Muscle Atrophy In Cancer Cachexia Research Articles

Astaxanthin Ameliorates Skeletal Muscle Atrophy in Mice With Cancer Cachexia

Astaxanthin (AST) is a natural marine carotenoid with a variety of biological activities. This study aimed to demonstrate the possible mechanisms by which AST improves skeletal muscle atrophy in cancer cachexia. In this study, the effects of different doses of AST (30 mg/kg b.w., 60 mg/kg b.w. and 120 mg/kg b.w.) on skeletal muscle functions were explored in mice with cancer cachexia. The results showed that AST (30, 60 and 120 mg/kg b.w.) could effectively protect cachexia mice from body weight and skeletal muscle loss. AST dose-dependently ameliorated the decrease in myofibres cross-sectional area and increased the expression of myosin heavy chain (MHC). AST treatment decreased both the serum and muscle level of IL-6 but not TNF-α in C26 tumor-bearing cachexia mice. Moreover, AST alleviated skeletal muscle atrophy by decreasing the expression of two muscle-specific E3 ligases MAFBx and MuRF-1. AST improved mitochondrial function by downregulating the levels of muscle Fis1, LC3B and Bax, upregulating the levels of muscle Mfn2 and Bcl-2. In conclusion, our study show that AST might be expected to be a nutritional supplement for cancer cachexia patients.

Paeoniflorin alleviated muscle atrophy in cancer cachexia through inhibiting TLR4/NF-κB signaling and activating AKT/mTOR signaling

Cancer cachexia is a progressive wasting syndrome, which is mainly characterized by systemic inflammatory response, weight loss, muscle atrophy, and fat loss. Paeoniflorin (Pae) is a natural compound extracted from the dried root of Paeonia lactiflora Pallas, which is featured in anti-inflammatory, antioxidant, and immunoregulatory pharmacological activities. While, the effects of Pae on cancer cachexia had not been reported before. In the present study, the effects of Pae on muscle atrophy in cancer cachexia were observed both in vitro and in vivo using C2C12 myotube atrophy cell model and C26 tumor-bearing cancer cachexia mice model. In the in vitro study, Pae could alleviate myotubes atrophy induced by conditioned medium of C26 colon cancer cells or LLC Lewis lung cancer cells by decreasing the expression of Atrogin-1 and inhibited the decrease of MHC and MyoD. In the in vivo study, Pae ameliorated weight loss and improved the decrease in cross-sectional area of muscle fibers and the impairment of muscle function in C26 tumor-bearing mice. The inhibition of TLR4/NF-κB pathway and the activation of AKT/mTOR pathway was observed both in C2C12 myotubes and C26 tumor-bearing mice treated by Pae, which might be the main basis of its ameliorating effects on muscle atrophy. In addition, Pae could inhibit the release of IL-6 from C26 tumor cells, which might also contribute to its ameliorating effects on muscle atrophy. Overall, Pae might be a promising candidate for the therapy of cancer cachexia.

ActRIIB small molecule inhibitor 2–67 ameliorates muscle atrophy in cancer cachexia by inhibiting the myostatin/Smad pathway

Muscle atrophy is one of the major clinical features of cancer cachexia, a multifactorial complex syndrome complicated by malignancy that remains a major challenge in clinical treatment. The myostatin/Smad signaling pathway, an important pathway for muscle protein degradation, is aberrantly activated in muscle atrophy mainly by the binding of myostatin to ActRIIB kinase. Here, a series of novel small molecule compounds were screened using a molecule-level screening model for ActRIIB kinase inhibitory activity and a cell-level screening model for C26 tumor cell conditioned medium-induced C2C12 myotube atrophy. Among the compounds, compound 2–67 exhibited significant ActRIIB kinase inhibition and myotubular atrophy alleviation effects. Furthermore, 2–67 dose-dependently attenuated myocyte atrophy induced by cachexic tumor cell conditioned medium or myostatin by inhibiting the activation of the myostatin/Smad signaling pathway in C2C12 myotubes. In a C26 tumor-bearing mouse cancer cachexia model, 2–67 treatment effectively alleviated cancer cachexia symptoms. 2–67 ameliorated the loss in body weight, improved appetite, alleviated muscle loss, and preserved muscle grip strength. 2–67 also inhibited the myostatin/Smad signaling pathway in muscle tissues in cancer cachexia mice. Therefore, novel small molecule inhibitors of ActRIIB kinase may be a new option for the treatment of cancer cachexia-induced muscle atrophy, and the present work provides an important reference for the subsequent development and application of related inhibitors.

