Effect of flemiphilippinin A on autophagy in non-small cell lung cancer cells based on ERK/mTOR pathway
Objective To investigate the effect of flemiphilippinin A on autophagy in non-small cell lung cancer(NSCLC)cells through the ERK/mTOR signaling pathway.Methods A549 cells were divided into control,flemiphilippinin A low,medium and high concentration(20,25 and 30 μmol·L-1)groups or control,flemiphilippinin A 30 μmol·L-1,SCH772984 10 μmol·L-1+flemiphilippinin A 30 μmol·L-1 and SCH772984 10 μmol·L-1 concentration groups.The cells in the control group were cultured in RMPI 1640 medium containing 1％fetal bovine serum,and the cells in the other groups were pretreated with flemiphilippinin A or SCH772984 for 1 h to achieve the corresponding concentration,and the cells were continuously cultured for 12 h.Cell viability was detected by the MTT assay.Cell colony formation assay was used to detect cell colony formation.Autophagy staining detection kit was used to detect autophagy in cells.The expression of the LC3B protein was detected by immunofluorescence.The protein expression levels of p-ERK,p-mTOR,Beclin 1,and P62 were detected by Western blotting.Results The results of MTT assay showed that each concentration of flemiphilippinin A could significantly inhibit the viability of A549 cells(P＜0.05,0.001).SCH772948(10 μmol·L-1)could reverse the inhibition of flemiphilippinin A-induced proliferation of A549 cells(P＜0.01).The cell colony formation assay showed that flemiphilippinin A(20,25,and 30 μmol·L-1)could inhibit the colony formation of A549 cells(P＜0.01).The results of the autophagy staining assay kit showed that flemiphilippinin A(20,25,and 30 μmol·L-1)could inhibit autophagy in A549 cells,and SCH772948 10 μmolL-1 could reverse the flemiphilippinin A-mediated autophagy inhibition in A549 cells.Immunofluorescence results showed that the fluorescence of LC3B induced by flemiphilippinin A(30 μmol·L-1)was decreased,and SCH772948(10 μmol·L-1)could reverse the decrease of LC3B fluorescence induced by flemiphilippinin A.The results of Western blotting showed that the expression levels of p-ERK,p-mTOR,and P62 proteins in A549 cells were significantly increased,and the expression level of Beclin 1 protein was significantly decreased of flemiphilippinin A(20,25,and 30 μmol·L-1)(P＜0.001).10 μmol·L-1 SCH772948 could significantly reverse the increase of p-ERK,p-mTOR,and P62 protein expression levels and the decrease of Beclin 1 protein in A549 cells induced by flemiphilippinin A(P＜0.001).Conclusion Flemiphilippinin A can inhibit autophagy and cause cell death in non-small-cell lung cancer cells through the ERK/mTOR pathway.
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