The Role of Artocarpin In Inhibiting Wnt/Β-Catenin Signalling Pathway Through Its Binding to Tcf-4/Β-Catenin Complex in H460-Derived Lung Cancer Stem Cells

The presence of cancer stem cells (CSCs) in lung cancer could contribute to cancer development and unsuccessful treatment. β-Catenin protein has been found to play a crucial role in cell survival. As artocarpin has been postulated to exert important antitumor properties, this research was conducted to elucidate the effect of artocarpin in the suppression of CSCs through modulating the β-Catenin signalling pathway. Using isolated CSCs from lung adenocarcinoma cell lines (H460), the potential of artocarpin in suppressing CSC was assessed using MTT assay. The stemness gene expression was analysed using real-time quantitative polymerase chain reaction; meanwhile, the assessment of artocarpin binding affinity against the β-Catenin active site was performed using docking analysis and validated with RT-qPCR. A 62.5% of lung CD166⁺CD44⁺ CSCs cells and 37.5% of lung CD166^-CD44^- non-CSCs cells were identified in the H460 cell line. The results revealed that artocarpin exerted the highest cytotoxic value against lung CD166⁺CD44⁺ CSCs with an IC₅₀ value of 5.07 µg/mL compared to CD166^-CD44^- non-CSCs and H460 cells which were 8.67 µg/mL and 9.07 µg/mL, respectively. Additionally, the expression of stemness genes such as KLF4, SOX2, and NANOG was found to be significantly reduced (P< 0.05) in the treated lung CSCs with fold change (FC) values of 0.025, 0.104, 0.074, respectively in 10 µM artocarpin. The molecular docking analysis showed that artocarpin possessed the best-docked complexes with β-Catenin and WNT protein with high binding energy values (-7.04 kcal/mol and -14.91 kcal/mol), respectively compared to other WNT/β-Catenin inhibitors including isorhamnetin, fisetin, genistein, silibinin, catechin, luteolin, coumestrol and β-naphthoflavone. These findings support the inhibitory activity of artocarpin on lung CSCs with low WNT and β-Catenin gene expression levels and FC values (0.155, P<0.01 and 0.129, P<0.05) in 10 µM artocarpin compared to untreated cells, respectively. Therefore, based on this study, it is suggested that artocarpin has the potential to be developed as a therapeutic agent to inhibit stem cell regeneration in lung cancer via the WNT/β-Catenin signalling pathway through inhibition of TCF-4/β-Catenin complex formation.