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.

Effects of tumor necrosis factor-alpha on muscle-protein turnover in female Wistar rats.

BACKGROUND: Many cancer patients experience a wasting syndrome (cachexia) characterized by weight loss and abnormalities in carbohydrate, protein, and lipid metabolism. Recent experimental studies suggest that the development of cancer cachexia involves the host's production of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha). PURPOSE: Our goal was to evaluate in rats the effects of an 8-day TNF-alpha treatment on overall protein metabolism in the liver, diaphragm, heart, and hind-leg muscles. METHODS: Four experimental groups corresponding to specific tissues (liver, diaphragm, heart, and hind-leg muscles) in female Wistar rats (100-150 g) were studied. Each group consisted of 25 TNF-alpha-treated and 25 control female Wistar rats. The TNF-alpha-treated rats were given intraperitoneal injections of recombinant-derived human TNF-alpha (0.5 mL) that was administered in two daily injections of 50 micrograms/kg (total dose of 100 micrograms/kg per day) for 8 days. Control animals followed the same injection schedule as the treatment group and received 0.5 mL of physiological saline instead of TNF-alpha. All rats were radioactively labeled with NaH14CO3 24 hours prior to TNF-alpha treatment. At 0, 1, 2, 4, and 8 days during TNF-alpha treatment, five rats per group were killed to measure the radioactive decay of labeled protein in specific tissues in order to estimate fractional protein turnover. During necropsy, the liver, hind-leg muscles (soleus muscle analyzed separately on 8th day only), heart, and diaphragm were rapidly weighed, and each was homogenized. Total protein content and total DNA were also determined. Total protein radioactivity and specific protein radioactivity (per milligram of protein) were evaluated for liver, diaphragm, heart, and hind-leg muscles. Radioactivity was counted in a liquid scintillation counter. Fractional rates of protein synthesis, protein degradation, total protein, and protein accumulation or loss were calculated. RESULTS: The TNF-alpha treatment administered to female Wistar rats for 8 days resulted in a transient decrease in food intake and body weight 24 hours after the beginning of the TNF-alpha treatment. In all types of tissues studied, TNF-alpha treatment resulted in increases in both the protein synthesis and protein degradation, with a greater increase in the protein degradation that resulted in a reduced protein accumulation following TNF-alpha treatment. This reduction in protein accumulation was directly associated with a decreased soleus muscle mass on day 8 of the treatment. CONCLUSIONS: Data suggest that TNF-alpha enhances muscle degradation in experimental situations where body weight loss is not apparent.

Human prolactin (hPRL) antagonists inhibit hPRL-activated signaling pathways involved in breast cancer cell proliferation.

The involvement of human prolactin (hPRL) in breast cancer has been recently reconsidered based on its autocrine/paracrine proliferative effect described in human mammary tumor epithelial cells. Therefore, there is growing interest in the development of potent hPRL antagonists that may inhibit this effect. We previously designed hPRL analogs displaying antagonistic properties in a human transcriptional bioassay. We now report that the most potent of those analogs, G129R-hPRL, antagonizes all hPRL-induced effects analysed in various breast cancer cell lines, including cell proliferation. The analog per se lacks intrinsic agonistic activity on PRL receptor-activated signaling cascades, cell proliferation and apoptosis, indicating that its mode of action only occurs through competitive inhibition of hPRL. We provide some molecular basis of this antagonistic effect by demonstrating that G129R-hPRL competitively inhibits hPRL-activation of the JAK-STAT and MAPK pathways, two signaling cascades involved in the mitogenic effect of hPRL in mammary epithelial cells. This competitive inhibition persists for at least 48 h, as evidenced by long term analysis of STAT5b activation or of progression through cell cycle. These results are the first demonstration at the molecular level that hPRL antagonists interfering with receptor dimerization disrupt signaling events in breast cancer cells, which prevents hPRL-induced cell proliferation.

S179D-human PRL, a pseudophosphorylated human PRL analog, is an agonist and not an antagonist.

