Inhibition of the SK-N-MC human neuroblastoma cell line in vivo and in vitro by a novel nutrient mixture.

Neuroblastoma, a peripheral nervous system cancer that can be highly invasive and metastatic, accounts for 8-10% of all solid childhood tumors in children under the age of 15 years. Despite multiple clinical efforts, prognosis remains poor for this enigmatic disease. A nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract has shown significant antitumor effects. Using the nude mouse xenograft model, we investigated the efficacy of NM. We also tested the effect of NM in vitro, evaluating cell viability, secretion of the matrix metalloproteinases (MMP)-2 and MMP-9, tissue inhibitor of metalloproteinase (TIMP)-2 secretion, Matrigel invasion and cellular apoptosis and morphology. Athymic nude mice 5-6 weeks of age were inoculated with 3x106 SK-N-MC neuroblastoma cells 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. NM inhibited the growth of xenograft tumors by 22% (P=0.04); and, in vitro, NM induced dose-dependent inhibition of cell proliferation with a decrease of 27% (P=0.001) and 36% (P=0.002) at 500 and 1000 µg/ml NM compared to the control, respectively. Zymography revealed MMP-2 secretion in normal cells and PMA (100 ng/ml)­induced MMP-9 secretion. NM inhibited the secretion of both MMPs with total blockage at a concentration of 100 µg/ml. Reverse zymography demonstrated a dose-dependent increase in TIMP-2 expression by NM. Notable, SK-N-MC human neuroblastoma cells were not invasive through Matrigel. NM induced dose-dependent apoptosis of SK-N-MC cells. The results suggest that NM may have therapeutic potential in treating neuroblastoma.

Suppression of metastasis of intratesticular inoculation of B16FO melanoma cells by a novel nutrient mixture in male athymic nude mice.

Metastasis, commonly to the lung, is the major cause of mortality from testicular cancer. The aim of the present study was to examine the effect of a novel nutrient mixture (NM) containing ascorbic acid, amino acids and green tea extract on the inhibition of melanoma growth and metastasis using a model of intratesticular inoculation of B16FO cells into nude mice. Male athymic mice (n=12), 10-12 weeks of age, were inoculated with 5×10(5) B16FO melanoma cells in 100 µl of PBS into the right testis, while the left testis was left untreated. Following inoculation, the mice were randomly divided into two groups. The control group (n=6) was fed a regular mouse chow diet and the NM 1% group (n=6) the same diet, but supplemented with 1% NM. Four weeks later the mice were sacrificed and the abdominal cavity was opened. Mice in the control group exhibited extensive metastasis in the peritoneal cavity and severely enlarged right testes and necrotic seminiferous tubules. By contrast, in the NM 1% fed group there was no evidence of peritoneal metastasis in 50% of the animals and mild metastasis in the remaining 50%. The right testes were enlarged and seminiferous tubules in the area of invasion showed evidence of degeneration. No metastasis to the liver, kidney or spleen were evident in either group. However, severe lung metastasis was observed in 2 of 6 mice in the control group and mild metastasis in 2 of 6 mice in the NM 1% group. In conclusion, these results confirm earlier studies and verify the anti-metastatic potential of NM.

Chemopreventive effect of a novel nutrient mixture on lung tumorigenesis induced by urethane in male A/J mice.

AIMS AND BACKGROUND: Lung cancer, a leading cause of cancer death, is associated with exposure to inhalation carcinogens, most commonly those found in tobacco smoke. We investigated the in vivo effect of dietary supplementation with a nutrient mixture containing lysine, proline, arginine, ascorbic acid, green tea extract, N-acetyl cysteine, selenium, copper and manganese on the development of urethane-induced lung tumors in male A/J mice. METHODS: After one week of isolation, seven-week-old male A/J mice (n = 25) weighing 17-19 g were randomly divided into three groups: group A (n = 5), group B (n = 10), and group C (n = 10). Mice in groups B and C were each given a single intraperitoneal injection of urethane (1 mg/g body weight) in saline, whereas group A mice received an injection of saline alone. Groups A and B were fed a regular diet, whereas group C was fed the same diet supplemented with 0.5% nutrient mixture. After 20 weeks, mice were sacrificed, lungs were excised and weighed, and tumors were counted and processed for histology. RESULTS: Urethane-challenged mice developed tumors. However, the mean number of tumors and the mean lung weights in the mice on the supplemented diet were significantly reduced, by 49% (P < 0.0001) and 18% (P = 0.0025), respectively, compared to mice on the control diet. We observed neither significant differences in body weight gains nor in diet consumption among the mice. Pulmonary lesions were morphologically similar for both the groups (adenomas), but lesions were smaller in the test group. CONCLUSIONS: The results suggest that nutrient mixture has inhibitory potential on the development of mouse lung tumors induced by urethane.

