The intracellular platinum content of T-ALL/LBL cells treated with EG-Se/Pt was increased compared with that of T-ALL/LBL cells treated with CDDP. with CDDP, and induced apoptosis and cell cycle arrest. The intracellular platinum content of T-ALL/LBL cells treated with EG-Se/Pt was increased compared with that of T-ALL/LBL cells treated with CDDP. EG-Se/Pt-induced apoptosis was mediated by caspase and ROS levels through the activation of the mitochondrial signaling pathway. The results of the present study suggest that EG-Se/Pt is a potential therapeutic candidate for the treatment of T-ALL/LBL. and (11,12). ROS have been reported to induce apoptosis via a series of downstream signaling pathways including a mitochondrial cascade (13,14). Furthermore, increased ROS levels in cancer cells serve a role in the selective killing of cancer cells by antitumor agents (12,15). Chemists from Tsinghua University (Beijing, China) have developed a novel compound, EG-Se/Pt, based on the coordination of Se-containing small molecules (EG-Se) and CDDP, which demonstrates broad-spectrum anticancer activity in breast, lung and liver cancer cell lines, and selectivity of tumor cells (12). The present study demonstrates that EG-Se/Pt kills T-LBL/ALL cells by inducing cell cycle arrest and ROS-mediated apoptosis through the mitochondrial signaling pathway. Materials and methods Cells and cell culture The human T-ALL/LBL cell lines Jurkat and Molt-4 were obtained from the American Type Culture Collection (Manassas, VA, USA), and were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 2 mM L-glutamine, 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 100 units/ml penicillin and 100 g/ml streptomycin. Cells were routinely cultured at 37C in a humidified incubator containing 5% CO2 and were passaged between every 2 and SB 525334 3 days. Antibodies and reagents Mouse monoclonal antibodies specific for cytochrome (1:200; cat. no. sc-13156) and -actin (1:200; cat. no. sc-47778) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit monoclonal antibodies against apoptosis regulator Bcl-2 (1:1,000; cat. no. 4223) and cleaved caspase-3 (1:1,000; cat. no. 9664), and rabbit polyclonal antibodies against apoptosis regulator Bax (1:1,000; cat. no. 2772), cleaved caspase-9 (1:1,000; cat. no. 9505) and cleaved poly(ADP-ribose) polymerase (PARP; 1:1,000; cat. no. 9542) were from Cell Rabbit polyclonal to ASH2L Signaling Technology, Inc. (Danvers, MA, USA). Rabbit SB 525334 monoclonal antibody against apoptotic protease-activating factor 1 (Apaf-1; 1:1,000; cat. no. ab32372) was from Abcam (Cambridge, UK). IRDye 800CW-conjugated goat polyclonal anti-rabbit and anti-mouse immunoglobulin (IgG) secondary antibodies (cat. nos. 925-32211 and 925-32210, respectively; both 1:10,000) were from LI-COR Biosciences (Lincoln, SB 525334 NE, USA). EG-Se/Pt was produced in-house. To examine the involvement of caspases in EG-Se/Pt-induced apoptosis, the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (z-VAD-FMK; Selleck Chemicals, Houston, TX, USA) was added at a concentration of 20 M for 3 h at 37C prior to treatment with EG-Se/Pt. To determine the involvement of ROS in EG-Se/Pt-induced apoptosis, cells were pretreated with 10 mM N-acetyl-L-cysteine (NAC) (Beyotime Institute of SB 525334 Biotechnology, Haimen, China) for 3 h at 37C prior to treatment with EG-Se/Pt. Cell viability assay The Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to study cell viability according to the manufacturer’s protocol. A cell suspension was inoculated into a 96-well plate (4104 cells/well). EG-Se/Pt was added to the wells of the plate at 5,10,15,25,35,50.75 and 100 M, and the plate was incubated at 37C for 12, 24, 48 or 72 h. Cells were also treated with CDDP (cat. no. 15663; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and EG-Se at the same concentrations, and left untreated as a negative control. Following treatment, 10 l CCK-8 solution was added to each well and the plate was incubated for 3 h at 37C with 5% CO2. Absorbance was measured at 450 nm using a microplate reader. The assay was performed SB 525334 using six replicates (n=6) for each group and repeated at least three times. Cell cycle assay Cells were inoculated into 6-well plates (1106 cells/well) and treated with EG-Se/Pt at 5, 15 and 35 M in Jurkat cells and at 1,12.5,25 M in Molt-4 cells. Following treatment, the cells were collected, washed with ice-cold PBS and fixed in 70% ethanol overnight at 4C..