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Resumen:
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La caquexia es un síndrome frecuentemente asociado al crecimiento tumoral, así como también a otros estados patológicos como son sepsis, SIDA (síndrome de inmunodeficiencia adquirida), diabetes, etc. La caquexia se caracteriza por una importante y progresiva pérdida de peso corporal debida principalmente a la desaparición de las reservas de grasa y a la disminución de masa muscular. Está acompañada también de anorexia, náuseas, astenia, debilidad, alteración de la homeostasis hormonal e inmunodepresión.
Sobre los factores de transcripción que podrían estar involucrados en el proceso de desgaste muscular observado en la caquexia (in vivo), se sabe muy poco, pero aparecen algunos factores como posibles candidatos tanto en el desencadenamiento como en la regulación de este proceso. NF-kappa-B y AP-1 son dos factores de transcripción que están activados en distintos tejidos en procesos inflamatorios. Se ha descrito que NF-kappa-B y AP-1 tienen una regulación diferencial en músculo esquelético de rata durante el proceso de sepsis. El mismo grupo ha determinado que C/EBP, otro factor de transcripción involucrado en el proceso de inflamación, aumenta su actividad de unión al DNA en músculo de ratas sépticas, y que este proceso es dependiente de glucocorticoides.
Los objetivos de este estudio son: 1) analizar la regulación de algunos factores de transcripción en el músculo esquelético durante la caquexia inducida por el crecimiento tumoral. 2) revertir el desgaste muscular asociado al crecimiento tumoral mediante estrategias basadas en factores de transcripción desde un punto de vista farmacológico. 3) aproximaciones a una terapia contra la caquexia mediante un adenovirus para la transferencia génica de un dominante negativo de AP-1.
Los resultados obtenidos nos permiten concluir que el factor de transcripción AP-1 presenta una regulación diferencial en el músculo esquelético durante el crecimiento tumoral, lo que sugiere que este factor está implicado en el desarrollo de la caquexia asociada al cáncer. Por el contrario, otros factores de transcripción estudiados (NF-kappa-B, C/EBP y Sp-1) no parecen estar implicados directamente en el desarrollo del proceso caquéctico, al menos a nivel muscular.
El factor miogénico MyoD presenta importantes cambios en sus niveles en el músculo esquelético de diferentes modelos experimentales de tumores caquécticos, así como en el músculo de pacientes con cáncer de páncreas. La administración aguda de LPS a ratas también afecta marcadamente los niveles musculares de MyoD. Estos datos sugieren una implicación de dicho factor en el desarrollo de la caquexia asociada al crecimiento tumoral y otros procesos patológicos.
La administración de SP100030, inhibidor de NF-kappa-B y AP-1, logra revertir parcialmente los efectos asociados al crecimiento del hepatoma ascítico Yoshida AH-130. Este efecto está asociado a una inhibición de la actividad de unión de AP-1 al DNA, un aumento en la expresión de MyoD, y una disminución en la proteólisis muscular.
El tratamiento con GW1929, agonista de PPAR-gamma, induce una recuperación en el peso de los músculos extensor digitorum longus de ratones portadores de carcinoma pulmonar de Lewis, asociado a un restablecimiento de los niveles de MyoD. Este compuesto también induce una disminución en la proteólisis de estos músculos in vitro.
La sobreexpresión de Tam67, dominante negativo de AP1, revierte totalmente los efectos causados por la adición de TNF-alfa al medio de cultivo de mioblastos C2C12 en diferenciación. Este efecto es producido por el restablecimiento del programa de diferenciación muscular, mediado por una disminución de ciclina D1, una recuperación de los niveles de MyoD y un aumento de la proteína MHC en estas células.
La transferencia génica in vivo de dominante negativo de AP-1 Tam67 mediante adenovirus, produce una recuperación en el peso de los músculos y una atenuación en la caída de los niveles de MyoD en el músculo gastrocnemius de animales portadores de tumor. |
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Abstract:
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(ENGLISH)
One of the most common manifestation of advanced malignant disease is the development of cancer cachexia. Indeed, cachexia occurs in the majority of cancer patients before death, and it is responsible for the death of 22% of cancer patients. The abnormalities associated with cancer cachexia include anorexia, weight loss, muscle loss and atrophy, anemia and alterations in carbohydrate, lipid and protein metabolism. The degree of cachexia is inversely correlated with the survival time of the patient and it always implies a poor prognosis. Perhaps one of the most relevant characteristics of cachexia is that of asthenia (or lack of muscular strenght), which reflects the important muscle waste that takes place in the cachectic cancer patient. Asthenia is also characterized by a general weakness as well as physical and mental fatigue. In addition, lean body mass depletion is one of the main trends of cachexia and it involves not only skeletal muscle but it also affects cardiac proteins, resulting in important alterations in heart performance.
