Z-ajoene from Crushed Garlic Alleviates Cancer-Induced Skeletal Muscle Atrophy

Abstract

Skeletal muscle atrophy is one of the major symptoms of cancer cachexia. Garlic (Allium sativum), one of the world's most commonly used and versatile herbs, has been employed for the prevention and treatment of diverse diseases for centuries. In the present study, we found that ajoene, a sulfur compound found in crushed garlic, exhibits protective effects against muscle atrophy. Using CT26 tumor-bearing BALB/c mice, we demonstrate in vivo that ajoene extract alleviated muscle degradation by decreasing not only myokines secretion but also janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) and SMADs/forkhead box (FoxO) signaling pathways, thereby suppressing muscle-specific E3 ligases. In mouse skeletal myoblasts, Z-ajoene enhanced myogenesis as evidenced by increased expression of myogenic markers via p38 mitogen-activated protein kinase (MAPK) activation. In mature myotubes, Z-ajoene protected against muscle protein degradation induced by conditioned media from CT26 colon carcinoma cells, by suppressing expression of muscle specific E3 ligases and nuclear transcription factor kappa B (NF-κB) phosphorylation which contribute to muscle atrophy. Moreover, Z-ajoene treatment improved myofiber formation via stimulation of muscle protein synthesis. These findings suggest that ajoene extract and Z-ajoene can attenuate skeletal muscle atrophy induced by cancer cachexia through suppressing inflammatory responses and the muscle wasting as well as by promoting muscle protein synthesis.

Keywords: Allium sativum; C2C12 cells; Z-ajoene; cancer; skeletal muscle atrophy.

Figure 1

(A) Tumor volumes of CT26 tumor-bearing mice; (B) total muscle weight at the time of euthanasia; (C) representative hematoxylin and eosin (H&E) staining (magnification × 100) (left) and average value for cross sectional area (right) of the quadriceps muscle. Values are presented as the mean ± SEM. Statistical significance was evaluated by one-way ANOVA followed by the Duncan’s multiple range test. Means with different superscript letter are significantly different at p < 0.05 (B) and p < 0.0001 (C). C, control; TC, tumor control; A5, 5 mg/kg ajoene extract; A10, 10 mg/kg ajoene extract (n = 10 per group for A and B; n = 4 for C).

Figure 2

Effects of ajoene extract on myokines secretion in CT26 tumor-bearing mice. (A,B) mRNA expression of interleukin-6 (IL-6), interleukin-6 receptor (IL-6R), and myostatin in the quadriceps muscle; (C) representative contour plots showing the proportions of macrophages (top) and MDSCs (bottom) in the spleen. Values are presented as mean ± SEM. Statistical significance was evaluated by one-way ANOVA followed by the Duncan’s multiple range test or Student’s t-test. Means with different superscript letters are significantly different at p < 0.05 (A) and p < 0.001 (B). * p < 0.05 compared with the C group; # p < 0.05 and ## p < 0.01 compared with the TC group. MDSCs, myeloid-derived suppressor cells; C, control; TC, tumor control; A5, 5 mg/kg ajoene extract; A10, 10 mg/kg ajoene extract (n = 5 per group).

Figure 3

Effects of ajoene extract on muscle atrophy-associated markers in CT26 tumor-bearing mice. The mRNA expression levels of genes associated with myotube synthesis (A), muscle degradation (B), JAK/STAT3 (C), and SMADs/FoxO signaling pathways (D). Values are presented as the mean ± SEM. Statistical significance was evaluated by one-way ANOVA followed by the Duncan’s multiple range test or Student’s t-test. Means with different superscript letters are significantly different at p < 0.05 (A, C, D) and p < 0.01 (B). # p < 0.05 compared with the TC group. C, control; TC, tumor control; A5, 5 mg/kg Z-ajoene; A10, 10 mg/kg Z-ajoene (n = 5 per group).

Figure 3

Effects of ajoene extract on muscle atrophy-associated markers in CT26 tumor-bearing mice. The mRNA expression levels of genes associated with myotube synthesis (A), muscle degradation (B), JAK/STAT3 (C), and SMADs/FoxO signaling pathways (D). Values are presented as the mean ± SEM. Statistical significance was evaluated by one-way ANOVA followed by the Duncan’s multiple range test or Student’s t-test. Means with different superscript letters are significantly different at p < 0.05 (A, C, D) and p < 0.01 (B). # p < 0.05 compared with the TC group. C, control; TC, tumor control; A5, 5 mg/kg Z-ajoene; A10, 10 mg/kg Z-ajoene (n = 5 per group).

Figure 4

Effect of Z-ajoene on myoblast differentiation. (A) Structure of Z-ajoene. (B) C2C12 myoblasts were differentiated using differentiation medium (DM) supplementary with Z-ajoene (0.1, 1, 10, or 100 nM) for 3 days and then collected for immunostaining of myosin heavy chain (MHC) (red) and 4’,6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 200 μm. (C) Differentiated C2C12 cells in the presence of Z-ajoene were subjected to Western blot analysis to determine the expression level of MHC. (D) Day-dependent effect of Z-ajoene (100 nM) on expression levels of myogenic factors during myoblast differentiation.

Figure 5

Effect of Z-ajoene on p38 MAPK activation during myoblast differentiation. (A) C2C12 cells were treated with Z-ajoene (100 nM) during days 1–3 of differentiation (D1–D3). Collected cell lysates were subjected to immunoblot. (B) C2C12 cells were pre-treated with (+) or without (-) SB203580 (10 μM) prior to Z-ajoene and differentiated in DM for 2 days. Cell lysates were subjected to Western blot analysis and immunostained with MHC (red) and DAPI (blue). Scale bar = 200 μm.

Figure 6

Preventive effect of Z-ajoene in conditioned media (CM) from CT26 colon carcinoma-induced myotube loss. C2C12 myoblasts were differentiated into myotubes for 3 days using differentiation medium (DM). Differentiated myotubes were pre-treated with Z-ajoene (1 μM) for 3 h, and then treated with (+) or without (-) 30% CM for 24 h. Cells were collected and the muscle specific E3 ligases expression was assessed by Western blot analysis (A) and quantitative real-time polymerase chain reaction (PCR) (C). Data are means ± SD of triplicated experiments. # p < 0.0001 compared with group treated only with DM; * p < 0.0001 compared with cells treated with CM. (B) Collected cells were fixed and immunostained with MHC (red) and DAPI (blue). Scale bar = 200 μm. (D) Cell lysates were subjected to Western blot analysis to analyze MHC, phosphorylated nuclear transcription factor kappa B (NF-κB), E3 ligases, myostatin, and phosphorylated mammalian target of rapamycin (mTOR).

Figure 7

Summary of the effects of Z-ajoene from Allium sativum on muscle atrophy and related molecular mechanisms in vivo and in vitro.