Acute myeloid leukemia is known as one of the most malignant diseases. agent for leucocythemia treatment. L., widely distributed in the temperate and tropical zones of the world, has been used as both foods and medicines. Its aerial part (Chinese AZD2281 name Ma-Chi-Xian) has been applied for the treatment of diarrhea, urinary tract infection and diabetes for a rather long history in China. It has wide pharmacological properties, such as antibacterial, regulating lipidemia, anti-aging, anti-oxidative, anti-inflammatory, wound-healing, analgesic and AZD2281 antitumor activities [3,4]. A cerebroside compound named as portulacerebroside A from L. shows property of antitumor . In this study, we attempted to explore the effects of PCA on the proliferation, cell cycle distribution, apoptosis, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) of human human leukemia HL60 cells and clarify the possible mechanisms involved in. Methods and materials Cell culture Human HL60 cell line was obtained from Shanghai Institute of Cell Biology (Shanghai, China). Cells were cultured in RPMI-1640 medium with 10% FBS (Gibco BRL, Rockville, MD, USA), 100 U/ml penicillin G and 100 g/mL streptomycin in an incubator (37C, 100% humidity and 5% CO2). PCA Portulacerebroside A (PCA) was isolated and purified from the aerial parts of L. according to the previous report and appeared as white powder . It was dissolved in an appropriate amount of dimethylsulfoxide (DMSO) and diluted to the desired concentrations before utilization, with the final concentration of DMSO kept below 0.5%. MTT assay The MTT assay AZD2281 was used to assess the effect of PCA on cell viability. In brief, the cells were seeded in 96-well culture plates and treated without or with PCA (1, 2, 5, 10, 25, 50 and 100 M) for 6, 12, 24 and 48 h. Subsequently the cell viability was evaluated by MTT assay. The absorbance was measured at 490 nm test wavelength and 570 nm reference wavelength with an automated Bio-Rad 550 microtiter plate reader (Rome, Italy). DNA fragmentation assay Following treatment with PCA (5, 10 and 50 M) for 24 h, the cells were harvested and fixed for 5 minutes in 3% Para formaldehyde in phosphate buffered saline. After air dying, cells were stained for 10 minutes with Hoechst 33258 (10 mL), mounted in 50% glycerol containing 20 mmol/L citric acid and 50 mmol/L orthophosphate, and stored at -20C before analysis. Nuclear morphology was evaluated using a fluorescence microscope (DMI3000B, AZD2281 Leica, Gemany). Measurement of apoptotic cells by flow cytometry HL60 cells were collected after treatment with PCA (5, 10 and 50 M) for 24 h and fixed with 75% ethanolover night at 4C, and stained with Annexin V and propidium iodide (PI). Cell apoptosis was evaluated using FAC flow cytometry (San Jose, CA). Mitochondria membrane potential (MMP) Rhodamine-123 (Rho-123) dye (Sigma) was used to detect the changes in MMP. Cells (5 104 cells/well) were cultured in 24-well plate. After a period of exposure (24 h) with various concentrations of PCA (5, 10 and 50 M), cells were washed with PBS, incubated with Rho-123 (10 mg/mL) and subsequently subjected to flowcytometry. Detection of reactive oxygen species (ROS) Detection of ROS was performed by flow cytometric analysis as described previously. In brief, (5 104 cells/well) were cultured in 24-well plate, after a period of exposure (12 h) with various concentrations of PCA (5, 10 and 50 M), cells were washed with PBS and resuspended in complete medium followed by incubation with 0.5 M dihydrorhodamine 123 (Sigma) for 30 min at 37C. ROS fluorescence intensity was determined by cytometry with excitation at 490 nm and emission at 520 nm. Western blot assay Cells were seeded at a density of 5 105 cells per well in 6-well plates, cultured overnight and then treated with PCA (5, 10 and 50 M) for 1 and 24 h. Cell protein lysates were separated in 10% sodium dodecyl sulfate-polyacrylamide gels, electroblotted onto to a polyvinylidene fluoride membrane (Roche Diagnostics, Mannheim, Germany), then detected with JNK, phosphorylated (P-) JNK, p38 MAPK, P-p38 MAPK, Bax, Bcl-2, casepase-3 and caspase-8 proteins. Protein loading was estimated using mouse anti-GAPDH monoclonal antibody. Lab Works Image Acquisition and Analysis Software (UVP, Upland, CA, USA) were used to quantify band intensities. Hhex Antibodies were purchased from Univ-bio Inc (Shanghai, China). Fluorescent quantitative reverse transcription-PCR (FQRT-PCR) AZD2281 Total mRNA was isolated from HL60 cells using TRIzol Reagent (Gibco-BRL, Gaithersburg, MD) according to the previous report (11). Briefly, a 4 g aliquot of RNA was reversely transcribed to cDNA by Thermoscript RT-PCR System reagent (Gibco-BRL). The primers and probes were.