RESUMEN
Cholangiocarcinoma (CCA) is an aggressive, metastatic bile duct cancer. CCA is difficult to diagnose, and responds poorly to current radio- and chemo-therapy. Piperlongumine (PL) is a naturally-occurring small molecule selectively toxic to cancer cells by targeting reactive oxygen species (ROS). In this study, we demonstrated the potential anticancer activity of PL in CCA. PL markedly induced death in CCA cell lines in a dose- and time-dependent manner through the activation of caspase-3 and PARP. PL also stimulated ROS accumulation in CCA. Co-exposure of PL with the ROS scavenger N-acetyl-L-cysteine or GSH completely blocked PL-induced apoptosis in CCA cell lines. Increased p21 via the p53-independent pathway in PL-treated CCA cells led to G2/M phase arrest and cell apoptosis. In addition, the study showed that PL trigger CCA cell lines death through JNK-ERK activation. Furthermore, the different antioxidant capacity of CCA cell lines also indicates the susceptibility of the cells to PL treatment. Our findings reveal that PL exhibits anti-tumor activity and has potential to be used as a chemotherapeutic agent against CCA.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Dioxolanos/farmacología , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Piper/química , Especies Reactivas de Oxígeno/agonistas , Acetilcisteína/farmacología , Antineoplásicos Fitogénicos/antagonistas & inhibidores , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Conductos Biliares/efectos de los fármacos , Conductos Biliares/metabolismo , Conductos Biliares/patología , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Dioxolanos/antagonistas & inhibidores , Dioxolanos/aislamiento & purificación , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Glutatión/farmacología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/agonistas , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H2O2. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.
Asunto(s)
Acetilcisteína/farmacología , Depuradores de Radicales Libres/farmacología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Acetilcisteína/metabolismo , Antineoplásicos Fitogénicos/antagonistas & inhibidores , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Catalasa/genética , Catalasa/metabolismo , Línea Celular Tumoral , Cromanos/antagonistas & inhibidores , Cromanos/metabolismo , Cromanos/farmacología , Citomegalovirus/enzimología , Dioxolanos/antagonistas & inhibidores , Dioxolanos/farmacología , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Depuradores de Radicales Libres/metabolismo , Humanos , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , Oxidantes/antagonistas & inhibidores , Oxidantes/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/metabolismo , Estabilidad Proteica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Proteínas Ubiquitinadas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Inhibition of the enzyme neutral metalloendopeptidase potentiates responses to atrial natriuretic factor and elicits reductions of blood pressure in desoxycorticosterone acetate sodium hypertensive rats. The present study evaluated the role of atrial natriuretic factor and bradykinin in the antihypertensive response to neutral metalloendopeptidase inhibition through the use of antibodies and antagonists, respectively. In addition, the pharmacokinetic mechanism by which neutral metalloendopeptidase inhibition interferes with atrial natriuretic factor metabolism was explored. The antihypertensive response to the neutral metalloendopeptidase inhibitor SCH 34826 was abruptly reversed by i.v. injection of a polyclonal antiserum to atrial natriuretic factor. In contrast, the antihypertensive response to SCH 34826 was unaffected by injection of the bradykinin antagonist Thi5,8-D-Phe7 bradykinin. The renal response to atrial natriuretic factor, SCH 34826, and phosphoramidon was inhibited by the bradykinin antagonist. The NEP inhibitor SCH 39370 significantly delayed the disappearance of TCA precipitable radioactivity from plasma following i.v. bolus dosing with 125I-labelled ANF 99-126. The effects were enhanced in the presence of the C receptor ligand. The results indicate that atrial natriuretic factor, but not bradykinin, plays an important role in the antihypertensive response to SCH 34826. Bradykinin plays a permissive role in the diuretic responses to atrial natriuretic factor and inhibitors of neutral metalloendopeptidase. Lastly, neutral metalloendopeptidase inhibition significantly alters the clearance and metabolism of tracer quantities of atrial natriuretic factor.
Asunto(s)
Factor Natriurético Atrial/farmacología , Metaloendopeptidasas/antagonistas & inhibidores , Animales , Antihipertensivos/farmacología , Factor Natriurético Atrial/metabolismo , Presión Sanguínea/efectos de los fármacos , Bradiquinina/antagonistas & inhibidores , Bradiquinina/fisiología , Desoxicorticosterona , Dioxolanos/antagonistas & inhibidores , Dioxolanos/farmacología , Dipéptidos/antagonistas & inhibidores , Dipéptidos/farmacología , Sinergismo Farmacológico , Hipertensión/inducido químicamente , Hipertensión/tratamiento farmacológico , Radioisótopos de Yodo , Cininas/antagonistas & inhibidores , Masculino , Ratas , Ratas Endogámicas , Tiorfan/farmacologíaRESUMEN
1. The inhibitory effects of lanthanide cations (Ln3+) on mechanical responses and 45Ca uptake in guinea-pig ileal longitudinal smooth muscle were studied. 2. Ln3+ strongly inhibited the phasic and tonic component of the response to the muscarinic agonist cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide (CD) the two components being affected to the same extent. Inhibition was also obtained for the responses evoked by high K+ but here the effect was mainly on the phasic response, the tonic component was merely delayed. 3. Other members of the Ln3+ series, with the exception of cerium, were found to be more effective than lanthanum in their ability to inhibit the CD response. Thulium, Tm3+, the thirteenth member of the series was the most effective cation. 4. Analysis of 170Tm uptake revealed at least two components. The concentration-dependence of one component, saturating at 2-5 x 10(-6) Tm, corresponded closely to that of the inhibitory effect of Tm3+ on contraction. 5. 170Tm uptake as a function of time showed a secondary rise after 30 min of exposure to the lanthanide. 6. Although 2-5 x 10(-6) M-Tm3+ produced 90% inhibition of the CD and the high K+ induced responses significant reduction of 45Ca uptake by the muscle was only detected when much higher Tm3+ concentrations (greater than or equal 10(-3) M-Tm3+) were used. 7. It is concluded that Ln3+ combine with membrane sites specifically involved in Ca2+ translocation during excitation-contraction coupling.