A Brief Review on the Experimental Aspects of Bojungikki-Tang in Cancer

Bojungikki-tang (BJIKT, Hochuekkito in Japanese, Buzhong-yi-qi-tang in Chinese) has been widely used as a traditional herbal formula in Korea, Japan, and China. BJIKT was first described in Pi Wei Lun (Treatise on Spleen and Stomach) written by Li Gao (1180–1251 A.D. in the Chinese Yuan Dynasty)1. Li Gao stated that conditions based on psychological or physical problems, such as excessive emotional changes, immoderate drinking, irregular eating pattern and overwork, that lead to qi deficiency are recommended for BJIKT to treat symptoms like dyspepsia, anorexia, and fatigue2,3. The crude ingredients typically include Angelicae Gigantis radix, Astragali radix, Atractylodis rhizome, Bupleuri radix, Cimicifugae rhizome, Citriunshiipericarpium, Ginseng radix alba and Glycyrrhizae radix4. This herbal prescription has been identified as an effective drug for its potential impact on improving the function of digestive systems, the quality of life and nutritional status of patients5–8. BJIKT is useful to treat conditions such as general fatigue, poor appetite, spontaneous sweating, and intermittent fever9.Numerous research studies have reported the immunomodulatory and antiinflammatory effects of BJIKT. Kiyohara’s group have reported that oral administration of BJIKT, to early aged BALB/c mice given a intranasal inoculation of influenza hemagglutinin vaccine, results in stimulating the mucosal immune system of upper respiratory tract10. Yang’s group shows that BJIKT treatment suppress the responses of polymorphonuclear neutrophils to IL-4-stimulation in patients with perennial allergic rhinitis11. Gou’s group have suggested that BJIKT inhibits the apoptosis and necrosis induced by 5-fluorouracil in mouse intestinal mucosal epithelia by reducing the inflammatory factors12. BJIKT have also gained much attention in recent years as adjuvant treatment for cancer13. Kuroda’s group demonstrate that BJIKT had clinical effects on cachexia for genitourinary cancer patients 14. Kim’s group Abstract

status of patients [5][6][7][8] . BJIKT is useful to treat conditions such as general fatigue, poor appetite, spontaneous sweating, and intermittent fever 9 .Numerous research studies have reported the immunomodulatory and antiinflammatory effects of BJIKT. Kiyohara's group have reported that oral administration of BJIKT, to early aged BALB/c mice given a intranasal inoculation of influenza hemagglutinin vaccine, results in stimulating the mucosal immune system of upper respiratory tract 10 . Yang's group shows that BJIKT treatment suppress the responses of polymorphonuclear neutrophils to IL-4-stimulation in patients with perennial allergic rhinitis 11 . Gou's group have suggested that BJIKT inhibits the apoptosis and necrosis induced by 5-fluorouracil in mouse intestinal mucosal epithelia by reducing the inflammatory factors 12 . BJIKT have also gained much attention in recent years as adjuvant treatment for cancer 13  reports the radioprotective effects of BJIKT in mouse exposed with high and low doses of gamma-rays 15 . Jeong's group shows that BJIKT has beneficial effects on patients with cancer-related fatigue and quality of lives in cancer patients 9 . Currently there are numerous basic and clinical studies to find anticancer drugs from herbal medicine. Modern biomolecular tools and approaches have contributed to understand the role and mechanism of action of herbal medicine that exhibited anticancer activity. This review will introduce the experimental approaches to understand the pharmacological actions of BJIKT in cancer.

