Objective        To determine the effect of liensinine on the autophagic phenotypes in breast cancer MDA-MB-231 cells in order to investigate its regulative role on autophagic functions in the cells.       Methods       Transmission electron microscopy (TEM) was used to observe the ultrastructural changes in MDA-MB-231 cells after liensinine treatment (20 μmol/L for 24 h). Confocal laser scanning microscopy was employed to determine the formation of autophagosome and autophagic flux after the cells were transfected with pEGFP-LC3 or ptfLC3. Western blot analysis was used to detect the expression of autophagy related proteins

including LC3B

SQSTM1/p62 and Beclin1.       Results       TEM displayed there were more autophagic vacuoles and some fission mitochondria

but no high density osmiophilic autolysosomes in the MDA-MB-231 cells after liensinine treatment. Confocal laser scanning microscopy confirmed that there were more EGFP-LC3 puncta in the cells after pEGFP-LC3 transfection (P0.01). Western blotting indicated that the expression levels of LC3B-Ⅱ and SQSTM1/p62 were increased in a dose- and time-dependent manner (20 μmol/L or 24 h vs control

P0.01)

and those of LC3B-Ⅰ and Beclin1 had no obvious change (20 μmol/L vs control

P0.05) in the cells after pEGFP-LC3 transfection. While

in the cells after mRFP-EGFP-LC3

there were only a few of autophagosomes and autolysosomes in the control cells

but a lot of autophagosomes in the cells treated with liensinine and bafilomycin A1

but no autolysosomes. Western blotting indicated that bafilomycin A1 had no effect on liensinine induced enhancement of LC3B-Ⅱ and SQSTM1/p62 (P0.05).       Conclusion        Liensinine suppresses the maturation of autophagosome

and therefore blocks the autophagic degradation in MDA-MB-231 cells.