Transcriptomic and proteomic insight into the effects of a defined European mistletoe extract in Ewing sarcoma cells reveals cellular stress responses

Background: The hydrophobic triterpenes, oleanolic and betulinic acid as well as the hydrophilic mistletoe lectins and viscotoxins possess anticancer properties. They do all occur in combination in European mistletoe ( Viscum album L.). Commercial Viscum album L. extracts are aqueous, excluding the insolub le triterpenes. We have previously shown that mistletoe lectins and triterpene acids are effective against Ewing sarcoma in vitro, ex vivo and in vivo. Methods: We recreated a total mistletoe effect (viscumTT) by combining an aqueous extract (viscum) and a triterpene extract (TT) solubilised with cyclodextrins and analysed the effects of viscumTT and the single extracts on TC-71 Ewing sarcoma cells in vitro by transcriptomic and proteomic profiling. Results: Treatment with the extracts strongly impacted Ewing s arcoma cell gene and protein expression. Apoptosis- associated and stress-activated genes were upregulated, proteasomal protein abundance enhanced and ribosomal and spliceosomal proteins downregulated. The mec hanism of action of viscum, TT and viscumTT in TC-71 and MHH-ES-1 cells suggests the involvement of the unfolded protein response. While viscum and viscumTT extract treatment indicate response to oxidative stress and activation of stress-mediated MAPK signalling, TT extract treatment suggests the involvement of TLR signalling and autophagy. Conclusions: Since the combinatory extract viscumTT exerts highly effective pro-apoptotic effects on Ewing sarcoma cells in vitro, this phytopolychemotherapy could be a promisi ng adjuvant therapeutic option for paediatric patients with Ewing sarcom