Suppressive Effect of Juzentaihoto on Vascularization Induced by B16 Melanoma Cells In Vitro and In Vivo

Juzentaihoto (JTT) is well known to be one of Japanese herbal medicines, and used for the supplemental therapy of cancer patients with remarkable success. The present study, therefore, was undertaken to examine the possible therapeutic mechanisms of JTT on cancer using B16 melanoma cell (B16 cell)/experimental mouse system. JTT was well mixed with rodent chow at 3.0% concentrations, and was administered orally ad libitum. Administration of JTT was started one week before tumor cell injection and continued throughout the experiment. Administration of JTT into mice significantly inhibited tumor metastasis in lungs after intravenous injection of 2 × 105 B16 cells in a volume of 50 μL. JTT also significantly suppressed enlargement of tumor size in hind footpad after the subcutaneous injection of 2 × 105 (50 μL) B16 cells. In the second part of experiments, the chamber that containing B16 cells was buried in the murine back. In JTT administrated group, vascular endothelial growth factor (VEGF) of chamber internal fluid significantly decreased, and vascularization of chamber circumference was also inhibited. These results strongly suggest that oral administration of JTT caused decrease in the generation of VEGF, which is responsible for vascularization, and results in inhibition of B16 cell metastasis.


Introduction
Cancer patients generally undergo surgical therapy, chemotherapy, radiotherapy, or a combination of these treatments. While the effects of these treatments are significant, it is a fact that most patients suffer from side effects, such as high fever, general fatigue, loss of appetite, pancytopenia, and many kinds of infections.
In Japan and China, herbal medicines, including Juzentaihoto (JTT), Hochuekkito, and among others, are used as a supplemental therapy for many kinds of chronic diseases such as loss of appetite, anemia, and chilliness of the arms and legs with remarkable success [1,2]. Recent reports clearly showed that when herbal medicines are used for cancer treatment, many patients experience fewer or diminished side effects induced by western medicine, such as chemotherapy and radiotherapy, and the survival period is longer [3,4]. It is also reported that herbal medicine can prevent the progression of colon carcinoma, gastric and breast cancer as well as the prevention of these cancer metastasis to the liver, lung, and bone [3,4]; moreover, hepatocellular carcinoma has been shown to become smaller without severe side effects after the treatment with herbal medicine [5]. Although these reports strongly suggest that herbal medicines will be a good candidate for the treatment of several types of cancer, the mechanisms by which herbal medicines could improve clinical status, including cancer metastasis, of cancer patients.
Angiogenesis is involved early in tumor progression as well as in sustained growth, invasion of established tumors, and the development of metastasis [6]. The regulation of angiogenesis is closely related to specific angiogenic factors and their receptors [7]. The predominant angiogenic factor is vascular endothelial growth factor (VEGF), a glycoprotein stimulating mitosis of endothelial cells and their migration [7]. Moreover, VEGF increases capillary permeability responsible for cancer metastases [7][8][9].
Our previous work clearly showed that oral administration of JTT for 21 days inhibited metastasis of B16 melanoma cells (B16 cell) on lung surfaces through the enhancement 2 Evidence-Based Complementary and Alternative Medicine of NK-cell activities. However, the influence of JTT on angiogenesis was not clear at present [10].
In this study, we examined the effects of JTT on the vascularization of B16 cell, as well as its effects on the VEGF production in B16 cells.

Materials and Methods
2.1. Animals. Specific pathogen-free C57BL/6 male mice, 6-week-old, were purchased from Japan CLEA Co., Ltd. (Tokyo, Japan). The animals were maintained at 25 ± 2 • C, humidity 50 ± 2%, and a light and dark cycle of 12 hours in our animal facilities. They were randomly divided into groups of 10 mice and fed chow containing 3% JTT [11] or regular diet (control). This study was approved by the Ethics Committee of Showa University for animal experiments (no. 01067).

