RESUMO
In this study, we show that triazoloacridinone derivative C-1305, a potent antitumor compound, in human lymphoblastic (MOLT4) and promyelocytic (HL60) leukemia cells induces G2/M arrest followed by apoptosis. In both type of cells, C-1305 at biological relevant concentrations corresponding to EC(90) value, induced a significant increase in the fraction of G2/M cells. The cell cycle perturbations were accompanied by the appearance of sub-G1 fraction, which can be considered as the apoptotic cells population. In both human leukemia cells apoptosis was additionally proved by appearance of DNA fragmentation, activation of caspase-3, PARP cleavage, externalization of phosphatydilserine as well as decrease of the mitochondrial transmembrane potential DeltaPsi(m) and ATP depletion. Treatment of lymphoblastic MOLT4 cells with the C-1305 at EC(90) concentration, caused massive death by apoptosis. Compared to MOLT4 cells, the capacity of HL60 cells to execute apoptosis after C-1305 treatment at equitoxic dose was significantly weaker, but very effective at high concentration (4x EC(90)). These differences could originate from different sensitivity of both cell types to cytotoxic action of C-1305 (EC(50) value for MOLT4 cells was 8 times lower than for HL60 cells and the EC(90) value was 14 times lower, respectively). Collectively, these results show that C-1305 is a novel and potent compound which induces G2/M arrest and subsequent apoptosis of human leukemia cells. This strong ability to induce apoptosis of tumor cells support the view that C-1305 could be consider as a new potent and promising antitumor agent.
Assuntos
Acridinas/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Triazóis/farmacologia , Acridinas/química , Trifosfato de Adenosina/metabolismo , Antineoplásicos/química , Western Blotting , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Eletroforese em Gel de Ágar , Citometria de Fluxo , Células HL-60 , Humanos , Marcação In Situ das Extremidades Cortadas , Membranas Intracelulares/efeitos dos fármacos , Leucemia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Triazóis/químicaRESUMO
C-1748 is a DNA-binding agent with potent antitumor activity, especially towards prostate and colon carcinoma xenografts in mice. Here, we elucidated the nature of cellular response of human colon carcinoma HCT8 and HT29 cells to C-1748 treatment, at biologically relevant concentrations (EC(90) and their multiplicity). Cell cycle analysis showed gradual increase in HCT8 cells with sub-G1 DNA content (25% after 72h) considered as apoptotic. Hypodiploid cell population increased up to 60% upon treatment with 4x EC(90) concentration of the drug. Compared with HCT8 cells, the fraction of sub-G1 HT29 cells did not exceed 14%, even following 4-fold dose escalation. Morphological changes and biochemical markers such as: phosphatydylserine externalization, apoptotic DNA breaks, mitochondrial dysfunction and caspase activation confirmed the presence of considerable amount of apoptotic HCT8 cells but only a low amount of apoptotic HT29 cells. Next, we demonstrated that HCT8 cells surviving after exposure to C-1748 were in the state of senescence, based on altered cell morphology and expression of a pH 6-dependent beta-galactosidase. On the contrary, no beta-galactosidase staining was observed in HT29 cells after C-1748 treatment. Moreover, prolonged drug incubation (up to 168h) resulted in massive detachment of cells from culture plates, which together with Annexin V/PI results, indicated that necrosis was the main response of HT29 cells to C-1748 treatment. We also determined the ability of C-1748 to induce reactive oxygen species (ROS) in colon cancer cells and demonstrated, that generation of ROS was not essential for C-1748-induced apoptosis and cytotoxic activity of this drug.