Sosiho‑tang ameliorates cachexia‑related symptoms in mice bearing colon 26 adenocarcinoma by reducing systemic inflammation and muscle loss

  • Authors:
    • Aeyung Kim
    • Minju Im
    • Jin Yeul Ma
  • View Affiliations

  • Published online on: December 28, 2015     https://doi.org/10.3892/or.2015.4527
  • Pages: 1841-1850
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Cachexia accompanied by muscle wasting is a key determinant of poor prognosis in cancer patients and cancer‑related death. Previous studies have demonstrated that inflammatory cytokines such as interleukin‑6 (IL‑6), tumor necrosis factor‑α (TNF‑α), IL‑1 and interferon‑γ (IFN‑γ) secreted from host cells and tumor cells participate in skeletal muscle wasting followed by severe loss of body weight. Therefore, blockade of the inflammatory response is thought to be a logical target for pharmacological and nutritional interventions to preserve skeletal muscle mass under cachectic conditions. Sosiho‑tang (SO; Xiaocharihu‑tang in Chinese and Sho‑saiko‑to in Japanese) is an Oriental herbal medicine that has been used to treat chronic hepatic diseases and to control fever. In recent studies, SO inhibited the production of inflammatory cytokines in lipopolysaccharide (LPS)‑stimulated macrophages, prevented thrombus formation and suppressed cancer progression. However, the anti‑cachectic activity of SO in tumor‑bearing mice has not yet been examined. In the present study, we characterized the effect of SO administration on cancer‑induced cachexia in CT‑26‑bearing mice, and elucidated the anti‑cachectic mechanisms. Daily oral administration of SO at doses of 50 and 100 mg/kg to CT‑26‑bearing mice significantly retarded tumor growth and prevented the loss of final body weight, carcass weight, heart weight, gastrocnemius muscle, and epididymal fat, compared with saline‑treated control mice. In addition, serum IL‑6 levels elevated by cancer were decreased by SO administration. In the J774A.1 macrophage cell line, SO efficiently suppressed LPS‑mediated increases in inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO), and procachectic inflammatory cytokine production through inhibition of nuclear factor‑κB (NF‑κB) and p38 activation. In addition, SO attenuated muscle atrophy caused by cancer cells by affecting myoblast proliferation and differentiation, and C2C12 myotube wasting. Taken together, these results suggest that SO is a safe and useful anti‑cachectic therapy for cancer patients with severe weight loss.

Introduction

Cancer cachexia is a severe, debilitating and life-threatening syndrome, characterized by a marked loss of skeletal muscle and adipose tissue and appetite, a reduction in physical functions, and profound involuntary weight loss despite adequate nutritional intake (13). Weight loss is more frequently experienced by 80–90% of pancreatic and gastrointestinal cancer patients than by sarcoma and breast cancer patients, and severe cachexia with >10% weight loss is closely correlated with high mortality, impaired quality of life and poor response to chemotherapy or radiotherapy (4). In addition, cachectic patients are more prone to chemotherapy-related toxic side effects. Therefore, it is important to control the cachectic state in cancer patients, since cachexia is the main cause of cancer-related death in approximately 25–30% of patients.

Although the mechanisms of cancer cachexia are not completely known, recent studies have indicated that persistent production of proinflammatory cytokines and catabolic factors secreted from both tumor cells and host cells, such as macrophages, plays an essential role in the induction and progression of cancer cachexia (5,6). Among procachectic cytokines, interleukin-6 (IL-6) is considered a key mediator in the pathogenesis of cancer cachexia. In previous studies, it has been reported that IL-6-secreting cells induce wasting of both muscle and fat, and IL-6 is responsible for muscle wasting in cachectic mice with CT-26 or Yomoto uterine cancer (7). Treatment with monoclonal antibodies to IL-6, or to the IL-6 receptor, markedly suppresses the development of cachexia in tumor-bearing mice and in patients with IL-6-overexpressing lung cancer, suggesting that blocking IL-6 function might be an effective intervention for the management of cachectic patients (8,9).

Several clinical trials to treat cancer cachexia have been performed, involving drugs that stimulate appetite, reduce proinflammatory cytokine production and inhibit tumor-induced protein degradation. However, current medical treatments are limited due to low efficacy and high toxicity. Among them, medroxyprogesterone acetate, approved in Europe for treatment of cancer and acquired immune deficiency syndrome (AIDS)-related cachexia, is widely used in the treatment of hormone-related cancer as a supportive therapy (10,11). However, this treatment also has unwanted side effects, including diabetes, osteoporosis, mood swings and thromboembolism (12). Therefore, it is essential to find novel agents capable of preventing cancer-induced cachexia with minimal adverse effects.

Sosiho-tang (SO, known as Xiaochaihu-tang in Chinese, and as Sho-saiko-to in Japanese) is an Oriental traditional herbal formula comprised of seven medicinal herbs, including Bupleurum root, Glycyrrhizae radix et rhizoma, Ginseng, radix, Pinellia tuber, Scutellaria root, Zingiberis rhizoma crudus and Zizyphi fructus. SO has long been used to treat chronic liver diseases such as hepatitis and liver cirrhosis in Korea, China and Japan (13). In addition, SO prevents inflammation and cancer progression (1417), suggesting that it might also prevent cancer-induced cachexia through suppression of inflammatory responses caused by cancer.

