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.

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Autophagy is induced as a homeostatic process by energy shortage and cellular stress but can also be influenced by drugs. Poor autophagic function contributes to carcinogenesis but autophagy also promotes survival of tumours. We performed immunohistochemistry for LC3, pAMP kinase, p62, p53 on 15 samples from breast cancer and 14 from pancreatic cancer. P53 mutations were detected using CDGE. Autophagy was active (LC3 puncta in ≥ 30% of cells) in 5/15 breast carcinomas and 7/14 pancreatic carcinomas. This was associated with energy shortage (strong pAMP kinase expression) in 4/5 in breast samples and 5/7 in those from pancreas, often clearly colocalized when expression was variable. In contrast, activation of AMP kinase was not associated with autophagy in 4/8 breast and 2/7 pancreatic cancers. In 2/5 breast samples and 2/7 from pancreas impaired autophagic flux was suggested by absent cytoplasmic LC3 staining and strong expression of p62. Nuclear expression of p53 was seen in 10/15 breast cancer samples and of those 3 were mutated, 11/14 pancreas samples showed nuclear staining for p53, none mutated. Three out of 7 non-mutated p53-protein-positive breast cancers had low autophagy. Autophagy was thus often associated with energy shortage, can clearly also be induced by other pathways, but p53 appears not to be involved. Lack of autophagy in the presence of energy shortage suggests defects in induction. There is, therefore, considerable variability in autophagic pathways and activity in cancer and this will have to be taken into account when considering therapeutic intervention in the autophagic process. Citation Format: Mar Egilsson, Ulfur Thoroddsen, Jon Gunnlaugur Jonasson, Margret Helga Ogmundsdottir, Helga M. Ögmundsdottir. Autophagic pathways and activity in breast and pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 311. doi:10.1158/1538-7445.AM2014-311

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