RESUMO
Se describe un caso reciente de acrometástasis en partes blandas de mano y se realiza la primera revisión bibliográfica de esta excepcional presentación de las acrometástasis. Se trata de un varón de 74 años, fumador de 20 cigarrillos por día desde los 18 años, que consultó por una lesión exofítica sangrante en cara dorsolateral del índice izquierdo. Se realizó biopsia y extirpación del 2º eje del dedo (rayo). La radiografía fue normal y la anatomía patológica confirmó una metástasis de carcinoma espinocelular diferenciado con indemnidad ósea. En el estudio clínico se descubrió un carcinoma pulmonar que luego se complicó con una fístula esofago-traqueal tratada mediante un stent. Falleció un mes después en su domicilio por una hematemesis, 12 meses posteriores a la primera consulta. La acrometástasis fue la primera manifestación de una neoplasia y se produjo en los tejidos blandos de la mano, una forma muy rara de presentación. Las metástasis en los huesos de las manos y pies son raras. La incidencia en mano es de 0,25-0,60 porciento entre los tumores que dan metástasis óseas. Las acrometástasis en tejidos blandos son aún más raras y se han comunicado muy pocos casos. El comportamiento biológico de las metástasis pulmonares en los huesos y su extrema rareza no han sido dilucidados. En general ocurren durante la diseminación terminal de las neoplasias e indican un mal pronóstico. El tiempo promedio de sobrevida es habitualmente menor de un año. Ocasionalmente pueden ser la forma de presentación de un blastoma. Los tumores más frecuentemente producen estas lesiones son los de pulmón, riñón, mama y colon. Los tumores no carcinomatosos son causa excepcional de estas lesiones. A pesar de su rareza, estas acrometástasis pueden ser importantes por dos motivos: primero, pueden ser la primera manifestación de una neoplasia oculta. segundo, su sintomatología puede ser confundida y se demora el diagnóstico. Pueden simular enfermedades inflamatorias o enfermedades subungueales benignas. La biopsia convencional asegura el diagnóstico. La amputación quirúrgica suele ser el mejor tratamiento paliativo y la radioterapia puede a veces ser efectiva
Assuntos
Humanos , Masculino , Idoso , Cirurgia Geral , Mãos , Neoplasias Pulmonares , Metástase Neoplásica , Radioterapia , Neoplasias de Tecidos MolesRESUMO
Se describe un caso reciente de acrometástasis en partes blandas de mano y se realiza la primera revisión bibliográfica de esta excepcional presentación de las acrometástasis. Se trata de un varón de 74 años, fumador de 20 cigarrillos por día desde los 18 años, que consultó por una lesión exofítica sangrante en cara dorsolateral del índice izquierdo. Se realizó biopsia y extirpación del 2º eje del dedo (rayo). La radiografía fue normal y la anatomía patológica confirmó una metástasis de carcinoma espinocelular diferenciado con indemnidad ósea. En el estudio clínico se descubrió un carcinoma pulmonar que luego se complicó con una fístula es
Assuntos
Humanos , Masculino , Idoso , Metástase Neoplásica , Neoplasias de Tecidos Moles , Neoplasias Pulmonares , Mãos/patologia , Cirurgia Geral , RadioterapiaRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative illness and the most frequent cause of dementia in the elderly. The identification of activated microglia within neuritic plaques, coupled with the presence of numerous inflammatory proteins, suggests that inflammation is an integral part of the pathogenetic process in AD. In the present paper we have investigated the levels of circulating inflammatory mediators as potential AD biomarkers concentrating essentially on (a) soluble CD40 (sCD40), a member of the tumor necrosis factor receptor superfamily lacking the membrane-associated endodomain by alternative splicing, and (b) transforming growth factor (TGF)-beta 1, a cytokine deeply involved in AD and playing a protective role on CNS. Decrease of TGF-beta1 in AD patients could enhance the effects of pro-inflammatory cytokines produced by activated microglia as well as the expression of factors, such as the CD40/CD40 ligand complex, by microglia and astrocytes. Total venous blood samples were obtained from 33 patients with clinical diagnosis of possible late-onset AD, 40 healthy age-matched and 11 healthy young individuals. A significant increase of sCD40 levels plasma of AD patients versus healthy controls was measured, concomitantly with a decrease in TGF-beta1 concentration. These variations, however, showed no correlation with the expression of ApoE epsilon 4 allele, which was determined in order to assess the different frequency of this risk factor between AD and control groups. Since no comparable modifications were detected in patients affected by Parkinson's disease or non-AD-based dementia, we propose that sCD40 and TGF-beta1 plasma levels might represent possible differential biomarkers of AD, and be useful pre-mortem to support the clinical diagnosis of late-onset AD.
