Prevention of human cell transformation by CAPE (caffeic acid phenethyl ester): Role of NF-κB factors.

B105

A chronic (8-week) exposure to 0.1 μM arsenite (As), a human environmental carcinogen, transforms human osteosarcoma (HOS) cells. When cells are concomitantly treated with As and 0.5 μM CAPE, cell transformation does not occur. Gene arrays and Western blots showed that in comparison to parental HOS cells, As causes many changes in gene expression, including down-regulation of inflammatory cytokine mRNAs, p53, and antioxidant enzyme mRNAs, as well as their protein products. Concurrently, levels of NF-κB p50/p105 mRNAs and proteins were also severely suppressed, which could explain a decrease in inflammatory factors, while p65 was not significantly changed quantitatively. However, small differences in molecular weight of p65 became apparent between the parental and transformed-HOS cells suggesting post-transcriptional modification. When HOS cells were exposed to As+CAPE for 8 weeks, cells did not grow in soft agar and CAPE preserved about 25-40% of p50/p105 levels present in HOS cells treated only with CAPE. Parental HOS cells contained p100 but not p52. Cells treated with As showed a strong increase in p52 and about a 4-fold decrease in p100. CAPE partially reversed As-mediated changes by restoring p100 and decreasing p52 about 2-fold. These results show that when p50/p105 of NF-κB1 are depleted, inflammatory factors, such as cytokines and chemokines, are also decreased, however, p52 of NF-κB2 is strongly up-regulated and likely plays an anti-apoptotic role. Co-treatment with CAPE reverses As-induced changes, including cell transformation, NF-κB1 & 2, p53, SOD1, NQO1, and induces apoptosis of transformed cells. Knockdown of p50 in parental HOS cells using p50 siRNA caused 60-70% decrease in p50 but had no effect on any other NF-κB factors. Our results proved that p50 is responsible for the regulation of inflammatory cytokines or their receptors, IL-8 (chemotactic & angiogenic factor), EGF, MIP, and some MMPs, all of which were decreased in the p50-knockout cells, while VEGF (vascular endothelial growth factor), a factor inducing angiogenesis, was increased. These data mimic many As-mediated changes. In summary, our data reflect a two-pronged CAPE-mediated NF-κB modulation: transcriptional regulation and post-transcriptional modification, which counteracts arsenite-induced transformational changes. These properties point to CAPE as a potential preventive and/or therapeutic agent that is effective at very low doses in controlling signal transduction pathways and cell transformation. [Supported in part by NIH grants ES10344 & ES00260]