Citrus unshiu peel extract alleviates cancer-induced weight loss in mice bearing CT-26 adenocarcinoma

Skeletal muscle atrophy is a critical feature of cancer-induced cachexia, caused by pro-cachectic factors secreted by host cells and tumor cells. Therefore, blockade of these factors has considered a reasonable target for pharmacological and nutritional interventions to prevent skeletal muscle loss under cancer-induced cachexia. Citrus unshiu peel (CUP) has been used for treating the common cold, dyspepsia, and bronchial discomfort and reported to have pharmacological activities against inflammation, allergy, diabetes, and viral infection. In the present study, we observed that daily oral administration of water extract of CUP (WCUP) to male BALB/c mice bearing CT-26 adenocarcinoma remarkably reduced the losses in final body weight, carcass weight, gastrocnemius muscle, epididymal adipose tissue, and hemoglobin (Hb), compared with saline treatment. The levels of serum IL-6 and muscle-specific E3 ligases elevated by tumor burden were also considerably reduced by WCUP administration. In an in vitro experiment, WCUP efficiently suppressed the production of pro-cachectic cytokines in immune cells as well as cancer cells. In addition, WCUP treatment attenuated C2C12 skeletal muscle cell atrophy caused by cancer cells. These findings collectively suggest that WCUP is beneficial as a nutritional supplement for the management of cancer patients with severe weight loss.


WCUP treatment in CT-26 tumor-bearing mice attenuates cancer-induced weight loss and cachexia symptoms.
Cancer cachexia is a complex syndrome characterized by anorexia, loss of skeletal muscle and adipose tissue, and involuntary weight loss despite a nutritional supply, and it is closely correlated with high mortality and poor prognosis in cancer patients 17 . To examine whether WCUP is effective against cancer-induced cachexia, CT-26 tumor-bearing mice showing reductions in body weight and food intake were orally administered WCUP on a daily basis and monitored. Body weight increased steadily in normal healthy mice but not in CT-26 tumor-bearing mice, resulting in a difference in body weight of approximately 10% regardless of tumor weight on day 10 after tumor injection (Fig. 1A). Over the course of the whole experiment lasting 27 days, normal mice increased their body weight by 19.58%, while saline-treated control mice increased it by 4.47%. Mice treated with WCUP at doses of 250 and 500 mg/kg increased their body weight by 9.62% and 13.46%, showing recovery of their weights to approximately 92.38% and 94.76% of that of normal mice, respectively. The average food intakes/mouse/day during the entire experimental period for normal, control, and 250 mg/kg WCUP-treated and 500 mg/kg WCUP-treated mice were 3.74 ± 0.08, 3.33 ± 0.16, 3.39 ± 0.20, and 3.31 ± 0.21, respectively, indicating that tumor burden induces a loss of appetite, and WCUP treatment does not improve appetite in CT-26 tumor-bearing mice (Fig. 1B). Tumor growth was not suppressed by WCUP administration, and tumor weights were similar among control, 250 mg/kg WCUP-treated, and 500 mg/kg WCUP-treated mice at 3.58 ± 0.91, 3.41 ± 0.97, and 3.53 ± 0.92, respectively (Fig. 1C,D). At the time of sacrifice, significant reductions in final body weight (p < 0.0001), carcass weight (p < 0.0001), epididymal adipose tissue (p = 0.0001), gastrocnemius muscle (p < 0.0001), and heart weight (p < 0.0001) were observed in the control mice compared with normal mice. In addition, Hb levels in control mice were also decreased by approximately 40% compared with the levels in normal mice (p < 0.0001). The oral administration of WCUP at doses of 250 and 500 mg/kg for 17 consecutive days starting on day 10 significantly prevented the losses in final body weight (F = 