Effect of Sipjeondaebo-Tang on Cancer-Induced Anorexia and Cachexia in CT-26 Tumor-Bearing Mice

Cancer-associated anorexia and cachexia are a multifactorial condition described by a loss of body weight and muscle with anorexia, asthenia, and anemia. Moreover, they correlate with a high mortality rate, poor response to chemotherapy, poor performance status, and poor quality of life. Cancer cachexia is regulated by proinflammatory cytokines such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α). In addition, glucagon like peptide-1 (GIP-1), peptide YY (PYY), ghrelin, and leptin plays a crucial role in food intake. In this study, we investigated the therapeutic effects of one of the traditional herbal medicines, Sipjeondaebo-tang (Juzen-taiho-to in Japanese; SJDBT), on cancer anorexia and cachexia in a fundamental mouse cancer anorexia/cachexia model, CT-26 tumor-bearing mice. SJDBT was more significantly effective in a treatment model where it was treated after anorexia and cachexia than in a prevention model where it was treated before anorexia and cachexia on the basis of parameters such as weights of muscles and whole body and food intakes. Moreover, SJDBT inhibited a production of IL-6, MCP-1, PYY, and GLP-1 and ameliorated cancer-induced anemia. Therefore, our in vivo studies provide evidence on the role of SJDBT in cancer-associated anorexia and cachexia, thereby suggesting that SJDBT may be useful for treating cancer-associated anorexia and cachexia.


Introduction
Cancer anorexia and cachexia correlate with a high mortality rate, poor response to chemotherapy, poor performance status, and poor quality of life (QOL), particularly in physical, psychological, and social functions in cancer patients [1,2]. Anorexia, which is characterized by the loss of desire to eat or loss of appetite, causes weight loss and malnutrition in cancer patients [3,4]. Cachexia is a multifactorial condition described by muscle loss, which is in part associated with loss of body weight, anorexia, asthenia, and anemia [5,6]. Cachexia occurs in about half of all cancer patients, and lung and upper gastrointestinal tract cancer patients have the highest frequency of cachexia, whereas patients with breast and lower gastrointestinal tract cancer have the lowest frequency of cachexia [7]. In addition, cachexia affects an ability of cancer patients to sustain chemotherapy or radiotherapy [8].
Therefore, it is important to manage anorexia and cachexia in cancer patients. However, current therapies for cancer patients with anorexia and cachexia are limited due to both poor efficacies and side effects of chemotherapeutics [9]. So, different therapeutic options are required to prevent cancer anorexia and cachexia.
Sipjeondaebo-tang (Juzen-taiho-to in Japanese and Shi-Quan-Da-Bu-Tang in Chinese; SJDBT), commonly used traditional herbal medicines in Korea, China, and Japan [10], is composed of 10 species of herbs. SJDBT is prescribed for patients suffering from anemia, fatigue, anorexia, scaly skin, and dryness of the mouth [11][12][13]. In addition, SJDBT has been known to have anticancer effects [14][15][16][17][18][19]. Nevertheless, its effect on cancer cachexia is poorly understood. Here, we examined the effect of SJDBT on key parameters of cancer anorexia and cachexia and found that SJDBT ameliorated cancer anorexia and cachexia in vivo by altering body and 2  muscle weights, food intakes, and levels of cytokines, hormones, RBC, Hb, and HCT in CT-26 tumor-bearing mice suffering from anorexia and cachexia, thereby suggesting that SJDBT may be useful for treating cancer anorexia and cachexia.

Preparation of SJDBT.
SJDBT were prepared by and obtained from Hanpoong Pharmaceutical Company (Jeonju, Korea) following the good manufacturing practices (GMP) procedures. SJDBT is manufactured as dried powder of hot water extract obtained from 10 herbs (320 g of Angelica gigas, 320 g of Astragalus membranaceus, 320 g of Atractylodes japonica, 320 g of Cinnamomum cassia, 320 g of Cnidium officinale, 160 g of Glycyrrhiza uralensis, 320 g of Paeonia lactiflora, 320 g of Panax ginseng, 320 g of Poria cocos, and 320 g of Rehmannia glutinosa; average yield = 28.85%). The dried powders were lyophilized and then dissolved the three different doses (low dose: 6.784 mg/kg, middle dose: 67.84 mg/kg, and high dose: 678.4 mg/kg) in distilled water.

