RESUMO
Galectin-3 (Gal-3) has a long, aperiodic, and dynamic proline-rich N-terminal tail (NT). The functional role of the NT with its numerous prolines has remained enigmatic since its discovery. To provide some resolution to this puzzle, we individually mutated all 14 NT prolines over the first 68 residues and assessed their effects on various Gal-3-mediated functions. Our findings show that mutation of any single proline (especially P37A, P55A, P60A, P64A/H, and P67A) dramatically and differentially inhibits Gal-3-mediated cellular activities (i.e., cell migration, activation, endocytosis, and hemagglutination). For mechanistic insight, we investigated the role of prolines in mediating Gal-3 oligomerization, a fundamental process required for these cell activities. We showed that Gal-3 oligomerization triggered by binding to glycoproteins is a dynamic process analogous to liquid-liquid phase separation (LLPS). The composition of these heterooligomers is dependent on the concentration of Gal-3 as well as on the concentration and type of glycoprotein. LLPS-like Gal-3 oligomerization/condensation was also observed on the plasma membrane and disrupted endomembranes. Molecular- and cell-based assays indicate that glycan binding-triggered Gal-3 LLPS (or LLPS-like) is driven mainly by dynamic intermolecular interactions between the Gal-3 NT and the carbohydrate recognition domain (CRD) F-face, although NT-NT interactions appear to contribute to a lesser extent. Mutation of each proline within the NT differentially controls NT-CRD interactions, consequently affecting glycan binding, LLPS, and cellular activities. Our results unveil the role of proline polymorphisms (e.g., at P64) associated with many diseases and suggest that the function of glycosylated cell surface receptors is dynamically regulated by Gal-3.
Assuntos
Galectina 3/química , Galectina 3/metabolismo , Polissacarídeos/metabolismo , Prolina/metabolismo , Sítios de Ligação , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Carboidratos , Galectina 3/genética , Galectinas , Glicosilação , Humanos , Ligação ProteicaRESUMO
Fibrosis, stiffening and scarring of an organ/tissue due to genetic abnormalities, environmental factors, infection, and/or injury, is responsible for > 40% of all deaths in the industrialized world, and to date, there is no cure for it despite extensive research and numerous clinical trials. Several biomarkers have been identified, but no effective therapeutic targets are available. Human galectin-3 is a chimeric gene product formed by the fusion of the internal domain of the collagen alpha gene [N-terminal domain (ND)] at the 5'-end of galectin-1 [C-terminal domain (CRD)] that appeared during evolution together with vertebrates. Due to the overlapping structural similarities between collagen and galectin-3 and their shared susceptibility to cleavage by matrix metalloproteases to generate circulating collagen-like peptides, this review will discuss present knowledge on the role of collagen and galectin-3 as biomarkers of fibrosis. We will also highlight the need for transformative approaches targeting both the ND and CRD domains of galectin-3, since glycoconjugate binding by the CRD is triggered by ND-mediated oligomerization and the therapies targeted only at the CRD have so far achieved limited success.
Assuntos
Colágeno , Fibrose , Galectina 3 , Animais , Humanos , Biomarcadores , Colágeno/genética , Colágeno/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Vertebrados , Glicoconjugados , Metaloproteinases da MatrizRESUMO
In the past decade, the development of immune checkpoint inhibitors in oncological clinical settings was in the forefront. However, the interest in musculoskeletal tumor patients as candidates for checkpoint inhibition remains underserved. Here, we are forwarding evidence proposing that galectin-3 (Gal-3) is an additional immune factor in the checkpoint processes. This review is the result of a large-scale cohort study depicting that overexpression of Gal-3 was widely prevalent in patients with musculoskeletal tumors, whereas T cell infiltrations were generally suppressed in the tumor microenvironment. Targeting Gal-3 would serve as a novel immune checkpoint inhibitor candidate in patients afflicted with aggressive musculoskeletal tumors.
Assuntos
Galectina 3 , Neoplasias , Estudos de Coortes , Humanos , Inibidores de Checkpoint Imunológico , Neoplasias/tratamento farmacológico , Linfócitos T , Microambiente TumoralRESUMO
The E3 ubiquitin (Ub) ligase gp78 plays an important role in endoplasmic reticulum (ER)-associated degradation (ERAD) and regulation of lipid biogenesis. Although a variety of substrates of gp78 have been described, the regulation of the degradation of gp78 itself remains poorly understood. To address this problem, we used co-immunoprecipitation-coupled liquid chromatography-tandem mass spectrometry (Co-IP/LC-MS/MS) to identify novel proteins interacting with gp78. One of the proteins identified in this study is the deubiquitylating (DUB) enzyme USP34 (Ub-specific protease 34). We demonstrate that knockdown of USP34 facilitates proteasomal degradation of gp78 and consequently impairs the function of gp78 in regulating lipid droplet formation. This study unveils a previously unknown function of USP34 in regulating the metabolic stability of gp78 and adds to our understanding of the relevance of partnering of DUBs and E3s in regulation of protein ubiquitylation.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Hepatócitos/metabolismo , Receptores do Fator Autócrino de Motilidade/genética , Proteases Específicas de Ubiquitina/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Células HEK293 , Hepatócitos/citologia , Humanos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores do Fator Autócrino de Motilidade/metabolismo , Transdução de Sinais , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Over the last few decades galectin-3, a carbohydrate binding protein, with affinity for N-acetyllactosamine residues, has been unique due to the regulatory roles it performs in processes associated with tumor progression and metastasis such as cell proliferation, homotypic/heterotypic aggregation, dynamic cellular transformation, migration and invasion, survival and apoptosis. Structure-function association of galectin-3 reveals that it consists of a short amino terminal motif, which regulates its nuclear-cytoplasmic shuttling; a collagen α-like domain, susceptible to cleavage by matrix metalloproteases and prostate specific antigen; accountable for its oligomerization and lattice formation, and a carbohydrate-recognition/binding domain containing the anti-death motif of the Bcl2 protein family. This structural complexity permits galectin-3 to associate with numerous molecules utilizing protein-protein and/or protein-carbohydrate interactions in the extra-cellular as well as intracellular milieu and regulate diverse signaling pathways, a number of which appear directed towards epithelial-mesenchymal transition and cancer stemness. Self-renewal, differentiation, long-term culturing and drug-resistance potential characterize cancer stem cells (CSCs), a small cell subpopulation within the tumor that is thought to be accountable for heterogeneity, recurrence and metastasis of tumors. Despite the fact that association of galectin-3 to the tumor stemness phenomenon is still in its infancy, there is sufficient direct evidence of its regulatory roles in CSC-associated phenotypes and signaling pathways. In this review, we have highlighted the available data on galectin-3 regulated functions pertinent to cancer stemness and explored the opportunities of its exploitation as a CSC marker and a therapeutic target.
Assuntos
Biomarcadores Tumorais/metabolismo , Galectina 3/metabolismo , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Proteínas Sanguíneas , Galectina 3/antagonistas & inibidores , Galectinas , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacosRESUMO
The skeleton is frequently a secondary growth site of disseminated cancers, often leading to painful and devastating clinical outcomes. Metastatic cancer distorts bone marrow homeostasis through tumor-derived factors, which shapes different bone tumor microenvironments depending on the tumor cells' origin. Here, we propose a novel insight on tumor-secreted Galectin-3 (Gal-3) that controls the induction of an inflammatory cascade, differentiation of osteoblasts, osteoclasts, and bone marrow cells, resulting in bone destruction and therapeutic failure. In the approaching era of personalized medicine, the current treatment modalities targeting bone metastatic environments are provided to the patient with limited consideration of the cancer cells' origin. Our new outlook suggests delivering individual tumor microenvironment treatments based on the expression level/activity/functionality of tumor-derived factors, rather than utilizing a commonly shared therapeutic umbrella. The notion of "Gal-3-associated bone remodeling" could be the first step toward a specific personalized therapy for each cancer type generating a different bone niche in patients afflicted with non-curable bone metastasis.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Galectina 3/metabolismo , Microambiente Tumoral , Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Remodelação Óssea , Proteínas de Transporte/metabolismo , Comunicação Celular , Diferenciação Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Citocinas/metabolismo , Feminino , Galectina 3/genética , Humanos , Masculino , Células Mieloides/citologia , Células Mieloides/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Ligação Proteica , Transdução de Sinais , Microambiente Tumoral/genéticaRESUMO
Galectin-3, a member of ß-galactoside-binding gene family is a multi-functional protein, which regulates pleiotropic biological functions such as cell growth, cell adhesion, cell-cell interactions, apoptosis, angiogenesis and mRNA processing. Its unique structure enables it to interact with a plethora of ligands in a carbohydrate dependent or independent manner. Galectin-3 is mainly a cytosolic protein, but can easily traverse the intracellular and plasma membranes to translocate into the nucleus, mitochondria or get externalized. Depending on the cell type, specific experimental conditions in vitro, cancer type and stage, galectin-3 has been reported to be exclusively cytoplasmic, predominantly nuclear or distributed between the two compartments. In this review we have summarized the dynamics of galectin-3 shuttling between the nucleus and the cytoplasm, the nuclear transport mechanisms of galectin-3, how its specific interactions with the members of ß-catenin signaling pathways affect tumor progression, and its implications as a therapeutic target.
