RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Genisteína/farmacologia , Neoplasias Laríngeas/tratamento farmacológico , MicroRNAs/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patologia , MicroRNAs/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Proteína Supressora de Tumor p53/genéticaRESUMO
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.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Estresse do Retículo Endoplasmático , Melanoma/patologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Tunicamicina/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Células HEK293 , Proteínas de Choque Térmico/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
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.