In vitro Studies
Kao's group studied the Granulocyte Colony-Stimulating-Factor (G-CSF) and Tumor Necrosis Factor-a (TNF-α) production by Peripheral Blood Mononuclear Cells (PBMC) isolated from healthy volunteers and hepatocellular carcinoma patients 16 .
Various concentrations of BJIKT were added to the prepared mononuclear cells 16 . BJIKT stimulated PBMC to produce G-CSF and TNF-α, that are beneficial to the biological defensive system 16 . Kao's group also reported that BJIKT suppressed the proliferation of 3 human hepatoma cell lines, Hep3B, HepG2, and HA22T cells 17 . BJIKT induced cell cycle arrest at the G0/G1 phase and apoptosis in Hep3B cells to inhibit tumor cell proliferation 17 . Interestingly, growth-inhibitory effects were higher in BJIKT compared to individual major compounds of BJIKT 17 . Kao's group suggest that major compounds from BJIKT might act in a synergistic or additive pathway to inhibit Hep3B proliferation 17 . Zhu's group evaluated growth inhibition of 4 herbal medicine (Sho-saiko-to, Hochu-ekki-to, Juzen-taiho-to, and Ninjin-yoei-to) on six human ovarian cancer cells (KF-1, MN-1, A2780 and their respective cisplatinresistant sublines KF-r, MN-r, A2780cp) 18 . However, BJIKT even at high concentrations did not show growth inhibition and apoptosis on all six cancer cell lines 18 . Kuo's group investigated the anti-tumor effect of BJIKT, and two other herbal formulas in human gastric cancer MKN-74 cells 19 . BJIKT most highly induced cytotoxicity in combination with mitomycin C(MMC) through a non-apoptotic mechanism 19 . Exposure to BJIKT first, followed by MMC treatment induced more cell death compared to MMC first, followed by BJIKT treatment 19 . From these data, BJIKT and chemotherapy administration sequence may alter cytotoxicity and cell death 19 . Sato's group studied the effect of BJIKT in low doses (50 µg/ml) to prevent non-specific cytotoxic effects attributed to saponins or detergent-like compounds 20 .
The present study observed that BJIKT augment the apoptotic impact of cisplatin by increasing caspase-3 activation and Bax/Bcl-2 ratio in human cervical cancer cell-line HeLa cells 20 . Data shows that BJIKT followed by cisplatin decreased protein levels of p-Akt, a cell survival factor, and increased protein levels of p53, a tumor suppressor 20 . Moreover, the interplay between Akt and p53 would explain the stimulation of cisplatininduced cell death by BJIKT in HeLa cells 20 . Yu's group also reported that BJIKT enhanced the cytotoxicity of cisplatin innon-small cell lung cancer (NSCLC) cells 21 . The study focused on the cultured human lung carcinomacisplatin resistant variant A549/DDP cells, BJIKT and cisplatin co-treatment induced apoptosis and autophagy via accumulation of reactive oxygen species (ROS) 21 . BJIKT and cisplatin co-treatment increasedBax/Bcl-2 ratio, cleaved caspase 3, and PARP cleavage in A549/DDP cells 21 . In addition z-VAD-FMK, a broad spectrum caspase inhibitor, partially limited the BJIKT and cisplatin-induced cell death 21 . Taken together, these data indicate that BJIKT and cisplatin cotreatment mediates cell death partially through caspasedependent intrinsic pathway 21 . Also, BJIKT and cisplatin co-treatment induced cytotoxic autophagy by increasing in LC3 fluorescent puncta formation, autophagic vacuoles, and LC3-II and ATG7 levels 21 . Therefore, the results suggest that both autophagy and caspasedependent apoptosis contribute to the antitumor effect of BJIKT and cisplatin co-treatment 21 .

In vivo studies
Harada's group treated mice bearing the syngeneic fibrosarcoma, Meth-A, with BJIKT and demonstrated that the enhancement of cytostatic activity mediated antitumor immunity 22 . Li's group using the B16 murine melanoma cell line xenograft on C57BL/6 (B6) mice, under restraint stress, reported that BJIKT administration normalized the serum levels of corticosterone, IL-12, and the expression of CD80 and CD86 23 . These data support that BJIKT augmented antitumor immune response in stress-burdened, tumor-bearing mice 23 . Onogi's group performed endometrial carcinogenesis, in ICR mice, induced by N-Methyl-N-Nitrosourea (MNU) into the left uterine tube and normal saline was injected into the opposite side 24 . Diet with 17ß-estradiol (E2) alone, E2 plus BJIKT, was given and incidence of prenoplastic and neoplastic mouse endometrial lesions were measured 24 . The incidence of adenocarcinoma was significantly less in BJIKT treated mice 24 . In addition, it was suggested that decrease in expression of c-Jun, TNF-α, ER-α and ER -β mediates the inhibitory effect of BJIKT on endometrial carcinogenesis 24 . Tsuneoka's group studied on the effect of BJIKT on N-nitrosobis (2-oxopropyl) amine (BOP) induced biliary carcinomas in bilioenterostomized hamsters 25 . Histological data show that BJIKT administration significantly decreased the average number of intrahepatic bile duct carcinomas per animal from 11.4 to 3.9, and also the total percentage of animals with carcinoma from 88% to 47% 25 . PCNA labeling index of the biliary epithelium was measure, and BJIKT significantly decreased cell proliferation from 9.6% to 6.4% 25 . The study shows the cancer preventative effect of BJIKT on BOP-induced biliary carcinogenesis in hamsters 25 . Yae's group used BALB/c mice bearing the Colon-26 (C26) adenocarcinoma to evaluated the effect of BJIKT 26 . The data shows that BJIKT did not suppress tumor growth, but significantly decreased serum IL-6 level, and increased the serum triglyceride level, weights of the gastrocnemius muscle and fat tissue around the testes in tumor-bearing mice in the terminal stage 26 .IL-6 expression from tumor tissues was evaluated by immunohistochemistry, and indicates that BJIKT treatment did not alter the IL-6 expression in cancer cells 26 . Interestingly, immunohistochemical analysis of IL-6 production by macrophages in the tissues surrounding tumors was significantly reduced in BJIKT treated C26-bearing mice 26 . Moreover, BJIKT inhibits IL-6 secretion from cultured THP-1 and RAW264.7 macrophage cell lines 26 .

