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

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.


Introduction
Cancer is one of the leading causes of morbidity and mortality. More than 18 million new cases were estimated to have been diagnosed in 2018 [1]. Around 50-80% of cancer patients, particularly those with gastrointestinal cancer including colon cancer, suffer from cachexia, which is a complex metabolic syndrome. Cachexia is characterized by weight loss mainly as a result of progressive loss of skeletal muscle, leading to inevitable functional impairment [2][3][4]. Cancer cachexia is also associated with poor responses to chemotherapy and quality of life, thereby contributing to shortened survival times [3,4]. Since at least 20-40% of cancer deaths are attributable to cachexia [3,5], there is an urgent need to find effective strategies for preventing and treating these symptoms in cancer patients.
Skeletal muscle atrophy is considered to be the representative manifestation of cancer cachexia and results from an imbalance of protein synthesis and degradation [4,6]. Specifically in cachexia,

Mouse Model of Cancer Cachexia
Six-week-old male BALB/c mice were obtained from Orient Bio (Sungnam, Republic of Korea). The animals were housed at 22 ± 1 • C under a 12 h light/dark cycle with ad libitum access to chow diet with water for 1 week. CT26 murine colon carcinoma cells were purchased from the Korean Cell Line Bank (KCLB; Seoul, Republic of Korea) and cultured in Dulbecco's modified Eagle's medium (DMEM) (WelGENE, Daegu, Republic of Korea) with 10% fetal bovine serum (Gibco BRL Life Technology, Grand Island, NY, USA). To induce cancer cachexia, BALB/c mice received subcutaneous injections of CT26 cells into the right flank of CT26 cells (5 × 10 5 per mouse). After inoculation, the AIN-76A diet (Research Diets, Inc., New Brunswick, NJ, USA) was provided and body weight, food intake, and tumor volume were measured twice per week. The estimated tumor volume (V) was calculated based on the formula W 2 × L × 0.5 (W, the largest tumor diameter in centimeters; L, the next largest tumor diameter in centimeters) as previously described [26]. When tumors reached 80-200 mm 3 on day 15, mice were randomly assigned to three groups and received vehicle (saline; tumor control, TC), 5 mg/kg ajoene extract (A5), or 10 mg/kg ajoene extract (A10) intraperitoneally for a week (n = 10 per group). The experimental design is presented in Figure S2. At necropsy, tissues and serum were snap frozen in liquid nitrogen and stored at −80 • C until further experiments. This animal study was approved by the Institutional Animal Care and Use Committee of Sookmyung Women's University (SMWU-IACUC-1702-049-03) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals developed by the Institute of Laboratory Animal Resources of the National Research Council [27].

Assessment of Muscle Cross-Sectional Area
The quadriceps muscles were fixed in 4% paraformaldehyde and stained with hematoxylin and eosin (H&E) to measure the muscle fiber cross sectional area. After staining, 250 muscle fiber areas in a muscle section were averaged. Images were acquired by using Camera Nikon DS-Ri2 and analyzed using NIS-Elements BR 4.50.00 (Nikon, Tokyo, Japan).

Flow Cytometry
Red blood cells (RBC) were removed from splenocytes using RBC lysing buffer (Sigma-Aldrich, St. Louis, MO, USA). The cells were incubated with antibodies for 30 min. The antibodies used for flow cytometry were as follows: CD45 (Tonbo Biosciences, San Diego, CA, USA), Gr-1, and CD11b (eBioscience, San Diego, CA, USA). Samples were acquired on a FACSCanto II (BD Biosciences, San Jose, CA, USA) using the Diva software. Data analysis was performed with the FlowJo software (Tree Star Inc., Ashland, OR, USA).

