RESUMO
Recent epidemiological studies indicate that the antidiabetic drug metformin has chemosensitizing and chemopreventive effects against carcinogenesis. Here, we demonstrate that metformin exerts varying degrees of antitumor activity against human leukemia cells, as reflected by differences in growth inhibition, apoptosis, and alterations to metabolic enzymes. In metformin-sensitive cells, autophagy was not induced but rather it blocked proliferation by means of arresting cells in the S and G2/M phases which was associated with the downregulation of cyclin A, cyclin B1, and cdc2, but not that of cyclin E. In 10E1-CEM cells that overexpress Bcl-2 and are drug-resistant, the effect of metformin on proliferation was more pronounced, also inducing the activation of the caspases 3/7 and hence apoptosis. In all sensitive cells, metformin decreased the Δψ m and it modified the expression of enzymes involved in energy metabolism: PKCε (PKCepsilon) and PKCδ (PKCdelta). In sensitive cells, metformin altered PKCε and PKCδ expression leading to a predominance of PKCε over PKCδ which implies a more glycolytic state. The opposite occurs in the nonresponsive cells. In conclusion, we provide new insights into the activity of metformin as an antitumoral agent in leukemia cells that could be related to its capability to modulate energy metabolism.
RESUMO
BACKGROUND: Bexarotene is the first synthetic retinoid X receptor-selective retinoid (rexinoid) approved for the treatment of cutaneous T-cell lymphoma (CTCL). However, little is known about the signalling pathways by which it exerts its anticarcinogenic effect. OBJECTIVES: To characterize the effects of bexarotene in CTCL cell lines and elucidate the underlying molecular pathways of its antineoplastic effect. METHODS: The cell lines Hut-78, HH and MJ were used. Cell viability was assessed with the XTT assay. The self-renewal potential of cells after bexarotene treatment was studied with the methylcellulose clonogenic assay. Flow cytometry was used to analyse the effects on cell cycle, Ki-67 expression and apoptosis induction. Cell cycle and apoptosis-related protein expression were determined by Western blot and immunofluorescence. RESULTS: Bexarotene induced a loss of viability and more pronounced inhibition of clonogenic proliferation in HH and Hut-78 cells, whereas the MJ line exhibited resistance. Bexarotene upregulated and activated Bax in sensitive lines, although not enough to signal significant apoptosis. Instead, all data point to the inhibition of proliferation, rather than apoptosis, as the main mechanistic action of the rexinoid. Bexarotene signals both G(1) and G(2)/M arrest by the modulation of critical checkpoint proteins. We further found that bexarotene activates p53 by phosphorylation at Ser15, which influences the binding of p53 to promoters for cell cycle arrest, induces p73 upregulation, and, in concordance, also modulates some p53/p73 downstream target genes, such as p21, Bax, survivin and cdc2. Bexarotene-mediated ataxia telangiectasia mutated protein (ATM) activation in all studied lines suggests that ATM is likely to be the p53/p73 upstream activator. CONCLUSIONS: Our data indicate for the first time that bexarotene exerts its effect in CTCL mainly by triggering the p53/p73-dependent cell cycle inhibition pathway, probably by upstream ATM activation. Therefore, bexarotene-modulated genes represent potential biomarkers to assess the response to treatment of patients with CTCL.
Assuntos
Anticarcinógenos/farmacologia , Proteínas de Ligação a DNA/metabolismo , Linfoma Cutâneo de Células T/patologia , Proteínas Nucleares/metabolismo , Neoplasias Cutâneas/patologia , Tetra-Hidronaftalenos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Bexaroteno , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Humanos , Linfoma Cutâneo de Células T/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Nucleares/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteínas Supressoras de Tumor/efeitos dos fármacosRESUMO
Previous results have shown that the metastatic colonization with B16F10 melanoma in vivo increased after in vitro treatment of the cells with IL-2 or IL-6. To further investigate the mechanisms underlying this effect, we have studied adhesion, invasion, and proliferation properties of B16 melanoma, using two sublines with different metastatic ability. Adhesion of tumor cells to Matrigel coats increased using IL-6, which also induced upregulation of VLA-4 expression in both sublines. Unexpectedly, invasion through Matrigel filters was almost completely inhibited by IL-6 and decreased in the presence of IL-2. Cell growth was not affected by these interleukins; however, IL-6 could partially overcome the proliferation blockade induced by stress conditions. Taken together, these results suggest that upregulation of adhesion properties and/or the protective effect induced by IL-6 could account for the enhancement of metastasis exerted by this interleukin.
