Caspase-independence and characterization of bisnaphthalimidopropyl spermidine induced cytotoxicity in HL60 cells

Abstract

Bisnaphthalimides are DNA intercalators of potential use as chemotherapeutics but for which the range of mechanism of action is only gradually being elucidated. Using human promyelocytic HL-60 cells, we extend characterization of the cytotoxicity of bisnaphthalimidopropylspermidine (BNIPSpd) and examine the relationship with caspase-activity. Within 4 h exposure, BNIPSpd (1-10 μM) induced significant DNA strand breakage. Evidence of apoptosis was progressive through the experimental period. Within 6 h, BNIPSpd increased the proportion of cells exhibiting plasma membrane phosphatidylserine exposure. Within 12 h, active caspase expression increased and was sustained with 5 and 10 μM BNIPSpd. Flow cytometric analysis revealed caspase activity in cells with and without damaged membranes. By 24 h, 5 and 10 μM BNIPSpd increased hypodiploid DNA content and internucleosomal DNA fragmentation (DNA ladders) typical of the later stages of apoptosis. 1 μM BNIPSpd exposure also increased hypodiploid DNA content by 48 h. Polyamine levels decreased by 24 h BNIPSpd exposure. The pan-caspase inhibitor, z-VAD-fmk, significantly decreased DNA degradation (hypodiploid DNA and DNA ladders) and cytotoxicity. Despite this, cell growth and viability remained significantly impaired. We propose that BNIPSpd cytotoxicity arises through DNA damage and not polyamine depletion and that cytotoxicity is dominated by but not dependent upon caspase driven apoptosis.

Novel Synthetic Approaches for Bisnaphthalimidopropyl (BNIP) Derivatives as Potential Anti-Parasitic Agents for the Treatment of Leishmaniasis

“…Particular analogues may also inhibit topoisomerase II activity directly, exert post DNA damage effects on ataxia telangiectasia-mutated activated DNA damage signaling pathways, or induce lysosomal permeability [25,[30][31][32]. Cell cycle arrest and apoptosis occur as a result of BNIP's anticancer effect, as verified in different in vitro cell models [22,27,[32][33][34][35].…”