Chen et al. Journal of Experimental & Clinical Cancer Research 2010, 29:13 http://www.jeccr.com/content/29/1/13 Page 4 of 8 marked apoptosis was observed in pcDNA3.1-CONTROL (6.1%) and B16-F10 groups (5.3%). Our finding mentioned above indicates that the long-term IGFBP7 expression possibly establishes a desirable basis for the therapeutic effect in vitro. Effect of pcDNA3.1-IGFBP7 on IGFBP7 expression and growth of MM homeograft in vivo To evaluate the therapeutic potential of pcDNA3.1- IGFBP7 on B16-F10 MM homeograft in vivo, we per- formed intratumoral injection of pcDNA3.1-IGFBP7 to study the effect on carcinogenesis. The results showed that pcDNA3.1-IGFBP7 inhibited tumor growth, at the time of killing, the volumes of MM in B16-F10 cell group and pcDNA3.1-CONTROL group were 587 ± 35 mm3 and 566 ± 34 mm3, respectively, being about 6-fold increase over the starting volume; whereas the volume of B16-F10 tumors injected with pcDNA3.1-IGFBP7 were 256 ± 25 mm3, with the volume increase being only 2.8- fold. The delay in tumor growth was statistically significant (P < 0.001). To evaluate the expression of IGFBP7 in tumor homeograft, the proteins were determined by wes- tern blotting. IGFBP7 expression in the pcDNA3.1- IGFBP7 group was significantly higher than in pcDNA3.1- CONTROL and B16-F10 cells groups (p < 0.01), whereas there was no significant difference in IGFBP7, expression was found between pcDNA3.1-CONTROL and B16-F10 cells groups (p > 0.05). Transfection of pcDNA3.1-IGFBP7 in vivo not only inhibited MM growth in C57BL/6J mice, but also prolonged C57BL/6J mice survival bearing B16- F10 melanoma tumor. Effect of pcDNA3.1-IGFBP7 on IGFBP7, caspase-3, VEGF and apoptosis expression in vivo To investigate the effect of pcDNA3.1-IGFBP7 on IGFBP7, caspase-3, VEGF expression, and MM apoptosis in vivo, we performed fluorescent immunohistochemistry and cytometry. As shown in Fig. 1. IGFBP7 and caspase-3, VEGF were mainly expressed in the cytoplasm of tumor cells. IGFBP7 was determined by fluorescent immunohis- tochemistry, positive staining of TRITC labeled IGFBP7 protein is red and localized in the cytoplasm, while GFP protein expressed by plasmids is green. The expression of caspase-3 and VEGF visualization is based on AEC stain- ing. 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 signifi- cant difference in IGFBP7 and caspase-3. VEGF expression is found between the pcDNA3.1-CONTROL and B16-F10 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 pcDNA3.1-CONTROL (K) and B16-F10 cell groups (L), while increased apoptosis in the B16-F10 tissue in mice treated with pcDNA3.1-IGFBP7 (J) (red arrow shows deep blue cells). cells groups (P > 0.05). According to these results deter- mined 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 charac- ters 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, and cell apoptosis rate (rs = 0.704, rs = 0.806 respectively, 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