Fangchinoline accumulates autophagosomes by inhibiting autophagic degradation and promoting TFEB nuclear translocation

Abstract

Autophagy, an evolutionarily conserved cellular self-digestive process, is associated with different diseases and can be inhibited or induced by a series of agents. In this study, we reported that fangchinoline (FCL), an alkaloid from Stephania tetrandra S. Moore, increased the expression of LC3-II and the formation of GFP-LC3 puncta in non-small cell lung cancer (NSCLC) cells. Numerous yellow puncta were observed in mRFP–EGFP-LC3 stably expressed NSCLC cells under FCL treatment and the FCL-increased expression of LC3-II was not further increased after co-treatment with bafilomycin A1, an autophagy inhibitor, suggesting that FCL inhibits autophagic flux. Results of co-localization of GFP-LC3 with LysoTracker Red or lysosomal-associated membrane protein 1 indicated that FCL inhibits autophagosomes–lysosomes fusion. Furthermore, FCL decreased the activities of cathepsin B and cathepsin D and affected the cellular acidification. Interestingly, FCL also increased the nuclear translocation of transcription factor EB (TFEB), a master regulator of autophagic and lysosomal genes, and the mRNA expressions of TFEB-targeted genes, such as SQSTM1, MAP1LC3B, and UVRAG. Knockdown of TFEB by using small inference RNA decreased the FCL-induced expression of LC3-II and the formation of GFP-LC3 puncta. Overall, we reported that FCL increases the expressions of autophagy markers via both inhibition (inhibition of autophagosomes–lysosomes fusion and dysfunction of lysosome) and induction (promotion of TFEB nuclear translocation) of autophagy, providing insights into the better understanding of the complexity of agent-mediated autophagy.