Muscle myostatin signalling is enhanced in experimental cancer cachexia.

BACKGROUND/AIMS: Myostatin belongs to the transforming growth factor-beta superfamily and negatively regulates skeletal muscle mass. Its deletion induces muscle overgrowth, while, on the contrary, its overexpression or systemic administration cause muscle atrophy. The present study was aimed at investigating whether muscle depletion as occurring in an experimental model of cancer cachexia, the rat bearing the Yoshida AH-130 hepatoma, is associated with modulations of myostatin signalling and whether the cytokine tumour necrosis factor-alpha may be relevant in this regard. MATERIALS AND METHODS: Protein levels of myostatin, follistatin (myostatin endogenous inhibitor) and the activin receptor type IIB have been evaluated in the gastrocnemius of tumour-bearing rats by Western blotting. Circulating myostatin and follistatin in tumour hosts were evaluated by immunoprecipitation, while the DNA-binding activity of the SMAD transcription factors was determined by electrophoretic-mobility shift assay. RESULTS: In day 4 tumour hosts muscle myostatin levels were comparable to controls, yet follistatin was reduced, and SMAD DNA-binding activity was enhanced. At day 7, both myostatin and follistatin increased in tumour bearers, while SMAD DNA-binding activity was unchanged. To investigate whether tumour necrosis factor-alpha contributed to induce such changes, rats were administered pentoxifylline, an inhibitor of tumour necrosis factor-alpha synthesis that partially corrects muscle depletion in tumour-bearing rats. The drug reduced both myostatin expression and SMAD DNA-binding activity in day 4 tumour hosts and up-regulated follistatin at day 7. CONCLUSIONS: These observations suggest that myostatin pathway should be regarded as a potential therapeutic target in cancer cachexia.

Tumor necrosis factor-alpha mediates changes in tissue protein turnover in a rat cancer cachexia model.

Rats bearing the Yoshida AH-130 ascites hepatoma showed enhanced fractional rates of protein degradation in gastrocnemius muscle, heart, and liver, while fractional synthesis rates were similar to those in non-tumor bearing rats. This hypercatabolic pattern was associated with marked perturbations of the hormonal homeostasis and presence of tumor necrosis factor in the circulation. The daily administration of a goat anti-murine TNF IgG to tumor-bearing rats decreased protein degradation rates in skeletal muscle, heart, and liver as compared with tumor-bearing rats receiving a nonimmune goat IgG. The anti-TNF treatment was also effective in attenuating early perturbations in insulin and corticosterone homeostasis. Although these results suggest that tumor necrosis factor plays a significant role in mediating the changes in protein turnover and hormone levels elicited by tumor growth, the inability of such treatment to prevent a reduction in body weight implies that other mediators or tumor-related events were also involved.

Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2-adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteolytic pathway.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

Novel investigational drugs mimicking exercise for the treatment of cachexia.

INTRODUCTION: Cachexia is a syndrome characterized by body weight loss, muscle wasting and metabolic abnormalities, that frequently complicates the management of people affected by chronic diseases. No effective therapy is actually available, although several drugs are under clinical evaluation. Altered energy metabolism markedly contributes to the pathogenesis of cachexia; it can be improved by exercise, which is able to both induce anabolism and inhibit catabolism. AREAS COVERED: This review focuses on exercise mimetics and their potential inclusion in combined protocols to treat cachexia. The authors pay with particular reference to the cancer-associated cachexia. EXPERT OPINION: Even though exercise improves muscle phenotype, most patients retain sedentary habits which are quite difficult to disrupt. Moreover, they frequently present with chronic fatigue and comorbidities that reduce exercise tolerance. For these reasons, drugs mimicking exercise could be beneficial to those who are unable to comply with the practice of physical activity. Since some exercise mimetics may exert serious side effects, further investigations should focus on treatments which maintain their effectiveness on muscle phenotype while remaining tolerable at the same time.

