Comparison of the effects of β-Arbutin on the NF-κB pathway in two-dimensional (2D) and three-dimensional (3D) colorectal cancer cell lines

Colorectal cancer (CRC) ranks as the third most prevalent cancer worldwide and is associated with significant mortality, primarily due to metastatic spread and resistance to therapy. In approximately half of CRC cases, the NF-κB signaling pathway is dysregulated, contributing to tumor progression, cell survival, and invasive behavior. β-Arbutin, a natural β-glucoside compound, has demonstrated potential anticancer activity. This study investigated the suppressive impact of β-Arbutin on NF-κB signaling in CRC, utilizing both conventional 2D and advanced 3D cell culture systems. HT-29 colorectal cancer cells were grown using both two-dimensional (2D) monolayer cultures and three-dimensional (3D) alginate bead models. Cell viability was assessed via WST-1 assay to establish the half-maximal inhibitory concentration (IC₅₀) values for β-Arbutin and the reference drug Sulfasalazine. Flow cytometry was employed to quantify apoptotic cell populations, Caspase 3/7 enzymatic activity, and related protein expression. Immunofluorescence staining further validated the protein levels detected by flow cytometry. All data were statistically analyzed using GraphPad Prism software, with a p-value threshold of <0.05 considered significant. β-Arbutin exhibited a more pronounced reduction in cell viability in 3D culture systems compared to conventional 2D cultures. The compound induced significantly higher apoptosis rates in 3D models (56.46 %) versus 2D cultures (22.11 %; p < 0.0001). Similarly, Caspase 3/7 activity was markedly elevated in β-Arbutin-treated 3D cells (53.56 %) relative to their 2D counterparts (34.04 %; p < 0.0001). Furthermore, β-Arbutin treatment resulted in a more substantial decrease in target protein expression levels in 3D cultures compared to 2D systems. The 3D CRC models demonstrated significantly greater sensitivity to β-Arbutin than 2D cultures, with more robust NF-κB pathway suppression and apoptotic response. This differential efficacy underscores the superior biomimetic properties of 3D culture systems. Our results position β-Arbutin as a potent NF-κB-targeting agent and validate its potential for clinical translation in colorectal cancer therapy.