Phenylbutyrate-induced apoptosis is associated with inactivation of NF-kappaB IN HT-29 colon cancer cells

• Published in: Cancer chemotherapy and pharmacology 2002-01, Vol.49 (1), p.27-34
• Main Authors: Feinman, Rena, Clarke, Kevin O, Harrison, Lawrence E
• Format: Article
• Language: English
• Subjects: Annexin A5 - metabolism; Apoptosis - drug effects; Blotting, Western; Caspase 3; Caspases - metabolism; Cell Nucleus - metabolism; Cytoplasm - metabolism; DNA - metabolism; Electrophoresis; Flow Cytometry; HT29 Cells; Humans; Luciferases - genetics; Luciferases - metabolism; Membrane Potentials - drug effects; Mitochondria - drug effects; NF-kappa B - antagonists & inhibitors; Phenylbutyrates - pharmacology; Time Factors; Transfection
• ISSN: 0344-5704

Cytotoxic chemotherapy has been used to treat patients with metastatic colorectal cancer with limited success. Therefore novel chemotherapeutic approaches are needed. Based on encouraging preclinical data, there has been an interest in developing derivatives of butyrate as clinically applicable agents. The purpose of this study was to investigate the effects of phenylbutyrate (PB), a butyrate analogue, on the cell growth and apoptosis in a colon cancer cell model. Growth curves, flow cytometric studies, Western blotting, DNA binding assays and transient transfection experiments were performed in vitro using the colon cancer cell line HT-29 after exposure to PB. Exposure of HT-29 colon cancer cells to PB resulted in growth inhibition and induction of apoptosis as measured by annexin V staining. This increase in apoptosis was associated with a decrease in mitochondrial membrane potential, an increase in caspase-3 activity and a decrease in intact PARP protein levels. Since NF-kappaB plays a pivotal role in the regulation of apoptosis, we explored the effects of PB on the DNA binding and transcriptional activity of this transcription factor. After PB treatment, NF-kappaB-DNA binding was markedly decreased and specifically, this decreased DNA binding was observed in the p50:p65 heterodimer. The decreased NF-kappaB DNA binding was observed as early as 3 h after PB treatment, while no apparent changes in annexin V binding were detected until 12 h after PB treatment. Untreated HT-29 cells transfected with a kappaB-luciferase reporter plasmid demonstrated significant constitutive activity of the kappaB binding site, which was markedly decreased after treating the cells with PB. These results suggest that PB-induced apoptosis may be partly regulated through the inactivation of NF-kappaB. PB, an oral butyrate analogue, may have therapeutic potential in colon cancer.