Animal Study for Cancer Cachexia.
Male BALB/c mice were purchased from Central Lab Animal Inc. (Seoul, Korea) at 6 weeks of age, and all mice were kept in pathogenfree environment. Mice were randomized into two groups (prevention or therapy model of cachexia). Each group was divided into subgroups (5 prevention groups: normal, control, low dose SJDBT (L-SJDBT of 6.784 mg/kg), middle dose SJDBT (M-SJDBT of 67.84 mg/kg), and high dose SJDBT (H-SJDBT of 678.4 mg/kg); 6 treatment groups: normal, control, L-SJDBT, M-SJDBT, H-SJDBT, and megestrol acetate-(MA-) treated). Animal studies were approved by the animal care center of Kyung-Hee University (KHUASP (SE)-12-048). The murine CT-26 tumor-bearing mouse was widely used for cancer cachexia model [20]. CT-26 colon carcinoma cell was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and from Dr. Suk-Chan Lee (Department of Genetic Engineering, Sungkyunkwan University, Suwon, Korea). Cells were cultured in RPMI-1640 medium with 10% fetal bovine serum and 1% antibiotics. The schematic experimental procedure is described in Figure 1.

Measurement of Food Intakes and Weights of Whole Body
and Muscles. Body weight and food intake were measured every day using an electronic scale. The measured quantity of food intake was divided by the number of mice to determine each intake per animal per day. At the time of sacrifice, the gastrocnemius muscles were dissected and weighted.

Measurement of Levels of Cytokines and
Hormones and Blood Analysis. Whole blood samples were collected by cardiac puncture, and serum was obtained after being

2.5.
Statistics. Data were presented as the mean and standard deviation. values less than 0.05 in two-tailed student's t-test or one-way ANOVA were considered statistically significant.

SJDBT Improves Cancer-Induced Weight Loss and Anorexia in CT-26 Tumor-Bearing Mice.
To examine whether SJDBT prevents cancer-induced cachexia or treats cancerinduced cachexia, we divided into two groups (Figure 1). A day when CT-26 tumor cells were injected into mice was designated as day 0. We found that control groups in each experimental set compared to the normal group showed cancer-induced anorexia and cachexia in a therapy model. As shown in Figure 2(a), we found that L-SJDBT and H-SJDBT compared to the control increased body weights slightly by approximately 11.1% and 8.9%, respectively, while being not statistically significant. Otherwise, when SJDBT were administered into mice after cancer-induced weight loss, M-SJDBT-treated mice compared to the control significantly increased the weight by approximately 36.6% (Figure 2(b)). Therefore, SJDBT appeared to treat tumor-induced loss of body weight. Next, we performed experiments to determine whether SJDBT improves the appetite in a model of either prevention or treatment. L-SJDBT and H-SJDBT, compared to the control, ameliorated cancer-induced anorexia by approximately 25.3% and 25.1%, respectively, in the prevention model (Figure 2(c)). In addition, M-SJDBT compared to the control significantly increased food intake in the therapy model ( Figure 2(d)). The control group compared to the normal decreased muscle weight by approximately 70% at 22 days after tumor cell injection and by approximately 87.5% at 43 days after tumor cell inoculation (Figures 2(e) and 2(f)). Both L-SJDBT and M-SJDBT prevented tumor-induced loss of muscle weight in the therapy model, since muscle weights of tumor-bearing mice treated with each of them were higher than those of tumor-bearing mice untreated by approximately 4-fold (Figure 2(f)). However, those were not effective in the prevention model (Figure 2(e)). Those results indicate that SJDBT may be effective for treating anorexia and for attenuating cachectic phenomenon, loss of muscle weight. Furthermore, our data showed that M-SJDBT was much more effective than MA in the treatment of cancerinduced reduction of food intake, while MA has been used to treat anorexia.