Suppression of growth and hepatic metastasis of murine B16FO melanoma cells by a novel nutrient mixture.

Highly metastatic melanoma is resistant to existing therapies. Our main objective was to investigate the effect of a nutrient mixture (NM) on B16FO tumor growth and hepatic metastasis. Tumor growth was studied in athymic nude male mice, 5-6 weeks old, inoculated with 10(6) B16FO melanoma cells subcutaneously and fed either a regular diet or one supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and their tumors excised, weighed and processed for histology. Metastasis was studied in C57BL/6 mice, which received 10(6) B16FO melanoma cells by intrasplenic injection, as well as a regular or 0.5% NM-supplemented diet for 2 weeks. Survival was studied in C57BL/6 mice receiving 10(6) B16FO melanoma cells intraperitoneally (i.p.) followed by the regular, NM-supplemented, or regular diet in addition to being administered with 2 mg NM injection 3 times per week. NM inhibited the growth of B16FO melanoma cells by 50%. Lesions in the two groups were consistent with malignant melanoma. Mice were injected with B16FO cells in the spleen. Those fed the regular diet developed large black spleens and livers indicating growth in the spleen and metastasis to the liver. In contrast, mice supplemented with NM showed less growth in spleen, but also reduced metastasis to the liver. The survival time of mice receiving NM supplementation and B16FO cells i.p. was greater than in mice which were fed the regular diet. To confirm effects in vivo, we investigated the effect of NM on murine B16FO melanoma cells in vitro, including cell proliferation by MTT assay, morphology by hematoxylin and eosin (H&E) staining and apoptosis using live green caspase detection kit. In vitro, NM was not toxic at 100 microg/ml concentration, but exhibited 44% toxicity over the control at 500 and 1000 microg/ml. H&E did not indicate any changes up to 100 microg/ml. NM induced slight apoptosis at 100 microg/ml, moderate at 500 and extensive at 1000 microg/ml concentration.

Modulation of N-methyl-N-nitrosourea induced mammary tumors in Sprague-Dawley rats by combination of lysine, proline, arginine, ascorbic acid and green tea extract.

INTRODUCTION: The limited ability of current treatments to control metastasis and the proposed antitumor properties of specific nutrients prompted us to examine the effect of a specific formulation (nutrient supplement [NS]) of lysine, proline, arginine, ascorbic acid, and green tea extract in vivo on the development of N-methyl-N-nitrosourea (MNU)-induced mammary tumors in rats. METHODS: A single intraperitoneal dose of MNU was injected into each of 20 female Sprague-Dawley rats (aged 50 days) to induce tumors. Two weeks after MNU treatment, a time by which the animals had recovered from MNU-induced toxicity, the rats were divided into two groups. Rats in group 1 (n = 10) were fed Purina chow diet, whereas those in group 2 (n = 10) were fed the same diet supplemented with 0.5% NS. After a further 24 weeks, the rats were killed and tumors were excised and processed. RESULTS: NS reduced the incidence of MNU-induced mammary tumors and the number of tumors by 68.4%, and the tumor burden by 60.5%. The inhibitory effect of NS was also reflected by decreased tumor weight; the tumor weights per rat and per group were decreased by 41% and 78%, respectively. In addition, 30% of the control rats developed ulcerated tumors, in contrast to 10% in the nutrient supplemented rats. CONCLUSION: These findings suggest that the specific formulation of lysine, proline, arginine, ascorbic acid, and green tea extract tested significantly reduces the incidence and growth of MNU-induced mammary tumors, and therefore has strong potential as a useful therapeutic regimen for inhibiting breast cancer development.