Antitumor Effects of Saffron-Derived Carotenoids in Prostate Cancer Cell Models
Figure 4
(a) Immunostaining for MMP-9 (A, D, G, J), MMP-2 (B, E, H, K), and uPA (C, F, I, L) performed in PC3 tumors treated or not with SE (D, E, F), CR (G, H, I), and CCT (J, K, L). Untreated PC3 tumors (A, B, C) show high expression of MMP-9, MMP2, and uPA. Indirect immunoperoxidase staining of tumor xenograft samples was performed on paraffin-embedded tissue sections (4 μm). SE, CR, and CCT significantly reduced the expression of these proteases. Black arrows show mitotic figures (mean 16 ± 4 mitotic figures/100x microscopic field), which are high in CTR and were reduced after SE (about 40% versus CTRL), CR (70% versus CTRL) or CCT (90% versus CTRL). Green arrows show condensed/pyknotic nuclei. This appearance is evident in SE- (1–4 pyknotic nuclei/100x microscopic field), CR- (2–8 pyknotic nuclei/100x microscopic field), or CCT-treated tumor bearing mice (>10 pyknotic nuclei/100x microscopic field). Red arrows show aberrant mitosis, which is more evident in CCT-treated tumors. Scale bar is 100 μm. (b) Zymography for gelatinases, (a) and (b), and plasminogen activator performed in PC3 cells treated with SE (0.4 mg/mL), CR (0.4 mM), and CCT (0.1 mM). Cell cultures were allowed to grow in complete medium for 8 days in presence of SE, CR, and CCT. Next, medium was changed and serum-free medium containing SE, CR, and CCT was added for additional 24–48 hr. (c) Migration assay performed by using filters with 8 μm pores coated with 0.1% gelatin and using as chemoattractant the NI3T3-conditioned medium. Cells were cultured for 8 days in complete medium with SE, CR, and CCT, then harvested by trypsinization, and added to the upper compartment of the Boyden chambers at the above-mentioned concentrations. Five 10x microscopic fields were counted for each replicate. Data (±SD) are representative of three different analyses. Treatment reduced significantly the migration of PC3 and 22rv1 cells (). (d) Invasion assay performed by using filters with 8 μm pores coated with 50 μL (12.5 μg/mL) Matrigel and using as a chemoattractant the mouse fibroblast NI3T3-conditioned medium. Cells cultured for 6 days with SE, CR, and CCT were added to the upper compartment of Boyden chambers at the above-mentioned concentrations. Five 10x microscopic fields were counted for each replicate. Data (±SD) are representative of three different analyses. We considered as significant ().
