Neferine, is not inducer but blocker for macroautophagic flux targeting on lysosome malfunction

Area of Science:

• Pharmacology
• Cell Biology
• Biochemistry

Background:

• Neferine, an alkaloid from Lotus seeds, has known anti-cancer, antioxidant, and anti-arrhythmic properties.
• It was previously identified as a macroautophagy inducer in cancer cells via AMPK/mTOR signaling suppression.

Purpose of the Study:

• To investigate the precise effect of neferine on autophagy signaling and lysosomal function in cancer cells.
• To clarify the mechanism by which neferine influences cellular degradation pathways.

Main Methods:

• Cell culture and treatment with neferine.
• Analysis of autophagy markers (LC3-II, p62/SQSTM1) and ATG5 knockdown.
• Electron microscopy (EM) for ultrastructural analysis of cellular compartments.
• Assessment of lysosomal maturation markers (cathepsin D) and cargo degradation.

Main Results:

• Neferine treatment inhibited autophagy signaling, contrary to previous findings.
• Accumulation of LC3-II and p62/SQSTM1 was observed, with ATG5 knockdown failing to reverse LC3-II accumulation.
• Electron microscopy revealed accumulation of multi-vesicular bodies (MVBs) and impaired lysosome maturation.
• Neferine reduced cathepsin D maturation and hindered the degradation of autophagic and phagocytic cargo, indicating lysosomal dysfunction.

Conclusions:

• Neferine impairs lysosomal degradation rather than stimulating autophagic flux.
• The study demonstrates that neferine blocks degradation within phagolysosomes by inhibiting lysosome maturation.
• These findings provide new insights into the anti-cancer mechanisms of neferine, highlighting its role in disrupting cellular waste clearance.