Cucurbitacin IIb attenuates cancer cachexia induced skeletal muscle atrophy by regulating the IL-6/STAT3/FoxO signaling pathway.

The main features of cancer cachexia include skeletal muscle atrophy, which can significantly reduce the quality of life of patients. Clinical treatment of cancer cachexia is mainly based on nutritional therapy and physical exercise; medication only improves appetite but does not reverse the symptoms of skeletal muscle wasting. In this work, we systematically studied the underlying molecular mechanisms by which cucurbitacin IIb (CuIIb) ameliorates muscle wasting in cancer cachexia both in vitro and in vivo. CuIIb significantly ameliorated the chief features of cancer cachexia in vivo, alleviating weight loss, food intake, muscle wasting, adipose tissue depletion, and organ weight reductions. In vitro, CuIIb (10 and 20 μM) dose-dependently attenuated conditioned medium (CM)-induced C2C12 myotube atrophy. Collectively, our findings demonstrated that CuIIb prevented the upregulation of the E3 ubiquitin ligase muscle atrophy Fbox protein (MAFbx), myosin heavy chain (MyHC), and myogenin (MyoG) and impacted protein synthesis and degradation. In addition, CuIIb decreased the phosphorylation of Tyr705 in STAT3 by regulating the IL-6/STAT3/FoxO pathway to reduce skeletal muscle atrophy in cancer cachexia.

Cancer cachexia‐mediated regulation of EcSOD synthesis requires Nrf2 activation by p62 phosphorylation

Oxidative stress plays an important role in skeletal muscle atrophy in cancer cachexia. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of antioxidant gene transcription. However, a potential role for Nrf2 in protection against muscle atrophy in cancer cachexia remains to be determined. Here, we demonstrated that Lewis lung carcinoma (LLC) cells injection in subcutaneous of C57BL/6 mice induces severe muscle atrophy in glycolytic muscle, as well as oxidative marker (i.e., malondialdehyde proteins) and atrogenes (i.e., atrogin-1 and MuRF1). Basal level of antioxidants expression in glycolytic extensor digitorum longus muscle was lower than in oxidative soleus muscle. LLC injection increased extracellular superoxide dismutase (EcSOD) protein expression in association with Nrf2 activation in extensor digitorum longus muscles. Muscle-specific loss of Nrf2 [i.e., Nrf2 muscle-specific knockout (mKO) mice] induced more severe muscle atrophy by cachexia in extensor digitorum longus muscle than in soleus muscle. LLC injection-mediated enhanced EcSOD protein expression in extensor digitorum longus muscle was blunted in Nrf2 mKO mice. Interestingly, LLC injection-induced p62 phosphorylation enhanced EcSOD protein expression in extensor digitorum longus muscle was blunted in p62 mKO mice. Collectively, these findings indicate that muscle Nrf2 activation by p62 phosphorylation plays an important for antioxidant defense system in skeletal muscle and protect against cachexia-induced muscle atrophy.

