Identification of biomarkers of tumour blood vessels for therapeutic targeting

Abstract

Cancer is a disease that develops as a result of unregulated cell growth, whereby damaged cells grow and divide uncontrollably forming lumps of tissue called tumours. Tumours can either be benign, wherein the tumour is confined within a single cluster and cannot spread to neighbouring tissues, or malignant wherein the tumour is mixed with normal cells and has the ability to invade and grow in normal tissues at a different site (Plank and Sleeman, 2003). The growth and development of tumours is dependent on a continuous supply of blood to ensure a sufficient stream of nutrients and oxygen to the tumour and also the removal of metabolic waste products (Neal and Berry, 2006; Neufeld and Kessler, 2006; DuBois and Demetri, 2007). This is achieved through a process called angiogenesis, the growth of a new network of blood vessels from pre-existing vessels (Bouis et al., 2006; Bhat and Singh, 2008). The aim of the study was to investigate the expression of biomarkers in vascular tumour endothelial (endothelioma) cells and to correlate the biomarker expression with endothelial cell growth and migration, important processes in angiogenesis and tumour progression. Molecules which interfere with cell function, LY294,002 and PF573,228 were employed in this study. LY294,002 is an inhibitor of Phosphatidylinositol 3-kinase (PI3K) signalling, which is involved in the regulation of cell proliferation, cell growth (size) and survival. PF573,228 is an inhibitor of focal adhesion kinase (FAK). FAK forms a scaffold for the attachment of cells at the extracellular matrix and is an important mediator of cell migration and invasion. The MTT assay was used to determine the viability of the cells after treatment with the drugs. Real time cell analysis was also used to determine the migration and invasion of endothelioma cells in response to basic fibroblast growth factor, a proangiogenic factor. Light and electron microscopy were employed to investigate morphological changes of the cells following treatment, while caspase activity was measured using an enzyme linked immunosorbent assay (ELISA). Array studies were undertaken to investigate the expression of angiogenic markers in control and drug-treated cells. A dose-dependent decrease in cell viability and migration were observed in treated cells, with LY294,002 showing a higher potency compared to PF573,228 in both instances. Further studies were undertaken using LY294,002 due to its potency. Light microscopy studies showed that treated cells displayed signs of apoptosis such as an irregular shape and chromatin condensation attributes which were not observed in non-treated cells. This was confirmed by electron microscopy which further revealed signs of apoptosis such as chromatin condensation and margination as well as membrane blebbing. An increase in caspase activity in treated endothelioma cells also confirmed the apoptotic effects of LY294,002. Alteration of the expression levels of various pro- and anti-angiogenic markers in treated cells was observed. Biomarkers that have been suppressed by LY294,002 in treated cells could offer great insight into cancer progression. Also, these markers can serve as targets for cancer therapy in targeted drug delivery.

Kanker is 'n siekte wat as gevolg van ongereguleerde selgroei ontwikkel. Beskadigde selle groei en verdeel sonder beheer en vorm knoppe (tumours) in weefsels. Tumours kan of goedaardig wees, waar die tumour beperk is in 'n enkele eenheid en verspreiding na naburige weefsel nie plaasvind nie, of kwaadaardig waar kanker en normale selle gemeng is, en die moontlikheid bestaan om normale weefsel in te dring en te groei in 'n ander ligging (Plank en Sleeman, 2004). Groei en ontwikkeling van tumours is afhanklik van 'n onafgebroke bloedtoevoer om voldoende nutriente en suurstof te verskaf en om metaboliese afbreek produkte te verwyder (Neal en Berry, 2006; neufeld en Kessler, 2006; duBois en Demetri, 2007). Dié word bereik deur 'n proses genoem angiogenese, gedefinieer as die groei van 'n nuwe netwerk bloedvate van reeds bestaande bloedvate (Bouis et al., 2006; Bhat en Singh, 2008). Die doel van die studie was om die voorkoms van biologiese merkers in vaskulêre tumour endoteelselle (?endothelioma? selle) te ondersoek en dit te korrelleer met endoteel selgroei en migrasie, belangrike prosesse in angiogenese en tumour ontwikkeling. Molekules wat selfunksie hinder, LY294,002 en PF573,228 was in hierdie studie gebruik. LY294,002 inhibeer die fosfatidielinositol 3-kinase (PI3K) seinstelsel, wat betrokke is in die regulering van selproliferasie, selgroei (grote) en oorlewing. PF573,228 is 'n inhibeerder van fokale adhesie kinase (FAK). FAK vorm 'n raam vir die aanhegging van selle by die ekstrasellulêre matriks en is 'n belangrike bemiddellaar van selmigrasie en indringing. Die "MTT" toets was gebruik om die prolifererasie van selle na behandeling met sekere middels te bepaal. "Real time" sel annaliese was ook gebruik om die migrasie en indringing van endoteelselle in response tot basiese fibroblaste groei faktor, 'n proangiogeniese faktor, te bepaal. Lig en elektron mikroskopie was gebruik om die morfologiese sel veranderinge na behandeling te ondersoek. Kaspase aktiwiteit was gemeet deur gebruik te maak van ELISA ("enzyme linked immuno assay") "Array" studies was gebruik om die voorkoms van angiogeniese merkers in kontrole en behandelde selle te bepaal. 'n Dosis afhanklike afname in sel proliferasie en migrasie was opgemerk in behandelde selle, met 'n meer merkbare effek van LY294,002, in dié opsig, in vergelyking met PF573,228 in albei gevalle. Met hierdie abservasie in gedagte was verdere studies beperk tot LY294,002. Ligte mikroskoop studies het bewys dat behandelde endoteelselle tekens van apoptose getoon het bv. oneweredige vorm en kromatien kondensering, eienskappe wat nie in die kontrole selle waargeneem was nie. Dié is bevestig deur middel van elektron mikroskopie waar tekens van apoptose soos kromatien kondensie, marginasie en membraan "blebbing" gesien was. Verdere bevestiging van die apoptotiese vermoë van LY294,002 was die verhoging in kaspase aktiwiteit in behandelde endoteelselle. Veranderings in die voorkomsvlakke van verskeie pro- en anti-angiogeniese merkers vir behandelde selle was waargeneem. Biologiese merkers wat deur LY294,002 onderdruk is in behandelde selle kan groter insig gee in die aard van kanker ontwikkeling. Hierdie merkers kan ook as teikens dien vir kankerterapie in geteikende middellewering.