For many years, our group has been involved in the development of human PRL antagonists. In two recent publications, S179D-human PRL, a human PRL analog designed to mimic a putative S179-phosphorylated human PRL, was reported to be a highly potent antagonist of human PRL-induced proliferation and signaling in rat Nb2 cells. We prepared this analog with the aim of testing it in various bioassays involving the homologous, human PRL receptor. In our hands, S179D- human PRL was able to stimulate 1) the proliferation of rat Nb2 cells and of human mammary tumor epithelial cells (T-47D), 2) transcriptional activation of the lactogenic hormone response element-luciferase reporter gene, and 3) activation of the Janus kinase/signal transducer and activator of transcription and MAPK pathways. Using the previously characterized antagonist G129R-human PRL as a control, we failed to observe any evidence for antagonism of S179D-human PRL toward any of the human PRL-induced effects analyzed, including cell proliferation, transcriptional activation, and signaling. In conclusion, our data argue that S179D-human PRL is an agonist displaying slightly reduced affinity and activity due to local alteration of receptor binding site 1, and that the antagonistic properties previously attributed to S179D-human PRL cannot be confirmed in any of the assays analyzed in this study.

Tumour necrosis factor-alpha increases the ubiquitinization of rat skeletal muscle proteins.

An acute intravenous administration of 100 micrograms/kg body weight of recombinant tumour necrosis factor-alpha (TNF) resulted in a time-dependent increase in the levels of both free and conjugated ubiquitin in rat skeletal muscle. The effects of the cytokine were more pronounced in the red muscle soleus than in the white muscle EDL. In the former muscle type, TNF-treatment also resulted in a time-dependent increase in the percentage of free ubiquitin. The results suggest that the ubiquitin system for non-lysosomal protein degradation could have a very important role in the mechanism triggered by TNF which is responsible for enhanced muscle proteolysis in sepsis and other pathological states.

Ubiquitin gene expression is increased in skeletal muscle of tumour-bearing rats.

Rats bearing the fast-growing AH-130 Yoshida ascites hepatoma showed a marked cachectic response which has been previously reported [Tessitore et al. (1987) Biochem. J. 241, 153-159]. Thus tumour-bearing animals showed significant decreases in body and muscle weight (soleus and gastrocnemius) as compared to both pair-fed and ad libitum-fed animals. These decreases were related to an enhanced proteolytic rate in the muscles of the tumour-bearing animals as measured by the tyrosine released in in vitro assays. In an attempt to elucidate which proteolytic system is directly responsible for the decrease in muscle mass, we have studied both lysosomal and non-lysosomal (ATP-dependent) proteolytic systems in this animal model. While the enzymatic activities of the main cathepsin (B and B + L) systems were actually decreased in gastrocnemius muscles of tumour-bearing rats, thus indicating that lysosomal proteolysis was not involved, the ubiquitin pools (both free and conjugated) were markedly altered as a result of tumour burden. These were associated with an increased ubiquitin gene expression in muscle of tumour-bearing rats, over 500% in relation to non-tumour bearers, thus suggesting that the ATP-dependent proteolytic system may be responsible for the muscle proteolysis and wastage observed in this animal tumour model. The fact that we have previously shown that TNF enhances the ubiquitinization of muscle proteins [García-Martínez et al. (1993) FEBS Lett. 323, 211-214], together with the high circulating levels of TNF detected in rats bearing the Yoshida hepatoma allows us to suggest that the cytokine may be responsible, most probably indirectly, for the activation of the referred proteolytic system in tumour-bearing rats.

Impaired response to interferon-gamma in activated macrophages due to tyrosine nitration of STAT1 by endogenous nitric oxide.

1. Inducible NO synthase (iNOS) expression and activity were measured in the mouse macrophage cell line J774 after exposure to bacterial lipopolysaccharide (LPS) with or without interferon-gamma (IFN-gamma). 2. Inhibition of NOS activity by concomitant N(G)-monomethyl-L-arginine (L-NMMA) treatment further increased iNOS protein levels, with a substantial increase in iNOS half-life. 3. Western blotting and ELISA demonstrated that several cell proteins were tyrosine-nitrated when iNOS activity was high. 4. Rapid IFN-gamma-induced phosphorylation of STAT1 was reduced by about 40% when cells were pretreated to induce iNOS, unless L-NMMA was present during the pretreatment period. 2D gel electrophoresis demonstrated the presence of nitrotyrosine in STAT1 after iNOS induction, and confirmed the reduction in phospho-STAT1 on subsequent stimulation with IFN-gamma for 15 min and its partial restoration when L-NMMA was present during the pretreatment period. 5. We did not detect tyrosine nitration of the upstream kinase JAK2 in LPS+IFN-gamma pretreated cells, but JAK2 activity was also impaired, and was partially restored by concomitant L-NMMA pretreatment. 6. We conclude that endogenous production of NO induces feedback inhibition of signalling pathways activated by IFN-gamma, at least in part by nitrating tyrosine residues in STAT1 which prevents phosphorylation.

