Ghrelin relieves cancer cachexia associated with the development of lung adenocarcinoma in mice

Abstract

Cancer cachexia is a multifactorial, critical illness syndrome characterized by an ongoing loss of skeletal muscle and adipose tissue. The reductions in body weight and skeletal muscle mass are important prognostic indicators for cancer patients that are refractory to current therapies. Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is produced in the stomach, stimulates food intake and growth hormone secretion, suppresses inflammation, and prevents muscle catabolism. We investigated the pharmacological potential of ghrelin in the treatment of cancer cachexia by using urethane-treated, bronchioalveolar epithelium-specific Pten-deficient mice that developed lung adenocarcinomas. Ghrelin or phosphate-buffered saline was given to mice daily for four weeks beginning at five months after urethane injection, which corresponded to the time point of lung adenocarcinoma formation. Ghrelin inhibited the inductions of C-reactive protein, tumor necrosis factor-α, interleukin-1β, and interleukin-6, mitigated the reduction of food intake and fat mass, and consequently ameliorated body weight loss in the mouse model of lung adenocarcinoma. We also demonstrated that skeletal muscle mass and muscle contraction force in both fast-twitch muscle and slow-twitch muscle were retained in ghrelin-treated mice in conjunction with an upregulation of local insulin-like growth factor 1/Akt signaling. In addition, ghrelin administration reduced the expressions of phosphorylated-p38 mitogen-activated protein kinase, phosphorylated-nuclear factor-kappa B, Forkhead box protein O1, muscle RING-finger protein-1, and F-Box protein 32 in the lysates of skeletal muscle in the tumor-bearing state. Our results indicate that ghrelin administration exerts a protective effect against cancer cachexia by ameliorating skeletal muscle wasting and regulating systemic inflammation.

Introduction

Cancer cachexia affects up to 80% of patients with advanced cancers and accounts for nearly 30% of cancer-related deaths (Acharyya et al., 2005, Fearon, 2008). A key feature of cachexia is significant reduction in body weight resulting predominantly from progressive depletion of skeletal muscle mass (Fearon et al., 2012). Muscle atrophy leads to general muscle weakness, impairment of activity of daily life, and eventually death through respiratory failure. The mechanisms of cancer cachexia are multifactorial, and cannot be fully reversed by nutritional support alone. Various hormones, proinflammatory cytokines, and tumor-derived factors have been shown to influence muscle protein synthesis/degradation balance through several major intracellular signal-transduction systems, including the insulin-like growth factor 1 (IGF1)/insulin receptor substrate 1/Akt pathway and the Forkhead box protein O1 (FoxO1)/muscle RING-finger protein-1 (MuRF1)/F-Box protein-32 (Atrogin1) pathway (Zhou et al., 2010). Ideal interventions against cancer cachexia should exert their effects both upstream (antagonizing key mediators of systemic inflammation) and downstream (blocking catabolic pathways or stimulating anabolic pathways in skeletal muscle) (Fearon et al., 2012). Despite recent advances in understanding the pathological mechanisms of cancer cachexia, few therapeutic options are currently available. In addition, there is no ideal rodent cancer cachexia model for replicating the condition in humans.

Ghrelin is a 28-amino-acid peptide initially isolated from the human and rat stomach as an endogenous ligand for the growth hormone secretagogue (GHS)-receptor (Kojima et al., 1999). While ghrelin has a potent orexigenic effect independently of growth hormone (GH) secretion, ghrelin is also known to have multifaceted effects on energy metabolism, including decrease of energy expenditure (Yasuda et al., 2003), stimulation of adiposity (Tschop et al., 2000), and prevention of muscle catabolism (Koshinaka et al., 2011, Sugiyama et al., 2012). In addition, ghrelin inhibits the expression of proinflammatory anorectic cytokines in human monocytes and T cells (Dixit et al., 2004). These observations suggest that ghrelin might improve cachectic conditions. Previous studies using tumor implantation models have reported that ghrelin administration resulted in significant increases in food intake and body weight (DeBoer et al., 2007, Hanada et al., 2003). However, the therapeutic effect and molecular mechanisms of ghrelin treatment against cancer cachexia, including muscle wasting, remain unknown, especially in the context of a model of cancer development.

We previously reported that almost all mice with a bronchioalveolar epithelium-specific null mutation of Pten, a tumor suppressor gene mutated in many human cancers, including lung adenocarcinoma (Marsit et al., 2005, Tang et al., 2006), spontaneously developed lung adenocarcinomas (Yanagi et al., 2007). This animal model of lung adenocarcinoma is highly reproducible and accelerates cancer formation over a relatively short time course by administration of urethane, a well-known initiator of lung carcinomas (Malkinson and Beer, 1983). In this study, we showed that there were marked upregulations of markers of muscle atrophy and proinflammatory cytokines as well as significant reduction in body weight and loss of skeletal muscle mass in urethane-treated, bronchioalveolar epithelium-specific Pten-deficient mice. To investigate the efficacy of ghrelin treatment against the syndrome of cancer cachexia in the present study, we used this mouse model of lung adenocarcinoma.