An important aspect of our drug discovery strategy required a test which would distinguish between specific EGFR-TKI-mediated effects on cell growth and non-target-specific cell growth inhibition or cytotoxicity. For this purpose we selected the human KB cell line derived from a vulval squa-mous tumour (Wakeling et al. 1997). KB cells grow well in basal conditions, that is in growth medium supplemented with 5% charcoal-treated calf serum (to deplete serum growth factors), and redouble their growth rate upon addition of EGF to the growth medium. Iressa inhibited EGF-stimulated growth much more potently than basal growth (IC50 0.054 mM compared with 8.8 mM) demonstrating that Iressa specifically inhibits EGF-stimulated tumour cell growth (Wakeling et al. 2002).
Selectivity of cell growth inhibition by Iressa was also exemplified in studies with normal tissue-derived human cells (HUVEC, human umbilical vein endothelial cells) which are stimulated to grow by several different growth factors. Iressa inhibited EGF-stimulated HUVEC cell growth with a potency similar to that recorded for EGF-stimulated KB cells (<0.1 mM) whereas at least tenfold greater concentrations were needed to significantly affect FGF- or VEGF-stimulated HUVEC growth (Wakeling et al. 2002).
Numerous investigators have shown that Iressa inhibits the proliferation of many different tumour cell lines; this growth inhibition is accompanied by inhibition of EGFR phosphorylation and of downstream signalling through the ERK MAP kinase(s) and PKB/Akt pathways, but tumour cell sensitivity to Iressa was not determined by the level of EGFR expression (Ciardiello et al. 2000; Albanell et al. 2001; Anderson et al. 2001; Moasser et al. 2001; Barnes et al. 2003; Vicentini et al. 2003). Although Iressa does not directly inhibit ErbB2 phosphorylation, it effectively inhibits the growth of ErbB2-overexpressing tumour cells, and inhibitory efficacy did not depend on the relative levels of expression of ErbB2 and EGFR (Anderson et al. 2001; Moasser et al. 2001; Moulder et al. 2001). Inhibition of ErbB2 signalling by Iressa may be mediated by induction of inactive ligand-hetero-meric receptor complexes containing ErbB2 and ErbB3 (Lichtner et al. 2001; Anido et al. 2003). Thus, Iressa may inhibit heregulin-mediated tumour cell growth as well as that mediated by EGFR ligands. As well as inhibiting tumour cell proliferation, Iressa treatment increases apoptosis (Ciardiello et al. 2000; Gilmore et al. 2002; Normanno et al. 2002), reduces motility and inva-siveness ( Fujimura et al. 2002; Barnes et al. 2003) and decreases angiogene-sis in some tumour cells (Ciardiello et al. 2001; Hirata et al. 2002). Iressa is a particularly effective inhibitor of the growth of anti-oestrogen-resistant breast cancer cells (McLelland et al. 2001; Knowlden et al. 2003), of taxane-resistant, hormone-independent breast cancer cells (Ciardiello et al. 2002) and enhances the anti-tumour activity of cytotoxic drugs (Ciardiello et al.
2001, 2002; Magne et al. 2002) and of radiation treatment (Bianco el 2002; Huang et al. 2002; Williams et al. 2002) in vitro.
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