Hl 60

HL-60 (acute promyelocytic leukemia cell line), THP-1 (acute monocytic leukemia cell line), OCI-AML2 (acute myelomonocytic leukemia cell line) and OCI-AML3 (acute myelomonocytic leukemia cell line) cell lines were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences. KASUMI-1 (acute myeloblastic leukemia cell line) cell line was gifted by Professor Chen Saijuan (Shanghai Institute of Hematology, Shanghai, China). These cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37 °C in a humidified incubator containing 5% CO2. MV4-11(acute myelomonocytic leukemia cell line) and MOLM-13(acute monocytic leukemia cell line) cell lines were a kind gift from Professor Ravi Bhatia (City of Hope National Medical Center, Duarte, CA, USA). These two cell lines were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% (FBS). Diagnostic AML patient samples were purified by standard Ficoll‐Hypaque (Sigma-Aldrich) density centrifugation, then cultured in RPMI 1640 with 10% FBS.

HL-60, K-562, MOLT-4, ACHN, OS-RC-2 and 786-O cells were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. The ACHN cells were grown and maintained in an MEM medium with 10% FBS, while the other cells were grown in a RPMI1640 medium with 10% FBS. The cell viability was determined using a CCK-8 (Dojindo) assay [23 (link)]. The cells were seeded at a density of 400 to 800 cells/well in 384-well plates, and were then treated with various concentrations (50, 10, 2, 0.4 and 0.08 μM) of compounds or a solvent control. After 72 h of incubation, the CCK-8 reagent was added, and the absorbance of the triplicate tests was measured at 450 nm using an Envision 2104 multi-label reader (Perkin Elmer). The dose–response curves were plotted in order to determine the IC₅₀ using Prism 5.0 (GraphPad Software Inc.).

Compounds 3−16 were tested for their cytotoxicity against several cancer cell lines, H1975 (human lung adenocarcinoma), U937 (human lymphocytic leukemia), K562 (human chronic myelogenous leukemia), BGC823 (human gastric adenocarcinoma), MOLT-4 (human acute T lymphoblastic leukemia), MCF-7 (human breast carcinoma), A549 (human lung adenocarcinoma), Hela (human cervical carcinoma), HL60 (human promyelocytic leukemia), and Huh-7 (human hepatocarcinoma), according to the reported CCK-8 method [23 (link)], which were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. In brief, these cell lines were incubated in in RPMI or DMEM media with 10% FBS and 1% penicillin/streptomycin under a Thermo/Forma Scientific CO₂ Water Jacketed Incubator with 5% CO₂ in air at 37 °C. Then they were treated with various concentration of compounds or control at a density of 400–800 cells/well in 384-well plates. Cell viability assay was determined by CCK-8 assay. After 72 h incubation, CCK-8 reagent [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)] (Dojindo, Japan) was added, and absorbance was measured at 450 nm using Envision 2104 multi-label Reader (Perkin Elmer, Waltham, MA, USA). Dose response curves were plotted to determine the IC₅₀ values using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA).