Potent anticancer activity of a novel iridium metallodrug via oncosis

Abstract

Oncosis (from Greek ónkos, meaning "swelling") is a non-apoptotic cell death process related to energy depletion. In contrast to apoptosis, which is the main form of cell death induced by anticancer drugs, oncosis has been relatively less explored but holds potential to overcome drug resistance phenomena. In this study, we report a novel rationally designed mitochondria-targeted iridium(III) complex (OncoIr3) with advantageous properties as a bioimaging agent. OncoIr3 exhibited potent anticancer activity in vitro against cancer cells and displayed low toxicity to normal dividing cells. Flow cytometry and fluorescence-based assays confirmed an apoptosis-independent mechanism involving energy depletion, mitochondrial dysfunction and cellular swelling that matched with the oncotic process. Furthermore, a Caenorhabditis elegans tumoral model was developed to test this compound in vivo, which allowed us to prove a strong oncosis-derived antitumor activity in animals (with a 41% reduction of tumor area). Indeed, OncoIr3 was non-toxic to the nematodes and extended their mean lifespan by 18%. Altogether, these findings might shed new light on the development of anticancer metallodrugs with non-conventional modes of action such as oncosis, which could be of particular interest for the treatment of apoptosis-resistant cancers.

Keywords: Anticancer agents; Antitumor activity; Caenorhabditis elegans; Iridium metallodrug; Oncosis.

Fig. 1

Chemical structure and photophysical characterization of OncoIr3. a The reported oncosis-like neutral inducers OncoIr1, OncoIr2 and our current work OncoIr3. b Absorption and emission spectra of OncoIr3 in water (1% DMSO). (c) Emission spectra of OncoIr3 in DMSO/water mixtures with different water fractions (fw). λ_exc = 405 nm

Fig. 2

a Confocal images from the co-localization assay of OncoIr3 (5 µM for 0.5 h) and Mitotracker Green (MTG; 0.1 µM for 0.5 h) in HeLa cancer cells. Scale bar = 20 µm. b Intracellular iridium content in A2780 cells after 2 h incubation with OncoIr3 as determined by ICP-MS (mean ± SD from three independent experiments). Statistical significance *p < 0.05 from unpaired t-test. c Half-maximal inhibitory concentration (IC₅₀) values for OncoIr3 in cancer cell lines

Fig. 3

a Detection of cell swelling (white arrows) and cell blebbing (yellow arrows) in HeLa cells after 24 h treatment with OncoIr3 (5 µM) Scale bar = 20 µm. b Cell size (FSC) vs. cell complexity (SSC) flow cytometry plots from A2780 cells after treatment with 5 µM cisplatin or OncoIr3 for 24 h. c Mitochondrial membrane potential evaluation after treatment with cisplatin (5 µM), antimycin A (Ant. A; 50 µM) or OncoIr3 determined with JC-1 dye by flow cytometry. d Relative levels of ATP in A2780 cells after 24 h treatment with cisplatin (5 µM) or OncoIr3. Statistical significance control vs. treatment *p < 0.05, **p < 0.01, ***p < 0.001 from One-Way ANOVA test

Fig. 4

a Membrane integrity test of A2780 cells determined by propidium iodide entry after treatment with OncoIr3 (*p < 0.05, ** p < 0.01, ***p < 0.001; unpaired t test). b Detection of cell membrane rupture and permeabilization in cancer cells following OncoIr3 (5 µM) treatment by fluorescence microscopy using propidium iodide staining (red). Scale bar = 20 µm. c Percentage of A2780 cells (mean ± SD from three independent experiments) in sub-G₁, G₁, S, and G₂/M phases of the cell cycle after treatment with cisplatin (5 µM) or OncoIr3 (*p < 0.05, ***p < 0.001; unpaired t test). d Representative flow cytometry dot plots of A2780 cells stained with Annexin V-FITC/Propidium iodide labelling method after treatment with cisplatin or OncoIr3 (5 µM) for 24 h

Fig. 5

a Wild-type gonads of the C. elegans strain N2 visualized with differential interference contrast (DIC) and stained with acridine orange (AO). Scale bars 50 μm. b Tumoral gonads of the C. elegans strain JK1466. Scale bars 50 μm. c, d Detection of OncoIr3 inside tumoral JK1466 nematodes Scale bar: 200 μm

Fig. 6

a In vivo tumor size evaluation upon OncoIr3 treatment. Data are represented as mean ± S.D (n = 10 per treatment group), **significantly different at p ≤ 0.05 by ANOVA test. b Mean lifespan of JK1466 strain treated with different concentrations of OncoIr3. Data are represented as mean lifespan ± S.E, **significantly different at p ≤ 0.05 by Log Rank test. c, d Representative images of extracted gonads of tumoral animals stained with acridine orange and treated with 100 μM of cisplatin (c) or with OncoIr3 (d). Scale bars: 50 μm