Baoyuan Jiedu Decoction Alleviates Cancer-Induced Myotube Atrophy by Regulating Mitochondrial Dynamics Through p38 MAPK/PGC-1α Signaling Pathway

Cancer cachexia is a multifactorial syndrome characterized by continuous body wasting and loss of skeletal muscle. Impaired mitochondria function is closely associated with muscle atrophy in cancer cachexia. Our previous study confirmed the effectiveness of Baoyuan Jiedu decoction (BJD) in inhibiting cancer-induced muscle atrophy in an in vivo model. However, little is known about its mechanisms in regulating mitochondria dysfunction. In this study, we evaluated the therapeutic effect and action mechanisms of BJD against atrophy both in the Lewis-conditioned medium induced C2C12 myotube atrophy model and in a BALB/c mice xenograft model using mouse colon cancer C26 cells. The mitochondrial content was tested by 10-Non-ylacridine orange staining. Expressions of related proteins and mRNAs were detected by western blotting (WB) and qPCR, respectively. As a result, 18 major components were identified in BJD by ultra-high performance liquid chromatography-quadrupole (UHPLC-Q) Exactive analysis. As shown in the in vitro results, BJD treatment prevented prominent myotube atrophy and increased the myotube diameter of C2C12 cells. Besides, BJD treatment increased mitochondrial content and ATPase activity. Furthermore, the protein and mRNA expressions that were related to mitochondrial functions and generation such as cytochrome-c oxidase IV, Cytochrome C, nuclear respiratory factor 1, and mitochondrial transcription factor A were significantly increased in BJD treatment compared to the control group. The in vivo results showed that BJD treatment prevented body weight loss and improved the gastrocnemius index in cachexia mice. Moreover, the expressions of Atrogin-1 and muscle RING-finger protein-1 were decreased by BJD treatment. Mechanically, BJD increased the expression of peroxisome proliferator-activated receptor-gamma coactivator 1, and consistently, inhibited the expression of p38 MAPK and its phosphorylation both in vivo and in vitro. Taken together, this study identified that BJD effectively relieved cancer-induced myotube atrophy and provided a potential mechanism for BJD in regulating mitochondrial dynamics through p38 MAPK/PGC-1α signaling pathway.

Muscle‐specific Nrf2 deficiency exacerbates cancer cachexia‐induced skeletal muscle atrophy

Skeletal muscle atrophy is a hallmark of several debilitating diseases such as cancer, thereby contributing to morbidity and mortality. Oxidative stress, which is caused by excessive production of reactive oxygen species and/or decreased antioxidant defense, plays an important role in skeletal muscle atrophy in cancer cachexia. Nuclear factor erythroid 2–related factor 2 (Nrf2) is a master regulator of antioxidant gene transcription. However, a potential role for Nrf2 in protection against muscle atrophy in cancer cachexia remains to be determined. To investigate this, C57BL/6 mice were given a subcutaneous injection in the left flank with 107 3LL lung cancer cells or PBS control. A grip strength test was performed at 25–27 days after 3LL injection using a grip strength meter. At 4 weeks after 3LL injection, soleus, plantaris, gastrocnemius, and extensor digitorum longus muscles were harvested to assess muscle weight and expression of select proteins. Muscle‐specific Nrf2 knockout (Nrf2mKO) and wild‐type littermates (WT) mice were also injected with 3LL cells and their skeletal muscles were harvested within the same time frame. Our results demonstrated that grip strength in 3LL injected C57BL/6 mice was significantly lower than in control mice. Gastrocnemius and extensor digitorum longus muscle weights in 3LL injected mice were significantly lower than control mice. Protein carbonylation, a surrogate of oxidative stress, was significantly increased in 3LL injected mice compared with control mice. Extracellular superoxide dismutase (EcSOD) protein expression was also increased in 3LL injected mice. Gastrocnemius and extensor digitorum longus muscle weight in 3LL injected Nrf2mKO mice were significantly lower than in 3LL injected WT mice. Loss of Nrf2 in skeletal muscle also blunted the 3LL injection‐induced increase in EcSOD protein expression. These findings indicate that endogenous muscle Nrf2 is a key protective molecule against atrophy likely by limiting the oxidative stress burden of cancer cachexia.Support or Funding InformationThis study was funded by Grant‐in‐Aid for Scientific Research (B) (15H03080) (18H03153) to Mitsuharu Okutsu.

