Tougu Xiaotong capsules inhibit lung cancer and gefitinib-resistant cells by blocking ERBB2/PI3K-AKT-mTOR signaling and triggering ferroptosis.

[BACKGROUND] Tougu Xiaotong Capsule (TXC), a traditional formulation used for osteoarthritis, comprises Sarcandra glabra, Ligusticum chuanxiong, Paeonia lactiflora, and Morinda officinalis. Since these constituents exhibit antitumor activity, we hypothesized that TXC could combat drug resistance through multiple mechanisms, including ferroptosis and erythroblastic oncogene B2‌ (ERBB2) signaling.

[PURPOSE] This study investigated the efficacy and molecular mechanisms of TXC in treating lung cancer, specifically in the context of gefitinib resistance.

[STUDY DESIGN] This study employed an integrated pharmacological approach combining phytochemical profiling, in vitro phenotypic and mechanistic assays on gefitinib-sensitive and -resistant lung cancer cells, proteomic analysis for target identification, and in vivo validation using a Lewis lung carcinoma (LLC) mouse model.

[METHODS] The chemical profile of TXC was characterized via ultra-performance liquid chromatography‒tandem mass spectrometry (UPLC‒MS/MS). In vitro assays were used to evaluate the antiproliferative, apoptotic, and antimigratory effects of TXC and its constituents on lung and breast cancer cell lines, including gefitinib-resistant cells. High-performance liquid chromatography-MS (HPLC-MS)-based proteomic analysis identified potential targets, subsequently validating the ability of TXC to modulate the ERBB2/PI3K-AKT-mTOR pathway and ferroptosis through Western blotting, transmission electron microscopy, and reactive oxygen species (ROS)/lipid peroxidation (LPO) assessment. Finally, a subcutaneous LLC mouse model confirmed the in vivo antitumor efficacy and safety profile of TXC, demonstrating its potential as a therapeutic agent.

[RESULTS] TXC had stronger inhibitory effects on lung cancer cells than did its components. In vitro, it reduced viability and proliferation in lung and breast cancer cells, induced cycle arrest and apoptosis in lung cancer cells, and inhibited migration. Proteomics revealed ERBB2 downregulation and heme oxygenase 1 (HMOX1) upregulation. TXC suppressed the ERBB2/PI3K-AKT-mTOR pathway and induced ferroptosis via HMOX1 upregulation and glutathione peroxidase 4 (GPX4) downregulation, causing iron accumulation, LPO, and ROS elevation. In vivo, oral TXC inhibited tumor growth in LLC mice via similar mechanisms. It also suppressed gefitinib resistance in cells and synergized with gefitinib. TXC combats lung cancer by blocking ERBB2/PI3K-AKT-mTOR signaling and triggering ferroptosis through HMOX1/GPX4 modulation, overcoming gefitinib resistance.

[CONCLUSION] This study elucidates a novel dual mechanism of action of TXC against lung cancer, particularly gefitinib-resistant phenotypes, characterized by the inhibition of the ERBB2/PI3K-AKT-mTOR signaling axis and the simultaneous induction of ferroptosis. These findings provide evidence supporting the potential utility of TXC as an adjuvant therapy to circumvent gefitinib resistance in clinical oncology.