Assuntos
Doença de Alzheimer/diagnóstico , Antígenos CD40/sangue , Fator de Crescimento Transformador beta/análise , Adulto , Idoso , Doença de Alzheimer/sangue , Doença de Alzheimer/genética , Apolipoproteínas E/genética , Biomarcadores/sangue , Diagnóstico Diferencial , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Pessoa de Meia-Idade , Doença de Parkinson/sangue , Doença de Parkinson/diagnóstico , Fatores de Risco , Fator de Crescimento Transformador beta1RESUMO
Many studies have attempted to define the state of differentiation of melanoma cells and to correlate it with other critical parameters of malignancy such as the tumorigenic and metastatic nature of the cells. In the present paper we focused on the possible relationships between the novel protein kinase C isoform nPKCdelta, melanin synthesis and proliferative capacity in a primary human melanoma cell line WM115. Cells were transfected to produce overexpression of this isoform and the effects on melanin synthesis, cyclin-E dependent kinase (cdk2) activity and cyclin E expression were studied. It was shown that translocation of nPKCdelta into the nucleus affects melanin synthesis and inhibits cdk2 activity. As a compensatory effect, the level of cyclin E increases. In view of these results we suggest a model for the role of nPKCdelta in melanoma cells that may offer a new therapeutic perspective.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Isoenzimas/fisiologia , Melaninas/biossíntese , Melanoma/patologia , Proteína Quinase C/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transporte Ativo do Núcleo Celular , Animais , Western Blotting , Ciclo Celular , Divisão Celular , Tamanho Celular , Ciclina E/biossíntese , Ciclina E/genética , Quinase 2 Dependente de Ciclina , Indução Enzimática , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas/genética , Camundongos , Monofenol Mono-Oxigenase/biossíntese , Monofenol Mono-Oxigenase/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteína Quinase C/genética , Proteína Quinase C-delta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Células Tumorais Cultivadas/metabolismoRESUMO
A variety of inflammatory proteins have been identified in brains of Alzheimer's disease (AD) patients, including inflammatory cytokines, acute phase proteins and complement components. In the present paper we have investigated the levels of circulating inflammatory mediators as potential biomarkers of this disease, concentrating mostly on transforming growth factor beta (TGF-beta1) in plasma and on nitric oxide synthase (NOS) activity in leukocytes. Plasma and leukocytes were isolated from 48 sporadic AD and 23 healthy control subjects of same age and sex. Since alpha2-Macroglobulin (alpha2M), an acute phase protein possibly involved in AD, is an important modulator of TGF-beta1 activity, binding and targeting this cytokine to its appropriate site of action, we have investigated the possible complex between TGF-beta1 and alpha2M in plasma of the same subjects. The results demonstrate a significant reduction of TGF-beta1 levels in plasma of AD patients. A complex between alpha2M and TGF-beta1 occurred in AD as well as healthy elderly control subjects, however, with no significant differences. Moreover, alpha2M appeared to bind only the inactive form of this cytokine. In contrast, NOS activity increased significantly in leukocytes of AD patients. Therefore, we suggest the combined determination of TGF-beta1 in the plasma and of NOS activity in the leukocytes as biomarkers of AD.