The Pathway to Cancer Cachexia: MicroRNA-Regulated Networks in Muscle Wasting Based on Integrative Meta-Analysis.

Cancer cachexia is a multifactorial syndrome that leads to significant weight loss. Cachexia affects 50%–80% of cancer patients, depending on the tumor type, and is associated with 20%–40% of cancer patient deaths. Besides the efforts to identify molecular mechanisms of skeletal muscle atrophy—a key feature in cancer cachexia—no effective therapy for the syndrome is currently available. MicroRNAs are regulators of gene expression, with therapeutic potential in several muscle wasting disorders. We performed a meta-analysis of previously published gene expression data to reveal new potential microRNA–mRNA networks associated with muscle atrophy in cancer cachexia. We retrieved 52 differentially expressed genes in nine studies of muscle tissue from patients and rodent models of cancer cachexia. Next, we predicted microRNAs targeting these differentially expressed genes. We also include global microRNA expression data surveyed in atrophying skeletal muscles from previous studies as background information. We identified deregulated genes involved in the regulation of apoptosis, muscle hypertrophy, catabolism, and acute phase response. We further predicted new microRNA–mRNA interactions, such as miR-27a/Foxo1, miR-27a/Mef2c, miR-27b/Cxcl12, miR-27b/Mef2c, miR-140/Cxcl12, miR-199a/Cav1, and miR-199a/Junb, which may contribute to muscle wasting in cancer cachexia. Finally, we found drugs targeting MSTN, CXCL12, and CAMK2B, which may be considered for the development of novel therapeutic strategies for cancer cachexia. Our study has broadened the knowledge of microRNA-regulated networks that are likely associated with muscle atrophy in cancer cachexia, pointing to their involvement as potential targets for novel therapeutic strategies.

PDK4 drives metabolic alterations and muscle atrophy in cancer cachexia

Cachexia is frequently accompanied by severe metabolic derangements, although the mechanisms responsible for this debilitating condition remain unclear. Pyruvate dehydrogenase kinase (PDK)4, a critical regulator of cellular energetic metabolism, was found elevated in experimental models of cancer, starvation, diabetes, and sepsis. Here we aimed to investigate the link between PDK4 and the changes in muscle size in cancer cachexia. High PDK4 and abnormal energetic metabolism were found in the skeletal muscle of colon-26 tumor hosts, as well as in mice fed a diet enriched in Pirinixic acid, previously shown to increase PDK4 levels. Viral-mediated PDK4 overexpression in myotube cultures was sufficient to promote myofiber shrinkage, consistent with enhanced protein catabolism and mitochondrial abnormalities. On the contrary, blockade of PDK4 was sufficient to restore myotube size in C2C12 cultures exposed to tumor media. Our data support, for the first time, a direct role for PDK4 in promoting cancer-associated muscle metabolic alterations and skeletal muscle atrophy.-Pin, F., Novinger, L. J., Huot, J. R., Harris, R. A., Couch, M. E., O'Connell, T. M., Bonetto, A. PDK4 drives metabolic alterations and muscle atrophy in cancer cachexia.

Transcriptomic analysis of the development of skeletal muscle atrophy in cancer-cachexia in tumor-bearing mice.

