Effects of a nutrient mixture on immunohistochemical localization of cancer markers in human cervical cancer HeLa cell tumor xenografts in female nude mice

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M.W. Roomi, T. Kalinovsky, J. Cha, N.M. Roomi,  A. Niedzwiecki and M. Rath
Dr. Rath Research Institute, 1260 Memorex Drive, Santa Clara, CA 95050
Experimental & Therapeutic Medicine 2014, DOI: 10.3892/etm.2014.2127

Although fully treatable in the early stages, once cervical cancer has metastasized, patient outcome is poor. The main objective of this study was to examine the effect of dietary supplementation with a nutrient mixture (NM) containing lysine, ascorbic acid, proline, green tea extract and other micronutrients on HeLa cell xenografts in nude female mice. Tumor growth was measured and immunohistochemical staining was evaluated for the following cancer markers: Ki67 (proliferation); matrix metalloproteinase (MMP) 2 and  9 (invasion/metastasis); vascular endothelial growth factor (VEGF) (angiogenesis); terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and B-cell lymphoma 2 (Bcl-2) (apoptosis); cyclooxygenase 2 (COX 2) and inducible nitric oxide synthase (iNOS) (inflammation); and glutathione S-transferase π (GSTπ) (a general cancer marker).

Following housing for a week, 5/6 week old female athymic nude mice (n=12) were inoculated subcutaneously with 3x106 HeLa cells in 0.2 ml phosphate buffered saline and 0.1 ml Matrigel™ and randomly divided into two groups; control group mice were fed regular mouse chow and NM group mice the regular diet supplemented with 0.5% NM (w/w). After four weeks, the mice were sacrificed and their tumors were excised and processed for histology. The NM strongly inhibited the growth of HeLa xenografts in nude mice. The mean tumor weight was reduced to 59% (P=0.001) in the mice fed the NM compared with the tumor weight in the controlled diet mice. Ki67, MMP 2 and  9, VEGF, TUNEL, Bcl 2, COX 2, iNOS and GSTπ all showed a lower intensity and frequency of staining in the NM group compared with that in the control group. In conclusion, NM supplementation strongly inhibited tumor growth and cancer markers in female nude mice injected with HeLa xenografts.

Running title: 
Nutrients suppress cervical cancer tumor growth and markers

Key words: 
HeLa, nutrient mixture, tumor growth, immunohistochemistry, Ki67, matrix metalloproteinase-2, matrix metalloproteinase-9, vascular endothelial growth factor, terminal deoxynucleotidyl transferase dUTP nick end labeling, B-cell lymphoma-2, cyclooxygenase 2, inducible nitric oxide synthase, glutathione S-transferase π