Little is known about the transcription factors that may be involucrated in cancer cachexia at the level of skeletal muscle. Among the main candidates that could have a role in such pathological state are nF-kB abd AP-1. These two transcription factors have already been involucarted in skeletal muscle during sepsis.
The objectives of the present study were: 1) to analyze the regulation of some transcription factors in skeletal muscle during cancer cacchexia. 2) reverse muscle wasting associated with tumour burden using strategies based on transcription factors. 3) to introduce an approximation to a therapy against cachexia based on the introduction of an adenovirus using the gene transfer of a dominant negative of AP-1.
The results obtained clearly demonstrate that while AP-1 and Myo D are clearly associated with muscle wasting during cancer cachexia, others such as NF-kB,C/EBP and Sp-1 do not seem do be involved.
Daily treatment of rats bearing the cachectic Yoshida AH-130 ascites hepatoma with the double inhibitor (NF-kB and AP-1) SP 100030 at a dose of 1 mg/kg of body weight resulted in a clear amelioration of the cachectic effect, especially at the level of skeletal muscle. Thus, tumour-bearing rats treated with SP100030 showed a significant recovery in the weights of gastrocnemius, EDL, tibialis and cardiac muscles. In addition, treatment with the inhibitor also affected both liver and kidney weights. The amelioration in muscle weight was accompanied by an increase in MyoD gene expression -the main transcription factor of muscle tissue involved in muscle differentiation-- in gastrocnemius muscle. At the dose used in this study SP 100030 was an effective inhibitor of AP-1, however, the nF-kB transcription factor was not affected. Interestingly, the effects of the inhibitor seem to be at the level of proteolysis since lower total proteolytic rates were found when incubating of isolated rat muscles in the presence of SP100030. Interestingly, the inhibitor influenced the gene expression of the E2 enzyme in skeletal muscle of tumour-bearing rats; this protein seems to be the main regulator of the activity of the main proteolytic system during cancer cachexia, the ubiquitin-proteasome system. In conclussion, treatment of cachectic tumour-bearing rats with SP 100030 results in an amelioartion of the muscle wasting effect suggesting that the AP-1 signalling cascade plays a very important role in the signalling of muscle wasting associated with disease.
The aim of the overexpression of TAM-67 study was to investigate a possible role of the AP-1 signaling cacscade in the process of wasting associated with cancer cachexia at the level od skeletal muscle. Bearing this in mind, an experimental design involucrating adenoviral trasnduction of the TAM-67 protein -a blocker of the AP.1 protein was used in both in vivo and in vitro experimental setups. Interestingly, the injection of virus expressing the TAM-67 protein to tumour-bearing rats, resulted in a significat recovery of the muscle mass -which is dramatically reduced as a result of tumour burden--, therefore suggesting that AP-1 is certainly involved in the signalling associated with muscle protein accretion. Although, several mediators have been suggested for the muscle wasting associated with cancer cachexia, many investigations suggest an important role for TNF (Tumour Necrosis factor). Indeed, addition of TNF to C2C12 cells resulted in a decreased content of both MyoD and MHC. Interestingly, in this muscle cell system, it was also observed that blockage of the AP-1 signalling cascade buy means of adenovirus (containing TAM-67) infection resulted in an improvement in the amount of myofibrillar protein (MHC) following TNF treatment, therefore suggesting that AP-1 signalling is involved in the intraceellular action of the cytokine oin skeletal muscle. In fact, from the results presented here, it may be suggested that AP-1 is involved in skeletal muscle proliferation since blocking the trancription factor sems to lead to an increased differentiation. In conclussion, the gene therapy approach presented here clearly suggests an important role for AP-1 in muscle signalling during catabolic states and may constitute the basis for a future sucessful therapeutic approach. |