Cancer-cachexia (CC) is a wasting condition directly responsible for 20-40% of cancer-related deaths. The mechanisms controlling development of CC-induced muscle wasting are not fully elucidated. Most investigations focus on the postcachectic state and do not examine progression of the condition. We recently demonstrated mitochondrial degenerations precede muscle wasting in time course progression of CC. However, the extent of muscle perturbations before wasting in CC is unknown. Therefore, we performed global gene expression analysis in CC-induced muscle wasting to enhance understanding of intramuscular perturbations across the development of CC. Lewis lung carcinoma (LLC) was injected into the hind-flank of C57BL6/J mice at 8 wk of age with tumor allowed to develop for 1, 2, 3, or 4 wk and compared with PBS-injected control. Muscle wasting was evident at 4 wk LLC. RNA sequencing of gastrocnemius muscle samples showed widespread alterations in LLC compared with PBS animals with largest differences seen in 4 wk LLC, suggesting extensive transcriptomic alterations concurrent to muscle wasting. Commonly altered pathways included: mitochondrial dysfunction and protein ubiquitination, along with other less studied processes in this condition regulating transcription/translation and cytoskeletal structure. Current findings present novel evidence of transcriptomic shifts and altered cellular pathways in CC-induced muscle wasting.

The rationale for preventing cancer cachexia: targeting excessive fatty acid oxidation.

Cachexia commonly occurs at the terminal stage of cancer and has largely unclear molecular mechanisms. A recent study published in Nature Medicine, entitled “Excessive fatty acid oxidation induces muscle atrophy in cancer cachexia,” reveals that cachectic cancer cells can secrete multiple cytokines that induce excessive fatty acid oxidation, which is responsible for muscle loss in cancer cachexia. Inhibition of fatty acid oxidation using etomoxir can increase muscle mass and body weight in cancer cachexia animal models. The usage of stable cachexia animal models is also discussed in this research highlight.

Excessive fatty acid oxidation induces muscle atrophy in cancer cachexia.

Cachexia is a devastating muscle-wasting syndrome that occurs in patients who have chronic diseases. It is most commonly observed in individuals with advanced cancer, presenting in 80% of these patients, and it is one of the primary causes of morbidity and mortality associated with cancer. Additionally, although many people with cachexia show hypermetabolism, the causative role of metabolism in muscle atrophy has been unclear. To understand the molecular basis of cachexia-associated muscle atrophy, it is necessary to develop accurate models of the condition. By using transcriptomics and cytokine profiling of human muscle stem cell-based models and human cancer-induced cachexia models in mice, we found that cachectic cancer cells secreted many inflammatory factors that rapidly led to high levels of fatty acid metabolism and to the activation of a p38 stress-response signature in skeletal muscles, before manifestation of cachectic muscle atrophy occurred. Metabolomics profiling revealed that factors secreted by cachectic cancer cells rapidly induce excessive fatty acid oxidation in human myotubes, which leads to oxidative stress, p38 activation and impaired muscle growth. Pharmacological blockade of fatty acid oxidation not only rescued human myotubes, but also improved muscle mass and body weight in cancer cachexia models in vivo. Therefore, fatty acid-induced oxidative stress could be targeted to prevent cancer-induced cachexia.

Notch Signaling Mediates Skeletal Muscle Atrophy in Cancer Cachexia Caused by Osteosarcoma.

Skeletal muscle atrophy in cancer cachexia is mediated by the interaction between muscle stem cells and various tumor factors. Although Notch signaling has been known as a key regulator of both cancer development and muscle stem cell activity, the potential involvement of Notch signaling in cancer cachexia and concomitant muscle atrophy has yet to be elucidated. The murine K7M2 osteosarcoma cell line was used to generate an orthotopic model of sarcoma-associated cachexia, and the role of Notch signaling was evaluated. Skeletal muscle atrophy was observed in the sarcoma-bearing mice, and Notch signaling was highly active in both tumor tissues and the atrophic skeletal muscles. Systemic inhibition of Notch signaling reduced muscle atrophy. In vitro coculture of osteosarcoma cells with muscle-derived stem cells (MDSCs) isolated from normal mice resulted in decreased myogenic potential of MDSCs, while the application of Notch inhibitor was able to rescue this repressed myogenic potential. We further observed that Notch-activating factors reside in the exosomes of osteosarcoma cells, which activate Notch signaling in MDSCs and subsequently repress myogenesis. Our results revealed that signaling between tumor and muscle via the Notch pathway may play an important role in mediating the skeletal muscle atrophy seen in cancer cachexia.

