Helenalin Acetate, a Natural Sesquiterpene Lactone with Anti-inflammatory and Anti-cancer Activity, Disrupts the Cooperation of CCAAT Box/Enhancer-binding Protein β (C/EBPβ) and Co-activator p300

Abstract

Edited by Joel GottesfeldRecent work has demonstrated pro-oncogenic functions of the transcription factor CCAAT box/enhancer-binding protein ␤ (C/EBP␤) in various tumors, implicating C/EBP␤ as an interesting target for the development of small-molecule inhibitors. We have previously discovered that the sesquiterpene lactone helenalin acetate, a natural compound known to inhibit NF-B, is a potent C/EBP␤ inhibitor. We have now examined the inhibitory mechanism of helenalin acetate in more detail. We demonstrate that helenalin acetate is a significantly more potent inhibitor of C/EBP␤ than of NF-B. Our work shows that helenalin acetate inhibits C/EBP␤ by binding to the N-terminal part of C/EBP␤, thereby disrupting the cooperation of C/EBP␤ with the co-activator p300. C/EBP␤ is expressed in several isoforms from alternative translational start codons. We have previously demonstrated that helenalin acetate selectively inhibits only the full-length (liver-enriched activating protein* (LAP*)) isoform but not the slightly shorter (LAP) isoform. Consistent with this, helenalin acetate binds to the LAP* but not to the LAP isoform, explaining why its inhibitory activity is selective for LAP*. Although helenalin acetate contains reactive groups that are able to interact covalently with cysteine residues, as exemplified by its effect on NF-B, the inhibition of C/EBP␤ by helenalin acetate is not due to irreversible reaction with cysteine residues of C/EBP␤. In summary, helenalin acetate is the first highly active small-molecule C/EBP␤ inhibitor that inhibits C/EBP␤ by a direct binding mechanism. Its selectivity for the LAP* isoform also makes helenalin acetate an interesting tool to dissect the functions of the LAP* and LAP isoforms.

The natural anti-tumor compound Celastrol targets a Myb-C/EBPβ-p300 transcriptional module implicated in myeloid gene expression

“…Expression vectors for chicken and mouse C/EBPβ, a C/EBPβ mutant lacking all cysteine residues (CallA) and chicken C/EBPα have been described [ 16 , 29 , 30 , 35 ]. Expression vectors for full-length human p300 and truncated p300 constructs p300/1751-2370 and p300/1751-1947 have been described [ 33 ].…”

“…Expression vectors for the p300/1751-1947 mutations C1789A, C1790A and C1789,1790A (CC) were generated by oligonucleotide-directed mutagenesis. Plasmids encoding Gal4-C/EBPβ, p300-VP16, Gal4-VP16 and C/EBPβ-YFP fusion proteins were described before [ 29 , 33 ].…”

“…We recently became aware that our cell-based screening assay also responds to inhibition of C/EBPβ, as exemplified by the sesquiterpene lactone helenalin acetate, a compound that inhibits Myb-induced mim-1 expression but was shown to be a potent inhibitor of C/EBPβ instead of a Myb inhibitor [ 28 , 29 ]. We showed before that Myb requires C/EBP family members, such as C/EBPα or C/EBPβ, as essential cooperation partners to stimulate the mim-1 gene [ 30 , 31 ], explaining why inhibition of C/EBPβ has a similar effect as the inhibition of Myb in our screening system.…”

Transforming growth factor β (TGFβ) cross-talk with the unfolded protein response is critical for hepatic stellate cell activation

“…Error bars, S.E. end, we utilized helenalin acetate, a sesquiterpene lactone reported to disrupt the interaction between C/EBP␤ and p300 (31). Co-treatment of LX-2 cells with TGF␤ and helenalin acetate (1 M) disrupted TGF␤ induction of fibronectin, procollagen 1␣1, procollagen 1␣2, and ␣SMA mRNA levels, as well as increased PPAR␥ mRNA (Fig.…”

“…LIP overexpression also inhibits collagen I expression (38). Thus, targeting C/EBP␤ or a specific isoform, such as the full-length LAP1, which can bind to p300, could reduce the activated HSC population in vivo (31).…”