Each group of Mice bearing LLC was s.c. injected intratumorally with corresponding treatment as described in “”Methods”". Treatment with combination of cisplatin and Ad-Endo resulted in the marked inhibition of tumor growth and S3I-201 datasheet longer life span(P < 0.05). Inhibition of tumor-induced angiogenesis and increase of apoptosis in vivo Angiogenesis within tumor tissues was estimated in terms of microvessel density (by counting the number of microvessels) on the section stained with anti-mouse
CD31 antibody. The apoptotic tumor cells were determined by the TUNEL assay. Tumors of the SIS3 concentration control groups, treated with Ad-null or NS, showed larger microvessel count than those of the other groups submitted to cisplatin or/and Ad-Endo, especially the combination group (P < 0.05) (Figure 3). There was no difference in apoptotic index among all groups, but more apoptotic cells were seen in the group of chemotherapy or adenovirus treatment alone. Furthermore, the combination group showed the largest apoptotic index (Figure 4). Figure 3 Inhibition of angiogenesis within tumor estimated by CD31 immunohistochemical analysis. (A) were representative sections from each group. a: Ad-hEndo+ cisplatin;
b: Ad-hEndo; c: cisplatin; d: Ad-null; e: NS. (B) Vessel density was determined via counting the number of the microvessels per high-power field within hot spot area. Values were expressed as means ± SE (5 high power fields/slide). Tumors of the combination group showed smaller number of microvessel count than that of the other groups submitted to cisplatin or Ad-Endo alone, especially the NS (P < 0.05). a: Ad-hEndo+cisplatin; b: MG-132 in vivo Ad-hEndo; c: cisplatin; d: Ad-null; e: NS. Figure 4 Detection of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining of tumor tissues. (A) Sections after treatment were stained with the TUNEL analysis to detect apoptotic cells. (B) Apoptotic index was determined by calculating the percentage of apoptotic cells among tumor cells (5 high power fields/slide). The combination group showed the highest apoptotic
index. a: Ad-hEndo+ cisplatin; b: Ad-hEndo; c: cisplatin; d: Ad-null; tuclazepam e: NS. Inhibition of angiogenesis in the alginate encapsulation assay We examined the effect of endostatin on angiogenesis in vivo by the alginate encapsulation assay. Alginate beads containing lewis lung cancer cells were implanted s.c. on the back of C57BL/6 mice. Different treatments were performed in recipient mice. 14 d later, alginate implants containing LLC cells showed strong vascularization in the group of Ad-null or NS under the stereomicroscope. The FITC-dextran uptake was 62–77% higher in the group of Ad-null or NS than in the group of Ad-hEndo alone or in the combination treatment group and 11% more than in the group of cisplatin alone (Figure 5). Figure 5 Inhibition of antiangiogenesis assay by alginate bead in vivo. (A) Representative alginate beads from each group.