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| 4.2 mAR expression in 2 different colon cancer animal models
The findings provided so far indicate that mAR are expressed in colon cancer cell lines Caco2 and HCT-116 in vitro. [Gu S, et al. 2009] Thus, we aimed to further evaluate the in vivo effects of albumin-conjugated androgens in colon cancer animal models. To this end we first estimated the expression of mAR in colon tumors generated in Balb/c mice and APC mice. As shown in figure 7, using testosterone-HSA-FITC we detected specific, FITC-related fluorescence in membrane specimens of Balb/c mice colon tumors (Fig. 7 A, a, a1), and APC mice colon tumors (Fig.8). No apparent staining could be identified in tissues labeled with HSA-FITC (Fig. 7A, b) and no apparent staining could be found in healthy tissue labeled with testosterone-HSA-FITC (Fig. 7B). Figure 7: In vivo testosterone-HSA expression in BALB/c mice A) Confocal laser scanning microscopic analysis of BALB/c colon tumor frozen sections stained with testosterone-HSA-FITC (a, a’), showing specific FITC related fluorescence at the cell membranes. No apparent membrane fluorescence was shown in control samples stained with HSA-FITC (b). B) Confocal laser scanning microscopic analysis of BALB/c colon tumor and healthy frozen sections stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes of tumor sections. |
Membrane staining of mAR in colon tumors and colon cancer cells. Confocal laser scanning microscopic analysis of WCL2 (a-c) and MCL2 (d-f) colon tumor specimens and HCT116 cells (g-j) stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes. Control staining with HSA-FITC showed no apparent membrane fluorescence.
A) Confocal laser scanning microscopic analysis of Caco2 cells (a-d) stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes (a, b). No apparent membrane fluorescence was shown in control samples stained with HSA-FITC (c, d). Visualization of nuclei was evident by DRAQ5™ staining. Magnification, ×100. B) Confocal laser scanning microscopic analysis of IEC 06 cells (e-f) stained with testosterone-HSA-FITC, showing no apparent membrane fluorescence at the cell membrane. conjugated androgens in colon cancer animal models. To this end, we first estimated the expression of mAR in colon tumors generated in Balb/c mice. As shown in fig. 7A and fig. 7B, using testosterone-HSA-FITC we detected specific, FITC-related fluorescence in membrane specimens of Balb/c mice colon tumors (Fig. 7Aa, a'), while no apparent staining could be identified in tissues labeled with HSA-FITC (Fig. 7Ab). Interestingly, mAR staining was very low in healthy colon tissue specimens of Balb/c mice (Fig. 7B) further supporting earlier findings pointing to cancer tissue specificity of mAR [31]. Having a clear indication for mAR-expression, we assessed the 12-week tumor incidence of colon tumors generated in Balb/c mice by chemical carcinogenesis (see Materials and Methods) in the presence or absence of continuous testosterone- HSA treatment. In vivo testosterone-HSA effects on tumor incidence in BALB/c mice. A) Confocal laser scanning microscopic analysis of BALB/c colon tumor frozen sections stained with testosterone-HSA-FITC (a, a'), showing specific FITC related fluorescence at the cell membranes. No apparent membrane fluorescence was shown in control samples stained with HSA-FITC (b). Visualization of nuclei was evident by DRAQ5™ staining. Magnification, ×100. (B) Confocal laser scanning microscopic analysis of BALB/c colon tumor and healthy frozen sections stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell membranes of tumor sections (a). Very low membrane fluorescence was shown in healthy colon sections stained with testosterone-HSA-FITC |
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