Assay for Tumor Cell Metastasis.
In the first experiments, B16 cells (2 × 10 5 cells) were injected intravenously into recipient mice in a volume of 50 μL. After 21 days, mice were killed under ether anesthesia and the number of tumor colonies on the lung surface was counted under a dissecting microscope (SZ-60; OLYMPUS Co. Ltd., Tokyo, Japan).
In the second experiments, B16 cells (2 × 10 5 cells) were injected subcutaneously into the left hind pad in a volume of 50 μL. After 21 days, the tumor was removed in the knee under a dissecting microscope (OLYMPUS Co. Ltd., Tokyo, Japan). The volume of tumor lumps (cutting departments) was measured by a water displacement method, using a plethysmometer (model no. TK-101P; Muromachi kikai, Tokyo, Japan) [12]. These mice were then maintained for further 21 days and the black dots, showing tumor colony formation on the lung surface, were counted under a dissecting microscope (OLYMPUS Co. Ltd.). In these two experiments, mice were given food-containing JTT and tap water ad libitum for two or three weeks starting 7 days before tumor cell injection.

2.5.
Assay for Cytotoxic Activity of JTT. B16 cells (5 × 10 5 cells) were introduced into each well of 24 well culture plates that contained 10% and 25% serum prepared from mice treated with JTT for 21 days in triplicate. After 24 hours, the numbers of viable cells were counted with a Countess Automated Cell Counter (Invitrogen Co., Tokyo, Japan) in the presence of trypan blue.
2.6. Millipore Chamber (MPC) Implantation. Millipore Chambers (MPCs) [13,14] containing B16 cells (1 × 10 6 cells) were implanted into the different experimental groups. The components of a MPC consisted of a plastic holding chamber (Millpore UK Ltd., Watford, UK), two fixation discs, and two 0.45 μm pore size Millipore Filters of 14 mm diameter (Millipore UK Ltd.). B16 cells cultured were placed in the holding chambers which were then closed by sealing a Millipore Filter at both ends using the fixation discs. Recipient mice were anaesthetized using an intraperitoneal sodium pentobarbital. A dorsal longitudinal incision was made and a subcutaneous plane developed into which was inserted a chamber containing B16 cells, and the incision closed with a subcutaneous suture.

2.7.
Assay for VEGF Production from B16 Cells. VEGF level in culture supernatants and the MPCs internal fluid was examined using commercially available mouse VEGF enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Inc., Minneapolis, Minn, USA) according to the manufacturer's recommendations. The sensitivity of VEGF assay kit was 3 pg/mL.

Determination of the Effect of JTT on In Vivo Angiogenesis.
Angiogenesis was induced by implantation of the MPCs containing B16 cells (1×10 6 cells/animal) hypodermically on the shaven dorsum skin mouse [15]. After 21 days of MPCs implantation, all animals were sacrificed and B16 cells and body fluid in the chamber were collected. At the same time, the skin adhering to the chamber and blood were collected. The body fluid and serum were used for the estimation of VEGF using ELISA kits according to the manufacturer's recommendation. Dorsum skins removed were washed with PBS and the length of tumor-directed blood vessels per cm 2 around the tumor was measured using a dissecting microscope (OLYMPUS Co. Ltd.) [16]. The images of mouse hypodermis were magnified to 250 diameters, and the blood vessels length using Digital Scale (FS-DSC101; Firestar Co. Ltd., Hiroshima, Japan) were measured. In addition, these skins were dipped in 4% formalin and used for immunohistological staining [14].
2.9. Immunohistochemistry for Angiogenesis. The experimental and control skins were stained with the rat primary antibody for mouse CD31 (PECAM-1; BD Biosciences, Tokyo, Japan) to examine the presence of the neogenesis blood vessels in the hypodermis [17,18]. The shin tissues were washed several times with saline to remove unwanted materials (e.g., blood and connective tissues, etc.), fixed in 4% paraformaldehyde-PBS, followed by 5%, 15%, and 30% sucrose-PBS; specimens were then embedded in Tissue-Tek (Sakura Finetechnical Co. Ltd., Tokyo, Japan) and cut into 10 μm sections. These sections were treated with 3% hydrogen peroxide in PBS to eliminate endogenous peroxidase activity and treated with blocking buffer (PBS  [20,21]. Relative quantification (RQ) studies [22] were made from collected data (threshold cycle numbers, referred to as Ct) with ABI Prism 7900HT Sequence-Detection System (SDS) software 2.3 (Applied Biosystem).

