Lysosome-targeted cyclometalated iridium(III) complexes: JMJD inhibition, dual induction of apoptosis, and autophagy.

{"full_text": " Metallomics, 14, 2022, mfac068\n https://doi.org/10.1093/mtomcs/mfac068\n Advance access publication date: 8 September 2022\n Paper\n\n\n\nLysosome-targeted cyclometalated iridium(III)\ncomplexes: JMJD inhibition, dual induction of apoptosis,\nand autophagy\nJun-Jian Lu1 ,\u2021 , Xiu-Rong Ma1 ,\u2021 , Kai Xie1 , Mei-Ru Chen1 , Bo Huang2 , \u2217 , Rong-Tao Li1 , \u2217 and Rui-Rong Ye 1 ,\u2217\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\n1\n Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, P. R. China and 2 Faculty of Chemistry and Chemical\nEngineering, Yunnan Normal University, Kunming 650500, P. R. China\n\u2217\n Correspondence: Yunnan Normal University, No. 1, Yuhua, Chenggong District, Kunming 650500, Yunnan, P. R. China. E-mail: huangbo15@foxmail.com (Bo\nHuang); Kunming University of Science and Technology, No. 727, South Jingming Road, Kunming 650500, Yunnan, P. R. China. E-mail: rongtaolikm@163.com\n(Rong-Tao Li); E-mail: yerr@mail2.sysu.edu.cn (Rui-Rong Ye)\n\u2021\n These authors contributed equally.\n\n\n\nAbstract\nA series of cyclometalated iridium(III) complexes with the formula [Ir(C\u02c6N)2 L](PF6 ) (C\u02c6N = 2-phenylpyridine (ppy, in Ir-1), 2-\n(2-thienyl)pyridine (thpy, in Ir-2), 2-(2,4-difluorophenyl)pyridine (dfppy, in Ir-3), L = 2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-\nyl)quinolin-8-ol) were designed and synthesized, which utilize 8-hydroxyquinoline derivative as N\u02c6N ligands to chelate the cofac-\ntor Fe2+ of the Jumonji domain-containing protein (JMJD) histone demethylase. As expected, the results of UV/Vis titration analysis\nconfirm the chelating capabilities of Ir-1\u20133 for Fe2+ , and molecular docking studies also show that Ir-1\u20133 can interact with the ac-\ntive pocket of JMJD protein, and treatment of cells with Ir-1\u20133 results in significant upregulation of trimethylated histone 3 lysine 9\n(H3K9Me3), indicating the inhibition of JMJD activity. Meanwhile, Ir-1\u20133 exhibit much higher cytotoxicity against the tested tumor cell\nlines compared with the clinical chemotherapeutic agent cisplatin. And Ir-1\u20133 can block the cell cycle at the G2/M phase and inhibit\ncell migration and colony formation. Further studies show that Ir-1\u20133 can specifically accumulate in lysosomes, damage the integrity\nof lysosomes, and induce apoptosis and autophagy. Reduction of mitochondrial membrane potential and elevation of reactive oxygen\nspecies also contribute to the antitumor effects of Ir-1\u20133. Finally, Ir-1 can inhibit tumor growth effectively in vivo and increase the\nexpression of H3K9Me3 in tumor tissues. Our study demonstrates that these iridium(III) complexes are promising anticancer agents\nwith multiple functions, including the inhibition of JMJD and induction of apoptosis and autophagy.\n\nKeywords: cyclometalated iridium(III) complexes, lysosome-targeted, 8-hydroxyquinoline, JMJD inhibition, apoptosis, autophagy\n\n\nGraphical abstract\n\n\n\n\nLysosome-targeted cyclometalated iridium(III) complexes exhibit multiple functions, including inhibition of JMJD and induction of\napoptosis and autophagy.\n\n\n\nIntroduction tivity and lower side effects through mechanisms of action differ-\n ent from those of platinum drugs.3\u20135 Among them, cyclometalated\nThe clinical success of platinum-based drugs has stimulated a\n iridium(III) complexes have emerged as promising alternatives\nstrong interest in finding other metal-based anticancer drugs.1 , 2\n to platinum-based drugs because they possess multiple anti-\nMany non-platinum anticancer drugs exhibit better antitumor ac-\n cancer modes, including perturbing the redox status of cells,6\u20139\n\n\n\nReceived: July 3, 2022. Accepted: August 28, 2022\n\u00a9 The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com\n\f2 | Metallomics\n\n\n\n\nScheme 1. Chemical structures of Ir-1\u20133.\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\ndamaging specific subcellular organelles10\u201313 and inhibiting cyclometalated iridium(III) complexes could mediate both apop-\nenzyme activities,14\u201316 and these antitumor modes can work tosis and autophagy, and exhibited excellent anticancer activity\ntogether synergistically to improve the efficacy of the drug.17\u201319 in vitro and in vivo.49\nMeanwhile, Jumonji domain-containing protein (JMJD) histone Herein, we designed and synthesized a series of cyclomet-\ndemethylases are important epigenetic regulators in cancer cells. alated iridium(III) complexes with the formula [Ir(C\u02c6N)2 L](PF6 )\nVarious studies have shown that JMJD histone demethylases are (C\u02c6N = 2-phenylpyridine (ppy, in Ir-1), 2-(2-thienyl)pyridine (thpy,\noverexpressed in a variety of cancer cells, including breast cancer, in Ir-2), 2-(2,4-difluorophenyl)pyridine (dfppy, in Ir-3), L = 2-(1H-\ncolorectal cancer, lung cancer, prostate cancer, etc., which is the imidazo[4,5-f][1,10]phenanthrolin-2-yl)quinolin-8-ol; Scheme 1),\nmain culprit of cancer cell proliferation, metastasis, and drug which utilize 8-hydroxyquinoline derivative as N\u02c6N ligands to\nresistance.20\u201325 Drug design targeting JMJD histone demethylase is chelate the cofactor Fe2+ of JMJD histone demethylase. First, the\nalso a focus of current antitumor drug development.26\u201331 Among inhibitory ability of Ir-1\u20133 on JMJD histone demethylase was con-\nthem, only one case of organometallic iridium complex with JMJD firmed by exploring their binding to Fe2+ using UV/Vis titration,\ninhibitory activity has been reported so far, which can effectively molecular docking, and analyzing their effects on trimethylated\ninhibit cell proliferation and induce apoptosis.17 histone 3 lysine 9 (H3K9Me3) expression levels. Next, we accessed\n Lysosomes (pH: 4.5\u20135.5) are cellular waste disposal units that the antiproliferative activity of Ir-1\u20133 against the tumor cells. Be-\ncontain different types of hydrolytic enzymes capable of degrad- sides, the anticancer mechanisms including cell cycle arrest, in-\ning almost all kinds of biomolecules in cells.32 , 33 Disruption of hibition of cell migration and colony formation, cellular localiza-\nthe lysosomal integrity, a process known as lysosomal membrane tion, cellular uptake mechanisms, lysosomal damage, induction\npermeabilization (LMP), can cause the release of lysosomal of apoptosis and autophagy, mitochondrial damage, and intracel-\ncomponents, such as cathepsins and other hydrolases, from lular ROS levels were elucidated. Finally, the antitumor activity\nthe lysosomal lumen to the cytosol.34 And the cathepsins and of Ir-1 in vivo was also evaluated. The results manifest that the\nother hydrolases initiate a cascade of biological events, including cyclometalated iridium(III) complexes Ir-1\u20133 are promising anti-\nsubstrate degradation, caspase activation, and reduction of cancer agents with multiple functions, including the inhibition of\nmitochondrial membrane potential (MMP), eventually leading to JMJD and induction of apoptosis and autophagy.\ncell death.35 As a result, the antitumor drugs that can facilitate\nLMP are worth designing and developing. For example, Liu et al.\nreported a series of half-sandwich iridium(III) complexes and\n Results and discussion\nruthenium(II) complexes that targeted the lysosome, disrupted\n Synthesis and photophysical characterization\nthe lysosomal integrity, increased intracellular reactive oxygen The intermediate compounds [Ir(C\u02c6N)2 (5,6-diamino-1,10-\nspecies (ROS) levels, and decreased MMP, ultimately induced the phenanthroline)](PF6 ) were synthesized following literature\nactivation of the mitochondrial apoptotic pathway.36 , 37 Recently, methods.50 Target compounds Ir-1\u20133 were obtained by heating\nChao et al. prepared a ruthenium complex with near-infrared ab- [Ir(C\u02c6N)2 (5,6-diamino-1,10-phenanthroline)](PF6 ) and 8-hydroxy-\nsorption for photodynamic therapy, which could induce cell apop- 2-quinolinecarboxaldehyde in methanol for 24 h, followed by\ntosis by generating singlet oxygen and damaging lysosomes.38 anion exchange with NH4 PF6 (Scheme S1). They were then puri-\n Damaging lysosomes may induce programmed cell death fied by column chromatography on silica gel using CH2 Cl2 /CH3 OH\n(RCD), including apoptosis, autophagy, and ferroptosis.39 RCD is a (30:1, v/v) as the eluent and characterized using ESI-MS (Figs.\nphysiological and active \u2018conscious suicide\u2019 of cells, which plays S1\u2013S3), 1 H NMR (Figs. S4\u2013S6), and elemental analysis.\nan important role in organismal development, homeostasis, and The UV/Vis absorption spectra of Ir-1\u20133 in PBS, CH3 CN, and\npathogenesis.40 Apoptosis, the most common form of RCD, can CH2 Cl2 were investigated at 298 K (Fig. S7A). In the UV region,\nbe initiated through death receptors on the cell surface (extrinsic Ir-1\u20133 displayed intense absorption bands at \u223c250\u2013420 nm orig-\npathway) or through mitochondria (intrinsic pathway), ultimately inated from intraligand \u03c0 \u2192\u03c0 * transitions (1 LC). The relatively\nleading to the activation of an initiator caspase.41 , 42 In tumor weak bands at \u223c420\u2013510 nm were attributed to metal-to-ligand\ntreatment, most chemotherapeutic drugs exert their efficacy charge-transfer absorption. Ir-1\u20133 exhibited green to orange phos-\nby inducing apoptosis in cancer cells.43 Besides, autophagy as phorescent emission upon excitation at 405 nm (Fig. S7B). And\na common form of RCD is a lysosomal degradation pathway, the emission intensity of Ir-1\u20133 was very sensitive to solvent\nwhich can induce autophagic death of cancer cells.44 , 45 A growing polarity. They were weakly emissive in PBS. However, the emis-\nnumber of studies have shown that the induction of multiple sion intensity was significantly enhanced in the nonpolar sol-\nantitumor mechanisms can improve antitumor efficacy more vent CH2 Cl2 . The photophysical data of Ir-1\u20133 are summarized in\nefficiently.13 , 36 , 46\u201348 Mao et al. reported that lysosome-targeted Table S1.\n\f Paper | 3\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\nFig. 1 UV/Vis spectra (2 \u00d7 10\u22125 M) of Ir-1 (A), Ir-2 (B), and Ir-3 (C) upon addition of Fe2+ in H2 O/DMSO (1:1, v/v) at 298 K.\n\n\n\n\nBinding of Ir-1\u20133 with Fe2+ blot. As shown in Fig. 3, the protein expression levels of H3K9Me3\n 2+\nAs Fe is an essential cofactor of the JMJD proteins, the possi- significantly increased in a dose-dependent manner upon treat-\nbility that Ir-1\u20133 could sequestrate Fe2+ to inhibit JMJD histone ment of HeLa cells with Ir-1\u20133. The result suggests that Ir-1\u20133 can\n inhibit the activity of JMJD.\ndemethylase activity was investigated by UV/Vis absorption spec-\ntra.51 , 52 As shown in Fig. 1, with the titration of Fe2+ , the absorp-\ntion of Ir-1\u20133 at 290\u2013405 nm decreased, accompanied by an in- Lipophilicity and in vitro cytotoxicity\ncrease of the absorption at 405\u2013430 nm. An isosbestic point was Lipophilicity (log Po/w ) can affect the cellular uptake, distribu-\nformed at around 405 nm, which indicated the formation of the tion, and cytotoxicity of metal-based complexes.54 Using a flask-\nnew species upon the addition of Fe2+ . Besides, iron shuttles be- shaking method, the log Po/w values of Ir-1\u20133 were determined as\ntween the ferrous and ferric forms in the cell,53 and the changes follows: Ir-3 (3.00) > Ir-2 (2.99) > Ir-1 (2.79). In addition, iridium is\nin UV/Vis absorption spectra of Ir-1\u20133 in the presence of Fe3+ were not an endogenous component of cells, therefore the cellular up-\nalso investigated (Fig. S8). In the presence of Fe3+ , the absorption take efficiency of iridium can be quantitatively determined using\nof Ir-1\u20133 underwent the same change phenomenon as Fe2+ . These inductively coupled plasma mass spectrometry (ICP-MS) analy-\nresults suggest that Ir-1\u20133 can effectively chelate not only Fe2+ but sis.15 Upon incubation with 20 \u03bcM Ir-1\u20133 for 2.5 h, the intracellu-\nalso Fe3+ . lar iridium contents of the compounds were in the following order:\n Ir-2 (948.54 ng/106 cells) > Ir-1 (786.00 ng/106 cells) > Ir-3 (721.66\nMolecular docking studies ng/106 cells) (Table S2). The cellular uptake efficiency of Ir-1\u20133 did\nIn order to investigate the bonding mode and bonding mecha- not correlate well with their lipophilicities, probably because the\nnism between Ir-1\u20133 and JMJD more intuitively, a molecular dock- overall outcome of cellular uptake is also influenced by other fac-\ning calculation was conducted. As shown in Fig. 2A, the terminal tors (e.g. molecular size).