Yoshida AH-130 Ascites Hepatoma Research Articles

Anabolic deficits and divergent unfolded protein response underlie skeletal and cardiac muscle growth impairments in the Yoshida hepatoma tumor model of cancer cachexia.

Cancer cachexia manifests as whole body wasting, however, the precise mechanisms governing the alterations in skeletal muscle and cardiac anabolism have yet to be fully elucidated. In this study, we explored changes in anabolic processes in both skeletal and cardiac muscles in the Yoshida AH-130 ascites hepatoma model of cancer cachexia. AH-130 tumor-bearing rats experienced significant losses in body weight, skeletal muscle, and heart mass. Skeletal and cardiac muscle loss was associated with decreased ribosomal (r)RNA, and hypophosphorylation of the eukaryotic factor 4E binding protein 1. Endoplasmic reticulum stress was evident by higher activating transcription factor mRNA in skeletal muscle and growth arrest and DNA damage-inducible protein (GADD)34 mRNA in both skeletal and cardiac muscles. Tumors provoked an increase in tissue expression of interferon-γ in the heart, while an increase in interleukin-1β mRNA was apparent in both skeletal and cardiac muscles. We conclude that compromised skeletal muscle and heart mass in the Yoshida AH-130 ascites hepatoma model involves a marked reduction translational capacity and efficiency. Furthermore, our observations suggest that endoplasmic reticulum stress and tissue production of pro-inflammatory factors may play a role in the development of skeletal and cardiac muscle wasting.

A Combination of Formoterol and the Histone Deacetylase Inhibitor AR42 has No Effects on Muscle Mass in Tumor-Bearing Rats

Background: Accelerated muscle and adipose tissue loss are two of the main aspects of cancer cachexia. β2-agonists seem to be successful in the treatment of cachexia in experimental animals. The aim if the present investigation was to study the effects on body weight loss in tumor-bearing animals of a combination of formoterol and AR-42, an inhibitor of histone deacetylase (HDAC). Methods: Rats were divided into two groups, namely controls (C) and tumor-bearing (T). TB group was further divided into four subgroups: untreated (saline as a vehicle), treated with Formoterol (F) (0,3 mg/kg body weight in saline, subcutaneous (s.c.), daily), treated with AR-42 (A) (20 mg/kg body weight in olive oil, intragastric (i.g.), only the last 4 days). and double-treated treated (TFA) with Formoterol (0,3 mg/kg body weight, subcutaneous (s.c.), daily) and AR-42 (20 mg/kg body weight in olive oil, intragastric (i.g.), only the last 4 days). 7 days after tumor transplantation, muscle weights, grip force and total physical activity were determined in all experimental groups. Results: The presence of the Yoshida AH-130 ascites hepatoma induced severe muscle wasting in rats. Treatment of the tumor-bearing animals with the beta2-agonist formoterol (0,3 mg/kg), resulted in a significant improvement in the cachectic state of the animals. Treatment of the tumor-bearing animals with AR42 did not result in any effects on muscle wasting in the cachectic rats. Furthermore, the combination of formoterol and AR42 showed no additional effects to those observed with just formoterol. Conclusion: The results presented question the previously described effects of AR42 on cancer cachexia, probably due to its effect on tumor growth.

Differential structural features in soleus and gastrocnemius of carnitine-treated cancer cachectic rats.

Muscle wasting is associated with chronic diseases and cancer. Elucidation of the biological mechanism involved in the process of muscle mass loss and cachexia may help identify therapeutic targets. We hypothesized that l-carnitine treatment may differentially revert muscle fiber atrophy and other structural alterations in slow- and fast-twitch limb muscles of rats bearing the Yoshida ascites hepatoma. In soleus and gastrocnemius of tumor-bearing rats (108 AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally) with and without treatment with l-carnitine (1 g/kg body weight for 7 days, intragastric), food intake, body and muscle weights, fiber typing and morphometry, morphological features, redox balance, autophagy and proteolytic, and signaling markers were explored. Levels of carnitine palmitoyl transferase were also measured in all the study muscles. l-Carnitine treatment ameliorated the atrophy of both slow- and fast-twitch fibers (gastrocnemius particularly), muscle structural alterations (both muscles), and attenuated oxidative stress, proteolytic and signaling markers (gastrocnemius). Despite that carnitine palmitoyl transferase-1 levels increased in both muscle types in a similar fashion, l-carnitine ameliorated muscle atrophy and proteolysis in a muscle-specific manner in cancer-induced cachexia. These data reveal the need to study muscles of different fiber type composition and function to better understand whereby l-carnitine exerts its beneficial effects on the myofibers in muscle wasting processes. These findings also have potential clinical implications, since combinations of various exercise and muscle training modalities with l-carnitine should be specifically targeted for the muscle groups to be trained.

