Research Article

Studies on Multifunctional Effect of All-Trans Retinoic Acid (ATRA) on Matrix Metalloproteinase-2 (MMP-2) and Its Regulatory Molecules in Human Breast Cancer Cells (MCF-7)

Figure 1

Cell viability assay: MCF-7 (300,000 cells/1.5 mL) (Figure 1(a)) and A375 (300,000/mL) (Figure 1(b)) cells were grown in serum-free culture medium (SFCM) in absence (Control) and in presence of 30  M ATRA (Treated) for 24 hours (Figure 1(a)) and 48 hours (Figure 1(b)). Cells were collected and 0.4% trypan blue solution was added in both control and ATRA-treated cell suspension. Number of blue cells and nonblue cells were counted and -test was done to obtain the -values. Zymographic analysis of pro-MMP-2 activity: MCF-7 (300,000 cells/1.5 mL) cells were grown in serum-free culture medium (SFCM) in absence (control) (lane C) and in presence of 10 (lane 1), 20 (lane 2), 30 (lane 3) M ATRA for 24 hours (Figure 1(c)). The culture supernatants were collected and MMPs were concentrated using Gelatin-sepharose 4B beads. Gelatinases were eluted from bead with sample buffer and subjected to zymography on 7.5% SDS-PAGE copolymerised with 0.1% gelatin. The zymogram was treated with 2.5% triton X-100 for 30 minutes followed by incubation in reaction buffer for 20 hours and stained with coomassie blue. The quantitative measurement of the zymogram (Figure 1(c)) was performed by using Image J Launcher (version 1.4.3.67). (C) shows the pro-MMP-2 activity in the SFCM of control cells. (1) is the representative of pro-MMP-2 activity in the SFCM of 10  M ATRA-treated cells. (2) and (3) represents the pro-MMP-2 activity in the SFCM of 20 and 30  M ATRA-treated cells (for 24 hours), respectively.
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(a)
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(b)
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(c)