Meanwhile, the inhibition of HDAC enzymatic http://www.selleckchem.com/products/Imatinib(STI571).html activity is involved in the effect of lycorine on K562 cells. Further in depth in vivo studies are presently under investigation in our laboratory. Materials and methods Cell culture and drugs The human CML cell line K562 was purchased from American Type Culture Collection and cultivated in RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum, 100 U/mL streptomycin, and 100 U/mL penicillin at 37 C in a humidified atmosphere with 5% CO2. Cells were diluted at a ratio of 1 3 every 1 d to 2 d. Lycorine was dissolved at 0. 034 M in dimethyl sulfoxide as a stock solution and diluted in serum free RPMI 1640 medium just before use. The maximum final concentration of DMSO in medium was less than 0. 02%.
Cell counting To examine the anti proliferative effect of lycorine, growth curves were protracted by manual cell counting. Exponentially growing K562 cells treated with different concentrations of lycorine or without lycorine were cultivated at 5 105 cells/mL in a culture flask. After appropriate culture, viable cells were counted manually and continuously for up to 3 d. Cell viability and cytotoxicity assay Cell viability and cytotoxicity were measured with 2 3 5 2H tetrazolium monosodium salt assay as described previously. Briefly, exponentially grow ing K562 cells treated with various concentrations of lycorine or without lycorine were cultivated at 1. 25 104 cells/well in a 96 well tissue cul ture plate at a total volume of 100 uL per well.
After cells were incubated for 24 and 48 h, 10 uL of CCK 8 solution was added to each well and incubation of cells was performed for another 4 h at 37 C. The relative cell viability was determined by scanning with an ELISA reader with a 450 nm filter and calculated by CCK 8 assay. Detection of HDAC activities A HDAC colorimetric assay kit was applied to determine HDAC enzymatic activities in the cell nu cleus according to the manufacturers instructions. Briefly, proteins were extracted from K562 cells treated with different concentrations of lycorine or without lycorine for 24 h using a nuclear and cyto plasmic protein extraction kit according to manufacturer recommendations. About 50 ug of nuclear protein from each group was added to a 96 well tissue culture plate at a final volume of 100 uL per well.
After incubation, HDAC activities were measured by scanning with an ELISA reader with a 450 nm filter. Values were expressed as the percentage of HDAC activ ities relative Carfilzomib to the untreated cell extract. Flow cytometry Flow cytometry was used to detect the cell cycle distri bution and quantitatively measure the apoptotic rate. After K562 cells treated with lycorine or with out lycorine were cultivated at 5 105 cells/mL in each culture flask for 24 h, 1 106 cells were har vested and washed with PBS.