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

• Published in: Biochimica et biophysica acta Vol. 1830; no. 6; pp. 3584 - 3592
• Main Authors: Ha Thi, Huyen Trang, Lim, Hee-Sun, Kim, Jooyoung, Kim, Young-Mi, Kim, Hye-Youn, Hong, Suntaek
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-06-2013
• Subjects: Apoptosis; Apoptosis Regulatory Proteins - biosynthesis; Apoptosis Regulatory Proteins - genetics; Bcl-2-Like Protein 11; Bim; c-Jun NH2-terminal kinase; caspases; Caspases - genetics; Caspases - metabolism; Cell Line, Tumor; cell viability; death; Female; flow cytometry; gene activation; gene expression; Gene Expression Regulation, Neoplastic; genes; Humans; JNK Mitogen-Activated Protein Kinases - genetics; JNK Mitogen-Activated Protein Kinases - metabolism; luciferase; Male; MAP Kinase Signaling System; Membrane Proteins - biosynthesis; Membrane Proteins - genetics; mitogen-activated protein kinase; NAD ADP-ribosyltransferase; necrosis; neoplasm cells; pro-apoptotic proteins; Protein Stability - drug effects; Proto-Oncogene Proteins - biosynthesis; Proto-Oncogene Proteins - genetics; quantitative polymerase chain reaction; reverse transcriptase polymerase chain reaction; signal transduction; Smad3; Smad3 Protein - genetics; Smad3 Protein - metabolism; stomach neoplasms; Stomach Neoplasms - genetics; Stomach Neoplasms - metabolism; Stomach Neoplasms - pathology; transcription (genetics); Transcription, Genetic - drug effects; Transcription, Genetic - genetics; transforming growth factor beta; Transforming Growth Factor beta - genetics; Transforming Growth Factor beta - metabolism; Transforming Growth Factor beta - pharmacology; Transforming growth factor-β; tumor necrosis factor-alpha; Tumor Necrosis Factor-alpha - genetics; Tumor Necrosis Factor-alpha - metabolism; Tumor Necrosis Factor-alpha - pharmacology; Tumor necrosis factor-α; Western blotting; c-Jun NH2-terminal kinase; JNK; Bim; PARP; TRAF2; Tumor necrosis factor-α; MAPK; TNFR1; NF-κB; CHX; Smad3; TNF-α; Transforming growth factor-β; SBE; RIP; TGF-β; ERK; Apoptosis
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2013.03.006

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.
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.
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.
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.
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.
•TGF-β sensitizes gastric cancer cell in TNF-α-induced caspase-dependent apoptosis.•Bim is a key mediator of TGF-β and TNF-α-induced apoptosis in gastric cancer cell.•Smad3 enhances the expression of Bim via transcriptional regulation.•JNK signaling stabilizes the Bim protein at post-translational step.•Crosstalk between Smad3 and JNK pathway is important for cancer cell death.