\nbenzene ring on N\u02c6N ligand L of Ir-1\u20133 could insert the pocket of The antiproliferative activity of complexes Ir-1\u20133 against\nJMJD (PDB code: 5ANQ) and interact with the active center Fe2+ HeLa, human lung adenocarcinoma epithelial (A549), cisplatin-\nthrough Fe2+ \u2013\u03c0 interaction. Meantime, the hydroxyl oxygen in the resistant A549 (A549R), human hepatocellular liver carcinoma\nterminal benzene could coordinate with Fe2+ . On the other hand, (HepG2), and human normal liver (LO2) cells were evaluated\nthe Ir(C\u02c6N)2 moiety with the large steric hindrance of octahedral by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\ngeometry could be firmly stuck outside the pocket, blocking the (MTT) assay, and cisplatin was also tested as a reference. As\nexit of the cavity of JMJD. As a result, the substrate of JMJD could shown in Table 1, the viability of these cancer cell lines was sig-\nnot enter the pocket, serving to inhibit the JMJD activity. In ad- nificantly inhibited by Ir-1\u20133, with IC50 values ranging from 4.7 to\ndition, the bottom of the active pocket of JMJD was a negatively 18.1 \u03bcM. Furthermore, they exhibited acceptable selectivity\ncharged region, and Ir-1\u20133 were positively charged ions, resulting toward human cancer cells over non-cancerous cells, and the\nin an electrostatic matching of the two, which improves the bind- selectivity fold varied with the auxiliary ligands of iridium(III)\ning stability. complexes, indicating that the auxiliary ligands of iridium(III)\n Based on the energy analysis, the binding energies of Ir-1\u20133 complexes may affect the selectivity of iridium(III) complexes\nand JMJD were \u20134.93, \u20134.95, and \u20134.89 kcal/mol, respectively. Be- toward human cancer cells over non-cancerous cells. More impor-\ncause the binding modes and the interactions between Ir-1\u20133 tantly, Ir-1\u20133 exhibited higher antiproliferative activity compared\nand JMJD were basically similar, the binding energies were also to cisplatin. Meanwhile, Ir-1\u20133 also exhibited \u223c5- to 10-fold\nsimilar. In conclusion, Ir-1\u20133 showed a good binding ability to higher cytotoxic effect on A549R cells than cisplatin, indicating\nJMJD and could be used to inhibit the demethylation activity of that these compounds have potential to overcome cisplatin\nJMJD. resistance.\n\n\nUpregulation of the histone-methylation level Inhibition of cell migration and colony formation\nJMJD can lead to the downregulation of H3K9Me3 and oncogene JMJD histone demethylase has been reported to play a role in\nactivation, thus the expression levels of H3K9Me3 can be used to promoting the metastasis of cancer cells.25 , 55 Meanwhile, inhi-\nmonitor the activity of JMJD.22 , 25 To further validate the ability of bition of cancer cell metastasis is a critical issue in tumor ther-\nIr-1\u20133 to inhibit JMJD, we examined their effects on H3K9Me3 ex- apy.56 Given the ability of Ir-1\u20133 to inhibit JMJD activity and in-\npression levels in human cervical cancer (HeLa) cells by Western crease the expression of histone methylation in cells, the effects\n\f4 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\nFig. 2 Docking results of Ir-1\u20133 with JMJD (PDB code: 5ANQ). (A) Docked conformations for Ir-1 (a), Ir-2 (b), and Ir-3 (c) with JMJD. The red sphere\nrepresents the active center Fe2+ of JMJD. (B) Electrostatic surface diagrams of Ir-1 (a), Ir-2 (b), and Ir-3 (c) are bound to JMJD. The blue color in the\nelectrostatic surface represents the positive charge, the red color represents the negative charge, and the white color represents the region with very\nlow or no charge, i.e. the non-polar region.\n\n\n\n\nFig. 3 Dose-dependent effects of Ir-1\u20133 on H3K9Me3 after 24 h of treatment.\n\n\n\nof the compounds on tumor cell migration were also evaluated. sults demonstrate that Ir-1\u20133 can effectively inhibit cancer cell\nAs shown in the wound healing assay (Fig. 4A), after treating HeLa metastasis.\ncells with 2 \u00d7 IC50 concentration of Ir-1\u20133, shorter migration dis-\ntances were found at 24 and 36 h compared to vehicle-treated Cell cycle arrest\ncells. And after incubation for 36 h, Ir-1\u20133 could efficiently impede It has been reported that the JMJD inhibitors can inhibit cell pro-\nHeLa cells\u2019 migration at a wound closure ratio of 2.5\u20132.7%, which liferation via inducing cell cycle arrest.58 , 59 Hence, the effects of\nwas much lower than that of the control group (17.8%) (Fig. 4B). Ir-1\u20133 on cell cycle distribution were examined via flow cytom-\nIn addition, colony formation is a key feature of malignant cancer etry with propidium iodide (PI) staining. As shown in Fig. 5 and\ncells,57 thereby the inhibition of colony formation induced by Ir- Table S3, Ir-1 could effectively arrest the cell cycle at the G2/M\n1\u20133 was assessed. As shown in Fig. 4C and D, the colony formation phase in a concentration-dependent manner, whereas Ir-2 and\nwas effectively suppressed upon incubation with Ir-1\u20133. These re- Ir-3 arrested the cell cycle at the G2/M phase only at higher\n\f Paper | 5\n\n\nTable 1. IC50 values of the tested compounds against different cell As shown in Fig. 6A, Ir-1\u20133 could be effectively taken up by HeLa\nlinesa cells and emitted intense dot-like luminescence in the cytoplasm\n after 2.5 h incubation at 37\u00b0C. Meanwhile, the phosphorescence\n IC50 (\u03bcM)\n signals of Ir-1\u20133 inside the cells highly overlapped with the punc-\nCompounds HeLa A549 A549R HepG2 LO2 tate structures of LysoTracker Deep Red FM (LTDR), and Pearson\u2019s\n colocalization coefficients were between 0.65 and 0.78. In con-\nIr-1 5.1 \u00b1 0.5 12.9 \u00b1 2.2 18.1 \u00b1 1.9 11.5 \u00b1 0.1 17.8 \u00b1 0.4 trast, the luminescent regions of Ir-1\u20133 had little overlap with\nIr-2 4.7 \u00b1 0.3 5.5 \u00b1 1.1 8.9 \u00b1 0.4 7.8 \u00b1 0.4 6.9 \u00b1 1.9 MitoTracker Deep Red FM (MTDR) (Fig. 6B). These results in-\nIr-3 8.1 \u00b1 1.4 10.0 \u00b1 2.7 12.0 \u00b1 1.9 10.2 \u00b1 0.7 9.8 \u00b1 1.6\n dicate that Ir-1\u20133 mainly distribute in lysosomes instead of\nCisplatin 23.4 \u00b1 0.7 21.5 \u00b1 1.7 89.3 \u00b1 3.1 21.5 \u00b1 1.4 27.2 \u00b1 0.8\n mitochondria.