After treatment, the cells were washed with PBS (phosphate buffered saline) and fixed by the addition of 3?ml of ice cold 70% ethanol. and 8, cytochrome c, p53, p21, p27 and Bax. There was also a decrease in the expression of Bcl-2, cyclin D1, CD90 and CD44?proteins. Conclusion The overall results showed that DODAC/PHO-S liposomes Carzenide were more effective than PHO-S alone, in promoting cytotoxicity Hepa1c1c7 tumor cells, activating the intrinsic and extrinsic pathways of programmed cell death. Keywords: Liposomes, Nanomedicine, Hepatocellular carcinoma, Antitumoral alkylphospholipids Background Despite great advances in the research and the development of new therapeutic strategies, cancer remains one of the leading causes of death worldwide. In 2014, there were an estimated 1,665,540 new cancer cases diagnosed and 585,720 deaths were expected in the United States of America in 2014, and it is expected to increase to over 24 million by 2035 [1, 2]. With regards to the limiting factors of the therapies currently available for the treatment of malignancy, new treatments that are more effective and less harmful are necessary. Therefore, antineoplastic phospholipids (AFTs) and lipid precursors have emerged as a promising new classes of antitumor brokers that do not target the DNA, however they change the plasma membrane Carzenide turnover, inducing cell death, with a high selectivity for cancer cell [3, 4]. Edelfosine, miltefosine, perifosine, erucylphosphocholine and erufosine, represent this new class of AFTs, structurally related antitumor brokers [5C7]. Synthetic phosphoethanolamine (PHO-S), an lipid precursor, amino-ethyl phosphoric ester, has been previously synthesized by our group [8C13]. We exhibited that the treatment of B16F10 cells with PHO-S was able to inhibit cell proliferation and induce G2/M cell cycle arrest [13]. In another study, PHO-S caused anti-proliferative effects on HUVEC, by reducing cyclin D1 mRNA, VEGFR1 gene transcription and VEGFR1 receptor expression [10, 12]. In vitro studies exhibited that PHO-S induced cytotoxicity and apoptosis via mitochondrial pathways, in leukemia cells. The results showed that PHO-S was able to provide antiproliferative effects on acute promyelocytic leukemia (APL) cell lines. PHO-S exhibited its antiproliferative effect on APL cell lines, decreasing CD177+ and Gr-17+ in immature myeloid cells in bone marrow, spleen and liver [11]. Additionally, the PHO-S has exerted anti-tumor activities in several tumor cell lines, such B16F10 cells; Skmel-28 and Mewo cells (human melanoma); MCF-7 cells (human breast malignancy) and ehrlich ascites tumor [8C10, 12, 13]. Recently, PHO-S was encapsulated in DODAC (Dioctadecyldimethylammonium Chloride) liposomes by our group and the TNFRSF4 liposomes were physico-chemically characterised [14, 15]. In vitro studies demonstrated the efficacy of DODAC/PHO-S liposomes in inducing cytotoxicity in B16F10 murine melanoma and Hepa1c1c7 murine hepatocellular carcinoma cells, with IC50% values significantly lower than PHO-S treatment. It was observed that Hepa1c1c7 cells display greater sensitivity to the DODAC/PHO-S formulation when compared with B16F10 and HUVEC cells. However, the molecular mechanism responsible for the anti-tumor properties of DODAC/PHO-S has not been exhibited [14, 15]. Consequently, our aim was to clarify the mechanism of cell death where DODAC/PHO-S liposomal formulation induces cytotoxicity in hepatocellular carcinoma Hepa1c1c17. Methods Liposomal formulation DODAC/PHO-S Liposomal formulation DODAC/PHO-S were formulated (1:1) in water, in accordance with procedures previously published [8C10, 12, 13]. After sonication, the liposomes were sterilized by filtration. Cell culture Hepa1c1c7 murine hepatocellular carcinoma (ATCC? CRL 2026) was cultured in MEM medium (LGC Biotecnologia, Cotia, SP, Brazil) and supplemented with 10% fetal bovine serum in a humidified incubator at 37?C and 5% CO2. The cell viability was verified using Trypan Blue exclusion Carzenide test. Hepa1c1c7 was chosen for the study because of its easy reproduction in vivo studies. Cell cycle phases distribution The Hepa1c1c7 cells at a cell density of 1 1??105 cells/well (80 to 90% confluence) were treated with PHO-S (0.3C2.0?mM), DODAC/PHO-S 1:1 (0.3C2.0?mM), and vacant DODAC (0.3C2.0?mM), for 12?h. After treatment, the cells were washed with PBS (phosphate buffered saline) and fixed by the addition of 3?ml of ice cold 70% ethanol. The cells.