Meg01

Normal PBMC were isolated from healthy donors. HeLa (cervical cancer), Jurkat (acute T cell leukemia), CML-T1, K562, MEG-01, and LAMA-84 cell lines (blast crisis of chronic myeloid leukemia) were purchased from DSMZ (Braunschweig, DE). Cell lines were authenticated routinely and were mycoplasma free. Cells were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA), supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 50 U/mL streptomycin and 50 U/mL penicillin (complete medium, CM), and maintained at 37 °C in 5% CO₂.

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).

The human leukemic cell lines KG1a, HL 60, and Meg 01 were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures), Braunschweig, Germany. The cells were cultured in RPMI-1640 medium (ATCC 30-2001) supplemented with 10% fetal bovine serum (ATCC, cat. number 3020) and 1% antibiotic mix (Sigma A5955) and maintained in a humidified atmosphere with 5% CO₂ at 37°C. The media were changed twice a week. For the cytotoxic assay, the cells were seeded in 24-well plates at a density of 7.5 × 10⁴ cells/mL and treated with various concentrations of Andosan (5.0% and 10.0%), or a matched concentration of PBS as a control, for 96 hrs. The total number and percent viable cells were counted by NucleoCounter using the NucleoCassette kit (ChemoMetec, Allerød, Denmark) according to the manufacturer's manual. For controls and each concentration of Andosan, the mean of five parallel measurements was noted. The results were converted to per cent of the number of viable cells in controls (100%).