RESUMEN
BACKGROUND Epithelial ovarian cancer is a major cause of mortality in women and one of the most common gynecologic disorders. Pterostilbene (PTS), a trans-3,5-dimethoxy-4'-hydroxystilbene, was chosen for this work due to its reported effectiveness as a chemotherapeutic agent in cancer studies. In this work, we studied underlying molecular mechanisms of PTS treatment in various ovarian cancer cell lines such as OVCAR8, OV1063, IGROV-1, and SKOV3. MATERIAL AND METHODS We used the cytometric bead array (CBA) method and real-time PCR analysis to analyze the secretion level of tumor necrosis factor alpha (TNF-α) and to measure the TNF-α mRNA expression. NF-kappa B (NF-κB) promoter analysis, Western blot analysis, electrophoresis mobility shift assay (EMSA), and immunostaining analyses were performed to measure the NF-κB activity and other relative proteins levels. RESULTS The PTS treatment decreased the release of TNF-α in IGROV-1 ovarian cancer cells. It also showed significant inhibitory effect on nuclear NF-κB p50, and NF-κB p65 protein levels. CONCLUSIONS From the results obtained, we suggest that PTS has the potential to treat ovarian cancer by reducing the level of TNF-α cytokine and to have a limited effect on NF-κB, AKT, and ERK signaling pathways.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Estilbenos/uso terapéutico , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , FN-kappa B/metabolismo , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Estilbenos/química , Estilbenos/farmacología , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To express the fusion protein of glutathione S-transferase (GST) and human Id-2 in E. coli and prepare the polyclonal antibodies against Id-2. METHODS: The coding sequence of Id-2 gene was amplified by RT-PCR from the total RNA of breast cancer tissue. The recombinant plasmid was identified by PCR, restriction endonuclease digestion analysis and sequencing. The fusion protein GST-Id-2 expressed in E. coli following IPTG induction was purified by glutathione-agarose affinity chromatography and used to immunize rabbits to prepare the polyclonal antibodies against GST-Id-2. RESULTS: PCR, restriction endonuclease digestion and sequence analyses showed that the Id-2 gene had been correctly inserted into pGEX-6P-1 vector, and the GST-Id-2 fusion protein expressed had a relative molecular mass of approximately 40,000 as shown by SDS-PAGE. The polyclonal antibodies obtained from the rabbit sera were found to specifically react with purified Id-2 by Western blotting, ELISA and agar gel immunodiffusion (AGP). CONCLUSION: The prepared polyclonal antibodies against Id-2 allow effective Id-2 detection and facilitate further investigation of the structure and antigen epitope of Id-2.