Survival was studied in C57BL/6 mice receiving 106 B16FO melanoma cells IP followed by regular diet, NM-supplemented diet, or regular diet in addition to NM 2mg injection 3x/week. NM inhibited the growth of B16FO melanoma cells by 50%. Lesions in both groups were consistent with malignant melanoma. Of mice injected with B16FO cells into the spleen, those provided with 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. Survival time of mice that received NM supplementation and B16FO cells IP was greater than in mice provided the regular diet. To confirm effects in vivo, we also investigated the effect of NM on murine B16FO melanoma cells in vitro, including cell proliferation by MTT assay, morphology by H&E staining and apoptosis using live green caspase detection kit. In vitro, NM was not toxic at 100 µg/ml concentration, but exhibited 44% toxicity over the control at 500 and 1000 µg/ml. H&E did not indicate any changes up to 100 µg/ml. NM induced slight apoptosis at 100 µg/ml, moderate at 500 and extensive at 1000 µg/ml concentration.
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