Murine breast carcinoma 4T1 cells are more sensitive to atranorin than normal epithelial NMuMG cells in vitro: Anticancer and hepatoprotective effects of atranorin in vivo Peter Sol  ar a, * , Gabriela Hr  ckov  a b , Lenka Kopta  síkov  a a , Samuel Velebný b , Zuzana Sol  arov  a c , Martin Ba  ckor a a Institute of Biology and Ecology, Faculty of Science, P.J.  Saf arik University in Ko sice, 040 01 Ko sice, Slovak Republic b Parasitological Institute of the Slovak Academy of Sciences, 040 01 Ko sice, Slovak Republic c Institute of Pharmacology, Faculty of Medicine, P.J.  Saf arik University in Ko sice, 040 01 Ko sice, Slovak Republic article info Article history: Received 28 November 2015 Received in revised form 9 February 2016 Accepted 7 March 2016 Available online 8 March 2016 Keywords: Lichen Atranorin 4T1 breast cancer NMuMG cells BALB/c mouse Lipid peroxidation abstract The aim of this study was to evaluate the anticancer effect of atranorin (ATR) on murine 4T1 breast carcinoma cells and compare its sensitivity with normal mammary epithelial NMuMG cells in vitro. Anti- tumor and hepatoprotective activity of ATR-therapy was examined on mouse model of 4T1-induced cancer disease. ATR significantly reduced clonogenic ability of carcinoma 4T1 cells at the concentra- tion of 75 mM, but clonogenicity of normal NMuMG cells was not affected by any of ATR concentrations tested. Moreover, flow cytometric and BrdU incorporation analysis did not confirm the inhibited entry into S-phase of the cell cyle after ATR incubation, and on the contrary, it induced apoptosis associated with the activation of caspase-3 and PARP cleavage in 4T1 cells. Although ATR did not cause any sig- nificant changes in Bcl-x L protein expression in NMuMG cells, an apparent depletion of Bcl-x L protein in 4T1 cells after 48 h ATR therapy was confirmed. Based on this result as well as the result of the total cell number decline, we can conclude that 4T1 cells are more sensitive to ATR therapy than NMuMG cells. ATR administration resulted in significantly longer survival time of BALB/c mice inoculated with 4T1 cells, what was associated with reduced tumor size and the higher numbers of apoptotic 4T1 cells. No differences were recorded in the number of BrdU-positive tumor cells between ATR-treated group and controls. Results indicate that ATR has rather proapoptotic than antiproliferative effect on 4T1 cells in vitro and in vivo and normal NMuMG cells are less sensitive to ATR. Furthermore, our studies revealed protective effect of ATR against oxidative stress in the livers of the tumor-bearing mice. © 2016 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Nowadays, the power of lichen extracts comes into consider- ation, while in natural medicine [7] it has been known for a long period of time. Lichens have been used for medicinal purposes throughout the ages [34] and some species, such as Cetraria islandica, Lobaria pulmonaria and Cladonia species were reputed to be effective in the treatment of pulmonary tuberculosis [54], even though their secondary metabolites are usually toxic to humans at the higher concentrations. Lichen secondary metabolites putatively protect lichens from a variety of environmental stress factors [53] and harmful light irradiation [38]. Lichens produce several classes of phenolic com- pounds, including: depsides, depsidones, dibenzofuranes, xan- thones, and anthraquinones [24]. Most of the secondary metabolites are formed as a part of the acetate-polymalonate biosynthetic pathway, but can also be produced by mevalonic acid and the shikimic acid pathways. Secondary metabolites are produced by the fungus alone and secreted onto the surface of li- chen's hyphae either in amorphous forms or crystal form [55]. Although there are hundreds of lichens secondary metabolites, their particular functions are almost unknown. Natural product atranorin (ATR) isolated from lichens is classi- fied as depside based on the presence of its a 2,4-dihydroxy-3- aldehyde-6-methylbenzoate moiety [15]. Except of ATR, other important members of the depside group are fumarprotocetraric * Corresponding author. Moyzesova 11, 040 01 Ko sice, Slovak Republic. E-mail address: peter.solar@upjs.sk (P. Sol ar). Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint http://dx.doi.org/10.1016/j.cbi.2016.03.012 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved. Chemico-Biological Interactions 250 (2016) 27e37