The influence of hypoxia and energy depletion on the response of endothelial cells to the vascular disrupting agent combretastatin A-4-phosphate

Combretastatin A-4 phosphate (CA4P) is a microtubule-disrupting tumour-selective vascular disrupting agent (VDA). CA4P activates the actin-regulating RhoA-GTPase/ ROCK pathway, which is required for full vascular disruption. While hypoxia renders tumours resistant to many conventional therapies, little is known about its influence on VDA activity. Here, we found that active RhoA and ROCK effector phospho-myosin light chain (pMLC) were downregulated in endothelial cells by severe hypoxia. CA4P failed to activate RhoA/ROCK/pMLC but its activity was restored upon reoxygenation. Hypoxia also inhibited CA4P-mediated actinomyosin contractility, VE-cadherin junction disruption and permeability rise. Glucose withdrawal downregulated pMLC, and coupled with hypoxia, reduced pMLC faster and more profoundly than hypoxia alone. Concurrent inhibition of glycolysis (2-deoxy-D-glucose, 2DG) and mitochondrial respiration (rotenone) caused profound actin filament loss, blocked RhoA/ROCK signalling and rendered microtubules CA4P-resistant. Withdrawal of the metabolism inhibitors restored the cytoskeleton and CA4P activity. The AMP-activated kinase AMPK was investigated as a potential mediator of pMLC downregulation. Pharmacological AMPK activators that generate AMP, unlike allosteric activators, downregulated pMLC but only when combined with 2DG and/or rotenone. Altogether, our results suggest that Rho/ROCK and actinomyosin contractility are regulated by AMP/ATP levels independently of AMPK, and point to hypoxia/energy depletion as potential modifiers of CA4P response.