Abstract A50: 7-Ketocholesterol loaded-phosphatidylserine liposome induces cell death, autophagy, and growth inhibition of melanoma and breast adenocarcinoma

• Published in: Clinical cancer research 2018-01, Vol.24 (1_Supplement), p.A50-A50
• Main Authors: Favero, Giovani Marino, Tortelli, Tharcisio Citrangulo, Fernandes, Daniel, Prestes, Ana Paula, Kmetiuk, Louise N.B., Otake, Andreia Hanada, Andrade, Luciana N.S., Faria, Daniele de Paula, Carneiro, Camila de Godoi, Garcez, Alexandre Teles, Marques, Fabio L.N., Chammas, Roger
• Format: Article
• Language: English
• ISSN: 1078-0432; 1557-3265
• DOI: 10.1158/1557-3265.TCM17-A50

Background: The exposure of phosphatidylserine (PS) is one of the first steps of programmed cell death. Phagocytosis on cancer microenvironment is well described in tumors and is associated with malignancy and poor prognosis. Tumor associated macrophages (TAMs) act suppressing the anticancer immune response. The tumor parenchymal cells are also capable of phagocytosis cells in apoptosis. In a previous study we observed that 7-ketocholesterol is capable of inducing autophagy on melanoma cell. Aims: Evaluate the activities of a 7-ketocholesterol loaded-phosphatidylserine liposome on autophagy and phagocytosis of tumor microenvironment. Methods: Liposomes were constituted by 20 mg commercial Phosphatidylserine (PS) and PS associated with 5 mg of 7-ketocholesterol extracted with chloroform/methanol (10: 1), dried, resuspended in 10 mL phosphate buffer, homogenized and sonicated for 6 minutes. The size and Zeta Pontencial (ZP) of liposomes were evaluated. Antiinflammatory activity of liposomes was evaluated by paw edema induced by carrageenan. A dependent-dose effect of liposomes on J774 macrophages, B16F10 melanoma cells, and 4T1 breast cancer cells was assessed by MTT. Cell death evaluations, for the same cells, were performed by flow cytometry with propidium iodide (PI) staining. The presence of acid vacuoles related to autophagy was evaluated by flow cytomery by acridine orange staining. The effects of the liposomes in vivo were evaluated by B16F10 melanoma-bearing C57/bl6 mice and 4T1 breast cancer-bearing Balb c mice. Endocytosis efficiency of the liposomes was observed by labeling it with PKH26 fluorescent staining and evaluated in 4T1 cells after 12 h. Liposomes were radiolabeled by adding 1 to 30 mCi of 99mTc radiopharmaceuticals (99mTcO4-, 99mTc-dextran-70, 99mTc-MIBI, 99mTc-DISIDA) and 18FDG; the solution was homogenized and sonicated for 6 minutes. The samples were centrifuged and part of the supernatant was added to an Amicon® filter (10kD) and concentrated, the concentrated was diluted with 400 uL of PBS and concentrated again. Liposome incorporation was determined by quotient of the radioactivity in the Amicon® by sum of Amicon® and filtrated solutions. Furthermore, lipophilicity (L), hydrophilicity (H), and charge (-/0/+) of the radioactive material were considered in the final analysis. Results: PS liposomes presented 141,9nm + 9,101 size with a -25,2 ZP; PS-7-ketocholesterol (PS/7KC) liposomes presented 153,9 nm + 10,35 size with a -29,1 ZP. T