Effects of ghrelin on doxorubicin-induced toxicity in skeletal muscle

Abstract

Doxorubicin is a potent anti-cancer drug that has been used for treating a wide range of cancers. However, doxorubicin causes multi-organ toxicity which has largely limited the clinical usage of it. There are more than 30% of cancer patients suffer from cancer cachexia which is characterized by significant weight loss and muscle wasting resulting in weakness and fatigue. Notably, application of doxorubicin may further compromise muscle function through the induction of muscle impairment in patients with cancer cachexia. Ghrelin is a peptide that has been proposed as a potential candidate for treating cancer cachexia due to its potent ability in stimulating growth hormone release and increasing food intake. Administration of ghrelin has been shown to preserve muscle mass during fasting-induced muscle atrophy. This study aimed to investigate whether ghrelin protects skeletal muscle from doxorubicin-induced damage. Wild type adult C57BL/6 mice were randomly assigned to saline control group (CON; n = 7), doxorubicin group (DOX; n = 7), doxorubicin with treatment of ghrelin group (DOX+Ghrelin; n = 7) and doxorubicin with treatment of ghrelin plus GHSR-1a antagonist [D-Lys3]-GHRP-6 group (DOX+Ghrelin+[D-Lys3]-GHRP-6; n = 5). Mice in all groups were injected with doxorubicin (15 mg/kg, i.p.) at the beginning except the mice in the CON group received saline as a placebo. Mice assigned to the DOX+Ghrelin and DOX+Ghrelin+[D-Lys3]-GHRP-6 groups then received ghrelin treatment (100 μg/kg, i.p.) every 12 hours while mice in both the CON and DOX groups received same volume of saline as a placebo. Mice in the DOX+Ghrelin+[D-Lys3]-GHRP-6 group had GHSR-1a antagonist [D-Lys3]-GHRP-6, (3.75mg/kg, i.p.) injected just before the administration of ghrelin. All mice were sacrificed 5 days after the first injection. Histological analysis revealed that abnormal high percentage of centronucleated fibers was only observed in doxorubicin-treated muscle. The increased abundance of cleaved-actin in doxorubicin-treated muscle was accompanied by the upregulation of Bax, increase in number of TUNEL positive nuclei and elevation of apoptotic DNA fragmentation whereas these changes were not found in mice receiving ghrelin treatment. Protein abundances of autophagic markers, including LC3 II/I ratio, Atg12-5 complex, Atg5 and Beclin-1 were not changed after doxorubicin administration but upregulated by ghrelin administration. Application of GHSR-1a antagonist did not blunt the anti-apoptotic effects of ghrelin in doxorubicin-treated muscle but further augmented the phosphorylation of Akt and protein abundances of autophagic markers. It is noted that the basal levels of apoptosis and autophagy were not affected by administration of ghrelin alone under normal physiological condition. It is also observed that autophagy was upregulated 24 hours after doxorubicin administration and return to basal level after 5 days. Collectively, our data suggest that ghrelin upregulates autophagy in the skeletal muscle following doxorubicin treatment in concomitant with the suppression of apoptosis and muscle protein degradation. It is worth noting that the modulating effects of ghrelin on apoptosis and autophagy in doxorubicin-treated skeletal muscle are possibly not mediated through the GHSR-1a signaling. These findings are consistent with the hypothesis that ghrelin protects skeletal muscle against the doxorubicin-induced injury.