Abstract 4108: The lichen compound usnic acid disturbs mitochondrial and lysosomal function by proton shuttling

Abstract

Differential intracellular pH plays an important role in organelle function and intracellular transport. The intracellular distribution of cancer drugs is also influenced by pH gradients. A wide range of biological activities has been described for the lichen compund usnic acid (UA) e.g., anti-inflammatory, anti-viral and anti-bacterial activity. UA has also been shown to reduce ATP production by liver cell mitochondria and inhibit growth and proliferation of several cancer cells. UA is a proton shuttle, and therefore we investigated its effects on the function of two pH-sensitive organelles, mitochondria and lysosomes, in several cancer cell lines and normal human fibroblasts. JC-1 staining was used to test for changes in inner mitochondrial membrane potential and ATP levels were determined by luminescence measurements. Western blotting was used to detect AMPkinase phosphorylation and degradation of the autophagosomal cargo p62 were assayed by Western blotting. Formation of autophagosomes was assessed by electron microscopy (EM) and immunostaining for the autophagosomal marker LC3. Lysosomal formation was estimated using lysotracker, a pH-sensitive lysosomal marker, and immunostaining for Lamp2. Further testing for autophagosomal-lysosomal fusion and acidification was performed using a tandem-tagged mRFP-GFP-LC3 fusion construct. A drop in inner membrane mitochondrial potential was demonstrated and reduced levels of ATP and AMPk activation were observed in UA-treated cancer cells. Clear signs of autophagy were seen after treatment with UA, observed by EM and an increased number of LC3-positive autophagosomes. Degradation of p62 was not detected. Staining with lysotracker revealed very marked effects in UA-treated cells, which have been interpreted as evidence of formation of large lysosomal aggregates by fusion. However, immunostaining for the lysosomal membrane protein Lamp2 revealed a different pattern of speckles. This may indicate that the lysotracker dye is found outside lysosomes. The retention of the dye is pH-dependent implying that intra-lysosomal pH may be affected by treatment with UA. Using cells containing the mRFP-GFP-LC3 fusion construct confirmed that acidification in the autophago-lysosomes was reduced following treatment with UA. Results indicate that UA disturbs the pH balance in cells and its effects on organelle function are mediated through its ability to shuttle protons across membranes. Clear signs of autophagy are seen after treatment with UA, but degradation of p62 does not occur and therefore no metabolic substrates are provided for the cell. UA has the potential to serve as a candidate for drug interaction along with other cancer drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4108. doi:1538-7445.AM2012-4108