Assuntos
Doença de Alzheimer/sangue , Leucócitos/enzimologia , Óxido Nítrico Sintase/sangue , Fator de Crescimento Transformador beta/sangue , Biomarcadores , Humanos , Fator de Crescimento Transformador beta1 , alfa-Macroglobulinas/análiseRESUMO
In this study we analysed the effect of overexpressing novel protein kinase C delta isoform (n-PKC delta) on melanin synthesis and metastatic potential in the highly metastatic BL6 murine melanoma cells. The proliferative capacity in vitro and into matrigel in vivo were also examined. Although murine melanocytes express the n-PKC delta isoform, BL6 cells do not express this isoform at levels detectable by Western blot analysis. In untransfected and transfected cells we also studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a modulator of specific isoforms of PKC, and of bryostatin 1, a potent immunomodulator and antineoplastic drug and a partial agonist of PKC. Our results demonstrate a pivotal role for this isoform in melanin synthesis and the close relationship between n-PKC delta expression and its association with the particulate fraction, melanogenesis and metastatic potential. In fact, heterogeneous BL6 cells overexpressing n-PKC delta and all the clones isolated showed increased intracellular melanin and metastatic capacity. TPA and bryostatin 1 decreased n-PKC delta expression, the intracellular melanin level and metastatic capacity in both cell lines. Therefore both treatments were able to abolish the effects of overexpressing n-PKC delta.
Assuntos
Isoenzimas/fisiologia , Melanoma Experimental/enzimologia , Metástase Neoplásica , Proteínas de Neoplasias/fisiologia , Isoformas de Proteínas/fisiologia , Proteína Quinase C/fisiologia , Adjuvantes Imunológicos/farmacologia , Animais , Antineoplásicos/farmacologia , Briostatinas , Ciclo Celular , Divisão Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Isoenzimas/biossíntese , Isoenzimas/genética , Lactonas/farmacologia , Macrolídeos , Melaninas/biossíntese , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteína Quinase C/biossíntese , Proteína Quinase C/genética , Proteína Quinase C-delta , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Células Tumorais Cultivadas/patologiaRESUMO
Transforming growth factor-beta (TGFbeta) contributes to the promotion of invasion, metastasis, angiogenesis and even immunosuppression. Since overexpression of the delta isoform of protein kinase C (nPKCdelta) in BL6 murine melanoma cells (BL6T cells) increases their metastatic capacity, we investigated the possible involvement of TGFbeta1 in this process. Immunohistochemical analysis demonstrated lower levels of TGFbeta1 in BL6T lung metastases compared with BL6 lung metastases. On the other hand, higher levels of this cytokine, in particular in its active form, occur in the plasma of BL6T metastasized animals, suggesting a nPKCdelta-dependent TGFbeta1 release. Therefore, nPKCdelta-dependent TGFbeta1 release and activation may be involved in the greater angiogenic and metastatic capacity of murine melanoma BL6T cells.
Assuntos
Isoenzimas/biossíntese , Melanoma/metabolismo , Proteína Quinase C/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/sangue , Animais , Western Blotting , Densitometria , Imuno-Histoquímica , Neoplasias Pulmonares/secundário , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Transplante de Neoplasias , Neovascularização Patológica , Isoformas de Proteínas , Proteína Quinase C-delta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1 , Células Tumorais CultivadasRESUMO
Bryostatin 1 and phorbol esters reduce the intracellular melanin level in high metastatic overexpressing nPKCdelta BL6 (BL6T) cells, thereby inducing white experimental metastasis in syngeneic mice. We evaluate here the possible differences between white and black metastases induced by both treatments on the proliferative and metastatic potential as well as on the expression of some cytokines involved in the metastatic process such as TGFbeta, IL-10 and IFNgamma. The level of expression of gelatinase A is also considered. White and black metastases induced after the injection of bryostatin 1- or phorbol ester-treated cells into the tail vein of syngenic mice were isolated and analysed for the levels of LDH usually used as markers of cytotoxicity, for the levels of cytokines and gelatinase A or dissociated and cultured in vitro for a few passages. The cultured cells were analysed in vitro for the proliferative capacity and the melanin synthesis. The same cells were also re-injected into syngeneic mice and the number of experimental metastases were counted after 17 days or injected with matrigel in order to quantify the proliferative capacity in vivo. The results show only one significant difference between bryostatin I and phorbol ester, namely the cells obtained from white bryostatin 1-treated cells return to a black phenotype after a few passages in culture. This suggests that PKC mediates many of the biological effects of bryostatin 1 but that its effect is lost in vitro. On the other hand, white and black metastases (at least for metastases induced by BL6T cells treated with phorbol ester) do appear significantly different. In vivo white metastases show lower levels of LDH, lower levels of proliferative capacity into matrigel, higher levels of TGFbeta and IFNgamma and, when re-injected into syngeneic mice, give big black metastases. Therefore, in murine melanoma cells, the treatment with bryostatin I induces the appearance of a white population expressing different levels of TGFbeta, IFNgamma, IL-10 and gelatinase A. Such a white population is more difficult to diagnose and is capable of turning into a more aggressive phenotype under suitable environmental conditions.