Interleukin-15 mediates reciprocal regulation of adipose and muscle mass: a potential role in body weight control.

Interleukin (IL)-15 is a cytokine which is highly expressed in skeletal muscle. Cell culture studies have indicated that IL-15 may have an important role in muscle fiber growth and anabolism. However, data concerning the metabolic effects of this cytokine in vivo are lacking. In the present study, IL-15 was administered to adult rats for 7 days. While IL-15 did not cause changes in either muscle mass or muscle protein content, it induced significant changes in the fractional rates of both muscle protein synthesis and degradation, with no net changes in protein accumulation. Additionally, IL-15 administration resulted in a 33% decrease in white adipose tissue mass and a 20% decrease in circulating triacylglycerols; this was associated with a 47% lower hepatic lipogenic rate and a 36% lower plasma VLDL triacylglycerol content. The decrease in white fat induced by IL-15 was in adipose tissue. No changes were observed in the rate of lipolysis as a result of cytokine administration. These findings indicate that IL-15 has significant effects on both protein and lipid metabolism, and suggest that this cytokine may participate in reciprocal regulation of muscle and adipose tissue mass.

Mice lacking TNFalpha receptors 1 and 2 are resistant to death and fulminant liver injury induced by agonistic anti-Fas antibody.

The liver is particularly susceptible to Fas-mediated cytotoxicity. Mice given an adequate parenteral dose of agonistic anti-Fas antibody (aFas) or of FasL are known to develop a devastating liver injury and to die in a few hours. The present work shows that mice lacking TNFR1 and TNFR2 (R(-)) both survive a single dose of aFas, otherwise rapidly lethal, and develop a mild form of hepatic damage, compared to the much more severe liver injury that in a few hours strikes wild-type mice (R(+)), eventually involving increased activity of proteases of different families (caspase 3-, 8-, and 9-like, calpains, cathepsin B). Neither the overall tissue levels of Fas and FasL nor Fas expression at the hepatocyte surface are altered in the liver of R(-) animals. The DNA-binding activity of the NF-kappaB transcription factor is enhanced after aFas treatment, but much more markedly in R(-) than in R(+) mice. Bcl2, while unchanged in untreated animals, is markedly upregulated in R(-) but not in R(+) mice challenged with aFas. The requirement of a normal TNFR1/TNFR2 phenotype for full deployment of the general and liver-specific aFas toxicity in mice most likely implies that treatment with aFas in some way results in activation of the TNFalpha-TNFRs system and that this activation synergizes with Fas-mediated signals in causing the fulminant liver injury and the animal death. The precise cellular and molecular details underlying this interplay between Fas- and TNFRs-mediated signaling systems in the general and liver-specific aFas toxicity largely remain to be clarified.

Rapid and extensive lethal action of clofibrate on hepatoma cells in vitro.

Clofibrate, for a long time in use as a hypolipidemic drug, is a well known peroxisomal proliferator (PP) and hepatocarcinogen in rodents. We show here that in vitro 1 mM clofibrate induces a rapid and massive death of rat AH-130 hepatoma cells. Cell death was prominent already after 4 h of treatment, with a characteristic ;apoptotic' pattern by conventional microscopy. This was further supported by the pronounced chromatin condensation detectable on 4',6-diamine-2'-phenylindole dihydrochloride (DAPI) staining, the clearcut internucleosomal DNA fragmentation on agarose-gel electrophoresis (ladder pattern), and the accumulation of markedly hypochromic cells observed in flow cytometric DNA histograms. Consistently with the apoptotic features of the process, some parameters commonly used to detect cell death, such as plasma membrane permeabilization to trypan blue or propidium iodide, lack of mitochondrial retention of rhodamine 123, or extracellular release of lactate dehydrogenase, were all virtually negative. However, these same parameters became markedly positive after 24 h of treatment, which was suggestive for the occurrence of ;secondary' necrosis among AH-130 cells. By a combination of flow cytometric parameters, after 4 h on 1 mM clofibrate only 41% of the AH-130 cells could still be categorized as viable (i.e., non-apoptotic and non-necrotic), while 46% of cells appeared apoptotic and 13% necrotic. At 24 h, 67% of cells were necrotic, 20% apoptotic and only 13% non-apoptotic and non-necrotic. Apoptosis was also extensive in AH-130 cells treated with another PP such as nafenopin at 1 mM concentration and in human hepatoma HepG2 cells treated with clofibrate. By contrast, clofibrate did not cause apoptosis on primary rat hepatocyte cultures. These observations indicate that: (i) apart from their well-known cell growth-promoting action, PPs such as clofibrate or nafenopin may exert a substantial cytotoxic action on targets such as the AH-130 or HepG2 hepatoma cells; (ii) this cell death evolves from an initial 'apoptotic' to an eventual ;necrotic' pattern; (iii) detection of cell death requires the adoption of a full panel of tests, adequate to cover the whole evolving death pattern, while such tests may even be substantially misleading whenever applied individually; (iv) the cytotoxicity of clofibrate and similar agents on normal and, particularly, tumoural cells may deserve careful reevaluation.