SJDBT Suppressed IL-6 and MCP-1 but Not TNF-in the Therapy Model.
As proinflammatory cytokines such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-(TNF-) derived from tumor cells were associated with cancer-related anorexia and cachexia [21,22], we next examined whether SJDBT affects serum levels of IL-6, MCP-1, and TNF-. Levels of IL-6 and MCP-1 significantly increased in the control compared to the normal by approximately 480-fold and 91-fold, respectively (Figures 3(a) and 3(b)). In addition, M-SJDBT significantly inhibited cancer-induced induction of IL-6 and MCP-1 by approximately 50.5% and 90.8%, respectively (Figures 3(a)  and 3(b)). Furthermore, MCP-1 level was uniquely altered in all tested doses of SJDBT. Whereas the serum level of TNF-increased slightly in the control compared to the normal by approximately 1.7-fold, M-SJDBT compared to the control reduced TNF-level by approximately 41.4% while it was not statistically significant (Figure 3(c)). These results suggest that M-SJDBT may ameliorate cancer-induced anorexia and cachexia by altering levels of cytokines such as IL-6 and MCP-1.

Effects of SJDBT on Levels of Gut Hormones and Leptin in the Therapy Model.
As anorexia involves appetite-regulating hormones such as gut hormones (GLP-1, PYY, and ghrelin) and leptin [23,24], we further examined whether SJDBT affects their levels in serum. Levels of GLP-1 and PYY significantly increased in the control compared to normal by approximately 1.3-fold and 20-fold, respectively (Figures 4(a)  and 4(b)). M-SJDBT significantly inhibited cancer-induced induction of both GLP-1 and PYY by approximately 63.2% and 44.9%, respectively (Figures 4(a) and 4(b)). However, SJDBT improved cancer cachexia in mice and inhibited the production of IL-6, MCP-1, PYY, and GLP-1. Therefore, we conclude that SJDBT improves cancer-induced cachexia including weight loss, anorexia, muscle wasting, anemia, and dysregulation of cytokines and hormones. levels of ghrelin and leptin were not altered by M-SJDBT. Those results suggest that M-SJDBT regulation of GLP-1 and PYY may affect cancer-associated anorexia and cachexia.

SJDBT Improves Cancer-Induced Anemia in CT-26
Tumor-Bearing Mice. Because cancer-induced anorexia and cachexia are a multifactorial syndrome including anemia [25], we measured levels of red blood cells in blood samples using HEMAVET 950 hematology analyzer. The control compared to the normal accrued anemia, which was measured by numbers of red blood cells (RBC; normal mice versus control mice; 9.04 ± 0.68 M/ L versus 6.53 ± 0.44 M/ L; = 0.02), hemoglobins (Hb; normal mice versus control mice; 16.91 ± 1.15 g/dL versus 10.6 ± 0.38 M/ L; = 0.0001), and hematocrits (HCT; percentage of red blood cells in whole blood; normal mice versus control mice; 52.9 ± 4.32% versus 32.53 ± 2.68%; = 0.03). L-SJDBT compared to the control significantly increased values of RBC, Hb, and HCT by approximately 18%, 34.7%, and 22%, respectively ( Figures  5(a), 5(b), and 5(c)). In addition, MA similarly affected those values, indicating that the treatment of anorexia might be linked to alterations of those values. While M-SJDBT compared to the control increased values of RBC, Hb, and HCT by approximately 7.6%, 24.5%, and 9.4%, respectively, those were not statistically significant. Therefore, those results indicate that L-SJDBT may be a good remedy for cancermediated anemia.