Assuntos
Interferon gama/metabolismo , Interleucina-10/metabolismo , Neoplasias Pulmonares/patologia , Metaloproteinase 2 da Matriz/metabolismo , Melanoma/secundário , Fator de Crescimento Transformador beta/metabolismo , Animais , Antineoplásicos/efeitos adversos , Briostatinas , Interferon gama/genética , Interleucina-10/genética , L-Lactato Desidrogenase/metabolismo , Lactonas/efeitos adversos , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Macrolídeos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/toxicidade , Fator de Crescimento Transformador beta/genéticaRESUMO
Recently, PKC has been shown to play a pivotal role in physiological brain functions, connected with the memorizing processes and their correspondent progressive decline with brain aging. We have studied the age-dependent changes of PKC isoforms activity in connection with NOS expression and activity in specific brain areas such as hippocampus, cortex and striatum. Starting from middle aged rats, a significant inactivation of c-PKC isoforms occurred, with respect to young animals, in all the brain areas analysed. However, in middle aged animals, no significant changes in the protein level of the main PKC isoforms expressed in brain were demonstrated. Moreover, in the hippocampus and in the cortex of middle aged rats, an increased level of NOS activity--a substrate of PKC whose phosphorylation by the kinase inhibits NOS activity--has been demonstrated, reaching the same level that occurs in striatum. However, only in the hippocampus, deeply implicated in learning and memory functions, an increase of nuclear c-PKC activity and of i- and n-NOS mRNA levels was shown. Taken together, these results indicate that down-regulation of c-PKC activity occurring in middle aged rats, leads to higher levels of NO that may contribute to cell damage and to alter the neuronal physiological functions as described in older animals. Moreover, in the hippocampus, our results suggest a relationship between the translocation of c-PKC to the nucleus and the enhancement of the expression of i- and n-NOS.
Assuntos
Envelhecimento/metabolismo , Córtex Cerebral/enzimologia , Corpo Estriado/enzimologia , Hipocampo/enzimologia , Óxido Nítrico Sintase/metabolismo , Proteína Quinase C/metabolismo , Envelhecimento/genética , Animais , Isoenzimas/metabolismo , Masculino , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Sprague-DawleyRESUMO
Protein kinase C (PKC) comprises a family of at least 11 isoforms that play a pivotal role in the angiogenic and metastatic process. In this study we analysed the effect of two PKC isoform-selective inhibitors, Gö6976 an inhibitor of c-PKCalpha and betaI, and bisindolylmaleimide (BIM) that prevents the effect of phorbol ester, on the gelatinolytic, angiogenic and metastatic capacity of the low metastatic B16F1 and the highly metastatic BL6 murine melanoma cells. The treatment with BIM did not modify these processes in either cell line, while Gö6976 induced a significant decrease in the angiogenic, gelatinase and metastatic potential in the BL6 cells. Both inhibitors inversely modulated VEGF and bFGF expression. Nitric oxide synthase (NOS), however, showed no change in activity. Taken together our results demonstrate an involvement of the c-PKCalpha isoform in the metastatic and angiogenic process, possibly by reducing the gelatinolytic activity. Thus, the c-PKCalpha isoform may be a novel target for therapy.
