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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

quantitative real-time RT-PCR. The gelatinases MMP-2 and MMP-9, matrilysin MMP- 7, and the collagenases MMP-1 and MMP-13 are believed to be important in colorectal cancer metastasis (8). Treatment with 10 μM retinol reduced MMP-2 mRNA levels to 63 ± 8% of control in SW620 cells but not in HCT-116 cells (Fig. 3.3A). In contrast, MMP-9 mRNA levels were reduced to 69 ± 4% of control by treatment with 10 μM retinol in HCT-116 cells (Fig. 3.3B). Treatment with 1 μM retinol reduced MMP-9 levels to 57 ± 1.5 % of control in the SW620 cell line but treatment with 10 μM retinol did not lower MMP-9 mRNA levels, indicating that different concentrations of retinol may exert varying effects on MMP-9 mRNA levels in this cell line. The mRNA level of MMP-7 was decreased in HCT-116 cells to 60 ± 11% of vehicle control by 10 μM retinol treatment, but MMP-7 mRNA was not affected by retinol treatment in SW620 cells (Fig. 3.3C). Treatment with 10 μM retinol decreased MMP-1 mRNA levels to 48 ± 8% and 44 ± 8% of vehicle control in HCT-116 and SW620 cells, respectively (Figure 3.3D). In contrast, retinol treatment of HCT-116 and SW620 cells did not change mRNA levels of MMP-13 (Fig. 3.3E). These data suggest that retinol may decrease cell invasion by reducing MMP mRNA levels in vitro. Retinol decreases gelatinase (MMP-2 and -9) enzyme activity We further investigated whether retinol decreased the enzyme activity and protein levels of the MMP whose transcription it altered. Retinol treatment decreased MMP-9 enzyme activity and total levels of active MMP-9 in the conditioned media obtained from both the HCT-116 and SW620 cell lines (Fig. 3.4). No pro-MMP-9 was detected in the 53

conditioned media (Fig. 3.4B and D). Retinol treatment also decreased MMP-2 activity in conditioned media from SW620 cells (Fig. 3.4C) but did not affect pro-MMP-2 levels in this cell line. Although MMP-2 mRNA was decreased by retinol treatment in HCT- 116 cells (Fig. 3.3), neither pro- nor active-MMP-2 levels were decreased by treatment with retinol in conditioned media from HCT-116 cells (Fig. 3.4A). Although retinol decreased the mRNA levels of MMP-1 and -7 and these enzymes are important for cell invasion, we were unable to detect MMP-1 and -7 enzyme activity or protein using the MMP-1 Biotrak Activity Assay System (GE Healthcare Bio-Sciences Corp., Piscataway, NJ), which detects both active and total MMP-1, or casein zymogel and western blotting of conditioned media, to detect MMP-7, in both cell lines (data not shown) potentially because these cell lines secrete low levels of MMP-1 and -7 (101,102). These data suggest that retinol may reduce the invasion of colon cancer cell lines in vitro by reducing the protein levels and activity of the gelatinases, MMP-2 and, particularly, MMP –9. MMP –2 and –9 are involved in colon cancer cell invasion Next, we verified that the MMP whose protein or activity levels were decreased by retinol treatment played a role in the invasion of these cell lines through Matrigel. Specific, neutralizing antibodies were used to block the activity of MMP-2 and -9 in in vitro invasion assays. Cell invasion was decreased to 19 ± 12% by an MMP-9 neutralizing antibody in the HCT-116 cell line (Fig. 3.5A). However, in the HCT-116 cell line, the MMP-2 neutralizing antibody only decreased cell invasion to 59 ± 10% of 54

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