Skeletal muscle wasting in tumor-bearing rats is associated with MyoD down-regulation.

Cachexia is a syndrome characterized by profound skeletal muscle wasting that frequently complicates malignancies. A number of studies indicate that protein hypercatabolism, largely mediated by classical hormones and cytokines, is the major component of muscle depletion. Impaired regeneration has been suggested to contribute to the reduction of muscle size. In particular, it has been shown that the expression of MyoD, a muscle-specific transcription factor, is down-regulated by cytokines such as TNFalpha and IFNgamma in a NF-kappaB-dependent posttranscriptional manner. The present study investigated whether modulations of the transcription factor MyoD are associated with the onset of muscle wasting in a well established model of cancer cachexia. Rats bearing the Yoshida AH-130 hepatoma develop a condition of muscle protein hypercatabolism, largely dependent on TNFalpha bioactivity. In the gastrocnemius of these animals the expression of MyoD was markedly reduced, paralleling the decrease of muscle weight. This pattern is associated with increased nuclear translocation of AP-1, while DNA-binding assays did not detect any change in NF-kappaB activity. This is the first observation demonstrating that muscle depletion in tumor-bearing rats is associated with a down-regulation of MyoD levels. Although the underlying mechanisms remain to be clarified, this change is compatible with the hypothesis that a reduced expression of molecules involved in the regulation of the regenerative response may concur to muscle wasting in cancer cachexia.

In vitro and in vivo conditional sensitization of hepatocellular carcinoma cells to TNF-induced apoptosis by taxol.

High mortality among hepatocellular carcinoma (HCC) patients reflects both late diagnosis and low curability, due to pharmacoresistance. Taxol (TAX) is toxic for many human HCC-derived cell lines, yet its clinical efficacy on HCCs is poor. Combining TAX with other drugs appears a promising possibility to overcome such refractoriness. We analyzed whether combining tumor necrosis factor (TNF) with TAX would improve their toxicity. Human HCC-derived cell lines were treated with TAX or TNF, alone or combined. Apoptosis was assessed by morphology and flow-cytometry. Several pro- and anti-apoptotic molecules were evaluated by western blotting and/or enzymatic assay. After a 24 hour treatment, TNF was ineffective and TAX modestly cytotoxic, whereas HCC cells were conditionally sensitized to TNF by TAX. Indeed some relevant parameters were shifted to a prodeath setting: TNF-receptor 1 was increased, SOCS3, c-FLIP and pSTAT3 were markedly downregulated. These observations provide a significant clue to critically improve the drug susceptibility of HCC cells by combining 2 agents, TAX and TNF. The sequential application of TAX at a low dosage followed by TNF for only a short time triggered a strong apoptotic response. Of interest, prior TAX administration could also sensitize to TNF-induced apoptosis in the Yoshida AH-130 hepatoma transplanted in mice. Therefore, scrutinizing the possibility to develop similar combination drug regimens in suitable preclinical models seems highly advisable.