RESUMO
The aim of the present paper was to analyse the effect of long-term inhibitory treatment, for at least 7 days, of individual protein kinase C (PKC) isoforms on the survival of LoVo human colon adenocarcinoma cells to doxorubicin exposure. The treatment for 2 h, after plating the cells, and after 3 days with 1 microM Gö6976, a specific inhibitor of protein kinase C (PKC)-alpha and -betal isoforms, induced on day 7 in LoVo cell lines (WT) a significant increased survival when these cells were exposed to increasing doxorubicin concentrations. In contrast, resistant LoVo cells (DX) did not show significant changes in the survival to doxorubicin exposure when incubated with the inhibitor of the same specific PKC isoforms. In addition, Gö6976 reduced the PKC-alpha activity (the main calcium-dependent PKC isoforms expressed) in both cell lines with contemporary increased expression. Under such conditions, an increased nuclear activity and an increased P-glycoprotein expression occurred only in WT-treated cells with respect to untreated cells. Taken together, our data indicate a specific relationship between PKC-alpha inhibition, the increased nuclear PKC-alpha activity as well as the increased expression of P-glycoprotein, possibly causing the acquisition of a resistant phenotype in WT LoVo cells.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Neoplasias do Colo/patologia , Doxorrubicina/farmacologia , Proteína Quinase C/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Sobrevivência Celular , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Isoenzimas , Fenótipo , Proteína Quinase C/metabolismo , Células Tumorais CultivadasRESUMO
The expression and subcellular distribution of liver cPKC alpha and beta, nPKC delta and aPKC zeta isoenzymes and the plasma levels of fibrinogen were measured in young, 2- and 6-month-old, and aged, 24-month-old, normal and turpentine-treated rats, to induce an aseptic inflammatory condition and the acute-phase response. In young and old rats a down-regulated expression of cPKC alpha and, to a lesser extent, beta isoenzymes, was observed 8 h after turpentine administration, i.e. at times preceding the maximal expression of fibrinogen mRNA. Under these conditions, the plasma fibrinogen levels peaked by 24 h in young animals, being up to 7-fold over the values of untreated controls at 72 h. By contrast, old untreated control rats showed 4-fold increases of basal plasma fibrinogen levels compared with young controls, with down-regulated expression of cPKC alpha. In old rats, treatment with turpentine increased up to 1.9-fold over the basal control values the fibrinogen concentration within 72 h. Levels similar to those of young turpentine-treated animals were reached at this time. The results of this study suggest a prominent role for cPKC alpha in eliciting the synthesis of fibrinogen after inducing an acute-phase response with turpentine administration in young as well as old rats. This isoform may act by regulating the serine phosphorylation of Stat3 transcription factor, whose activation is under IL-6 control, a multifunctional cytokine that is proving to be a major contributor to the acute-phase response. No evidence for a role of aPKC zeta or of nPKC delta was demonstrated under these conditions.
Assuntos
Reação de Fase Aguda/metabolismo , Envelhecimento/metabolismo , Fibrinogênio/metabolismo , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Animais , Masculino , Ratos , Ratos WistarRESUMO
Protein Kinase C (PKC) is a family of at least 11 closely related isoforms with different modality of activation, and intracellular and tissue distribution. The aim of the present work was to analyse the effect of treatment with 0.1 microM TPA as well as treatment with specific inhibitors of individual PKC isoenzymes (Gö6976 for c-PKC alpha and beta isoforms and BIM for c-PKCs and n-PKCs isoforms), on the NF-kB/IkB alpha pathway in the low and high metastatic B16F1 and BL6 murine melanoma cells. The DNA-binding activity of the transcription factors AP1, AP2, CREB and OTC was also considered. Different modality of activation for NF-kB and AP1 was demonstrated in the two cell lines with the possible specific involvement of c-PKCs isoforms. In fact, in the high metastatic BL6 cells the long-term treatment for 24 hours with TPA, with no c-PKC activation or the inhibition with Gö6976 as well as with BIM, induced an increased NF-kB and AP1 DNA-binding activity. In contrast, in the low metastatic B16F1 cells the short-term treatment with TPA, induced the activation of c-PKCs isoforms, and enhanced NF-kB and AP1 DNA-binding activity. No significant changes were demonstrated for AP2, CREB and OTC DNA-binding activity in both cell lines.