3.086, p = 0.0829), carcass weight (F = 12.11, p = 0.0013), and heart weight (F = 17.05, p = 0.0003), as well as the wasting of epididymal adipose tissue (F = 19.63, p = 0.0002) and gastrocnemius muscle (F = 26.80, p < 0.0001), in CT-26 tumor-bearing mice compared with saline-treated control mice. Furthermore, Hb levels in WCUP-treated mice were maintained at levels similar to those in normal mice (F = 24.86, p < 0.0001) (Figs 2 and S1). These results indicate that WCUP administration is helpful to maintain body weight in tumor-bearing mice and to ameliorate cachexia symptoms, with no influence on appetite or tumor growth. WCUP treatment in CT-26 tumor-bearing mice decreased serum IL-6 levels and muscle degradation-related protein expression. To further elucidate the mechanisms underlying the anti-cachectic effect of WCUP, we measured serum IL-6 levels in mice. Tumor burden caused a marked increase in the serum IL-6 level (p = 0.0008), and the administration of WCUP significantly decreased the IL-6 level in the serum of CT-26 tumor-bearing mice in a dose-dependent manner (F = 27.73, p = 0.0009) (Fig. 3A). It has been reported that, in cachexia, tumor-derived inflammatory cytokines such as IL-6, TNF-α , and IL-1 are critical inducers of muscle wasting and fat depletion 6 . To examine whether WCUP can reduce the production of tumor-derived cytokines, CT-26 cells were treated with 250, 500, and 1000 μg/mL WCUP, and then the levels of mRNA and IL-6 secretion were determined. WCUP up to 1000 μg/mL was non-cytotoxic and significantly reduced the level of IL-6 mRNA (Fig. S2). In addition, WCUP treatment suppressed the IL-6 production in CT-26 cells in a dose-dependent manner (F = 583.1, p = 0.0001) (Fig. 3B). The levels of TNF-α and IL-1β in the serum of mice and culture supernatants of CT-26 cells were below the detection limit in all groups (data not shown). To Scientific RepoRts | 6:24214 | DOI: 10.1038/srep24214 further characterize the cachexia induced by the tumor, the expression of the muscle-specific ubiquitin ligases MAFbx and MuRF-1 was assessed in gastrocnemius muscles. As reported previously 23 , MAFbx and MuRF-1 mRNA expression was dramatically elevated in the muscles of CT-26 tumor-bearing mice compared with normal mice (Fig. S2). Consistent with its preventive effect on muscle wasting, WCUP administration reduced the levels of MAFbx and MuRF-1 mRNA and protein in the muscles of CT-26 tumor-bearing mice (Fig. 3C,D). These findings indicate that WCUP efficiently suppresses CT-26 tumor-induced inflammatory responses and delays muscle breakdown.
WCUP downregulates LPS-induced inflammatory cytokine production in J774A.1 cells. The chronic host inflammatory response in cancer patients elicits muscle degradation even in the face of adequate Male BALB/c mice (n = 15) were subcutaneously injected with CT-26 cells in the abdominal region. On day 10 after tumor inoculation, the mice were divided randomly into three groups (n = 5 per group) and administered WCUP daily at doses of 250 and 500 mg/kg, or saline for 17 days. Control mice with no tumors (n = 5) were also administered an equal volume of saline daily during the experiment. Body weight (A), food intake (B), and tumor volume (C) were compared on days 14, 17, 20, 24, and 27. At the time of sacrifice, tumors were removed and weighed (D). Animal experiments were repeated three times, and representative results are shown. Data are presented as means ± SD. Statistical significance was determined with Student t-test. *p < 0.05, significantly different from the group of 'tumor + saline' in the same day (on day 17; tumor + WCUP 250 mg/kg, p = 0.0210, tumor + WCUP 500 mg/kg, p = 0.0234) (on day 20; tumor + WCUP 250 mg/kg, p = 0.0123, tumor + WCUP 500 mg/kg, p = 0.0193) (on day 24; tumor + WCUP 250 mg/kg, p = 0.0258).