In the present study, we examined the effects of oral administration of SO on the severity of key parameters of cachexia in CT-26-bearing mice. In addition, we also investigated the efficacy of SO on the production of proinflammatory cytokines and muscle wasting, and further elucidated the in vitro mechanism of its anti-cachectic activity using murine cell lines, including the CT-26 colon carcinoma, the J774A.1 macrophage, and the C2C12 myoblast cell lines.

Materials and methods

Cells

Murine colon carcinoma CT-26 cells and murine myoblast C2C12 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Murine macrophage-like J774A.1 cells [Korean Cell Line Bank (KCLB): no. 40067] were obtained from the KCLB (Seoul, Korea). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) or Roswell Park Memorial Institute (RPMI)-1640 medium (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% CO2 incubator at 37°C. To induce myogenic differentiation, the C2C12 cells at a density of 70–80% were cultured in DMEM containing 5% horse serum (HS; Gibco-BRL, Grand Island, NY, USA) for 5–7 days.

Animals

Six-week-old male BALB/c mice were purchased from Taconic Farms (Samtako Bio Korea, Osan, Korea) and housed under specific pathogen-free conditions under a 12-h light-dark cycle at 22±1°C and 55±5% humidity. All animal experiments were approved by the Animal Care and Use Committee of the Korea Institute of Oriental Medicine (KIOM, Daejeon, Korea) with reference numbers #13–100, #14–074 and #15–011. Experiments were performed according to the guidelines of the Animal Care and Use Committee at KIOM.

Antibodies and reagents

Antibodies against p21, cyclin-dependent kinase (CDK)2, cyclin D, p38, p-p38 (Thr180/Tyr182), p-IκBα (Ser32), p-IKKα/β (Ser176/180), signal transducer and activator of transcription (STAT)3, p-STAT3 (Tyr705), p65, p-p65 (Ser536), inducible nitric oxide synthase (iNOS), and TBP were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-α-tubulin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against myosin heavy chain was purchased from R&D Systems (Minneapolis, MN, USA). Horseradish peroxidase (hRP)-conjugated anti-mouse and anti-rabbit antibodies were purchased from Cell Signaling Technology. Recombinant murine tumor necrosis factor-α (rMu TNF-α) was purchased from Promokine (heidelberg, Germany).

Preparation of SO

The composition of herbal extract SO is listed in Table I. All herbs were obtained from Yeongcheon herbal Market (Yeongcheon, Korea), validated by Professor Ki hwan Bae (Chungnam National University, Daejeon, Korea), and stored in the herbal bank at KIOM prior to use. A total of 1,674.5 g of chopped SO was heat-extracted in 16.745 liters of distilled water for 3 h at 115°C using a Cosmos-600 Extractor (Gyungseo, Incheon, Korea). The decoction was filtered through standard testing sieves (150 µm; Retsch, haan, Germany), lyophilized, and stored in desiccators at 4°C. The final amount of lyophilized SO powder was 416.783 g, and the yield was 24.89%. For in vitro experiments, the SO powder was dissolved in 10% dimethyl sulfoxide to 50 mg/ml, filtered through a 0.22-µm disk filter, and then stored at -20°C prior to use.

Table I

Herbal composition of Sosiho-tang.

Table I

Herbal composition of Sosiho-tang.

Name of herbAmount
(g)
Location of origin
Bupleurum root12.00Yeongcheon, Korea
Glycyrrhizae radix et rhizoma2.00China
Ginseng radix4.00Geumsan, Korea
Pinellia tuber4.00Uiseong, Korea
Scutellaria root8.00Suncheon, Korea
Zingiberis rhizoma crudus1.49Yeongcheon, Korea
Zizyphi fructus2.00Yeongcheon, Korea
Total amount33.49
Experimental design of cancer cachexia

The CT-26 cells were subcutaneously inoculated into the abdominal region of 7-week-old male BALB/c mice (5×106 cells/mouse). To confirm the induction of cancer-mediated cachexia, the body weights and tumor volumes of the mice were measured once every 2 days throughout the experiment. Significant weight losses in the tumor-bearing mice were observed between 8–10 days after the tumor inoculations, and the mice were fed with saline or SO at doses of 50 and 100 mg/kg from day 10 to day 27 after tumor inoculation. The administered dose was calculated based on the amount used in human adults (33.49 g/60 kg of body weight/day) and the yield of powdered extract (24.89%). The age-matched healthy control mice having no tumors were treated with saline. During the experiments, food intake was calculated as the mean value of five mice per cage. At the time of sacrifice, mice were euthanized by intraperitoneal injection with a 2:1 mixture of zoletil (Virbac, Magny-en-Vexin, France) and rompun (Bayer, Seoul, Korea), and then the tumor, epididymal fat, gastrocnemius muscle, and heart were dissected and weighed. In addition, serum samples were obtained for measuring levels of IL-6, tumor necrosis factor (TNF)-α, and IL-1β. After exsanguination, the remaining viscera were removed and the carcass weight was measured.

