TNF-α-Induced Upregulation of ADAMTS-8 Expression in SW480 Cells: Implications for Intracellular Signaling Pathways and Transcription Factor Activity.

Tumor necrosis factor-alpha (TNF-α) is associated with the metastatic phenotype and regulates cellular communication in Colorectal Cancer (CRC). This study investigates the effects of TNF-α on the expression of the ADAMTS-8 gene in SW480 colorectal cancer cells, focusing on both mRNA and protein levels, as well as the underlying intracellular signaling pathways activated in response to TNF-α. SW480 cells were treated with TNF-α at varying concentrations (10, 20, and 40 ng/mL) for 24, 48, and 72 h. Utilizing the MTT assay, a significant reduction in cell viability was observed at 48 and 72 h across all doses, indicating a cytostatic effect of TNF-α. The transcriptional activity of ADAMTS-8 was assessed through transient transfection of full-length and truncated promoter constructs, revealing that TNF-α significantly upregulated ADAMTS-8 expression through specific promoter regions. Quantitative RT-PCR and Western blot analyses demonstrated a time- and dose-dependent increase in both mRNA and protein levels of ADAMTS-8 in cells treated with 10 and 20 ng/mL of TNF-α. TNF-α strongly increased the transcriptional activity of several truncated ADAMTS-8 promoter constructs. Further exploration of intracellular signaling pathways revealed the activation of NF-κB, PI3K, and SAPK/JNK pathways, with limited activation of p38/MAPK. Inhibition studies employing specific pathway inhibitors indicated that blockade of NF-κB, PI3K, p38/MAPK, and JNK signaling pathways significantly suppressed TNF-α-induced ADAMTS-8 expression at both mRNA and protein levels. Additionally, Chromatin Immunoprecipitation (ChIP) assays confirmed the binding of NF-κB, STAT-3, ELK-1, c-Jun, and ATF-1 to the ADAMTS-8 promoter in response to TNF-α treatment. These findings suggest a multifaceted regulatory mechanism wherein TNF-α promotes ADAMTS-8 expression by activating key transcription factors and associated signaling cascades—in silico analysis revealed that ADAMTS-8 is upregulated in colorectal adenocarcinoma and positively correlates with TNF expression, suggesting a potential link between inflammatory signaling and favorable prognostic outcomes. Knockdown of the ADAMTS-8 markedly promoted cell growth. When combined with ADAMTS-8 silencing, TNF-α attenuated the proliferation effect, indicating a potential interaction between TNF-α signaling and ADAMTS-8 pathways and supporting tumor suppressor function of ADAMTS-8.