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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

To determine if the

To determine if the ability of retinol to decrease PI3K activity was suppressed by PI, immunoprecipitated PI3K from non-retinol-treated HCT-116 cell lysates was incubated with 10 μg retinol and increasing concentrations of PI. As can be seen in Fig. 4.3C, the decrease in PI3K activity in response to retinol was suppressed by incubation with 50 μg of PI. Specifically, 10 μg retinol (0.6 μM retinol) decreased PI3K activity to 38.9 ± 15.9 % of vehicle control. However, when treated with 10 μg retinol and 50 μg PI, PI3K activity increased to 110.0 ± 20.8 % of vehicle control. These data suggest that retinol inhibits the interaction of PI3K with its substrate PI. Retinol and a PI3K inhibitor both decrease cell invasion. The PI3K inhibitor, LY294002, was used to determine if PI3K played a role in the invasion of the HCT-116 and SW620 cell lines. As can be seen in Fig. 4.4, HCT-116 and SW620 cell invasion was decreased following treatment with retinol and LY294002. Cell invasion was significantly decreased to 36 ± 6.2% by 10 μM retinol and to 37 ± 13.3 and 28 ± 10.3% by 10 and 50 μM LY294002, respectively, in the HCT-116 cell line (Fig. 4.4A). In the SW620 cell line, invasion was significantly decreased to 31.6 ± 5.8% by 10 μM retinol and to 20.5 ± 3.4 and 9.7 ± 0.8% by 10 and 50 μM LY294002, respectively (Fig. 4.4B). Previously, we showed that cell number was not affected after 24 h of treatment with retinol (144). These data indicate that PI3K is involved in the invasion of these ATRA-resistant colon cancer cell lines. These results, together with those in Fig. 4.3, suggest that retinol may decrease cell invasion by inhibiting the activity of PI3K. 81

Retinol and wortmannin exhibit similar electrostatic potential surfaces. Extensive molecular modeling in the form of both ab initio calculations and molecular dynamics stimulations were performed to provide a better understanding of the reactivity of retinol, PI and wortmannin. As expected the ab initio calculations revealed a number of non-zero atomic charges that were indicative of various degrees of polarity within the ligands. The most polar of the three ligands was the PI fragment as there were a number of acidic hydrogens on the alcohol groups of the cyclohexyl ring as well as a significant charge gradient near the phosphate moiety. Wortmannin also contained a number of electron dense areas, but fewer acidic hydrogens overall as the most reactive hydrogen was the one attached directly to the five-membered furan ring. These results parallel those reported by x-ray crystallography that suggested this particular hydrogen atom was directly involved with PI3K complexation (148). Retinol was observed to contain only one acidic hydrogen atom, the one located on the alcohol group, and thus based on polarity alone might be considered the least reactive ligand as a majority of the molecule is encompassed by a relatively non-polar hydrocarbon ring and hydrocarbon chain. The simulated annealing procedure generated conformational families for retinol that could be categorized by the distance between the terminal alcohol group and the ring, the angle the ring formed with the midpoint of the carbon chain and the hydroxyl group, and the torsion angles along the unsaturated hydrocarbon chain. The vast majority of the structures formed one major family that showed folding of the hydrocarbon chain inward toward the ring as the pi orbitals of the unsaturated carbon-carbon double bonds aligned to form a ring-like structure. The elongated conformation that left the hydroxyl 82

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