Ghrelin prevents tumour- and cisplatin-induced muscle wasting: characterization of multiple mechanisms involved

Abstract

Background: Cachexia and muscle atrophy are common consequences of cancer and chemotherapy administration. The novel hormone ghrelin has been proposed as a treatment for this condition. Increases in food intake and direct effects on muscle proteolysis and protein synthesis are likely to mediate these effects, but the pathways leading to these events are not well understood.

Methods: We characterized molecular pathways involved in muscle atrophy induced by Lewis lung carcinoma (LLC) tumour implantation in c57/bl6 adult male mice and by administration of the chemotherapeutic agent cisplatin in mice and in C2C12 myotubes. The effects of exogenous ghrelin administration and its mechanisms of action were examined in these settings.

Results: Tumour implantation and cisplatin induced muscle atrophy by activating pro-inflammatory cytokines, p38-C/EBP-β, and myostatin, and by down-regulating Akt, myoD, and myogenin, leading to activation of ubiquitin-proteasome-mediated proteolysis and muscle weakness. Tumour implantation also increased mortality. In vitro, cisplatin up-regulated myostatin and atrogin-1 by activating C/EBP-β and FoxO1/3. Ghrelin prevented these changes in vivo and in vitro, significantly increasing muscle mass (P < 0.05 for LLC and P < 0.01 for cisplatin models) and grip strength (P = 0.038 for LLC and P = 0.001 for cisplatin models) and improving survival (P = 0.021 for LLC model).

Conclusion: Ghrelin prevents muscle atrophy by down-regulating inflammation, p38/C/EBP-β/myostatin, and activating Akt, myogenin, and myoD. These changes appear, at least in part, to target muscle cells directly. Ghrelin administration in this setting is associated with improved muscle strength and survival.

Keywords: Cachexia; Cancer; Ghrelin; Growth hormone; Muscle.

Figure 1

Ghrelin improves tumour and cisplatin-induced cachexia. (A) Changes in body weight in the Lewis lung carcinoma (LLC)-induced cachexia model. HK-V represents the group injected with heat-killed (HK) LLC and vehicle. T-V (tumour + vehicle) and T-G (tumour + ghrelin) groups represent animals inoculated with LLC (10⁶ cells) receiving vehicle (saline) or ghrelin (0.8 mg/kg twice daily), respectively. The change is expressed as % from baseline. The carcass weight represents the weight after subtracting the tumour mass. (B) Changes in body weight in cisplatin-induced cachexia. V, vehicle-treated group; C, cisplatin-treated group; CG, cisplatin + ghrelin-treated group; G, ghrelin-treated group. The change expressed as % from baseline. (C) Quadriceps, gastrocnemius, soleus, tibialis anterioris (TA), and extensor digitalis longus (EDL) muscle mass normalized to baseline lean body mass (LBM) weight by nuclear magnetic resonance in the LLC-induced cachexia model. (D) Muscle mass in cisplatin-induced cachexia. (E) Grip strength % change from baseline compared to HK-V group in LLC-induced cachexia. (F) Grip strength change in cisplatin-induced cachexia. Unadjusted muscle weight changes in both models (not shown) follow the same pattern as adjusted muscle weight changes shown in Figure 1C and D.

Figure 2

Ghrelin increases protein synthesis markers and decreases proteolytic markers and proteasome activity induced by tumour or cisplatin administration. mRNA levels of MyoD, myogenin, atrogin-1/MAFbx, MuRF-1, and myostatin in Lewis lung carcinoma (LLC)-induced cachexia (A) and in cisplatin-induced cachexia (B). mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as fold change from controls. Protein levels measured by western blot (I) in LLC-induced cachexia (C) and in cisplatin-induced cachexia (D). Quantification of Atrogin-1/MAFbx (II) and Myostatin (III) was normalized to GAPDH, p-Akt (IV) to total Akt, and p-p38 (V) to total p38. Proteasome activity in LLC-induced cachexia (E) and in cisplatin-induced cachexia (F) expressed as arbitrary fluorescence units/µg protein. HK-V, heat-killed (HK) LLC + vehicle. T-V (tumour + vehicle) and T-G (tumour + ghrelin) groups represent animals inoculated with LLC (10⁶ cells) receiving vehicle (saline) or ghrelin (0.8 mg/kg twice daily), respectively. V, vehicle-treated group; C, cisplatin-treated group; CG, cisplatin + ghrelin-treated group; G, ghrelin-treated group.

Figure 3

Ghrelin improves cisplatin-induced myotubes breakdown. (A) Immunofluorescence staining for anti-myosin/myosin heavy chain (MHC) antibody in C2C12 myotubes. MHC staining outlines the myotubes (red). 4'6-Diamidino-2-phenylindole was used to stain the nuclei (blue). (B) Myotubes size expressed as % from vehicle. (C–D) Western blot of MHC and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in C2C12 myotubes. V, vehicle-treated group; C, cisplatin-treated group; CG, cisplatin + ghrelin-treated group.

Figure 4

Ghrelin prevents cisplatin-induced changes in protein synthesis and degradation in vitro. (A) mRNA levels for myoD, myogenin, atrogin-1/MAFbx, MuRF-1, and myostatin normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as fold change form vehicle. (B) Western blot of cisplatin- and ghrelin-treated C2C12 myotubes (I). Atrogin-1/MAFbx (II), Myostatin (III), and MyoD (IV) are normalized to GAPDH, p-Akt (V) to total Akt and p-p38 (VI) to total p38. V, vehicle-treated group; C, cisplatin-treated group; CG, cisplatin + ghrelin-treated group.

Figure 5

Mechanism of action of ghrelin in C2C12 myotubes. (A) Western blot of transcriptional factor in nuclear extracts. (I) Blot and quantification of C/EBP-β (II) and FoxO1 (III) in nuclear protein, Lamin A was used as the reference. (B) Luciferase assay of myostatin promoter. Map of myostatin promoter (top), which includes the FoxO, drosophila mothers against decapentaplegic protein (SMAD), and C/EBP-β DNA-binding sites and linked to a vector containing luciferase. The relative luciferase activity (bottom) is expressed as fold change from vehicle. (C) Luciferase assay of atrogin-1/MAFbx promoter. Top is the map of atrogin-1/MAFbx promoter, pA includes the C/EBP-β and FoxO DNA-binding sites. pA-C/EBP-β-M includes a mutant C/EBP-β site and a normal FoxO site. pA-FOXO-M includes a mutant FoxO site and a normal C/EBP-β site. The relative luciferase activity expressed as fold change from vehicle (bottom).V, vehicle-treated group; C, cisplatin-treated group; CG, cisplatin + ghrelin-treated group; G, ghrelin-treated group.

Figure 6

Ghrelin increases survival in Lewis lung carcinoma (LLC)-induced cachexia (Kaplan–Meyer curve). Days survived from when tumours were noted and ghrelin and vehicle injections started (approximately 7 days after LLC cell inoculation). T+V, tumour + vehicle; T+G, tumour + ghrelin.