Assuntos
Carbazóis/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , Indóis/farmacologia , Isoenzimas/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Isoenzimas/antagonistas & inibidores , Cinética , Camundongos , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Metástase Neoplásica , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C beta , Proteína Quinase C-alfa , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição AP-2 , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
The development of tumor metastasis is a multistep process. Key aspects of this process are the interaction of tumor cells with the extracellular matrix, digestion of, and motility through the basement membrane and the induction of angiogenesis. In this study, we analysed the effects of a low dose of TPA (12-tetradecanoylphorbol-13-acetate; 0.1 microM on angiogenesis, proteolytic activity and lung colonizing potential of both weakly metastatic B16F1 cells and highly metastatic BL6 murine melanoma cells. Our results demonstrated opposite effects of TPA in the two cell lines. TPA-treated B16F1 cells showed enhanced release of basic FGF (bFGF) and vascular endothelial growth factor (VEGF) and increased angiogenic capacity and lung colony formation in vivo. In contrast, TPA-treated BL6 cells demonstrated a dramatic reduction of angiogenic and gelatinolytic activity and metastatic capacity. However, both cell lines showed an induction of VEGF as well as bFGF expression by TPA treatment suggesting that in BL6 cells antagonistic factors, inhibiting the angiogenic and metastatic capacity, are induced by this treatment.
Assuntos
Carcinógenos/farmacologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/patologia , Metástase Neoplásica , Neovascularização Patológica , Acetato de Tetradecanoilforbol/farmacologia , Animais , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento de Fibroblastos/biossíntese , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/patologia , Linfocinas/biossíntese , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Phorbol esters, known activators of c- and n-protein kinase C (PKC) isoforms, play a pivotal role in tumor promotion. In order to better understand the relationships between PKC activation, the metastatic potential and the proliferative capacity, we have analyzed the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment on the proliferative as well as on the cell cycle distribution and on the cell size of low and high metastatic murine B16F1 and B16-BL6 (BL6) melanoma cells, respectively. TPA-treated B16F1 cells showed an increased proliferative capacity up to 72 h, the cytofluorimetric analysis revealing an increased number of cells in the S + G2-M phase of the cell cycle. In contrast, TPA-treated BL6 cells reached a plateau at 48 h and showed an increased cell volume in the G1 and S phases of the cell cycle, with a reduction in the percentage of cells in the S + G2-M phase. Taken together, our results indicate opposite effects of TPA treatment in murine melanoma cells of different metastatic potential. TPA could cause a block in the G1 phase of the cell cycle with enhanced cell volume in high metastatic BL6 cells. The same treatment, on the contrary, induced an increased entry into the cell cycle of low metastatic B16F1 cells, suggesting a relationship between cell proliferation and the metastatic potential of B16 murine melanoma cells. Moreover, under the present conditions, classical PKC isoforms were inactivated, suggesting the involvement of the TPA-dependent novel PKCs.
Assuntos
Carcinógenos/farmacologia , Tamanho Celular/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Fase S/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Melanoma/patologia , Melanoma/fisiopatologia , Camundongos , Metástase Neoplásica , Proteína Quinase C/metabolismo , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/fisiologiaRESUMO
Metastasis is a multistep process in which protein kinase C (PKC) appears to be significantly involved. We analysed the activity and expression of classical (alpha, beta, gamma) and novel PKC epsilon isoforms in B16-F1 and B16-BL6 melanoma cells maintained under different culture conditions in vitro. We used high and low concentrations of tyrosine and phenylalanine in different media (DMEM or RPMI 1640 respectively) that affect the metastatic potential and also the proliferative capacity of the cells. We also tested a weakly metastatic amelanotic B78-H1 melanoma cell line which is unaffected by the different culture conditions. In both B16 melanoma cell lines activation of PKC alpha (without increased expression) occurred under growth conditions permissive of metastasis (DMEM). In contrast, the weakly metastatic amelanotic B78-H1 cell line showed a substantial inactivation of this isoform in the two different culture media, suggesting a specific involvement of PKC alpha in the metastatic process. Moreover, in B16 melanoma cells, novel PKC epsilon was activated under culture conditions which stimulated growth but not metastasis (RPMI 1640). In order to define the relationship between PKC activation and the metastatic process we also determined the release of cathepsin B. No correlation between PKC activity and cathepsin B release in either B16 melanoma cell lines could be demonstrated.
