Suppressive effect of juzentaihoto on vascularization induced by b16 melanoma cells in vitro and in vivo

Abstract

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 × 10(5) 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 × 10(5) (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.

Figure 1

Influence of Juzentaihoto (JTT) on B16 melanoma cell metastasis in mice. C57BL/6 mice were orally administered JTT, which was started one week before injection of 2 × 10⁵ melanoma cells and killed 3 or 6 weeks later to count tumor cell colonies in the lungs. (a) Number of colonies in the lungs 3 weeks after intravenous injection of cells; (b) number of tumor colonies in the lungs 6 weeks after subcutaneous injection of cells. *P < 0.05 versus control (each group: n = 10, mean ± SEM).

Figure 2

Influence of JTT on tumor cell growth in vitro or in vivo. JTT administration animal exerts cytotoxic effects on B16 melanoma cells and results in the prevention of tumor cell metastasis. (a) Effect of JTT on paw swelling. *P < 0.05 versus control. (b) Effect of the serum principle of JTT administration animals on B16 cultured cells (each group: n = 10, mean ± SEM).

Figure 3

Effect of JTT on angiogenesis (MPCs). The ability of JTT to inhibit in vivo tumor-induced angiogenesis was examined by implantation of the MPCs containing B16 cells (1 × 10⁶ cells/animal) hypodermically intradermally on the shaven dorsum of animals. In the sham group, the vascularization occurred mildly. (a) The blood vessels images which were induced by B16-MPCs. (b) Neogenesis vascular length which was induced by B16-MPCs. *P < 0.05 (each group: n = 10, mean ± SEM).

Figure 4

CD31 is expressed on neogenesis blood vessels. Comparison of CD31 expression patterns (brown) with each experimental groups. “a–c” are high magnification of “A–C”. (A–C) Scale bars, 250 μm. (a–c) Arrows are the staining parts of cd31. Scale bars, 50 μm.

Figure 5

Influence of JTT on VEGF production from B16 cell. The experiment was undertaken to examine the influence of JTT on VEGF production from B16 cells. (a) The VEGF level contents in culture supernatants. (b) The VEGF level contents in the MPCs internal fluid. *P < 0.05 versus control (each sample: n = 10, mean ± SEM).

Figure 6

Effect of JTT on the VEGF mRNA expression. The experiment was carried out to examine whether oral administration of JTT could cause decrease in VEGF mRNA expression of the B16 cells. (a) The VEGF mRNA expression of B16 culture cells. (b) The VEGF mRNA expression of the B16 cells contained in the MPCs. *P < 0.05 versus control (each sample: n = 10, mean ± SEM).