Phytochemical Combinations of Lichen Evernia prunastri (L.) Ach reduce Drug Resistance to Temozolomide but not to Paclitaxel in vitro

Anastasiia Shcherbakova^1,2Linh Nguyen^1,3Anna Koptina^1,3Anders Backlund³Subhadip Banerjee⁴Evgeniy Romanov²Gudrun S. Ulrich-Merzenich^1*

• ¹Medical Clinic III, Synergy and Experimental Medicine Research Group, University Hospital Bonn, Bonn, Germany
• ²Volga State University of Technology, Institute of Forestry and Nature Management, Yoshkar-Ola 424000, Russia
• ³Pharmacognosy, Department of Pharmaceutical Biosciences, Uppsala University, SE-751 24 Uppsala, Sweden
• ⁴Mae Fah Luang University, Medicinal Plants Innovation Centre, Chiang Rai 57100, Thailand

Temozolomide (TMZ) and Paclitaxel (PXT), crucial anti-cancer drugs for glioblastoma (GBM) and primary breast cancer (BC), respectively, face drug resistance. Therefore, we investigated the adjuvant potential of characterized extracts of the lichens Evernia prunastri (L.) Ach. (Epr), Cladonia arbuscula (Wallr.) Flot (Car) and their metabolites, evernic acid (EA) and usnic acid (UA) alone or in combination with TMZ and PTX for their immunomodulatory and chemosensitivity increasing potential. TMZ-resistant U-87 cells, MCF7 BCcells, and normal human skin fibroblasts (HSKF) were treated with hexane (Hex), dichloromethane (DCM), and acetonitrile (ACN) extracts of Epr (EprDCM, EprACN), Car (CarHex, CarACN), and with EA and UA to measure cell metabolic activity. Molecular mechanisms were predicted using ChemGPS-NP and validated by Western blot, RNA sequencing, quantitative RT-PCR, and Wnt inhibitory factor 1 (WIF1) protein expression. Combinatory effects were calculated by Combination Index (CI) and Zero Interaction Potency methods (ZIP).Extracts and selected metabolites reduced concentration-dependent cellular metabolic activity in U-87 and MCF7 cells. EprACN and EA (U-87 cells: IC50 30 µg/ml), safe to HSKF, regulated key proteins in MAP kinases pathways, supporting predictions made by ChemGPS-NP. The combination EA-TMZ showed additive effects (TMZ-reduction: 3.4 fold), reduced transcription of Wnt pathway members, and increased in U-87 cells protein releases of WiF1, the central inhibitor of Wnt-signaling. Further gene expression data (GE) suggest involvement of IL-17 receptor and BDNF. In summary, the combination EA-TMZ interacts with the Wnt pathway regulation associated with sensitizing U-87 cells, without increasing GEs of proinflammatory cytokines. EA deserves further investigation as an adjuvant.