Sparstolonin B Suppresses Proliferation and Modulates Toll-like Receptor Signaling and Inflammatory Pathways in Human Colorectal Cancer Cells

Background: Sparstolonin B (SsnB), a natural compound with anti-inflammatory and anti-proliferative properties, was investigated for its effects on cell viability, apoptosis, and inflammatory pathways in human colorectal cancer cells (HCT-116) and healthy human fibroblasts (BJ). Phorbol 12-myristate 13-acetate (PMA), a tumor promoter and inflammatory activator, was used to stimulate proliferation and inflammatory pathways. Methods: HCT-116 and BJ cells were treated with SsnB (3.125-50 mu M) or PMA (1-10 nM) for 12-18 h. Cell viability was assessed using MTT analysis, while apoptosis was evaluated through cleaved caspase-3 staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry. Proliferation was analyzed through proliferating cell nuclear antigen (PCNA) staining. Toll-like receptor (TLR) signaling, cytokine expression, and sphingolipid levels were measured using immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry, respectively. Results: SsnB reduced HCT-116 cell viability in a dose- and time-dependent manner with minimal effects on BJ cells. SsnB (25 mu M, 12 h) decreased HCT-116 viability 0.6-fold, while PMA (10 nM, 12 h) increased it 2-fold (p < 0.01). No significant change was observed in BJ cells. PCNA fluorescence staining increased 2-fold with PMA and decreased 0.4-fold with SsnB (p < 0.001). PMA upregulated TLR2 and TLR4 mRNA and protein levels, with MyD88, p-ERK, and pNF-kappa B fluorescence increasing 2.1-, 1.5-, and 1.7-fold, respectively (p < 0.001). PMA elevated TNF-alpha, IL-1 beta, and IL-6 levels (p < 0.01). SsnB suppressed PMA-induced effects and promoted apoptosis, increasing cleaved caspase-3 levels by 1.5-fold and TUNEL staining by 1.9-fold (p < 0.01). Flow cytometry confirmed a significant increase in early and late apoptotic cells in the SsnB group. SsnB also increased ceramide (C18, C20, C22, and C24) levels (1.3- to 2.5-fold, p < 0.01) while reducing PMA-induced S1P and C1P increases (p < 0.01). Conclusions: SsnB selectively inhibits proliferation, induces apoptosis, and modulates inflammatory and sphingolipid pathways in colorectal cancer cells, with minimal toxicity to healthy fibroblasts, supporting its potential as a targeted therapeutic agent.