Abstract
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.
Highlights
Vascular disrupting agents (VDAs) cause tumour blood vessels to collapse and this strategy kills tumour cells indirectly by rapidly cutting off their blood supply[1]
Under extreme hypoxic conditions (0.1% O2 for 14 h), basal phospho-myosin light chain (pMLC) was reduced to very low levels and Combretastatin A-4 phosphate (CA4P) did not induce it further (Fig. 1a,f)
In this study we found that hypoxia and energy depletion, diminished the ability of endothelial cells to respond to CA4P
Summary
Vascular disrupting agents (VDAs) cause tumour blood vessels to collapse and this strategy kills tumour cells indirectly by rapidly cutting off their blood supply[1]. CA4P and other tubulin-binding VDAs have not yet gained FDA approval for the treatment of cancer despite resulting in some clinical responses when combined with additional treatments of chemotherapy or anti-angiogenic agents[7]. The binding of CA4P to β-tubulin disrupts endothelial microtubules, and this process rapidly activates molecular signalling pathways that modify the actin cytoskeleton and the morphology of endothelial cells, thereby severely compromising their barrier function[8,12]. Endothelial cells from normal as well as pathological tissues use glycolysis as a means of generating ATP and are less dependent on oxidative phosphorylation for their energy supplies[21] Both hypoxia and energy depletion are sensed by the master switch molecule adenosine monophosphate protein kinase (AMPK). We show that AMPK is strongly activated by hypoxia, glucose deprivation and inhibitors of endothelial metabolism, its activation per se is not sufficient to regulate CA4P signalling
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