Assuntos
Galectina 3/metabolismo , Neoplasias/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Galectina 3/antagonistas & inibidores , Galectina 3/química , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Multivalent protein-carbohydrate interactions that are mediated by sugar-binding proteins, i.e., lectins, have been implicated in a myriad of intercellular recognition processes associated with tumor progression such as galectin-mediated cancer cellular migration/metastatic processes. Here, using a modified ELISA, we show that glycodendrimers bearing mixtures of galactosides, lactosides, and N-acetylgalactosaminosides, galectin-3 ligands, multivalently affect galectin-3 functions. We further demonstrate that lactose functionalized glycodendrimers multivalently bind a different member of the galectin family, i.e., galectin-1. In a modified ELISA, galectin-3 recruitment by glycodendrimers was shown to directly depend on the ratio of low to high affinity ligands on the dendrimers, with lactose-functionalized dendrimers having the highest activity and also binding well to galectin-1. The results depicted here indicate that synthetic multivalent systems and upfront assay formats will improve the understanding of the multivalent function of galectins during multivalent protein carbohydrate recognition/interaction.
Assuntos
Galectina 1/metabolismo , Galectina 3/metabolismo , Glicosídeos/metabolismo , Sítios de Ligação , Dendrímeros/química , Dendrímeros/metabolismo , Ensaio de Imunoadsorção Enzimática , Galectina 1/química , Galectina 3/química , Glicosídeos/química , Humanos , Ligação ProteicaRESUMO
Galectin-3 is a member of the family of ß-galactoside-binding lectins characterized by evolutionarily conserved sequences defined by structural similarities in their carbohydrate-recognition domains. Galectin-3 is a unique, chimeric protein consisting of three distinct structural motifs: (i) a short NH2 terminal domain containing a serine phosphorylation site; (ii) a repetitive proline-rich collagen-α-like sequence cleavable by matrix metalloproteases; and (iii) a globular COOH-terminal domain containing a carbohydrate-binding motif and an NWGR anti-death motif. It is ubiquitously expressed and has diverse biological functions depending on its subcellular localization. Galectin-3 is mainly found in the cytoplasm, also seen in the nucleus and can be secreted by non-classical, secretory pathways. In general, secreted galectin-3 mediates cell migration, cell adhesion and cell-cell interactions through the binding with high affinity to galactose-containing glycoproteins on the cell surface. Cytoplasmic galectin-3 exhibits anti-apoptotic activity and regulates several signal transduction pathways, whereas nuclear galectin-3 has been associated with pre-mRNA splicing and gene expression. Its unique chimeric structure enables it to interact with a plethora of ligands and modulate diverse functions such as cell growth, adhesion, migration, invasion, angiogenesis, immune function, apoptosis and endocytosis emphasizing its significance in the process of tumor progression. In this review, we have focused on the role of galectin-3 in tumor metastasis with special emphasis on angiogenesis.
Assuntos
Carcinogênese/genética , Galectina 3/genética , Neoplasias/genética , Neovascularização Patológica/genética , Apoptose/genética , Proteínas Sanguíneas , Adesão Celular , Galectina 3/química , Galectina 3/metabolismo , Galectinas , Humanos , Metástase Neoplásica , Neoplasias/patologia , Precursores de RNA/genética , Splicing de RNA/genética , Transdução de SinaisRESUMO
By using lactose-functionalized poly(amidoamine) dendrimers as a tunable multivalent platform, we studied cancer cell aggregation in three different cell lines (A549, DU-145, and HT-1080) with galectin-3. We found that small lactose-functionalized G(2)-dendrimer 1 inhibited galectin-3-induced aggregation of the cancer cells. In contrast, dendrimer 4 (a larger, generation 6 dendrimer with 100 carbohydrate end groups) caused cancer cells to aggregate through a galectin-3 pathway. This study indicates that inhibition of cellular aggregation occurred because 1 provided competitive binding sites for galectin-3 (compared to its putative cancer cell ligand, TF-antigen on MUC1). Dendrimer 4, in contrast, provided an excess of ligands for galectin-3 binding; this caused crosslinking and aggregation of cells to be increased.
