Antiproliferative and Pro-Apoptotic Effects of Pseudevernia furfuracea (L.) Zopf Extract and Its Active Component Physodic Acid via Oxidative Stress and DNA Damage in Breast Cancer Cells

Dominika Sebova¹Simona Zilakova¹Viktoria Medvecova¹Michal Goga²Richard Frenak²Annamaria Bardelcikova¹Andrej Mirossay¹Ladislav Mirossay¹Ján Mojžiš¹Martin Kello^1*

• ¹Faculty of Medicine, University of Pavol Jozef Šafárik, Kosice, Slovakia
• ²Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University in Košice, Košice, Slovakia

Mammary gland malignancies are the most diagnosed oncological diseases in women. The currently available treatment faces several problems, including resistance to cytostatics and the relatively high recurrence rates. These limitations have led to an increasing interest in natural substances as potential anticancer agents. Therapeutic approaches using a combination of natural anticancer agent and conventional cytostatic drug could also be beneficial in minimizing the risk of chemotherapy.In the present study, we evaluated the anti-cancer effect of Pseudevernia furfuracea (L.) Zopf extract (PSE) and isolated secondary metabolite physodic acid (PHY) in in vitro models of breast cancer subtypes (ER+, HER2+, triple-negative).To investigate the effects of tested compounds, a range of assays were employed. BrdU and clonogenic assays were used to evaluate antiproliferative activity. The flow cytometry and Western blot were used to demonstrate apoptotic cell death, oxidative stress, DNA damage and immune checkpoint modulation in time-dependent manner (24, 48, 72 h).Furthermore, activation of intrinsic apoptotic pathway, involving changes in Bcl-2 family proteins, caspase 3/7 activity, caspase 9 cleavage, cytochrome c release and PARP cleavage, was detected in all BC cells. Moreover, we determined the PSE and PHY-mediated generation of ROS and RNS, which led to DNA damage and the activation of the DNA damage response.Treatment with PSE and PHY in BC cells resulted in mitochondrial apoptosis associated with oxidative stress and DNA damage. Furthermore, modulation of immune checkpoint PD-1/PD-L1 was demonstrated. Based on the results, we assume the use of PSE and PHY as promising targeted agents for BC.• PSE and PHY reduced the viability of the tested breast cancer cells (ER+, HER2+ and triplenegative).• PSE and PHY induced apoptosis.• PSE and PHY activated the mitochondrial apoptotic pathway.• PSE and PHY induced oxidative stress and DNA damage.• PSE and PHY modulate the expression of PD-L1.