Assuntos
Melanoma Experimental/enzimologia , Metástase Neoplásica , Proteína Quinase C/biossíntese , Animais , Catepsina B/metabolismo , Técnicas de Cultura de Células/métodos , DNA de Neoplasias/biossíntese , Ativação Enzimática , Isoenzimas/biossíntese , Camundongos , Fenilalanina , Células Tumorais Cultivadas , TirosinaRESUMO
PKC, a family of 11 related isoforms, appears deeply implicated in carcinogenesis and in the metastatic process, however, little being known on the specific role of each isoform in that process. In this work we analysed the subcellular distribution and the in situ expression of classical PKC (alpha and beta) isoforms and the expression of PKC delta in the tumour and lung-metastases induced in the rat using the 'resistant hepatocyte' model of diethylnitrosamine-induced hepatocarcinogenesis. With respect to control untreated liver, an activation and increased expression of PKC alpha was observed in tumour and lung metastatic nodules, while cytosolic and membrane PKC beta was undetectable. In contrast, nuclear PKC beta showed an increased activity in the tumour and a marked increased activity and expression in metastatic nodules, suggesting a possible role of such isoform in the metastatic process. The analysis of PKC delta expression revealed a down regulation in both tissues with respect to control untreated liver, suggesting the inhibitory role of such isoform on tumour cell proliferation in agreement with our previous observations. Taken together, these results point to a different role for PKC alpha and delta in the development of tumour and metastatic nodules using the 'resistant hepatocyte' model of liver carcinogenesis, and suggest a possible involvement of nuclear PKC beta in the development of secondary tumours.
Assuntos
Carcinógenos , Núcleo Celular/enzimologia , Dietilnitrosamina , Isoenzimas/metabolismo , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Pulmonares/enzimologia , Proteína Quinase C/metabolismo , Animais , Caderinas/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/secundário , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
The membrane and cytosolic protein phosphorylation patterns in the early stages of diethylnitrosamine-induced rat liver carcinogenesis, promoted by 2-acetylaminofluorene in the diet plus partial hepatectomy (DEN-AAF-PH), were analyzed by two-dimensional gel electrophoresis in animals fed a low protein (5% casein) diet, or the original high protein (24% casein) diet, in order to modulate the development of GST-P-positive preneoplastic lesions. Compared with untreated controls, membrane and cytosolic protein phosphorylation patterns changed only slightly in low protein-fed rats 7 days post-hepatectomy, with no appearance of enzyme-altered hyperplastic foci in the liver sections. By contrast, high protein-fed animals demonstrated GST-P-positive preneoplastic lesions 7 days post-hepatectomy and several acidic and more basic high M(r) phosphorylated membrane (between 97 and 116 kDa) as well as cytosolic (between 97 and 200 kDa) proteins could be detected. In the presence of enzyme-altered hepatocytes in the liver sections, low protein-fed rats demonstrated at 60 days post-hepatectomy cytosolic protein phosphorylation patterns remarkably similar to those shown by 24% casein-fed animals at 7 days post-hepatectomy, suggesting close correlation between protein phosphorylation patterns and development of preneoplastic lesions during the early stages of DEN-AAF-PH liver carcinogenesis. This may arise by a constitutive activation of one or more signal transduction pathways, possibly involving protein kinase C, during liver tumour promotion.