nutrition, and cytokines including IL-6 and TNF-α produced by the host immune cells in response to a tumor are major contributors to muscle wasting. In this study, we examined whether WCUP could inhibit the production of inflammatory cytokines under LPS stimulation and elucidated its underlying mechanism using murine macrophage J774A.1 cells. WCUP at 250, 500, and 1000 μg/mL suppressed LPS-induced NO production by 5.82%, 19.58%, and 26.10%, respectively, compared with that in the WCUP-untreated control (F = 30.37, p = 0.0001) (Fig. 4A). In addition, WCUP at 1000 μg/mL almost completely blocked the LPS-induced increase in iNOS expression at both the mRNA and protein levels (Fig. 4B). As shown in Supplementary Fig. S5, WCUP treatment significantly suppressed LPS-induced increase in the mRNA expressions of iNOS, IL-6, TNF-α , and IL-1β in J774A.1 cells with no apparent cytotoxicity at the concentrations evaluated (qRT-PCR, iNOS; F = 150.4, p < 0.0001, IL-6; F = 24.97, p = 0.0002, TNF-α ; F = 25.67, p = 0.0002, IL-1β ; F = 192.2, p < 0.0001). In addition, secreted protein levels of IL-6, TNF-α , and IL-1β in LPS-stimulated J774A.1 cells were efficiently decreased by WCUP treatment in a dose-dependent manner (IL-6; F = 75.29, p < 0.0001, TNF-α ; F = 127.8, p < 0.0001, IL-1β ; F = 250.1, p < 0.0001) (Fig. 4C). Since the activations of mitogen-activated protein kinase (MAPK), NF-κ B, and STAT3 are involved in the production of pro-inflammatory cytokines, we examined whether these proteins are regulated by WCUP treatment. The levels of phosphorylated p38, ERK, JNK, Iκ Bα , and STAT3 were significantly increased after LPS stimulation compared with the levels in untreated control cells, while WCUP treatment remarkably decreased the levels of phosphorylated p38, ERK, JNK, Iκ Bα , and STAT3 (Fig. 4D). In a time course experiment, the inhibitory effects of WCUP on MAPK and NF-κ B activation were demonstrated more clearly After sacrifice on day 27, the carcasses, epididymal adipose tissues, gastrocnemius muscles, and hearts of the mice were weighed. Hb levels in blood were determined using the ADVIA 2120i hematology system (n = 5 per group). Data are representative of three independent experiments and expressed as means ± SD. Statistical significance was evaluated with Student t-test. Significantly different from the group of normal mice with no tumor; # p < 0.001. Significantly different from the group of tumor + saline; *p < 0.05, **p < 0.01, ***p < 0.001. N, normal mice with no tumor; T, tumor-bearing mice.
( Fig. S6). Similar to observations in J774A.1 cells, WCUP efficiently suppressed LPS-induced NO production, iNOS expression, and pro-inflammatory cytokine production in primary peritoneal macrophages (Fig. S8). In addition, WCUP did not affect the viability of peritoneal macrophages, ruling out the possibility of cytotoxic effects.

Figure 3. Effect of WCUP on levels of serum IL-6 and muscle atrophy-related proteins in CT-26 tumorbearing mice. (A)
After sacrifice, serum IL-6 levels in mice were determined by ELISA (n = 3 per group). Statistical significance was evaluated with Student t-test. # p < 0.001 vs. normal mice with no tumor, *p < 0.05 and **p < 0.01 vs tumor + saline. (B) CT-26 cells were treated with the indicated concentrations of WCUP for 48 h, and then the IL-6 levels in culture supernatants were measured by ELISA (n = 3 per group). Statistical significance was evaluated with Student t-test. ***p < 0.001 vs. WCUP-untreated control cells. (C,D) mRNA and protein levels of MAFbx and MuRF-1 in gastrocnemius muscle were examined by RT-PCR and Western blotting, respectively (n = 2 per group). The levels of GAPDH and tubulin were used for normalization of the samples. Bar graph presents the means ± SD. Statistical significance was evaluated with Student t-test. # p < 0.05 vs. normal mice with no tumor, *p < 0.05 and **p < 0.01 vs tumor + saline. The full size gels and blots were shown in the Supplementary Fig. S3 and band of interest is indicated with an arrow. WCUP prevents CT-26-mediated suppression of C2C12 myoblast proliferation. In cancer cachexia, tumor-derived factors including pro-inflammatory cytokines, Mstn, activin, and PIF are inextricably linked to skeletal muscle wasting 24,25 . In previous studies, it was demonstrated that treatment with conditioned medium (CM) derived from CT-26 cells suppressed C2C12 myoblast proliferation by regulating cell cycle-related proteins. In addition, CT-26 CM attenuated C2C12 myoblast differentiation and promoted C2C12 myotube wasting by increasing the expression of E3 ligases in myotubes such as MAFbx and MuRF-1 and by elevating intracellular protein degradation through the ubiquitin-proteasome pathway 24 . To examine the effects of WCUP on CT-26-induced muscle wasting, we first examined C2C12 myoblast proliferation after incubation in WCUP-treated or -untreated CT-26 CM at dilutions of 1:3 and 1:5 in growth medium (GM) for 36 h. WCUP itself did not affect the viability of C2C12 myoblast cells (Fig. 5A). WCUP-untreated CT-26 control CM significantly inhibited C2C12 myoblast proliferation by approximately 71.06% and 64.33% at dilutions of 1:3 and 1:5, respectively, compared with GM (1:3 and 1:5 dilution, p < 0.0001), while 250, 500, and 1000 μg/mL WCUP-treated CT-26 CM did not severely retard C2C12 myoblast proliferation (1:3 dilution; F = 77.41, p < 0.0001, 1:5 dilution; F = 186.0, p < 0.0001). Interestingly, cells in WCUP-treated CT-26 CM at 1000 μg/mL were almost completely maintained, similar to cells in GM (Fig. 5B,C). Consistent with previous studies, exposure to CT-26 control CM significantly increased p21 expression and decreased CDK2 and cyclin D expression in C2C12 myoblast cells. However, changes in these proteins after exposure to WCUP-treated CT-26 CM were insignificant, in accordance with their effects on proliferation (Fig. 5D).
