Ym155 localizes to the mitochondria leading to mitochondria dysfunction and activation of AMPK that inhibits BMP signaling in lung cancer cells

Abstract

The imidazolium compound Ym155 was first reported to be a survivin inhibitor. Ym155 potently induces cell death of many types of cancer cells in preclinical studies. However, in phase II clinical trials Ym155 failed to demonstrate a significant benefit. Studies have suggested that the cytotoxic effects of Ym155 in cancer cells are not mediated by the inhibition of survivin. Understanding the mechanism by which Ym155 induces cell death would provide important insight how to improve its efficacy as a cancer therapeutic. We demonstrate a novel mechanism by which Ym155 induces cell death by localizing to the mitochondria causing mitochondrial dysfunction. Our studies suggest that Ym155 binds mitochondrial DNA leading to a decrease in oxidative phosphorylation, decrease in TCA cycle intermediates, and an increase in mitochondrial permeability. Furthermore, we show that mitochondrial stress induced by Ym155 and other mitochondrial inhibitors activates AMP-activated kinase leading to the downregulation to bone morphogenetic protein (BMP) signaling. We provide first evidence that Ym155 initiates cell death by disrupting mitochondrial function.

Figure 1

Structural properties of Ym155 are similar to Ethidium Bromide: (A) Structures of Ym155 and ethidium bromide with calculated topological polar surface area (tPSA), octanol–water partition coefficient (CLogP), and LogS. (B) Similarities of the electrostatic charge distributions of Ym155 and ethidium bromide determined with Molecular Operating Environment (MOE) software.

Figure 2

Ym155 binds mitochondrial DNA and disrupts mitochondrial function. (A) Dual immunofluorescent imaging of PicoGreen and mitochondrial marker TUMF in A549 cells. PicoGreen stained mitochondria are in the cytoplasm (arrows). Mitochondria staining both PicoGreen and TUMF are orange in merged images. (B) Representative images of PicoGreen mitochondrial staining with increasing doses of Ym155. (C) Quantification of PicoGreen cytoplasm fluorescence demonstrating Ym155 quenches PicoGreen staining. (D) ATP concentration of H1299 cells treated with Ym155 20 nM up to 24 h, presented as percent of control (n = 4). (E) Mean of 2 experiments for ATP concentration of H1299 treated increasing concentrations of Ym155 for 3 h. (F) TMRM assay of mitochondrial membrane potential and fluorescence quantification of H1299 cells treated with Ym155 for 3 h (20 × magnification). (G) Immunoblot of cytosol of H1299 cells treated with Ym155 for 24 h. To include results for only Ym155 treated cells the image was cropped (Supplementary Information). (H) Immunoblot for activated caspase-3 and PARP expression of A549 cell treated with Ym155 for 24 h. (I) Immunoblot of H1299 cells treated with Ym155 for 48 h. (J–K) Mean cell death of two experiments of H1299 and A549 cells treated with Ym155 for 24 h. with and without 50 μM Z-VAD-FMK (VAD) or 20 μM necrostatin. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Ym155 decreases mitochondrial respiration: The Seahorse XFe24 analyzer was used to monitor oxygen consumption rate (OCR) and examine oxidative phosphorylation of H1299 cells treated with Ym155 for 3 h. Inhibitors of ETC oligomycin (complex V), FCCP (uncoupler), Rotenone (complex I) were used to interrogate oxidative phosphorylation. (A) OCR curves of H1299 cells treated with Ym155. (B) Mean basal respiration, (C) ATP production, (D) maximal respiration, (E) proton leak, and (F) spare capacity from 3 experiments.

Figure 4

Ym155 decreases TCA cycle metabolite intermediates. Metabolite intermediates of glycolysis, TCA cycle, and pyrimidine intermediates were determined by MS/LS/MS of H1299 cells treated with Ym155 for 3 h. (A) Early and late glycolysis intermediates were increased with Ym155 as wells as the pentose pathway intermediate ribose phosphate. (B) TCA intermediates alpha-ketoglutarate, fumarate, and malate were decreased. Citrate and succinate metabolites were increased. (C) Pyrimidine intermediates were decreased following Ym155 treatment. CAD (carbamoyl aspartate dehydrogenase), DHODH (dihydroorotate dehydrogenase). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Ym155 decreases glucose 2-carbon transfer in the TCA cycle. H1299 cells were loaded with [U13C6] glucose for 2 h then treated with Ym155 for 3 h. (A, B) The number 3 carbons (M + 3) in glycolysis intermediates pyruvate and lactate was the same between Ym155 treated and control. (D) The TCA cycle intermediate isocitrate, alpha-ketoglutarate, fumarate, and malate had significantly lower M + 2 carbons in Ym155 treated cells compared to controls (C–F).

Figure 6

Ym155 activates AMPK and downregulates BMP signaling. (A) Immunoblot for BMPR2 in H1299 and A549 cells treated with Ym155 for 24 h. (B) Immunoblot for pAMPK (T172) and pACC (Ser79) of cells treated for 24 h. (C) Immunoblot for pAMPK (T172) and pACC (Ser79) of A549 control transfected cells (A549-puro) and A549 cells stably transfected with LKB1 (A549-LKB1) treated for 24 h. (D) Immunoblot of A549-puro and A549-LKB1 cells treated with Ym155 for 24 h.

Figure 7

Mitochondrial inhibitors activate AMPK and downregulate BMP signaling. H1299 and A549 cells were treated with inhibitors for 24 h. (A) Immunoblots for cells treated with atpenin, (B) cisplatin, or (C–D) phenformin.

Figure 8

Ym155 is more potent suppressing growth and inducing cell death compared to other mitochondrial inhibitors. H1299 and A549 cells were treated with inhibitor for 48 h. and the percent live and dead cells counted. The data represents the represents mean of 2 experiments treated with (A) Ym155, (B) Atpenin, (C) cisplatin, (D) or phenformin.