Cell Culture, Myoblast Differentiation and Collection of Conditioned Medium of CT26 Cancer Cells
C2C12 murine myoblast cells (American Type Culture Collection, Manassas, VA, USA) were maintained in growth medium (GM; DMEM containing 15% fetal bovine serum). When cells reached 95% confluence, GM was replaced with differentiation medium (DM, DMEM containing 2% horse serum) (differentiation day 0: D0). After 3 days (differentiation day 3: D3), cells were subjected to analytical experiments.
To prepare the CT26 murine colon cancer cell-conditioned medium (CT26-CM), CT26 cells were seeded. After 24 h, cells were washed three times with phosphate-buffered saline (PBS) and replaced with serum-free DMEM to exclude serum inflammatory factors, followed by an additional 24 h incubation. The resulting CT26-CM was centrifuged, sterilized by filtering with a 0.22-µm syringe filter, and diluted into fresh DM, with a final concentration of 30% for cell treatment.

RNA Extraction and Real-Time Quantitative Polymerase Chain Reaction Analysis (qRT-PCR)
Total RNA was extracted from mouse quadriceps muscle tissue and C2C12 cells using TRIzol reagent (Invitrogen™, Carlsbad, CA, USA). RNA purification and first-strand cDNA synthesis were performed following the manufacturer's recommendation (Labopass™ cDNA synthesis kit, Cosmogenetech, Seoul, Republic of Korea). The RT-qPCR reaction was conducted with the SYBR ® Green PCR Master Mix and performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA, USA). All mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. The primers used for the amplifications are presented in Table S1.

Statistical Analysis
Differences were assessed using Student's t-test or one-way analysis of variance (ANOVA) followed by the Duncan's multiple range test with SAS version 9.4 (SAS Institute, Inc., Cary, NC, USA). All experiments were performed in triplicate at least three times. Differences with a p value of less than 0.05 were considered statistically significant.

Ajoene Extract of Garlic Attenuates Cancer-Induced Muscle Atrophy in CT26 Tumor-Bearing Mice
To investigate the effects of ajoene extract on cancer-induced muscle atrophy, we examined the in vivo efficacy of ajoene extract treatment in CT26 tumor-bearing mice. We did not observe significant differences in tumor growth among the mice treated with 0, 5, and 10 mg/kg ajoene extract ( Figure 1A). Total muscle weight was significantly increased in the mice treated with 10 mg/kg of ajoene extract compared with the tumor control mice (p = 0.010) ( Figure 1B; Table 1). Measurements of the cross-sectional area of the quadriceps muscle revealed that ajoene extract treatment (5 and 10 mg/kg) significantly increased the muscle fiber area compared with the tumor control group (p < 0.0001) ( Figure 1C). In the cachexia groups, spleen and liver weights were increased compared with controls (p < 0.0001) and these did not differ among the three groups. The perirenal fat weight was reduced in the tumor control group, while it tended to increase in ajoene extract-treated groups (p = 0.020)  (Table 1). Accordingly, these results indicate that ajoene extract treatment alleviates muscle atrophy at concentrations that do not exhibit anti-cancer effects in tumor-bearing mice.

Ajoene Extract Suppresses Muscle Wasting by Reducing Myokines Secretion in CT26 Tumor-Bearing Mice
Recent studies have reported that myokines, which are secreted from myocytes, play an important role in muscle wasting [28,29]. Therefore, we investigated the effect of ajoene extract on the level of myokines such as IL-6 and myostatin in muscles of tumor-bearing mice. IL-6 mRNA expression showed the tendency to increase in the tumor control group and to decrease by ajoene extract treatment ( Figure 2A). We also determined the level of interleukin-6 receptor (IL-6R) since it is involved in IL-6 stabilization and IL-6 signaling activation [30]. Similarly, mRNA expressions of IL-6R and myostatin were increased in the tumor control group, whereas they were significantly reduced in response to ajoene extract treatment (IL-6R, p = 0.042; myostatin, p < 0.001) (Figure 2A,B). Given that IL-6 is released by myeloid cells including macrophages and myeloid-derived suppressor cells (MDSCs) associated with cachexia [31][32][33], we analyzed the proportions of macrophages and MDSCs in the spleen. The proportions of both were elevated in the tumor control group compared with the control group (p < 0.01), while they were significantly suppressed in the 10 mg/kg ajoene extract-treated mice (macrophages, p < 0.05; MDSCs, p < 0.01) ( Figure 2C). These results were in agreement with the reduced mRNA levels of IL-6 and IL-6R in respective groups ( Figure 2A). Taken together, our data suggest that ajoene extract effectively suppresses myokine secretion in muscles of tumor-bearing mice, thereby contributing to the protection against cancer-induced muscle wasting.