\n a\n IC50 values are drug concentrations necessary for 50% inhibition of cell viabil- We further examined the cellular uptake mechanisms of Ir-1\u2013\nity. The data are presented as mean \u00b1 standard deviation (SD) and cell viability 3 by confocal microscope. Incubation of HeLa cells with Ir-1\u20133 at\nis assessed after 48 h incubation.\n low temperature (4\u00b0C) or pretreatment with metabolic inhibitor\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\n carbonyl cyanide m-chlorophenyl hydrazone (CCCP) resulted in a\nconcentrations. Quantitative cell cycle distribution data showed significant decrease in cellular uptake efficiency (Fig. S9). While,\nthat cells were exposed to Ir-1\u20133 at the highest tested concentra- the cellular uptake levels of Ir-1\u20133 were not affected in cells pre-\ntion (4 \u00d7 IC50 ) for 24 h, and the percentage of cells in the G2/M treated with chloroquine, which regulates endocytosis by inhibit-\nphase increased from 16.7% (control) to 34.6% (Ir-1, 20.4 \u03bcM), ing the acidification of endosomes. The results infer that Ir-1\u20133\n25.3% (Ir-2, 18.8 \u03bcM), and 46.7% (Ir-3, 32.4 \u03bcM), respectively. are transported into the cell membrane primarily via an energy-\n dependent mechanism instead of an endocytic pathway, which\nCellular localization and uptake mechanisms is similar to other cyclometalated Ir(III) complexes previously re-\nThe subcellular localization and cellular transport pathways of ported.54 , 61\nmetal-based anticancer drugs can provide more hints on their an-\ntitumor mechanisms.12 , 60 Herein, the subcellular localization and Lysosomal damage\ncellular transport pathways of Ir-1\u20133 were monitored by tracking Metal complexes localized in lysosomes have been reported to\nthe luminescence of the complexes using confocal microscopy. induce lysosomal dysfunction, which is considered to be one of\n\n\n\n\nFig. 4 (A) Representative pictures of a wound healing assay of HeLa cells after treated with Ir-1\u20133 for 0 h, 24 h, and 36 h. The black lines indicate the\ndistance of the wound. (B) Quantitative data of wound healing. Wound closure (%) = [1\u2013(width at indicated time)/(width at 0 h)] \u00d7 100%. (C) Images of\ncolony formation after treatment with Ir-1\u20133. (D) Quantitative data of colony formation assays.\n\f6 | Metallomics\n\n\n meaning the acidity in the lysosomal microenvironment is altered\n (Fig. S10). These results validate that Ir-1\u20133 can increase the per-\n meability of lysosomes, therefore disrupting the acidic environ-\n ment of lysosomes.\n LMP can evoke the release of lysosomal hydrolases, such as\n cathepsin B, from lysosomes to the cytosol to initiate apoptosis.63\n The effect of Ir-1\u20133 on cathepsin B release was examined using the\n fluorogenic substrate Magic Red MR-(RR)2 .64 As depicted in Fig. 7B,\n the red fluorescence of Magic Red MR-(RR)2 was punctate in HeLa\n cells for the control cells, showing cathepsin B mostly aggregated\n in lysosomes. While, with the increase of concentration of Ir-1\u20133,\n the red fluorescence gradually showed a dispersive pattern in the\n cytoplasm, which indicated that Ir-1\u20133 induced lysosomal dam-\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\n age and released cathepsin B from the lysosomes to the cytosol.\n\n\n Apoptosis induction\n Lysosomal damage and JMJD inhibition have been reported to\n be closely related to RCD.17 , 62 , 65 Among them, apoptosis is the\n most common form of RCD. Cells undergoing apoptosis are asso-\n ciated with a series of defined morphological changes and biolog-\n ical events, such as cell shrinkage, plasma membrane blistering,\n nuclear fragmentation, apoptotic-body formation, accompanied\nFig. 5 Quantitative cell cycle distribution data for HeLa cells after\ntreatment with Ir-1\u20133 for 24 h.\n by phosphatidylserine externalization, and caspases activation,\n etc.66 , 67 To investigate whether Ir-1\u20133 induced apoptosis in HeLa\n cells, the changes in nuclei morphology of Ir-1\u20133-treated HeLa\nthe main toxicity paradigms of lysosome-targeted metal-based cells were first observed by 2\u0005 -(4-ethoxyphenyl)-5-(4-methyl-1-\nanticancer complexes.38 , 62 Thus, the lysosomal integrity of HeLa piperazinyl)-2,5\u0005 -bi-1H-benzimidazole trihydrochloride (Hoechst\ncells after treatment with Ir-1\u20133 was tested with acridine orange 33342) staining. As shown in Fig. 8A, the nuclei of vehicle-treated\n(AO) staining. AO is a common fluorescent indicator of acidic cells showed a round and homogeneous staining pattern. While\norganelles and can be used to study the integrity of lysosomes after treatment of HeLa cells with Ir-1\u20133, the proportion of nu-\nbecause it emits red fluorescence in lysosomes and green fluo- clei with apoptotic morphology (nuclear fragmentation, chro-\nrescence in cytosol and nuclei.34 As shown in Fig. 7A, vehicle- matin condensation, and apoptotic-body formation)68 increased\ntreated cells displayed distinct red fluorescence in lysosomes. progressively in a concentration-dependent manner.\nHowever, after HeLa cells were treated with Ir-1\u20133 for 6 h, the red Apoptosis induced by Ir-1\u20133 was further verified quantitatively\nfluorescence in cells remarkably decreased in a concentration- by flow cytometric analysis with Annexin V and PI double stain-\ndependent manner, implying that the lysosomes were seriously ing, with cisplatin as a control (Fig. 8B). The results showed that,\ndamaged. Additionally, upon treatment of HeLa cells with Ir-1\u20133 compared with the control cells, cells exposed to Ir-1\u20133 increased\nat the much higher dose (40 \u03bcM) for 5 h, LTDR was diffusely dis- the percentage of apoptotic cells in a concentration-dependent\ntributed in cells and lost its ability to image lysosomes in cells, manner. And the percentage of cells in the early and late\n\n\n\n\nFig. 6 The confocal microscopic images of HeLa cells co-labeled with Ir-1\u20133 (20 \u03bcM, 2.5 h) and LTDR (50 nM, 0.5 h) (A) or MTDR (150 nM, 0.5 h) (B) [\u03bbex =\n402 nm (for Ir-1\u20133), 633 nm (for MTDR and LTDR); \u03bbem = 520 \u00b1 20 nm (for Ir-3), 570 \u00b1 20 nm (for Ir-1 and Ir-2), and 665 \u00b1 20 nm (for MTDR and LTDR)].