Curcumin Inhibits Protease Activated Receptor 2-Induced ERK Phosphorylation Calcium Mobilization and Anti-Apoptotic Signaling in Inflammation-Driven Colorectal Cancer Cells.

Abstract

Background: Chronic inflammation drives colorectal cancer (CRC) progression, with PAR-2, a G-protein coupled receptor, linking extracellular inflammatory signals to tumor-promoting pathways via ERK1/2 phosphorylation, calcium mobilization, TNF-α upregulation, and apoptosis suppression. While curcumin has notable anti-inflammatory and anti-cancer properties, its effects on PAR-2 signaling in inflammation-driven CRC remain underexplored.

Objective: This study investigates how curcumin modulates PAR-2 expression and downstream oncogenic signaling in inflammation-driven CRC cells and explores its potential direct interaction with PAR-2 at the structural level.

Methods: HT 29 and Caco-2 CRC cell lines were exposed to lipopolysaccharide (LPS) to induce an inflammatory phenotype, followed by treatment with curcumin at 50 µM and 100 µM. PAR-2 and PAR-1 expression, along with downstream markers including ERK1/2, p-ERK, TNF-α, caspase-8, cleaved caspase-8, caspase-3, Bcl 2, and Bax, were analyzed by Western blot and quantitative PCR. Calcium mobilization was assessed using Fluo-4 dye-based fluorescence imaging. Apoptosis was quantified using MTT viability assays, AO/EtBr dual staining, and Annexin V/PI flow cytometry. In parallel, AlphaFold-predicted structural models of PAR-2 were used to perform molecular docking with curcumin using CB-Dock2, to identify potential binding pockets and assess binding energetics.

Results: Curcumin selectively downregulated PAR-2-but not PAR-1-at both transcript and protein levels in a dose-dependent manner. This downregulation was accompanied by suppression of ERK phosphorylation and calcium signaling, inhibition of TNF-α secretion, and reversal of the anti-apoptotic signaling axis (Bcl 2 downregulation and Bax and caspase-3/-8 upregulation). Functional assays confirmed enhanced apoptosis in curcumin-treated cells. Computational docking revealed a high-affinity binding interaction between curcumin and the transmembrane domain of PAR-2, supporting the hypothesis of direct G-Protein-Coupled Receptor (GPCR) modulation.

Conclusions: Our findings reveal that curcumin targets the PAR-2/ERK/TNF-α axis and reactivates apoptotic pathways in inflammation-driven CRC, establishing it as a potent, mechanistically validated candidate for therapeutic repurposing in CRC.