Ajoene Extract Suppresses Muscle Wasting by Reducing Myokines Secretion in CT26 Tumor-Bearing Mice
Recent studies have reported that myokines, which are secreted from myocytes, play an important role in muscle wasting [28,29]. Therefore, we investigated the effect of ajoene extract on the level of myokines such as IL-6 and myostatin in muscles of tumor-bearing mice. IL-6 mRNA expression showed the tendency to increase in the tumor control group and to decrease by ajoene extract treatment (Figure 2A). We also determined the level of interleukin-6 receptor (IL-6R) since it is involved in IL-6 stabilization and IL-6 signaling activation [30]. Similarly, mRNA expressions of IL-6R and myostatin were increased in the tumor control group, whereas they were significantly reduced in response to ajoene extract treatment (IL-6R, p = 0.042; myostatin, p < 0.001) (Figures 2A  and 2B). Given that IL-6 is released by myeloid cells including macrophages and myeloid-derived suppressor cells (MDSCs) associated with cachexia [31][32][33], we analyzed the proportions of macrophages and MDSCs in the spleen. The proportions of both were elevated in the tumor control group compared with the control group (p < 0.01), while they were significantly suppressed in the 10 mg/kg ajoene extract-treated mice (macrophages, p < 0.05; MDSCs, p < 0.01) ( Figure 2C). These results were in agreement with the reduced mRNA levels of IL-6 and IL-6R in respective groups ( Figure 2A). Taken together, our data suggest that ajoene extract effectively suppresses myokine secretion in muscles of tumor-bearing mice, thereby contributing to the protection against cancer-induced muscle wasting.

Ajoene Extract Inhibits Muscle Degradation by Down-Regulating JAK/STAT3 and SMADs/FoxO Signaling Pathways In CT26 Tumor-Bearing Mice
To investigate the effects of ajoene extract on muscle wasting in tumor-bearing mice, we determined the mRNA expression levels of genes associated with myotube synthesis and muscle degradation. Myosin heavy chain (MHC) expression was significantly decreased in the tumor group, while it tended to increase in the 10 mg/kg ajoene extract group (p = 0.039). The expression of MyoD, a myogenesis initiator, exhibited a similar pattern to MHC expression; however, the difference was not statistically significant ( Figure 3A). E3 ligases, MAFbx and MuRF1 associated with the development of muscle catabolism [34] were elevated in the tumor control group, whereas ajoene extract treatment significantly decreased the expressions of these markers (MAFbx, p = 0.004; MuRF1, p = 0.003) ( Figure 3B).
Mice treated with ajoene extract exhibited more obvious changes in muscle degradation than myotube formation. Therefore, we further investigated molecular mechanisms underlying the protective role of ajoene extract in attenuating muscle degradation. IL-6 is known to activate JAK/STAT3 signaling to induce muscle-specific E3 ligases [4,10]. We observed elevated levels of Jak and Stat3 mRNA in the tumor control group, which were significantly reduced in the 10 mg/kg ajoene extract-treated mice (Jak, p = 0.029; Stat3, p = 0.046) ( Figure 3C).
Furthermore, myostatin activates the assembly of SMAD2/3 and SMAD4, which both, in turn, relocate into nucleus to stimulate transcription of muscle atrophy-related genes [35,36]. Therefore, we also analyzed myostatin-related signaling as another upstream pathway of MAFbx and MuRF1. As with the genes involved in the JAK/STAT3 pathway, 10 mg/kg ajoene extract significantly suppressed the expression of Smad2, Smad3, and Smad4 compared with the tumor control group (Smad2, p = 0.012; Smad3, p = 0.049; Smad4, p < 0.001) ( Figure 3D). Ajoene extract also significantly down-regulated the expression of FoxO1, which is activated by Smad2 and Smad3 (p < 0.001) ( Figure 3D). Collectively, these results indicate that ajoene extract alleviates muscle atrophy by modulating JAK/STAT3 and SMADs/FoxO signaling pathways in tumor-bearing mice.