Interleukin-1 receptor antagonist (IL-1ra) is unable to reverse cachexia in rats bearing an ascites hepatoma (Yoshida AH-130).

The mechanisms leading to the development of cancer cachexia are still poorly understood. Recently, cytokines such as interleukin 1 and tumour necrosis factor-alpha have been involved as mediators of the tissue wasting consequent to tumour growth. The rat ascites hepatoma Yoshida AH-130 is a highly anaplastic tumour that causes in the host an early and marked depletion of both the skeletal muscle and the adipose tissue, mainly accounted for by a hypercatabolic state. Profound hormonal alterations and the release of tumour necrosis factor-alpha and interleukin 1 by the tumour cells likely concur in forcing the metabolic balance towards the catabolic side [1]. In order to possibly achieve the correction of this wasting condition, the AH-130 bearing rats were administered a daily s.c. dose of interleukin 1 receptor antagonist (IL-1ra; 2 mg/kg). This factor, however, was completely ineffective in either inhibiting tumour proliferation or in preventing the consequent tissue depletion and protein hypercatabolism. These observations suggest that interleukin 1 is not important, at least in this model system, for either the development of cachexia or tumour growth.

Enhanced leucine oxidation in rats bearing an ascites hepatoma (Yoshida AH-130) and its reversal by clenbuterol.

The growth of the rat ascites hepatoma Yoshida AH-130 causes marked tissue protein hypercatabolism and alterations of the hormonal homeostasis in the host. After a single intravenous tracer dose of L-[1-14C]leucine in vivo, 14CO2 release by tumour-bearing rats is significantly elevated with respect to the controls. Treatment of the tumour hosts with a beta-adrenergic agonist (clenbuterol) is able to prevent either the depletion of the skeletal muscle mass or the enhanced whole-body leucine oxidation. Incubation of soleus muscles in the presence of L-[1-14C]leucine indicates an increased ability of the muscle obtained from the tumour hosts to utilize the amino acid for oxidation. Similarly to what is observed in vivo, clenbuterol administration exerts a protective effect reducing the rate of leucine oxidation to the control levels.

alpha-Adrenergic receptors may contribute to the hypertriglyceridemia associated with tumour growth.

The inoculation of the Yoshida AH-130 ascites hepatoma to rats resulted in an important loss of adipose tissue associated with a decrease in lipoprotein lipase (LPL) activity. Tumour burden also resulted in an important hyperlipidemia which affected both triglyceride and free fatty acids. Administration of phentolamine (an alpha-adrenergic antagonist) to tumour-bearing rats did not influence LPL activity, but it reversed the increase in plasma triglycerides associated with tumour burden. It is suggested that the hypertriglyceridemia associated with tumour growth may be, in part, a consequence of the effect of catecholamines on hepatic triglyceride secretion, via alpha-adrenergic receptors.

Lack of effect of eicosapentaenoic acid in preventing cancer cachexia and inhibiting tumor growth.

It has been recently reported that a diet enriched in n-3 polyunsaturated fatty acids reduces the growth of different kinds of tumors as well as the host tissue hypercatabolic state frequently associated. The rat ascites hepatoma Yoshida AH-130 is a fast growing tumor that causes a rapid and progressive body weight loss in the host and tissue waste associated with a hypercatabolic condition. Plasma levels of classical hormones and humoral mediators (prostaglandin E2 and tumor necrosis factor-alpha) are early perturbed after tumor transplantation (Tessitore, L., Costelli, P. and Baccino, F.M. (1993) Humoral mediation for cachexia in tumour-bearing rats. Br. J. Cancer, 67, 16-23). Enhanced protein degradation rates and alteration of lipoprotein lipase activity mainly account for the wasting of protein and adipose mass, respectively. However, the daily intragastric administration of eicosapentaenoic acid (1.5 g/kg body wt) to AH-130 bearing rats was completely ineffective either in preventing tissue waste or in reducing tumor growth. The low degree of differentiation and the high growth rate of the AH0130 hepatoma probably account for this lack of effect.

