In vivo and in vitro effects of a nutrient mixture on breast 4T1 cancer progression

M.W. Roomi, T. Kalinovsky, N.M. Roomi, J. Cha, M. Rath, A. Niedzwiecki
Dr. Rath Research Institute, 1260 Memorex Drive, Santa Clara, CA 95050
International Journal of Oncology 2014, DOI: 10.3892/ijo.2014.2379

Long-term survival of patients with breast cancer remains poor, due to metastasis and recurrence. We investigated the effect of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract in vivo and in vitro on murine 4T1 breast cancer, a representative model for metastatic breast cancer. After one week of isolation, 5-6 week old female Balb/C mice were inoculated with 5x105 4T1 cells into the mammary pad and randomly divided into two groups; the Control group was fed a regular diet and the NM group a regular diet supplemented with 0.5% NM. After four weeks, the mice were sacrificed and their tumors, lungs, livers, kidneys, hearts and spleens were excised and processed for histology.

Dimensions (length and width) of tumors were measured using a digital caliper, and the tumor burden was calculated using the following formula: 0.5 x length x width. We also tested the effect of NM in vitro on 4T1 cells, measuring cell proliferation by MTT assay, MMP secretion by zymography, invasion through Matrigel , migration by scratch test and morphology by H&E staining. NM inhibited tumor weight and burden of 4T1 tumors by 50% (p =0.02) and 53.4% (p=<0.0001), respectively. Lung metastasis was profoundly inhibited by NM supplementation: mean number of colonies was reduced by 87% (p<0.0001) and mean weight of lungs by 60% (p=0.0001) compared to control mice. Metastasis to liver, spleen, kidney and heart was significantly reduced with NM supplementation. In vitro, NM exhibited 50% toxicity over the control at 250 and 500 µg/ml concentrations. Zymography demonstrated MMP-2 and MMP-9 secretion which was inhibited by NM in a dose dependent fashion, with virtual total inhibition of both at 1000 µg/ml. Migration by scratch test and Invasion through Matrigel were inhibited in a dose dependent manner with total block of invasion at 250 and of migration at 1000 µg/ml. These results suggest that NM has therapeutic potential in treatment of breast cancer.

Running title: 
Nutrients inhibit breast cancer progression

Key words: 
breast cancer 4T1 cell line, nutrients, tumor growth, metastasis, MMPs, migration, Matrigel invasion