Assuntos
Interleucina-2/farmacologia , Interleucina-6/farmacologia , Melanoma Experimental/secundário , Metástase Neoplásica/patologia , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Contagem de Células , Divisão Celular/efeitos dos fármacos , Colágeno/metabolismo , Combinação de Medicamentos , Matriz Extracelular/metabolismo , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Integrina alfa4beta1 , Integrinas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Laminina/metabolismo , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/fisiopatologia , Proteoglicanas/metabolismo , Receptores de Interleucina-2/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Células-Tronco/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.
Assuntos
Interleucina-2/fisiologia , Melanoma/imunologia , Melanoma/patologia , Anticorpos Monoclonais/imunologia , Comunicação Autócrina , Divisão Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Humanos , Interleucina-2/biossíntese , Interleucina-2/farmacologia , Metástase Neoplásica , Receptores de Interleucina-2/imunologia , Proteínas Recombinantes/farmacologia , Células Tumorais CultivadasRESUMO
Serum soluble interleukin-2 receptor (sIL-2R), intercellular adhesion molecule-1 (sICAM-1) and interleukin-10 (IL-10) have each been reported as useful markers for melanoma progression. To evaluate the clinical relevance of these three markers, we simultaneously analysed their serum levels in patients with melanoma. A longitudinal study with a 3-year follow-up was performed and different stages of the disease were considered. Mean values of sIL-2R were significantly higher than in normal controls in all stages and correlated with the disease progression. The prognosis of patients with levels > 529 U/ml of sIL-2R was significantly poorer than in patients with sIL-2R levels < 529 U/ml. Levels of sICAM-1 were also elevated in melanoma patients, specially at the time of the metastatic disease. Serum IL-10 levels were more frequently detectable in the patients that developed metastasis during follow-up, and the prognosis of patients with detectable IL-10 levels was significantly poorer than in those patients with IL-10 undetected levels. Statistical analysis based on Logistic and Cox regression models showed that only sex, stage and sIL-2R value are factors significantly associated with metastatic progression. Moreover, high levels of sIL-2R could be a risk factor for malignant progression in melanoma.
Assuntos
Biomarcadores Tumorais/sangue , Molécula 1 de Adesão Intercelular/sangue , Interleucina-10/sangue , Melanoma/sangue , Receptores de Interleucina-2/sangue , Adulto , Idoso , Feminino , Seguimentos , Humanos , Modelos Logísticos , Estudos Longitudinais , Masculino , Melanoma/patologia , Melanoma/secundário , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Solubilidade , Análise de SobrevidaRESUMO
In this work we have studied the behavior of some cell structures, such as actin, tubulin and chromatin during apoptosis induced in F9 cells after retinoic acid treatment. In this experimental model, all defined steps of morphological changes described for apoptosis are observed. The correlation between a partial maintenance of F-actin and microtubular structures and the spatial distribution of F-actin suggests a possible relationship between this molecule and the characteristic shape changes observed in apoptosis. Additionally, the disposition of monomeric G-actin suggests a possible relationship between the fragmentation of this molecule and the cleavage of DNA. The analysis of the U2af1-rs1 specific sequence shows that the internucleosomal fragmentation observed in this gene is randomly produced during apoptosis and is not dependent of demethylation status. The results obtained confirm that specific cleavage of these cell structures is inherent to the development of the apoptotic process and do not exclude the possibility that proteolysis of key actin and/or tubulin molecules or the cleavage of specific chromatin sequences other than the ones analyzed here, could control the different phases of the apoptotic process.
Assuntos
Apoptose , Citoesqueleto/ultraestrutura , Proteínas do Tecido Nervoso , Proteínas Nucleares , Ribonucleoproteínas , Actinas/metabolismo , Actinas/ultraestrutura , Animais , Diferenciação Celular , Cromatina/metabolismo , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica , Proteínas/genética , Fator de Processamento U2AF , Tretinoína/farmacologia , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestrutura , Células Tumorais CultivadasRESUMO
In the present study, the effect of in vitro cyclosporin A (CsA) treatment on IL-2R expression and the metastatic behavior of B16F10 melanoma cells has been reported. CsA treatment was found to increase the percentage of B16F10 cells expressing the alpha-subunit of IL-2R on the cell surface and also at the mRNA level. Moreover, CsA treated B16F10 cells also express the beta-subunit of IL-2. In vivo experiments showed that CsA increases the affinity of B16F10 metastazing cells for the liver and decreases that for the lung. CsA modulated the expression of MHC class I and class II antigens, but no significant differences in the resistance of CsA-treated B16F10 cells to NK lysis were observed. Finally, proliferation of B16F10 cells in the presence of several doses of CsA did not vary and CsA increased the amount of IL-1beta mRNA expression. These results suggest that CsA, through the modulation of cytokines and MHC antigen expression on B16F10 cells, could have an effect upon the metastatic progression of the B16F10 melanoma.
