R. Hoffman et al., PROTEIN-BINDING MODULATES INHIBITION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR KINASE AND DNA-SYNTHESIS BY TYRPHOSTINS, Cancer chemotherapy and pharmacology, 36(4), 1995, pp. 316-324
Inhibition of growth factor-stimulated DNA synthesis carried out in de
fined medium is often compared with inhibition of serum-stimulated DNA
synthesis so as to assess the selectivity of growth-factor-receptor t
yrosine kinase inhibitors such as tyrphostins. We investigated whether
protein binding may influence the interpretation of these experiments
. Protein binding of tyrphostins was determined by ultrafiltration, eq
uilibrium dialysis or spectrophotometer, and was quantitated by high-p
erformance liquid chromatography (HPLC). For growth factor-stimulated
DNA synthesis, we used the non-small-cell lung cancer cell line L23/P
stimulated by transforming growth factor alpha (TGF alpha). The epider
mal growth factor (EGF)-receptor kinase was assayed by phosphorylation
of a peptide substrate or by receptor autophosphorylation. Protein bi
nding of a number of tyrphostins ranged from 64% to 98%. There was a p
ositive correlation (r = 0.995) between the degree of protein binding
and the hydrophobicity. Inhibition of the EGF-receptor tyrosine kinase
activity by the highly protein-bound tyrphostin B56 [N-(4-phenylbutyl
)-3,4-dihydroxybenzylidene cyanoacetamide] was reduced by bovine serum
albumin (BSA), but BSA had less of an effect on inhibition of the EGF
-receptor kinase by the weakly protein-bound tyrphostin A47 (RG 50864;
3,4-dihydroxybenzylidene cyanothioacetamide). Tyrphostins B46 [N-(3-p
henylpropyl)-3,4-dihydroxybenzylidene cyanoacetamide] and B56 (both hi
ghly protein-bound) inhibited DNA synthesis of L23/P cells with approx
imately 3-fold greater potency in 0.5% serum than in 10% serum, but th
e inhibition of DNA synthesis in 0.5% serum was reduced by the additio
n of BSA. Tyrphostins B46 and B56 inhibited DNA synthesis stimulated b
y TGF alpha in defined medium to a greater extent than DNA synthesis s
timulated by serum. However, this apparent selectivity for inhibition
of TGF alpha-stimulated DNA synthesis was lost when the protein concen
tration in the defined medium was made equivalent to that in the serum
-containing medium. By contrast, BSA enhanced the selective inhibition
of TGF alpha-stimulated DNA synthesis by tyrphostin A47. These result
s demonstrate that protein binding accounts for the apparent selectivi
ty of some highly protein-bound tyrphostins for TGF alpha-stimulated D
NA synthesis of L23/P cells. Therefore, protein binding should be take
n into consideration in assessments of the selectivity of tyrphostins.