Louis, MO, USA). in China. EM possesses multiple pharmacological activities, including anticancer activity (Tabopda et al., 2008; Ooi et al., 2011, 2012), anti-protozoal activity (Gachet et al., 2010), melanogenesis inhibition activity (Hasegawa et al., 2010), bone regenerative activity (Ngueguim et al., 2012), and hepatoprotective activity (Lin et al., 1995). However, whether EM is an efficacious treatment option for human AML and CML remains unknown. In this study, we investigated the anti-leukemia properties and associated molecular mechanisms of EM23, a natural sesquiterpene lactone isolated from EM, in the K562 and HL-60 human CML and AML cell lines. Mechanistically, we demonstrated that EM23 inhibited the mammalian Trx system, interfered with cellular redox homeostasis and resulted in ROS-dependent apoptosis by regulating complex signaling pathways, including those governed by ASK1, MAPK, and NF-B. Materials and Methods Cell Culture and Reagents The Gpr68 human CML cell line K562, the human APL cell line HL-60, human liver cell line HL-7702 and mouse embryonic fibroblast cell line NIH/3T3 (NIH/swiss) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). K562 and HL-60 cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY, USA). HL-7702 and NIH/3T3 cells were grown in DMEM medium (Life Technologies, Grand Island, NY, USA). All cell lines were grown in specific media supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 g/mL streptomycin (Invitrogen, Carlsbad, CA, USA). The cells were grown in a 5% CO2 humidified atmosphere in incubators maintained at 37C. EM23 (Figure ?Figure1A1A) was isolated and purified from EM by our group. The chemical structure of EM23 was identified by 1H-NMR and 13C-NMR spectra data as described in our previous study (Liang et al., 2012). A stock solution of EM23 was dissolved in DMSO at concentration of 100 mM and diluted to the indicated final concentration in culture medium. DMSO was diluted to 0.1% in medium as a vehicle control. Open in a separate window FIGURE 1 EM23 inhibits cell proliferation and cell cycle progression. (A) Chemical structure of EM23. (B) Effects of EM23 on human myeloid leukemia cell proliferation. K562 and HL-60 cells were treated with various concentrations of EM23 for 48 and 72 h, respectively. Cell viability was measured using a CCK-8 assay. (C) Cytotoxic effects of EM23 on normal mammalian cells. HL-7702 and NIH/3T3 cells were treated with various concentrations of EM23 for 48 h. Cell viability was measured using a CCK-8 assay. (D) Effects of EM23 on cell cycle distribution. Following treatment with EM23 for 48 h, cells were fixed and stained with PI solution. Cell cycle distribution was measured by flow cytometry. All of data are presented as the mean SD of at least three independent experiments. ?< 0.05 and ??< 0.01. The reagents DAPI, DCFH-DA, PI, JC-1, and NAC were purchased from Sigma Chemical Co. (St. Louis, MO, USA). ERK inhibitor "type":"entrez-nucleotide","attrs":"text":"FR180204","term_id":"258307209","term_text":"FR180204"FR180204 was purchased from Merck Millipore (Bellerica, MA, USA). A PierceTM BCA Protein Assay Kit was ONO-4059 obtained from Thermo Fisher Scientific (Rockford, IL, USA). TNF- was obtained from Sino Biological Inc. (Beijing, China). A CCK-8, a TUNEL Apoptosis Detection Kit, dithiothreitol (DTT), a Nuclear and Cytoplasmic Extraction Kit, RIPA buffer and RNase were purchased from Beyotime (Shanghai, China). Phosphatase inhibitor cocktail tablets and protease inhibitor cocktail tablets were supplied by Roche (Mannheim, Germany). All other chemicals and solvents were of reagent or HPLC grade. Primary antibodies against TrxR, Trx, ASK1, p-ASK1 (Thr845), and Lamin B1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). GAPDH, -actin, caspase 3, caspase 9, cleaved-caspase 3, cleaved-caspase 9, cleaved PARP, p-p38 (Thr180/Tyr182), p-ERK1/2 (Thr202/Tyr204), p-JNK (Thr183/Tyr185), p38, ERK1/2, JNK, p-NF-B p-p65 (Ser536), NF-B p65, IB, anti-mouse, and anti-rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (CST, Beverly, MA, USA). Alexa Fluor? 568 phalloidin and ONO-4059 Alexa Fluor? 488 anti-rabbit fluorescent secondary antibodies were purchased from Life Technologies (Grand Island, NY, USA). Cell Proliferation Analysis A CCK-8 assay was ONO-4059 used to detect EM23-mediated inhibition of cellular proliferation. Cells in suspension were plated in 96-well plates at a density of 1 1 104 cells/well. Following this, the cells were treated with either vehicle (0.1% DMSO) or EM23. The highest concentration of EM23 used was 100 M; additional concentrations were tested following two-fold serial dilutions. The cells were treated for 48 h and then 10 L CCK-8 solution was added to each well, and the plate was incubated for an additional 4 h. The absorbance of the plate was measured at 450 nm using a microplate reader (Bio-Rad; Hercules, CA, USA), and the IC50.