M.W. Roomi, V. Ivanov, A. Niedzwiecki and M. Rath
Dr. Rath Research Institute, Oncology Division, 1260 Memorex Drive, Santa Clara, CA 95050

Presented at: 
46th Annual Meeting of The American Society for Cell Biology, San Diego December 9-13, 2006.

Published in: 
46th Annual Meeting of The American Society for Cell Biology, San Diego, December 9-13, 2006 Proceedings, Abstract # 1280.

Abstract

Introduction:
A novel nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract has exhibited anti-tumor activity in vivo and vitro. In this study we examined the effect of NM on melanogenesis in vivo and in vitro using B16-F0 melanoma cell line. In advanced stages, highly metastatic melanoma is resistant to existing therapies.

Objective:
We investigated the effect of NM on murine B16FO melanoma cells in vitro evaluating viability, MMP secretion, invasion, morphology and apoptosis. In vivo studies were carried out in athymic nude mice bearing B16-F0 xenografts.

Methods:
Athymic nude male mice, 5-6 weeks old, were inoculated with 1x106 B16-FO melanoma cells (ATCC) subcutaneously and randomly divided into two groups; group A was fed a regular diet and group B a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and their tumors were excised, weighed and processed for histology. We also tested the effect of NM in vitro, measuring cell proliferation by MTT assay, invasion through Matrigel, secretion of MMPs by gelatinase zymography, cell morphology by H&E staining and apoptosis using live green caspase detection kit (Molecular Probes).

Results:
NM inhibited the growth of B16-FO melanoma cells in vivo by 50%. Lesions, both in control and test groups were composed of cords and nests of large, irregularly round, pigmented cells consistent with a malignant melanoma. In vitro, NM was not toxic to the melanoma cells at 100 mg/ml concentration, but exhibited 50% toxicity over the control at 500 and 1000 mg/ml. H&E did not indicate any morphological changes up to 100 mg/ml. B16-F0 melanoma cells demonstrated no MMP secretion nor invasion through Matrigel. NM induced slight apoptosis at 100 mg/ml, moderate at 500 and extensive at 1000 mg/ml concentration.

Conclusion:
Taken together these results suggest that NM has many attractive features as a new anti-tumor agent.

Comment

Melanoma is a very serious and highly metastatic form of skin cancer, which causes the most skin cancer-related deaths. In its advanced stages melanoma is resistant to existing therapies. We investigated the effect of a unique nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract on murine B16FO melanoma cells in vitro and also in vivo by injecting these melanoma cells under the skin of nude mice. After 4 weeks of supplementation with NM growth of melanoma tumors in mice was inhibited by 50%. Melanoma cells exposed to 500 and 1000 mg/ml NM concentration in vitro, exhibited 50% toxicity over the control. At these concentrations a moderate and extensive apoptosis (natural cell death) was observed respectively. These results are significant as they suggest NM as a therapeutic agent for melanoma.