RESUMEN
The tumor suppressor p53 is activated in response to cellular stress to prevent malignant transformation by activation of the DNA repair machinery to preserve the cell, or by induction of apoptosis to eliminate the cell should the damage prove irrevocable. The gene encoding p53 frequently undergoes inactivating mutations in many human cancers, but WT p53 is often expressed at high levels in melanoma, which, as judged from the malignant nature of the disease, fails to act as an effective tumor suppressor. Here we show that p53 directly up-regulates microRNA-149* (miR-149*) that in turn targets glycogen synthase kinase-3α, resulting in increased expression of Mcl-1 and resistance to apoptosis in melanoma cells. Although deficiency in miR-149* undermined survival of melanoma cells and inhibited melanoma growth in a mouse xenograft model, elevated expression of miR-149* was found in fresh human metastatic melanoma isolates, which was associated with decreased glycogen synthase kinase-3α and increased Mcl-1. These results reveal a p53-dependent, miR-149*-mediated pathway that contributes to survival of melanoma cells, provides a rational explanation for the ineffectiveness of p53 to suppress melanoma, and identifies the expression of miR-149* as a mechanism involved in the increased expression of Mcl-1 in melanoma cells.
Asunto(s)
Melanoma/genética , MicroARNs/genética , Proto-Oncogenes/genética , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Células HCT116 , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Desnudos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVE: To establish a model of ER stress-induced apoptosis with tunicamycin and to examine whether Bim is dysregulated and its potential role in resistance of melanoma cells to apoptosis under endoplasmic reticulum (ER) stress. METHODS: A model of ER stress-induced apoptosis was established with tunicamycin. Apoptotic cells were quantitated using the annexin V/propidium iodide method by flow cytometry. Hoechst staining was also used to confirm the apoptotic cell death. Western blotting was used to measure the activation of caspase-3 and -9, and the expression of Bim, GRP78, CHOP, and Foxo1 at the protein level. The expression of Bim, CHOP and Foxo1 at the mRNA level was quantitated by qPCR. The siRNA technique was used to inhibit the expression of Bim. RESULTS: Treatment of the melanoma cells with tunicamycin did not induce significant apoptosis and activation of caspase cascade, whereas it caused marked activation of caspase-3 and -9, and apoptosis in HEK293 cells which were used as a control. With exposure to tunicamycin (3 µmol/L) for 12, 24, 36 hours the Bim protein levels were not increased in Mel-RM and MM200 cells. Its mRNA levels were 0.37 ± 0.05, 0.13 ± 0.02 and 0.02 ± 0.01 in Mel-RM cells, while 0.41 ± 0.06, 0.16 ± 0.04 and 0.21 ± 0.03 in MM200 cells, respectively. The expression of Bim mRNA was significantly reduced compared with that in the control groups of the two cell lines (P < 0.01). siRNA knockdown of Bim protected HEK293 cells against activation of caspase-3. The cell apoptosis of Bim siRNA group was (5.69 ± 0.38)%, significantly lower than that of the siRNA control group (40.32 ± 1.64)% and blank control group (35.46 ± 2.01)% (P < 0.01). In the melanoma cells after exposure to tunicamycin (3 µmol/L) for 6, 12, 24, and 36 hours the transcription factor CHOP at mRNA level were significantly increased and the expressions at protein level were also up-regulated. The expressions of another transcription factor Foxo1 at mRNA level significantly decreased and the expressions at protein level were down-regulated, too. CONCLUSIONS: The lack of Bim up-regulation contributes to the resistance of melanoma cells to ER stress-induced apoptosis and may be a mechanism by which melanoma cells adapt to ER stress conditions. Transcription factors CHOP and Foxo1 may be responsible for the dysregulation of Bim in melanoma cells upon ER stress.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Estrés del Retículo Endoplásmico , Melanoma/patología , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Tunicamicina/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Proteínas de Choque Térmico/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismoRESUMEN
Genistein, a plant isoflavone, is reported to have therapeutic potentials in multiple cancers, However, the molecular mechanism underlying promoting cell apoptosis in laryngeal cancer remains unclear. In this study, we report that miR-1469 was induced by genistein in laryngeal cancer. Elevated miR-1469 promoted cell apoptosis and inhibited Mcl1 expression. In addition, we also observed that tumor suppressor p53 was increased under genistein treatment. Elevation of p53 promoted miR-1469 expression, leading to miR-1469 increase and Mcl1 decrease. Therefore, our findings suggest that genistein can suppress laryngeal cancer cell survival through p53 -miR-1469-Mcl1pathway.