Cell internalization of 7-ketocholesterol-containing nanoemulsion through LDL receptor reduces melanoma growth in vitro and in vivo : a preliminary report

// Giovani M. Favero 1, 2 , Jessica L. Paz 1 , Andreia H. Otake 3, 4 , Durvanei A. Maria 5 , Elia G. Caldini 6 , Raphael S.S. de Medeiros 3, 4 , Debora F. Deus 7 , Roger Chammas 3, 4 , Raul C. Maranhao 7, 8 and Sergio P. Bydlowski 1 1 Laboratory of Genetics and Molecular Hematology (LIM31), Department of Hematology, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil 2 Department of General Biology, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil 3 Centro de Investigacao Translacional em Oncologia (LIM24), Departamento de Radiologia e Oncologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil 4 Instituto do Cancer do Estado de Sao Paulo (ICESP), SP, Brazil 5 Biochemistry and Biophysics Laboratories, Instituto Butantan, Sao Paulo, SP, Brazil 6 Laboratory for Cell Biology, Department of Pathology, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, Brazil 7 Laboratory of Metabolism and Lipids, Heart Institute (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil 8 Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Sao Paulo, SP, Brazil Correspondence to: Sergio P. Bydlowski, email: spbydlow@usp.br Keywords: 7-ketocholesterol; nanoemulsion; melanoma; cell death; LDL receptor Received: October 29, 2017      Accepted: January 25, 2018      Epub: February 04, 2018      Published: March 06, 2018 ABSTRACT Oxysterols are cholesterol oxygenated derivatives which possess several biological actions. Among oxysterols, 7-ketocholesterol (7KC) is known to induce cell death. Here, we hypothesized that 7KC cytotoxicity could be applied in cancer therapeutics. 7KC was incorporated into a lipid core nanoemulsion. As a cellular model the murine melanoma cell line B16F10 was used. The nanoparticle (7KCLDE) uptake into tumor cells was displaced by increasing amounts of low-density-lipoproteins (LDL) suggesting a LDL-receptor-mediated cell internalization. 7KCLDE was mainly cytostatic, which led to an accumulation of polyploid cells. Nevertheless, a single dose of 7KCLDE killed roughly 10% of melanoma cells. In addition, it was observed dissipation of the transmembrane potential, evidenced with flow cytometry; presence of autophagic vacuoles, visualized and quantified with flow cytometry and acridine orange; and presence of myelin figures, observed with ultrastructural microscopy. 7KCLDE impaired cytokenesis was accompanied by changes in cellular morphology into a fibroblastoid shape which is supported by cytoskeletal rearrangements, as shown by the increased actin polymerization. 7KCLDE was injected into B16 melanoma tumor-bearing mice. 7KCLDE accumulated in the liver and tumor. In melanoma tumor 7KCLDE promoted a >50% size reduction, enlarged the necrotic area, and reduced intratumoral vasculature. 7KCLDE increased the survival rates of animals, without hematologic or liver toxicity. Although more pre-clinical studies should be performed, our preliminary results suggested that 7KCLDE is a promising novel preparation for cancer chemotherapy.