Dual targeting of EGFR signaling and microtubule dynamics enhances replication-associated cellular stress and suppresses tumor growth in gefitinib-resistant non-small cell lung cancer

Acquired resistance to epidermal growth factor receptor (EGFR)-targeted therapy remains a major obstacle in the treatment of EGFR-mutant non-small cell lung cancer (NSCLC). This study investigated whether repurposing mebendazole (MBZ), a microtubule-disrupting agent, could restore sensitivity to gefitinib in gefitinib-resistant HCC827 GR5 cells. Gefitinib monotherapy showed minimal cytotoxicity, reducing cell viability only marginally (92.7 ± 1.5%), whereas MBZ significantly decreased viability to 79.7 ± 0.6% at 0.1 µM and 29.7 ± 0.6% at 0.5 µM (p < 0.001). Combination treatment (gefitinib 1 µM + MBZ 0.1 µM) further reduced viability to 37.7 ± 0.6% (p < 0.001). Mechanistically, alkaline comet assay revealed significantly increased single-strand DNA damage in the combination group (tail intensity 22.33 ± 0.56%, p < 0.001), while neutral comet assay confirmed elevated double-strand DNA breaks. MBZ also induced dose-dependent γ-H2AX activation (p < 0.001), supporting replication stress-associated DNA damage signaling. In vivo , combination therapy significantly suppressed tumor growth, prolonging tumor doubling time from 6.2 days in controls to 25 days and increasing growth delay to 18.8 days (p < 0.01), without substantial systemic toxicity. Collectively, these findings identify replication stress vulnerability as a therapeutically exploitable axis and support MBZ repurposing as an adjuvant strategy to overcome gefitinib resistance.