Preparation of CT-26 conditioned medium (CM)

The CT-26 cells were plated into 100-mm culture dishes at a density of 5×104 cells/cm2 and treated with or without SO for 24 h under complete medium conditions. After washing three times with phosphate-buffered saline (PBS), the cells were additionally washed twice with serum-free medium, and then incubated for another 24 h in serum-free DMEM. The resulting CM was centrifuged to remove debris, filtered using a 0.22-µm disk filter, and then stored in a freezer. The CM was diluted at a 1:3 or 1:5 ratio with either DMEM containing 10% FBS and antibiotics [growth medium (GM)] for myoblast treatment or DMEM containing 5% hS and antibiotics [differentiation medium (DM)] for the myotube treatment. Prior to dilution, an appropriate quantity of FBS, hS, or antibiotics for compensation were added to the CM.

Myoblast proliferation assay

To examine the C2C12 myoblast proliferation, cells were plated into a 96-well culture plate at a density of 1×103 cells/well, and then treated with SO-treated or -untreated CM at 37°C for 48 h. At the indicated time points, cell proliferation was determined using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's protocol.

Determination of NO production

The cells were pretreated with the indicated concentrations of SO for 1 h and then stimulated with LPS for 24 h. The collected culture supernatant was mixed with an equal volume of Griess reagent (1% sulfanil-amide, 0.1% naphthylethylenediamine dihydrochloride and 2.5% phosphoric acid), and incubated at room temperature for 5 min, and then the absorbance was measured at 570 nm.

Reverse transcription-polymerase chain reaction (RT-PCR)

The total RNA was isolated using an RNA extraction solution (BioAssay Co., Daejeon, Korea) according to the manufacturer's instructions, and the RNA concentrations were quantitated using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The RNA (3 µg) was reverse transcribed using a 1st Strand cDNA Synthesis kit (BioAssay Co.) and then the cDNA samples were analyzed by semiquantitative PCR using specific primers (Table II). PCR products were visualized by electrophoresis using agarose gels and staining with GreenLight™ (BioAssay Co.), and band intensities were analyzed using ImageJ software (National Institutes of health, Bethesda, MD, USA).

Table II

Primers used for PCR.

Table II

Primers used for PCR.

Target geneSequences
iNOSF: 5′-CCTCCTCCACCCTACCAAGT-3′
R: 5′-CACCCAAAGTGCTTCAGTCA-3′
IL-6F: 5′-CATGTTCTCTGGGAAATCGTGG-3′
R: 5′-AACGCACTAGGTTTGCCGAGTA-3′
TNF-αF: 5′-CATGTTCTCTGGGAAATCGTGG-3′
R: 5′-AACGCACTAGGTTTGCCGAGTA-3′
IL-1αF: 5′-GGTTAAATGACCTGCAACAGGA-3′
R: 5′-TCTTTGGTGGCAATAAACAGC-3′
GAPDHF: 5′-TCATGACCACAGTCCATGCC-3′
R: 5′-TCCACCACCCTGTTGCTGTA-3′

[i] F, forward; R, reverse.

Western blot analysis

For whole cell lysates, cells were harvested, washed and lysed in M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA). The cytosolic and nuclear fractions were obtained using NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Scientific). The lysates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, hercules, CA, USA), then blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline containing 0.05% Tween-20 (TBST) and immunoblotted using specific antibodies at 4°C overnight. After washing with TBST, the membranes were reacted with secondary antibodies conjugated with HRP for 1 h at room temperature, and the target proteins were visualized using the SuperSignal WestFemto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA) and the ImageQuant LAS 4000 Mini (GE healthcare, Piscataway, NJ, USA). The relative band intensities were measured using ImageJ software.

Enzyme-linked immunosorbent assay (ELISA)

The levels of murine IL-6, TNF-α and IL-1β in the culture supernatants and sera were determined using an ELISA antibody kit (eBioscience, San Jose, CA, USA) according to the manufacturer's instructions.

Statistical analysis

Data are presented as means ± standard deviation (SD). Differences between groups were analyzed by the Student's t-test using the SigmaPlot software (ver 8.0; SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Oral administration of SO alleviates cachexia symptoms in CT-26 tumor-bearing mice and suppresses tumor growth