Assuntos
Proteínas Alimentares/administração & dosagem , Neoplasias Hepáticas Experimentais/metabolismo , Proteínas de Membrana/metabolismo , Lesões Pré-Cancerosas/metabolismo , Proteínas/metabolismo , Animais , Dietilnitrosamina , Neoplasias Hepáticas Experimentais/induzido quimicamente , Masculino , Fosforilação , Ratos , Ratos Endogâmicos F344RESUMO
In a preclinical mouse model the plant ribosome-inactivating proteins (RIPs) pokeweed antiviral proteins PAP-1, and PAP-S and ricin A-chain (RTA) induced a pathological elevation of serum concentrations of glutamate pyruvate transaminase (GPT) and blood urea nitrogen (BUN) and had a significant immunosuppressive effect on B- and T-lymphocytes. The present analysis and comparison of the biodistribution and systemic/organ toxicity associated with RIP injection suggest a possible in vivo mechanism of action of PAP-1 and PAP-S and identify several limitations in the clinical use of these two toxins and RTA. When administered intravenously, PAP-1 and PAP-S consistently accumulated in kidneys and induced histologically documented damage to kidney and liver, with a LD50 of 3.3 mg/kg and 1.6 mg/kg for PAP-1 and PAP-S, respectively. In mice injected with PAP-S after chlorpromazine (CPZ) administration, GPT levels returned to normal between 24 and 72 h after toxin injection, while the BUN levels remained elevated. Mortality of the animals was delayed but all mice eventually succumbed. All the three toxins inhibited the expansion of anti-sheep red blood cells (SRBC) antibody-forming cells and the production of anti-SRBC antibody levels, although PAP-S showed the most potent activity. Despite the immunosuppressive activity, all toxins were highly immunogenic.
Assuntos
N-Glicosil Hidrolases , Proteínas de Plantas/toxicidade , Ribossomos/efeitos dos fármacos , Ricina/toxicidade , Alanina Transaminase/sangue , Animais , Nitrogênio da Ureia Sanguínea , Avaliação Pré-Clínica de Medicamentos , Feminino , Imunoconjugados , Rim/efeitos dos fármacos , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas a Pancreatite , Proteínas de Plantas/imunologia , Proteínas de Plantas/farmacocinética , Proteínas Inativadoras de Ribossomos Tipo 1 , Ricina/imunologia , Ricina/farmacocinética , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Distribuição TecidualRESUMO
The purpose of this study was to determine if decreasing dietary protein from 24% (high protein) to 5% casein (low protein), substituting sucrose and cornstarch isocalorifically for casein, would modify the activity of protein kinase C (PKC) alpha and beta isoenzymes, as well as the expression of PKC alpha, beta, delta and zeta subtypes in the particulate, soluble and nuclear fractions of rat liver, and the development of gamma-glutamyltranspeptidase (GGT)-positive foci and nodules in the early stages (4, 7 and 60 days post-hepatectomy) of diethylnitrosamine-induced carcinogenesis promoted by 2-acetylaminofluorene in the diet plus partial hepatectomy (DEN-AAF-PH). In rats fed the 5% casein diet, body and liver weights decreased significantly compared with 24% casein-fed animals. However, the PKC total activity was unmodified. In 5% casein-fed rats, over-expression of PKC delta in the liver particulate fraction was detected at 7 and 60 days post-hepatectomy, with no significant PKC alpha and beta isoform activation. These animals showed only scattered GGT-positive hepatocytes at 60 days post-hepatectomy, with no appearance of hyperplastic foci or preneoplastic nodules. In contrast, rats fed the 24% casein diet demonstrated a progressive loss of PKC delta expression in the particulate fraction during tumour promotion, with activation and increased membrane association of PKC alpha and beta subtypes. These animals developed hyperplastic cell foci and preneoplastic nodules at 7 and 60 days respectively. Taken together, the results of this study suggest that overexpression of PKC delta in the liver particulate fraction of low protein-fed rats may play a specific role in inhibiting the development of hepatocellular focal lesions in the early stages of DEN-AAF-PH-induced carcinogenesis and confirm the role for nuclear PKC beta in promoting the selective growth of carcinogen-initiated hepatocytes in high protein-fed animals. No evidence for a role of PKC zeta in the carcinogenic process could be demonstrated.