Identification of the main components in WCUP using HPLC. Six phytochemicals in WCUP were analyzed by HPLC: narirutin, naringin, hesperidin, neohesperidin, poncirin, and nobiletin. To achieve optimal separation, gradient elution of water and acetonitrile was applied. TFA was used to inhibit peak tailing and enhance peak shape, and the UV wavelengths of these six compounds were controlled based on the maximal UV absorption of each. Each compound in the WCUP was identified by comparing the retention time (t R ) and UV spectra of standard compounds. Narirutin (1, t R : 12.05 min), naringin (2, t R : 13.87 min), hesperidin (3, t R : 15.45 min), neohesperidin (4, t R : 17.92 min), poncirin (5, t R : 34.74 min), and nobiletin (6, t R : 34.67 min) were identified in WCUP (Fig. 7).

Discussion
Unintentional weight loss is regarded as a medically serious condition when more than 10% of body weight is lost over a 6-month period or 5% within the last month, despite adequate nutritional intake. Continuing weight loss may deteriorate into a wasting condition called cachexia. Symptoms in patients with advanced cachexia include severe skeletal muscle loss, anorexia, early satiety, lack of energy and strength, anemia, and alterations in immune function. Cachexia is considered a critical cause of morbidity and mortality in cancer patients, occurring in up to 80-90% of patients with advanced tumors of pancreatic and gastric origin and leading to cancer-related death in 20% of cases. Loss of muscle mass is a hallmark of cancer-induced cachexia and is closely related to poor were calculated after normalization to GAPDH and tubulin using ImageJ software. The full size gels and blots were shown in the Supplementary Fig. S4 and band of interest is indicated with an arrow. (C) The levels of IL-6, TNF-α , and IL-1β in culture supernatants collected as described in (A) were measured by ELISA. The data are representative of independent experiments performed in triplicate and expressed as means ± SD. Statistical significance was evaluated with Student t-test. # p < 0.001 vs. untreated control, *p < 0.05, **p < 0.01, and ***p < 0.001 vs. WCUP-untreated control cells. (D) J774A.1 cells were pretreated with 500 and 1000 μg/ mL WCUP for 12 h and then stimulated with 200 ng/mL LPS for 30 min. After extracting proteins, the levels of p38, ERK, JNK, Iκ Bα , STAT3, and their phosphorylated forms were detected by Western blotting. The band intensities relative to untreated control cells were calculated using ImageJ software after normalization to tubulin. The full size gels and blots were shown in the Supplementary Fig. S7 and band of interest is indicated with an arrow. efficacy of anti-cancer therapies and more severe side effects during chemotherapy 3 . Skeletal muscle catabolism is mediated by three major proteolytic pathways, including the lysosomal system, the calcium-activated system, and the ubiquitin-proteasome pathway. In the ubiquitin-proteasome proteolytic pathway, two muscle-specific E3 ligases, MAFbx and MuRF-1, play pivotal roles in the degradation of myofibrillar and intracellular proteins, and their expression during cancer cachexia is regulated by pro-inflammatory cytokines such as TNF-α and IL-6. In addition, tumor-derived factors such as Mstn and PIF have also been implicated in the development of skeletal muscle wasting 24,26,27 . Therefore, blockade of these pro-cachectic factors effectively reduces cachexia symptoms in tumor-bearing mice.