Discussion
Most of the data derived from in vitro studies used BJIKT combined with chemotherapeutic agent. Few studies have explored the chemopreventive/anti-tumor effects of BJIKT as a single agent. BJIKT, in human hepatoma cells, a dose-dependent anti-proliferative effect was achieved 17 . BJIKT-only treatment inhibits cell growth by 20% in A549/DDP cells at high dose (1000 μg/ml) 21 .
In contrast, BJIKT-only treated human ovarian cancer cells, even at high dose (1000 μg/ml) did not inhibit cell growth 18 . The reported differences might be explained by differences in the organ used by different groups. However, at high doses, BJIKT can have non-specific effects and cause cytotoxicity 20 . These issues need to be explored in larger studies to examine the role of BJIKT in growth inhibition. Studies examining the effects of BJIKT on cell death have reported contradictory results.
A majority of studies have reported on the apoptotic effect of BJIKT in Hep3B cells, HeLa cells, and A549/ DDP cells 17,20,21 . In contrast, apoptosis was not involved in the increased cell death by BJIKT in combination with MMC on MKN-74 cells 19 . Interestingly, a study by Yu's group reported that BJIKT and cisplatin co-treatment contribute to cell death by both apoptosis and autophagy in A549/DDP cells 21 . It provides an interesting link to the non-apoptotic mechanism found in MKN-74 cells as suggested by Yu's group 21 .
Studies identifying the effect of BJIKT in vivo are mostly based on chemical induced carcinogenesis and xenograft tumor models. However, the anti-tumor effect of BJIKT was evaluated mainly by histopathological evaluation, and the molecular studies from Onogi's group are based on ovarectomized mice. Understanding the anti-tumor effects of BJIKT and the underlying molecular mechanisms still remains to be investigated. Mouse models are particularly valuable for cancer research to examine interactions among tumor cells and between the tumor cells and their host environment 27 . Transgenic mouse tumor model enable researchers to develop novel targeted therapies and chemopreventive agents 27 . Unfortunately, despite the benefits of transgenic models, BJIKT has yet been conducted with genetic manipulation techniques.
It has been suggested by Li's group that BJIKT increased the antitumor immune responses under stressed conditions and by Yae's group that BJIKT ameliorate the serum triglyceride level and weight of fat tissue in tumor-bearing mice 23,26 . Interestingly, Ouyang's group reported that BJIKT enhanced chemotherapyrelated fatigue in 4T1 breast cancer mice 28  BJIKT and PTX co-treatment up-regulated muscle superoxide dismutase activity and decreased the levels of malondialdehyde compared to PTX treatment 28 . Growing evidence indicates that directly or indirectly production of ROS is a mechanism shared by numerous chemotherapeutic agents, and cause muscle weakness and develop of fatigue 28,29 . In the aggregate, these findings suggest that BJIKT could improve cancer induced fatigue and skeletal muscle wasting by preventing ROS generation.

Conclusion
The in vitro and in vivo studies based on BJIKT suggest that BJIKT is a potential therapeutic agent for cancer ( Table 1). The anti-tumor effect of BJIKT is maximized when combined with chemotherapeutic drug. Improving survival time, body weight and fatigue affected by cancer represent another promising role for BJIKT. A role that BJIKT, has been well defined from its long history of traditional use.