RESUMEN
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 4â¯h exposure, BNIPSpd (1-10⯵M) induced significant DNA strand breakage. Evidence of apoptosis was progressive through the experimental period. Within 6â¯h, BNIPSpd increased the proportion of cells exhibiting plasma membrane phosphatidylserine exposure. Within 12â¯h, active caspase expression increased and was sustained with 5 and 10⯵M BNIPSpd. Flow cytometric analysis revealed caspase activity in cells with and without damaged membranes. By 24â¯h, 5 and 10⯵M BNIPSpd increased hypodiploid DNA content and internucleosomal DNA fragmentation (DNA ladders) typical of the later stages of apoptosis. 1⯵M BNIPSpd exposure also increased hypodiploid DNA content by 48â¯h. Polyamine levels decreased by 24â¯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.
Asunto(s)
Daño del ADN , Sustancias Intercalantes/toxicidad , Quinolonas/toxicidad , Espermidina/análogos & derivados , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Células HL-60 , Humanos , Espermidina/metabolismo , Espermidina/toxicidadRESUMEN
New bisoxynaphthalimidopolyamines (BNIPOPut, BNIPOSpd and BNIPOSpm) were synthesized. Their cytotoxic properties were evaluated against breast cancer MCF 7 cells and compared with bisnaphthalimidopolyamines BNIPSpd and BNIPSpm. Among the bisoxynaphthalimido polyamines, BNIPOSpm and BNIPOSpd exhibited cytotoxic activity with an IC50 f 29.55 and 27.22 microM, respectively, while BNIPOPut failed to exert significant cytotoxicity after 48-h drug exposure. DNA binding was determined by midpoint of thermal denaturation (Tm) measurement, ethidium bromide displacement and DNA gel mobility. Both BNIPOSpm and BNIPOSpd exhibited strong binding affinities with DNA. BNIPOPut had the least effect. The results were compared with other cytotoxic bisnaphthalimido compounds (BNIPSpm and BNIPSpd) previously reported by us. Using the single cell gel electrophoresis assay, it was found that BNIPSpm and BNIPSpd caused substantial DNA damage to MCF 7 treated cells while BNIPOSpm showed no significant effect over a range of drug concentrations after 4-h drug exposure. However, after 12-h drug exposure, BNIPOSpm had induced significant DNA damage similar to that of BNIPSpm (after 4-h drug exposure). Fluorescence microscopic analysis revealed that at 1 microM drug concentration and after 6-h drug exposure, both BNIPSpm and BNIPSpd were located within the cell while the presence of BNIPOSpm, was not observed. Therefore, we conclude that BNIPSpd, BNIPSpm and BNIPOSpm induce DNA damage consistent with their rate of uptake into the cells.