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Copyright by Eunyoung Park 2007 - The University of Texas at Austin

Copyright by Eunyoung Park 2007 - The University of Texas at Austin

RHF/6-31G(d) model

RHF/6-31G(d) model chemistry to generate not only a starting geometry for further calculations but more importantly a set of atomic charges for each of the atoms. Molecular dynamics was employed to interrogate the conformational space of the ligands via a series of simulated annealing experiments. Since the compounds were all relatively small organics, all dynamics calculations were performed using an MM+ force field with electrostatic contributions to the potential energy term calculated using the atomic charges generated from the ab initio optimization. The annealing cycle was conducted using a time step of 0.0005 ps and composed of a 5 ps heating step where the structure was taken from 0°K to 800°K, followed by a 30 ps production phase at constant temperature, before a final 10 ps cooling phase from 800°K back to 0°K. This final cooled structure was then energy optimized and saved before becoming the starting structure for a subsequent annealing cycle. The procedure was repeated 100 times to generate 100 candidate conformations for retinol, wortmannin and the PI fragment. The 100 conformations were sorted based on distances, angles and torsion angles of various moieties to produce a set of conformational families for each compound. An electrostatic potential isosurface was generated for the lowest energy structure of each of these conformation families. The isosurface provided a means of assessing the electrostatic character of each compound and facilitates comparisons of their potential substrate interactions by highlighting locations of high reactivity. The ligands in this study were not docked to the p110 protein, but instead compared to each other to examine similarities that might help elucidate their potential interaction with PI3K. 77

Statistical Analysis Quantitation was performed with Biorad Quantity One software. Values shown are the mean ± SEM of three independent experiments. All data are expressed relative to vehicle control. All statistical tests were performed using SPSS (Apache Software Foundation, Wilmington, DE, version 12.0 for Windows). Data were analyzed using two-tailed t-tests comparing each retinol concentration to control (0 μM). Results were considered significantly different at P < 0.05. RESULTS Retinol decreases PI3K activity. Previously, we showed that retinol inhibited the invasion of HCT-116 and SW620 ATRA-resistant human colon cancer cell lines (144). Because PI3K plays a regulatory role in tumor metastasis (137,138), we examined the effect of retinol on PI3K activity in intact cells, treated with 0, 1 and 10 μM retinol for 30 min immediately prior to harvest. Retinol decreased PI3K activity in both the HCT-116 and SW620 cell lines (Fig. 4.1A and B). Specifically, in the HCT-116 cell line, 1 and 10 μM retinol significantly decreased the activity of PI3K to 59.8 ± 11.4 and 70.3 ± 7.7% of vehicle control, respectively (Fig. 4.1A). Treatment with 1 μM retinol trended to reduce PI3K activity in the SW620 cell line to 70.8 ± 11.4% of vehicle control (Fig. 4.1B). Treatment with 10 μM retinol also significantly reduced PI3K activity in the SW620 cell line to 71.6 ± 5.9% of vehicle control (Fig. 4.1B). There were no differences in PI3K activity between 78

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