Antitumor Effects of Saffron-Derived Carotenoids in Prostate Cancer Cell Models
Figure 3
(a) Hematoxylin/eosin (A, C, E, G) and Masson trichrome (B, D, F, H) staining of 22rv1 tumors treated with SE (C, D), CR (E, F), and CCT (G, H). Untreated 22rv1 tumors show tumor cell nests enveloped by dense blue-stained collagen deposits that are abundant in the peripheral areas (A). Tumor cell nests show their undifferentiated characteristics (B). SE seem to induce a differentiated phenotype with large tumor cells and prominent nuclei organizing in structures similar to glands (C, D). Vessels (D) appear dilated with unstructured capillary bed dispersed in collagen I deposits (azure staining). The differentiated appearance was greater after CR treatment (E, F) with smaller blood pervious vessels that follow the fibrous strands of collagen and surrounding the pseudoglandular acini (E). CR treatment induces deposition of fibrin clots (pale pink/orange staining) dispersed in dense collagen I deposits (fibrosis), enveloping tumor cell nests, as evolution of massive blood pouring. This appearance was not associated with presence of thrombotic vessels. CCT treatments (G, H) show a histological structure poorly differentiated (G) and rich in necrotic areas with the presence of numerous phagocytes (neutrophils and monocytes) dispensed in collagen I deposit resulting from a colliquative necrosis. Scale bar is 100 μm. (b) Immunohistochemical evaluation of E-cadherin in PC3 xenografts (I, J, K, L) treated or not with SE (J), CR (K), or CCT (L). Indirect immunoperoxidase staining of tumor xenograft samples was performed on paraffin-embedded tissue sections (4 μm). It is opportune to note the strong E-cadherin staining after SE and CR treatments whereas controls (CTRL) tissues were negative and CCT showed low levels of this antigen. Scale bar is 100 μm. (c) Western blotting performed on 22rv1 cell extracts derived from cultures treated for 8 days with SE (0.4 mg/mL), CR (0.4 mM), and CCT (0.1 mM). EMT markers (vimentin, β-catenin, and N-cadherin) were significantly reduced whereas epithelial markers (E-cadherin, K18, and PSA) were upregulated in 22rv1 cells. Interestingly, the expression levels of the truncated form (ligand-independent) of AR were significantly reduced after CR and CCT treatments whereas no changes were showed for full-length AR levels. (d) Densitometric analysis performed on E-cadherin and K18 expression in PC3 and 22rv1 cells treated with SE, CR, and CCT. (d) Time-dependent modulation of E-cadherin after treatments with SE (0.4 mg/mL), CR (0.4 mM), and CCT (0.1 mM) in the PC3 cell model (an in vitro assay). Western blots are representative of three different analyses.
