NF-kappaB inhibitor Parthenolide mediates DNA demethylation and histone acetylation in leukemia cells

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Purpose: Parthenolide has been shown to exhibit strong in-vitro and in-vivo anti-tumor activity through the inhibition of NF-κB activity, increase of reactive oxygen species (ROS) levels and enhance proapoptotic activation of p53. Parthenolide selectively kills primary human acute myeloid leukemia (AML), blast crisis chronic lymphomatic leukemia (bcCML) and B CML cells, while spares normal hematopoietic cells. However, its potential as an epigenetic modulator has not been investigated. Method: Leukemia cell lines MV4-11, Kasumi-1 and K562 were treated with 1-100 μM of parthenolide and MV4-11 xenografted nu/nu athymic mice was given i.v. parthenolide at 10 mg/kg, and the transcription and protein levels of DNMT1, SP1, HDAC1, acetylated H3 and H4, p53 and p21 were assessed by either RT-PCR or western blot. The inhibition of NF-κB was determined using Electrophoretic Mobility Shift Assays and the mechanism of down-regulation of DNMT1 transcription was investigated by chromatin immunoprecipitation. Hypomethylating effect of parthenolide on Kasumi-1 and K562 cells in-vitro and MV4-11 cells in-vivo was assessed by a LC-MS/MS method for global DNA methylation. The inhibition effect of parthenolide on DNMT1 and M. SssI activity was evaluated using an in-vitro fluorescent-based ELISA assay and the interaction of DNMT1 with parthenolide was depicted using DNMT1 homology molecular modeling and ligand docking. Results: We have found that parthenolide is an epigenetic modulator; it induces global DNA hypomethylation and enhances histone acetylation in a dose- and time-dependent manner in our test leukemia cells. Parthenolide inhibits M. SssI activity with an EC50 of 5.0 µM, depletes DNMT1 protein at 10 µM and down-regulates DNMT1 transcription in a time- and dose-dependent fashion. The docking model demonstrates that parthenolide exerts its inhibitive effect on DNMT activity possibly through covalent binding to the catalytic active site by the Michael addition of the cysteine of DNMT1 to the γ-methylene lactone in parthenolide. Moreover, we have found that parthenolide significantly increases histone acetylation without alteration of the enzyme level, suggesting that parthenolide may inhibit HDAC. Notably, the increased histone acetylation and DNA demethylation correlated to the up-regulation of p21 transcription. Conclusion: For the first time, we report parthenolide as a novel modulator of DNA methylation and histone acetylation. Ongoing studies combining this agent with other DNMT and HDAC inhibitors are underway. Supported by NIH-NCI-RO1-CA102031, U01-CA-76576 and the BioMedical Mass Spectrometry Laboratory.