Preventive effects of low-intensity exercise on cancer cachexia-induced muscle atrophy.

We hypothesized that low-intensity endurance exercise might be more effective in preventing cancer cachexia-induced muscle atrophy through both an increase in protein synthesis and a decrease in protein degradation. The purpose of present study was to evaluate the effects and to clarify the mechanism of low-intensity endurance exercise on cancer cachexia-induced muscle atrophy. Twenty-four male Wistar rats were randomly divided into 4 groups: control (Cont), Cont plus exercise (Ex), AH130-induced cancer cachexia (AH130), and AH130 plus Ex. Cancer cachexia was induced by intraperitoneal injections with AH130 Yoshida ascites hepatoma cells; we analyzed the changes in muscle mass and the gene and protein expression levels of major regulators or indicators of skeletal muscle protein degradation and synthesis pathway in the soleus muscles. Low-intensity exercise inhibited the muscle mass loss through a suppression of the ubiquitin-proteasome pathway, increased hypoxia-inducible factor- 1α and phosphorylated AMPK, and inhibited the deactivation of mammalian target of rapamycin pathway in the soleus muscle, which contributed to the prevention of cancer cachexia-induced muscle atrophy. These results suggest that low-intensity exercise has the potential to become an effective therapeutic intervention for the prevention of cancer cachexia-induced muscle atrophy.-Tanaka, M., Sugimoto, K., Fujimoto, T., Xie, K., Takahashi, T., Akasaka, H., Kurinami, H., Yasunobe, Y., Matsumoto, T., Fujino, H., Rakugi, H. Preventive effects of low-intensity exercise on cancer cachexia-induced muscle atrophy.

Effects of the beta2 agonist formoterol on atrophy signaling, autophagy, and muscle phenotype in respiratory and limb muscles of rats with cancer-induced cachexia

Muscle mass loss and wasting are characteristic features of patients with chronic conditions including cancer. Beta-adrenoceptors attenuate muscle wasting. We hypothesized that specific muscle atrophy signaling pathways and altered metabolism may be attenuated in cancer cachectic animals receiving treatment with the beta2 agonist formoterol. In diaphragm and gastrocnemius of tumor-bearing rats (intraperitoneal inoculum, 108 AH-130 Yoshida ascites hepatoma cells, 7-day study period) with and without treatment with formoterol (0.3 mg/kg body weight/day/7days, subcutaneous), atrophy signaling pathways (NF-κB, MAPK, FoxO), proteolytic markers (ligases, proteasome, ubiquitination), autophagy markers (p62, beclin-1, LC3), myostatin, apoptosis, muscle metabolism markers, and muscle structure features were analyzed (immunoblotting, immunohistochemistry). In diaphragm and gastrocnemius of cancer cachectic rats, fiber sizes were reduced, levels of structural alterations, atrophy signaling pathways, proteasome content, protein ubiquitination, autophagy, and myostatin were increased, while those of regenerative and metabolic markers (myoD, mTOR, AKT, and PGC-1alpha) were decreased. Formoterol treatment attenuated such alterations in both muscles. Muscle wasting in this rat model of cancer-induced cachexia was characterized by induction of significant structural alterations, atrophy signaling pathways, proteasome activity, apoptotic and autophagy markers, and myostatin, along with a significant decline in the expression of muscle regenerative and metabolic markers. Treatment of the cachectic rats with formoterol partly attenuated the structural alterations and atrophy signaling, while improving other molecular perturbations similarly in both respiratory and limb muscles. The results reported in this study have relevant therapeutic implications as they showed beneficial effects of the beta2 agonist formoterol in the cachectic muscles through several key biological pathways.

Formoterol attenuates increased oxidative stress and myosin protein loss in respiratory and limb muscles of cancer cachectic rats.

