Abstract 2194: Synergistic cytotoxicity of targeted liposomes containing doxorubicin and C6-ceramide against nucleolin-overexpressing ovarian cancer cells is supported by the downregulation of the Akt pathway

The acknowledgment that cancer stem cells (CSC) may originate from non-stem cancer cells, interconverting through an EMT-mediated process has turned these distinct cell subpopulations into two relevant therapeutic targets. Moreover, the PI3K/AKT/mTOR pathway is essential to CSC proliferation and survival and it is often over activated in ovarian cancer. One of the strategies to target these different tumor cell populations, relies on the combination of conventional chemotherapeutic drugs (as tumor debulking agents, targeting non-stem cancer cells) with sphingolipids targeting CSC (at the level of PI3K/Akt/mTOR). Nucleolin overexpression has been demonstrated on the surface of both breast CSC and non-stem breast cancer cells (Fonseca NA, Biomaterials 2015) and endothelial cells from tumor blood vessels. The pH-sensitive lipid-based nanoparticle, functionalized with the nucleolin-binding F3 peptide, was recently modified to encapsulate a synergistic combination of a sphingolipid (C6-ceramide) and doxorubicin (DXR). Following the promising results in breast cancer, the aim of this work was to assess the therapeutic potential of this strategy against nucleolin-overexpressing ovarian cancer cell lines, as well as the underlying mechanism of action at the molecular level. A 12.9-fold increase of association of fluorescently-labelled F3 peptide-targeted liposomes ([F3]L), relative to the non-targeted counterparts, into (bulk) ovarian cancer cell lines (SKOV-3, OVCAR-3 and TOV-112D) was observed by flow cytometry. Importantly, a similar pattern of association by putative CSC enriched population (CD44high/EpCAMhigh) was also observed. These results corroborated the marked increase of cytotoxicity (assessed by the resazurin reduction viability assay) enabled by the targeted drug combinations, relative to [F3]L containing only DXR. In fact, the targeted drug combination enabled a 90% death of SKOV-3 cancer cells, following an 8 h incubation, a level of cell killing not achieved by the counterpart containing only DXR. Against the OVCAR-3 and TOV-112D cell lines, the IC90 of the targeted drug combination, following 1 h of incubation, was 2-fold lower than the one resulting from the incubation with [F3]L containing only DXR. In addition, the effect of nucleolin-mediated intracellular delivery of C6-ceramide at the p-Akt protein levels was further discriminated by western blot analysis in SKOV-3 cells. The observed 3.2-fold downregulation of p-Akt levels supported the C6