Muscle atrophy in response to cytotoxic chemotherapy is dependent on intact glucocorticoid signaling in skeletal muscle.

Cancer cachexia is a syndrome of weight loss that results from the selective depletion of skeletal muscle mass and contributes significantly to cancer morbidity and mortality. The driver of skeletal muscle atrophy in cancer cachexia is systemic inflammation arising from both the cancer and cancer treatment. While the importance of tumor derived inflammation is well described, the mechanism by which cytotoxic chemotherapy contributes to cancer cachexia is relatively unexplored. We found that the administration of chemotherapy to mice produces a rapid inflammatory response. This drives activation of the hypothalamic-pituitary-adrenal axis, which increases the circulating level of corticosterone, the predominant endogenous glucocorticoid in rodents. Additionally, chemotherapy administration results in a significant loss of skeletal muscle mass 18 hours after administration with a concurrent induction of genes involved with the ubiquitin proteasome and autophagy lysosome systems. However, in mice lacking glucocorticoid receptor expression in skeletal muscle, chemotherapy-induced muscle atrophy is completely blocked. This demonstrates that cytotoxic chemotherapy elicits significant muscle atrophy driven by the production of endogenous glucocorticoids. Further, it argues that pharmacotherapy targeting the glucocorticoid receptor, given in concert with chemotherapy, is a viable therapeutic strategy in the treatment of cancer cachexia.

Concurrent evolution of cancer cachexia and heart failure: bilateral effects exist

Cancer cachexia is defined as a multifactorial syndrome of involuntary weight loss characterized by an ongoing loss of skeletal muscle mass and progressive functional impairment. It is postulated that cardiac dysfunction/atrophy parallels skeletal muscle atrophy in cancer cachexia. Cardiotoxic chemotherapy may additionally result in cardiac dysfunction and heart failure in some cancer patients. Heart failure thus may be a consequence of either ongoing cachexia or chemotherapy-induced cardiotoxicity; at the same time, heart failure can result in cachexia, especially muscle wasting. Therefore, the subsequent heart failure and cardiac cachexia can exacerbate the existing cancer-induced cachexia. We discuss these bilateral effects between cancer cachexia and heart failure in cancer patients. Since cachectic patients are more susceptible to chemotherapy-induced toxicity overall, this may also include increased cardiotoxicity of antineoplastic agents. Patients with cachexia could thus be doubly unfortunate, with cachexia-related cardiac dysfunction/heart failure and increased susceptibility to cardiotoxicity during treatment. Cardiovascular risk factors as well as pre-existing heart failure seem to exacerbate cardiac susceptibility against cachexia and increase the rate of cardiac cachexia. Hence, chemotherapy-induced cardiotoxicity, cardiovascular risk factors, and pre-existing heart failure may accelerate the vicious cycle of cachexia-heart failure. The impact of cancer cachexia on cardiac dysfunction/heart failure in cancer patients has not been thoroughly studied. A combination of serial echocardiography for detection of cachexia-induced cardiac remodeling and computed tomography image analysis for detection of skeletal muscle wasting would appear a practical and non-invasive approach to develop an understanding of cardiac structural/functional alterations that are directly related to cachexia.

Deacetylase Inhibitors Modulate the Myostatin/Follistatin Axis without Improving Cachexia in Tumor-Bearing Mice