Ajoene Extract Inhibits Muscle Degradation by Down-Regulating JAK/STAT3 and SMADs/FoxO Signaling Pathways In CT26 Tumor-Bearing Mice
To investigate the effects of ajoene extract on muscle wasting in tumor-bearing mice, we determined the mRNA expression levels of genes associated with myotube synthesis and muscle degradation. Myosin heavy chain (MHC) expression was significantly decreased in the tumor group, while it tended to increase in the 10 mg/kg ajoene extract group (p = 0.039). The expression of MyoD, a myogenesis initiator, exhibited a similar pattern to MHC expression; however, the difference was not statistically significant ( Figure 3A). E3 ligases, MAFbx and MuRF1 associated with the development of muscle catabolism [34] were elevated in the tumor control group, whereas ajoene extract treatment significantly decreased the expressions of these markers (MAFbx, p = 0.004; MuRF1, p = 0.003) ( Figure 3B).
Mice treated with ajoene extract exhibited more obvious changes in muscle degradation than myotube formation. Therefore, we further investigated molecular mechanisms underlying the protective role of ajoene extract in attenuating muscle degradation. IL-6 is known to activate JAK/STAT3 signaling to induce muscle-specific E3 ligases [4,10]. We observed elevated levels of Jak and Stat3 mRNA in the tumor control group, which were significantly reduced in the 10 mg/kg ajoene extract-treated mice (Jak, p = 0.029; Stat3, p = 0.046) ( Figure 3C).
Furthermore, myostatin activates the assembly of SMAD2/3 and SMAD4, which both, in turn, relocate into nucleus to stimulate transcription of muscle atrophy-related genes [35,36]. Therefore, we also analyzed myostatin-related signaling as another upstream pathway of MAFbx and MuRF1. As with the genes involved in the JAK/STAT3 pathway, 10 mg/kg ajoene extract significantly suppressed the expression of Smad2, Smad3, and Smad4 compared with the tumor control group (Smad2, p = 0.012; Smad3, p = 0.049; Smad4, p < 0.001) ( Figure 3D). Ajoene extract also significantly down-regulated the expression of FoxO1, which is activated by Smad2 and Smad3 (p < 0.001) ( Figure  3D). Collectively, these results indicate that ajoene extract alleviates muscle atrophy by modulating JAK/STAT3 and SMADs/FoxO signaling pathways in tumor-bearing mice.

Z-ajoene Stimulates Myogenesis
Normally, during differentiation after addition of differentiation medium (DM), mononucleated myoblasts become long and tubular myocytes. These myocytes fuse together to become multinucleated and adopted the cylinder-shape of myotubes [37], and mature myotubes can be detected by immunostaining for MHC and 4'-6-diamidino-2-phenylindole (DAPI). We observed increased myoD expression and a large number of mature myotubes after treatment of ajoene extract ( Figure  S3) indicating the myogenic effects. To investigate whether Z-ajoene ( Figure 4A), as an active ingredient of garlic, has myogenic properties, we added it to myoblasts during differentiation. Zajoene increased MHC expression and formation of multinucleated mature myotubes in a dosedependent manner (Figures 4B and 4C). Z-ajoene (100 nM) also day-dependently increased the expressions of MHC and myogenin as compared with respective day-specific control. The expression level of MyoD, a myogenic transcriptional factor, reached a maximum on differentiation day 2 (D2) in both control and Z-ajoene-treated cells. Exposure to Z-ajoene led to a 1.5-fold increase in the expression of MyoD as compared with control at D2 ( Figure 4D). Taken together, Z-ajoene stimulates myoblast differentiation.