\nScale bar: 20 \u03bcm.\n\f Paper | 7\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\nFig. 7 (A) Detection of lysosomal disruption upon Ir-1\u20133 treatment in HeLa cells by AO staining [\u03bbex = 488 nm, \u03bbem = 510 \u00b1 20 nm (green) and 625 \u00b1 20\nnm (red)]. (B) Observation of cathepsin B release from lysosomes to the cytosol upon Ir-1\u20133 treatment in HeLa cells using Magic Red MR-(RR)2 as a\nfluorogenic substrate (\u03bbex = 543 nm, \u03bbem = 630 \u00b1 20 nm). Scale bar: 20 \u03bcm.\n\n\n\n\napoptotic stages ranged from 7.42% (control) to 37.30% (Ir-1, diacetate (H2 DCFDA) as the fluorescent probe. H2 DCFDA is a\n20.4 \u03bcM), 19.30% (Ir-2, 18.8 \u03bcM), and 58.30% (Ir-3, 32.4 \u03bcM), non-fluorescent cell-permeable dye, but can turn into highly\nrespectively. fluorescent 2\u0005 ,7\u0005 -dichlorofluorescein (DCF) by the oxidation of\n The cleavage of Poly(ADP-ribose) polymerase (PARP), a sub- intracellular ROS.75 As depicted in Fig. 9B, compared to vehicle-\nstrate protein of caspase-3, has been identified as the biomarker of treated cells, a concentration-dependent increase in ROS levels\napoptosis.69 As a member of the cysteine protease family, caspase- was observed in HeLa cells treated with Ir-1\u20133. Flow cytometric\n3 plays a crucial significant role in mediating apoptosis under analysis (Fig. S11) also showed that, compared with the control\nvarious stimuli.70 Therefore, the cleavage of caspase-3 and PARP cells, the DCF mean fluorescence intensity of cells treated with\nwere assessed by Western blot. As depicted in Fig. 8C, caspase-3 Ir-1\u20133 at 4 \u00d7 IC50 increased by 2.1-, 2.4-, and 3.0-fold, respectively.\nand PARP were cleaved in a dose-dependent manner after incuba- Furthermore, N-acetyl-L-cysteine (NAC), a ROS scavenger, could\ntion with Ir-1\u20133. These results manifest that Ir-1\u20133 induce apop- lead to marked inhibition of cell death induced by Ir-1\u20133 (Fig.\ntotic cell death through caspase-dependent mechanisms, which is S12). These results demonstrate that ROS plays an important role\nalso the main pathway for iridium(III) complexes-induced apop- in Ir-1\u20133-induced cell death.\ntosis.71 , 72\n\n Autophagy induction\nMitochondrial dysfunction and cellular ROS Autophagy is a lysosomal degradation pathway through lyso-\nproduction somal hydrolases that allows the degradation of damaged or\nLMP can cause the release of cathepsins, which trigger substrate unnecessary cytoplasmic contents, including dysfunctional pro-\ndegradation, disruption of mitochondrial integrity, and caspase- teins and organelles.76 Autophagy plays an important role in cel-\ndependent cell death.35 The influence of Ir-1\u20133 on mitochondrial lular homeostasis, cancer prevention, and treatment.77 Besides,\nintegrity was monitored by detecting the changes in red/green lysosome-targeted metal-based compounds have been reported\nfluorescence intensity ratio of 5,5\u0005 ,6,6\u0005 -tetrachloro-1,1\u0005 -3,3\u0005 - to induce both apoptosis and autophagy in cancer cells.46 , 48 , 78\ntetraethyl-benzimidazolylcarbocyanineiodide (JC-1).73 In control Therefore, we tested whether Ir-1\u20133 could induce autophagy by\ncells, JC-1 mainly existed in the form of aggregates and emitted Western blot analysis. During autophagy, LC3-I will be converted\nred fluorescence, suggesting the high MMP (Fig. 9A). But after into LC3-II, which has been identified as an autophagy marker.79\ntreatment of HeLa with Ir-1\u20133 for 12 h, a prominent decrease in When cells undergo autophagy and lysosomal functions are in-\nMMP was observed, and the proportion of cells with depolarized tact, LC3-II levels are elevated while p62 levels are reduced. How-\nmitochondria increased from 4.3% (control) to 42.6% (Ir-1, 20.4 ever, when autophagy is blocked due to impaired lysosomal func-\n\u03bcM), 57.6% (Ir-2, 18.8 \u03bcM), and 63.8% (Ir-3, 32.4 \u03bcM), respectively. tions, LC3-II and P62 levels are increased.49 , 80 As depicted in Fig.\nThe results manifest that Ir-1\u20133 can cause mitochondrial dys- S13, the ratio of LC3-II to LC3-I increased significantly after treat-\nfunction and eventually activate the mitochondrial apoptotic ment with Ir-1, Ir-2, and rapamycin compared to the control.\npathway. Meanwhile, the ratio of LC3 (LC3-II/LC3-I) conversion induced by\n Mitochondrial dysfunction and abnormal production of ROS Ir-1 and Ir-2 showed a marked concentration-dependent behavior\nare two closely related events involved in cancer cell death caused (Fig. 10). In addition, Ir-1\u20133 could also activate p62, which signified\nby many anticancer agents.10 , 74 Intracellular ROS levels were in- that autophagy induced by Ir-1\u20133 was blocked owing to the dam-\nvestigated by confocal microscopy using 2\u0005 ,7\u0005 -dichlorofluorescein aged lysosomes.\n\f8 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\nFig. 8 (A) Hoechst 33 342 staining for the nuclei (\u03bbex = 405 nm, \u03bbem = 460 \u00b1 20 nm). Scale bar: 20 \u03bcm. The arrows indicate the apoptotic morphological\nnuclei. (B) Flow-cytometric quantification of Annexin V-FITC/PI labeled cells after treatment with Ir-1\u20133 for 24 h [\u03bbex = 488 nm, \u03bbem = 530 \u00b1 20 nm (for\nannexin V) and 620 \u00b1 20 nm (for PI)]. (C) Western blot analysis of the apoptosis-related protein (caspase-3, PARP) in cells after incubation with Ir-1\u20133\nfor 24 h (FL, full length; CF, cleaved form).\n\n\n\nIn vivo antitumor activity could also alter the expression of histone methylation levels in\nTo test whether iridium complexes could inhibit tumor growth in vivo. Therefore, the expression of H3K9Me3 in tumor tissues was\nvivo, we assessed the biological efficacy of Ir-1 in an H22 mouse detected by Western blot. As shown in Fig. 11D, Ir-1 could increase\nmodel. As shown in Fig. 11A and C, after intratumoral injection the expression of H3K9Me3, indicating the inhibition of JMJD ac-\nof 5 mg/kg Ir-1 into H22-bearing mice, the tumor volume was tivity.