Statistical Analysis.
Continuous variables were presented as the mean ± standard error of the mean SEM. The statistical significance between the control and the experimental groups was analyzed with analysis of variance followed by Fisher's protested least significant difference test. A P value of less than 0.05 was considered statistically significant.

Suppression of B16 Melanoma Cell Metastasis by JTT.
This experiment was undertaken to examine the influence of oral administration of JTT on tumor cell metastasis using different two types of experimental models. Mice pretreated with 3.0% JTT were injected intravenously with 2 × 10 5 B16 cells and were killed 21 days later to count the number of tumor cell colonies on the lung surfaces. As shown in Figure 1(a), oral administration of 3.0% JTT could cause significant suppression of B16 cell metastasis. We then examined whether oral administration of JTT could also prevent spontaneous tumor cell metastasis as in the case of intravenous administration of tumor cells. As shown in Figure 1(b), administration of 3.0% JTT into mice could prevent spontaneous B16 cell metastasis from right hind footpad to lung surfaces.

Effect of JTT on Paw
Swelling. This experiment was undertaken to examine the influence of oral administration of JTT on paw swelling caused by tumor cell growth. Tumor cell growth of mice treated with 3.0% JTT were injected into the palm with 2 × 10 5 B16 cells and the volume of the palm was measured after 21 days. As shown in Figure 2(a), oral administration of 3.0% JTT could cause significant suppression of paw swelling owing to the tumor growth.

Effect of Serum Obtained from JTT-Treated
Mouse on B16 Cell Viability. The present study was designed to determine whether the serum prepared from mice treated with JTT exerts cytotoxic effects on B16 cells and results in the prevention of tumor cell metastasis. B16 cells at a concentration of 5 × 10 5 cells/well were cultured in the mouse serum for 24 hours and the numbers of viable cells were counted with trypan blue dye exclusion test. As shown in Figure 2(b), JTT group could not suppress B16 cell growth even when cells were cultured in the presence of 25% mice serum: the number of cells in experimental cultures is almost equal (not significant; P > 0.05) to that observed in control cultures. 10 6 cells/animal) hypodermically on the shaven dorsum of animals. In the sham group, the vascularization occurred mildly. In the group that implanted B16-MPCs, the blood vessels, which were induced by the tumor, were significantly reduced in the JTT-treated group (Figure 3(a)). Control animals had the length of 10427.0 ± 1641.7 μm/cm 2 around the MPCs whereas JTT-treated animals had mere 6711.3 ± 345.4 μm/cm 2 (Figure 3(b)).

Immunohistochemistry for CD31.
Antiangiogenesis is a major anticancer mechanism. Therefore, the skins of MPCs contact hypodermis were evaluated in sections stained with CD31 to further investigate the antiangiogenic effect of JTT. As shown in Figure 4, the (a-c) were high magnification photographs of (A-C), the neogenesis blood vessels (CD31positive) of the control groups had increased as compared with the sham groups (the CD31-positive cells were shown in arrows). In contrast, vascularization was decreased in the JTT-treated groups.
3.6. Influence of JTT on VEGF Production from B16 Cells. The experiment was undertaken to examine the influence of JTT on VEGF production from B16 cells. The VEGF contents in culture supernatants and the MPCs internal fluid were examined by ELISA. As shown in Figure 5(a), JTT in culture supernatants suppressed the ability of B16 cells to produce VEGF. In addition, as shown in Figure 5(b), JTT treatment in the MPCs internal fluid suppressed the ability of B16 cells to produce VEGF.

Effect of JTT on the VEGF mRNA Expression.
The final experiment was carried out to examine whether oral administration of JTT could cause decrease in VEGF mRNA expression of the B16 cells. As shown in Figure 6, in the B16 cells contained in the MPCs, the VEGF mRNA expression of the JTT treatment groups had decreased significantly as compared with the control groups.

