The soluble form of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (sTRAIL) is a cancer cell-specific molecule exerting anti-tumor activity in a variety of transformed cells. sTRAIL elicits apoptosis by activating the pro-apoptotic receptors 1 (TRAIL-R1) and 2 (TRAIL-R2). However, clinical studies in cancer patients demonstrated that recombinant human sTRAIL showed short serum half-life that does not lead to a subsequent tumor accumulation over time. The aim of this work was to develop a novel liposomal formulation carrying the sTRAIL at the outer surface (sTRAIL-SL) in order to improve its pharmacokinetic features, while preserving its anti-tumor activity and increasing its in vivo efficacy. Neuroectodermal tumor cells (i.e neuroblastoma (NB) and melanoma) can be either sensitive or resistant to sTRAIL-driven death. In this regard, we investigated the possibility to combine our novel sTRAIL liposomal formulation with the proteasome inhibitor Bortezomib (BTZ), known to be able to sensitize tumor cells to sTRAIL-induced apoptosis, by up-regulating TRAIL-R2.
The expression of TRAIL receptors was validated by FACS analysis in a panel of human NB and melanoma cell lines. Receptors expression after BTZ treatment was also evaluated, confirming the BTZ-driven up-regulation of TRAIL-R2 in all the tumor cells tested. No effect on TRAIL-R1 expression was pointed out. Cellular association of sTRAIL-SL was evaluated by FACS analysis on both NB and melanoma cell lines. In vitro cytotoxicity and selective apoptosis induction by sTRAIL-SL were examined in both sTRAIL-sensitive and -resistant NB and melanoma cell lines, by using annexin V/PI assay. Treatments were carried out with sTRAIL, either free or coupled to SL, alone and in combination with BTZ. In sTRAIL-sensitive cells, we observed that sTRAIL-SL treatment resulted in a dose-dependent induction of apoptosis with an efficacy slightly more pronounced than free sTRAIL. Moreover, experiments of combination treatments showed that BTZ effectively sensitized cells to sTRAIL-induced apoptosis. Upon BTZ priming, sTRAIL-resistant cells undergo enhanced apoptotic death by triggering with sTRAIL-SL compared to treatment with free sTRAIL. In preliminary mechanistic experiments sTRAIL-induced cytotoxicity is dependent on β-catenin degradation and Caspase-3 and 8 activation. No effect was exerted on normal fibroblasts. In vivo experiments investigating the pharmacokinetic profile of sTRAIL when coupled to nanocarriers and evaluating its anti-tumor activity against both NB and melanoma animal models are on running. Our data show that sTRAIL, when coupled to nanocarriers preserved and improved its tumor-selective activity on both NB and melanoma, suggesting that it might be an efficient vehicle for sTRAIL delivery and, in combination with BTZ, a valid strategy for the treatment of sTRAIL-resistant tumors.
Citation Format: Monica Loi, Arianna Giacomini, Pamela Becherini, Fabio Pastorino, Mirco Ponzoni. Bortezomib enhanced cytotoxic effects of novel sTRAIL-targeted nanocarriers against neuroectodermal tumors . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5609. doi:10.1158/1538-7445.AM2013-5609