Background Ginger leaf (GL) has long been used as a vegetable,

Background Ginger leaf (GL) has long been used as a vegetable, tea and herbal medicine. inhibition by PD98059 attenuated GL-mediated ATF3 expression but not p38 inhibition by SB203580, indicating ERK1/2 pathway implicated in GL-induced ATF3 activation. Conclusions These findings suggest that the 722544-51-6 supplier reduction of cell viability and apoptosis by GL may be a result of ATF3 promoter activation and subsequent increase of ATF3 expression through ERK1/2 activation in human colorectal cancer cells. voucher number: PARK1003(ANH)) were kindly provided by the Bonghwa Alpine Medicinal Plant Experiment Station, Korea. One kilogram of ginger leaf was extracted with 1000?ml of 80% methanol with shaking for 24?h. After 24?h, the methanol-soluble fraction was filtered and concentrated to approximately 20? ml volume using a vacuum evaporator and then fractionated with petroleum ether and ethyl acetate in a separating funnel. The ethyl acetate fraction was separated from the mixture, evaporated by a vacuum evaporator, and prepared aseptically and kept in a refrigerator. Cell culture and treatment Human colorectal cancer cell lines (HCT116, SW480 and LoVo), human breast cancer cell lines (MCF-7 and MDA-MB231) and human hepatocellular carcinoma (HepG-2) were purchased from Korean Cell Line Bank (Seoul, Korea) and grown in DMEM/F-12 supplemented with 10% fatal bovine serum (FBS), 100 U/ml penicillin and 100?g/ml streptomycin. The cells were maintained at 37C under a humidified atmosphere of 5% CO2. The extracts of ginger leaf (GL) were dissolved in dimethyl sulfoxide (DMSO) and treated to cells. DMSO was used as a vehicle and the final DMSO concentration did not exceed 0.1% (v/v). Cell viability Cell viability was measured using MTT assay system. Briefly, cells were plated onto 96-well plated and grown overnight. The cells were treated with 0, 50, 100 and 200?g/ml of GL for 24 and 48?h. Then, the cells were incubated with 50?l of MTT solution (1?mg/ml) for an additional 2?h. The resulting crystals were dissolved in DMSO. The formation of formazan was measured by reading absorbance at a wavelength of 570?nm. Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was prepared using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA (1?g) was reverse-transcribed using a Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturers protocol for cDNA synthesis. PCR was carried out using PCR Master Mix Kit (Promega, Madison, WI, USA) with primers for human ATF3 and human GAPDH as follows: human ATF3: 5-gtttgaggattttgctaacctgac-3, and reverse 5-agctgcaatcttatttctttctcgt-3; huaman GAPDH: forward 5-acccagaagactgtggatgg-3 and reverse 5-ttctagacggcaggtcaggt-3. Transient transfections Transient transfections were performed using the PolyJet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) according to the manufacturers instruction. HCT116 and SW480 cells were plated in 722544-51-6 supplier 12-well 722544-51-6 supplier plates at a concentration of 2??105 cells/well. After growth Serpine1 overnight, plasmid mixtures containing 0.5?g of ATF3 promoter linked to luciferase and 0.05?g of vector were transfected for 24?h. The transfected cells were cultured in the absence or presence of GL for the indicated times. The cells were then harvested in 1??luciferase lysis buffer, and luciferase activity was normalized to the luciferase activity using a dual-luciferase assay kit (Promega, Madison, WI, USA). Transfection of small interference RNA (siRNA) The cells were plated in six-well plates and incubated overnight. HCT116 cells were transfected with control siRNA and ATF3 siRNA for 48?h at a concentration of 100 nM using TransIT-TKO transfection reagent (Mirus, Madison, WI, USA) according to the manufacturers instruction. Then the cells were treated with GL (100?g/ml) for 24?h. Cell death assay Cell death was performed using Cell Death Detection ELISAPLUS Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturers instruction. Briefly, HCT116 and SW480 cells were seeded in 12-well plate. After 24?h, cells were treated with 0, 25, 50 and 100?M of GL for 24?h. Cytosol was prepared using Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA). Equal amounts of cytosolic extracts, immunoreagent containing anti-histone-biotin, and anti-DNA-POD were added to microplate well and incubated for 2?h under shaking. After washing, the ABTS solution was added to each well for 20?min and then the ABTS stop solution was added. The absorbance was recorded at 405?nm and 490. SDS-PAGE and Western blot After GL treatment, cells were washed with 1??phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with 722544-51-6 supplier protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MD. USA).

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