To examine whether SO is an effective treatment for cancer-induced cachexia, mice were administered SO from 10 days after CT-26 cell inoculation, when mice showed reductions in body weight and food intake by ~10–12% compared to the normal mice (Fig. 1). Normal mice exhibited gradually increased body weight during the experimental period by 21.6%, while control mice had increased body weight by 4.3% (Fig. 2A). SO administration at doses of 50 and 100 mg/kg significantly increased body weights, by 13.1 and 13.7%, respectively, showing recovery of body weights to ~93.5 and 93.9%, respectively, of the normal mice. In previous studies, it has been reported that SO inhibits cell proliferation via cell cycle arrest at G0/G1, and induces apoptosis of various types of cancer cells, including hepatocellular carcinoma, Lewis lung carcinoma, cholangiocarcinoma, ovarian cell carcinoma and renal cell carcinoma (15,16). In the present study, SO administration at 50 and 100 mg/kg significantly retarded CT-26 tumor growth, by 34.29 and 38.81%, respectively, compared with the control mice on day 27. Control mice had a mean tumor weight of 2.88±0.89 g, while mice treated with 50 and 100 mg/kg of SO had mean tumor weights of 2.52±0.61 and 2.26±0.19 g, reflecting 12.56 and 21.37% reductions, respectively (Fig. 2B). The mean values of food intake/mouse/day during the experiment for normal, control, and 50- and 100-mg/kg SO-treated mice were 3.56±0.11, 3.13±0.24, 3.32±0.16 and 3.37±0.18 g, respectively, indicating that SO aids in improving appetite (data not shown). In addition, administration of SO significantly prevented the loss of final body weight, carcass weight, heart weight and wasting of epididymal adipose tissue and gastrocnemius muscle in the CT-26 tumor-bearing mice. Furthermore, serum IL-6 levels were markedly elevated in the tumor-bearing control mice compared to the non-tumor-bearing normal mice, and these values were significantly reduced by SO administration (Fig. 2C). The serum levels of TNF-α and IL-1β were below the detection limit in all groups (data not shown). These results indicate that SO reduces tumor burden and delays the process of CT-26 tumor-induced cachexia.

SO downregulates LPS-induced NO production, iNOS expression and inflammatory cytokine production in murine macrophage J774A.1 cells

Fig. 2C shows that the serum level of IL-6 was markedly decreased by SO administration in the CT-26-bearing mice compared to the control mice. In cancer patients, the tumor induces a chronic host inflammatory response, characterized by the production of cytokines such as IL-6, IL-1β, IFN-γ and TNF-α. These cytokines are known to be involved in the induction of cancer-related muscle wasting through activation of iNOS expression and NO production (1820). Therefore, we examined whether SO could suppress iNOS expression, NO generation and inflammatory cytokine production in the LPS-stimulated J774A.1 cells. SO almost completely blocked the LPS-induced increase in iNOS expression at both the mRNA and protein levels (Fig. 3A). Furthermore, SO at 10, 50 and 100 µg/ml inhibited the NO production by 59.62, 76.43 and 90.42%, respectively, in a dose-dependent manner, compared with the untreated controls (Fig. 3B). The mRNA levels of IL-6, IL-1α and TNF-α, as well as the protein levels of IL-6, IL-1β and TNF-α, were strongly increased by LPS stimulation, and were efficiently suppressed by the SO treatment (Fig. 3C and D). Similar to observations in J774A.1 cells, SO suppressed the LPS-induced NO production, iNOS expression, and cytokine production in peritoneal macrophages (data not shown). SO at the concentrations used in the experiments did not affect the viability of J774A.1 cells or primary peritoneal macrophages, excluding the possibility of cytotoxic effects.

Figure 3

The inhibitory effects of SO on lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inflammatory cytokine production in murine macrophage J774A.1 cells. (A) The J774A.1 cells were pretreated with indicated concentrations of SO for 1 h, and then stimulated with 200 ng/ml LPS for 24 h. The inducible nitric oxide synthase (iNOS) mRNA and protein levels were examined by RT-PCR and western blotting, respectively. The band intensities relative to LPS-stimulated cells were calculated using the ImageJ software. The levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin were measured for normalization. Data are representative of three independent experiments. (B) The culture supernatants were collected and analyzed for NO production. The control cells were treated with vehicle alone. The data are representative of three independent experiments performed in triplicate and are expressed as means ± SD. #P<0.05 vs. untreated control, *P<0.05 vs. SO-untreated control cells. (C) The J774A.1 cells pretreated with SO for 1 h were stimulated with 200 ng/ml LPS for 6 h. The mRNA levels for interleukin (IL)-6, IL-1α, and tumor necrosis factor (TNF)-α were analyzed by RT-PCR, and relative fold increases were calculated after normalization with GAPDH using ImageJ software. The data show means ± SD of two independent experiments. #P<0.05 vs. untreated control, *P<0.05 vs. SO-untreated control cells. (D) After stimulation of SO-pretreated cells with LPS for 24 h, the levels of IL-6, IL-1β, and TNF-α in the culture supernatants were quantitated by ELISA. Data are representative of three independent experiments performed in triplicate and are expressed as means ± SD. #P<0.05 vs. untreated control, *P<0.05 vs. SO-untreated control cells.

