ubiquitin ligases are involved in muscle atrophy of cancer cachexia : An in vitro and in vivo study

Muscle atrophy F-Box (MAFbx)/atrogin-1 and muscle ring-finger-1 (MuRF-1) have been identified as two muscle-specific E3 ubiquitin ligases that are highly expressed in skeletal muscle during muscle atrophy. However, the role of muscle-specific E3 ubiquitin ligases during the process of muscle atrophy of cancer cachexia remains largely unknown. In the present study, we analyzed the expression of atrogin-1 and MuRF-1 in the skeletal muscle of patients with malignant and benign disease. The possible mechanisms were studied both in a colon 26-induced cancer cachexia mouse model and in tumor necrosis factor-α (TNF-α) induced atrophy C2C12 cells. Our results demonstrated that atrogin-1 and MuRF-1 tended to be increased in the skeletal muscle of patients with malignant disease even before weight loss. Non-tumor body weights and gastrocnemius weights were significantly decreased while expression levels of ubiquitin proteasome pathway associated genes (atrogin-1, MuRF-1, ubiquitin and E2-14K) were upregulated in cancer cachexia mice. Significant myotube atrophy with atrogin-1 overexpression was observed in the C2C12 cells treated with TNF-α. Meanwhile, knockdown of atrogin-1 by small interfering RNA (siRNA) protected C2C12 cells from the adverse effect of TNF-α. In conclusion, muscle-specific E3 ubiquitin ligases were upregulated during cancer cachexia, and atrogin-1 may be a potential molecular target for treating muscle atrophy induced by cancer cachexia. Introduction Cancer cachexia, a condition of advanced protein calorie malnutrition, is characterized by loss of skeletal muscle protein, depletion of lipid storage, anorexia, poor performance and ultimately death (1-3). Cancer cachexia occurs in ~80% of cancer patients and is the primary cause of death for 22-30% of all cancer patients (3,4). However, cancer cachexia is difficult to diagnose until patients have lost more than 5-7% of their body weight (5,6). Furthermore, it was reported that 80% of patients with upper gastrointestinal cancers and 60% of patients with lung cancer had already experienced substantial weight loss at the moment of diagnosis (7). Therefore, it is essential to illuminate the early mechanisms in the development of cancer cachexia. The pathogenesis of cancer cachexia including alterations in energy and substrate metabolisms is multifunctional. Skeletal muscle protein depletion (i.e. muscle atrophy) due to hyper-catabolism is the most common characteristic of cancer cachexia. Cancer cachexia is a complex process that occurs as a consequence of a variety of stressors including neural inactivity, mechanical unloading, inflammation, metabolic stress and elevated glucocorticoids (1,8,9). Several proteolytic systems, such as the lysosome, the calcium-dependent systems and the ubiquitin-dependent systems are involved in degrading skeletal muscle (8). However, it was reported that ubiquitin dependent proteolysis is the main mechanism for the enhanced muscle protein degradation in cancer cachexia (10). In addition, cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6) and interferon-γ (IFN-γ) have been proposed as mediators for the cancer cachectic process (3,11,12). The ubiquitin-dependent proteasome pathway includes a series of reactions involving three classes of proteins: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin protein ligases (E3) (13). Muscle atrophy F-Box (MAFbx)/atrogin-1 and muscle ring-finger-1 (MuRF-1), identified over 10 years ago, are two muscle-specific E3 ubiquitin ligases that are increased in skeletal muscle under atrophy-inducing conditions such as humans under voluntary bed rest (14) or with spinal cord injury (15), amyotrophic lateral sclerosis (16), chronic obstructive pulmonary disease Muscle-specific E3 ubiquitin ligases are involved in muscle atrophy of cancer cachexia: An in vitro and in vivo study LEI YUAN1*, JUN HAN1*, QINGYANG MENG1, QIULEI XI1, QIULIN ZHUANG1, YI JIANG1, YUSONG HAN1, BO ZHANG1, JING FANG2 and GUOHAO WU1 1Department of General Surgery, Zhongshan Hospital, Fudan University; 2Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, P.R. China Received December 8, 2014; Accepted February 9, 2015 DOI: 10.3892/or.2015. 3845 Correspondence to: Professor Guohao Wu, Department of General Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, P.R. China E-mail: prowugh@163.com *Contributed equally