Rho-GTPase signaling and the vascular disrupting activities of combretastatin A-4-phosphate


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Chryso Kanthou, Leigh Williams, Gillian Tozer

University of Sheffield, UK

Abstract

Background

The vascular disrupting agent (VDA) combretastatin A-4-phosphate (CA-4-P), causes selective damage to tumour blood vessel networks followed by tumour cell necrosis. In vivo, production of nitric oxide (NO), via constitutive nitric oxide synthase (ecNOS), protects against the vascular damaging effects of CA-4-P through undefined mechanisms [1]. In vitro, CA-4-P activates Rho-GTPase/Rho kinase (ROCK) signaling, triggers rapid endothelial cytoskeletal remodelling, and causes a rise in monolayer permeability hypothesised to be important for tumour vascular disruption [2].

Results

Here we demonstrate that inhibition of ROCK, by administration of selective ROCK inhibitor Y27632 just before CA-4-P, had a profound protective effect on necrosis induction in SW1222 colorectal carcinomas in SCID mice, thus providing the first direct evidence for Rho signaling in driving the vascular damaging effects of CA-4-P in vivo. Active ROCK suppresses ecNOS activity by phosphorylation at Thr495 and downregulates ecNOS expression. CA-4-P phosphorylated eNOS at Thr495, and attenuated eNOS protein expression in endothelial cells, effects that were reversed by Y27632, raising the possibility that ROCK activation may also play a role in regulating ecNOS activity in vivo. cGMP, downstream of NO, and cAMP, inhibit Rho by PKG- and PKA-mediated phosphorylation respectively, and are therefore also potential CA-4-P activity modulators. cGMP/cAMP analogues suppressed the phosphorylation of ROCK target myosin light chain by CA-4-P and attenuated both cytoskeletal remodelling and elevated permeability in endothelial monolayers.

Conclusion

Our data demonstrate that interactions between NOS, cGMP/cAMP and Rho signaling influence the vascular disrupting activities of CA-4-P and suggest that NO, cAMP and cGMP can be further targeted to improve the efficacy of CA-4-P and other, related VDAs.

References

[1] Tozer et al (2009). Clin Cancer Res (in press).

[2] Kanthou & Tozer (2002). Blood 99:2060.

Acknowledgements
Funded by Cancer Research UK