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

Background: Sparstolonin B (SsnB), a natural compound with an-ti-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 prolifer-ation and inflammatory pathways. Methods: HCT-116 and BJ cells were treated with SsnB (3.125–50 μM) or PMA (1–10 nM) for 12–18 hours. Cell viability was assessed using viability assays, while apoptosis was evaluated by cleaved caspase-3 staining, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cy-tometry. Proliferation was analyzed through proliferating cell nuclear antigen (PCNA) staining. Toll-like receptor (TLR) signaling, cytokine expression, and sphingolipid lev-els were measured using immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry, respectively. Results: SsnB significantly reduced HCT-116 cell viability in a dose- and time-dependent manner, with minimal effects on BJ cells. PMA stimulated proliferation, PCNA expression, and inflammatory markers, including TLR2, TLR4, MyD88, phosphorylated extracellular signal-regulated kinase (p-ERK), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), and cytokines TNF-α (tumor necrosis factor-alpha), IL-1β (interleukin-1 beta), IL-6 (inter-leukin-6). SsnB suppressed PMA-induced effects and promoted apoptosis in HCT-116 cells, increasing cleaved caspase-3, TUNEL staining, and ceramide levels while de-creasing S1P (sphingosine-1-phosphate) and C1P (ceramide-1-phosphate). Conclu-sions: SsnB selectively inhibits proliferation, induces apoptosis, and modulates in-flammatory and sphingolipid pathways in colorectal cancer cells, with minimal tox-icity to healthy fibroblasts, supporting its potential as a targeted therapeutic agent.