Muscle mass loss and wasting are characteristic features of patients with chronic conditions including cancer. Therapeutic options are still scarce. We hypothesized that cachexia-induced muscle oxidative stress may be attenuated in response to treatment with beta2-adrenoceptor-selective agonist formoterol in rats. In diaphragm and gastrocnemius of tumor-bearing rats (108 AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally) with and without treatment with formoterol (0.3 mg/kg body weight/day for seven days, daily subcutaneous injection), redox balance (protein oxidation and nitration and antioxidants) and muscle proteins (1-dimensional immunoblots), carbonylated proteins (2-dimensional immunoblots), inflammatory cells (immunohistochemistry), and mitochondrial respiratory chain (MRC) complex activities were explored. In the gastrocnemius, but not the diaphragm, of cancer cachectic rats compared to the controls, protein oxidation and nitration levels were increased, several functional and structural proteins were carbonylated, and in both study muscles, myosin content was reduced, inflammatory cell counts were greater, while no significant differences were seen in MRC complex activities (I, II, and IV). Treatment of cachectic rats with formoterol attenuated all the events in both respiratory and limb muscles. In this in vivo model of cancer-cachectic rats, the diaphragm is more resistant to oxidative stress. Formoterol treatment attenuated the rise in oxidative stress in the limb muscles, inflammatory cell infiltration, and the loss of myosin content seen in both study muscles, whereas no effects were observed in the MRC complex activities. These findings have therapeutic implications as they demonstrate beneficial effects of the beta2 agonist through decreased protein oxidation and inflammation in cachectic muscles, especially the gastrocnemius.

Cancer cachexia-when proteasomal inhibition is not enough.

Cachexia is a life threatening syndrome associated with several diseases, such as end‐stage heart failure, end‐stage renal disease, chronic obstructive pulmonary disease, chronic inflammation (i.e. rheumatoid arthritis), acquired immune deficiency syndrome, and cancer.1, 2 Cachexia is found in 31–87% of cancer patients especially in advanced disease stages.3 It is characterized by progressive weight loss, metabolic alterations, fatigue, and persistent reduction of body cell mass in response to a malignant tumour.2, 4, 5, 6 The incidence of cachexia in cancer patients is dependent on the type and site of the tumour. While patients with non‐Hodgkin's lymphoma, breast cancer, and sarcomas show low incidences, rates up to 83% in pancreatic cancer patients, and over 85% in patients with gastric cancer have been found. Additionally, around 60% of small‐cell and non‐small‐cell lung cancer patients develop cachexia.7, 8, 9 Cancer cachexia affects the function of several organs such as muscle, adipose tissue, liver, brain, immune system, and heart, collectively decreasing patients' quality of life and worsening their prognosis. Therefore, cachexia must be considered as a true multi‐organ syndrome.10 Because cancer cachexia leads to a decrease in physical performance and quality of life,11 and is associated with poor survival (accounting for more than 20% of cancer deaths,7, 12, 13, 14, 15) it is of major clinical relevance. Even more so since cachectic patients show lower response rates to chemotherapy7 and a reduced tolerance to anticancer treatment.16 Despite its importance, weight loss in cancer patients is rarely recognized, assessed,17 or treated actively.18, 19 Thus, cancer cachexia represents an important underappreciated clinical syndrome.

Chronic Myeloid Leukemia and Hepatoblastoma: Two Cancer Models to Link Metabolism to Stem Cells.

Low oxygen tension is a critical aspect of the stem cell niche where stem cells are long-term maintained. In “physiologically hypoxic” stem cell niches, low oxygen tension restrains the clonal expansion of stem cells without blocking their cycling, thereby contributing substantially to favor their self-renewal. The capacity of stem cells, hematopoietic stem cells in particular, to reside in low oxygen is likely due to their specific metabolic profile. A strong drive to the characterization of this profile emerges from the notion that cancer stem cells (CSC), like normal stem cells, most likely rely on metabolic cues for the balance between self-renewal/maintenance and clonal expansion/differentiation. Accordingly, CSC homing to low oxygen stem cell niches is the best candidate mechanism to sustain the so-called minimal residual disease. Thus, the metabolic profile of CSC impacts long-term cancer response to therapy. On that basis, strategies to target CSC are intensely sought as a means to eradicate neoplastic diseases. Our “metabolic” approach to this challenge was based on two different experimental models: (A) the Yoshida’s ascites hepatoma AH130 cells, a highly homogeneous cancer cell population expressing stem cell features, used to identify, in CSC adapted to oxygen and/or nutrient shortage, metabolic features of potential therapeutic interest; (B) chronic myeloid leukemia, used to evaluate the impact of oxygen and/or nutrient shortage on the expression of an oncogenetic protein, the loss of which determines the refractoriness of CSC to oncogene-targeting therapies.

