Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA319

Poster Communications

Differential effects of Integrin-linked kinase knockdown and vascular endothelial growth factor receptor blockade on melanoma cell growth and angiogenesis

P. Mabeta1

1. Physiology, University of Pretoria, Pretoria, South Africa.

Angiogenesis is the process by which new blood vessels are formed from a pre-existing microvasculature [1]. It is also a key feature in the growth and progression of several cancers. Studies have identified vascular endothelial factor (VEGF) as an important regulator of angiogenesis in both the physiological and pathological settings [1,2]. In the context of tumours, VEGF signalling was shown to be impaired in several neoplasms. This discovery led to the development of therapies against VEGF [1-3]. While antiangiogenic VEGF-targeted therapy has resulted in increased cancer patient survival, the improvement has been marginal partly due to the development of resistance [2,3]. Therefore there is a need for the identification of alternative or complimentary therapeutic strategies. Integrin-linked kinase (ILK) is an effector of integrin-mediated cell adhesion which is involved in the regulation of the PI3k/Akt pathway [4]. The kinase has also been linked to the promotion of tumour-associated neovascularisation. The purpose of the present study was to study the effects of a multi-targeted therapeutic strategy comprising ILK-knockdown and the blockade of VEGF receptors -1,-2 or VEGFR-3 in human melanoma cells and endothelial cells. To evaluate the effects of ILK knockdown the human vein endothelial cells and melanoma cells, purchased from the American Type Culture Collection, were used. The effects of inhibiting ILK using siRNA alone and in combination with a broad spectrum tyrosine kinase inhibitor, Sunitinib, or in combination with a VEGFR-3 inhibitor MAZ-51, were evaluated on cell growth using the crystal violet nuclear staining assay. The effects of treatments on cell migration and invasion were studied using the xCELLigence Real Time Cell Analysis (RTCA) system. The expression of proangiogenic and antiangiogenic proteins were determined using protein array analysis. Protein expression was further studied using Western blotting. The combination of Sunitinib and Integrin linked kinase knockdown resulted in a more severe decrease in melanoma migration and invasion as well as angiogenesis when compared to the individual treatments or to ILK knockdown + VEGFR-3 blockade. Both combination treatments inhibited cell growth with comparable potency. Also, results from western blot and protein array studies revealed a decrease in the expression of several proangiogenic factors including platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), while a number of endogenous angiogenesis inhibitors such as Platelet Factor 4 (PF4) were upregulated. The combination approach of ILK-knockdown and Sunitinib may be useful in the elaboration of antiangiogenic therapy in melanoma, further studies are warranted.

Where applicable, experiments conform with Society ethical requirements