DMSO I group; Figure 2E and F) and the treatment groups (ZM198,615 or Montelukast vs. DMSO II group; Figure 2G and H). Figure 2 Effects of CysLT1R antagonists on HCT-116 xenograft tumor proliferation and apoptosis. The effects of the CysLT1R antagonists on tumor vascularization were studied by staining for CD31, an endothelial cell-specific antigen. We observed a slightly decreased vessel till number in the sections taken from the Pre-ZM group compared to the DMSO I group (46.1��6.7 vs. 56.0��7.9; Figure 3A and B). Vascular number is not the only parameter to indicate adequate tumor blood supply; vessel area is also a critical determinant of tumor blood flow . In tumor sections from the Pre-ZM group, we noticed that the vessels appeared smaller and thinner, and had less branching.
The tumor vessels in the DMSO I group appeared more mature with lumens, thick walls, and strong CD31 staining along their lengths. We therefore measured the CD31-positive staining areas. As shown in Figure 3C, tumors from the Pre-ZM198,615 group had a statistically significant (P<0.05) decreased mean of the CD31-positive area compared to tumors in the DMSO I group (2596��121.4 pixels vs. 3900��522.3 pixels, respectively), corresponding to a 33% reduction. There were no statistically significant differences in the mean number of vessels and vascular size among mice in the treatment groups (DMSO II vs. ZM198,615 or Montelukast; Figure 3D, E, and F). The reduced vascular size in the tumor sections taken from the Pre-ZM group indicated that CysLT1R antagonist treatment for 21 days could inhibit tumor vascularization and have a more pronounced effect on tumor progression.
Figure 3 Effects of CysLT1R antagonists on HCT-116 xenograft tumor angiogenesis. Next, the expression levels of selected proteins involved in the cell cycle, apoptosis, and angiogenesis were investigated. p21WAF/Cip1, a potential cell cycle inhibitor, was shown to be significantly upregulated in tumor samples from the Pre-ZM group compared to the DMSO I group (P<0.01; Figure 4A). We also observed moderately increased levels of cleaved caspase 3 fragments (Figure 4B) and significantly decreased expression levels of VEGF (P<0.05; Figure 4C) in tumors from the Pre-ZM group compared to the DMSO I group. Similar analysis were made for the treatment groups (ZM198,615 or Montelukast vs. DMSO II).
Significantly increased expression levels of p21WAF/Cip1 (P<0.01; Figure 3D) and decreased expression levels of VEGF (P<0.05; Figure 4D) could be observed for the Montelukast-treated group, but not for the ZM198,615-treated group compared to the DMSO II group. Increased levels of cleaved caspase 3 fragments were also observed in the treatment groups (ZM198,615 or Montelukast Dacomitinib vs. DMSO II) (Figure 4E). Figure 4 Effects of CysLT1R antagonists on cell cycle, apoptosis, and angiogenesis in HCT-116 xenograft tumors.