Novel o-naphthoquinones induce apoptosis of EL-4 T lymphoma cells through the increase of reactive oxygen species.
Novel β-lapachone analogs 2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ1), 2-p-tolyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ3) and 2-methyl-2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (NQ7), which have trypanocidal activity, were assayed for cytotoxic effects on murine EL-4 T lymphoma cells. The NQs inhibited the proliferation of EL-4 cells at concentrations above 1μM. Nuclear staining of the EL-4 cells revealed chromatin condensation and a nuclear morphology compatible with the induction of apoptosis. Flow cytometry assays with annexin V-FITC and propidium iodide confirmed the cell death by apoptosis. Using electron paramagnetic resonance (EPR), a semiquinone radical was detected in EL-4 cells treated with NQs. In addition, a decrease in the GSH level in parallel with reactive oxygen species (ROS) production was observed. Preincubation with n-acetyl-l-cysteine (NAC) was able to reverse the inhibitory effects of the NQs on cell proliferation, indicating that ROS generation is involved in NQ-induced apoptosis. In addition, the NQs induced a decrease in the mitochondrial membrane potential and increased the proteolytic activation of caspases 9 and 3 and the cleavage of Poly (ADP-Ribose) Polymerase (PARP). In conclusion, these results indicate that redox cycling is induced by the NQs in the EL-4 cell line, with the generation of ROS and other free radicals that could inhibit cellular proliferation as a result of the induction of the intrinsic apoptosis pathway.
Centro de Estudios Farmacológicos y Botánicos (CEFYBO), Facultad de Medicina, Universidad de Buenos Aires-CONICET, Paraguay 2155, Piso 16, C1121ABG Buenos Aires, Argentina.
María Emilia
ME
Di Rosso
María Laura
ML
Barreiro Arcos
Igal
I
Elingold
Helena
H
Sterle
Sabrina
S
Baptista Ferreira
Vitor Francisco
VF
Ferreira
Mónica
M
Galleano
Graciela
G
Cremaschi
Marta
M
Dubin
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001578:Benzopyrans / Q000037:antagonists & inhibitors / Q000494:pharmacology; D016227:Benzoquinones / Q000378:metabolism; D045744:Cell Line, Tumor; D053144:Cell Nucleus Shape / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D042002:Chromatin Assembly and Disassembly / Q000187:drug effects; D016166:Free Radical Scavengers / Q000494:pharmacology; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D016399:Lymphoma, T-Cell / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D009285:Naphthoquinones / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014344:Trypanocidal Agents / Q000037:antagonists & inhibitors / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects*
C588159:2-methyl-2-phenyl-3,4-dihydro-2H-benzo(h)chromene-5,6-dione; C588158:2-p-tolyl-3,4-dihydro-2H-benzo(h)chromene-5,6-dione; C588157:2-phenyl-3,4-dihydro-2H-benzo(h)chromene-5,6-dione; D000970:Antineoplastic Agents; D001578:Benzopyrans; D016227:Benzoquinones; D016166:Free Radical Scavengers; D009285:Naphthoquinones; D017382:Reactive Oxygen Species; D014344:Trypanocidal Agents; C025232:semiquinone radicals; D005978:Glutathione; D000111:Acetylcysteine
2,7-dichlorodihydrofluorescein diacetate; 2-methyl-2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione; 2-nitro-5-thiobenzoic acid; 2-p-tolyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione; 2-phenyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione; 5,5′-dithiobis-2-nitrobenzoic acid; AO; Apoptosis; DMFA; DTNB; EB; EPR; H2DCF-DA; MMP; N,N-dimethyl-formamide; NAC; NQ1; NQ3; NQ7; O-naphthoquinones; Oxidative stress; PARP; PI; PTP; Poly (ADP-Ribose) Polymerase; ROS; Rho-123; TCA; TNB; acridine orange; electron paramagnetic resonance; ethidium bromide; mitochondrial membrane potential; n-acetyl-l-cysteine; permeability transition pore; propidium iodide; reactive oxygen species; rhodamine-123; trichloroacetic acid