Images were examined with NIKON 80i microscope at MEK inhibitor 400× or 1000x magnification and captured with Spot Digital https://www.selleckchem.com/products/4egi-1.html Camera and Spot Advanced Software Package (Diagnostic Instruments, Sterling Heights, MI).
The percentage of cells with mitotic abnormalities was calculated by the number of the cells showing the abnormal mitotic figures (including chromosomal misalignment and formation of multipolar spindles) divided by the total number of mitotic cells counted. A minimum of 500 cells from randomly selected fields were scored per condition per experiment. Mouse xenograft model The procedure was adapted from published protocol [3] and were in accordance to the Institutional Animal Care and Use Committee of DCB. C.B-17 SCID mice (6-7 weeks, 21-24 g) (Biolasco,
Taipei, Taiwan) were used. Females were used for Colo-205 and Huh-7 while and males were for MDA-MB-231. Cells were injected subcutaneously into the flank in 50% matrigel solution (BD Biosciences, San Jose, CA). 1×107, 3×106, and 6×106 implanted cells/mouse was used for Huh-7, Colo-205, and MDA-MB-231, see more respectively. Treatment initiated when tumor volume reached 150 mm3. For Colo-205 and Huh-7, mice were treated with vehicle control (10% DMSO 25% PEG200) per oral PO/BID/28 cycles in total. For Huh-7, a dose increase was incurred on day 4 to increase efficacy. For Colo205, a dose decrease was incurred on day 13 to decrease body weight loss. For MDA-MB-231, mice were treated with vehicle control (5% DMSO, 10% Cremophor, 85% water for Injections (WFI)) per oral PO/BID/28 cycles in total, or TAI-1 formulated in vehicle (20 mg/kg intravenously IV/QDx28 cycles or 150 mg/kg per oral PO/BID/28 cycles in total). Tumor size were measured with digital calipers and volume calculated using the formula (L x W x W)/2, of which L and W represented the length and the width in diameter (mm) Methane monooxygenase of the tumor, respectively. Body weights and tumor growth were measured twice a week. Mean
tumor growth inhibition of each treated group was compared with vehicle control and a tumor growth inhibition value calculated using the formula: [1-(T/C) ×100%] (T: treatment group, C: control group tumor volume). Pilot toxicology study in mice A sub-acute toxicology study was performed for TAI-1. Female C.B-17 SCID mice (7 weeks old) were used in this study. Mice were divided into four treatment groups: vehicle control (10% DMSO, 25% PEG200, 65% double distilled H2O), test article (in vehicle) at 7.5, 22.5, and 75.0 mg/kg, and all mice were treated twice a day by oral administration for 7 days (n = 8 for each group). Body and organ weights were measured. Blood were collected by cardiac puncture and serum analyzed for complete blood count and biochemical indices. In vitro kinase assay Inhibition of kinase activity by test compound was estimated by [33P] labeled radiometric assay. 20 kinase assays (Millipore) were adapted.
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