Woo-Sik Jeong1, In-Wha Kim, Rong Hu, Ah-Ng Tony Kong. 1. Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA.
Abstract
PURPOSE: To study and compare effects of selected natural chemopreventive agents on the transcription activation of nuclear factor-kappa B (NF-kappaB) in human HT-29 colon cancer cells. METHODS: The natural chemopreventive compounds isothiocyanates (ITCs) found in cruciferous vegetables, flavonoids found in green tea, resveratrol (RES) and procyanidin dimers found in red wine, and curcumin (CUR) found in turmeric curry food were examined in this study. HT-29 cells were stably transfected with NF-kappaB luciferase construct, and stable clones were selected. One of the clones, HT-29 N9 cells, was selected and treated with various concentrations of the natural chemopreventive agents and subsequently challenged with NF-kappaB stimulator lipopolysaccharide (LPS), and the luciferase activities were measured. Western blot analysis of phosphorylated IkappaBalpha was performed after treatments with the natural chemopreventive agents. The effects of these agents on cell viability and apoptosis were also evaluated by a nonradioactive cell proliferation MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]. Trypan blue staining, and caspase assay. RESULTS: Treatments with the natural chemopreventive compounds resulted in different responses in the NF-kappaB-luciferase assay. ITCs such as phenethyl isothiocyanate (PEITC), sulforaphane (SUL), allyl isothiocyanate (AITC), and curcumin (CUR) strongly inhibited LPS-induced NF-kappaB-luciferase activations, whereas RES increased activation at lower dose, but inhibited activation at higher dose, and tea flavonoids and procyanidin dimers had little or no effects. ITCs, CUR, (-)-epigallocatechin-3-gallate (EGCG), and RES reduced LPS-induced IkappaBalpha phosphorylation. Furthermore, in the MTS assay, PEITC, SUL, and CUR also potently inhibited cell growth. Caspase-3 activity was induced by chemopreventive compounds, however, the kinetics of caspase-3 activation varied between these compounds within the 48-h time period. CONCLUSIONS: These results suggest that natural chemopreventive agents have differential biological functions on the signal transduction pathways in the colon and/or colon cancer.
PURPOSE: To study and compare effects of selected natural chemopreventive agents on the transcription activation of nuclear factor-kappa B (NF-kappaB) in humanHT-29 colon cancer cells. METHODS: The natural chemopreventive compounds isothiocyanates (ITCs) found in cruciferous vegetables, flavonoids found in green tea, resveratrol (RES) and procyanidin dimers found in red wine, and curcumin (CUR) found in turmeric curry food were examined in this study. HT-29 cells were stably transfected with NF-kappaB luciferase construct, and stable clones were selected. One of the clones, HT-29 N9 cells, was selected and treated with various concentrations of the natural chemopreventive agents and subsequently challenged with NF-kappaB stimulator lipopolysaccharide (LPS), and the luciferase activities were measured. Western blot analysis of phosphorylated IkappaBalpha was performed after treatments with the natural chemopreventive agents. The effects of these agents on cell viability and apoptosis were also evaluated by a nonradioactive cell proliferation MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]. Trypan blue staining, and caspase assay. RESULTS: Treatments with the natural chemopreventive compounds resulted in different responses in the NF-kappaB-luciferase assay. ITCs such as phenethyl isothiocyanate (PEITC), sulforaphane (SUL), allyl isothiocyanate (AITC), and curcumin (CUR) strongly inhibited LPS-induced NF-kappaB-luciferase activations, whereas RES increased activation at lower dose, but inhibited activation at higher dose, and tea flavonoids and procyanidin dimers had little or no effects. ITCs, CUR, (-)-epigallocatechin-3-gallate (EGCG), and RES reduced LPS-induced IkappaBalpha phosphorylation. Furthermore, in the MTS assay, PEITC, SUL, and CUR also potently inhibited cell growth. Caspase-3 activity was induced by chemopreventive compounds, however, the kinetics of caspase-3 activation varied between these compounds within the 48-h time period. CONCLUSIONS: These results suggest that natural chemopreventive agents have differential biological functions on the signal transduction pathways in the colon and/or colon cancer.
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