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

<b>Background:</b> 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. <b>Methods:</b> HCT-116 and BJ cells were treated with SsnB (3.125-50 μM) 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. <b>Results:</b> SsnB reduced HCT-116 cell viability in a dose- and time-dependent manner with minimal effects on BJ cells. SsnB (25 μM, 12 h) decreased HCT-116 viability 0.6-fold, while PMA (10 nM, 12 h) increased it 2-fold (<i>p</i> < 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 (<i>p</i> < 0.001). PMA upregulated TLR2 and TLR4 mRNA and protein levels, with MyD88, p-ERK, and pNF-κB fluorescence increasing 2.1-, 1.5-, and 1.7-fold, respectively (<i>p</i> < 0.001). PMA elevated TNF-α, IL-1β, and IL-6 levels (<i>p</i> < 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 (<i>p</i> < 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, <i>p</i> < 0.01) while reducing PMA-induced S1P and C1P increases (<i>p</i> < 0.01). <b>Conclusions:</b> 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.