Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide

• Published in: Biochimica et biophysica acta Vol. 1860; no. 5; pp. 617 - 629
• Main Authors: Pett, Luke, Kiakos, Konstantinos, Satam, Vijay, Patil, Pravin, Laughlin-Toth, Sarah, Gregory, Matthew, Bowerman, Michael, Olson, Kevin, Savagian, Mia, Lee, Megan, Lee, Moses, Wilson, W. David, Hochhauser, Daniel, Hartley, John A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.2017
• Subjects: A549 Cells; Animals; Antigens, Neoplasm - biosynthesis; Antigens, Neoplasm - genetics; CCAAT-Binding Factor - genetics; CCAAT-Binding Factor - metabolism; Chemosensitisation; DNA Damage; DNA Topoisomerases, Type II - biosynthesis; DNA Topoisomerases, Type II - genetics; DNA-binding polyamides; DNA-Binding Proteins - biosynthesis; DNA-Binding Proteins - genetics; Etoposide - adverse effects; Etoposide - pharmacology; Gene Expression Regulation, Enzymologic - drug effects; Gene Expression Regulation, Enzymologic - genetics; Gene modulation; Mice; NF-Y; NIH 3T3 Cells; Nylons - chemistry; Nylons - pharmacology; Promoter Regions, Genetic; Sequence selectivity; Topoisomerase IIα (topo IIα); Transcription factor-DNA interactions; NF-Y; Transcription factor-DNA interactions; Sequence selectivity; Gene modulation; Chemosensitisation; DNA-binding polyamides; Topoisomerase IIα (topo IIα)
• ISSN: 1874-9399; 0006-3002; 1876-4320; 1878-2434
• DOI: 10.1016/j.bbagrm.2016.10.005

Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®. Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5′-TACGAT-3′ on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1–2μM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage. Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents. Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani. [Display omitted] •Polyamide N1 functionalisation offers a strategy to improve drug-like properties.•Diamino Hx-polyamides contain an N1-alkyl amino group on the heterocyclic rings.•Incorporating an additional cationic group enhances solubility and cellular uptake.•Targeted inhibition of NF-Y:DNA binding by HxIP* re-stimulated topo IIα expression.•Pharmacological modulation of topo IIα increases chemosensitivity to topo II poisons.