YM155 inhibits topoisomerase function

Abstract

YM155 (sepantronium bromide) has been evaluated in clinical trials as a survivin suppressant, but despite positive signals from early work, later studies were negative. Clarification of the mechanism of action of YM155 is important for its further development. YM155 affects cells in a cell cycle-specific manner. When cells are in G1, YM155 prevented their progression through the S phase, leaving the cells at G1/S when exposed to YM155. Passage through mitosis from G2 is also defective following YM155 exposure. In this study, YM155 did not behave like a typical DNA intercalator in viscosity, circular dichroism, and absorption spectroscopy studies. In addition, molecular modeling experiments ruled out YM155 DNA interaction to produce DNA intercalation. We show that YM155 inhibited topoisomerase 2α decatenation and topoisomerase 1-mediated cleavage of DNA, suggesting that YM155 inhibits the enzyme function. Consistent with these findings, DNA double-strand break repair was also inhibited by YM155.

Figure 1

YM155 arrests cell cycle at G1/S or G2/M. H1299 non-small cell lung cancer cells were treated with volasertib (BI6727, 40 nM) to arrest cell cycle at G2/M before releasing into the media containing DMSO, BI6727 (40 nM), YM155 (40 nM) or YM/BI (40 nM each). Flow cytometry was performed at 4, 8, 24 hours later to determine the nuclear DNA contents (A). H1299 cells were synchronized with mimosine at G1/S. They were then released into media containing either DMSO (left panel) or YM155 (40 nM, right) on top. DNA content (DNA con) or BrdU incorporation (BrdU Inc) were monitored overtime at 4, 8, 24 hour. The percentages represented in the BrdU boxes are cells at S phase (upper) and G1 (lower) respectively (B). H1299 cells are arrested in M phase by nocodazole (50ng/ml) before released into medium containing DMSO (left column) or YM155 (40 nM, right) 4, 8, 12, 16, 20 and 24 hours. Passage through M and S were followed by flow cytometry (C). Cells were arrested in G1/S by mimosine (200 μM). Cells are released then into media with DMSO (left) or YM155 (40 nM, right). Cell cycle protein levels were followed with Western Blot at 0, 4, 8, 12, 16, 24 hours. Beta actin (ACTB) was used as loading control (D).

Figure 2

YM155 does not bind DNA (A) Viscometric titrations of calf thymus DNA with ethidium bromide (EtBr), Netropsin (Net), and YM155. The data are the average of three separate readings. Where η and η₀ represent the viscosity of the solution containing ligand-DNA complex and free DNA respectively. (B) Circular dichroism spectra of YM155 with hairpin duplex DNA at different DNA-drug ratios. The experiments were conducted in Tris-HCl buffer (50 mM Tris-HCl, 100 mM NaCl, 1mM EDTA, pH = 7.4) at 25 °C. (C–F) Energy minimized structures of YM155 at the 6–31G* (p,d) level of theory by Spartan 10 software. YM155 is represented as a ball and stick model (C), and space filling model with front (D), back (E), down (F) side view with carbon in grey, hydrogen in yellow, oxygen in red and nitrogen in blue.

Figure 3

YM155 (sepantronium) does not stimulate TOP1 or TOP2α activity. Unlike CPT (irinotecan, a TOP1 poison, lane 4 white arrow, 50 μM), increasing concentrations of YM155 did not increase the amount of open circular DNA (form II) formed (A). The distribution of topoisomers was shifted in lane 11 (Figure 3B) indicating induced inhibition of TOP1 by YM155 in a dose dependent manner. SC: Supercoiled, Rel: Relaxed, Lin: Linear. Unlike VP16 (etoposide, a TOP2α poison at 50 μM, lane 4, white arrow), increasing concentrations of YM155 did not induce the formation of linear DNA. Instead, formation of nicked circular (up) and relaxed circular (down) from the kDNA was inhibited (black arrows and parenthesis on right) in EtBr gel (C) and Non-EtBr Gel (D). Cat: Catenated, Dec: Decatenate kDNA. Lane 4, 5–11 contained topoisomerase.

Figure 4

YM155 suppresses TOP1 and TOP2α but not E. Coli Gyrase activity YM155 activities against purified TOP1 (A, B), TOP2α (C, D) and E Coli Gyrase (E, F) were performed using H331, a known TOP2α CIC, as control. YM155 inhibited TOP1 (B) when the concentration increased whereas H331 did not (A). SC: supercoil, Rel: relax, Lin: Linear. Compared with HU331 (C, Left middle), YM155 inhibited TOP2α more potently leading to substantial decrease in the formation of nicked circular (N.C) and relaxed circular (R.C) kDNA (D, right middle). Cat: Catenated kDNA, Decat: Decatenated kDNA. Neither HU331 nor YM155 had activity against E. Coli Gyrase in E and F in the bottom panel. Topoisomerases were included in lane 4, 6–12 in A, B; lane 4–12 in C, D; lane 3, 5–11 in E and 4–12 in F.

Figure 5

YM155 delays double strand break DNA repair H1299 cells were radiated with ironized radiation (2Gy) and are subsequently treated with DMSO or YM155 (40 nM) for 2, 6 and 12 hours. Cells were stained for γH2AX foci (green) to follow DNA damage and RAD51 foci (red) to follow the status of DNA repair. Quantitative analysis of γH2AX foci present as a function of time (A) or RAD51 foci (B).

Figure 6

Schematic description of the mechanism of action of YM155. Catenated DNAs are frequently encountered during DNA replication, repair, transcription and chromosomal separation. Continued presence of such sends signals through checkpoint cascade resulting in cell cycle arrest, unreplicated/damaged DNA, decreased DNA transcription and unseparated chromosome. Progression of the above processes depend on prompt resolution of catenated DNA at junction A and B. YM155 impairs TOP2α mediated DNA decatenation.