Chen et al. Journal of Experimental & Clinical Cancer Research 2010, 29:13 http://www.jeccr.com/content/29/1/13 Page 2 of 8 potential tumor suppressor role against colorectal carci- nogenesis. Moreover, Wajapeyee, [9] et al showed that recombinant IGFBP7 (rIGFBP7) induced apoptosis in melanoma cell lines, efficiently. These exciting data sug- gested that IGFBP7 may be an efficacious anticancer agent, since experiments have provided evidences that IGFBPs have both IGF-dependent and IGF-independent antitumoral actions [13,14]. Recent data also demonstrated that a prostatic carci- noma cell line stably transfected with IGFBP7 cDNA showed poor tumorigenicity both in vitro and in vivo [10]. Meanwhile, in our previous study, we found that IGFBP7 expression was low in B16-F10 cells. However, it is still unclear whether IGFBP7 cDNA inhibits prolif- eration of B16-F10 cells in vitro or B16-F10 MM growth in vivo. Therefore, in the present study, we constructed the pcDNA3.1-IGFBP7 plasmid as an antitumor agent to investigate whether it is effective in treating mice bearing B16-F10 melanoma tumor. Methods Plasmid construction The pcDNA3.1-IGFBP7 expression plasmid was con- structed. IGFBP7 gene (GenBank ID: 29817 No. AK156315.1) was amplified by RT-PCR from mRNA of splenocytes derived from C57BL/6J mice (IGFBP7 fw: 5’GAAGATCTATGGAGCGGCCGTCGCT-3’, IGFBP7 rev: 5’-CGGAATTCTTTATAGCTCGGCACCTTCA- CCT-3’). IGFBP7 cDNA was purified by Shanghai Biolo- gical Engineering Company. The eukaryotic vector expressing eGFP and IGFBP7 was termed as pcDNA3.1- IGFBP7, and pcDNA3.1-CONTROL only expressed eGFP. The inserted sequences were verified by DNA sequencing, and digested by restriction endonuclease (EcoRI, and Bgl II enzyme). Tumor cells and in vitro transfection with pcDNA3.1- IGFBP7 B16-F10 cells were purchased from the Institute of Cell Biology (Shanghai institute for biological sciences). Cells were seeded in six-well plates (2 × 105 cells per well), cultured overnight at 37°C in 5% CO2, and grown to 60% confluence prior to transfection. Transfection with pcDNA3.1-IGFBP7 was performed by Effectene Trans- fection Reagent (QIAGEN Companies) according to the manufacturer’s instructions. Cells transfected with pcDNA3.1-CONTROL and those without any transfec- tion served as controls. The experimental and two con- trol groups were termed pcDNA3.1-IGFBP7, pcDNA3.1- CONTROL and B16-F10 cells, respectively. All experi- ments were preformed in triplicate and repeated at least twice. RT-PCR and gelelectrophoresis Total RNA from 1 × 106 cultured cells was extracted using the TRIZOL reagent (Invitrogen, San Diego, U.S.A.). Then RNA was reversely transcribed and synthesized to cDNA using Reverse transcriptase (Rever TraAce-a-TM, TOYOBO, JAPAN). Primer sequences for IGFBP7 (fw: 5’-GTAAGGAGGACGCTGGAGAGT-3’, rev: 5’-CTGG- CTGTAATAAAGTGTTAGTGG-3’) and b-actin (fw: 5’-CCGTGAAAAGTGACCCAG-3’ rev: 5’-TAGCCAC- GCTCGGTCAGG-3’). PCR and gelelectrophoresis condi- tions were described as previous [3]. The expected size of fragment of IGFBP7 and b-actin was 255 bp, 136 bp, respectively. Analysis of Cell Viability Cell viability was determined by the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) and measured by microplate reader scanning at 450 nm as previously described elsewhere [15]. Quantification of cell apoptosis by flow cytometry B16-F10 cells were washed by PBS and collected after digestion by 0.25% trypsin, cell suspension was added dropwise to PBS while gently vortexed, then centrifuged at 1000 rpm at 4°C for 10 min. After resuspension of the cells in labeling buffer, 10 μl Annexin VFITC was added and then incubated in the dark. Following 150 μL of propidium iodide (PI) was added, the cells were incu- bated for 2 h at room temperature. Then cell apoptosis was measured by flow cytometry [16,17]. Mice Thirty-six six-week-old female Wild-type C57BL/6J mice weighing 18-25 g were treated in accordance with the guidelines of the National Institutes of Health for the humane treatment of animals, and all animal protocols were approved by Huazhong University of Science and Technology’s animal care and use committee. Mice were anesthetized with urethane (1.9 g/kg sc; 12.5 mg urethane/ ml 0.9% saline; Sigma Chemical, St. Louis, MO), and their temperature was maintained at 37°C[18]. 1 × 104 B16-F10 cells were injected subcutaneously in the lower backs of mice, where MM emerged after 1 week. Tumor volume (v) was calculated as follow, v = L × I2 × 0.52, where L and I represent the maximum and minimum tumor diameter measured weekly. All the mice were divided into three groups randomly (n = 12 each group), termed pcDNA3.1- IGFBP7, pcDNA3.1-CONTROL and B16-F10 cells groups respectively. Then Invivofectamine reagent-plasmid duplex complexes 200 μl (Reagent for in vivo plasmid delivery, Invitrogen, U.S.A), containing pcDNA3.1-IGFBP7 (1 μg), or pcDNA3.1-CONTROL (1 μg), DMEM 200 μl were