Z-ajoene Stimulates Myogenesis
Normally, during differentiation after addition of differentiation medium (DM), mononucleated myoblasts become long and tubular myocytes. These myocytes fuse together to become multi-nucleated and adopted the cylinder-shape of myotubes [37], and mature myotubes can be detected by immunostaining for MHC and 4'-6-diamidino-2-phenylindole (DAPI). We observed increased myoD expression and a large number of mature myotubes after treatment of ajoene extract ( Figure S3) indicating the myogenic effects. To investigate whether Z-ajoene ( Figure 4A), as an active ingredient of garlic, has myogenic properties, we added it to myoblasts during differentiation. Z-ajoene increased MHC expression and formation of multinucleated mature myotubes in a dose-dependent manner ( Figure 4B,C). Z-ajoene (100 nM) also day-dependently increased the expressions of MHC and myogenin as compared with respective day-specific control. The expression level of MyoD, a myogenic transcriptional factor, reached a maximum on differentiation day 2 (D2) in both control and Z-ajoene-treated cells. Exposure to Z-ajoene led to a 1.5-fold increase in the expression of MyoD as compared with control at D2 ( Figure 4D). Taken together, Z-ajoene stimulates myoblast differentiation. Nutrients 2019, 11, x FOR PEER REVIEW 4 of 18

Z-ajoene Activates p38 MAPK During Myogenesis
As the p38 mitogen-activated protein kinase (MAPK) activation is the most well-known mechanism in myoblast differentiation [38], we estimated the level of phosphorylated p38 MAPK during myogenesis. Phosphorylated-p38 MAPK continuously increased during the differentiation period and reached its highest level at D3. Z-ajoene treatment significantly activated p38 MAPK compared with control, demonstrating that its role in myoblast differentiation operates via activation of p38 MAPK ( Figure 5A).
Pre-treatment of SB203580 (10 M, an inhibitor of p38 MAPK) prior to Z-ajoene treatment inhibited p38 MAPK phosphorylation by 43% ( Figure 5A), and dramatically suppressed MHC expression and myotube formation ( Figure 5B) compared with Z-ajoene group. These results suggest that p38 MAPK activation contributes to the Z-ajoene-induced myogenesis.

Z-ajoene Activates p38 MAPK During Myogenesis
As the p38 mitogen-activated protein kinase (MAPK) activation is the most well-known mechanism in myoblast differentiation [38], we estimated the level of phosphorylated p38 MAPK during myogenesis. Phosphorylated-p38 MAPK continuously increased during the differentiation period and reached its highest level at D3. Z-ajoene treatment significantly activated p38 MAPK compared with control, demonstrating that its role in myoblast differentiation operates via activation of p38 MAPK ( Figure 5A).

Z-ajoene Prevents Myotube Protein Loss in Vitro
To investigate the preventive potential of Z-ajoene for muscle wasting, fully differentiated myotubes were treated with Z-ajoene (0.1 and 1 M) prior to further treatment with conditioned medium (CM) from CT26 murine colon cancer cells [39]. The CM of cancer cells is known to create an inflammatory condition, associated with the production of pro-inflammatory cytokines in vitro and in vivo. These environments can trigger NF-B activation following E3 ligases (MAFbx, MuRF1) expression to cause muscle atrophy [39].
CM decreased MHC level in differentiated myotubes, but 1 M Z-ajoene recovered MHC levels by 4.3-fold compared with CM alone ( Figure 6A). CM treatment increased the protein and mRNA levels of E3 ligases, MAFbx and MuRF1 in myotubes. However, pre-treatment of 1 M Z-ajoene significantly reduced protein and mRNA expression of MAFbx and MuRF1 (Figures 6A and 6C). However, 0.1 M Z-ajoene treatment could not suppress the expression of E3 ligases (Figures 6A and  6C). As shown in Figure 6B, Z-ajoene protected the loss of MHC expressing and multinucleated myotubes by attenuating the expression of MAFbx and MuRF1. Greater than 10 times higher concentration of Z-ajoene was needed for anti-myopathy activity when compared with concentration that elicited myogenic activity. The difference in effective concentrations of Z-ajoene may derive from the difference between normal differentiation condition and a damaged myotube environment.
As the balance between the rate of protein synthesis and protein degradation is important in the maintenance of skeletal muscle mass, we observed the effect of Z-ajoene on the expression of several factors that have been considered to mediate catabolism or anabolism of muscle proteins. Cancer mediated myotube atrophy is known to be induced by several catabolic mediators, such as E3 ubiquitin ligases, NF-B, and myostatin [40]. We found that these mediators were increased by CM treatment, but significantly diminished in Z-ajoene-treated myotubes ( Figure 6D). The mammalian

