Abstract
Benzyl isothiocyanate (BITC), a dietary isothiocyanate derived from cruciferous vegetables, inhibits the proliferation of colorectal cancer cells, most of which overexpress β-catenin as a result of mutations in the genes for adenomatous polyposis coli or mutations in β-catenin itself. Because nuclear factor-κB (NF-κB) is a plausible target of BITC signaling in inflammatory cell models, we hypothesized that it is also involved in BITC-inhibited proliferation of colorectal cancer cells. siRNA-mediated knockdown of the NF-κB p65 subunit significantly decreased the BITC sensitivity of human colorectal cancer HT-29 cells with mutated p53 tumor suppressor protein. Treating HT-29 cells with BITC induced the phosphorylation of IκB kinase, IκB-α and p65, the degradation of IκB-α, the translocation of p65 to the nucleus and the upregulation of NF-κB transcriptional activity. BITC also decreased β-catenin binding to a positive cis element of the cyclin D1 promoter and thus inhibited β-catenin-dependent cyclin D1 transcription, possibly through a direct interaction between p65 and β-catenin. siRNA-mediated knockdown of p65 confirmed that p65 negatively affects cyclin D1 expression. On the other hand, when human colorectal cancer HCT-116 cells with wild-type p53 were treated with BITC, translocation of p65 to the nucleus was inhibited rather than enhanced. p53 knockout increased the BITC sensitivity of HCT-116 cells in a p65-dependent manner, suggesting that p53 negatively regulates p65-dependent effects. Together, these results identify BITC as a novel type of antiproliferative agent that regulates the NF-κB pathway in p53-deficient colorectal cancer cells.
Highlights
Phosphorylation of p65 at Ser[536] is an indicator of nuclear factor-κB (NF-κB) activation.[13]
We previously demonstrated that benzyl ITC (BITC), an ingredient in papaya,[20] inhibits cell proliferation by inducing cell cycle arrest and apoptosis through the mitogen-activated protein kinase pathways in human T-lymphocytic leukemia Jurkat cells.[21]
These results indicate that NF-κB has a significant role in cell growth inhibition, rather than cell death by BITC in human colorectal cancer cells
Summary
Phosphorylation of p65 at Ser[536] is an indicator of NF-κB activation.[13]. Cross talk between β-catenin and NF-κB has a significant role in regulating the expression of their target genes.[14,15,16] For example, p65 is recently reported to inhibit βcatenin binding on the positive cis element TCF-binding element[0] (TBE0) site of the cyclin D1 promoter and its Isothiocyanates (ITCs), mainly derived from cruciferous vegetables such as broccoli, wasabi (Japanese horseradish) and watercress, are highly effective in chemoprevention and have antitumor activities in vitro and in vivo.[18]. We investigated the role of NF-κB in BITC-inhibited colorectal cancer cell proliferation. We demonstrate that NF-κB sensitizes to BITC-induced antiproliferation in human colorectal cancer HT-29 cells. Our results indicate that BITC inhibits β-catenin-dependent cyclin D1 transcription and cell proliferation by causing p65 to accumulate in the nucleus. Experiments with two other colorectal cancer cell lines (HCT-116 p53+/+ and HCT-116 p53−/ − ) revealed that p53 tumor suppressor protein negatively regulates the effects of p65 in BITC signaling. This study provides the evidence showing that NF-κB represents a novel therapeutic target of the ITC-based prevention of colorectal cancer with p53 mutation and β-catenin overexpression
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