Tumour necrosis factor-alpha alters the blood compartmentation of amino acids in the rat.

The work presented focuses on the importance of studying the distribution of blood amino acids in both the cellular and plasmatic fraction when performing studies concerning the effects of cytokines on amino acid metabolism. Tumour necrosis factor treatment resulted in important changes in blood amino acid compartmentation between plasma and red blood cells in rats. The animals showed a change in compartmentation with an increase in the concentration of most amino acids in the cellular fraction with the exception of phenylalanine, glutamate, aspartate and tyrosine.

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.

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.

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.

Comparative effects of beta2-adrenergic agonists on muscle waste associated with tumour growth.

The implantation of the Yoshida AH-130 ascites hepatoma (a fast growing tumour) to rats resulted in a dramatic loss of both white adipose tissue and muscle (skeletal and cardiac) mass. Administration of beta2-adrenergic agonists to tumour-bearing rats resulted in a partial recovery of skeletal muscle and heart mass. Treatment of the tumour-bearing animals with the different drugs (salbutamol, salmeterol and clenbuterol) did not influence tumour growth or food intake so it can be suggested that the effects were solely due to metabolic changes. In addition, while the three drugs had clear effects on gastrocnemius muscles, clenbuterol and salbutamol had also an effect on soleus, and salbutamol had a clear effect on cardiac muscle. It is suggested that any of the studied beta2-adrenergic agonists (but perhaps, particularly salmeterol) could be used clinically in the treatment of cancer cachexia.

Protein turnover in skeletal muscle of tumour-bearing transgenic mice overexpressing the soluble TNF receptor-1.

The implantation of the Lewis lung carcinoma (a fast-growing mouse tumour that induces cachexia) to both wild-type and transgenic mice for the soluble TNF receptor type I protein (sTNF-R1) resulted in a considerable loss of carcass weight in both groups. However, while in the wild-type mice there was a loss of both fat and muscle, in the transgenic mice muscle waste was not affected to the same extent as in the wild-type group. Muscle waste in wild-type mice was accompanied by an increase in the fractional rate of protein degradation, while no changes were observed in protein synthesis. The result was a decreased rate of protein accumulation which accounted for the muscle weight loss observed as a result of the tumour burden. In contrast, transgenic mice did not have such low rates of protein accumulation after tumour implantation. The increase in protein degradation in the tumour-bearing transgenic mice was accompanied by a similar increase in protein synthesis which compensated for the loss of muscle protein by degradation. Both tumour-bearing groups showed an enhanced expression of ubiquitin and proteasome C8 subunit genes, all of them related to the activation of the ATP-dependent proteolytic system in skeletal muscle. It is suggested that TNF may, in part, be responsible for the loss of protein in skeletal muscle of tumour-bearing mice.

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.

Involvement of prolactin in breast cancer: redefining the molecular targets.

The mammary gland is the major target tissue of prolactin (PRL) in mammals. Although this pituitary hormone has been long suspected to be involved in the progression of human breast cancer, the failure of clinical improvement by treatment with dopamine agonists (which lower circulating levels of PRL) rapidly reduced the interest of oncologists concerning a potential role of PRL in the development of breast cancer. Within the last few years, however, several studies reported first, that PRL is also synthesized by mammary epithelial cells, and second that it may exert a proliferative action in an autocrine/paracrine manner. In agreement with a recent epidemiological study, these observations have led to a reconsideration of the role of PRL as an active participant in breast cancer, and are an impetus to redefine the molecular targets of anti-prolactin strategies since dopamine analogs are assumed to be inefficient on extrapituitary PRL synthesis. In this review, we briefly summarize the current knowledge of PRL effects on both normal and tumor mammary cells, and we discuss the most relevant articles supporting the autocrine-paracrine action of PRL in the breast. With the aim of defining putative new molecular targets, we propose an overview of the main PRL receptor signaling cascades known to be triggered by PRL in mammary epithelial cells or, when not available, in other cell types. Finally, because proteolytic fragments of rat PRL have been shown to inhibit the angiogenic process, which may be relevant for preventing the progression of solid tumors such as breast tumors, we discuss the hypothesis that the enzymatic cleavage of human PRL could also represent a new molecular target in the search for alternative strategies in the treatment of breast cancer.

