RESUMEN
Synthetic triterpenoids are multitarget compounds exhibiting promise as preventative and therapeutic agents for cancer. Their proposed mechanism of action is by forming Michael adducts with reactive nucleophilic groups on target proteins. Our previous work demonstrates that the 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivatives promote B-lymphoid cell apoptosis through a mitochondria-mediated pathway linked to mitochondrial protein aggregation. As one function of the Lon protease is to eliminate abnormal mitochondrial proteins, we hypothesized that CDDO-induced protein aggregation and lymphoma apoptosis occur by inactivating this enzyme. Here, we show that CDDO and its derivatives directly and selectively inhibit Lon. CDDO blocks Lon-mediated proteolysis in biochemical and cellular assays, but does not inhibit the 20S proteasome. Furthermore, a biotinylated-CDDO conjugate modifies mitochondrial Lon. A striking common phenotype of CDDO-treated lymphoma cells and Lon-knockdown cells is the accumulation of electron-dense aggregates within mitochondria. We also show that Lon protein levels are substantially elevated in malignant lymphoma cells, compared with resting or activated B cells. Finally, we demonstrate that Lon knockdown leads to lymphoma cell death. Together, these findings suggest that Lon inhibition plays a contributory role in CDDO-induced lymphoma cell death, and support the concept that mitochondrial Lon is a novel anticancer drug target.
Asunto(s)
Linfoma/enzimología , Mitocondrias/enzimología , Ácido Oleanólico/análogos & derivados , Proteasa La/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Activación Enzimática/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Cuerpos de Inclusión/efectos de los fármacos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Linfoma/genética , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Ácido Oleanólico/síntesis química , Ácido Oleanólico/metabolismo , Ácido Oleanólico/farmacología , Proteasa La/antagonistas & inhibidores , Proteasa La/genética , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Unión Proteica , Regulación hacia ArribaRESUMEN
OBJECTIVE: Ligands for the transcription factor peroxisome proliferator-activated receptor gamma (PPAR gamma) are emerging as a new class of antitumor agents. Herein, we investigated the triterpenoid CDDO, a PPAR gamma ligand, for its potential as an anticancer agent on human diffuse large B-cell lymphoma (DLBCL) cells. METHODS: The ability of CDDO to induce apoptosis in human DLBCL cells of both the germinal center and activated B-cell subtypes was determined by MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay, (3)H-thymidine incorporation, and Annexin-V/propidium iodide staining. Small molecule antagonists of PPAR gamma, transfection assays, DNA binding assays, immunofluorescence, Western blotting, and NF-kappaB inhibitors were utilized to determine the contribution of PPAR gamma and NF-kappaB to the cytotoxic effects of CDDO. RESULTS: Human DLBCL cells express PPAR gamma and PPAR gamma is activated by CDDO. In both subtypes of DLBCL cells CDDO inhibited proliferation, was cytotoxic, and induced apoptosis. The ability of CDDO to kill DLBCL cells was found to be independent of PPAR gamma activation. Interestingly, CDDO exposure resulted in activation of the p50 and p65 subunits of NF-kappaB. Moreover, the combination of CDDO with NF-kappaB inhibitors resulted in enhanced DLBCL cell death, indicating that NF-kappaB activation was a prosurvival signal. CONCLUSION: These findings support the potential of CDDO, alone or in combination with NF-kappaB inhibitors, as a novel therapy for patients with DLBCL.