The tumor vascular targeting agent combretastatin A-4-phosphate induces reorganization of the actin cytoskeleton and early membrane blebbing in human endothelial cells.
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.
Kanthou
Chryso
C
Tumour Microcirculation Group, Gray Cancer Institute, Mount Vernon Hospital, PO Box 100, Northwood, Middlesex HA6 2JR, UK. kanthou@gci.ac.uk
Tozer
Gillian M
GM
D000199:Actins / Q000187:drug effects* / Q000378:metabolism; D000209:Acute-Phase Proteins / Q000187:drug effects / Q000494:pharmacology / Q000502:physiology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002199:Capillary Permeability / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D003599:Cytoskeleton / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000648:ultrastructure*; D022001:Focal Adhesions / Q000187:drug effects; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000494:pharmacology / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000494:pharmacology / Q000502:physiology; D009218:Myosins / Q000494:pharmacology / Q000502:physiology; D009336:Necrosis; D013267:Stilbenes / Q000494:pharmacology*; D022502:Stress Fibers / Q000187:drug effects
D000199:Actins; D000209:Acute-Phase Proteins; D000972:Antineoplastic Agents, Phytogenic; D013267:Stilbenes; C010781:acute-phase protein rho; D009219:Myosin-Light-Chain Kinase; D020928:Mitogen-Activated Protein Kinases; D009218:Myosins; C058728:fosbretabulin