Molecular Mechanism Analysis of Cinnamomi Ramulus in Improving Drug Resistance of Non-small Cell Lung Cancer Using Cell Experiments and Network Pharmacology

OBJECTIVE To investigate the effect of traditional Chinese medicine Cinnamomi Ramulus on the proliferation, metastasis, and apoptosis of drug-resistant cell line H1650 in non-small cell lung cancer(NSCLC), and reveal the function and potential molecular mechanism of Cinnamomi Ramulus in improving the resistance of H1650 to gefitinib.

METHODS H1650 cells were treated with Cinnamomi Ramulus extract, gefitinib, or two in combination. Using MTT, wound healing assay, and flow cytometry to study H1650 cells’ effect on cell viability, migration, and apoptosis. Then, network pharmacology was used to predict the effective active components of Cinnamomi Ramulus extract and the possible molecular targets relating to NSCLC, and to conduct the relationship network of "Cinnamomi Ramulus-active ingredient-targets-disease". GO and KEGG analysis were subsequently performed on the potential molecular targets of Cinnamomi Ramulus, and the core targets and signaling pathways that are closely related to the active components of Cinnamomi Ramulus are further predicted. Finally, Western blotting was used to detect the changes in protein expression and protein phosphorylation level of the predicted core molecules after treatment with Cinnamomi Ramulus extract and gefitinib.

RESULTS  In H1650, Cinnamomi Ramulus extract inhibited cell viability and migration and prompted cell apoptosis. Compared with the single-drug group with gefitinib, those two drugs in combination reduced the IC₅₀ of gefitinib inhibiting cell proliferation. Network pharmacology predicted 7 active components, 252 molecular targets related to NSCLC disease, and a number of signaling pathways closely related to the initiation and development of tumors. In particular, Src, PI3K, MAPK1, and their related signaling pathways might be the core molecular targets affecting NCSLC cell function and drug resistance. At the molecular level, Cinnamomi Ramulus extract significantly reduced the protein expression of Src and the downstream molecules in the Src signaling pathway(phosphorylation level of Akt and mTOR) in H1650 cells.

CONCLUSION  Cinnamomi Ramulus improves gefitinib resistance in H1650 cells presumably by inhibiting Src/Akt/mTOR signaling pathway.