Assuntos
Dendrímeros/metabolismo , Galectina 3/metabolismo , Lactose/metabolismo , Mucina-1/metabolismo , Neoplasias/metabolismo , Antígenos Glicosídicos Associados a Tumores/metabolismo , Linhagem Celular Tumoral , Dendrímeros/química , Humanos , Lactose/análogos & derivados , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de ProteínasRESUMO
Galectin-3 meditates cell surface glycoprotein clustering, cross linking, and lattice formation. In cancer biology, galectin-3 has been reported to play a role in aggregation processes that lead to tumor embolization and survival. Here, we show that lactose-functionalized dendrimers interact with galectin-3 in a multivalent fashion to form aggregates. The glycodendrimer-galectin aggregates were characterized by dynamic light scattering and fluorescence microscopy methodologies and were found to be discrete particles that increased in size as the dendrimer generation was increased. These results show that nucleated aggregation of galectin-3 can be regulated by the nucleating polymer and provide insights that improve the general understanding of the binding and function of sugar-binding proteins.
RESUMO
Galectin-3 is a chimeric carbohydrate-binding protein, which interacts with cell surface carbohydrate-containing molecules and extracellular matrix glycoproteins and has been implicated in various biological processes such as cell growth, angiogenesis, motility, and metastasis. It is expressed in a wide range of tumor cells and is associated with tumor progression. The functions of galectin-3 are dependent on its localization and post-translational modifications such as cleavage and phosphorylation. Recently, we showed that galectin-3 Tyr-107 is phosphorylated by c-Abl; concomitantly, it was also shown that galectin-3 can be cleaved at this site by prostate-specific antigen (PSA), a chymotrypsin-like serine protease, after Tyr-107, resulting in loss of galectin-3 multivalency while preserving its carbohydrate binding activity. Galectin-3 is largely a monomer in solution but may form a homodimer by self-association through its carbohydrate recognition domain, whereas, in the presence of a ligand, galectin-3 polymerizes up to pentamers utilizing its N-terminal domain. Oligomerization is a unique feature of secreted galectin-3, which allows its function by forming ordered galectin-glycan structures, i.e. lattices, on the cell surface or through direct engagement of specific cell surface glycoconjugates by traditional ligand-receptor binding. We questioned whether Tyr-107 phosphorylation by c-Abl affects galectin-3 cleavage by PSA. The data suggest a role for galectin-3 in prostate cells associated with increased activity of c-Abl kinase and loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. In addition, the ratio of phosphorylated/dephosphorylated galectin-3 might be used as a complementary value to that of PSA for prognosis of prostate cancer and a novel therapeutic target for the treatment of prostate cancer.
Assuntos
Galectina 3/química , Galectina 3/metabolismo , Antígeno Prostático Específico/metabolismo , Proteólise , Tirosina/metabolismo , Linhagem Celular Tumoral , Quimiotaxia , Espaço Extracelular/metabolismo , Humanos , Modelos Moleculares , Neovascularização Patológica/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Conformação Proteica , Proteínas Proto-Oncogênicas c-abl/metabolismoRESUMO
Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus-cytoplasm transport of galectin-3, which is a ß-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the ß-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the ß-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to ß-catenin to inhibit transcriptional activity. Reduced expression of ß-catenin target genes is consistent with the Nup98 reduction and the galectin-3-nucleus translocation rate. Overall, the results show Nup98's involvement in nuclear-cytoplasm translocation of galectin-3 and ß-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.
