Centipeda minima extract sensitizes lung cancer cells to DNA-crosslinking agents via targeting Fanconi anemia pathway

Background: Intrinsic and acquired chemoresistance remains a critical challenge in lung cancer chemotherapy. Fanconi anemia (FA) pathway plays an important role in antagonizing the cytotoxic effects of chemotherapeutics by repairing DNA damage. We recently demonstrated that the traditional Chinese medicinal herb, Centipeda minima (C. minima), possessed anti-inflammatory and antioxidant properties. However, the potential anticancer application of C. minima and the underlying mechanisms remain unclear. Purpose: We aimed to investigate the combined anticancer effects of the ethanol extract of C. minima (ECM) and DNA-crosslinking agents on non-small cell lung cancer (NSCLC) and elucidate the underlying mechanisms. Methods: Cell viability and flow cytometry assay were performed to determine the synergistic cytotoxicity of ECM and DNA-crosslinking agents, cisplatin (CDDP) or mitomycin C (MMC), in NSCLC cells. Western blotting and immunofluorescence were conducted to examine the effects of ECM on protein expression in DNA damage repair pathway. Comet assay was applied to evaluate DNA damage levels. Subcutaneous xenografts of NSCLC were established to evaluate the combined anticancer effects of ECM and CDDP. Results: Combined treatments with ECM and DNA-crosslinking agents exhibited synergistic cytotoxic effects against A549 and H1299 cells. FANCD2 was highly expressed in NSCLC that correlates with poor prognosis of NSCLC patients, based on the online database analysis. ECM significantly inhibited DNA damage-induced monoubiquitination and nuclear foci formation of FANCD2, thereby sensitizing NSCLC to CDDP- or MMCinduced DNA damage and apoptosis, as evidenced by increased expression of gamma-H2AX, increased cleavage of caspases-3 and PARP, and enhanced Annexin V-FITC/PI staining. Further, ECM can also decrease the protein level of FANCD2 that contributes to the chemosensitizing effects. Moreover, ECM significantly attenuated CDDPmediated S-phase arrest by antagonizing the activation of ATR/Chk1 pathway in NSCLC cells. Animal experiments further demonstrated that ECM and CDDP combination treatment synergistically inhibited tumor growth by decreasing FANCD2 protein level in tumor tissues. Conclusion: Our results demonstrated that ECM can inhibit DNA-crosslinking agents-induced activation of FA pathway by attenuating both the expression and monoubiquitination of FANCD2. ECM and CDDP combination therapy exhibited synergistic anticancer effects both in vitro and in vivo, indicating that ECM and its active components might serve as novel anticancer drugs in the combination chemotherapy.