SO strongly blocks LPS-induced p38, NF-κB, and STAT3 activation in J774A.1 cells

Because mitogen-activated protein kinase (MAPK), NF-κB, and STAT3 activations are closely related with proinflammatory cytokines, we examined whether these pathways were affected by SO treatment. After LPS stimulation, the levels of phosphorylated p38, IκBα, IKKαβ, and STAT3 were significantly decreased by SO treatment, in a dose-dependent manner (Fig. 4A). However, the levels of phosphorylated ERK and JNK were not affected (data not shown). Because NF-κB activation requires nuclear translocation of p65, we measured p65 levels in the cytosolic and nuclear fractions. In control cells, the p65 subunit translocated from the cytosol to the nucleus by LPS stimulation, whereas SO treatment effectively prevented p65 nuclear translocation in a dose-dependent manner (Fig. 4B).

SO attenuates CT-26-mediated skeletal muscle atrophy in murine C2C12 myotubes

In cancer cachexia, proinflammatory cytokines including IL-6, IL-1β, IFN-γ and TNF-α have been implicated in the progression of skeletal muscle wasting (21). In addition to these humoral factors, tumor-derived factors such as myostatin and proteolysis-inducing factor collectively promote skeletal muscle wasting (2224). In previous studies, it has been reported that CT-26 CM inhibited C2C12 myoblast proliferation and differentiation and stimulated C2C12 myotube wasting, while myostatin secreted from tumors acted as a key contributor (22). To examine the effects of SO on tumor-induced muscle wasting, we evaluated C2C12 myoblast proliferation after exposure to SO-treated or -untreated CT-26 CM at 1:3 or 1:5 dilutions with GM. Control CM significantly retarded C2C12 myoblast growth by ~65 and 55% at 1:3 and 1:5 dilutions, respectively, compared to GM (Fig. 5A). However, compared with the control CM, SO-treated CM at 100 µg/ml did not cause a significant inhibition of C2C12 myoblast proliferation, resulting in 28.2 and 7.7% inhibition at 1:3 and 1:5 dilutions, respectively, compared to GM. Since CT-26 CM inhibits myoblast proliferation by cell cycle arrest, we next examined the effect of SO-treated or -untreated CM on the expression of cell cycle-related proteins in C2C12 myoblasts. Consistent with previous studies, the level of p21 was dramatically upregulated after exposure to control CM, while the levels of CDK2 and cyclin D were decreased. However, changes in these proteins in C2C12 myoblasts exposed to SO-treated CM were insignificant, consistent with their effects on cell proliferation (Fig. 5B). Notably, the levels of IL-6 in CT-26 CM were significantly decreased by SO treatment, in a dose-dependent manner (Fig. 5C). We next examined whether SO attenuated the CT-26-mediated inhibition of C2C12 myoblast differentiation. Fig. 6A shows that C2C12 myoblasts differentiating in control CM (diluted to a ratio of 1:5 with DM) exhibited reduced myotube numbers as compared with the untreated control, whereas SO-treated CM slightly attenuated the impairment of C2C12 myoblast differentiation. The immunoblot analysis revealed that Myh expression in myoblasts differentiating in control CM was markedly decreased compared with DM, while SO significantly prevented a CT-26-mediated reduction in Myh expression (Fig. 6B). To examine the effect of SO on C2C12 myotube wasting, differentiated C2C12 myotubes were incubated in SO-treated or -untreated CT-26 CM at 1:5 dilutions with DM for 48 h. The myotubes incubated with control CM exhibited a considerable muscle wasting appearance, whereas myotubes incubated with SO-treated CM did not exhibit muscle wasting, maintaining almost intact myotubes in a similar manner to the DM-treated controls (Fig. 6C). The Myh expression in myotubes was also reduced by control CM, while it was significantly conserved in myotubes incubated with SO-treated CM compared with DM-treated controls. In addition, control CM increased p65 phosphorylation in myotubes, while SO-treated CM did not (Fig. 6D). Previous studies reported that skeletal muscle atrophy was induced by TNF-α, as shown by a significant decrease in cell surface myotubes derived from C2C12 cells and Myh expression (25). We confirmed that TNF-α induced myotube wasting and SO significantly attenuated TNF-α-mediated skeletal muscle atrophy in C2C12 cells (Fig. 6C and D).

Discussion

Cachexia is a complex metabolic syndrome characterized by anorexia, loss of skeletal muscle mass and adipose tissue, significant reduction in body weight and asthenia. It occurs in many chronic diseases, including cancer, AIDS, renal failure, diabetes and chronic obstructive pulmonary disease. Half of all cancer patients, and 80–90% of patients with tumors of pancreatic and gastric origin, exhibit a cachexia syndrome, which has a profound impact on quality of life, and causes more severe chemotherapy-related adverse events and a decreased lifespan. Most importantly, the majority of terminal cancer patients suffer from cachexia, and 22% of them die as a result of this disorder (13,26).

Numerous cytokines secreted from the tumor or the host inflammatory cells, including TNF-α, IL-1, IL-6 and IFN-γ, have been postulated to act as procachectic factors that mimic leptin signaling and suppress ghrelin signaling, leading to sustained anorexia. Furthermore, these cytokines are also involved in the induction of cancer-mediated skeletal muscle wasting by inhibition of protein synthesis and/or acceleration of protein degradation. Skeletal muscle depletion is closely associated with worse outcomes in patients undergoing surgery, chemotherapy, and radiotherapy; therefore, reversal of skeletal muscle mass and body weight loss by inhibiting procachectic cytokines may be useful for the management of cancer patients and for an overall increase of well-being (3,6,1820).