Z-ajoene Prevents Myotube Protein Loss in Vitro
To investigate the preventive potential of Z-ajoene for muscle wasting, fully differentiated myotubes were treated with Z-ajoene (0.1 and 1 µM) prior to further treatment with conditioned medium (CM) from CT26 murine colon cancer cells [39]. The CM of cancer cells is known to create an inflammatory condition, associated with the production of pro-inflammatory cytokines in vitro and in vivo. These environments can trigger NF-κB activation following E3 ligases (MAFbx, MuRF1) expression to cause muscle atrophy [39].
CM decreased MHC level in differentiated myotubes, but 1 µM Z-ajoene recovered MHC levels by 4.3-fold compared with CM alone ( Figure 6A). CM treatment increased the protein and mRNA levels of E3 ligases, MAFbx and MuRF1 in myotubes. However, pre-treatment of 1 µM Z-ajoene significantly reduced protein and mRNA expression of MAFbx and MuRF1 ( Figure 6A,C). However, 0.1 µM Z-ajoene treatment could not suppress the expression of E3 ligases ( Figure 6A,C). As shown in Figure 6B, Z-ajoene protected the loss of MHC expressing and multinucleated myotubes by attenuating the expression of MAFbx and MuRF1. Greater than 10 times higher concentration of Z-ajoene was needed for anti-myopathy activity when compared with concentration that elicited myogenic activity. The difference in effective concentrations of Z-ajoene may derive from the difference between normal differentiation condition and a damaged myotube environment.
As the balance between the rate of protein synthesis and protein degradation is important in the maintenance of skeletal muscle mass, we observed the effect of Z-ajoene on the expression of several factors that have been considered to mediate catabolism or anabolism of muscle proteins. Cancer mediated myotube atrophy is known to be induced by several catabolic mediators, such as E3 ubiquitin ligases, NF-κB, and myostatin [40]. We found that these mediators were increased by CM treatment, but significantly diminished in Z-ajoene-treated myotubes ( Figure 6D). The mammalian target of rapamycin (mTOR) signaling pathway has been suggested to be an important anabolic pathway in muscle to increase skeletal muscle mass and fiber size [7]. Pre-treatment of Z-ajoene restored the decreased level of phosphorylated mTOR by CM damage.
These data suggested that pre-treatment with Z-ajoene effectively prevented the CM-induced myotube loss, via regulation of both catabolic and anabolic pathways.
Nutrients 2019, 11, x FOR PEER REVIEW 6 of 18 target of rapamycin (mTOR) signaling pathway has been suggested to be an important anabolic pathway in muscle to increase skeletal muscle mass and fiber size [7]. Pre-treatment of Z-ajoene restored the decreased level of phosphorylated mTOR by CM damage. These data suggested that pre-treatment with Z-ajoene effectively prevented the CM-induced myotube loss, via regulation of both catabolic and anabolic pathways.

