Recombinant human trail

Multilamellar liposomes were prepared using the thin film method as described previously [21 (link)]. Respective volumes were taken from the stock lipid solutions and gently mixed in a glass test tube and left under vacuum overnight. The lipid pellet was hydrated using 700 μL of liposome buffer (20 mM HEPES, 150 mM NaCl, pH 7.5). The multilamellar liposomes were generated using 10 cycles of freeze (2 min) and thaw (3 min). To prepare unilamellar liposomes, the liposomes were subjected to 10 extrusion cycles at 55 °C using two different size (200 nm and 100 nm) of polycarbonate membranes. For this study, we prepared two liposomal-treatment groups: control (liposomes that do not contain proteins on their surface) and TRAIL-liposomal therapy (E-selectin and TRAIL conjugated liposomes). Recombinant human E-selectin (R&D Systems) and recombinant human TRAIL (Enzo) were reconstituted to a final concentration of 500 μg/mL and 1 mg/mL and then stored at -80 °C. Freshly prepared liposomes were incubated with E-selectin and TRAIL to a final concentration of 71.43 nM and 250 nM for 15 min at 37 °C and then overnight at 4 °C on a rotator to prepare TRAIL-liposomal therapy [21 (link)]. The liposomes were characterized using a DLS-Malvern Zetasizer and a NanoSight NS300. This TRAIL-liposomal formulation has been previously used, characterized and validated in a previous study [21 (link)].

SUM149 and SUM190 cells (obtained from Asterand, Inc., Detroit, MI, USA) were cultured as previously described.^{16 (link), 59 (link)} Asterand characterizes cell lines using short-tandem repeat polymorphism analysis. Cells were banked upon receipt and cultured for no more than 6 months before use in this study. rSUM149 and rSUM190 are isogenic, acquired resistance cell lines established in the laboratory.^{16 (link)}Transient cell transfections were performed using the Mirus TransIT 2020 transfection reagent (Mirus Bio, Madison, WI, USA) according to the manufacturer's instructions. rSUM149 and rSUM190 cells were transfected with a plasmid containing XIAP-targeting short hairpin RNA and 48 h post transfection, viable cells were used in the ADCC assay. Effective knockdown was confirmed by western immunoblot analysis.
For caspase activity and cell viability, cells were treated with indicated doses of recombinant human TRAIL (Enzo Life Sciences, Farmingdale, NY, USA) for 24 h. Cell viability was determined by trypan blue exclusion as previously described.^{59 (link)} For proliferation measurement, cells were seeded in a 96-well plate and treated with indicated doses of antibodies for 72 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich, St. Louis, MO, USA) assay reagent was added and cellular proliferation measured as previously described.^{60 (link), 61 (link)}

RAJI (Burkitt's lymphoma), U-937 (non-Hodgkin lymphoma), JURKAT (ALL), K-562 (chronic myeloid leukemia, CML), HL-60 (promyelocytic leukemia) MEG-01 (CML in megakaryocytic blast crisis), PC-3 (prostate cancer) cells were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). SH-SY5Y (neuroblastoma) and MDA-MB-231 (breast adenocarcinoma) were obtained from the American tissue culture collection (Manassas, VA, USA). All cell lines were cultured in RPMI 1640 medium (Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal calf serum (Lonza) and 1% antibiotic–antimycotic (Lonza). Peripheral blood mononuclear cells (PBMCs) from human healthy donors were isolated and cultured as previously described [19 (link)]. All experiments were performed on cells in the exponential growth phase. Iso-3 was purified from Aplysina aerophoba [17 (link)] and dissolved in DMSO. Epigallocatechin gallate (EGCG), DAC, Necrostatin-1, VP-16, PP242 and bafilomycin A₁ were purchased from Sigma-Aldrich (Bornem, Belgium). SAHA and Z-VAD-FKM were purchased from Cayman Bio-connect (Huissen, The Netherlands) and from Millipore (Merck, Brussels, Belgium), respectively. All drugs were dissolved in DMSO. Recombinant human TRAIL was purchased by Enzo Life Science (Antwerpen, Belgium).