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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

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that exhibited by control cells, indicating that MMP-2 may play a smaller role in the invasion of this cell line through Matrigel than MMP-9. This data reflects that shown in Fig. 3.4A where MMP-2 activity was not decreased, but MMP-9 activity was decreased, by retinol treatment in HCT-116 cells. In the SW620 cell line, invasion was decreased to 26 ± 7% and 29 ± 27% by MMP-2 -and -9 neutralizing antibodies, respectively (Fig. 3.5B), suggesting that MMP-2 and –9 are involved in SW620 colon cancer cell invasion through Matrigel. Similarly, both MMP-2 and –9 activity were decreased by retinol treatment in SW620 cells (Fig. 3.4B). Retinol increases TIMP-1 protein levels in conditioned media Because MMP-9 activity is regulated by TIMP-1, western blot analysis of cell lysates and conditioned medium as well as quantitative real-time RT-PCR were used to examine the effect of retinol on TIMP-1 protein and mRNA levels. Retinol treatment increased TIMP-1 protein levels in media but not in cell lysates (Fig. 3.6A and B). Unlike other TIMPs, TIMP-1 is inducible. TIMP-1 protein concentration is controlled at the level of transcription, mRNA stability, protein degradation, and endocytosis (85). Retinol slightly decreased TIMP-1 mRNA levels to 86 ± 5% and 85 ± 4% of control in HCT-116 cells treated with 1 and 10 μM retinol, respectively, but not in SW620 cells (Fig. 3.6C-D). Importantly, retinol does not increase TIMP-1 mRNA or protein levels in the cell lysates (Fig. 3.6C-D) and the inhibitory effect of retinol on cell invasion is not blocked by actinomycin D or cycloheximide treatment (Fig. 3.2). Therefore, if TIMP-1 55

is mediating the inhibitory effect of retinol on cell invasion, retinol may be increasing extracellular TIMP-1 levels by decreasing TIMP-1 degradation or endocytosis. DISCUSSION This study demonstrates that retinol decreases the invasion of human colon cancer cell lines in vitro (Fig. 3.1). The inhibitory effects of retinol are not dependent upon increased gene transcription via RAR or mRNA translation (Fig. 3.2). Depending on cell line, retinol treatment also decreased the mRNA levels of MMP-1, 2, -7 and -9 (Fig. 3.3) as well as the activity of MMP-2 and -9 and total media MMP-9 protein levels (Fig. 3.4). The specific MMP affected varied with cell line but the ability of retinol to decrease MMP-9 protein and activity levels was consistent between both cell lines. TIMP-1 binds to pro-MMP-9, inhibiting the activation of pro-MMP-9 (9). TIMP-1 levels were also increased by retinol treatment in the media collected from both cell lines (Fig. 3.6). Taken together, these data suggest that retinol, the form of vitamin A derived from the diet, inhibits the invasion of colon cancer cell lines in vitro by decreasing their ability to digest ECM proteins. It is important to note that the colon cancer cell lines used in the present study produce little (HCT-116) or no (SW620) ATRA (95). Other groups have also found that colon cancer cell lines lack the ability to synthesize ATRA from retinol (103). In addition, the colon cancer cell lines used in the present study do not convert retinol to 4- oxoretinol or anhydroretinol, two naturally occurring retinoids capable of inhibiting cell growth (95). Therefore, we hypothesize that retinol itself, and not a bioactive 56

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