The current management strategies for cancer cachexia using appetite stimulants, progestational agents and orexigenic agents are limited due to their poor in vivo efficacy and high toxicity. Instead, herbal medicines have received increasing attention for use as adjuvants to enhance the efficacy and diminish complications during chemotherapy or radiation therapy. Recent studies have shown that administration of Rikkunshito, a Japanese Kampo medicine composed of eight medicinal plants, attenuated anorexia-cachexia, prolonged survival and promoted anticancer efficacy by potentiating ghrelin signaling in tumor-bearing mice (27,28). Hochuekkito, a Kampo formula comprised of 10 medicinal plants, significantly attenuated marked reductions in carcass weight, food and water intake, and weight of gastrocnemius muscle and epididymal fat tissue caused by the CT-26 adenocarcinoma, by suppressing IL-6 production in macrophages (29). In addition, Sipjeondaebo-Tang (Shi-Quan-Da-Bu-Tang in Chinese and Juzen-Taiho-To in Japanese), composed of 10 species of herbs, showed therapeutic effects on cancer anorexia and cachexia by modulating body and muscle weight, food intake and cytokine and hormone production (30). Some natural herbs and their components, including Rhizoma coptidis, berberine and quercetin showed anti-cachectic effects by inhibiting tumor growth and suppressing inflammation (31,32).

SO is a traditional Oriental herbal prescription that has long been used to cure alternating chills and fever from half-exterior, half-interior lesser yang disease, and has been used to treat patients with liver diseases, including hepatic fibrosis, chronic hepatitis and hepatocellular carcinoma (1416). SO is currently prescribed for the management of fever and sore throat. Previous studies demonstrated that SO prevents depressive-like behavior in rodents by elevating serotonin and 5-hydroxyindoleacetic acid levels in the prefrontal cortex and hippocampus, inhibits thrombus formation by anti-platelet activity, and suppresses anaphylactic reaction in mast cells (3335). Recently, we demonstrated that SO has anti-inflammatory activity in LPS-stimulated RAW 264.7 cells, by suppression of NF-κB activation and MAPK phosphorylation (17).

The present study examined whether SO administration exerted inhibitory effects on the induction of cachexia in CT-26 adenocarcinoma-bearing mice, and then determined the mechanism zof action for this anti-cachectic activity. Our data showed that SO oral administration significantly inhibited tumor growth and recovered body weight compared with saline-treated control mice. SO prevented loss of skeletal muscle/fat tissue and increase in serum IL-6 levels caused by the CT-26 tumor. We also observed that SO suppressed the production of procachectic inflammatory cytokines, including IL-6, IL-1 and TNF-α, in macrophages through inhibition of NO generation and suppression of p38, NF-κB and STAT3 activation. In addition, SO prevented CT-26-mediated muscle atrophy involving proliferation, differentiation and wasting in murine C2C12 myoblasts and myotubes.

In summary, the present results demonstrate that SO reduces tumor burden and systemic inflammatory responses, followed by prevention of muscle and fat degradation, indicating that SO is a safe and effective herbal medicine for treating cancer patients with cachexia by protecting against loss of skeletal muscle mass during catabolic conditions.

Acknowledgments

The present study was supported by Grant K15280 awarded to the Korea Institute of Oriental Medicine (KIOM) from the Ministry of Science, ICT and Future Planning (MSIP), Republic of Korea

References

1 

Fearon KC, Voss AC and Hustead DS; Cancer Cachexia Study Group: Definition of cancer cachexia: Effect of weight loss, reduced food intake, and systemic inflammation on functional status and prognosis. Am J Clin Nutr. 83:1345–1350. 2006.PubMed/NCBI

2 

Tazi E and Errihani H: Treatment of cachexia in oncology. Indian J Palliat Care. 16:129–137. 2010.

3 

Aoyagi T, Terracina KP, Raza A, Matsubara H and Takabe K: Cancer cachexia, mechanism and treatment. World J Gastrointest Oncol. 7:17–29. 2015.PubMed/NCBI

4 

Ozola Zalite I, Zykus R, Francisco Gonzalez M, Saygili F, Pukitis A, Gaujoux S, Charnley RM and Lyadov V: Influence of cachexia and sarcopenia on survival in pancreatic ductal adenocarcinoma: A systematic review. Pancreatology. 15:19–24. 2015. View Article : Google Scholar

5 

Grabiec K, Burchert M, Milewska M, Błaszczyk M and Grzelkowska-Kowalczyk K: Systemic and local mechanisms leading to cachexia in cancer. Postepy hig Med Dosw (Online). 67:1397–1409. 2013.In Polish. View Article : Google Scholar

