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-α (TNF-α) and transforming growth factor-β (TGF-β) are well known as central signaling molecules in natural antitumor mechanisms. However, some cancer cells are resistant to TNF-α or TGF-β-induced death signaling. Herein, we investigated synergistic activities of TGF-β and TNF-α 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-β in combination with TNF-α. 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-β sensitizes SNU620 cells undergoing TNF-α-induced caspase-dependent apoptosis. TNF-α and TGF-β synergistically induced the degradation of poly(ADP-ribose) polymerase (PARP) and caspase cascade activation. We also confirmed that c-Jun NH2-terminal kinase (JNK) and Smad3 play critical roles in the apoptotic pathway. In addition, a pro-apoptotic protein Bim was critical for apoptosis and was regulated by TGF-β and TNF-α 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-β and TNF-α 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-β and TNF-α act in concert to activate apoptosis in gastric cancer cell through crosstalk between Smad and JNK signaling pathways.