Huang-qin decoction increases the sensitivity of EGFR-TKIs to NSCLC cells by regulating stat3/GPX4 to induce redox ratio and ROS to inhibit CSCs.

[OBJECTIVES] Our aim is to explore the combined effects and the potential mechanism between Huang-qin decoction (HQD) and epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC) cells and .

[METHODS] Ultra-high performance liquid chromatography (UPLC) was done to detect the consistency of ingredients in different batches of HQD. Cell counting kit-8 (CCK-8) assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay and Annexin V-FITC/PI assay were performed to detect the anti-cancer effects of HQD and EGFR-TKIs (1st generation EGFR-TKI gefitinib or 3rd generation EGFR-TKI Osimertinib) in different NSCLC cell lines. ATP assay was applied to detect the effect of HQD and EGFR-TKIs in NSCLC cell lines and organoids in three-dimensional (3D) culture. The mRNA expression levels of lung cancer stem cell markers makers and redox related genes, such as sex determining region Y-box 2 (SOX2), aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), glutathione peroxidase 4 (GPX4), dual oxidase 1 (DUOX1) and dual oxidase 2 (DUOX2), were detected by using quantitative real-time PCR assay. Western blot methods were done to detect the effects of EGFR-TKIs and HQD on the protein expression levels of SOX2, ALDH1A1, GPX4, signal transducer and activator of transcription 3 (stat3) and p-stat3. Besides, the alteration of intracellular total reactive oxygen species (ROS) levels of NSCLC cells in 2D culture and 3D culture after the treatments were detected using DCFH-DA staining assay and fluorimetric intracellular total ROS activity assay, respectively. Femtosecond laser labeling free imaging (FLI) method was used to detect the redox ratio of NSCLC cells in 3D culture. experiments included subcutaneous mice xenografts of PC-9-PIK3CA-M cells to validate the anti-cancer effect, mechanism and safety of gefitinib and HQD .

[RESULTS] The consistency of ingredients in different batches of HQD was confirmed. HQD enhanced the anti-proliferation and pro-apoptosis effect of gefitinib or Osimertinib in NSCLC cell lines and organoids both in 2D culture and 3D culture . The combination of HQD and EGFR-TKIs could regulate stat3/GPX4 signal pathway to induce redox ratio, thus increasing ROS levels to inhibit CSC markers . Moreover, the drug safety, anti-cancer effect and potential mechanism of the therapy of HQD and EGFR-TKIs were confirmed .

[CONCLUSIONS] HQD enhances the anti-cancer effect of EGFR-TKIs in NSCLC through ROS-mediated CSC makers inhibition by suppressing stat3/GPX4 axis to induce redox ratio, providing a novel strategy for the treatment of NSCLC patients.