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

Abstract

ObjectivesThe 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. MethodsTumors 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. ResultsCompared 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. ConclusionsLpLnUM15 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 SourcesUSDA National Institute of Food and Agriculture, CONACyT fellowship.