CUP is dried skin from the Korean citrus fruit Citrus unshiu Markov., which belongs to the Rutaceae family and is used as a traditional herbal medicine to treat the common cold, gastrotympanites, nausea, vomiting, and dyspepsia. Previous studies reported that ethyl acetate extracts of CUP and its constituent nobiletin markedly inhibited hepatitis C virus infection in MOLT-4 cells (a human lymphoblastoid leukemia cell line) 28 . In addition, CUP strongly suppressed tumor growth in mice exposed to renal carcinoma cells by boosting cytokines such as IFN-γ and TNF-α , and it showed potent anti-tyrosinase activity in B16F10 cells as well as anti-osteoporosis activity in ovariectomized (OVX) rats [29][30][31] . Recently, it has been demonstrated that ethanol extract of CUP has the potential to ameliorate hyperglycemia and hepatic steatosis in type 2 diabetic db/db mice by modulating the levels of anti-inflammatory cytokines (e.g., adiponectin and IL-10) and pro-inflammatory cytokines (e.g., IL-6,  monocyte chemotactic protein-1, IFN-γ , and TNF-α ) in the liver and plasma 22 . Moreover, water extract of CUP inhibited pro-inflammatory cytokines in LPS-stimulated RAW 264.7 macrophages through the suppression of NF-κ B activation and MAPK phosphorylation 21 .
In the current study, we found that, in CT-26 tumor-bearing mice, body weight recovered considerably by oral administration of WCUP compared with saline treatment, while tumor growth and food intake were similar between saline-and WCUP-treated mice. Skeletal muscle mass, fat mass, and hemoglobin levels were also increased by WCUP administration, suggesting beneficial effects of WCUP on cancer-induced cachexia symptoms without affecting appetite or tumor growth (Figs 1 and 2). In normal mice with no tumor, repeated administration of WCUP at doses of 250 or 500 mg/kg for 15 days did not cause significant difference in body weight (data not shown). Next, WCUP administration prevented increases in serum IL-6 levels and muscular E3 ligase levels caused by CT-26 tumor burden (Fig. 3), resulting in the recovery of skeletal muscle mass and lean body mass. In J774A.1 and peritoneal macrophages under LPS stimulation, we observed that WCUP dramatically inhibited the production of NO as well as pro-cachectic cytokines, including IL-6, TNF-α , and IL-1β , via suppression of MAPK phosphorylation and NF-κ B/STAT3 activation (Fig. 4). Furthermore, WCUP decreased the CT-26 cell-derived production of IL-6 and myostatin, by which WCUP-treated CT-26 CM showed less impairment of C2C12 myoblast proliferation and differentiation and prevention of tumor-induced C2C12 myotube wasting, compared with WCUP-untreated control CT-26 CM (Figs 5 and 6). These findings indicate that WCUP suppresses the levels of humoral and tumor-derived pro-cachectic factors and efficiently prevents cancer-induced severe weight loss. Certain compounds identified from WCUP including narirutin, naringin, hesperidin, and nobiletin have been reported to exhibit potent anti-inflammatory activities [32][33][34] . In addition, hesperidin has been demonstrated to promote MyoD-induced myogenic differentiation and to accelerate injury-induced muscle regeneration in mice 35 . These results suggest that these compounds in WCUP may contribute to its anti-cachectic effects in CT-26 tumor-bearing mice. In the further study, we will identify active compounds in WCUP and elucidate the mechanisms involved in the anti-cachectic effects.
Currently, dietary supplementations, including herbal medicines, have received increasing attention as adjuvants to diminish complications in tumor-bearing mice. Treatment with fish oil and selenium prevented increases in IL-6, TNF-α , and myostatin levels in mice receiving chemotherapy with docetaxel and significantly attenuated skeletal muscle atrophy 36 . Some natural herbs and their components, including sophocarpine and matrine from Sophora flavescens Ait. (Kushen) and berberine and quercetin from Rhizoma coptidis, prevented cachexia-related symptoms in mice bearing Colon-26 adenocarcinoma 37,38 . In a recent study, we reported that Sosiho-tang (SO) ameliorated cachexia-related symptoms in tumor-bearing mice by retarding tumor growth and reducing systemic inflammation and muscle loss, suggesting that it may be very useful for cancer patients with severe weight loss 39 .
In summary, the present study demonstrated that WCUP reduces systemic inflammation in tumor-bearing mice and suppresses the production of pro-cachectic factors in tumors, followed by the prevention of skeletal muscle atrophy and weight loss. Identification of the active ingredients in WCUP responsible for the improvements in skeletal muscle atrophy and inflammation is now underway. Furthermore, to confirm that WCUP can act as a safe and potent anti-cancer adjuvant, its preventive/therapeutic effects against chemotherapy-induced muscle loss and its synergistic anti-cancer efficacy with chemotherapeutic agents are under investigation.

