B47
Biological effects of the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin (DZNep) and analogues in breast cancer cell lines
Annette L Hayden, Peter WM Johnson, Graham K Packham, Simon J Crabb
University of Southampton, UK
Background
The S-adenosylhomocysteine hydrolase inhibitor DZNep is a novel chromatin remodelling compound, proposed to cause demethylation of histones by inhibiting enhancer of zeste homolog 2 (EZH2). EZH2 is part of polycomb repressive complex 2 (PRC2) with histone 3 lysine 27 (H3K27) methyltransferase activity, inducing H3K27 hypermethylation and epigenetic silencing. EZH2 over-expression is linked to poor prognosis in breast cancer. EZH2 knockdown inhibits cellular proliferation and cell cycle progression.
Method
We assessed biological effects of DZNep and the analogues 3-deazaadenosine (DZA) and neplanocin A (Nep A) in MCF7 and MDA-MB-231 breast cancer cell lines. Techniques included immunoblotting (for protein expression), MTS assays (cell proliferation), nile red staining of lipid droplets (differentiation) and flow cytometry (cell cycle analysis, cell death). We used fractional effect analyses to assess interactions with the histone deacetylase inhibitor trichostatin A (TSA).
Results
10μM DZNep downregulated EZH2 protein expression in breast cancer cells. MCF7 and MDA-MB-231 cells underwent dose dependent inhibition of proliferation in response to DZNep. G2/M cell cycle arrest occured by 48 hours in response to 10μM DZNep. MCF7 cells then underwent apoptosis at 72 hours. MDA-MB-231 cells were relatively resistant but underwent apoptosis in response to 10μM DZNep by 144 hours. Both cell lines exhibited a differentiated phenotype with lipid droplet accumulation after 72 hours DZNep treatment. DZNep analogues where active in proliferation assays in both cell lines but potency varied (MCF7 cell IC50 values: DZNep 298.2nM; DZA 54.6nM; Nep A 6042.5nM). DZA also induced G2/M cell cycle arrest. Finally, DZNep and TSA induced a synergistic inhibition of cell proliferation at IC50 doses.
Conclusion
DZNep inhibits proliferation and induces G2/M cell cycle arrest, apoptosis and differentiation in breast cancer cells. DZNep analogues are also active with differing potency. Targeting EZH2 and synergistic interactions with HDAC inhibition might be of therapeutic value in breast cancer.
