cells harvested, washed and resuspended in 4% paraformaldehyde. Cells were Wxed at room temperature for 30 min then washed twice. Fixed cells were resuspended in 0.5 ml of 0.1% Triton X100, incubated on ice to permeabilise the cells, and washed. TUNEL reaction mixture was prepared according to the kit Celastrol instructions, and each cell pellet was resuspended in 50 l. Cells were incubated at 37°C for 1 h, washed and resuspended in FACS FLOW solution for FACS analysis using a FACScan system . In vivo xenograft studies Experiments were conducted at EPO GmbH, Berlin Buch, Germany and approved by the Institutional Animal Care and Use Committee at EPO. These standards are equivalent to the UKCCCR guidelines for the welfare of animals in experimental neoplasia .
Female Ncr:nu/nu mice were injected sub cutaneously with 107 HCT116 cells, and treatment began at day 6 when tumour size Pimecrolimus molecular weight reached around 100 mm3. At this stage, animals were randomised into groups of ten for experiments. PXD101 was formulated in L arginine/ isotonic sterile saline to give a Wnal concentration suYcient for a dose of 100 mg/ml. 5 FU for clinical use was diluted with isotonic sterile saline and used at a suYcient concentration for a dose of 15 mg/ml. PXD101 was administered for 5 days each week for 2 weeks in the morning followed by 5 FU in the afternoon as appropriate. Tumour diameters were measured at 3 day intervals using calliper measurements and estimated assuming spherical geometry according to the formula: /2, until day 31 when the study was terminated.
Relative tumour volumes compared to pre treatment are also shown to account for variations in starting tumour volumes. Following on from the HCT116 xenograft experiments, a further Erlotinib price study was performed using HT29 cells and an increased dose of 5 FU and PXD101 at either 60 or 100 mg/kg. Study treatment began on positive tumour take , at which point, animals were randomised into groups of 8 and continued until day 35. The 5 FU treatment was stopped after the Wrst 5 day cycle due to weight loss and diarrhoea. Two toxic deaths occurred in the 100/30 mg/kg PXD101/5 FU combination group and the data is not presented here. Mice were therefore administered 60 mg/kg PXD101 for 5 days each week in the morning followed by 30 mg/kg 5 FU in the afternoon during the Wrst week only. Tumour volumes were calculated and are presented as above.
Results PXD101 and 5 FU are potent inhibitors of HCT116 dyphylline ic50 cell proliferation in vitro The WST1 assay tests the viability and/or proliferation of cells by measuring mitochondrial activity by cleavage of a tetrazolium salt to formazan dye by the electron coupling reaction. Using this assay, PXD101 and 5 FU titration curves were produced and gave EC50 values of 0.28 and 7.46 M for incubations over 48 h, respectively signs . EVect of PXD101 on TS expression in vitro As it has been previously reported that HDACi can down regulate TS we therefore investigated the eVects of treatment of HCT116 cells with PXD101. HCT116 cells were incubated with 0.9 M PXD101 for up to 24 h and immunoblotting of whole cell lysates performed. PXD101 down regulated TS protein levels after 6 h incubation, with no expression being detectable after 24 h . Treatment of HCT116 cells with 0.9 M PXD101 for 24 h also signiWcantly diminished .