Liposomes Loaded With Soybean Lunasin and Amaranth Unsaponifiable Matter Promoted Apoptosis Through Caspase 3 and Cell Proliferation Arrest in an In Vivo Melanoma Model

OBJECTIVES: The objective was to assess the mechanism of action of liposomes loaded with amaranth unsaponifiable matter and lunasin (LpLnUM) in melanoma tumors developed in C57BL/6 mice. METHODS: Tumors were developed in C57BL/6 using the melanoma cell line B16-F10 and treated every other day for 22 days using LpLnUM with 15 (LpLnUM15) or 30 (LpLnUM30) mg lunasin/kg body weight (BW) via either topically (T) or subcutaneously (S) (treated groups, TG), or lunasin solution at 30 mg/kg BW. Tumors were excised, fixed, and embedded in paraffin, cut with a microtome (5 μm thick), and fixed in microscope slides. For immunohistochemistry, glycogen synthase kinase (GSK)-3β, caspase 3 (C3), and Ki-67 antibodies were used, with horseradish peroxidase and 3,3’-diaminobenzidine as a substrate to reveal protein expression. Hematoxylin and eosin assay assessed tissue structure. Images were captured with a NanoZoomer and analyzed at a 20x magnification. RESULTS: Compared with the tumor-untreated control (UC), T and S applied liposomes inhibited the tumor in 61 and 71%, respectively. No difference between lunasin concentrations (p < 0.05). The smaller the tumor volume, the higher C3 expression (r = −0.819, p < 0.05). Tumors showed a C3 overexpression in those groups S treated with LpLnUM15 (1.41-fold, p < 0.01) and LpLnUM30 (1.53-fold, p < 0.01) compared with the UC. In contrast, lunasin solution (without liposomes) T applied at 30 mg/kg BW had no difference with the UC, but it was different (p < 0.05) when compared with the groups treated with only S applied liposomes. GSK-3β suggested that even though the expression decreased in the TG there was no difference among controls and treatments. The proliferation marker Ki-67 showed an under expression of the marker between the TG (p < 0.001) and the UC. The Ki-67 expression in mice T treated with LpLnUM30 and LpLnUM15 was inhibited 89 and 76%, respectively. In those mice with S application, the Ki-67 inhibition was 81 and 93% when treated with LpLnUM30 and LpLnUM15, respectively. These last results suggest an arrest in the cell cycle. CONCLUSIONS: LpLnUM15 and LpLnUM30 prevented melanoma tumor development by promoting cell apoptosis via C3 expression and cell cycle arrest regardless of lunasin concentration and application method in mice. FUNDING SOURCES: USDA National Institute of Food and Agriculture, CONACyT fellowship.