Discussion
Muscle atrophy is defined as reduced muscle fiber cross-sectional area, protein content, muscle strength, and insulin sensitivity [4,6,41]. Currently, the incidence of muscle atrophy is expected to increase due to increases in the elderly population (sarcopenia) and prevalence of chronic diseases and sedentary lifestyles (cachexia) [42]. About 20-40% of cancer patients die from muscle loss generated by cancer, not from the cancer itself [3,5]. Cachexia is caused by cancer, diabetes, obstructive pulmonary disease, acquired immune deficiency syndrome (AIDS), and chronic kidney failure. The main symptoms of cachexia are skeletal muscle wasting, anorexia, and unintentional weight loss, leading to progressive functional impairments [2][3][4]. To date, no effective treatment for the pharmacological management of cachexia exists due to multiple underlying biological mechanisms [43]. Sarcopenia is an age-related muscle wasting condition which was recently recognized as a new clinical disease by International Classification of Disease in 2016. To overcome muscle atrophy, we need to discover agents that can protect muscle against cachexic stress and/or enhance the differentiation of myoblasts into myotubes.
CT26 colorectal adenocarcinoma bearing mice are commonly used to induce cancer cachexia with several accompanying symptoms including hepatic functional impairments, adipose and skeletal muscle wasting, and an increase in IL-6 concentration [44,45]. Our in vivo results showed that ajoene extract (14.2% w/w ajoene, 10 mg/kg body weight) effectively decreased muscle atrophy by reducing secretion of myokines such as IL-6 and myostatin and down-regulating JAK/STAT3 and SMADs/FoxO signaling pathways, thereby suppressing muscle-specific E3 ligases. These results were observed at the dose that did not exhibit anti-cancer effects in CT26 tumor-bearing mice, indicating muscle-specific activity of ajoene extract.
Ajoene extract was prepared from garlic bulb through crushing, heat treatment and organic solvent extraction. We analyzed composition of the extract and identified Z-ajoene as the main constituent (11.1% w/w) that might also contain linear polysulfides and vinyldithiin [46]. We previously reported several biological activities of Z-ajoene, including activation of Nrf2 and suppression of NK-kB signaling that could contribute to anti-myopathy potential of Z-ajoene. [22,24]. In order to confirm the preventive activity and uncover the mechanisms behind the effects of ajoene extract on muscle atrophy, we used pure Z-ajoene as the main component of the extract. Conditioned medium (CM) from cancer cell culture has been well-established as an inducer of cachexic conditions. We treated mature myotubes with CM derived from CT26 colon carcinoma cells to mimic in vitro cachexia. Z-ajoene protected against cachexic damage by modulating catabolism or anabolism of muscle proteins. Z-ajoene protected against muscle degradation through suppressing the levels of E3 ubiquitin ligases (MAFbx, MuRF1) [47], NF-κB, and myostatin [40]. Under cachexic conditions, myo-proteins become substrates for NF-κB-mediated E3 ubiquitin ligases resulting in proteasomal degradation. Although many studies report that inhibitors of proteasomal degradation can attenuate myo-protein degradation, there are no clinical applications of these inhibitors for treatment of muscle wasting diseases [34,48,49]. Myostatin, a TGFβ family member, functions in an autocrine-manner to balance muscle growth under normal conditions [50,51]. As excessive levels of myostatin mediate muscle atrophy [35], myostatin inhibition may prevent muscle loss in cachexic conditions. Many studies have demonstrated that NF-κB upregulates myostatin gene expression via direct binding to promoter regions in cachexic conditions [52,53].
Additionally, Z-ajoene positively regulated an anabolic pathway of protein synthesis. Z-ajoene restored the decreased level of phosphorylated mTOR by CM-induced decreases in mature myotubes. Taken together, the results from in vivo and in vitro studies indicate that ajoene extract and Z-ajoene can prevent cancer-induced muscle atrophy by suppressing cellular pathways associated with muscle protein degradation.
As a strategy for muscle regeneration in atrophic conditions, we evaluated the effect of Z-ajoene on myoblast differentiation into myotubes. Activated myogenic satellite cells undergo myogenesis to become myoblasts, and then fuse together to form myotubes. The myotubes then cluster into myofibers to form muscle. Considering the role of satellite cells on muscle generation, myogenesis-stimulating compounds may be able to function as potential therapeutic agents for treating muscle atrophy. C2C12 cells are mouse skeletal myoblasts derived from muscle satellite cells, and can be differentiated by serum starvation medium. The myoD and myogenic regulatory factor (Myf)-5 not only contribute to myogenic lineage specification of muscle stem cells, but also induce the expression of myogenin and myogenic regulatory factors (MRFs), leading to terminal differentiation [54,55]. In particular, the interaction of MyoD as a myogenic transcriptional factor with non-muscle specific muscle proteins is essential to the expression of myogenin and MHC, following new myofiber formation. Z-ajoene enhanced the expression of myogenic factors including MyoD, MHC or myogenin both in vivo and in vitro and increased the formation of multinucleated myotubes during myoblast differentiation. To clarify the mechanism of Z-ajoene-mediated myogenesis, we investigated the p38 mitogen-activated protein kinase (MAPK) that is considered as one of the key regulators of myoblast differentiation. It has been shown that binding of phosphorylated E proteins, SWI/SNF subunit BAF60, or Mef2 by p38 MAPK to myoD contributes to expression of myogenic factors [56]. Consistent with other studies, p38 MAPK phosphorylation gradually increased with differentiation day and was potentiated by Z-ajoene treatment, while SB203580, a p38MAPK inhibitor counteracted the Z-ajoene-induced myogenesis.

