Chlorotoxin-Fc Fusion Inhibits Release of MMP-2 from Pancreatic Cancer Cells
Figure 2
(a) Gelatinase activities of conditioned media (CM) from cultured PANC-1 cells. Cells were treated with 0, 12, 60, and 300 nM human IgG, chlorotoxin (CTX), and M-CTX-Fc at 1, 3, 6, 12, and 24 h. Each 15 μL of the CM were subjected to gelatin zymography. (b) PANC-1 cells were seeded onto 12-well plates at a density of 1 × 105 cells/well and cultured. After 20 h of culture, M-CTX-Fc at a range of 0–300 nM was added to each well for 24 h, and then the cells were trypsinized and counted. (c) M-CTX-Fc decreased MMP-2 release in PANC-1 cells. PANC-1 cells were treated with incremental concentrations of human IgG, CTX, and M-CTX-Fc for 24 h. The CM were collected and concentrated. The protein in the CM was separated using 10% SDS-PAGE, transferred to a PVDF membrane, and probed with monoclonal MMP-2 antibody. (d) qRT-PCR analysis of MMP-2 expression in the absence and presence of M-CTX-Fc. qRT-PCR analysis was performed using primers for MMP-2. Primers specific for GAPDH were used as an internal control. PCR products were separated using 2% TAE agarose gel electrophoresis.
