ells , breast cancer cells , leukemia cells , osteosarcoma cells , cervical cancer cells , hepatoma cells , fibrosarcoma cells , and mouse colon carcinoma LuM1 cells derived from colon 26 tumor were also used in this study. HT29, LS174T, SW480, SW620 and MDA-MB-468 were purchased from ATCC. MCF7 and HT1080 cells were obtained from the JCRB Cell Bank. THP-1 and K562 cells were kindly provided by Dr. Y. Honma, Shimane University Faculty of 2883-98-9 site Medicine. Gastric cancer cell lines were supplied by the Department of Pathology, Shimane University Faculty of Medicine. Other cell lines were supplied by Dr. A. Nakagawara, Chiba Cancer Center Research Institute. Characteristics of the cell lines used in this study are described elsewhere. Leukemia cell lines were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum and 40 g/ml gentamicin. Other cell lines were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS and 40 g/ml gentamicin in a humidified atmosphere with 21% O2/5% CO2 or 1% O2/5% CO2. Hypoxic culture conditions were achieved in a humidified automatic PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19668191 O2/CO2 incubator. Reagents -Shogaol and -gingerol were purchased from TOKIWA PHYTOCHEMICAL CO., Ltd.. Preparation of ginger extract Dry powder PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19667238/ of the root parts of Syussai Shoga, which was cultivated in the Hikawa area in Izumo, Shimane prefecture, was extracted with ethanol for 20 min in a sonication water bath. Ethanol was evaporated at 80C to yield a crude ethanol extract of the ginger. The extract was weighed and dissolved in ethanol or dimethylsulfoxide at the desired concentration. Cell growth and viability assay Cell growth and viability was measured by using the MTT 2,5-diphenyltetrazolium bromide) assay. Briefly, cells were cultured in 96-well tissue culture plates and treated in triplicate in 100 l DMEM/10% FBS containing different concentrations of SSHE or solvent alone for the indicated period. At the end of 
the incubation, 10 l of MTT was added to the wells to allow formation of MTT formazan crystals for 4 h. After the medium was removed, the crystals were solubilized in 100 l of DMSO. Absorbance was recorded at 550 nm. Cell viability was also assayed by a trypan blue dye exclusion test. Cell cycle analysis Panc-1 cells treated with SSHE for 20 h were fixed in 70% ethanol and stored at -20C until use. The fixed cells were washed with Dulbecco’s PBS and incubated with 100 g/ml RNase A and 50 g/ml PI. The cells were then subjected to flow cytometric analysis using a FACSCalibur flow cytometer. Measurement of mitochondria membrane potential Mitochondria membrane potential was monitored by the JC-1 Mitochondrial Membrane Potential Assay Kit. For this assay, 100 l/ml of the JC-1 Staining Solution was added to Panc-1 cells treated with SSHE for 20 h in 6-well plates, and the 3 / 22 Antitumor Activity of Ginger Extract against Pancreatic Cancer cells were incubated for 15 min. Afterwards, the cells were detached by trypsinization, washed twice with the assay buffer, and then subjected to flow cytometry. Annexin V/Propidium iodide staining The Annexin V-FITC Apoptosis Detection Kit was used to detect annexin V and/or PI-positive cells. Briefly, Panc-1 cells were washed with ice-cold DPBS and then stained with Annexin V-FITC for 15 min at room temperature in the dark and PI in ice-cold Binding Buffer. Annexin V and/or PI-positive cells were counted using a FACS Calibur flow cytometer. Measurement of ROS generation The production of ROS was monitored