Conclusions
In the present study, ajoene extract from crushed garlic (Allium sativum) ameliorates muscle atrophy by down-regulating not only myokines secretion but JAK/STAT3 and SMADs/FoxO signaling pathways, contributing to the preservation of muscle mass in a mouse model of cancer-induced cachexia. Z-ajoene not only attenuates myo-protein degradation under cancer-induced muscle wasting, but also stimulates myogenesis (Figure 7). Therefore, we suggest that Z-ajoene extracted from garlic has potential as a nutritional supplement for the prevention and treatment of muscle atrophy for cancer patients. differentiated by serum starvation medium. The myoD and myogenic regulatory factor (Myf)-5 not only contribute to myogenic lineage specification of muscle stem cells, but also induce the expression of myogenin and myogenic regulatory factors (MRFs), leading to terminal differentiation [54,55]. In particular, the interaction of MyoD as a myogenic transcriptional factor with non-muscle specific muscle proteins is essential to the expression of myogenin and MHC, following new myofiber formation. Z-ajoene enhanced the expression of myogenic factors including MyoD, MHC or myogenin both in vivo and in vitro and increased the formation of multinucleated myotubes during myoblast differentiation. To clarify the mechanism of Z-ajoene-mediated myogenesis, we investigated the p38 mitogen-activated protein kinase (MAPK) that is considered as one of the key regulators of myoblast differentiation. It has been shown that binding of phosphorylated E proteins, SWI/SNF subunit BAF60, or Mef2 by p38 MAPK to myoD contributes to expression of myogenic factors [56]. Consistent with other studies, p38 MAPK phosphorylation gradually increased with differentiation day and was potentiated by Z-ajoene treatment, while SB203580, a p38MAPK inhibitor counteracted the Z-ajoene-induced myogenesis.

Conclusions
In the present study, ajoene extract from crushed garlic (Allium sativum) ameliorates muscle atrophy by down-regulating not only myokines secretion but JAK/STAT3 and SMADs/FoxO signaling pathways, contributing to the preservation of muscle mass in a mouse model of cancerinduced cachexia. Z-ajoene not only attenuates myo-protein degradation under cancer-induced muscle wasting, but also stimulates myogenesis (Figure 7). Therefore, we suggest that Z-ajoene extracted from garlic has potential as a nutritional supplement for the prevention and treatment of muscle atrophy for cancer patients.