Chen et al. Journal of Experimental & Clinical Cancer Research 2010, 29:13 http://www.jeccr.com/content/29/1/13 Page 6 of 8 we studied IGFBP7, caspase-3, VEGF expression and apoptosis in tumor homograft tissues. The results of the immunohistochemistry and TUNEL showed that, IGFBP7 and caspase-3 expression in pcDNA3.1-IGFBP7 group are significantly higher than in pcDNA3.1-CONTROL and B16-F10 cells groups, but VEGF expression in the pcDNA3.1-IGFBP7 group were significantly lower than both in control groups, and no significant difference in IGFBP7 and caspase-3, VEGF expression were found between the pcDNA3.1-CONTROL and B16-F10 cells groups. According those results determined by immuno- histochemistry, there were significantly more apoptotic cells in the pcDNA3.1-IGFBP7 group than in control groups. This was considered possibly to relate to IGFBP7 promote apoptosis effectiveness. However, our finding contrasted with the results of Adachi [28] et al, who found that high expression of IGFBP7 in invasive tumor cells was associated with poor prognosis. This discrepancy may be due to the difference in the immunohistochemical scor- ing [20,29]. We used the composite score to evaluate the expression of IGFBP7, which seems to be one of the most promising and accurate scoring systems currently defined. Futhermore, we demonstrated that the expression of IGFBP7 is positive correlation with caspase-3, and cell apoptosis rate. In addition, there is negative correlation between IGFBP7 and VEGF. Those results suggested that pcDNA3.1-IGFBP7 can up-regulate IGFBP7, caspase-3 expression, and down-regulate VEGF expression in vivo to inhibit the proliferation of MM cells, which resulted in slowing down of MM growth, as shown in additional files 4. Angiogenesis is essential for tumor development, and the increasing evidences show that IGF-I plays a crucial role in tumor growth by up-regulating the VEGF expres- sion and neovascularisation [30]. A recent study indicated that IGFBP7 might exhibit angiogenesis-modulating prop- erties, reducing VEGF expression by regulating IGF avail- ability in body fluids and tumor tissues and modulating combination of IGF-I to its receptors [30,31]. Moreover the reduction of VEGF-induced tube formation by IGFBP7 could be mainly mediated by inhibition of MAP kinase cascade through c-Raf, and BRAF-MEK-ERK sig- nalling [32], Although our research implied IGFBP7 blocks VEGF-induced angiogenesis and VEGF expression by interfering with IGF-I, its role in tumor angiogenesis remains poorly understood. The mechanisms by which IGFBP7 induced apoptosis and inhibit neovascularization should be further explored. Conclusion Our data show that increasing IGFBP7 expression by using the pcDNA3.1-IGFBP7 plasmid suppresses MM growth, induces apoptosis and reduces VEGF in vitro and in vivo. Intratumoral injection of pcDNA3.1- IGFBP7 holds promise as a clinical gene therapy approach for MM, which provide a framework for further studies of its broader applicability to a range of human tumors. However, there are several insufficien- cies on this therapeutics. Firstly, it would be difficult to make uniform distribution of pcDNA3.1-IGFBP7 in tumor tissue by intratumoral injection of invivofectamin, and a transferrin-polyethylenimine (Tf-PEI) delivery sys- tem (our previous studies) needs to be used in the further study. Secondly, there are no suitable MM cell lines available that express high level of IGFBP7 to prove the specificity of anti-tumor effect of pcDNA3.1- IGFBP7. Moreover, many biological roles of pcDNA3.1- IGFBP7 remain to be elucidated. Additional file 1: pcDNA3.1-IGFBP7 plasmid checked by restriction enzyme analysis, and transfection with Effectene authenticated by immunofluorescence. Restriction enzyme analysis of pcDNA3.1-IGFBP7 plasmid by EcoR I and Bgl II manifested that the obtained plasmid was the objective one with predicted length. Plasmid transfection with Effectene was successful, authenticated by immunofluorescence. Click here for file [ http://www.biomedcentral.com/content/supplementary/1756-9966-29- 13-S1.PDF ] Additional file 2: Effect of pcDNA3.1-IGFBP7 plasmid on IGFBP7 expression in vitro. Higher concentration of pcDNA3.1-IGFBP7 plasmid led to higher IGFBP7 mRNA and protein expression in B16-F10 melanoma cells, detected by RT-PCR and western blot. pcDNA3.1-IGFBP7 transfection led to reduction of B16-F10 cells viability, determined by the Cell Counting Kit-8. Click here for file [ http://www.biomedcentral.com/content/supplementary/1756-9966-29- 13-S2.PDF ] Additional file 3: Effect of different plasmids on tumor cell apoptosis rate detected by flow cytometry and laser scanning confocal microscopy. Apoptosis rate detected by flow cytometry of B16 melanoma resulted in an obvious increase in pcDNA3.1-IGFBP7 group than those in pcDNA3.1-CONTROL and B16 groups, consistent with laser confocal display of tumor sections of the three groups, suggested significant effects of in- vitro and in-vivo pcDNA3.1-IGFBP7 transfection on B16 apoptosis. Click here for file [ http://www.biomedcentral.com/content/supplementary/1756-9966-29- 13-S3.PDF ] Additional file 4: In-vivo anti-tumor effect of pcDNA3.1-IGFBP7 plasmid. Survival curves and tumor volumes showed different effects of the three groups. pcDNA3.1-IGFBP7 group has a significantly higher survival rate and smaller tumor size, compared to pcDNA3.1-CONTROL and B16-F10 groups. Click here for file [ http://www.biomedcentral.com/content/supplementary/1756-9966-29- 13-S4.PDF ] Abbreviations IGFBP7: insulin-like growth factor binding protein 7; MM: malignant melanoma; VEGF: vascular endothelial growth factor; TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end labelling. Acknowledgements We thank Ming jian Yang for technique guidance, and Hoi Lun Lau for editing the manuscript. This project was supported by the National Science Fund Program from the National Natural Science Foundation of China (No. 30700717).