Muscle wasting, as occurring in cancer cachexia, is primarily characterized by protein hypercatabolism and increased expression of ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1. Myostatin, a member of the TGFbeta superfamily, negatively regulates skeletal muscle mass and we showed that increased myostatin signaling occurs in experimental cancer cachexia. On the other hand, enhanced expression of follistatin, an antagonist of myostatin, by inhibitors of histone deacetylases, such as valproic acid or trichostatin-A, has been shown to increase myogenesis and myofiber size in mdx mice. For this reason, in the present study we evaluated whether valproic acid or trichostatin-A can restore muscle mass in C26 tumor-bearing mice. Tumor growth induces a marked and progressive loss of body and muscle weight, associated with increased expression of myostatin and ubiquitin ligases. Treatment with valproic acid decreases muscle myostatin levels and enhances both follistatin expression and the inactivating phosphorylation of GSK-3beta, while these parameters are not affected by trichostatin-A. Neither agent, however, counteracts muscle atrophy or ubiquitin ligase hyperexpression. Therefore, modulation of the myostatin/follistatin axis in itself does not appear sufficient to correct muscle atrophy in cancer cachexia.

Attenuation of skeletal muscle atrophy in cancer cachexia by d-myo-inositol 1,2,6-triphosphate

To determine the effectiveness of the polyanionic, metal binding agent D-myo-inositol-1,2,6-triphosphate (alpha trinositol, AT), and its hexanoyl ester (HAT), in tissue wasting in cancer cachexia. The anti-cachexic effect was evaluated in the MAC16 tumour model. Both AT and HAT attenuated the loss of body weight through an increase in the nonfat carcass mass due to an increase in protein synthesis and a decrease in protein degradation in skeletal muscle. The decrease in protein degradation was associated with a decrease in activity of the ubiquitin-proteasome proteolytic pathway and caspase-3 and -8. Protein synthesis was increased due to attenuation of the elevated autophosphorylation of double-stranded RNA-dependent protein kinase, and of eukaryotic initiation factor 2alpha together with hyperphosphorylation of eIF4E-binding protein 1 and decreased phosphorylation of eukaryotic elongation factor 2. In vitro, AT completely attenuated the protein degradation in murine myotubes induced by both proteolysis-inducing factor and angiotensin II. These results show that AT is a novel therapeutic agent with the potential to alleviate muscle wasting in cancer patients.

Effect of branched-chain amino acids on muscle atrophy in cancer cachexia.

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2alpha (eukaryotic initiation factor 2alpha) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G-eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70(S6k) (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70(S6k), caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G-eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.

Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and d-α-tocopherol (10 μM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin–proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-κB (NF-κB), through inhibition of phosphorylation of the NF-κB inhibitor protein (I-κB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 μM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 μM), a specific inhibitor of cytosolic phospholipase A 2, U-73122 (5 μM) and D609 (200 μM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 μM) and NSC23766 (10 μM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 μM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 μM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with d-α-tocopherol (1 mg kg − 1 ) attenuated protein degradation and increased protein synthesis in skeletal muscle.

Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase.

Atrophy of skeletal muscle is due to a depression in protein synthesis and an increase in degradation. Studies in vitro have suggested that activation of the dsRNA-dependent protein kinase (PKR) may be responsible for these changes in protein synthesis and degradation. In order to evaluate whether this is also applicable to cancer cachexia the action of a PKR inhibitor on the development of cachexia has been studied in mice bearing the MAC16 tumour. Treatment of animals with the PKR inhibitor (5 mg kg−1) significantly reduced levels of phospho-PKR in muscle down to that found in non-tumour-bearing mice, and effectively attenuated the depression of body weight, with increased muscle mass, and also inhibited tumour growth. There was an increase in protein synthesis in skeletal muscle, which paralleled a decrease in eukaryotic initiation factor 2α phosphorylation. Protein degradation rates in skeletal muscle were also significantly decreased, as was proteasome activity levels and expression. Myosin levels were increased up to values found in non-tumour-bearing animals. Proteasome expression correlated with a decreased nuclear accumulation of nuclear factor-κB (NF-κB). The PKR inhibitor also significantly inhibited tumour growth, although this appeared to be a separate event from the effect on muscle wasting. These results suggest that inhibition of the autophosphorylation of PKR may represent an appropriate target for the attenuation of muscle atrophy in cancer cachexia.