated in this study that germline variant of gal-3H(64) increases nuclear accumulation of ß-catenin and promotes TCF transcriptional activity and enhances more the galectin-3's role in gastric cancer progression.
Assuntos
Núcleo Celular/metabolismo , Galectina 3/genética , Mutação/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , beta Catenina/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Western Blotting , Proliferação de Células , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Células Germinativas , Histidina/química , Histidina/genética , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Prolina/química , Prolina/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Fator de Transcrição 4 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , beta Catenina/genéticaRESUMO
BACKGROUND & AIMS: Galectin-3 is a beta-galactoside-binding protein that increases gastric cancer cell motility in response to integrin signaling and is highly expressed in gastric tumor cells. Galectin-3 induces cytoskeletal remodeling to increase cell motility, but the mechanisms of this process are not understood. We investigated the effects of galectin-3 on fascin-1, an actin-bundling protein. METHODS: We collected malignant and normal tissues from gastric cancer patients and examined the expression levels of galectin-3 and fascin-1. We silenced galectin-3 expression in human gastric cancer cell lines using small interfering RNA and lenti-viral constructs and determined the effects on fascin-1 expression, cell motility, and invasion. RESULTS: Malignant gastric tissues expressed high levels of galectin-3 and fascin-1, compared with normal gastric tissues. Silencing of galectin-3 resulted in altered cancer cell morphology, reduced fascin-1 expression, decreased cell motility, and reduced malignant cell invasion. Galectin-3 overexpression reversed these effects. Silencing of fascin-1 also reduced cell motility and caused changes in cell shape, as did silencing of galectin-3. Furthermore, galectin-3 silencing inhibited the interaction between glycogen synthase kinase (GSK)-3beta, beta-catenin, and T-cell factor (TCF) 4, and the binding of beta-catenin/TCF-4 to the fascin-1 promoter. Nuclear localization of GSK-3beta and beta-catenin were not detected when galectin-3 was silenced. Overexpression of mutated galectin-3 (with mutations in the GSK-3beta binding and phosphorylation motifs) did not increase fascin-1 levels, in contrast to overexpression of wild-type galectin-3. CONCLUSIONS: Galectin-3 increases cell motility by up-regulating fascin-1 expression. Galectin-3 might be a potential therapeutic target for the prevention and treatment of gastric cancer progression.
Assuntos
Proteínas de Transporte/metabolismo , Movimento Celular , Galectina 3/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neoplasias Gástricas/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Forma Celular , Galectina 3/genética , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Proteínas dos Microfilamentos/genética , Mutação , Invasividade Neoplásica , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/secundário , Fatores de Transcrição TCF/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição , Transfecção , Regulação para Cima , beta Catenina/metabolismoRESUMO
Galectin-3 is known to modulate cell proliferation and apoptosis and is highly expressed in human cancers, but its function in gastric cancer is still controversial. Here, we examined the role of galectin-3 in gastric cancer cells by silencing it with synthetic double-stranded siRNA. After silencing of galectin-3, cell numbers decreased and cell shape changed. Galectin-3 siRNA treatment also induced G(1) arrest. DNA microarray analysis was used to assess changes in gene expression following galectin-3 silencing. We found that silencing of galectin-3 caused changes in gene expression. RT-PCR and real-time PCR were utilized for validation of the changes found in microarray studies. Western blot analysis confirmed changes in the expression of proteins of interest: cyclin D1, survivin, XIAP, XAF, PUMA, and GADD45alpha. Generally, it tended to increase the expression of several pro-apoptotic genes, and to decrease the expression of cell cycle progressive genes. We also confirmed that changes in the expression of these genes were caused by galectin-3 overexpression. Finally, we demonstrated that silencing of galectin-3 enhanced apoptosis induction with chemotherapeutic agents by further reducing the expression of anti-apoptotic and/or cell survival molecules such as survivin, cyclin D1, and XIAP, and increasing the expression of pro-apoptotic XAF-1. We conclude that galectin-3 is involved in cancer progression and malignancy by modulating the expression of several relevant genes, and inhibition of galectin-3 may be an approach to improve chemotherapy of gastric cancers.
