Silymarin sensitizes human colorectal cancer cells to 5-ALA-mediated photodynamic therapy by enhancing cytotoxicity and apoptotic signaling in vitro.

BACKGROUND: Photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) generates reactive oxygen species (ROS) that can kill tumor cells, but treatment efficacy may be limited by cellular antioxidant defenses. Silymarin, a flavonolignan complex from Silybum marianum, exhibits context-dependent antioxidant/pro-oxidant activity and has been suggested to modulate cancer redox homeostasis. This study evaluated whether silymarin pre-treatment enhances PDT-induced cytotoxicity in human colorectal cancer cells in vitro. METHODS: HCT-116 human colorectal carcinoma cells were cultured under standard conditions. Cells were pretreated with silymarin (50–400 µg/mL) for 24 h, incubated with 1 mM 5-ALA for 4 h to induce intracellular protoporphyrin IX accumulation, and then irradiated with a 635-nm diode laser (50 mW/cm²) at light doses of 0–20 J/cm². Cell viability was quantified 48 h post-treatment using the MTT assay. Drug–light interactions were assessed using the Bliss independence model to determine synergy scores across the dose matrix. Apoptosis-related gene expression (BAX, Bcl-2, caspase-3, -8, and − 9) was measured by RT-qPCR and analyzed as log2 fold change relative to controls. RESULTS: PDT alone reduced HCT-116 viability in a light-dose–dependent manner (IC₠₀ ≈ 8 J/cm²), while silymarin alone produced moderate concentration-dependent cytotoxicity with a high IC₠₀ (~ 787 µg/mL), indicating limited single-agent potency. Combining silymarin with PDT consistently produced greater loss of viability than either treatment alone. Bliss analysis showed uniformly negative synergy scores (− 10.17 to − 29.62), confirming synergistic enhancement of PDT by silymarin, strongest at low silymarin concentrations and moderate light doses; the most synergistic condition was 50 µg/mL silymarin plus 7.5 J/cm² PDT. Mechanistically, combination therapy markedly increased pro-apoptotic BAX expression and further suppressed anti-apoptotic Bcl-2 compared with monotherapies. Caspase-8, caspase-9, and caspase-3 were all upregulated by the combination, indicating activation of both extrinsic and intrinsic apoptotic pathways. CONCLUSION: Silymarin pre-treatment significantly potentiates 5-ALA-PDT cytotoxicity in HCT-116 colorectal cancer cells through a synergistic interaction that amplifies apoptosis-related signaling. These findings support silymarin as a promising, low-toxicity adjuvant to improve PDT efficacy against colorectal carcinoma, warranting further validation in additional models and in vivo studies.