Chemical sympathectomy increases neutrophil-to-lymphocyte ratio in tumor-bearing rats but does not influence cancer progression.

The sympathetic nervous system regulates many immune functions and modulates the anti-tumor immune defense response, too. Therefore, we studied the effect of 6-hydroxydopamine induced sympathectomy on selected hematological parameters and inflammatory markers in rats with Yoshida AH130 ascites hepatoma. We found that chemically sympathectomized tumor-bearing rats had significantly increased neutrophil-to-lymphocyte ratio, leukocyte-to-lymphocyte ratio, and plasma levels of tumor necrosis factor alpha. Although our findings showed that sympathetic denervation in tumor-bearing rats led to increased neutrophil-to-lymphocyte ratio, that is an indicator of the disease progression, we found no significant changes in tumor growth and survival of sympathectomized tumor-bearing rats.

A differential pattern of gene expression in skeletal muscle of tumor‐bearing rats reveals dysregulation of excitation–contraction coupling together with additional muscle alterations

Cachexia is a wasting condition that manifests in several types of cancer. The main characteristic of this condition is a profound loss of muscle mass. By using a microarray system, expression of several hundred genes was screened in skeletal muscle of rats bearing a cachexia-inducing tumor, the AH-130 Yoshida ascites hepatoma. This model induced a strong decrease in muscle mass in the tumor-bearing animals, as compared with their healthy counterparts. The results show important differences in gene expression in EDL skeletal muscle between tumor-bearing animals with cachexia and control animals. The differences observed pertain to genes related to intracellular calcium homeostasis and genes involved in the control of mitochondrial oxidative phosphorylation and protein turnover, both at the level of protein synthesis and proteolysis. Assessment of these differences may be a useful tool for the design of novel therapeutic strategies to fight this devastating syndrome.

English

The present study shows the protective effects of the Ganoderma lucidum aqueous crude extract (GLE) against mice liver cancer. The mice were randomized into four groups, namely, controls and tumor bearers that were G. lucidum extract- treated or untreated. Tumor bearers were inoculated intraperitoneally (ip) with 108 Yoshida AH-130 ascites hepatoma cells, whereas the controls received saline alone. The treatment began 1 day after tumor cell inoculation, and continued for 14 days. Administration of G. lucidum extract reduced the tumour weight of G. lucidum-treated groups in a dose dependent way (1.627 ± 0.135, 1.142 ± 0.144 versus 2.163 ± 0.316) as compared to model control group. The inhibition rate was 24.84, and 47.31 by G. lucidum extract administration at concentrations of 200 and 400 mg/kg body weight, respectively. Results of this investigation justify continuing with further studies of G. lucidum extract components to develop chemoprevention strategies as an option in the treatment of cancer.   Key words: Liver cancer, mice, Ganoderma lucidum, interleukin-2, antitumour.

Megestrol acetate treatment influences tissue amino acid uptake and incorporation during cancer cachexia

Summary Background & aims Cachexia is a multi-organic syndrome associated with cancer, characterized by body weight loss, muscle and adipose tissue wasting and inflammation, being often associated with anorexia. The aim of the present investigation was to study the effects of megestrol acetate (MA) on the rate of leucine incorporation into muscle tissue and on the uptake of AIB (a non-metabolizable analogue of alanine) in both skeletal muscle and liver tissue. Methods The effects of MA (100 mg/kg) were tested in cachectic tumour-bearing rats (Yoshida AH-130 ascites hepatoma). Tissue amino acid uptake was assessed by means of the alanine analogue α-amino-3H-isobutyrate (AIB). 14C-leucine oxidation and tissue protein incorporation were also determined. Results Administration of MA to tumour-bearing rats resulted in an important reversal of the muscle wasting process, as reflected by individual muscle weights. Treatment with the progestogen resulted in an increased AIB uptake together with an increased leucine incorporation in skeletal muscle. MA treatment significantly decreased AIB uptake by the liver. Conclusions MA treatment results in both a significant increased uptake of neutral amino acids (AIB) by skeletal muscle and a significant incorporation of the branched-chain amino acid leucine into muscle protein, indicating a clear anabolic action of the drug on muscle tissue.

