Targeting acquired resistance via promoting stemness of EGFR mutant mitigates lung adenocarcinomas progression: Intervention by harpagoside (Xuandanqingjin decoction)

Abstract

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide. Resistance to EGFR TKIs and paclitaxel-based chemotherapies in EGFR-mutant lung cancer patients leads to poor prognosis. Effective targeted therapies alone or in combination remain a major clinical challenge. Harpagoside, derived from the Chinese traditional medicine Xuandanqingjin (XDQJ) decoction, has shown potential anti-cancer effects in various tumors. In this study, we used two experimental models to investigate the anti-cancer effect of harpagoside on lung adenocarcinoma progression. We successfully established PC9GR cells resistant to gefitinib. RNA sequencing revealed differential expression of genes related to cancer stem cells and inflammation between PC9 and PC9GR cells. PC9GR formed significantly more and larger colonies compared to PC9 cells. Expression analysis showed reduced E-cadherin and increased Vimentin in PC9GR cells relative to PC9 cells. Tumors in mice treated with both harpagoside and gefitinib had significantly smaller volumes compared to the model and gefitinib-only groups. Additionally, we found that paclitaxel (PTX) in combination with low-concentration harpagoside could sensitize lung cancer cells to PTX treatment, exhibiting synergistic antitumor activity, as indicated by reduced cell proliferation and induced apoptosis. Further findings showed that, in addition to apoptotic cell death, harpagoside combined with PTX significantly triggered ferroptosis in cancer cells, as evidenced by elevated ROS production, lipid peroxidation, and intracellular Fe₂⁺ levels, while decreasing GSH contents. Moreover, harpagoside and PTX combinational treatments markedly reduced cell migration and invasion processes, and such synergistic effects were confirmed in vivo by restrained liver metastasis. Animal analysis also identified suppressive effects of harpagoside and PTX co-treatments on tumor growth in a xenograft mouse model. To gain deeper mechanistic insights into the combinational therapy, RNA-seq assays were conducted. The findings revealed that harpagoside and PTX co-treatments modulated multiple signaling pathways, particularly the Nrf2, apoptosis, and ferroptosis pathways, to exert their anti-tumor potential in cancer cells. Importantly, we found that the synergistic effects of harpagoside and PTX combination on the modulation of proliferation, apoptosis, ferroptosis, and epithelial-to-mesenchymal transition (EMT) events were almost abrogated in cancer cells with Nrf2 overexpression, suggesting that Nrf2 suppression might be required for the combinational therapy-induced cytotoxicity. Collectively, these results suggest that combinational therapy of harpagoside and PTX exhibits promising antitumor activity in lung cancer through depressing Nrf2 signaling.