Materials and Methods
Cell culture and preparation of conditioned medium (CM). CT-26 murine colon carcinoma cells (ATCC R CRL-2638 TM ) and C2C12 murine myoblast cells (ATCC R CRL-1772 TM ) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). J774A.1 murine macrophage-like cells (KCLB no. 40067) were purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were maintained in RPMI 1640 or DMEM (Lonza, Walkersville, MD, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Cellgro, Manassas, VA, USA) and penicillin (100 U/mL)/streptomycin (100 μg/mL) (Cellgro) in a humidified 5% CO 2 incubator at 37 °C. For CM collection, CT-26 cells were plated in 100-mm culture dishes at a density of 5 × 10 4 /cm 2 and treated with or without WCUP for 24 h in complete media conditions. After washing completely, cells were incubated for 24 h in serum free DMEM and the resulting CMs were centrifuged to remove debris and filtered using a 0.22 μm disk filter. CMs were diluted with either DMEM containing 10% FBS and antibiotics (growth medium; GM) or DMEM containing 5% horse serum (HS; Gibco-BRL, Grand Island, NY, USA) and antibiotics (differentiation medium; DM) for myoblast or myotube treatment, respectively. Prior to dilution, an appropriate quantity of FBS, HS, or antibiotics were compensated. Myogenic differentiation was induced by incubating C2C12 myoblast at a density of 70-80% in DMEM containing 5% HS for 5-7 days.

Preparation of water extract of CUP (WCUP). The identity of CUP obtained from Yeongcheon Oriental
Herbal Market (Yeongcheon, Korea) was first confirmed by Professor Ki Hwan Bae (College of Pharmacy, Chungnam National University, Daejeon, Korea) and stored in the KIOM herbal bank. To prepare WCUP, dried CUP (50 g) was soaked in distilled water (1000 mL) and then heat-extracted at 115 °C for 3 h in a Cosmos-600 Extractor (Gyeonseo Co., Incheon, Korea). After filtration through standard testing sieves (150 μm, Retsch, Haan, Germany), WCUP was freeze-dried and kept in desiccators at 4 °C. The amount of WCUP powder collected was 11.89 g, and the yield was 23.78%. For in vitro experiments, WCUP powder was dissolved in 10% DMSO to a final concentration of 50 mg/mL, filtered through a 0.22-μm disk filter, and then stored at −20 °C until use. Mouse cancer cachexia model. To induce cancer-mediated cachexia, CT-26 cells (2 × 10 6 per mouse) were subcutaneously inoculated into the abdominal region of 7-week-old male BALB/c mice. Body weight, tumor volume, and food intake were measured twice a week during the experiment. Food intake was calculated as the mean value of five mice per cage. On day 10 after tumor inoculation, when significant decreases in body weight and food intake were observed in tumor-bearing mice, the mice were divided randomly into three groups and orally administered saline or WCUP daily at doses of 250 and 500 mg/kg. WCUP was suspended in the saline and administered orally in a volume of 100 μL using disposable animal feeding needles purchased from Fuchigami (Kyoto, Japan). Age-matched healthy control mice having no tumors were treated with an equal volume of saline. On day 27 after tumor inoculation, the mice were sacrificed by intraperitoneal injection with a 2:1 mixture of Zoletil (Virbac, Magny-en-Vexin, France) and Rumpun (Bayer, Seoul, Korea) (200 μL per mouse). The tumor, heart, gastrocnemius muscle, and epididymal fat were resected, and blood samples were collected. Whole-blood and serum samples were examined for hemoglobin (Hb) levels using the ADVIA 2120i hematology system (Siemens Healthcare Diagnostics, Tarrytown, NY, USA) and IL-6 levels using the ELISA antibody kit (eBioscience, San Jose, CA, USA), respectively. For measurement of carcass weight, the remaining viscera and blood were completely removed and clearly wiped out with gauze pad.

Reverse transcription and polymerase chain reaction (RT-PCR).
Total RNA was extracted using RNA extraction solution (BioAssay Co., Daejeon, Korea) according to the manufacturer's instruction. RNA concentrations were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and 3 μg RNA was reverse transcribed using 1 st Strand cDNA Synthesis kit (BioAssay Co.). Quantitative real-time PCR was performed using QuantStudio 6 Flex Real-Time PCR system (Thermo Scientific, Rockford, IL, USA) as described previously 40 . For each gene, the Ct values were normalized to those of β -actin and then presented as fold differences compared to untreated control cells. cDNA aliquots were also analyzed by semi-quantitative PCR as described previously 21 , and the PCR products were visualized by electrophoresis on 1% agarose gels and staining with GreenLight TM (BioAssay Co.). The band intensity was measured using ImageJ software (National Institute of Health, USA).