Effect of lipid mimetics of GM3 and lyso-GM3 dimer on EGF receptor tyrosine kinase and EGF-induced signal transduction

Abstract

The tyrosine kinase activity associated with epidermal growth factor receptor (EGFR) has been a target in studies of pharmacological reagents to inhibit growth of cancer cells, which are mostly of epidermal origin. Lyso-GM3 dimer showed stronger inhibitory effect on the tyrosine kinase of EGFR than GM3, with minimal cytotoxicity [Y. Murozuka, et al. Lyso-GM3, its dimer, and multimer: their synthesis, and their effect on epidermal growth factor-induced receptor tyrosine kinase. Glycoconj. J. 24 (2007) 551–563]. Synthesis of lipids with sphingosine requires many steps, and the yield is low. A biocombinatory approach overcame this difficulty; however, products required a C 12 aliphatic chain, rather than the sphingosine head group [Y. Murozuka, et al. Efficient sialylation on azidododecyl lactosides by using B16 melanoma cells. Chemistry & Biodiversity 2 (2005) 1063–1078]. The present study was to clarify the effects of these lipid mimetics of GM3 and lyso-GM3 dimer on EGFR tyrosine kinase activity, and consequent changes of the A431 cell phenotype, as follows. (i) A lipid mimetic of lyso-GM3 dimer showed similar strong inhibitory effect on EGF-induced EGFR tyrosine kinase activity, and similar low cytotoxicity, as the authentic lyso-GM3 dimer. (ii) A lipid mimetic of lyso-GM3 dimer inhibited tyrosine phosphorylation of EGFR or its dimer to a level similar to that of the authentic lyso-GM3 dimer, but more strongly than GM3 or a lipid mimetic of GM3. (iii) Associated with the inhibitory effect of a lipid mimetic of lyso-GM3 dimer on EGF-induced EGFR kinase activity, only Akt kinase activity was significantly inhibited, but kinases associated with other signal transducers were not affected. (iv) The cell cycle of A431 cells, and the effects of GM3 and a lipid mimetic of lyso-GM3 dimer, were studied by flow cytometry, measuring the rate of DNA synthesis with propidium iodide. Fetal bovine serum greatly enhanced S phase and G 2/M phase. Enhanced G 2/M phase was selectively inhibited by pre-incubation of A431 cells with a lipid mimetic of lyso-GM3 dimer, whereas GM3 had only a minimal effect.