Cellular Mechanisms of the Anticancer Effects of the Lichen Compound Usnic Acid

Abstract

The lichen compound usnic acid is used for its antimicrobial activities in cosmetic products and is also a component of slimming agents. Its effect against cancer cells was first noted over 30 years ago. In this study possible mechanisms of this effect were investigated using two human cell lines, the breast cancer cell line T-47D and the pancreatic cancer cell line Capan-2. Pure (+)-usnic acid from Cladonia arbuscula and (−)-usnic acid from Alectoria ochroleuca were shown to be equally effective inhibitors of DNA synthesis, with IC₅₀ 4.2 µg/mL and 4.0 µg/mL for (+) and (−)-usnic acid against T-47D, and 5.3 µg/mL and 5.0 µg/mL against Capan-2, respectively. Flow cytometric analysis confirmed the inhibited entry into the S-phase and showed reduction in cell size. Classical apoptosis, as assessed by TUNEL staining, was not observed. Necrosis, measured by LDH release, was seen only in Capan-2 after exposure for 48 hours. Staining with the mitochondrial dye JC-1 demonstrated dose-dependent loss of mitochondrial membrane potential following treatment with usnic acid in both cell lines. In conclusion, usnic acid had a marked inhibitory effect on growth and proliferation of two different human cancer cell lines and led to loss of mitochondrial membrane potential. Cell survival was little affected; late necrosis was seen in one of the cell lines. No difference was noted between the two enantiomers.

Key words

usnic acid - human cancer cells - antiproliferative effect - growth inhibition - mitochondrial membrane potential

References

• 1 Nash T H. Lichen biology. Cambridge; Cambridge University Press 1997
• 2 Ingolfsdottir K. Usnic acid.  Phytochemistry. 2002;  61 729-736
• 3 Durazo F A, Lassman C, Han S H B, Saab S, Lee N P, Kavano M, Saggi B, Gordon S, Farmer D G, Yersiz H, Goldstein R L, Ghobrial M, Busutil R W. Fulminant liver failure due to usnic acid for weight loss.  Am J Gastroenterol. 2004;  99 950-952
• 4 Sanchez W, Maple J T, Burgart L J, Kamath P S. Severe hepatotoxicity associated with use of a dietary supplement containing usnic acid.  Mayo Clin Proc. 2006;  81 541-544
• 5 Kupchan S M, Kopperman H L. l-Usnic acid: tumor inhibitor isolated from lichens.  Experientia. 1975;  31 625-752
• 6 Takai M, Uehara Y, Beisler J A. Usnic acid derivatives as potential antineoplastic agents.  J Med Chem. 1979;  22 1380-1384
• 7 Cardarelli M, Serino G, Campanella L, Ercole P, De Cicco Nardone F, Alesiani O, Rossiello F. Antimitotic effects of usnic acid on different biological systems.  Cell Mol Life Sci. 1997;  53 667-672
• 8 Kristmundsdottir T, Aradottir H A, Ingolfsdottir K, Ogmundsdottir H M. Solubilization of the lichen metabolite (+)-usnic acid for testing in tissue culture.  J Pharm Pharmacol. 2002;  54 1447-1452
• 9 Bezivin C, Tomasi S, Rouaud I, Delcros J G, Boustie J. Cytotoxic activity of compounds from the lichen: Cladonia convoluta.  Planta Med. 2004;  70 874-877
• 10 Koparal A T, Tuylu B A, Turk H. In vitro cytotoxic activities of (+)-usnic acid and (−)-usnic acid on V79, A549, and human lymphocyte cells and their non-genotoxicity on human lymphocytes.  Nat Prod Res. 2006;  20 1300-1307
• 11 Mayer M, O'Neill M A, Murray K E, Santos-Magalhaes N S, Carneiro-Leao A M, Thompson A M, Appleyard V C. Usnic acid: a non-genotoxic compound with anti-cancer properties.  Anticancer Drugs. 2005;  16 805-809
• 12 da Silva Santos N P, Nascimento S C, Wanderley M S O, Pontes-Filho N T, da Silva J F, de Castro C M, Pereira E C, da Silva N H, Honda N K, Santos-Magalhaes N S. Nanoencapsulation of usnic acid: an attempt to improve antitumour activity and reduce hepatotoxicity.  Eur J Pharm Biopharm. 2006;  64 154-160
• 13 Huneck S, Yoshimura I. Identification of lichen substances. Berlin; Springer Verlag 1996: 228-229
• 14 Ingolfsdottir K, Chung G A, Skulason V G, Gissurarson S R, Vilhelmsdottir M. Antimycobacterial activity of lichen metabolites in vitro.  Eur J Pharm Sci. 1998;  6 141-144
• 15 Casey G, Lo-Hsueh M, Lopez M E, Vogelstein B, Stanbridge E J. Growth suppression of human breast cancer cells by the introduction of a wild-type p 53 gene.  Oncogene. 1991;  6 1791-1797
• 16 Eisold S, Linnebacher M, Ryschich E, Antolovic D, Hinz U, Klar E, Schmidt J. The effect of adenovirus expressing wild-type p 53 on 5-fluorouracil chemosensitivity is related to p 53 status in pancreatic cancer cell lines.  World J Gastroenterol. 2004;  10 3583-3589
• 17 Haraldsdóttir S, Gudlaugsdóttir E, Ingólfsdóttir K, Ögmunsdóttir H M. Anti-proliferative effects of lichen-derived lipoxygenase inhibitors on twelve human cancer cell lines of different tissue origin in vitro.  Planta Med. 2004;  70 1098-1100
• 18 Smith H J. Smith and Williams' introduction to the principles of drug design and action. Amsterdam; Harwood Academic Publishers 1988: 98-165
• 19 Bazin M A, Le Lamer A C, Delcros J G, Rouaud T, Uriac P, Boustei J, Corbel J C, Tomasi S. Synthesis and cytotoxic activities of usnic acid derivatives.  Bioorg Med Chem. 2008;  16 6860-6866
• 20 Luo Z J, Saha A K, Xiang X Q, Ruderman N B. AMPK, the metabolic syndrome and cancer.  Trends Pharmacol Sci. 2005;  26 69-76
• 21 Jendrossek V, Handrick R, Belka C. Celecoxib activates a novel mitochondrial apoptosis signaling pathway.  FASEB J. 2003;  17 1547-1549
• 22 Han D, Matsumaru K, Rettori D, Kaplowitz N. Usnic acid-induced necrosis of cultured mouse hepatocytes: inhibition of mitochondrial function and oxidative stress.  Biochem Pharmacol. 2004;  67 439-451
• 23 Yamaguchi T, Miki Y, Yoshida K. Protein kinase C delta activates I kappa B-kinase alpha to induce the p 53 tumor suppressor in response to oxidative stress.  Cell Signal. 2007;  19 2088-2097
• 24 Hsu L M, Huang Y S, Chang F Y, Lee S D. “Fat burner” herb, usnic acid, induced acute hepatitis in a family.  J Gastroenterol Hepatol. 2005;  20 1138-1139