1. IGFBP7 and caspase-3, VEGF were mainly expressed in the cytoplasm of tumor cells. IGFBP7 was determined by fluorescent immunohistochemistry, positive staining of TRITC labeled IGFBP7 protein is red and Enzalutamide chemical structure localized in the cytoplasm, while GFP protein expressed by plasmids is green. The expression of caspase-3 and VEGF visualization is based on AEC staining. The results are consistent with our hypothesis, as show in Fig. 1. A-F that IGFBP7 and caspase-3 expression in the pcDNA3.1-IGFBP7 group is significantly higher in the pcDNA3.1-CONTROL
and B16-F10 cells groups (IGFBP7 P < 0.002, caspase-3 p < 0.004), but VEGF expression in the pcDNA3.1-IGFBP7 group is significantly lower in the pcDNA3.1-CONTROL and B16-F10 cells groups (P < 0.006) (Fig. 1. G-I) respectively, and no significant difference in IGFBP7 and caspase-3. VEGF expression
is found between the pcDNA3.1-CONTROL selleck chemical and B16-F10 cells groups (P > 0.05). According to these results determined by immunohistochemistry, there were significantly more apoptotic cells in the pcDNA3.1-IGFBP7 group than in the pcDNA3.1-CONTROL and B16-F10 cells groups (p < 0.031). As shown in Fig. 1. J-L, morphological characters of apoptotic cells are cell shrinkage, deformation, and loss of contact with neighbouring cells. Fig. 1. J shows more apoptotic cells in the pcDNA3.1-IGFBP7 group than in the pcDNA3.1-CONTROL (Fig. 1. K), and B16-F10 cells groups (Fig. 1. L), which contained almost the same numbers of apoptotic cells. The expression of IGFBP7 is positively correlated with caspase-3, Urocanase and cell apoptosis rate (rs = 0.704, rs = 0.806 respectively, LY3039478 molecular weight p < 0.01). However there is negative correlation between IGFBP7 and VEGF rs = -0.564, p < 0.01).
These results suggested that pcDNA3.1-IGFBP7 inhibited the proliferation of MM cells by up-regulating IGFBP7 and caspase-3 expression and down-regulating VEGF expression in vivo, resulting in slowing down of MM growth. Figure 1 Detection of IGFBP7, caspase-3, VEGF, and apoptosis expressed in homeograft tumors sections with original magnification × 100 in A-F, and ×400 in G-L. A shows significantly higher IGFBP7 expression in pcDNA3.1-IGFBP7. B demonstrates the successful transfection of pcDNA3.1 plasmid. C shows the physiological expression of IGFBP7 in melanoma (red color, as blue arrows indicate). D-F shows the effect of pcDNA3.1-IGFBP7 on caspase-3 expression in the cytoplasm of tumor sections, with strong expression in pcDNA3.1-IGFBP7 group seen in D, while weak expression in the pcDNA3.1-CONTROL and B16-F10 cell groups seen in E, F. G-I shows the expression of VEGF in vivo, with negative expression in most of cells in the pcDNA3.1-IGFBP7 group seen in G, while strong expression in the cytoplasm of pcDNA3.1-CONTROL and B16-F10 cell groups (red arrow represented) showed in H, I. J-L shows tumor apoptosis in vivo, with few apoptotic cells in pcDNA3.