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

Presented at: 
46th Annual Meeting of the Society of Toxicology; Charlotte, NC; March 25-29, 2007

Published in: 
The Toxicologists (suppl Toxicological Sciences), Abstract #610, pg 126, 2007.

Abstract

Introduction: 
A novel nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract has exhibited anti-tumor activity in vivo and in vitro. In this study we investigated the effect of NM on HepG2, human hepatoma cell line.

Objective: 
NM has been shown to possess a wide range of pharmacological properties and is one of the most promising chemo preventive agents for cancer. To provide a better understanding of the preventive effect of NM on liver cancer, we examined NM for its effect on proliferation, MMP secretion, invasion, morphology, and apoptosis.

Methods: 
HepG2 cells (ATCC) were cultured in MEM medium supplemented with 10% FBS and antibiotics. The cells were then challenged with NM at 0, 10, 50, 100, 500 and 1000 µg/ml in triplicate at each concentration. Cell proliferation was assessed by MTT assay, MMP activity by gelatinase zymography, invasion through Matrigel, morphology by H&E staining, and apoptosis using live green apoptosis detection kit. Cells were also treated with PMA for MMP-9 induction.

Results:
NM was slightly toxic at 100-µg/ml concentration and significant at 500 and 1000 µg/ml concentrations. NM showed a dose dependent inhibition of MMP-9 in PMA-induced cells, with virtual total inhibition at 500-µg/ml concentration. Invasion through Matrigel was inhibited by 30%, 80% and 100% at 50, 100 and 500 µg/ml. H&E staining showed no morphological changes below 500 µg/ml. HepG2 cells demonstrated moderate apoptosis at 100 µg/ml NM and profound at 500 and 1000 µg/ml NM.

Conclusions: 
These results suggest that NM has therapeutic potential as a new anti-tumor agent.

Comment

To provide a better understanding of the preventive effect of NM (a unique nutrient mixture containing lysine, proline, ascorbic acid and green tea extract) on liver cancer, we examined NM on HepG2 cell proliferation, MMP secretion, invasion, morphology, and apoptosis. NM showed dose-dependent inhibition of MMP-2, with virtual total inhibition at 500 µg/ml and dose-dependent inhibition of Matrigel invasion and migration of human hepatocellular carcinoma HepG2 with 97% inhibition at 500 µg/ml and total blockage at 1000 µg/ml. HepG2 cells showed no morphological changes below 500 µg/ml and obvious apoptosis at higher concentrations. Moderate apoptosis was observed at 100 µg/ml NM and profound at 500 and 1000 µg/ml NM. These results are significant since they suggest NM has therapeutic potential in liver cancer by inducing apoptosis and inhibiting invasive parameters.