Discussion
As herbal medicine therapy has been known to be beneficial with high efficacy and safety, market of herbal medicine therapy is growing in Korea, China, and Japan as well as in the United States [26]. Especially, herbal medicines are used for clinical trial and therapy in cancer patients [27,28]. However, biomedical scientists have limitations to use the herbal medicines due to the lack of evidence even in lab-based experimental studies [28]. For example, SJDBT is widely used for cancer patients suffering from symptoms such as loss of appetite, fatigue, and anemia in Korea, China, and Japan [10,12,13,29]. However, its effect on the loss of either weight or food intake is still unknown. In this present study, we showed SJDBT effect on cancer anorexia and cachexia.
Cancer-induced anorexia and cachexia involve proinflammatory cytokines such as IL-6, MCP-1, and TNF- [21,22]. Furthermore, it has been suggested that MCP-1 rather than TNF-is more important for cancer cachexia [22,30,31]. Circulating level of IL-6 is known to be important for keeping body weight and for survival in cancer patients [32][33][34]. In our study, SJDBT reduced IL-6 but increased MCP-1, while it did not affect TNF-. Thus, it is plausible that SJDBT may modulate effects of cytokines on cancer-induced anorexia and cachexia. Meanwhile, GLP-1, PYY, ghrelin, and leptin are known as readouts for anorexia/cachexia [35]. SJDBT significantly suppressed GLP-1 and PYY levels, whereas it did not affect ghrelin and leptin. In sum, our data suggest that SJDBT ameliorates cancer-associated anorexia/cachexia by regulating cytokines (IL-6 and MCP-1) and hormones (GLP-1 and PYY). Anemia is tone of common features of cancer cachexia [6,8,36]. Especially, anemia is linked to the fatigue and QOL in cancer patients [36]. In addition, Hb level is supposed to be related to QOL [37]. SJDBT increased values of RBC, Hb, and HCT, which further indicates that SJDBT is beneficial for treating cancer anorexia/cachexia.

Mediators of Inflammation
Cancer-associated anorexia is related to central mechanisms governing food intake [38]. Food intake is controlled by neuroendocrine paths through central nervous system (CNS) [39]. Short-term reaction for food intake is mediated by peptides produced by enteroendocrine cells in the gastrointestinal tract [39]. Incretin hormones such as GLP-1 mediate a response of the pancreas to nutrients, resulting in the reduction of food intake. Likewise, ghrelin is secreted from the stomach and promotes appetite. Leptin mediates long-term reaction for food intake. It is produced mainly from the adipose and stimulates food intake through CNS [40]. Furthermore, cancer-associated anorexia is caused by chronic stimulations via cytokines released from cancer cells or from host cells responding to cancer cells [41]. Cytokines have been known to interrupt with appetite-stimulating signaling [42]. Furthermore, cytokines impair muscle and fat metabolisms, which is tightly linked to cancer-associated anorexia [42,43]. While cytokines such as TNF-and IL-6 have been known to mediate cancer cachexia [42], SJDBT reduced IL-6 level without altering TNF-. IL-6 is known to cause the loss of lean body weight in CT-26 tumor-bearing cachectic mice by inducing proteolytic pathways [44]. In addition, MCP-1, the circulating level of which was altered by SJDBT, has been known to regulate leptin level in the adipose and to mediate cachexia [22,45]. Therefore, it is plausible that SJDBT may target cytokine production in cancer cells, which may result in modulations of hormone levels. However, we could not exclude possibilities that SJDBT may target multiple sources including cancer cells, cancerassociated immune cells, adipocytes, and hypothalamic neurons. Recent studies have revealed functional relationships between cytokines (IL-6 and MCP-1) and hormones (GLP-1 and PYY) [46][47][48][49][50][51]. Thus, it remains to be deciphered molecular and cellular mechanisms of SJDBT to ameliorate cancerassociated anorexia and cachexia.

Conclusion
Our study demonstrates that the SJDBT ameliorates cancerinduced anorexia and cachexia in CT-26 tumor-bearing mouse model by altering the production of IL-6, MCP-1, PYY, and GLP-1. Our in vivo studies first provide evidence on the role of SJDBT in cancer-associated anorexia/cachexia, which suggests that SJDBT may be useful for patients with cancerassociated anorexia/cachexia.