All cell lines were grown as monolayers of up to 80% confluence in RPMI 1640 supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37°C, 5% 4-Hydroxytamoxifen CO2 and humidified air. Growth inhibition experiments To assess antiproliferative effects, the total protein sulforhodamine B (SRB) assay was used as described previously [15]. In brief, cells were seeded in 96 well plates at a cell line specific density to ensure exponential growth throughout the whole period of the assay. These cell numbers were determined previously by cell growth kinetics. After 24 h, exponentially growing cells were exposed to serial dilutions

of each drug alone or drug combinations for the indicated times continuously. To investigate the influence of drug schedules drug A was added 24 h after cell seeding followed by drug B another 24 h later or vice versa. Corresponding control plates with single agents were treated in parallel. After 120 h total assay time, media was Selleck EPZ5676 removed and cells were fixed with 10% TCA and processed according to the published SRB assay protocol [15]. Absorbency was measured at 570 nm using a 96-well plate reader (Rainbow, SLT, Germany). DNA gel electrophoresis To detect apoptosis by DNA gel electrophoresis the

floating cells after drug treatment with an IC90 of FWGE for 48 h were used. After washing cells twice with PBS they were lysed in lysis-Alpelisib research buy buffer (100 mM TRIS-HCL (pH8.0), 20 mM EDTA, 0,8% SDS). Subsequent to treatment with RNaseA for 2 h at 37°C and proteinase K (Roche Molecular Biochemicals) overnight at 50°C, lysastes were mixed with DNA loading buffer. To separate DNA fragments, probes were run on a 1.5% agarose

gel followed by ethidium bromide staining and rinsing with destilled water. DNA ladders were visualized under UV light and Glutathione peroxidase documented on a BioDocAnalyse instrument (Biometra). Data analysis Dose response curves were generated by Sigma Plot (Jandel Scientific, San Rafael, CA) and IC50 values were calculated based on the Hill equation. Drug interaction was assessed using the model of Drewinko [16]. In brief, a hypothetical curve was calculated by multiplying the ratio of treated and untreated control with the dose response data points of the single drug curve. Synergy could be assumed if the hypothetical curve runs above the combination curve and antagonism is indicated if the hypothetical curve runs below the combination curve. In case of additivity both curve were superimposed. Statistical significance was probed with the two tailed, unpaired student’s t-test. Significance was assumed at a p-value < 0.05.