The western blot showed that pcDNA3.1-IGFBP7 increased the expression of IGFBP7. Results are consistent with previous determined by RT-PCR. According to these results detected by RT-PCR and western blot, the IGFBP7 expressed in the pcDNA3.1-IGFBP7 group were significantly higher in the pcDNA3.1-CONTROL and B16-F10 cells groups (p < 0.03), as shown in additional files 2, Figure S2. pcDNA3.1-IGFBP7 suppresses B16-F10 cells growth in vitro The proliferation of pcDNA3.1-IGFBP7-transfected cells was significantly suppressed compared with control cells (P
< 0.01). The highest suppression effect of pcDNA3.1-IGFBP7 was found at 48 h post-transfection, and no significant difference in proliferation between pcDNA3.1-CONTROL and untransfected cells was observed (P > 0.05), indicating that transfection of pcDNA3.1-IGFBP7 #CHIR-99021 molecular weight randurls[1|1|,|CHEM1|]# blocks the proliferation of B16-F10 cells by increasing IGFBP7 synthesis and secretion, as shown in additional files 2, Figure S3. To evaluate apoptosis-induced effect of pcDNA3.1-IGFBP7 in melanoma cells, B16-F10 cells at 48 h post-transfection was monitored by FCM. The apoptosis rate in pcDNA3.1-IGFBP7 group (24.6%) was significantly higher than that in control groups (P < 0.01). However, no 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 learn more desirable basis for the therapeutic effect in vitro. Effect of pcDNA3.1-IGFBP7 Celastrol 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 performed 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 western 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.