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

OBJECTIVES: 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. Wefurther examined whether the induction of tumor necrosisfactor-alpha (TNF-alpha) might mediate these effects. METHODS: EAC lines were cultured under standard and serum-free conditions to study the effects of PDB on cell kinetics. Cell proltferation was determined by cell count using a hemacytometer and by incorporation of3H thymidine into 100 % trichloracetic acid-precipitable DNA. Apoptosis was determined by measuring cytoplasmic histone-associated DNAfragments. 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. RESULTS: Activation ofthe 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 expressio