Synergistic Anticancer Effects of Lenvatinib Combined with N-butylidenephthalide in Human Colorectal Cancer Cells

Background: Colorectal cancer (CRC) is one of the most prevalent and deadly gastrointestinal malignancies worldwide. Lenvatinib, a multi-tyrosine kinase inhibitor, has shown limited clinical benefit as a monotherapy for CRC. Therefore, combining lenvatinib with N-butylidenephthalide (BP), a known anticancer and adjuvant agent, has been explored to enhance therapeutic outcomes. This study investigates the effects of lenvatinib and BP, individually and in combination, on HCT15 and HCT116 CRC cell lines. Methods: Cell proliferation was assessed by MTT assay. Cell cycle distribution and apoptosis were measured with PI staining and annexin V-FITC staining, respectively, analyzed by flow cytometry. Reactive oxygen species (ROS) levels were determined using the DCFDA assay. Mitochondrial membrane potential (MMP) was evaluated with the JC-10 assay. Oxidative DNA damage was quantified by measuring 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels using an ELISA kit. Immunofluorescence staining was performed to evaluate γ-H2AX foci expression. Protein expression levels related to apoptosis and cell cycle regulation were analyzed by western blotting. Results: The combination of lenvatinib and BP exhibited synergistic cytotoxicity effect, promoting apoptosis by disrupting MMP in CRC cells. Additionally, this combination increased ROS accumulation, leading to oxidative DNA damage via 8-OHdG induction. Furthermore, the combination treatment induced G2/M phase cell cycle arrest by modulating the ATM-Chk2 signaling pathway. Conclusions: This study demonstrates that the combination of lenvatinib and BP represents a promising therapeutic strategy for CRC by enhancing apoptosis and cell cycle arrest through ROS-mediated DNA damage.