Muscle hypercatabolism during cancer cachexia is not reversed by the glucocorticoid receptor antagonist RU38486.

In rats into which a fast growing ascites hepatoma (Yoshida AH-130) had been transplanted, tumor growth elicited a marked loss of body weight and tissue waste, particularly of the skeletal muscle. This depletion has been associated with enhanced rates of protein breakdown, mainly due to hyperactivation of the ATP-ubiquitin-dependent proteolytic system [Llovera, M., García-Martínez, C., Agell, N., Marzábal, M., López-Soriano, F.J. and Argilés, J.M. (1994) FEBS Lett., 338, 311-318]. Profound alterations of the hormonal status and the production of tumor necrosis factor have been involved in the development of such wasting syndrome [Tessitore, L., Costelli, P. and Baccino, F.M. (1993) Br. J. Cancer, 67, 15-23]. In the present study, the role of glucocorticoids in muscle hypercatabolism was investigated using the glucocorticoid receptor antagonist RU38486. The treatment with this drug was unable to interfere with the development of cachexia in the AH-130 hosts with regard to tissue weight as well as to muscle protein turnover rates. As one would expect, the RU38486 was also ineffective in lowering both the expression of ubiquitin mRNA and the degree of muscle protein ubiquitinization in AH-130 bearers. These data allow us to exclude that glucocorticoids play a direct crucial role in the development of cachexia in this tumor model.

Different cytokines modulate ubiquitin gene expression in rat skeletal muscle.

Intravenous administration of different cytokines caused important changes in the expression of ubiquitin genes in skeletal muscle. Tumour necrosis factor-alpha caused a 2.2- and 1.9-fold increase in the expression of the 2.4 and 1.2 kb transcripts, respectively. Administration of interferon-gamma also caused a 2.2- and 1.8-fold increase in the 2.4 and 1.2 kb transcripts, respectively. While administration of leukaemia inhibitory factor and interleukin-6 resulted in no changes in ubiquitin gene expression, interleukin-1 administration also caused an increase in both ubiquitin gene transcripts (2.8- and 1.9-fold for the 2.4 and 1.2 kb transcripts, respectively). The results suggest that some of the cytokine effects on the ubiquitin system gene expression could be related to the enhanced skeletal muscle proteolysis found during cancer cachexia and other pathological states.

Tumour growth and nitrogen metabolism in the host (Review).

Tumour growth is frequently associated with massive loss of body weight and negative nitrogen balance leading to cachexia, one of the worst features of malignancy accounting for nearly two thirds of cancer deaths. At the metabolic level, cachexia is associated with depletion of body lipid stores as well as loss of skeletal muscle protein, which is manifested as an excessive nitrogen loss. The present study reviews the modulations of nitrogen metabolism in tumour-bearing conditions. In addition, the role of different mediators (such as cytokines) in the development of protein wasting is discussed. Particular emphasis has been given to tumour necrosis factor-alpha (TNF-alpha) which seems to have a key role in mediating changes related to nitrogen metabolism in the tumour-bearing host.

Branched-chain amino acid catabolism and cancer cachexia (review).

The present study focuses on the role of branched-chain amino acids (BCAA) (leucine, isoleucine and valine) in cancer cachexia. This is a frequent and devastating complication of cancer occurring in more than two thirds of patients who die with advanced cancer, and in up to 22% of cancer deaths it is the only abnormality found at autopsy. BCAA are essential amino acids often increased in cancer patients and this phenomenon may be related with increased protein degradation in skeletal muscle and consequently, cancer cachexia. The relevance of this association in medical terms is evident, future investigations may focus on therapeutical strategies based on the administration of BCAA to cachectic patients.