\nmuch less than those of the solvent control group. After 15 days,\nthe tumor volume of Ir-1-treated mice decreased by \u223c57% com-\npared with the solvent control group. Importantly, no significant Experimental section\ndifference was found in body weight among the solvent control Materials and instruments\ngroup and the drug administration group (Fig. 11B), suggesting IrCl3 \u00b7nH2 O, ppy, thpy, dfppy, NAC, NH4 PF6 , chloroquine, CCCP,\nthat Ir-1 has no severe side effects in these conditions. Having and 5,6-diamino-1,10-phenanthroline were obtained from Alfa\nseen that iridium complexes could increase the expression of hi- Aesar. 8-hydroxy-2-quinolinecarboxaldehyde was purchased\nstone methylation levels in vitro, we next assessed whether Ir-1 from Innochem. Cisplatin, MTT, DMSO, AO, H2 DCFDA, and\n\f Paper | 9\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\nFig. 9 (A) The loss of MMP examined by flow cytometry with JC-1 staining after treatment with Ir-1\u20133 for 12 h [\u03bbex = 488 nm, \u03bbem = 530 \u00b1 20 nm for\nJC-1 monomer (green) and 585 \u00b1 20 nm for JC-1 aggregates (red)]. (B) The intracellular ROS levels were examined by confocal microscopy with\nH2 DCFDA staining after treatment with Ir-1\u20133 for 6 h (\u03bbex = 488 nm, \u03bbem = 530 \u00b1 20 nm). Scale bar: 20 \u03bcm.\n\n\n\n\nFig. 10 Western blot analysis of autophagy-related protein (LC3, p62) in HeLa cells treated with Ir-1\u20133 for 24 h.\n\n\n\nHoechst 33342 were obtained from Sigma Aldrich. Cell Cycle Preparation of iridium(III) complexes\nand Apoptosis Analysis Kit, Annexin V-FITC Apoptosis Detection Three Ir(III) chloro-bridged dimers [Ir(ppy)2 Cl]2 ,81 [Ir(thpy)2 Cl]2 ,82\nKit, PBS, crystal violet, 4% paraformaldehyde, and JC-1 were [Ir(dfppy)2 Cl]2 83 and the intermediate compounds [Ir(C\u02c6N)2 (5,6-\npurchased from Beyotime Biotechnology. MTDR and LTDR were diamino-1,10-phenanthroline)](PF6 )50 were synthesized following\npurchased from Life Technologies. Magic Red MR-(RR)2 was the literature methods. The synthetic routes of Ir-1\u20133 were illus-\npurchased from Immunochemistry Tech. Dulbecco\u2019s modified trated in Scheme S1. And Ir-1\u20133 were converted into hexafluo-\nEagle medium (DMEM), Fetal bovine serum, and penicillin- rophosphate by ammonium hexafluorophosphate for the biologi-\nstreptomycin were obtained from Gibco. Primary antibodies cal test.\nagainst H3K9Me3, caspase-3, PARP, p62, and LC-3 were ob- Synthesis of Ir-1: [Ir(ppy)2 (5,6-diamino-1,10-phenanthroline)\ntained from Cell Signaling Technology. Male BALB/c mice were ](PF6 ) (0.1 g, 0.117 mmol) and 8-hydroxy-2-\npurchased from Beijing Weitong Lihua Experimental Animal quinolinecarboxaldehyde (0.024 g, 0.117 mmol) was refluxed\nCenter. in methanol for 24 h at 65\u00b0C in the dark. Upon completion, the\n NMR spectra were recorded on a Bruker Avance 600 spectrom- above solvent was added to excess saturated NH4 PF6 aqueous so-\neter. ESI-MS spectra were carried out using an LCQ DECA XP spec- lution and stirred for another 6 h. The solvent was then removed\ntrometer. Cell viability measurements were carried out using a by a rotary evaporator. The crude solid was purified using column\nSpetraMax M2 plate reader. Cell imaging images were conducted chromatography on silica gel by elution with CH2 Cl2 /CH3 OH\non a Nikon A1R/A1 laser-scanning confocal microscope, and flow (30:1, v/v). Yield: 0.094 g (orange powder), 80%. 1 H NMR (600 MHz,\ncytometric analyses were conducted on a CyFlow Space flow cy- [D6 ] DMSO) \u03b4 9.84 (s, 1H), 9.15 (d, J = 31.4 Hz, 2H), 8.57 (d, J = 8.5\ntometer. Hz, 1H), 8.48 (d, J = 8.5 Hz, 1H), 8.31 (d, J = 8.1 Hz, 2H), 8.26\u20138.09\n\f10 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\nFig. 11 Antitumor activity of Ir-1 in H22 xenograft-bearing mice. The average tumor volume (A) and body weight (B) of BALB/c mice in solvent control\nand Ir-1 (5 mg/kg) groups (n = 5) (** P < 0.01). (C) Photographs of tumors removed from mice. (D) Western blot of H3K9Me3 expression in tumor tissues.\n\n\n(m, 4H), 8.00 (d, J = 7.7 Hz, 2H), 7.91 (t, J = 7.5 Hz, 2H), 7.65\u20137.40 were continuously added, and the changes in UV/Vis absorp-\n(m, 4H), 7.18 (d, J = 4.0 Hz, 1H), 7.11\u20136.96 (m, 6H), 6.32 (d, J = 7.2 tion spectra were measured. The excitation wavelength of Ir-1\u20133\nHz, 2H). ESI-MS (CH3 OH): m/z 432.6064 [M-PF6 + H]2+ , 864.2040 was 405 nm.\n[M-PF6 ]+ . Anal. Calcd for C44 H29 F6 IrN7 OP (%): C, 52.38; H, 2.90; N,\n9.72; found: C, 52.18; H, 3.01; N, 9.59.\n Synthesis of Ir-2: The compound Ir-2 was synthesized by a Molecular docking\nsimilar method to Ir-1 except that [Ir(ppy)2 (5,6-diamino-1,10- Dockings of Ir-1\u20133 and JMJD were carried out by AutoDock 4.2,\nphenanthroline)](PF6 ) was replaced by [Ir(thpy)2 (5,6-diamino- using the Lamarckian genetic algorithm method.84 The crystal\n1,10-phenanthroline)](PF6 ). Yield: 0.096 g (reddish brown powder), structure of JMJD (PDB ID: 5ANQ) was retrieved from the protein\n82%. 1 H NMR (600 MHz, [D6 ] DMSO) \u03b4 9.79 (s, 1H), 9.12 (d, J = 38.0 data bank (PDB). A docking box of 60 \u00d7 60 \u00d7 60 points with a grid\nHz, 2H), 8.53 (d, J = 8.5 Hz, 1H), 8.44 (d, J = 8.4 Hz, 1H), 8.17 (d, spacing of 0.375 \u00c5 was used in each calculation. The center of\nJ = 28.2 Hz, 4H), 7.81\u20137.76 (m, 4H), 7.73 (d, J = 4.6 Hz, 2H), 7.49 (s, the box was set as the metal ion in active site. The number of ge-\n2H), 7.41 (s, 2H), 7.13 (s, 1H), 6.85 (t, J = 5.2 Hz, 2H), 6.30 (d, J = 4.3 netic algorithm calculations was set as 1000. All other parameters\nHz, 2H). ESI-MS (CH3 OH): m/z 438.5591 [M-PF6 + H]2+ , 876.1091 were used as default settings. For each of the docking cases, the\n[M-PF6 ]+ . Anal. Calcd for C40 H25 F6 IrN7 OPS2 (%): C, 47.06; H, 2.47; conformation with the lowest energy was selected as the bind-\nN, 9.60; found: C, 47.38; H, 2.27; N, 9.43. ing mode for analysis according to the Autodock scoring func-\n Synthesis of Ir-3: The compound Ir-3 was synthesized by a tion. The output from AutoDock was rendered with the PyMOL\nsimilar method to Ir-1 except that [Ir(ppy)2 (5,6-diamino-1,10- program.