Assuntos
Galectina 3/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte Ativo do Núcleo Celular/genética , Proliferação de Células , Progressão da Doença , Células HeLa , Humanos , Células MCF-7 , Neoplasias/metabolismo , Neoplasias/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética , Transdução de Sinais , beta Catenina/metabolismoRESUMO
Studies on lymph node metastasis of soft tissue sarcomas are insufficient because of its rarity. In this study, we examined the expressions of vascular endothelial growth factor (VEGF)-C and VEGF-D in soft tissue sarcomas metastasized to lymph nodes. In addition, the effects of the two molecules on the barrier function of a lymphatic endothelial cell monolayer against sarcoma cells were analyzed. We examined 7 patients who had soft tissue sarcomas with lymph node metastases and who had undergone neither chemotherapy nor radiotherapy before lymphadenectomy. Immunohistochemistry revealed that 2 of 7 sarcomas that metastasized to lymph nodes expressed VEGF-C both in primary and metastatic lesions. On the other hand, VEGF-D expression was detected in 4 of 7 primary and 7 of 7 metastatic lesions, respectively. Interestingly, 3 cases that showed no VEGF-D expression at primary sites expressed VEGF-D in metastatic lesions. Recombinant VEGF-C at 10(-8) and VEGF-D at 10(-7)and 10(-8)g/ml significantly increased the random motility of lymphatic endothelial cells compared with controls. VEGF-D significantly increased the migration of sarcoma cells through lymphatic endothelial monolayers. The fact that VEGF-D induced the migration of fibrosarcomas through the lymphatic endothelial monolayer is the probable reason for the strong relationship between VEGF-D expression and lymph node metastasis in soft tissue sarcomas. The important propensities of this molecule for the increase of lymph node metastases are not only lymphangiogenesis but also down-regulation of the barrier function of lymphatic endothelial monolayers, which facilitates sarcoma cells entering the lymphatic circulation.
Assuntos
Sarcoma/patologia , Fator C de Crescimento do Endotélio Vascular/biossíntese , Fator D de Crescimento do Endotélio Vascular/biossíntese , Idoso , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Excisão de Linfonodo , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Sarcoma/metabolismo , Células Tumorais CultivadasRESUMO
Galectin-3 is the only chimeric representative of the galectin family. Although galectin-3 has ubiquitous regulatory and physiological effects, there is a great number of pathological environments where galectin-3 cooperatively participates. Pectin is composed of different chemical structures, such as homogalacturonans, rhamnogalacturonans, and side chains. The study of pectin's major structural aspects is fundamental to predicting the impact of pectin on human health, especially regarding distinct molecular modulation. One of the explored pectin's biological activities is the possible galectin-3 protein regulation. The present review focuses on revealing the structure/function relationship of pectins, their fragments, and their biological effects. The discussion highlighted by this review shows different effects described within in vitro and in vivo experimental models, with interesting and sometimes contradictory results, especially regarding galectin-3 interaction. The review demonstrates that pectins are promissory food-derived molecules for different bioactive functions. However, galectin-3 inhibition by pectin had been stated in literature before, although it is not a fully understood, experimentally convincing, and commonly agreed issue. It is demonstrated that more studies focusing on structural analysis and its relation to the observed beneficial effects, as well as substantial propositions of cause and effect alongside robust data, are needed for different pectin molecules' interactions with galectin-3.
Assuntos
Galectina 3 , Pectinas , Galectina 3/metabolismo , Galectinas , Humanos , Pectinas/químicaRESUMO
The accepted notion of dNTP transport following cytoplasmic biosynthesis is 'facilitated diffusion'; however, whether this alone is sufficient for moving dNTPs for DNA synthesis remains an open question. The data presented here show that the MYH9 gene encoded heavy chain of non-muscle myosin IIA binds dNTPs potentially serving as a 'reservoir'. Pull-down assays showed that MYH9 present in the cytoplasmic, mitochondrial and nuclear compartments bind to DNA and this interaction is inhibited by dNTPs and 2-deoxyribose-5-phosphate (dRP) suggesting that MYH9-DNA binding is mediated via pentose sugar recognition. Direct dNTP-MYH9 binding was demonstrated by ELISA and a novel PCR-based method, which showed that all dNTPs bind to MYH9 with varying efficiencies. Cellular thermal shift assays showed that MYH9 thermal stability is enhanced by dNTPs. MYH9 siRNA transfection or treatment with myosin II selective inhibitors ML7 or blebbistatin decreased cell proliferation compared to controls. EdU labeling and cell cycle analysis by flow cytometry confirmed MYH9 siRNA and myosin II inhibitors decreased progression to S-phase with accumulation of cells in G0/G1 phase. Taken together, our data suggest a novel role for MYH9 in dNTP binding and DNA synthesis.