Assuntos
Ciclosporina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma Experimental/patologia , Metástase Neoplásica , Proteínas de Neoplasias/biossíntese , Receptores de Interleucina-2/biossíntese , Animais , Feminino , Masculino , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , Receptores de Interleucina-2/genética , Organismos Livres de Patógenos Específicos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Elevated soluble IL-2 receptor (sIL-2R) and IL-6 serum concentrations have been reported as adverse prognostic factors in several types of cancer. In order to determine whether these factors are predictive of metastatic progression in melanoma, sIL-2R and IL-6 levels were measured in sera from 172 patients with melanoma and 60 in healthy controls. Mean sIL-2R values were significantly higher in the patients than in normal controls and the highest values were observed in those that developed metastasis during follow-up. However, no correlation was found with the stage of the disease. Serum IL-6 levels were found to be correlated with age and sex, but not correlated with sIL-2R levels. Statistical analysis was based on logistic and Cox regression models. The factors considered were age, sex, stage, disease-free interval and serum sIL-2R and IL-6 levels. The analysis showed that only the sIL-2R value is significantly linked to metastatic progression. This finding suggests that high serum levels of sIL-2R could be a predictive factor of metastatic progression in malignant melanoma.
Assuntos
Interleucina-6/sangue , Melanoma/sangue , Receptores de Interleucina-2/sangue , Neoplasias Cutâneas/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
We had previously shown that murine B16F10 melanoma cells express the receptor for IL-2, transcribe the gene for IL-2 and respond to its factor by increasing their proliferation. In the present work we have investigated the effect of in vitro IL-2 treatment on the metastatic ability of B16F10 cells. In vivo experiments showed that the metastatic colonization of the liver was notably higher after intrasplenic inoculation of IL-2-treated cells. However, no change was observed when cells were intravenously inoculated. In vitro, cells became more resistant to NK lysis although the cytometric analysis of class 1 MHC molecules revealed a decrease in H-2Kb expression. In contrast IL-2 induced a two fold increment in the expression of la antigen. On the other hand slot-blot analysis showed that IL-2 gene expression could be upregulated, however no free IL-2 was released into the culture medium of B16F10 cells. We conclude that IL-2 increases the ability of B16F10 cells to metastase to the liver. The increase in the resistance to NK activity and in la antigen expression could be involved in the mechanisms underlying this effect.
Assuntos
Interleucina-2/farmacologia , Neoplasias Hepáticas Experimentais/secundário , Melanoma Experimental/patologia , Animais , Antígenos de Histocompatibilidade/análise , Interleucina-2/genética , Interleucina-2/metabolismo , Células Matadoras Naturais/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análiseRESUMO
In this study, we analyzed interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression in murine B16F10 melanoma and studied the effect of recombinant IL-2 (rIL-2) on the proliferation of these cells. Flow cytometry analysis revealed the presence of the IL-2R alpha subunit in B16F10 melanoma, with a mean positivity rate of 30%. Using confocal microscopy, the expression of this chain could be visualized on the surface of B16F10 cells and in intracellular compartments when the cells were permeabilized with ethanol. In addition to the alpha subunit, the IL-2R beta subunit was also expressed in B16F10 cells as shown by reverse transcription and polymerase chain reaction analysis. The functionality of the IL-2R on B16F10 cells was shown by the fact that cell proliferation increased dose-dependently with the addition of rIL-2 to the culture medium. We also detected expression of the IL-2 gene in B16F10 cells. In Northern blot assays, a typical band of 0.9 kb corresponding to IL-2 mRNA was observed, although supernatants from B16F10 cultures had no detectable IL-2 activity. Furthermore, the addition of neutralizing antibody (anti-IL-2) to cell cultures had no effect on cell proliferation. From these results, we concluded that an IL-2 signalling system is present in murine B16F10 melanoma cells and that IL-2 favors B16F10 cell proliferation, suggesting a role for this cytokine in the tumoral activity of these cells.
