Proton-Shuttling Lichen Compound Usnic Acid Affects Mitochondrial and Lysosomal Function in Cancer Cells

<div><p>The lichen compound usnic acid (UA) is a lipophilic weak acid that acts as a proton shuttle and causes loss of mitochondrial inner membrane potential. In the current study we show that UA treatment induced the formation of autophagosomes in human cancer cells, but had minimal effects on normal human fibroblasts. However, autophagic flux was incomplete, degradation of autophagosomal content did not occur and acidification was defective. UA-treated cells showed reduced ATP levels and activation of AMP kinase as well as signs of cellular stress. UA is thus likely to trigger autophagosome formation both by energy depletion and stress conditions. Our findings indicate that the H⁺-shuttling effect of UA operates not only in mitochondria as previously shown, but also in lysosomes, and have implications for therapeutic manipulation of autophagy and pH-determined drug distribution.</p> </div

UA caused decline in cellular ATP and activation of AMP kinase.

<p>(A) Levels of ATP, measured in a luminometer, were decreased in T47D cells after treatment with UA (10 µg/mL; DMSO 0.2%) for 24 hours. Data are presented as luminescence/cell of each group compared with DMSO control. Error bars indicate standard error of the mean, *p<0.05. (B) Phosphorylation of AMP kinase, verified by Western blotting, was detected in T47D cells after treatment with UA (10 µg/mL; DMSO 0.2%) for 24 and 48 hours.</p

UA-induced formation of autophagosomal vaculoes was not followed by autosomal maturation and substrate degradation.

<p>(A) No degradation of p62 was detected, by Western blotting, in T47D and MCF7 cells after treatment with UA (5.0 and 10 µg/mL, 24 h; DMSO 0.1%). (B) Lysotracker, detected by fluorescence microscopy, shows diffuse staining in T47D cells after treatment with UA (10 µg/mL; DMSO 0.2%) for 24 h and 72 h. (C) Lysotracker intensity per cell was quantified by ImageJ and data presented as mean fluorescence value of each group compared to DMSO control. Error bars indicate standard error of the mean, **p<0.001. The scale bar shown represents 100 µm and applies to all panels. (D) No effects on Lamp2 immunostaining were detected, after treatment with UA <i>(</i>10 µg/mL; DMSO 0.2%) for 24 hours. The scale bar shown represents 100 µm and applies to all panels. (E) A plasmid expressing mRFP-GFP-LC3 was transfected into T47D cells. Lack of autophagolysosomal acidification was seen after treatment with UA (10 µg/mL; DMSO 0.2%) for 24 hours by detection of distinct GFP puncta. The scale bar shown represents 20 µm and applies to all panels.</p

UA induced formation of autophagosome vacuoles.

<p>Induction of autophagic vacuoles, with double membranes characteristic of autophagosomes, was detected by electron microscopy in T47D cells after treatment with UA (2.5 and 5.0 µg/mL; DMSO 0.1%) for 24 hours. Black arrows indicate autophagic vacuoles, white arrows indicate double membrane formation.</p

UA induced formation of LC3 puncta.

<p>(A) An increase in LC3 puncta was detected, by immunofluorescence in T47D and MCF7 cells after treatment with UA (10 µg/mL) for 24 hours. No effect was seen in normal fibroblasts. The scale bar shown represents 20 µm and applies to all panels. (B) LC3 puncta per cell were counted and quantified by ImageJ and data represented as LC3 puncta/cell of each group compared with DMSO control. Error bars indicate standard error of the mean, *p<0.05, **p<0.001. (C) Increase in LC3 I and LC3 II, verified by Western blotting, was detected in T47D cells after treatment with UA (10 µg/mL; DMSO 0.2%) for 24 hours.</p