L-Carnitine: An adequate supplement for a multi-targeted anti-wasting therapy in cancer

Tumour growth is associated with weight loss resulting from both adipose and muscle wasting. Administration of L-carnitine (1 g/kg body weight) to rats bearing the AH-130 Yoshida ascites hepatoma, a highly cachectic rat tumour. The treatment results in a significant improvement of food intake and in muscle weight (gastrocnemius, EDL and soleus). These beneficial effects are directly related to improved physical performance (total physical activity, mean movement velocity and total travelled distance). Administration of L-carnitine decreases proteasome activity and the expression of genes related with this activity, such as ubiquitin, C8 proteasome subunit and MuRF-1. Interestingly, L-carnitine treatment also decreases caspase-3 mRNA content therefore suggesting a modulation of apoptosis. Moreover, addition of 50 μM of L-carnitine to isolated EDL muscles results in a significant decrease in the proteolytic rate suggesting a direct effect. It can be concluded that L-carnitine supplementation may be a good approach for a multi-targeted therapy for the treatment of cancer-related cachexia.

Theophylline is able to partially revert cachexia in tumour-bearing rats

Background and aimsThe aim of the present investigation was to examine the anti-wasting effects of theophylline (a methylxantine present in tea leaves) on a rat model of cancer cachexia.MethodsThe in vitro effects of the nutraceuticals on proteolysis were examined on muscle cell cultures submitted to hyperthermia. Individual muscle weights, muscle gene expression, body composition and cardiac function were measured in rats bearing the Yoshida AH-130 ascites hepatoma, following theophylline treatment.ResultsTheophylline treatment inhibited proteolysis in C2C12 cell line and resulted in an anti-proteolytic effect on muscle tissue (soleus and heart), which was associated with a decrease in circulating TNF-alpha levels and with a decreased proteolytic systems gene expression. Treatment with the nutraceutical also resulted in an improvement in body composition and cardiac function.ConclusionTheophylline - alone or in combination with drugs - may be a candidate molecule for the treatment of cancer cachexia.

Formoterol treatment downregulates the myostatin system in skeletal muscle of cachectic tumour-bearing rats

Cachexia is a common systemic manifestation. Additionally, myostatin is known to be a negative regulator of skeletal muscle development. The present study aimed to investigate whether formoterol down-regulates the myostatin system in skeletal muscle of tumour-bearing rats. Real-time PCR and Western blotting were used for the analysis. Results showed that rats bearing the Yoshida AH-130 ascites hepatoma, a cachexia-inducing tumour, exhibited marked muscle wasting that affected the mass of the muscles studied. The cachectic animals exhibited a significant increase in the mRNA levels of the myostatin receptor (ActIIB) in gastrocnemius muscles. Notably, the expression of the various forms of follistatin, a protein with the opposite effects to those of myostatin, was significantly reduced as a result of the implantation of the tumour. When the animals were treated with formoterol, a β-agonist with anti-cachectic potential, increases in skeletal muscle weights were observed. The β-agonist significantly increased levels of various follistatin isoforms and significantly decreased the expression levels of the myostatin receptor. In addition, formoterol treatment resulted in a significant decrease of the myostatin protein content of the gastrocnemius muscle. In conclusion, the results presented indicate that certain anabolic actions of formoterol on the skeletal muscle of cachectic animals may be mediated via the myostatin system.

Sirtuin 1 in skeletal muscle of cachectic tumour‐bearing rats: a role in impaired regeneration?

Background:In advanced malignant disease, cachexia and muscle wasting appear to be among the most common manifestations. This phenomenon is partially related with a decreased muscle regeneration capacity, as previously described in our laboratory.Methods and results:Rats bearing the Yoshida AH-130 ascites hepatoma were used in the experiments. The animals experienced a marked weight loss with decreases in skeletal muscle weights (13% gastrocnemius, 18% extensor digitorum longus, and 12% tibialis muscles). Muscle gene expression was measured using real-time polymerase chain reaction. Skeletal muscle from cachectic tumour-bearing rats is associated with a decreased expression of genes involved in regeneration such as Pax-7 (39%), myogenin (24%), and MyoD (17%). mRNA levels of Sirt1 increased (91%) in cachectic skeletal muscle. The Sirt1 gene has been shown to be associated with changes in muscle myoblast differentiation. Treatment of the tumour-bearing animals with formoterol—a beta2-agonist—normalizes the expression of genes involved in regeneration (i.e., increase of Pax7 (139%)), at the same time as it does with that of Sirt1 (42% decrease).Conclusions:It is suggested that the lack of muscle regeneration observed during muscle wasting in tumour-bearing animals is linked to the action of Sirt-1, possibly via PGC-1α. These factors may constitute possible targets of pharmacological treatment against muscle loss, thus potentially contributing to the understanding and mitigation of muscle atrophy associated with disease.

