Figure 3.

Angiopoietin2, BiP, and Hsp90α are key mediators of the anti-angiogenic effect of simvastatin. (A) CRC cells were incubated with 0.2 μM simvastatin for 3 days, and cell lysates were subjected to the immunoblot assay. Angiopoietin2 and Hsp90α were decreased in all eight CRC cells, and BiP also tended to decrease, except in RKO, SW48, and SW403. (B) HUVEC invasion assay using conditioned media, and HUVEC invasion assay after addition of angiopoietin2 (Ang2) (0.1 μg ml⁻¹), BiP (1 μg ml⁻¹), or Hsp90α (1 μg ml⁻¹) active protein. Addition of these proteins reversed the anti-angiogenic effect of simvastatin. (C) Conditioned media of CRC cells transfected with 20 nM siRNAs suppressed HUVEC invasion. The immunoblot analysis confirmed loss of angiopoietin2, BiP, and Hsp90α protein expression (size bar, 400 μm).