FIGURE 4.
Induction of tumor cell death by PIIBNP is integrin αv-dependent but substrate-independent. A, 24-well plates were coated with type I collagen. 4 μm GST, PIIBNP, or mutant PIIBNP was incubated with hCh-1 cells in serum-free medium for 3 days. Cells were imaged by light microscopy using a Q Capture Retiga 2000R camera (×20 magnification). The cells were also analyzed by trypan blue exclusion assays as described under “Experimental Procedures,” and the percentage of cell death was calculated and presented. hCh-1 cells (B) and MDA-MB231 cells (C) were incubated with different concentrations of PIIBNP (♦), mutant PIIBNP (□), or GST (▴) for 90 and 41 h. The values represent means ± S.D. for three independent assays. D, hCh-1 cells, HeLa cells, and MB231 cells were plated on type I collagen, fibronectin, or matrigel and incubated with 4 μm GST (white bar) or PIIBNP (black bar) for 90 h (hCh-1), 24 h (HeLa), or 41 h (MB-231). Cell viability was analyzed as described above. The values represent means ± S.D. for three independent assays. E, the control siRNA-treated and anti-integrin αv siRNA-treated hCh-1 cells were plated in 24-well plates previously coated with type I collagen and incubated without or with 4 μm GST, PIIBNP, or mutant PIIBNP in a serum-free medium for 90 h. PIIBNP reduced only control siRNA-treated cell number (white bar) and not the anti-siRNA integrin αv-treated cell number (black bar). F, the integrin αv expression levels were analyzed by Western blotting for hCh-1, anti-integrin αv siRNA-treated hCh-1, control siRNA-treated hCh-1, HeLa, MDA-MB231, and human chondrocytes.