Lipid metabolism in tumour-bearing mice: studies with knockout mice for tumour necrosis factor receptor 1 protein.

The implantation of the Lewis lung carcinoma (a fast-growing mouse tumour that induces cachexia) to both wild-type and gene-deficient mice for the tumour necrosis factor (TNF) receptor type I protein (Tnfr1(0)/Tnfr1(0)), resulted in a considerable loss of carcass (26%) and white (77%) and brown adipose (37%) tissue weights in the wild-type mice, while it induced much less marked effects in the gene-deficient mice. Tumour burden also inflicted an important decrease in total lipoprotein lipase (LPL) activity in epididymal white adipose tissue (50%) in the wild-type mice while no changes were observed in the knockout mice. In addition, all tumour-bearing animals were clearly hypertriglyceridaemic (80% increase in circulating triacylglycerols in wild-type and 36% in knockout mice). It is concluded that although TNF seems to be to some extent responsible for adipose waste, LPL changes and hyperlipaemia (via receptor I), the role of other cytokines (alone or in combination with TNF) in promoting changes in lipid metabolism during cancer cachexia cannot be discarded.

Role of TNF receptor 1 in protein turnover during cancer cachexia using gene knockout mice.

The implantation of the Lewis lung carcinoma (a fast-growing mouse tumour that induces cachexia) to both wild-type and gene-deficient mice for the TNF-alpha receptor type I protein (Tnfr1 degree/Tnfr1 degree), resulted in a considerable loss of carcass weight in both groups. However, while in the wild-type mice there was a loss of both fat and muscle, in the gene-knockout mice muscle wastage was not affected to the same extent. In both groups, tumour burden resulted in significant increases in circulating TNF-alpha, a cytokine which, as we have previously demonstrated, can induce protein breakdown in skeletal muscle. Muscle wastage in wild-type mice was accompanied by an increase in the fractional rate of protein degradation, while no changes were observed in protein synthesis. The result is a decreased rate of protein accumulation that accounts for the muscle weight loss observed as a result of tumour burden. In contrast, gene knockout mice did not have significantly lower rates of protein accumulation as a result of tumour implantation. The increase in protein degradation in the tumour-bearing wild mice was accompanied by an enhanced expression of both ubiquitin and proteasome subunit genes, all of them related to the activation of the ATP-dependent proteolytic system in skeletal muscle. Tumour-bearing gene-deficient mice did not show any increase in gene expression. It is concluded that TNF-alpha (alone or in combination with other cytokines) is responsible for the activation of protein breakdown in skeletal muscle of tumour-bearing mice.

Ubiquitin gene expression is increased in human muscle undergoing neurogenic involvement.

Histological features of neurogenic muscle involvement included type grouping, muscle fiber atrophy, and target fibers. In muscles with myofiber atrophy and target fibers, we found an increased expression of the genes encoding for the ubiquitin-ATP-dependent proteolytic system. Thus, in patients with target fibers, a 5.2- and a 3.9-fold increase were observed for the 2.4 and 1.2 kb transcripts, respectively, while in those with atrophic angulated hyperoxidative fibers, a 3.9- and a 4.4-fold increase were observed for the 2.4 and 1.2 kb transcripts, respectively. It is suggested that the activation of this proteolytic system may be responsible for the skeletal muscle alterations that often accompany human muscle neurogenic involvement.

Effect of solvents on the fumonisins analysis by high-performance liquid chromatography with AccQ.Fluor as the derivatizing reagent.

The effect of several solvent systems on the chromatographic response of fumonisin B1 and B2 derived with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ.Fluor) is described. Naturally contaminated corn samples were extracted and purified by a standard method. Then, samples were dissolved in different solvents, derived with AccQ.Fluor reagent and analysed using HPLC. Results were solvent dependent, methanol being the best one among all assayed solvents for both fumonisins studied and acetonitrile the poorest. o-Phthaldialdehyde (OPA) reagent was used as a reference method.