Transcriptional and post-translational regulation of Bim is essential for TGF-β and TNF-α-induced apoptosis of gastric cancer cell

Background: Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) are well known as central signaling molecules in natural antitumor mechanisms. However, some cancer cells are resistant to TNF-alpha or TGF-beta-induced death signaling. Herein, we investigated synergistic activities of TGF-beta and TNF-alpha and molecular mechanisms involved in apoptosis of gastric cancer cells. Methods: SNU620, a human gastric carcinoma cell line was tested for cell viability by treatment of TGF-beta in combination with TNF-alpha. Cell apoptosis, proliferation, caspase activation and gene expression were tested using flow cytometry, Western blot, MTT assay, luciferase assay and real-time qRT-PCR analysis. Knockdown of target genes were performed using lentiviral shRNA system. Results: TGF-beta sensitizes SNU620 cells undergoing TNF-alpha-induced caspase-dependent apoptosis. TNF-alpha and TGF-beta, synergistically induced the degradation of poly(ADP ribose) polymerase (PARP) and caspase cascade activation. We also confirmed that c-Jun NH2-terminal kinase (INK) and Smad3 play critical roles in the apoptotic pathway. In addition, a pro-apoptotic protein Elm was critical for apoptosis and was regulated by TGF-beta and TNF-alpha at the transcriptional and post-translational levels. Expression of Bim was induced at the transcriptional level by Smad3 while Bim protein stability was maintained by a JNK-mediated pathway. Conclusion: By understanding the synergistic activation of TGF-beta and TNF-alpha in apoptosis, we may have a chance to identify good therapeutic approaches for the treatment of cancers that are resistant to death signals. General significance: Our results indicate that TGF-beta and TNF-alpha act in concert to activate apoptosis in gastric cancer cell through crosstalk between Smad and JNK signaling pathways. (C) 2013 Elsevier B.V. All rights reserved.