A new semisynthetic cardenolide analog 3β-[2-(1-amantadine)- 1-on-ethylamine]-digitoxigenin (AMANTADIG) affects G2/M cell cycle arrest and miRNA expression profiles and enhances proapoptotic survivin-2B expression in renal cell carcinoma cell lines

// Elke Nolte 1 , Sven Wach 1 , Izabella Thais Silva 2, 6 , Sabine Lukat 1 , Arif B. Ekici 3 , Jennifer Munkert 4 , Frieder Muller-Uri 4 , Wolfgang Kreis 4 , Claudia Maria Oliveira Simoes 2 , Julio Vera 5 , Bernd Wullich 1 , Helge Taubert 1 , Xin Lai 5 1 Department of Urology, University Hospital Erlangen, Erlangen, Germany 2 Department of Pharmaceutical Sciences, Universidade Federal de Santa Catarina, Florianopolis, Brazil 3 Institute of Human Genetics, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Germany 4 Department of Biology, Chair of Pharmaceutical Biology, Friedrich-Alexander-University Erlangen-Nurnberg, Erlangen, Germany 5 Laboratory of Systems Tumor Immunology, Department of Dermatology, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nurnberg, Erlangen, Germany 6 Department of Pharmacy, Federal University of Minas Gerais, Belo Horizonte, Brazil Correspondence to: Xin Lai, email: xin.lai@uk-erlangen.de Keywords: 3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin, cardiac glycoside analog, human renal cell carcinoma cells, miRNA, cell cycle Received: June 21, 2016      Accepted: December 24, 2016      Published: January 14, 2017 ABSTRACT Cardiac glycosides are well known in the treatment of cardiovascular diseases; however, their application as treatment option for cancer patients is under discussion. We showed that the cardiac glycoside digitoxin and its analog AMANTADIG can inhibit the growth of renal cell carcinoma (RCC) cell lines and increase G2/M cell cycle arrest. To identify the signaling pathways and molecular basis of this G2/M arrest, microRNAs were profiled using microRNA arrays. Cardiac glycoside treatment significantly deregulated two microRNAs, miR-2278 and miR-670-5p. Pathway enrichment analysis showed that all cardiac glycoside treatments affected the MAPK and the axon guidance pathway. Within these pathways, three genes, MAPK1, NRAS and RAC2, were identified as in silico targets of the deregulated miRNAs. MAPK1 and NRAS are known regulators of G2/M cell cycle arrest. AMANTADIG treatment enhanced the expression of phosphorylated MAPK1 in 786-O cells. Secondly, we studied the expression of survivin known to be affected by cardiac glycosides and to regulate the G2/M cell phase. AMANTADIG treatment upregulated the expression of the pro-apoptotic survivin-2B variant in Caki-1 and 786-O cells. Moreover, treatment with AMANTADIG resulted in significantly lower survivin protein expression compared to 786-O control cells. Summarizing, treatment with all cardiac glycosides induced G2/M cell cycle arrest and downregulated the miR-2278 and miR-670-5p in microarray analysis. All cardiac glycosides affected the MAPK-pathway and survivin expression, both associated with the G2/M phase. Because cells in the G2/M phase are radio- and chemotherapy sensitive, cardiac glycosides like AMANTADIG could potentially improve the efficacy of radio- and/or chemotherapy in RCCs.