RESUMEN
Transforming growth factor-ß (TGF-ß) exerts apoptotic effects on various types of malignant cells, including liver cancer cells. However, the precise mechanisms by which TGF-ß induces apoptosis remain poorly known. In the present study, we have showed that threonine 32 (Thr32) residue of FoxO3 is critical for TGF-ß to induce apoptosis via Bim in hepatocarcinoma Hep3B cells. Our data demonstrated that TGF-ß induced FoxO3 activation through specific de-phosphorylation at Thr32. TGF-ß-activated FoxO3 cooperated with Smad2/3 to mediate Bim up-regulation and apoptosis. FoxO3 (de)phosphorylation at Thr32 was regulated by casein kinase I-ε (CKI-ε). CKI inhibition by small molecule D4476 could abrogate TGF-ß-induced FoxO/Smad activation, reverse Bim up-regulation, and block the sequential apoptosis. More importantly, the deregulated levels of CKI-ε and p32FoxO3 were found in human malignant liver tissues. Taken together, our findings suggest that there might be a CKI-FoxO/Smad-Bim engine in which Thr32 of FoxO3 is pivotal for TGF-ß-induced apoptosis, making it a potential therapeutic target for liver cancer treatment.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/biosíntesis , Carcinoma Hepatocelular/genética , Factores de Transcripción Forkhead/genética , Neoplasias Hepáticas/genética , Proteínas de la Membrana/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Factor de Crecimiento Transformador beta/genética , Apoptosis/genética , Proteína 11 Similar a Bcl2 , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proteína Forkhead Box O3 , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/patología , Treonina/genéticaRESUMEN
PURPOSE: Bax and Bak are regarded as key mediators for cytochrome c (Cyt c) release and apoptosis. Loss of Bax or Bak is often reported in human cancers and renders resistance of these cancerous cells to chemotherapy. Here, we investigated that phenylarsine oxide (PAO) could induce Bax/Bak-independent apoptosis. EXPERIMENTAL DESIGN: Annexin V/propidium iodide (PI) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and caspase activation assays were conducted to detect apoptosis in Bax/Bak-deficient mouse embryonic fibroblasts (MEF) and HCT116 bax(-/-) colorectal cancer cells. Cyt c release and Bim expression were assessed by Western blotting and immunostaining. Bim was stably knocked down by short hairpin RNA. Immunoprecipitation was applied to detect the interaction between Bim and Bcl-2. Both subcutaneous and colorectal orthotopic tumor implantation models were used in nude mice to investigate the effect of PAO in vivo. RESULTS: PAO triggered Cyt c release and apoptosis in a Bax/Bak-independent manner. Bim and Bcl-2 were both involved in this process. PAO augmented the expression of Bim and strengthened the interaction between Bim and Bcl-2. Furthermore, PAO attenuated the growth of Bax-deficient cancer cells in vivo. CONCLUSIONS: Our results showed that PAO induced apoptosis in chemotherapy-resistant cancer cells, which suggests that PAO has the potential to serve as a chemotherapeutic agent for Bax- and Bak-deficient cancers.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Neoplasias Colorrectales/patología , Proteínas de la Membrana/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/fisiología , Proteína X Asociada a bcl-2/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Western Blotting , Células Cultivadas , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Ratones Desnudos , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Overwhelming evidence indicates that Bax and Bak are indispensable for mediating cytochrome c release from mitochondria during apoptosis. Here we report a Bax/Bak-independent mechanism of cytochrome c release and apoptosis. We identified a natural diterpenoid compound that induced apoptosis in bax/bak double knock-out murine embryonic fibroblasts and substantially reduced the tumor growth from these cells implanted in mice. Treatment with the compound significantly increased expression of Bim, which migrated to mitochondria, altering the conformation of and forming oligomers with resident Bcl-2 to induce cytochrome c release and caspase activation. Importantly, purified Bim and Bcl-2 proteins cooperated to permeabilize a model mitochondrial outer membrane; this was accompanied by oligomerization of these proteins and deep embedding of Bcl-2 in the membrane. Therefore, the diterpenoid compound induces a structural and functional conversion of Bcl-2 through Bim to permeabilize the mitochondrial outer membrane, thereby inducing apoptosis independently of Bax and Bak. Because Bcl-2 family proteins play important roles in cancer development and relapse, this novel cell death mechanism can be explored for developing more effective anticancer therapeutics.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/biosíntesis , Apoptosis/efectos de los fármacos , Diterpenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Proteína X Asociada a bcl-2/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Línea Celular Transformada , Citocromos c/genética , Citocromos c/metabolismo , Regulación de la Expresión Génica/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Membranas Mitocondriales/metabolismo , Permeabilidad/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
PURPOSE: Both CD44 and CD133 were reported as putative markers for isolating colorectal cancer stem cells (CSC). It remains to be resolved if both of these markers are of functional importance for colorectal CSC. EXPERIMENTAL DESIGN: The expression of CD44 and CD133 in normal colonic tissues and primary colorectal cancer was assessed by immunohistochemistry in a series of 60 patients on tissue microarray sections. Both in vitro clonogenic and in vivo tumorigenic assay were applied to measure CSC activities from the cells isolated from patients. Lentiviral RNA interference was used to stably knock down CD44 or CD133 in colorectal cancer cells from patients. RESULTS: We found that CD44(+) cells displayed clustered growth and they did not colocalize with CD133(+) cells within colorectal cancer. As few as 100 CD44(+) cells from a patients' tumor initiated a xenograft tumor in vivo. A single CD44(+) cell from a tumor could form a sphere in vitro which has characteristic stem cell properties and was able to generate a xenograft tumor resembling the properties of the primary tumor. Knockdown of CD44, but not CD133, strongly prevented clonal formation and inhibited tumorigenicity in xenograft model. CONCLUSIONS: These results indicate that CD44 is a robust marker and is of functional importance for colorectal CSC for cancer initiation.