Assuntos
Cadeias Pesadas de Miosina , Miosina não Muscular Tipo IIA , Proteínas do Citoesqueleto , DNA/genética , Desoxirribose , Humanos , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II , Miosina não Muscular Tipo IIA/genética , Miosina não Muscular Tipo IIA/metabolismo , Pentoses , Fosfatos , RNA Interferente Pequeno , AçúcaresRESUMO
Galectin-3, a beta-galactoside-binding lectin, is found in cellular and extracellular location of the cell and has pleiotropic biological functions such as cell growth, cell adhesion and cell-cell interaction. It may exhibit anti- or pro-apoptotic activity depending on its localization and post-translational modifications. Two important post-translational modifications of galectin-3 have been reported: its cleavage and phosphorylation. Cleavage of galectin-3 was reported to be involved with angiogenic potential and apoptotic resistance. Phosphorylation of galectin-3 regulates its sugar-binding ability. In this report we have identified novel tyrosine phosphorylation sites in galectin-3 as well as the kinase responsible for its phosphorylation. Our results demonstrate that tyrosines at positions 79, 107 and 118 can be phosphorylated in vitro and in vivo by c-Abl kinase. Tyrosine 107 is the main target of c-Abl. Expression of galectin-3 Y107F mutant in galectin-3 null SK-Br-3 cells leads to morphological changes and increased motility compared to wild type galectin-3. Further investigation is needed to better understand the functional significance of the novel tyrosine phosphorylated sites of galectin-3.
Assuntos
Galectina 3/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-abl/metabolismo , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Movimento Celular , Galectina 3/genética , Humanos , Imunoprecipitação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Mutação , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-abl/genética , Especificidade por Substrato , Tirosina/genética , Tirosina/metabolismoRESUMO
Emerging evidence suggests that the transcription factor Forkhead Box M1 (FoxM1) is associated with aggressive human carcinomas, including breast cancer. Because elevated expression of FoxM1 has been observed in human breast cancers, FoxM1 has attracted much attention in recent years as a potential target for the prevention and/or therapeutic intervention in breast cancer. However, no information is currently available regarding how downregulation of FoxM1 could be achieved for breast cancer prevention and therapy. Here, we report for the first time that 3,3'-diindolylmethane (DIM), a nontoxic dietary chemopreventive agent could effectively downregulate FoxM1 in various breast cancer cell lines. Using gene transfection, real-time reverse transcription-PCR, Western blotting, invasion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, we found that DIM could enhance Taxotere-induced growth inhibition of breast cancer cells, and decreased invasive capacity of breast cancer cells was observed after either treatment alone or the combination. These effects were associated with downregulation of FoxM1. We also found that knock down of FoxM1 expression by small interfering RNA (siRNA) transfection increased DIM-induced cell growth inhibition, whereas over-expression of FoxM1 by cDNA transfection attenuated DIM-induced cell growth inhibition, suggesting the mechanistic role of FoxM1. Most importantly, the combination treatment significantly inhibited tumor growth in severe combined immunodeficiency (SCID) mice, and the results were correlated with the downregulation of FoxM1 in tumor remnants. We conclude that inactivation of FoxM1 and its target genes by DIM could enhance the therapeutic efficacy of Taxotere in breast cancer, which could be a useful strategy for the prevention and/or treatment of breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Fatores de Transcrição Forkhead/genética , Indóis/farmacologia , Taxoides/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Docetaxel , Regulação para Baixo , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatic cancer is a leading cause of cancer-related mortality and often has a poor prognosis because of its late diagnosis, aggressive local invasion, early metastasis and poor response to chemotherapy. The chemotherapeutic agent gemcitabine is effective for treating advanced pancreatic cancer, but its efficacy remains less than satisfactory. It is expected that further investigation of pancreatic cancer cell invasion and development of strategies to block this process should improve the disease prognosis. In this study, we tested our hypothesis that galectin-3 (gal-3), a multifunctional member of the ß-galactoside-binding protein family, may regulate pancreatic cancer cell motility and silencing of it inhibit cell motility. Previous studies demonstrated that this protein is associated with tumor cell adhesion, proliferation, differentiation, angiogenesis, apoptosis and metastasis. Here, we used gal-3 small interfering RNA (siRNA) to silence its expression in various pancreatic cancer cell lines to determine whether gal-3 regulates cell proliferation, migration and invasion in vitro. We found that silencing gal-3 reduced cellular migration and invasion, but failed to affect proliferation. In gal-3 siRNA-transfected cells, we detected a decrease in ß-catenin expression, an important signal for cancer cell invasion, which was caused by downregulation of phosphorylated Akt and GSK-3ß. We also found that matrix metalloproteinase (MMP)-2 expression was reduced by gal-3 silencing. These results indicate that gal-3-mediated invasion via MMP-2 regulated by ß-catenin degradation is initiated by Akt phosphorylation in pancreatic cancer cells. Our results suggest that gal-3 can be a novel therapeutic target in pancreatic cancer.