Formoterol and cancer muscle wasting in rats: Effects on muscle force and total physical activity

Cancer cachexia occurs in the majority of cancer patients before death, and it is responsible for the death of 22% of cancer patients. One of the most relevant characteristics of cachexia is that of asthenia, which reflects significant muscle wasting noted in cachectic cancer patients The aim of the present study was to assess whether the β(2)-adrenergic agonist formoterol is associated with an improvement in physiological parameters such as grip force and total physical activity in cachetic rats. Administration of the β(2)-agonist formoterol (0.3 mg/kg for 7 days) in rats bearing Yoshida AH-130 ascites hepatoma tumors, a model which induces a strong loss of both body and muscle weight, resulted in a significant reversal of the muscle wasting process, as reflected by individual muscle weights. The anti-wasting effects of the drug were also observed in terms of total physical activity and grip force, thus resulting in an improvement in physical performance in cachectic tumor-bearing rats.

Cancer cachexia: Physical activity and muscle force in tumour-bearing rats

Rats bearing the Yoshida AH-130 ascites hepatoma are subjected to substantial weight loss, which is accompanied by anorexia at the end of the tumour cycle. Total physical activity (measured using the IR Actimeter system and Actitrack software) was determined during 11 days in control and tumour-bearing animals, skeletal muscle strength being also by the grip-strength test. The results presented clearly show that the presence of the tumour induces an earlier decrease in physical performance, which affects both skeletal muscle force and physical activity (both locomotor movements and stereotyped movements and distance travelled, among others parameters).

Nutraceutical inhibition of muscle proteolysis: A role of diallyl sulphide in the treatment of muscle wasting

The abnormalities associated with cancer cachexia include anorexia, weight loss, muscle loss and atrophy, anaemia and alterations in carbohydrate, lipid and protein metabolism. The aim of the present investigation was to examine the anti-wasting effects of some nutraceuticals such as genistein, resveratrol, epigallocatechin gallate and diallyl sulphide (DAS). The in vitro effects of these nutraceuticals on proteolysis were examined in muscle cell cultures submitted to hyperthermia. The in vivo effects of DAS were also tested in cachectic tumour-bearing rats (Yoshida AH-130 ascites hepatoma). Although all the nutraceuticals tested inhibited muscle proteolysis, the most promising effects were related with DAS. In vivo administration of DAS only leads to a small improvement in tibialis muscle and heart weights; however, administration of DAS to healthy animals increased all muscle weights, this being associated with a decreased gene expression of proteolytic systems components. It may be suggested that DAS could be used to improve muscle mass during healthy conditions.

Megestrol acetate: Its impact on muscle protein metabolism supports its use in cancer cachexia

Cachexia is a multiorganic syndrome associated with cancer, characterized by body weight loss, muscle and adipose tissue wasting and inflammation, being often associated with anorexia. The aim of the present investigation was to examine the effect of megestrol acetate (MA) in cachectic tumour-bearing animals analyzing changes in muscle proteolysis and in parameters related with quality of life. The effects of MA (100mg/kg) were tested in cachectic tumour-bearing rats (Yoshida AH-130 ascites hepatoma). Administration of MA to tumour-bearing rats resulted in an important reversal of the muscle wasting process, as reflected by individual muscle weights. MA also decreased the rate of protein degradation in incubated isolated skeletal muscles. Real-time PCR analysis revealed that MA treatment resulted in a decrease in ubiquitin, E2 and atrogin-1 mRNA content in muscles, therefore suggesting that the main anti-proteolytic action of the drug may be based on an inhibition of the ATP-ubiquitin-dependent proteolytic system. The drug also improves appetite, weight loss, total physical activity and grip force. The results indicate that treatment with megestrol acetate increases appetite, weight loss, physical performance and muscle force in tumour-bearing rats suggesting that MA is a good candidate for muscle wasting treatment.