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

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.

Production of the Cytotoxic Cardenolide Glucoevatromonoside by Semisynthesis and Biotransformation of Evatromonoside by a Digitalis lanata Cell Culture

“…However, especially for the Caki-1 renal cell carcinoma cell line and the PC3 prostate cancer cell line, GEV (4) was the most bioactive and cytotoxic compound. It was already shown that cardiac glycosides might have a different impact on various cancer cell lines [36]. In case of, non-small cell lung cancer A549 cells, EV seems a very promising candidate [12], and a well-established synthesis for this compound is an additional advantage of this study.…”

“…The effect of the synthesized EV (2), GEV (3), and N-GEV (4) on the viability of renal cell carcinoma (Caki-1, 786-O) and prostate cancer (DU-145 and PC3) cell lines was investigated and compared to DTG (1) determined by [35,36]. All cardiac glycosides exhibited cytotoxic effects against renal and prostate cancer cell lines (▶ Table 2), however, they differed in their impact on the investi-gated cell lines.…”

“…IC 50 values for cytotoxicity are in nM (48 h) and IC 50 values for the α1, 2, 3 subunit of the porcine Na + /K + -ATPase inhibition are in µM. The IC 50 values represent the mean from three experiments each consisting of three technical replicates; values for DTG were evalutated by * [35] and † [36].…”

“…Enzymatic activities of the Na + /K + -ATPase α1, 2, 3 subunit of porcine cortex (Sigma) were assayed as described by [36,43]. Detailed information of the assay conditions is provided within the Supporting Information.…”

Pathways, Processes, and Candidate Drugs Associated with a Hoxa Cluster-Dependency Model of Leukemia

“…The key pathway associated with the Hoxa del signature was transcriptional misregulation in cancer. Several drugs with anti-cancer properties reported for solid tumors including, Mefloquine in glioblastoma [47], Digitoxigenin in renal carcinoma [48] and Emetine in bladder cancer [49] were associated with the Hoxa del signature. Furthermore, some of these drugs e.g., Emetine and Cephaline were recently shown to be highly active against primary chronic lymphocytic leukemia cells by repressing HIF-1α and disturbing intracellular redox homeostasis [50].…”