Chen et al. Journal of Experimental & Clinical Cancer Research 2010, 29:13 http://www.jeccr.com/content/29/1/13 Page 5 of 8 and down-regulating VEGF expression in vivo, resulting in slowing down of MM growth. As to show the exactitude of our experiment design, we used pcDNA3.1-IGFBP7 simultaneously expressed GFP and IGFBP7 rather than pcDNA3.1 plasmid con- taining only IGFBP7 gene. That was because, if we used pcDNA3.1 plasmid only containing IGFBP7 gene, we could not estimate the transfection efficiency in-vivo experiments, and moreover, we could not discriminate whether high level of IGFBP7 expression in xenograft sections dued to plasmid transfection or physiological IGFBP7 synthesis of melanoma. Well, pcDNA3.1- IGFBP7 simultaneously expressed GFP and IGFBP7 could solve both of the problems, as shown in additional files 3, Figure S2. We evaluated apoptosis-induced effect in melanoma cells of pcDNA3.1 only containing IGFBP7 gene, and in those of pcDNA3.1-IGFBP7 simultaneously expressed GFP and IGFBP7, finding out that insersion of GFP would not affect the expression of IGFBP7, as shown in additional files 3, Figure S1. Discussion It has been confirmed that transfection with anti-tumor plasmids is more specific, more efficient, and longer last- ing for anti-tumor therapy than recombinant protein. Transfection of anti-tumor plasmids may have some advantages over the application of rIGFBP7, namely the less danger of immunological rejection and the low cost of synthesis and purification [3]. In addition, MM cells transfected with eukaryotic expression plasmids could have stable and effective expression of IGFBP7 gene. Our research demonstrated that pcDNA3.1-IGFBP7 vector promotes expression of IGFBP7 specifically and have a long-lasting effect. However, it is conflicting to our hypothesis that IGFBP7 expression should ascensus, but it was attenuate over time. The possible explanation for this phenomenon was attributed to the high performance of PCMV promoter contained in pcDNA3.1-IGFBP7, which would exhaust and be toxic to tumor cells since it ad infinitum synthesized IGFBP7. Meanwhile augmenta- tion of IGFBP7 in cell supernatant would induce apopto- sis of part of tumor cells and therefore, the synthesis of IGFBP7 also decreases with reduction of tumor cells. To determine therapeutic potential of pcDNA3.1- IGFBP7 in vitro, we analyzed cells viability and apoptosis rates by the Cell Counting Kit-8 and FCM. Our results are consistent with the research of Sprenger [13], which indi- cated that the growth of a tumorigenic SV40 prostate cell line, M12, was suppressed by transfecting the IGFBP-rP1 cDNA. Also, prostatic carcinoma cells were stably trans- fected with IGFBP7 cDNA and showed poor tumorigeni- city [21]. Moreover, IGFBP7 which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce apoptosis [9], but it is contradictory to some researcher’s findings, as they indicated that IGFBP7 was highly overexpressed in glioma tissues, med- iateing glioma cell growth, and migration [22]. In addition, the expression pattern of IGFBP7 varies with tumor types. Both up-regulated expression [23] and down-regulated expression [24,25] of IGFBP7 is observed in different types of cancer. In our previous study, we found that IGFBP7 expression was low in B16-F10 cells. Vladislava [26] also indicated that unlike human melanomas, the murine mela- noma cell lines (B16-F10) did not have activating muta- tions in the Braf oncogene at exon 11 or 15, however, there were distinct patterns of mutation in the ras gene. RAS proteins are membrane-bounded small G proteins, and RAF, MEK, and ERK are cytosolic protein kinases that form a tiered protein kinase cascade downstream of RAS, whereas ARAF and CRAF are not mutated because their regulation is fundamentally different from that of BRAF. As a consequence, RAS is mutated in melanoma, the cells (B16-F10) switch their signaling from BRAF to CRAF [27], then IGFBP7 expression is decreased, enabling the cells to escape from senescence and resulting in uncontrolled pro- liferation. Accordingly, RAS-CRAF-MEK-ERK pathways contribute to the development of murine melanoma. Transfection of pcDNA3.1-IGFBP7 into B16-F10 cells, upgraded the expression of IGFBP7, which inhibits CRAF- MEK-ERK signaling through an autocrine/paracrine path- way, thereby restraining proliferation and activates apopto- sis. Together, these results suggest that IGFBP7 plays different roles in different tumor or host environments. Therefore, we need to evaluate the therapeutic potential of pcDNA3.1-IGFBP7 on B16-F10 in vivo. Although the apoptosis-inducing effect of pcDNA3.1- IGFBP7 in cultured cells was shown for in vitro applica- tions, its therapeutic applications in vivo represent an alto- gether more daunting challenge. To elevate transfection efficiency, we employed Invivofectamine (a new in vivo plasmid delivery reagent) to carry pcDNA3.1-IGFBP7 transfected into tumors tissue. Fortunately, our data clearly showed that intratumoral injection of the Invivofec- tamine pcDNA3.1-IGFBP7 complex was able to slow down the growth of B16-F10 MM homograft, and its transfection efficiency was about 70%. Most importantly, it had a lasting effect on tumor development, being effective for at least 20 days, because stable expression of IGFBP7 by using pcDNA3.1-IGFBP7. We focused on the therapeu- tic mechanisms of the Invivofectamine pcDNA3.1-IGFBP7 complex in B16-F10 MM homograft. The antitumor research of IGFBP has provided evidence that IGFBPs may have both IGF-dependent and independent actions. We hypothesized that IGFBP7 can inhibit MM gowth by IGF-dependent way [14], and reduce VEGF expression through preventing IGF-Ibinding to its receptors. In addi- tion, IGFBP7 induces MM apoptosis through a novel IGF-independent pathway. To confirm the presumption,