Mechanism of Central Nervous System Leukemia Figure 2. Increased in vitro BBB permeability due to MMP-2 and -9 secreted by SHI-1, HL-60 and U937 cells. (A) Morphological changes of BMVECs affected by SHI-1, HL-60 and U937 cells treated with or without GM6001. (B) Rates of leukemic cell invasion of co-culture with BMEVCs compared with those of monoculture (without BMEVCs) (P..05) and those treated with GM6001 (*P,.001). Results are shown as the mean 6 SD of three independent experiments. (C) Zymography gelatinase secretion in SHI-1, HL-60 and U937 cells treated with or without GM6001. Original magnification: (A)6600. doi:10.1371/journal.pone.0020599.g002 SHI-1 cell’s invasion impaired by knock-down of MMP-2 or -9 genes and decreased disruption of ZO-1, claudin-5 and occludin with alleviative permeability of in vitro BBB To elucidate the specific contribution of MMP-2 and -9 in the role of the BBB opening, we silenced the genes expression of MMP-2 and -9 in SHI-1 cells by means of RNAi. The down- regulation of the mRNA expression was still effective with levels between 60% and 65% when determined 24 h after treatment with the respective siRNAs (Figure 4A). These results were confirmed by zymographic analysis of cultured supernatants and demonstrated successful blockage of MMP-2 and -9 productions (Figure 4B). Down-regulation of MMP-2 and -9 also impaired the invasive capacity of SHI-1 cells (data not shown). Furthermore, to examine the BBB disruption related to MMP-2 and -9 secreted by SHI-1 cells, RNAi-treated SHI-1 cells were cultured with BMVECs in the in vitro model of BBB. As shown in Fig. 4C, the rate of invasion of SHI-1 cells reduced when MMP-2 or -9 genes were knocked-down, which showed the relative weaker disruption of the BBB (P,.001). Consistently, immunofluorescence for ZO-1, Figure 3. Confocal imaging showed protein localization of ZO-1, claudin-5 and occludin in human BMEVCs. (A) Confocal imaging of ZO-1, claudin-5 and occludin of human BMEVCs; (B, C, D) BMEVCs co-cultured with SHI-1, HL-60 and U937 cells with or without treatment by GM6001. (E) BMEVCs of co-cultured with SHI-1 cells after knock-down of the MMP-2/MMP-9 gene. Data in all panels are representative of at least 3 separate experiments on 3 distinct cultures. (Scale bars: 10 mm). doi:10.1371/journal.pone.0020599.g003 PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e20599