\nphenanthroline)](PF6 ) was replaced by [Ir(dfppy)2 (5,6-diamino-\n1,10-phenanthroline)](PF6 ). Yield: 0.091 g (yellow powder), 78%. 1 H\nNMR (600 MHz, [D6 ] DMSO) \u03b4 9.88 (s, 1H), 9.24 (d, J = 26.7 Hz, 2H), Western blot analysis\n8.60 (d, J = 8.6 Hz, 1H), 8.52 (d, J = 8.5 Hz, 1H), 8.33 (d, J = 9.0 HeLa cells cultured in 100 mm cell culture dishes were exposed\nHz, 4H), 8.17 (d, J = 33.7 Hz, 2H), 8.01 (t, J = 7.6 Hz, 2H), 7.64 (d, to different concentrations of Ir-1\u20133 for 24 h. Cells were collected,\nJ = 10.1 Hz, 2H), 7.54 (d, J = 7.2 Hz, 2H), 7.23 (d, J = 6.4 Hz, 1H), 7.12 washed twice with pre-cooled PBS, and lysed with RIPA buffer.\n(t, J = 6.7 Hz, 2H), 7.09\u20137.03 (m, 2H), 5.74 (d, J = 7.3 Hz, 2H). ESI- The protein concentration was quantified using the BCA assay\nMS (CH3 OH): m/z 468.5877 [M-PF6 + H]2+ , 936.1667 [M-PF6 ]+ . Anal. kit. Equal amounts of cellular total proteins were separated\nCalcd for C44 H25 F10 IrN7 OP (%): C, 48.89; H, 2.33; N, 9.07; found: C, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis\n49.12; H, 2.09; N, 8.86. (SDS-PAGE) and then transferred to the polyvinylidene difluoride\n membrane. After blocking, the membranes were incubated with\nBinding of Ir-1\u20133 with iron ions primary antibodies against H3K9Me3, caspase-3, PARP, LC-3,\nThe solution (2 \u00d7 10\u22125 M) of Ir-1\u20133 in H2 O/DMSO (1:1, v/v) was and p62 at 4\u00b0C overnight and then incubated with secondary\nprepared, and then the aqueous solutions of FeCl2 and FeCl3 antibodies.\n\f Paper | 11\n\n\nLipophilicity cal microscopy immediately. The excitation wavelengths of Ir-1\u20133\nThe lipophilicity of Ir-1\u20133 was determined according to a previous and LTDR/MTDR were 405 nm and 633 nm, respectively. The emis-\nprocedure85 and presented as log Po/w values. Log Po/w is defined sion was collected at 520 \u00b1 20 nm for Ir-3, 570 \u00b1 20 nm for Ir-1\nas the logarithmic ratio of the concentration of Ir-1\u20133 in n-octanol and Ir-2, and 665 \u00b1 20 nm for LTDR and MTDR.\nto that in the aqueous phase.\n Cellular uptake mechanism studies\nICP-MS measurement HeLa cells were incubated with the Ir(III) under different\nHeLa cells were seeded in 100 mm dishes and cultured overnight. conditions, such as different temperatures and pretreatment\nThe medium was replaced with a fresh medium containing Ir-1\u2013 with metabolic inhibitors or endocytic inhibitors. To investi-\n3 (20 \u03bcM). After 2.5 h incubation, the treated cells were washed gate the effect of temperature on cellular uptake, HeLa cells\nwith PBS, trypsinized, and collected. The cells were counted and were treated with Ir-1\u20133 (20 \u03bcM) for 2.5 h at 4\u00b0C or 37\u00b0C.\ndigested with 60% HNO3 for over 24 h. Intracellular iridium con- For inhibitors, HeLa cells were pretreated with CCCP (30 \u03bcM)\ntent was measured using the ELAN DRC II ICP-MS (USA). or chloroquine (50 \u03bc\u039c) for 1 h at 37\u00b0C, then exposed to Ir-\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\n 1\u20133 (20 \u03bcM) for 2.5 h at 37\u00b0C. After incubation, the cells\nIn vitro cytotoxicity assay were washed three times with PBS and visualized by confocal\n microscopy.\nThe cytotoxicity of complexes Ir-1\u20133 against HeLa, A549, A549R,\nHepG2, and LO2 cell lines was evaluated by MTT assay, and cis-\nplatin was included as a control. Briefly, exponentially grown cells\n AO staining\nwere seeded in 96-well plates and allowed to attach for 24 h. Cells After HeLa cells were seeded in confocal dishes and cultured\nwere treated with a series of concentrations of the tested com- overnight, the indicated concentrations of Ir-1\u20133 were added and\npounds for 44 h. Then, 20 \u03bcl MTT (5 mg/ml) was added to each incubated for 6 h. The cells of each dish were washed with PBS\nwell and incubated for another 4 h. The medium was removed, and incubated with AO (5 \u03bcM) for 15 min. After incubation, the\nand the formazan products were dissolved in DMSO (150 \u03bcl/well) cells were washed again with PBS to remove the excess probe\nand shaken for 10 min. The cell viability was measured at 595 nm and visualized by confocal microscopy. Emission was collected at\nusing a SpetraMax M2 plate reader. 510 \u00b1 20 nm (green) and 625 \u00b1 20 nm (red) upon excitation at\n 488 nm.\nWound healing assay\nExponentially grown HeLa cells were seeded in 12-well plates. Af- Detection of cathepsin B release\nter the cells reached \u223c80\u201390% confluence, the cross lines on the Cathepsin B activity was monitored using the fluorogenic sub-\ncenter of each well were carefully made by a 200 \u03bcl sterile pipette strate Magic Red MR-(RR)2 according to the manufacturer\u2019s in-\ntip. The scratched cells were washed away by PBS. Then, the cells structions. Briefly, HeLa cells were seeded in confocal dishes and\nwere cultured in a serum-free culture medium containing the in- cultured overnight, then treated with the indicated concentra-\ndicated concentrations of Ir-1\u20133. The wound closure was moni- tions of Ir-1\u20133 for 8 h. Upon completion, the fluorogenic substrate\ntored and photographed by an inverted microscope at 0, 24, and Magic Red MR-(RR)2 was added and cultured for another 1 h. The\n36 h. media was removed, and the cells were rinsed twice with PBS be-\n fore confocal imaging (\u03bbex = 543 nm, \u03bbem = 630 \u00b1 20 nm).\nColony forming assay\nHeLa cells were placed at a density of 600 cells per well in six- Hoechst 33 342 staining assay\nwell plates. After 24 h, the medium was removed and replaced HeLa cells were seeded in confocal dishes and cultured overnight.\nwith a fresh medium containing the indicated concentrations of After treated with different concentrations of Ir-1\u20133 for 24 h,\nIr-1\u20133 for another 24 h. Then, the medium containing the tested the cells were washed twice with PBS carefully before fixed with\ncomplexes was removed again, and the cells were maintained in 4% paraformaldehyde, and then stained with Hoechst 33342 (5\nthe fresh medium for 1 week. Cell colonies were fixed with 4% \u03bcg/ml). Eventually, the morphology of the cell nuclei was visu-\nparaformaldehyde for 15 min and stained with 0.005% crystal vi- alized by a confocal microscope (\u03bbex = 405 nm, \u03bbem = 460 \u00b1\nolet for 30 min. The images of colonies were recorded with a digital 20 nm).