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Figure 8: In vivo testosterone-HSA expression in APC mice

Confocal laser scanning microscopic analysis of APCMin/+ colon tumor frozen sections stained with testosterone-HSA-FITC, showing specific FITC-related fluorescence at the cell membranes. Visualization of nuclei was evident by DRAQ5™ staining. Magnification, ×100.

4.3 mAR activation by testosterone-HSA was followed by extensive reduction of tumor incidence in vivo

Having a clear indication for mAR-expression, the 12-week tumor incidence of colon tumors generated in Balb/c mice was assessed by chemical carcinogenesis (see Experimental Procedures) in the presence or absence of continuous testosterone-HSA treatment. The animals used for these studies were divided in two groups comprising 5 and 7 animals. One group (7 animals) was treated subcutaneously (3 times/week, for 12 weeks) with 5mg/kg testosterone-HSA, whereas the other group (5 animals) remained untreated. The results (Figure 9, A) show that testosterone-HSA-treatment produced a clear and significant reduction of tumor incidence by 65%. The histological analysis of tumors by Tunel assay (Figure 9, B) confirmed that apoptotic cells were present in significant numbers predominantly at the tumors of animals treated with testosterone-HSA, while they were significantly less in the non-treated animals. These results collectively show that mAR is a functional target that may be used for the selective elimination of colon cancer cells in vivo.

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. Visualization of nuclei was evident by DRAQ5™ staining.

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. The animals used for these studies were divided in two groups comprising of 5 and 7 animals respectively. One group (7 animals) was treated subcutaneously (3 times/week for 12 weeks) with 5 mg/kg testosterone- HSA, whereas the other group (5 animals) remained untreated. The results (Fig. 7C) show that testosterone- HSA treatment produced a clear and significant reduction of tumor incidence by 65%. The histological analysis of tumors by TUNEL assay confirmed that apoptotic cells were present in significant numbers predominantly in the tumors of animals treated with testosterone- HSA (Fig. 7D, middle panels), while they were significantly less either in the non-treated animals (Fig. 7D, right panels), or in healthy tissues of treated animals (Fig. 7D, left panels). These results collectively show that mAR is a functional and specific target that may be used for the selective elimination of colon cancer cells in vivo.

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