Effect and mechanism of gracillin-induced autophagy in lung cancer A549 cells
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.
Reineckia carneagracillinnon-small cell lung cancer cellsautophagyFAM102API3K/Akt signaling pathway
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