Effects of rapamycin on proliferation and epithelial-mesenchymal transition of lung cancer A549 cells-derived spheres

Objective: To study the effect of rapamycin on lung cancer stem cell-derived spheres, and explore its related mechanism.Methods: The lung cancer A549 cell-derived spheres were obtained by culture with tumor small-cell lung carcinoma conditioned medium. The proportion of CD133+ CD44+ cells in A549 cell-derived spheres was detected by flow cytometry. Then the spheres were treated with three concentrations (20, 50 and 100 ng/mL) of rapamycin, respectively. After that, the inhibition effect of rapamycin on the expression of phospho-mammalian target of rapamycin (p-mTOR) was detected by Western blotting, and the optimal inhibition concentration of rapamycin was selected for further research. After A549 cell-derived spheres were treated with 100 ng/mL rapamycin for 24 h, the expression levels of E-cadherin, vimentin, phospho-signal transducer and activator of transcription 3 (p-STAT3), phospho-p70 ribosomal protein S6 kinase (p-p70S6K) and phospho-4E-binding protein 1 (p-4EBP1) were detected by Western blotting, while the mRNA levels of SRY (sex-determining region Y)-box 2 (Sox2), Oct4, CD133 and Nanog were examined by real-time fluorescent quantitative-PCR. The proliferative activities of A549 cells after rapamycin treatment for 1-7 days were detected by MTT method, and the size of the spheres after rapamycin treatment for 14 d was observed by contrast microscopy.Results: The A549 cell-derived spheres were successfully obtained by suspension culture, and proportion of CD133+ CD44+ cells in the spheres was (75.0±8.7)%. All three concentrations of rapamycin (20, 50 and 100 ng/mL) inhibited the expression of p-mTOR protein, and 100 ng/mL was the optimal inhibition concentration (P < 0.05). After treatment with 100 ng/mL rapamycin for 24 h, the expression level of E-cadherin protein in A549 cell-derived spheres was obviously increased (P < 0.05), but the expression levels of vimentin, p-STAT3, p-p70S6K and p-4EBP1 proteins were decreased (P < 0.05), and the levels of Sox2, Oct4, CD133 and Nanog mRNAs were also decreased (P < 0.05). Rapamycin could significantly inhibit the proliferation of A549 cells during 3-7 days, and the size of the spheres on the 14th day was smaller than that of the spheres without rapamycin treatment.Conclusion: Rapamycin can inhibit the proliferation of A549 cell-derived spheres, the sphere-formation ability and epithelial-mesenchymal transition. The mTOR-STAT3 and p-70S6K signaling pathways may play important roles in these processes. DOI:10.3781/j.issn.1000-7431.2015.11.633