Bis((1,4-dimethyl-9H-carbazol-3-yl)methyl)amine-mediated anticancer effect triggered by sequence-specific cleavage of DNA leading to programmed cell death in the human U87 cell line

Abstract

Bis((1,4-dimethyl-9H-carbazol-3-yl)methyl)amine (DMCM) has been designed as a novel anti-cancer agent which is believed to have a sequence-specific groove binding interaction with DNA, leading to anticancer effects. DMCM was synthesized, and the sequence-specific interaction of DNA with DMCM was elucidated with restriction enzyme digestion and docking analysis. The interaction of DMCM with calf thymus (CT-DNA) and poly[d(A–G).(C–T)]₂ has been demonstrated using biophysical techniques and the topoisomerase assay. The anti-cancer activity of the compound was studied by cell viability, DNA fragmentation, apoptosis and flow cytometry assays in the human glioma U87 cell line. DMCM-induced cleavage in pGEM and pBluescript vectors with different restriction enzymes showed cleavage at GACGTC sequences related to the Sal I restriction site. The docking analysis of DMCM with (GACGTCGACGTC)₂ showed interaction of the pyrrole ring and the exocyclic NH of bis-carbazole involving A₅–T₈ sequences in the minor groove. According to FT-IR spectroscopy, DMCM induced small perturbations in CT-DNA at thymine (T)–adenine (A) base pairs, deoxyribose rings and phosphate groups. The binding constants (K_b) of DMCM with poly[d(A–G).(C–T)]₂ and CT-DNA were 2.89 × 10⁷ M⁻¹ and 0.68 × 10⁷ M⁻¹, respectively, and the topoisomerase assay result revealed the groove binding interaction of DMCM with DNA. DMCM-induced cytotoxic effects (IC₅₀ = 13.34 μM) in the human U87 MG cell line were superior to those of the standard drugs carmustine (IC₅₀ = 18.24 μM) and temozolomide (IC₅₀ = 100 μM). DMCM treatment leads to accumulation of cells in the G2/M stage of the cell cycle, which is consistent with the apoptosis results observed in DMCM induced fragmentation of DNA by the TUNEL assay. Precisely, the results exemplify that DMCM induced G2/M cell cycle arrest leading to apoptosis, mediated through cleavage of GACGTC sequences in the minor groove of DNA; thus, DMCM possesses the potential to be developed as a novel anticancer agent.