Facultad de Ciencias de la Salud

A series of compounds containing the sulfonamide scaffold were synthesized and screened for their in vitro anticancer activity against a representative panel of human cancer cell lines, leading to the identification of N-(2-methyl-1H-indol-5-yl)-1-naphthalenesulfonamide (8e) as a compound showing a remarkable activity across the panel, with IC50 values in the nanomolar-to-low micromolar range. Cell cycle distribution analysis revealed that 8e promoted a severe G2/M arrest, which was followed by cellular senescence as indicated by the detection of senescence-associated b-galactosidase (SA-b-gal) in 8e-treated cells. Prolonged 8e treatment also led to the onset of apoptosis, in correlation with the detection of increased Caspase 3/7 activities. Despite increasing c-H2A.X levels, a well-established readout for DNA double-strand breaks, in vitro DNA binding studies with 8e did not support interaction with DNA. In agreement with this, 8e failed to activate the cellular DNA damage checkpoint. Importantly, tubulin staining showed that 8e promoted a severe disorganization of microtubules and mitotic spindle formation was not detected in 8e-treated cells. Accordingly, 8e inhibited tubulin polymerization in vitro in a dose-dependent manner and was also able to robustly inhibit cancer cell motility. Docking analysis revealed a compatible interaction with the colchicine-binding site of tubulin. Remarkably, these cellular effects were reversible since disruption of treatment resulted in the reorganization of microtubules, cell cycle re-entry and loss of senescent markers. Collectively, our data suggest that this compound may be a promising new anticancer agent capable of both reducing cancer cell growth and motility.

La leucemia aguda linfoblástica (LAL) es una enfermedad linfoproliferativa caracterizada por la presencia de alteraciones cromosómicas y mutaciones que hacen de esta enfermedad una de las principales causas de mortalidad dentro de las neoplasias hematológicas. El Imatinib (IM) forma parte integral de la terapia de primera línea en leucemias Ph+ al ser un inhibidor específico de Bcr-Abl. Sin embargo, en LAL-B Ph+ resulta muy poco efectivo al expresarse la oncoproteína Bcr-Abl p190. Por otra parte, resultados previos de nuestro grupo demuestran que Bcr-Abl induce la degradación proteasomal del inhibidor del ciclo celular p27kip1, y la no degradación de esta proteína es suficiente para detener la progresión del ciclo celular. Además, las células que expresan Bcr-Abl son más sensibles a la inducción de apoptosis por la inhibición del proteasoma con Bortezomib (Btz) que células que no expresan dicha oncoproteína. Asimismo, el Btz es capaz de detener el crecimiento celular e inducir la apoptosis en células de LAL-T al inhibir la expresión de genes diana de Notch1. Por todo ello, en este trabajo hemos explorado el uso del inhibidor del proteasoma Btz en células de leucemia aguda linfoblástica, tanto de células B con cromosoma Philadelphia (LAL-B Ph+) como de células T (LAL-T), demostrando que este inhibidor causa parada de ciclo celular e induce apoptosis por activación de la ruta de las caspasas. Además el Btz induce una menor expresión del gen antiapoptótico Bcl-2, así como una expresión diferencial de los genes pro-supervivencia y pro-apoptoticos entre las líneas celulares estudiadas que se correlaciona directamente con la diferente sensibilidad al Btz. También, hemos visto que la resistencia al Btz tiene una relación directa con una mayor expresión del proteasoma constitutivo. Finalmente, analizamos el efecto combinado de IM y Btz en células de LAL-B Ph+ y descubrimos que el IM aumenta la sensibilidad al Btz, de tal forma que la administración secuencial del IM seguido del Btz produce un efecto sinérgico mayor que si se administran de manera conjunta los dos fármacos. Con nuestros resultados proponemos el uso del Btz para inducir apoptosis en células de LAL, especialmente cuando hay mayor expresión del inmunoproteasoma, y la administración consecutiva de IM seguido de Btz para mejorar el tratamiento de leucemias Bcr-Abl positivas. Acute Lymphoblastic Leukemia (ALL) is a lymphoproliferative disease characterized by the presence of chromosomal abnormalities and mutations that make this disease one of the leading causes of death in hematological malignancies. Imatinib (IM) has become an integral part of front-line therapy for Ph+ leukemias being a specific inhibitor of Bcr-Abl. However, it has proven to be far less efficacious in the treatment of ALL-B Ph+ expressing the p190 form of Bcr-Abl. Previous results of our group suggest that Bcr-Abl regulates proteasomal degradation of the cell-cycle inhibitor p27kip1, and the no degradation of this protein is sufficient to induce a cell-cycle arrest. In addition, Bcr-Abl-expressing cells are more sensitive to apoptosis induction by the proteasome inhibitor Bortezomib (Btz) than cells no expressing Bcr-Abl. Thus, Btz induces a cell-cycle arrest and also apoptosis in Bcr-Abl-expressing cells and not in control cells. In this study we focused on the use of the proteasome inhibitor Btz in B-cell Philadelphia chromosome positive and T-cell Acute Lymphoblastic Leukemia (ALL-B Ph+ and ALL-T) cell lines. Our results showed that Btz induced cell-cycle arrest and caspase-dependent apoptosis. Furthermore, Btz significantly induced the down-regulation of the anti-apoptotic protein BCL-2 and a differential expression of pro- and anti-apoptotic proteins between the cell lines tested, which correlated with sensitivity to Btz treatment. In line with that, we also found an increased expression of the constitutive proteasome in the Btz less sensitive cell lines. Finally, we explored the possible synergistic effects between Btz and IM in ALL-B Ph+ cell lines. We observed that IM increases the sensitivity to Btz and that the administration of IM followed by Btz produces a synergistic effect greater than when the two drugs were administrated at the same time. Based on our results we propose that Btz induces apoptosis in ALL cell lines, especially in those with an increased expression of the immunoproteasome. We also propose a sequential administration of IM followed by Btz to improve the treatment in patients with Bcr-Abl-positive leukemias.