Reversal of the transformed phenotype and inhibition of peptidylglycine alpha-monooxygenase in Ras-transformed cells by 4-phenyl-3-butenoic acid

Abstract

Recent studies have shown that the proliferation of some tumor cells is dependent on autocrine growth loops that require amidated autocrine growth factors. Peptidylglycine alpha-monooxygenase (PAM) is required for amidation of these growth factors and, therefore, this enzyme is an attractive target for anti-tumor compounds. 4-Phenyl-3-butenoic acid (PBA) is an irreversible turnover-dependent inhibitor of PAM in vitro and has been shown to decrease lung cancer cell proliferation by inhibiting the synthesis of amidated growth factors. We show here that PBA (0.1 mg/mL) inhibits the growth of Ras-transformed epithelial cells (WB-Ras) but has little effect on the proliferation of normal epithelial cells (WB-Neo). The methyl ester derivative of PBA (PBA-Me) at 10-fold lower concentration also exhibits a selective inhibition of Ras-transformed cell growth compared to normal epithelial cell growth. In addition, PBA produces a significant upregulation of gap junctional communication between WB-Ras cells following 2-5 day treatments, with a corresponding increase in the degree of connexin 43 phosphorylation and an increase in the number of connexin 43-containing plasma membrane gap junction plaques. Western blot analyses indicate no effect of PBA on the proportion of p21 Ras in the membrane versus cytosolic fractions or on p44/42 MAP kinase phosphorylation. Furthermore, the cell morphology of PBA-treated WB-Ras cells is altered, so as to more closely resemble that of non-transformed WB-Neo cells. PAM activity was assayed in both WB-Ras and WB-Neo cells, and we demonstrate that PBA at long treatment times (4 days) inhibits PAM activity in both cell types at concentrations that produce selective growth inhibition of WB-Ras cells. Shorter PBA treatment times (24 h), however, inhibit PAM activity in WB-Ras but not WB-Neo cells, an effect that was mimicked by PBA-Me. Taken together, these results clearly demonstrate that PBA returns Ras-transformed cells to a more normal phenotype, a finding consistent with the known increased dominance of the Ras signaling pathway in transformed epithelial cells.