6 

Onesti JK and Guttridge DC: Inflammation based regulation of cancer cachexia. BioMed Res Int. 2014:1684072014. View Article : Google Scholar : PubMed/NCBI

7 

Tamura S, Ouchi KF, Mori K, Endo M, Matsumoto T, Eda H, Tanaka Y, Ishitsuka H, Tokita H and Yamaguchi K: Involvement of human interleukin 6 in experimental cachexia induced by a human uterine cervical carcinoma xenograft. Clin Cancer Res. 1:1353–1358. 1995.PubMed/NCBI

8 

Enomoto A, Rho MC, Fukami A, hiraku O, Komiyama K and hayashi M: Suppression of cancer cachexia by 20S,21-epoxy-resi bufogenin-3-acetate-a novel nonpeptide IL-6 receptor antagonist. Biochem Biophys Res Commun. 323:1096–1102. 2004. View Article : Google Scholar : PubMed/NCBI

9 

Ando K, Takahashi F, Kato M, Kaneko N, Doi T, Ohe Y, Koizumi F, Nishio K and Takahashi K: Tocilizumab, a proposed therapy for the cachexia of Interleukin6-expressing lung cancer. PLoS One. 9:e1024362014. View Article : Google Scholar : PubMed/NCBI

10 

Madeddu C, Macciò A, Panzone F, Tanca FM and Mantovani G: Medroxyprogesterone acetate in the management of cancer cachexia. Expert Opin Pharmacother. 10:1359–1366. 2009. View Article : Google Scholar : PubMed/NCBI

11 

Mantovani G, Macciò A, Lai P, Massa E, Ghiani M and Santona MC: Cytokine involvement in cancer anorexia/cachexia: Role of megestrol acetate and medroxyprogesterone acetate on cytokine downregulation and improvement of clinical symptoms. Crit Rev Oncog. 9:99–106. 1998. View Article : Google Scholar

12 

Inui A: Cancer anorexia-cachexia syndrome: Current issues in research and management. CA Cancer J Clin. 52:72–91. 2002. View Article : Google Scholar : PubMed/NCBI

13 

Lee JK, Kim JH and Shin HK: Therapeutic effects of the oriental herbal medicine Sho-saiko-to on liver cirrhosis and carcinoma. Hepatol Res. 41:825–837. 2011. View Article : Google Scholar : PubMed/NCBI

14 

Shimizu I: Sho-saiko-to: Japanese herbal medicine for protection against hepatic fibrosis and carcinoma. J Gastroenterol Hepatol. 15:D84–D90. 2000. View Article : Google Scholar : PubMed/NCBI

15 

Mizushima Y, Kashii T, Tokimitsu Y and Kobayashi M: Cytotoxic effect of herbal medicine Sho-saiko-to on human lung-cancer cell-lines in vitro. Oncol Rep. 2:91–94. 1995.PubMed/NCBI

16 

Yano H, Mizoguchi A, Fukuda K, Haramaki M, Ogasawara S, Momosaki S and Kojiro M: The herbal medicine Sho-saiko-to inhibits proliferation of cancer cell lines by inducing apoptosis and arrest at the G0/G1 phase. Cancer Res. 54:448–454. 1994.PubMed/NCBI

17 

Oh YC, Cho WK, Jeong YH, Im GY, Lee KJ, Yang HJ and Ma JY: Anti-inflammatory effect of Sosihotang via inhibition of nuclear factor-κB and mitogen-activated protein kinases signaling pathways in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. Food Chem Toxicol. 53:343–351. 2013. View Article : Google Scholar

18 

Barton BE: IL-6-like cytokines and cancer cachexia: Consequences of chronic inflammation. Immunol Res. 23:41–58. 2001. View Article : Google Scholar : PubMed/NCBI

19 

MacDonald N: Cancer cachexia and targeting chronic inflammation: A unified approach to cancer treatment and palliative/supportive care. J Support Oncol. 5:157–162. 2007.PubMed/NCBI

20 

Deans C and Wigmore SJ: Systemic inflammation, cachexia and prognosis in patients with cancer. Curr Opin Clin Nutr Metab Care. 8:265–269. 2005. View Article : Google Scholar : PubMed/NCBI

21 

Bonetto A, Aydogdu T, Kunzevitzky N, Guttridge DC, Khuri S, Koniaris LG and Zimmers TA: STAT3 activation in skeletal muscle links muscle wasting and the acute phase response in cancer cachexia. PLoS One. 6:e225382011. View Article : Google Scholar : PubMed/NCBI

22 

Lokireddy S, Wijesoma IW, Bonala S, Wei M, Sze SK, McFarlane C, Kambadur R and Sharma M: Myostatin is a novel tumoral factor that induces cancer cachexia. Biochem J. 446:23–36. 2012. View Article : Google Scholar : PubMed/NCBI

23 

Costelli P, Muscaritoli M, Bonetto A, Penna F, Reffo P, Bossola M, Bonelli G, Doglietto GB, Baccino FM and Rossi Fanelli F: Muscle myostatin signalling is enhanced in experimental cancer cachexia. Eur J Clin Invest. 38:531–538. 2008. View Article : Google Scholar : PubMed/NCBI

