The tumor vascular targeting agent combretastatin A–4-phosphate induces reorganization of the actin cytoskeleton and early membrane blebbing in human endothelial cells

Abstract

Combretastatin A–4-phosphate (CA-4-P) is a tubulin-binding compound currently in clinical trial as a tumor vascular-targeting agent. In endothelial cells, CA-4-P is known to cause microtubule depolymerization, but little is known about its subsequent effects on cell morphology and function. Here, we demonstrate that within minutes of endothelial cell exposure to CA-4-P, myosin light chain (MLC) was phosphorylated, leading to actinomyosin contractility, assembly of actin stress fibers, and formation of focal adhesions. These cytoskeletal alterations appeared to be a consequence of Rho activation, as they were abolished by either the Rho inhibitor C3 exoenzyme or Rho-kinase inhibitor Y-27632. In response to CA-4-P, some cells rapidly assumed a blebbing morphology in which F-actin accumulated around surface blebs, stress fibers misassembled into a spherical network surrounding the cytoplasm, and focal adhesions appeared malformed. Blebbing was associated with decreased cell viability and could be inhibited by Rho/Rho-kinase inhibitors or by blocking the CA-4-P–mediated activation of stress-activated protein kinase-2/p38. The extracellular-regulated kinases 1 and 2 (ERK-1/2) were shown to protect against blebbing since blebbing was attenuated on ERK-1/2 stimulation and was up-regulated by specific inhibition of ERK-1/2 activation. The use of MLC kinase (MLCK) and myosin adenosine triphosphatase inhibitors led us to propose a role for MLCK and myosin activity independent of MLC phosphorylation in regulating the blebbing process. CA-4-P–mediated contractility and blebbing were associated with a Rho-dependent increase in monolayer permeability to dextrans, suggesting that such functional changes may be important in the rapid response of the tumor endothelium to CA-4-P in vivo.