Preferential inhibition of platelet-derived growth factor-stimulated DNA synthesis and protein tyrosine phosphorylation by nordihydroguaiaretic acid.

J Domin,T Higgins,E Rozengurt

doi:10.1016/s0021-9258(17)37188-0

Abstract

Nordihydroguaiaretic acid (NDGA), a reportedly specific lipoxygenase inhibitor, was found to selectively inhibit platelet-derived growth factor (PDGF)-stimulated DNA synthesis in Swiss 3T3 cells. Maximal inhibition of PDGF-induced [3H]thymidine incorporation (96%) was observed using 4 microM NDGA (IC50 = 1.5 microM). No effect of NDGA was observed upon DNA synthesis stimulated with either fetal bovine serum, bombesin, or epidermal growth factor (EGF) in the presence of insulin, or with the potent mitogen Pasteurella multocida toxin. The selective inhibition of PDGF-stimulated DNA synthesis by NDGA was also observed in diploid murine cells, rat, and human fibroblasts. Furthermore, 4 microM NDGA also inhibited PDGF-stimulated anchorage-independent colony growth of rat-1 cells by 76%. Using Swiss 3T3 cells, we found that PDGF-stimulated arachidonic acid mobilization and prostaglandin E2 production was abolished by NDGA in a dose-dependent manner. Inhibition of PDGF-stimulated arachidonic acid mobilization by NDGA could not, however, explain its potent inhibitory effect upon PDGF-stimulated DNA synthesis. Our results showed that NDGA also selectively inhibited PDGF receptor tyrosine phosphorylation in a dose-dependent manner in intact cells. Protein tyrosine phosphorylation stimulated by EGF or bombesin was not altered by NDGA treatment. Crucially, NDGA inhibited in vitro the tyrosine kinase activity of anti-phosphotyrosine and anti-PDGF receptor immunoprecipitates prepared from cultures stimulated with PDGF. This inhibition of receptor tyrosine phosphorylation in a cell-free system confirmed that NDGA acts directly at the level of the PDGF receptor tyrosine kinase domain. These results suggest that the potent and selective inhibitory effect of NDGA on PDGF-stimulated DNA synthesis results from its inhibitory action on tyrosine phosphorylation.

Highlights

Nordihydroguaiaretic acid (NDGA) inhibited in vitro the tyrosine kinase activity of anti-phosphotyrosine and anti-platelet-derived growth factor (PDGF) receptor immunoprecipitates prepared from cultures stimulated with PDGF
These results suggest that the potent and selective inhibitory effect of NDGA on PDGF-stimulatedDNA synthesis results from its inhibitory action on tyrosine phosphorylation
We found that this phenolic plant lignan dramatically inhibited PDGF-stimulatedDNA synthesis in a selective mannerS. urprisingly, themechanism by which this effect is achieved involves inhibition of PDGF receptor protein tyrosine kinase activity

Summary

EXPERIMENTAL PROCEDURES

Cell Culture-Stock cultures of Swiss 3T3 cells were propagated as previously described [32]. After 40 h, the mediumwasremoved, and the cultures were Assay ofprotein KinaseActivity-Immunoprecipitates prepared from washed twice with Tris saline, pH 7.4, at 4 "C, fixed twice (for 5 and 2 1.5 x lo cells as described above were washed three times with lysis min) with 5% trichloroaceticacid at 4 "C, and washed three times with buffer and twice with 50 m~ HEPES, pH 7.4,O.l m~ EDTA, 0.01%Brij, 70% ethanol. ARer a 10-min other factors, as described for eachexperiment, to give 5 x 1D"cells per incubation, immunoprecipitates were washed twice with lysis buffer ml. One hour prior to the start of the experiment, was a generous gift of Dr Thomas Parsons, University of Virginia, NDGA was added as indicated After this time, the cells were washed twice with DMEM and incubated in 1ml of the same medium in the absenceor presence of PDGF and NDGAat 37 "C.

RESULTS

FBS PDGF INS

None NDGA

Findings

DISCUSSION