Effects of Phorbol Dibutyrate on Cell Proliferation, Apoptosis, and Tumor Necrosis Factor-alpha Expression in Human Endometrial Adenocarcinoma Cells

Abstract

Recent evidence suggested that protein kinase C (PKC), a major cell cycle regulator in endometrial models, mimics progesterone withdrawal by inducing downstream signals. In the current study we examined the hypothesis that the PKC activator phorbol 12,13 dibutyrate (PDB) would inhibit cell proliferation and induce apoptosis in two endometrial adenocarcinoma cell (EAC) lines, HEC-1B and Ishikawa cells. We further examined whether the induction of tumor necrosis factor-alpha (TNF-alpha) might mediate these effects. EAC lines were cultured under standard and serum-free conditions to study the effects of PDB on cell kinetics. Cell proliferation was determined by cell count using a hemacytometer and by incorporation of (3)H thymidine into 10% trichloracetic acid-precipitable DNA. Apoptosis was determined by measuring cytoplasmic histone-associated DNA fragments. Conditioned media concentrations of TNF-alpha were measured by a commercially available enzyme-linked immunosorbent assay (ELISA). EACs were transfected with a -125-bp TNF-alpha promoter luciferase construct and treated with PDB to evaluate transcriptional activation. Activation of the PKC system with PDB (10 nM) decreased cell proliferation and mitogenesis in EACs. PDB induced apoptosis in both EAC lines. EACs exhibit basal TNF-alpha gene expression and protein secretion and these were increased potently by PDB. However, neutralization of TNF-alpha by addition of anti-TNF-alpha antibodies did not prevent the suppression of mitogenesis, induction of apoptosis, or activation of TNF-alpha gene expression by PDB. Activation of the PKC system leads to inhibition of cell proliferation, induction of apoptosis, and TNF-alpha expression in EACs. However, apoptosis in this setting does not appear to require TNF-alpha action. EACs provide an informative model to investigate aspects of endometrial epithelial remodeling that may occur under physiologic conditions of progesterone withdrawal.