Effect of two aliphatic aldehydes, methylglyoxal and 4-hydroxypentenal, on the growth of Yoshida ascites hepatoma AH-130.

The influence of a ketoaldehyde, methylglyoxal (MG), and a hydroxyalkenal, 4-hydroxypentenal (HPE), on the growth of a highly-deviated tumour has been investigated. MG and HPE, administered intraperitoneally, strongly depressed in rats the proliferative activity of the Yoshida ascites hepatoma AH-130, reducing its mitotic and labelling indices as well as the proportion of cycling cells (growth fraction). Monitoring the effects on the cell cycle by the labelled mitoses method showed that the percentage of labelled mitoses was markedly lowered after either aldehyde, which is indicative for a blocking effect in the S phase. In addition, the mean cell cycle time was slightly prolonged by MG, probably due to accumulation of cells in G1, whereas HPE delayed the first mitotic peak and increased the mean DNA synthetic period without modifying the overall cycle time. The effects of HPE on the cell cycle were prevented by pretreatment with polyamines. Repeated doses of MG significantly increased the fraction of tumour-bearing rats surviving at 90 days ('indefinite' survivors) as well as the survival time of those which succumbed, implying that the carcinostatic effect of MG persisted over several cell cycles. By contrast, HPE did not significantly modify the survival of AH-130-bearing rats, suggesting that its influence on tumour growth was rapidly reversible.

Seizures during concomitant treatment with theophylline and ranitidine: a case report.

H2-antagonists such as cimetidine and ranitidine are metabolized by cytochrome P-450. In this way they may interfere with theophylline metabolism. Cimetidine is known to have this effect and frequently to induce a theophylline toxic effect, while data concerning ranitidine are more uncertain. In this paper, we report the case of a 67-year-old woman with non-insulin dependent diabetes. She was taking aminophylline for respiratory failure and after ranitidine infusion exhibited generalized convulsions. Theophylline values which were monitored within the therapeutic range, increased toxic levels after ranitidine therapy and epileptic episodes. The increase in theophylline levels was associated with a further reduction in the clearance rate of the bronchodilator. We think that ranitidine may combine with other clinical factors known to reduce theophylline metabolism mainly in the elderly and severely ill patients. Theophylline-induced seizures may occur when theophylline serum levels are slightly above the therapeutic range, as in our case report.

[The neuroendocrine aspects of chronic alcoholism: the effect of alcohol intake and its withdrawal]. / Aspetti neuroendocrini dell'etilismo cronico: effetto dell'assunzione di alcool e della sua sospensione.

Neuroendocrine dysfunctions, in part similar to those found in depression, are present in chronic alcoholism. The aim of this investigation was to evaluate the effects of chronic alcohol intake on cortisol secretion in basal conditions, after dexamethasone (DXT) suppression or corticotropin (ACTH) stimulation in 10 alcoholic men, during active drinking and after two weeks of alcohol withdrawal. The 24-hour, day- and night-time urinary cortisol and melatonin levels, and the effects of thyrotropin releasing hormone (TRH) on thyrotropin (TSH) and prolactin (PRL) secretions were studied in the same subjects. The data were correlated to the scores obtained by the Hamilton Rating Scale for depression and compared to those found in healthy subjects. Increased cortisol levels and the lack of DXT suppression of cortisol secretion are considered to be alcohol-dependent inasmuch as they disappear in most patients after alcohol withdrawal. The cortisol response to ACTH 1-24 infusion measured before and after alcohol withdrawal was similar in the patients we studied; moreover no significant difference was found between patients and controls. The increment of urine free cortisol levels in active alcoholics was not statistically significant. Urine cortisol levels became similar to those of the control subjects after alcohol withdrawal. The increased diurnal values of urine melatonin and the inversion of the physiological ratio between nocturnal and diurnal levels observed during alcohol intake became normal upon alcohol withdrawal. The TSH and PRL responses after the administration of 50 or 200 micrograms TRH were higher in alcoholics than in controls, while a blunted response is known to occur in depression.