K. Majumder1, R. Chugh1, S. Modi1, N. Arora1, S. Banerjee1, R. Dawra1, A. Saluja1, V. Dudeja1 1University Of Minnesota,Surgery,Minneapolis, MN, USA
Introduction: Triptolide, a diterpene triepoxide from the Chinese plant Tripterygium wilfordii, is markedly effective against pancreatic cancer cells both in vivo and in vitro. Minnelide, a water soluble analog of triptolide, is currently in phase I clinical trials. While our data suggests that downregulation of transcription factors HSF-1 (Heat shock factor -1), Sp-1 and NFκB contribute to the effect of triptolide on cancer cells, the mechanism of action of triptolide remains elusive. There is increased understanding of epigenetics in the pathogenesis of cancer and altered histone modifications can play a role in aberrant protein expression profile in cancer cells. In general post-translational modification of histones such as methylation represses gene transcription whereas acetylation activates gene transcription. For example binding of histone-3 trimethylated at lysine-9 residue (tri-methylated H3K9) leads to repression of transcription. In the current work we have evaluated whether epigenetic modulation plays a role in triptolide’s mechanism of action.
Methods: Pancreatic cancer cells (MIA Paca2 and S2-VP10) were treated with triptolide (0-200nM). Protein and mRNA were extracted and analyzed by western blotting, qPCR and PCR array for histone methylation/acetylation and levels of chromatin modifying enzymes. CHIP qPCR was performed to analyze binding of tri-methylated histones to the promoter region of genes regulated by HSF-1 (e.g. HSP70), Sp-1 (FGFR-1) and NFκB (TNFα).
Results: Dose dependent increase of tri-methyl histones (H3Lys27, H3Lys9 and H3Lys4) and a concurrent decrease of acetylated histones (Acetyl histone H3Lys9 and H3Lys18) were observed on western blotting suggesting that triptolide has widespread impact on histone post-translational modification. Western blotting and qPCR showed time and dose dependent decrease in levels of both the demethylases and methyltransferases with triptolide. CHIP qPCR showed increased binding of trimethylated H3K9 but not trimethylated H3K27 to promoter of FGFR-1, HSP70 and TNFα in triptolide treated Mia Paca2 cell lines as compared to untreated cells (% Fold change in occupancy of H3K9me3 on triptolide 200nM treatment when compared to no treatment: HSP70 promoter = 321%, FGFR-1 promoter = 262% and TNFα promoter= 208%).
Conclusion: Triptolide alters histone methylation and acetylation profile suggesting an epigenetic component to its action. Corresponding to these changes there is an increased binding of tri-methylated H3K9, to the response element of the transcription factors (HSF-1, Sp-1and NFκB) which are downregulated by triptolide. This is the first report suggesting that epigenetic modulation may be the mechanism of anti-tumor action of triptolide. Dissecting this mechanism further may lead to elucidation of novel therapeutic target in pancreatic cancer.