RESUMO
Thapsigargin (SERCA ATPase inhibitor) inhibited the S100A4 metastatic marker expression in MDA-MB231 breast cancer cells. We found that S100A4 gene transcription is regulated by Ca2+ signaling pathways. We found that the synthesis of S100A4 mRNA and S100A4 protein in MDA-MB231 cells was effectively suppressed by thapsigargin at a concentration of 0.4-4 µM with retaining cell viability. We assume that the change in the gene transcription in response to disturbance of Ca2+ homeostasis is directly involved in the remodeling of Ca2+ signaling pathways.
Assuntos
Neoplasias da Mama/patologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Retículo Sarcoplasmático/enzimologia , Tapsigargina/farmacologia , Linhagem Celular Tumoral , Humanos , Proteína A4 de Ligação a Cálcio da Família S100/genética , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
Reduced expression of metastatic marker protein S100A4 in triple-negative breast cancer cells MDA-MB-231 leads to a decrease in the migration ability of cells and increases the sensitivity of the modified cells to docetaxel therapy. Cells capable of migration differ from the immotile cells in the content of the S100A4 protein in the cell, and this difference persists after the treatment of cells with the agents that reduce the intracellular level of S100A4. The presence of exogenous S100A4 protein in culture medium reduces the content of this protein in breast cancer cells. The results of the study show that the ability of breast cancer cells to migrate depends on the S100A4 protein concentration in the cell.
Assuntos
Movimento Celular , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Proteína A4 de Ligação a Cálcio da Família S100/biossíntese , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Proteínas de Neoplasias/genética , Proteína A4 de Ligação a Cálcio da Família S100/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The sensitivity of MDA-MB231 breast cancer cells to the effects of pharmacological agents was evaluated by their motility and viability. Dexamethasone, doxorubicin, or docetaxel administered separately in their effective concentration suppressed cell motility (in 16 h) and caused cell death (in 48 h). The strength of the effects increased in the following order: dexa methasoneAssuntos
Neoplasias da Mama/metabolismo
, Dexametasona/farmacologia
, Docetaxel/farmacologia
, Doxorrubicina/farmacologia
, Proteína A4 de Ligação a Cálcio da Família S100/metabolismo
, Morte Celular/efeitos dos fármacos
, Linhagem Celular Tumoral
, Movimento Celular/efeitos dos fármacos
, Feminino
, Humanos
RESUMO
We studied the effect of PGRPs-Hsp70 cytotoxic complex that is analogous to natural complex secreted by cytotoxic lymphocytes and the antitumor drug paclitaxel on the development of M3 melanoma in DBA mice. Significant inhibition of tumor growth was observed in all experimental groups by days 20 and 35 of observation; paclitaxel monotherapy was less effective than administration of PGRPs-Hsp70 cytotoxic complex and its combination with paclitaxel. Pairwise comparison of Kaplan-Meier curves showed that survival was maximum in the group receiving combined therapy with PGRPs-Hsp70 cytotoxic complex and paclitaxel in comparison with groups receiving monotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Melanoma/tratamento farmacológico , Paclitaxel/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Animais , Proteínas de Choque Térmico HSP70/metabolismo , Estimativa de Kaplan-Meier , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos DBARESUMO
S100A4 is a Ca2+-binding protein that performs an important role in metastasis. It is also known for its antitumor functions. S100A4 is expressed by a specialized subset of CD4+CD25+ lymphocytes and is present on those cell's membranes along with peptidoglycan recognition proteins (PGRPs). There, by interacting with major heat shock protein Hsp70, S100A4 plays an important cytotoxic role. The resulting stably formed complex of PGRPs, S100A4 and Hsp70 is required for the identification and binding between a lymphocyte and a target cell. Here, we investigated the S100A4 functions in CD4+CD25+PGRPs+S100A4+ lymphocyte cytotoxicity against target cells. We demonstrated that those lymphocytes do not form a stable complex with the tumor target cells that themselves have S1004A on their surface. That observation can be explained by our finding that S100A4 precludes the formation of a stable complex between PGRPs, S100A4 (on the lymphocytes' surface), and Hsp70 (on the target cells' surface). The decrease in S100A4 level in CD4+CD25+PGRPs+S100A4+ lymphocytes inhibits their cytotoxic activity, while the addition of S100A4 in the medium restores it. Thus, the resistance of target cells to CD4+CD25+PGRPs+ S100A4+ lymphocyte cytotoxicity depends on their S100A4 expression level and can be countered by S100A4 antibodies.
