Résumé
OBJECTIVE To investigate the effect and mechanism of gracillin from Reineckia carnea on autophagy in non- small cell lung cancer A549 cells. METHODS Using A549 cells as subjects, the effects of different concentrations of gracillin (0.25, 0.5, 1, 2, 4 μmol/L) on the proliferation of cells were detected by CCK-8 after being treated for different time (12, 24, 48 h). Compared with the control group without medication, the effect of gracillin (2 μmol/L) on the formation of autophagosomes in cells was observed by transmission electron microscope after 24 h of exposure. The aggregation of GFP-LC3 on autophagosome membrane was detected by GFP-LC3 plasmid transfection after being treated with gracillin (0.25, 0.5, 1, 2 μmol/L) for 24 h. Quantitative real-time PCR and Western blot assay were used to detect the mRNA and protein expressions of family with sequence similarity 102 member A(FAM102A), the expressions of autophagy-related proteins [p62, Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)], and the expressions of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway-related proteins in A549 cells after being treated with gracillin (0.25, 0.5, 1 and 2 μmol/L) for 24 h. RESULTS Gracillin significantly inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. The IC50 was 2.55 μmol/L at 24 h. After 24 h of gracillin treatment, autophagosomes with bilayer membrane structure were found in the cell cytoplasm, and GFP-LC3 green fluorescent spots on autophagosome membrane were obvious, representing an increasing trend as drug concentration. Compared with the control group, mRNA and protein expressions of FAM102A (0.5, 1, 2 μmol/L groups), protein expression of Beclin-1 (1, 2 μmol/L groups) and LC3B-Ⅱ/LC3B-Ⅰ ratio (2 μmol/L group) were significantly increased in different concentrations of gracillin groups, while the protein expression of p62 (1, 2 μmol/L groups), and the protein phosphorylations of Akt (1, 2 μmol/L groups) and PI3K (2 μmol/L group) were all decreased significantly (P<0.05 or P<0.01). CONCLUSIONS Gracillin can promote excessive autophagy in A549 cells by up-regulating mRNA and protein expressions of FAM102A and inhibiting PI3K/Akt signaling pathway, thus inhibiting cell proliferation.