(P21-003-24) Soy Lunasin and Amaranth Unsaponifiables Loaded in Liposomes Prevented Melanoma Tumor Development by Arresting Cell Cycle, and Upregulating P16, P21, P27, and P53 Inhibitors

Objectives: The objective was to determine the prevention of melanoma development in a mouse model treated with liposomes loaded with soy lunasin and amaranth unsaponifiables as squalene source (UM+LunLip) by assessing markers for cell cycle arrest and cell cycle progression.

Methods: Liposomes UM+LunLip were prepared using the thin film-hydration method with 1,2-dioleoyl-sn-glycerol-3-phospho-rac-(1-glycerol)-sodium-salt:1,2-dioleoyl-sn-glycerol-3-phosphocholine (phospholipids) (1:1 molar ratio), phospholipids:unsaponifiables (1:14 w/w), and lunasin (49%) relative to total lipids. In an in vivo melanoma model, after a 7-day acclimation period of C57BL/6 mice, melanoma B16-F10 cells were transplanted in the rear right flank. Mice were randomized to a control untreated group (n=4) and two treated groups (UM+LunLip, lunasin 30 mg/kg body weight): topically applied (n=8) and subcutaneously applied (n=8). Treatments were started on the same day of melanoma transplantation and applied every other day for 21 days. After tumor excision, tumors were dehydrated, paraffin-embedded, and sliced for immunohistochemistry detection of the markers p16, p21, p27, and p53 for cell cycle inhibition and Ki-67, CDK6 and cyclin D1 for cell cycle progression. GraphPad Prism was used for statistical analysis.

Results: The UM+LunLip had an average size of 121.3 ± 4.8 nm, a squalene encapsulation efficiency (EE) of 59.0 ± 0.1%, lunasin EE of 82.1 ± 3.3, and −79.2 ± 11.2 mV zeta potential. Tumor development was prevented with either subcutaneous (98%, p < 0.0001) or topical (65%, p < 0.01) UM+LunLip application. The overexpression of Ki-67 in both groups vs the control (p < 0.05) suggested cell cycle arrest. CDK6 and cyclin D1 expression were not significantly inhibited, but up to 1.3 and 2.5 fold changes (FC, p > 0.05) respectively, were observed. The markers of cell cycle inhibition p16, p21, p27, and p53 showed an over-expression (up to 442 FC, 119 FC, 46 FC, and 76 FC, respectively, p < 0.01) in treated mice, particularly with subcutaneous application, compared to tumor-bearing untreated control.

Conclusions: The observed results support the hypothesis that UM+LunLip induced melanoma cell cycle arrest by upregulating cell cycle inhibitors.

Funding Sources: USDA∖Pulse Crop Health Initiative’s funding 58-3060-9-045