Assuntos
Antígenos CD4/metabolismo , Proteínas de Transporte/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Linfócitos/imunologia , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Citotoxicidade Imunológica , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos , Paclitaxel/farmacologiaRESUMO
Naïve non-activated lymphocytes are capable of releasing the chemoattractant complex Tag7-Mts1 and can migrate along the gradient of its concentration. After activation of these cells by IL-2, they acquire the abilities to kill tumor cells and to release the cytotoxic Tag7-Hsp70 complex, which is accompanied by a loss of both the Tag7-Mts1-mediated lymphocyte chemotaxis and the ability to release this chemoattractant into the conditioned medium.
Assuntos
Quimiotaxia/imunologia , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Citocinas/imunologia , Imunidade Celular , Interleucina-2/imunologia , Linfócitos/imunologia , Neoplasias/imunologia , Humanos , Células K562 , Ativação LinfocitáriaRESUMO
The effect of the transcription factor Oct-1 (POU2F1) on the expression of the tumor cell marker metastasin (Mts1/S100A4) was studied. Comparative analysis of various tumor lines showed no clear correlation between the expression level of Mts1/S100A4 and the content of Oct-1. However, at stable transfection of tumor cells with Oct-1A, Oct-1L, and Oct-1X isoforms we detected an elevated level of Oct-1, which stimulated Mts1/S100A4 secretion. These findings extend our understanding of the molecular mechanisms of the tumorigenic effect of Oct-1.
Assuntos
Neoplasias/metabolismo , Fator 1 de Transcrição de Octâmero/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Far-Western Blotting , Linhagem Celular Tumoral , Meios de Cultura , Feminino , Humanos , Isoformas de ProteínasRESUMO
PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7-Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4(+) and CD8(+) lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.
Assuntos
Quimiotaxia de Leucócito/imunologia , Inibidor p16 de Quinase Dependente de Ciclina/imunologia , Citocinas/imunologia , Imunidade Inata , Células Matadoras Naturais/imunologia , Imunidade Adaptativa , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Separação Celular , Quimiotaxia de Leucócito/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/farmacologia , Citocinas/genética , Citocinas/farmacologia , Escherichia coli , Regulação da Expressão Gênica , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Cultura Primária de Células , Ligação Proteica , Proteínas Recombinantes , Transdução de SinaisAssuntos
Antígenos HLA/metabolismo , Células Matadoras Ativadas por Linfocina/imunologia , Linfócitos T Citotóxicos/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Células HeLa , Humanos , Interleucina-2/farmacologia , Células K562 , Células Matadoras Ativadas por Linfocina/classificação , Ativação Linfocitária , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/classificaçãoRESUMO
Peptidoglycane-recognizing protein Tag7 formed a complex with S100A4 (a representative of S100 protein family), the apparent dissociation constants in the absence and presence of Ca2+ were 2 x l0(-8) M and 10(-9) M, respectively. Analysis of fluorescence spectra of hydrophobic fluorescent probe 2-toluidinyl naphthalene-6-sulfonate in the presence of S100A4 and Tag7 proteins showed that extensive area or several sites are involved into the complex formation between these proteins. The formation of Tag7-S100A4 complex had virtually no effect on the role of S100A4 in the regulation of intracellular Ca2+ metabolism. Removal of not only Tag7, but also S100A4 from neutrophil conditioned medium reduced lysis of E. coli cell, while addition of the Tag7-S100A4 complex to the medium restored antibacterial activity.
