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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

10 μM retinol reduced

10 μM retinol reduced SW620 cell migration to 58.7 ± 11.6%, 36.5 ± 8.9%, and 36.4 ± 7.2% of vehicle control, respectively (Fig. 3.1A). Treatment with retinol for 8 h did not decrease cell number (data not shown). Because cell migration is only one facet of metastatic potential, we determined whether retinol could inhibit the ability of colon cancer cells to digest and move through Matrigel, a basement membrane-like protein matrix, in vitro. The ability of HCT-116 cells to invade through Matrigel was decreased to 52.6 ± 15.9% and 40.0 ± 6.7% of vehicle control by 1 and 10 μM retinol, respectively (Fig. 3.1B). SW620 cell invasion was decreased by 1 and 10 μM retinol to 81.2 ± 19.0% and 46.0 ± 15.0% of vehicle control, respectively (Fig. 3.1B). Cell number was not affected after 24 h of treatment with retinol (data not shown). These data show that retinol inhibits both cell motility and invasion through an ECM. The effect of retinol on cell invasion is RAR-independent To elucidate whether retinol mediated cell invasion through increased gene transcription or mRNA translation, HCT-116 and SW620 cells were treated with cycloheximide or actinomycin D. HCT-116 cells treated with 1 μM retinol and actinomycin D were significantly less invasive than cells treated with 1 μM retinol alone, indicating that transcription may be required for invasion in this cell line (Fig. 3.2B). Importantly, treatment with cycloheximide (Fig. 3.2A) or actinomycin D (Fig. 3.2B) did not block the inhibitory effect of retinol on cell invasion in either cell line, suggesting that 51

increased gene transcription and translation do not mediate the inhibitory effect of retinol in cell invasion. In most ATRA-sensitive cells, the actions of retinol are mediated by its metabolite, ATRA, which acts by binding to RARs and inducing gene expression via RAREs. However, HCT-116 cells lack all RAR (90) and, while SW620 cells may express some RARs (93,99), we have shown that retinol inhibits HCT-116 and SW620 colon cancer cell growth independent of ATRA and the RARs (95). To determine if the inhibitory effect of retinol on cell invasion was also RAR-independent and to confirm our actinomycin D data, we used the RAR pan-antagonist, AGN193109, to block RAR signaling and ensure that any RARs present would be inactive. This antagonist, when added at 10 times the concentration of agonist, blocks the ability of agonist to bind to RAR (100). Cells were treated with 0 or 1 μM retinol with and without 10 μM AGN 193109. The 10 μM concentration of retinol was not examined in this experiment because 100 μM AGN193109 was toxic to the cells. As can be seen in Fig. 3.2C, the RAR-antagonist did not alleviate the inhibitory effect of retinol on cell invasion. These data show that retinol inhibits the invasion of colon cancer cells independent of RAR and new protein or mRNA synthesis. Retinol decreases MMP mRNA levels MMP facilitate the cell invasion by digesting the ECM. To determine whether the retinol-induced decrease in cell invasion was due solely to lower cell motility or also to a decrease in ECM digestion, we examined the mRNA levels of five MMP using 52

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