The effect of Sparstolonin B on toll-like receptor signaling in colon cancer cells

Objective: Colorectal cancer is among the most common malignancies worldwide and ranks third in cancer-related mortality. Increased expression of Toll-like receptors 2 and 4 (TLR2/4) in colorectal cancer enhances the inflammatory response and promotes tumor development. This study investigated the effects of Sparstolonin B (SsnB), a TLR2/4 antagonist, on cell viability, inflammatory signaling pathways, apoptotic processes, and sphingolipid metabolism in human colorectal cancer cells (HCT-116). Methods: HCT-116 and healthy fibroblast (BJ) cells were treated with SsnB (3.125-50 µM) and/or phorbol 12-myristate 13-acetate (PMA; 1-10 nM). The effects of these treatments on cell survival were evaluated in a dose- and time-dependent manner using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The impact of SsnB on proliferation was assessed by proliferating cell nuclear antigen (PCNA) enzyme-linked immunosorbent assay (ELISA) and immunofluorescence analysis. Apoptotic effects were determined using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), flow cytometry, and cleaved caspase-3 immunofluorescence analysis. TLR signaling, cytokine expression, and sphingolipid levels were measured by quantitative real-time polymerase chain reaction (q-RT PCR), ELISA, immunofluorescence staining, and mass spectrometry (LC-MS/MS). Results: SsnB reduced HCT-116 cell viability in a dose- and time-dependent manner, with minimal effects on BJ cells. While PMA treatment enhanced proliferation, SsnB suppressed this effect. Cleaved caspase-3 activity and the proportion of TUNEL-positive cells increased in the SsnB-treated groups. PMA-induced increases in TLR2/4, MyD88, p-ERK, and p-NF-κB levels were markedly suppressed by SsnB. Moreover, SsnB reduced pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), increased ceramide species (C18, C20, C22, and C24), and decreased S1P and C1P levels associated with tumor progression. Conclusion: SsnB selectively inhibits proliferation, induces apoptosis, and modulates TLR-mediated inflammatory responses and sphingolipid metabolism in colorectal cancer cells. Its lack of significant toxicity in healthy fibroblasts highlights SsnB as a promising targeted therapeutic agent for colorectal cancer treatment. Keywords: Sparstolonin B; colorectal cancer; apoptosis; toll-like receptor; inflammation; sphingolipid