Assuntos
Interleucina-2/metabolismo , Melanoma Experimental/patologia , Receptores de Interleucina-2/metabolismo , Animais , Sequência de Bases , Divisão Celular , Primers do DNA/química , Técnica Indireta de Fluorescência para Anticorpo , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Neoplásico/genética , Células Tumorais CultivadasAssuntos
Melanoma Experimental/imunologia , Receptores de Interleucina-2/metabolismo , Animais , Sequência de Bases , Membrana Celular/imunologia , Ciclosporina/farmacologia , Primers do DNA/genética , Imunossupressores/farmacologia , Interleucina-2/farmacologia , Melanoma Experimental/genética , Melanoma Experimental/secundário , Camundongos , Reação em Cadeia da Polimerase , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina-2/química , Receptores de Interleucina-2/genética , Células Tumorais CultivadasRESUMO
Maintaining B16F10 tumor cells in stirring culture for 48 h leads to an increase in lung and liver colonizing capacity in comparison with cells in adherent culture. Parallel to the increased metastatic capacity, we have observed a decrease in the proliferative rate of tumor cells (as the percentage of proliferating cell nuclear antigen-positive cells) and an increase in the population of tumor cells expressing Ia antigen. These results are not exclusive to B16F10 cells, since the same results were obtained when we analyzed 3LL cells maintained in identical culture conditions. In all the tumor lines tested, we found an association between the nonproliferating and the Ia-positive cell populations. We induced Ia expression by treating B16F10 cells in adherent culture with the lectin concanavalin A and again, coincident with an increase in metastatic capacity, we found the same association between the two parameters analyzed--nonproliferating state and Ia antigen expression. In addition, it was found that B16F10 cells induce lymphocytic proliferation, and a direct relationship was established between the number of Ia+ cells and lymphocytic proliferation.
Assuntos
Antígenos de Histocompatibilidade Classe II/análise , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Melanoma Experimental/imunologia , Animais , Concanavalina A/farmacologia , Meios de Cultura , Ativação Linfocitária , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mitomicinas/farmacologia , Antígeno Nuclear de Célula em Proliferação/análise , Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
The expression of various protooncogenes (myc, Ki-ras, Ha-ras, erbB, fms, sis, jun and fos) and a gene implicated in multidrug resistance (mdr1) was investigated in cell sublines, isolated from a rat rhabdomyosarcoma cell line, and in the corresponding tumors induced by injection of these cells into syngeneic rats. These cell lines and tumors, selected or not by treatment with chlorozotocin (Czt) or adriamycin (Adr), differed in their tumorigenicities and metastatic potentials. Our results showed that 1) an increased expression of some protooncogenes could be correlated with the metastatic potential of tumors; 2) such a correlation was not observed in the cultured cells from which these tumors were derived; 3) mdr1 expression, similarly to that of protooncogenes, was correlated with metastatic potential in all tumors except the Adr-selected tumors.
Assuntos
Resistência a Medicamentos/genética , Proto-Oncogenes , Rabdomiossarcoma/genética , Animais , Northern Blotting , Linhagem Celular , Células Clonais , Doxorrubicina/farmacologia , Expressão Gênica , Metástase Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Ratos , Rabdomiossarcoma/patologia , Transcrição GênicaRESUMO
We have examined the anatomical-functional sites within mouse liver where phenylhydrazine (PHZ)-induced hematopoietic foci, and M5076 reticulum cell sarcoma, B16F10 melanoma and Lewis lung-carcinoma cells specifically develop as colonies after intrasplenic injection. Cancer foci occurred predominantly in the 2.4 to 4.0 segment of the sinusoidal pathway, corresponding to hepatic acinar zone I. No significant differences were detected between different types of tumor, including their different tendencies to spontaneously metastasize liver, or as a result of the different procedures used for obtaining foci or metastases. In addition, PHZ-treatment of mice previously injected with tumor cells, resulted in double colonization of the liver tissue by both hematopoietic and cancer cells, predominantly in zone I. This spatial coincidence indicates that non-cancer-specific mechanisms operate in zone I, either promoting implantation and/or growth of cell colonies or, alternatively, inhibiting these processes in the region surrounding the central vein (Rappaport zone 3). Our observations failed to reveal mutual displacement of cancer or hematopoietic foci by potential competition for development sites in zone I. Enumeration and diameter measurements of cancer foci in PHZ-treated animals showed that the presence of hepatic hematopoietic foci coincided with a significant increase in the hepatic metastasis volume. However, the fact that no significant differences in pulmonary metastases occurred in both the PHZ-treated and control mice given tail-vein injection of cancer cells, and that PHZ reduces cancer cell proliferation in vitro, reveal evidence of local interactions with hematopoietic foci which promote growth of cancer foci in liver.