Assuntos
Citocinas/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas S100/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados , Citocinas/genética , Humanos , Neutrófilos/citologia , Neutrófilos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/genéticaRESUMO
S100A4 protein is present in low concentrations (2.1-15.7 ng/10(6) cells) in lymphocyte and neutrophil culture medium. Addition of stimulants to the cells did not lead to an appreciable increase in the content of this protein. The initial content of S100A4 is significantly higher (92-447 ng/10(6) cells) in culture media of highly metastatic KSML-100 adenocarcinoma and M3 and B16 melanoma cells. The release of S100A4 by these cells significantly increased after addition of lymphocytes and Tag7/Hsp70 cytotoxic complex. Repeated injection of antibodies to S100A4 to mice with transplanted M3 melanoma inhibited tumor growth.
Assuntos
Linfócitos/metabolismo , Neoplasias/metabolismo , Proteínas S100/metabolismo , Animais , Anticorpos/imunologia , Biomarcadores Tumorais/metabolismo , Humanos , Células K562 , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias/patologia , Proteína A4 de Ligação a Cálcio da Família S100Assuntos
Proteínas de Transporte/toxicidade , Proteínas de Transporte/uso terapêutico , Proteínas de Choque Térmico HSP70/toxicidade , Proteínas de Choque Térmico HSP70/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Proteínas de Transporte/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/administração & dosagem , Humanos , Camundongos , Transplante de Neoplasias , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Proteínas Recombinantes/toxicidadeAssuntos
Citocinas/imunologia , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico HSP70/imunologia , Células Matadoras Ativadas por Linfocina/imunologia , Receptor fas/imunologia , Animais , Brefeldina A/farmacologia , Células Cultivadas , Citocinas/química , Citocinas/isolamento & purificação , Citocinas/metabolismo , Citotoxicidade Imunológica , Ácido Egtázico/farmacologia , Eletroforese , Complexo de Golgi/imunologia , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/isolamento & purificação , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Células K562 , Células Matadoras Ativadas por Linfocina/citologia , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Camundongos , Inibidores da Síntese de Proteínas/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Receptor fas/genéticaAssuntos
Proteínas de Transporte/imunologia , Citocinas/imunologia , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/imunologia , Adenosina Trifosfatases/metabolismo , Animais , Western Blotting/métodos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Cromatografia em Gel , Citocromos c/metabolismo , Citocinas/química , Citocinas/genética , Citocinas/metabolismo , Citotoxicidade Imunológica , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Camundongos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
The response of two cell lines, CSML-0 (does not express metastasin) and CSML-100 (high expression of metastasin) to cytolytic action of glucocorticoid was studied. Dexamethasone (1 microM) induced apoptosis of CSML-0 cells, while CSLM-100 cells were resistant to its cytolytic action. Apoptotic death of CSLM-100 cells was induced by incubation with dexamethasone in the presence of Ca-ATPase inhibitors, vanadate or thapsigargin. Metastasin, a proteins of the S-100 family, activated Ca-ATPase and ATP-dependent Ca2+ transport in plasmolemmal fraction of CSML-100 cells. Experiments showed that metastasin-induced activation of Ca-ATPase is a possible mechanisms of CSML-100 cell resistance to cytolytic dexamethasone action.
Assuntos
Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , Proteínas S100/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos Hormonais/farmacologia , Apoptose/fisiologia , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Eletroforese , Inibidores Enzimáticos/farmacologia , Camundongos , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/farmacologia , Tapsigargina/farmacologia , Células Tumorais Cultivadas , Vanadatos/farmacologiaAssuntos
Citocinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Apoptose/fisiologia , Humanos , Immunoblotting , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Camundongos , Peso Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/isolamento & purificação , Receptores de Citocinas/química , Receptores de Citocinas/isolamento & purificação , Receptores de Citocinas/metabolismo , Células Tumorais CultivadasRESUMO
We show that various nonlymphoid cultured cells can activate the production of cytotoxic factors in response to direct contact with cells of a different kind. Accumulation of cytotoxic factors in the medium was detected 1 h after contact of K562 and L929 cells or after contact of L929 cells with purified membranes of K562 cells. TNF-alpha or immunologically related proteins, or both, but not Fas-ligand or lymphotoxin, were also accumulated in membranes of K562 and L929 cells shortly after these cells had been allowed to contact each other. The cytotoxic factors expressed by nonlymphoid cells trigger apoptosis of target cells. These observations strongly suggest that nonlymphoid cells possess molecular mechanisms controlling cellular compatibility.