\ncamera.\n Annexin V/PI staining\nCell cycle analysis Analysis was performed according to the manufacturer\u2019s instruc-\nHeLa cells seeded in six-well plates were treated with Ir-1\u20133 for tions. HeLa cells were seeded in six-well plates and cultured\n24 h. The cells were harvested and fixed with 1 ml pre-cooled 70% overnight. After treated with the indicated concentrations of Ir-1\u2013\naqueous ethanol. After storage at 4\u00b0C for 24 h, the cells were cen- 3 for 24 h, cells were harvested, re-suspended in 195 \u03bcl Annexin-\ntrifuged at 800 g for 15 min and washed twice with pre-cooled binding buffer, then labeled with 5 \u03bcl Annexin V and 10 \u03bcl PI for\nPBS. DNA staining was achieved by re-suspending the immobi- 15 min in the dark. Subsequently, the samples were analyzed by\nlized cells in PBS containing PI (50 \u03bcg/ml) and RNAse (100 \u03bcg/ml) flow cytometry [\u03bbex = 488 nm, \u03bbem = 530 \u00b1 20 nm (for annexin V)\nand then examined using flow cytometry. and 620 \u00b1 20 nm (for PI)].\n\n\nIntracellular localization assay MMP assessment\nHeLa cells were incubated with Ir-1\u20133 (20 \u03bcM) at 37\u00b0C for 2.5 h and HeLa cells were seeded in six-well plates in a humidified atmo-\nfurther co-incubated with the commercial mitochondrial probe sphere and allowed to adhere overnight. The cells were treated\nMTDR (150 nM) or lysosomal probe LTDR (50 nM) for another 0.5 with the concentrations of 2 \u00d7 IC50 , 3 \u00d7 IC50 , and 4 \u00d7 IC50 of Ir-\nh. Cells were rinsed three times with PBS and visualized by confo- 1\u20133 for 12 h. After incubation, cells were harvested and stained\n\f12 | Metallomics\n\n\nwith JC-1 (5 \u03bcg/ml) in the dark for 20 min, followed by washing Supplementary material\nwith a blank buffer. The cells were centrifuged for flow cytomet-\n Supplementary data are available at Metallomics online.\nric testing. The excitation was at 488 nm, and the dual emission\nwas collected at 530 \u00b1 20 nm for JC-1 monomer (green) and 585 \u00b1\n20 nm for JC-1 aggregates (red). Acknowledgements\n This work was supported by the National Natural Science Foun-\nMeasurement of intracellular ROS dation of China (21967014, 22007042), Applied Basic Research\nHeLa cells were seeded in confocal dishes and cultured overnight. Projects of Yunnan Province (202001AT070036), the Innovative\nAfter treated with Ir-1\u20133 at the indicated concentrations for 6 h, Team of Yunnan Province (2019HC018), and High-level Scientific\nthe cells were incubated with H2 DCFDA (10 \u03bcM) for 20 min at 37\u00b0C Research Foundation for Talent Introduction of Kunming Univer-\nin the dark. Then, the cells were washed twice with serum-free sity of Science and Technology (KKKP201826008).\nDMEM to remove the excess probe. The fluorescence intensity of\nDCF in cells was immediately detected by confocal microscopy\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/14/9/mfac068/6694002 by guest on 12 May 2026\n(\u03bbex = 488 nm, \u03bbem = 530 \u00b1 20 nm). Conflicts of interest\n The authors declare no competing financial interests.\nEvaluation of antitumor activities in vivo\nMale BALB/c mice aged 4\u20135 weeks were bred in compliance with Data availability statements\nthe guidelines of the Institutional Animal Care and Use Commit-\n The data underlying this article are available in the article and in\ntee of the Kunming University of Science and Technology. All stud-\n its online supplementary material.\nies involving animals were approved by the Animal Ethics Com-\nmittee of Kunming University of Science and Technology. H22 cells\n(2 \u00d7 106 ) were suspended in 100 \u03bcl PBS, and the xenografts were References\nestablished by subcutaneous injection. When the tumor reached\n 1. L. Kelland, The resurgence of platinum-based cancer\n\u223c80\u2013120 mm3 , the mice were randomly assigned to two groups\n chemotherapy, Nat. Rev. Cancer, 2007, 7 (8), 573\u2013584.\n(n = 5) before the experiment. For the solvent control group, each\n 2. S. Medici, M. Peana, V. M. Nurchi, J. I. Lachowicz, G. Crisponi and\nmouse was injected with 100 \u03bcl PET diluent [6% poly(ethylene\n M. A. Zoroddu, Noble metals in medicine: latest advances, Coord.\nglycol) 400, 3% ethanol, 1% Tween 80, and 90% PBS]. For the ex-\n Chem. Rev., 2015, 284 (1), 329\u2013350.\nperimental groups, mice were injected intratumorally with Ir-1 (5\n 3. J. Karges, M. Tharaud and G. Gasser, Polymeric encapsulation of\nmg/kg, dissolved in 100 \u03bcl PET) every 3 days. Besides, tumor size\n a Ru(II)-based photosensitizer for folate-targeted photodynamic\nand body weight of mice were also recorded every 3 days. After 15\n therapy of drug resistant cancers, J. Med. Chem., 2021, 64 (8), 4612\u2013\ndays of treatment, the mice were sacrificed, and the tumors were\n 4622.\nseparated. The calculation formula of tumor volume (V) was as\n 4. K. C. Tong, C. N. Lok, P. K. Wan, D. Hu, Y. M. E. Fung, X. Y. Chang, S.\nfollows: V = ab2 \u00d7 0.5, where a and b were the longest and short-\n Huang, H. Jiang and C. M. Che, An anticancer gold(III)-activated\nest diameters of the tumor, respectively.\n porphyrin scaffold that covalently modifies protein cysteine thi-\n ols, Proc. Natl. Acad. Sci. U.S.A., 2020, 117 (3), 1321\u20131329.\nStatistical analysis 5. W. Y. Zhang, S. Banerjee, G. M. Hughes, H. E. Bridgewater, J. I. Song,\nAll biological experiments were performed at least twice with trip- B. G. Breeze, G. J. Clarkson, J. P. C. Coverdale, C. Sanchez-Cano, F.\nlicates in each experiment. The quantitative data were presented Ponte, E. Sicilia and P. J. Sadler, Ligand-centred redox activation\nas means \u00b1 SD. of inert organoiridium anticancer catalysts, Chem. Sci., 2020, 11\n (21), 5466\u20135480.\n 6. S. Kuang, F. Wei, J. Karges, L. Ke, K. Xiong, X. Liao, G. Gasser,\nConclusions L. Ji and H. 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