Roomi MW, Kalinovsky T, Niedzwiecki A, Rath M
Breakthroughs in Melanoma Research, ed. Y. Tanaka, In Tech Publisher, 2011, Ch 26, pp 559-574
Abstract:
Consumption of a plant-based diet has been associated with prevention of the development and progression of cancer. We have developed strategies to inhibit cancer development and its spread by targeting common mechanisms used by all types of cancer cells that decrease stability and integrity of connective tissue. Our approach to strengthening of collagen and connective tissue compromised in cancer metastasis , tumor growth and angiogenesis has been based on using specific natural components, among them lysine, proline, ascorbic acid and green tea extract (NM). We have documented synergistic effects of this micronutrient mixture and its effectiveness in controlling key mechanisms of cancer in vivo and in vitro in a wide variety of cancer cell lines.
We investigated the in vivo and in vitro effects of NM on growth and metastasis of melanoma, a highly aggressive skin cancer. We studied the effects of NM administered in the diet and by intravenous injections on pulmonary metastasis and the effects of dietary NM on hepatic metastasis of B16FO cells in a murine model. Since proteases, especially MMP-2 and MMP-9, play key roles in tumor cell invasion and metastasis, we also investigated the effects of NM in vitro on melanoma A2058 MMP-2 and -9 secretion (by gelatinase zymography) and invasion through Matrigel.
The effect of NM on xenograft tumor growth of melanoma A2058 cells was studied in male athymic mice. In vitro studies included evaluation of NM effects on melanoma A2058 cell growth (by MTT assay) and induction of apoptosis (by live green caspases).
Pulmonary metastasis was studied in female C57BL/6 mice, which were injected via tail vein with B16FO melanoma cells (5 X 104/mouse), and treated with NM intravenously (IV), intraperitoneally (IP) and/or orally for two weeks. Lungs isolated from mice injected with 5 X 104 B16FO cells and fed NM 0.5% diet had fewer metastatic colonies (by 63%, p<0.0001) than did the lungs of mice fed the control diet. Pulmonary colonization was inhibited by 86% (p<0.0001) in mice receiving NM by IP and IV injections. Hepatic metastasis was studied in athymic nude mice, which received 106 B16FO melanoma cells by injection into the spleen and received either a control diet or an NM (0.5%)-supplemented diet for two weeks. Intrasplenic tumor growth and hepatic metastasis were reduced significantly (by 64%, p=0.001 and 55%,p=0.006, respectively) in the NM-supplemented group compared to the control group.
The effect of dietary NM 0.5% on tumor growth was studied in athymic male nude mice using the model of melanoma xenografts (3x106 cells). NM supplementation demonstrated significant reduction in tumor weight (by 57%, p<0.0001 over control). Histological evaluation noted inhibition of MMP-9 and VEGF secretion and mitotic index in mice fed the NM diet.
In vitro results correlated to the in vivo results with NM. Gelatinase zymography revealed MMP-2 secretion by normal A2058 melanoma cells and induction of MMP-9 secretion with phorbol 13-myristate 12-acetate. NM inhibited both MMP-2 and MMP-9 in a dose-dependent fashion with complete blockage of MMP-9 at 100 μg/mL and MMP-2 at 500 μg/mL. Matrigel invasion of melanoma A2058 cells was inhibited by 100% (p<0.0001) at 100 μg/mL. NM inhibited cell proliferation by 64% (p<0.0001) at 500 μg/mL. Dose-dependent induction of apoptosis in melanoma A2058 cells was confirmed with NM challenge.
Our results suggest that NM is an excellent candidate for therapeutic use in the treatment of melanoma by inhibiting critical steps in cancer progression and metastasis.
Keywords:
melanoma; micronutrient mixture; metastasis, invasive parameters, tumor growth, apoptosis