Briefly, the STE was analyzed by Hitachi L 6200 with an L 4500 selleck chem KPT-330 Diode Array detector with a PE Sciex Qstar Pulsar ESI TOF mass spectrometer. Samples were injected onto a Merck LiChrospher 100 RP 18 column. The column was equilibrated in 0. 05% acetic acid water and elution of the compo nents was achieved by increasing the concentration of solution B from 0 to 100% in 30 min at a flow rate of 1 ml min. Absorbance was monitored at 254 nm. The molecular masses of the peaks were deter mined from electro spray ionization mass spectra using a multiply charged ion profile based on the modified method of Chang et al. For subsequent experi ments, the STE powder was dissolved in dimethyl sulfate to achieve designed concentrations.

Cell and cell culture A human nasopharyngeal carcinoma cell line from ATCC, HONE 1 cells, was cultured in RPMI 1640 medium, 10% fetal bovine serum, 2 mM glutamine, 100 U ml penicillin, and 100 ug ml streptomycin. All cell cultures were maintained at 37 C in a humidified atmosphere of 5% CO2. For STE treatment, appropriate amounts of stock solution of STE were added into the culture medium to achieve the indicated concentrations. The cells were then incubated for the indicated time periods. Dimethyl sulfox ide solution without STE was used as blank reagent. Analysis of cell viability To evaluate the cytotoxicity of STE, an MTT colorimet ric assay was performed to determine cell viability. Cells were seeded in 24 well plates at a density of 1��105 cells per well and treated with 0, 25, 50, 75, 100, 150 and 200 ug mL of STE at 37 C in 5% CO2 for 24 h and 48 h.

At the end of the exposure period, the cells were washed with PBS and incubated with 0. 8 mL of MTT per well at 37 C in 5% CO2 for 4 h. The viable cell number was directly proportional to the pro duction of formazan following solubilization with iso propanol, which was measured spectrophotometrically at 563 nm. Cell migration and invasion assays Cell migration and invasion were assayed according to the methods described by Chu et al. After treatment with STE for 24 h, the surviving HONE 1 cells were harvested and seeded to a Boyden chamber at 104 cells per well in serum free medium, and then incubated for 24 h at 37 C. To deter mine cell migration, the cells were seeded into the Boyden chamber on membrane filters that were not coated with Matrigel.

The filters were then air dried for 5 h in a lam inar flow hood. The migrating cells were fixed with methanol and stained with Giemsa. The GSK-3 cell numbers were counted by light microscopy. For the invasion assay, 10 uL Matrigel was applied to 8 um pore size polycarbonate membrane filters. The bottom cham ber contained standard medium. The invasion of cells treated or untreated with STE was measured as in the migration assay.