Discussion
Herbal medicine is used frequently as a supplemental therapy for many kinds of chronic diseases with remarkable success [1]. In cases of treatment for cancer, herbal medicine is reported to be able to prevent the progression of colon carcinoma, gastric and breast cancer as well as the prevention of the cancer metastasis to the liver, lung, and bone [23].
However, the precise mechanisms by which herbal medicine can alleviate the clinical symptoms of cancer patients such as tumor metastasis, are not well defined. The present study, therefore, was undertaken to examine the possible therapeutic mechanisms of herbal medicine on cancer through the choice of JTT and B16 cell/mouse system in vivo and in vitro. The present results clearly showed that oral administration of JTT inhibited B16-cell colony formation on the lung surface, when the recipient mice were given tumor cells intravenously (Figure 1(a)). JTT also suppressed spontaneous B16 tumor cell metastasis from hind footpad to the lung surface (Figure 1(b)). The prevention of tumor cell growth and metastasis is well accepted to be through diverse mechanisms, including tumor cell death, apoptosis, and immune-mediated cancer regression. Our results clearly showed the absence of cytotoxic effects of JTT on B16 cells (Figure 2(b)). It is also showed that oral administration of JTT suppressed the paw swelling by B16 cell growth, 8 Evidence-Based Complementary and Alternative Medicine suggesting that neovascularized mechanisms to promote metastasis are responsible for the prevention of tumor cell colony formation on the lung surface.
In the next study, we focused on the role of JTT during the initial growth phase in the spontaneous metastasis of B16 cells; tumor-induced neovascularization and tumor growth. In the present study, we observed the decrease of neovascularization on tumor tissues treated by JTT administration, especially in the MPCs implanted mice (Figures 3(a)  and 3(b)). The experiments of the MPCs implanted B16 cells clearly demonstrate that the high density of CD31positive vessels in the site of dorsal skin is detectable by immunohistochemical staining. The endothelial cell proliferation (CD31 positive cells) was also inhibited by oral JTT administration. It is important to know about the antiangiogenic effect with JTT administration, because good candidates for antiangiogenic drugs include those available for oral administration for a long term without severe systemic side effects [24]. JTT has satisfied these conditions and could be a candidate for an antiangiogenic material for patients with cancer.
Angiogenesis is composed of several processes: dissociation of pericytes from preexisting vessels, digestion of extracellular matrix with proteases, proliferation, migration and invasion of endothelial cells, and tube formation, and then finally remodeling occurs. VEGF is considered to be secreted from tumor cells in a paracrine fashion to induce blood vessel growth. Even though VEGF is a potent mitogenic stimulation of endothelial cells, several studies have demonstrated the ability of VEGF to function as a survival factor for endothelial cells [25]. Since VEGF is generated from a variety of tumors, it is the most important angiogenic factor associated closely with induction and maintenance of the neovasculature in human tumors [26,27]. We observed the decrease in VEGF levels in the MPCs contents and culture supernatants treated by JTT ( Figure 5). In addition, JTT decreased VEGF mRNA expression in B16 cells ( Figure 6) prepared from mice pretreated with JTT for 21 days, but did not culture B16 cells with JTT-treated mouse serum. The reasons for this discrepancy in in vitro experiments are not dear at present. The process of protein synthesis requires two different steps. In the first step, socalled transcription, mRNA is synthetized from DNA in the nucleus. mRNA formed then comes out through nuclear membrane into cytoplasm where it attaches to mRNAbinding site on ribosome and starts protein synthesis which is called translocation step. Therefore, there is a possibility that short-term (within 24 h) exposure of B16 cells with metabolized JTT in vitro could inhibit only translocation step. Further experiments are needed to clarify this point. These antiangiogenic activities of oral JTT administration may be responsible for the prevention of B16 tumor cell metastasis.
We reported that oral administration of JTT into mice caused B16 cell metastasis through the enhancement of IFNγ functions [10]. It is also showed that VEGF-A secretion from corneal fibroblasts induced by proinflammatory cytokine (e.g., TNF-α and IL-1) stimulation was suppressed by IFN-γ [28]. Taken together, it is suggested that JTT may possess novel pharmacologic properties that interfere with angiogenesis triggered by VEGF secreted from B16 primary oncocytes through the enhancement of IFN-γ mechanism.