24 

Jespersen J, Kjaer M and Schjerling P: The possible role of myostatin in skeletal muscle atrophy and cachexia. Scand J Med Sci Sports. 16:74–82. 2006. View Article : Google Scholar : PubMed/NCBI

25 

Langen RC, Schols AM, Kelders MC, Wouters EF and Janssen-Heininger YM: Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-kappaB. FASEB J. 15:1169–1180. 2001. View Article : Google Scholar : PubMed/NCBI

26 

Donohoe CL, Ryan AM and Reynolds JV: Cancer cachexia: Mechanisms and clinical implications. Gastroenterol Res Pract. 2011:6014342011. View Article : Google Scholar : PubMed/NCBI

27 

Fujitsuka N, Asakawa A, Amitani H, Hattori T and Inui A: Efficacy of ghrelin in cancer cachexia: Clinical trials and a novel treatment by Rikkunshito. Crit Rev Oncog. 17:277–284. 2012. View Article : Google Scholar : PubMed/NCBI

28 

Fujitsuka N and Uezono Y: Rikkunshito, a ghrelin potentiator, ameliorates anorexia-cachexia syndrome. Front Pharmacol. 5:2712014. View Article : Google Scholar : PubMed/NCBI

29 

Yae S, Takahashi F, Yae T, Yamaguchi T, Tsukada R, Koike K, Minakata K, Murakami A, Nurwidya F, Kato M, et al: Hochuekkito (TJ-41), a kampo formula, ameliorates cachexia induced by colon 26 adenocarcinoma in mice. Evid Based Complement Alternat Med. 2012:9769262012. View Article : Google Scholar

30 

Choi YK, Jung KY, Woo SM, Yun YJ, Jun CY, Park JH, Shin YC, Cho SG and Ko SG: Effect of Sipjeondaebo-tang on cancer-induced anorexia and cachexia in CT-26 tumor-bearing mice. Mediators Inflamm. 2014:7365632014. View Article : Google Scholar : PubMed/NCBI

31 

Iizuka N, Miyamoto K, hazama S, Yoshino S, Yoshimura K, Okita K, Fukumoto T, Yamamoto S, Tangoku A and Oka M: Anticachectic effects of Coptidis rhizoma, an anti-inflammatory herb, on esophageal cancer cells that produce interleukin 6. Cancer Lett. 158:35–41. 2000. View Article : Google Scholar : PubMed/NCBI

32 

Camargo CA, da Silva ME, da Silva RA, Justo GZ, Gomes-Marcondes MC and Aoyama H: Inhibition of tumor growth by quercetin with increase of survival and prevention of cachexia in Walker 256 tumor-bearing rats. Biochem Biophys Res Commun. 406:638–642. 2011. View Article : Google Scholar : PubMed/NCBI

33 

Kim HM, Kim YY, Moon HS, Lee EH, Moon SJ and An NH: Inhibitory effect of anaphylactic reaction of Sosiho-Tang. Immunopharmacol Immunotoxicol. 20:567–578. 1998. View Article : Google Scholar : PubMed/NCBI

34 

Lee JJ, Kim T, Cho WK and Ma JY: Antithrombotic and anti-platelet activities of Soshiho-tang extract. BMC Complement Altern Med. 13:1372013. View Article : Google Scholar

35 

Su GY, Yang JY, Wang F, Xiong ZL, Hou Y, Zhang K, Song C, Ma J, Song SJ, Teng HF, et al: Xiaochaihutang prevents depressive-like behaviour in rodents by enhancing the serotonergic system. J Pharm Pharmacol. 66:823–834. 2014.

Related Articles

Journal Cover

March-2016
Volume 35 Issue 3

Print ISSN: 1021-335X
Online ISSN:1791-2431

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Kim A, Im M and Ma JY: Sosiho‑tang ameliorates cachexia‑related symptoms in mice bearing colon 26 adenocarcinoma by reducing systemic inflammation and muscle loss. Oncol Rep 35: 1841-1850, 2016
APA
Kim, A., Im, M., & Ma, J.Y. (2016). Sosiho‑tang ameliorates cachexia‑related symptoms in mice bearing colon 26 adenocarcinoma by reducing systemic inflammation and muscle loss. Oncology Reports, 35, 1841-1850. https://doi.org/10.3892/or.2015.4527
MLA
Kim, A., Im, M., Ma, J. Y."Sosiho‑tang ameliorates cachexia‑related symptoms in mice bearing colon 26 adenocarcinoma by reducing systemic inflammation and muscle loss". Oncology Reports 35.3 (2016): 1841-1850.
Chicago
Kim, A., Im, M., Ma, J. Y."Sosiho‑tang ameliorates cachexia‑related symptoms in mice bearing colon 26 adenocarcinoma by reducing systemic inflammation and muscle loss". Oncology Reports 35, no. 3 (2016): 1841-1850. https://doi.org/10.3892/or.2015.4527