Subject(s)
Apoptosis , Pancreatic Neoplasms/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Cell Line, Tumor , Humans , Inhibitor of Apoptosis Proteins/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , RNA Interference , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor/genetics , Signal Transduction/genetics , Survivin , Time Factors , Transfection , bcl-X Protein/geneticsABSTRACT
Signal transducer and activator of transcription 3 (STAT3) is a key regulator of cytokine signaling pathways that regulates gene expression. In pancreatic cancer, constitutive activation of STAT3 contributes to oncogenesis by preventing apoptosis through upregulation of anti-apoptotic proteins. We have examined the inhibition of STAT3 as a potential therapeutic approach in pancreatic cancer. siRNA targeting STAT3 was used to evaluate the role of STAT3 in modulating the expression of Survivin/BIRC5 and BCL-xL in the pancreatic cancer cell lines PANC-1 and BxPC-3 and induction of apoptosis. Expression of STAT3, Survivin/BIRC5, and BCL-xL on mRNA and protein level was measured by real-time RT-PCR and Western blot analysis 24, 48, and 72 h after transfection. STAT3 downregulation resulted in a decrease of cell viability in both cell lines and induced apoptosis in BxPC-3 cells. Despite significant inhibition of STAT3, the expression of the anti-apoptotic genes Survivin/BIRC5 and BCL-xL were not subsequently downregulated. Even more, the cell line BxPC-3 shows a significant increase of Survivin/BIRC5 and BCL-xL mRNA after 48-72 h as a result of STAT3 downregulation. Inactivation of STAT3 in pancreatic cancer cell lines induces apoptosis but also may promote the expression of anti-apoptotic genes.
Subject(s)
Apoptosis , Gene Expression Regulation, Neoplastic , Pancreatic Neoplasms/pathology , STAT3 Transcription Factor/physiology , Cell Line, Tumor , Cell Survival , Down-Regulation , Humans , Inhibitor of Apoptosis Proteins/analysis , Inhibitor of Apoptosis Proteins/genetics , Pancreatic Neoplasms/metabolism , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/genetics , Survivin , bcl-X Protein/analysis , bcl-X Protein/geneticsABSTRACT
PURPOSE: Increased expression of survivin has been identified as a negative prognostic marker in a variety of human cancers. We have previously shown that survivin is a radiation-resistance factor and that the therapeutic effect of survivin knock-down might result from an impaired DNA repair capacity. In this study, we aimed to elucidate an interrelationship between survivin's cellular localization and DNA double-strand break repair. METHODS AND MATERIALS: Survivin's cellular distribution and nuclear complex formation were assayed by Western blotting of subcellular fractions, by immunofluorescence staining, and co-immunoprecipitation in SW480 colorectal cancer cells. DNA repair capacity was analyzed by kinetics of gamma-H2AX foci formation, and by DNA-dependent protein kinase (DNA-PKcs) assays in the presence of survivin-specific or nonspecific control siRNA. RESULTS: Following irradiation, we observed a rapid nuclear accumulation of survivin and subsequent phosphorylation of the protein in the nucleus. Co-immunoprecipitation analyses from nuclear extracts revealed an interaction among survivin, Ku70, gamma-H2AX, MDC1, and DNA-PKcs that was confirmed by immunofluorescence co-localization in nuclear foci. Survivin knock down by siRNA resulted in an impaired DNA double strand break repair, as demonstrated by an increased detection of gamma-H2AX foci/nucleus at 60 min and a higher amount of residual gamma-H2AX foci at 24 hr postirradiation. Furthermore, we detected in survivin-depleted cells a hampered S2056 autophosphorylation of DNA-PKcs and a significantly decreased DNA-PKcs kinase activity. CONCLUSION: These data indicate that nuclear survivin is linked to DNA double-strand break repair by interaction with members of the DNA double-strand breaks repair machinery, thus regulating DNA-PKcs activity.
