Etoposide vp 16

A549 and NCI-H1650 (H1650) cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/mL streptomycin, and 100 units/mL penicillin. Cells were grown to 80% confluence and then serum-deprived for 12 h prior to etoposide (VP16) (Sigma–Aldrich, St Louis, MO, USA) stimulation. Stable H2AX knockdown A549 cells and stable control cells were passaged in our laboratory as described before [4 (link)].

Cell viability was recorded applying CCK-8 indirect viability assay (CCK-8, Dojindo Laboratories, Kumamoto, Japan). The kit was used according to the producer's manual and the absorbance was measured at 450 nm wavelength in a microplate reader (Spectramax M2, Molecular devices, San José, USA). A treatment with 5 µM and 10 µM of Etoposide (VP16, Sigma-Aldrich) or 5 µM and 10 µM of Cisplatin (Cispl, Sigma-Aldrich) was done 3 days after seeding of neuroblastoma cells for a total of 96 hours. The results were compared with conventional 2D culture system. As controls (CTRL), the cells treated with vehicle (DMSO) were considered.

For the initial screen, a smart chemical library was obtained from Prestwick Chemicals (http://www.prestwickchemical.com/prestwick-chemical-library.html). The library consisted of 1280 small molecules and natural compounds approved by the FDA, EMA and other agencies. These compounds were chemically and pharmacologically diverse with known efficacy and bio-safety in humans. To confirm the effect of β-escin observed in the screen, subsequent studies utilised β-escin from an alternate source (Sigma Aldrich, E-1378). The pan caspase OPH inhibitor, QVD was used to inhibit apoptosis (R&D systems). To compare Escin to current chemotherapeutic agents, Temozolomide (TMZ, 50–100 μM, Sigma-Aldrich), Etoposide VP16 (20 μM, Sigma-Aldrich) and Cisplatin (10 μM, Tocris) were used.

The bulk of the data has been generated with the SCLC cell line NCI-H69, with additional data from the colorectal cancer cell lines SW-480 and SW-620, and breast cancer cell line MDA-231 which also possess mutated p53 gene. The cells were grown in 12 well plates (Scientific Laboratory Supplies Limited, UK) in RPMI 1640 (Gibco, UK) supplemented with 10% FBS (Autogen Bioclear, UK) and 10,000 U/ml penicillin and 10 mg/ml streptomycin (Sigma, UK) at 37 °C under 5% CO₂ and treated with 10, 20, 50, 100 and 150 µM of DNA damaging agent Etoposide (VP-16) (Sigma, UK) in the presence or absence of p38MAPK inhibitor SB203580 (Tocris Chemical Co., UK) or MK2 inhibitor MK2.III (Merck, UK) in concentrations of 0, 0.3125, 0.625, 1.25, 2.5, and 5.0 µM. Etoposide, SB203580 and MK2.III were all dissolved in 0.2% of DMSO before use. Cells treated only with the carrier molecule DMSO (0.2%) served as a control.

Human acute leukemia cell lines Reh (non-B non-T ALL) [44 (link)], Sup-B15 (B-ALL) [45 (link)], Jurkat (T-ALL) [46 (link)], and Kasumi-1 (AML, FAB M2) [47 (link)] were obtained from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Bone marrow was collected from healthy adult donors after they had provided informed consent, and the hBM-MSCs obtained were cultured in DMEM-low glucose (Gibco/Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), identified as described in our previous report [48 (link)]. Bone marrow mononuclear cells, of which more than 90% were malignant cells, from primary and refractory/relapsed acute leukemia (non-M3 AML and ALL) patients were purified by Ficoll-Paque isodensity gradient centrifugation (Tianjing, China). Idarubicin (IDA) and the Wnt signaling-specific inhibitor ICG-001 were obtained from Pfizer Inc., (New York, NY, USA) and from Selleck Chemicals, (Houston, TX, USA), respectively, and dissolved in phosphate-buffered saline (PBS) and dimethylsulfoxide (DMSO). Etoposide (VP-16) and the PI3K/Akt signaling inhibitor Ly294002 were purchased from Sigma-Aldrich (St Louis, MO, USA), and dissolved in DMSO. All of them were stored at −20°C.