Mitochondria-targeted cyclometalated iridium (III) complexes: Dual induction of A549 cells apoptosis and autophagy.

{"full_text": " Journal of Inorganic Biochemistry 249 (2023) 112397\n\n\n Contents lists available at ScienceDirect\n\n\n Journal of Inorganic Biochemistry\n journal homepage: www.elsevier.com/locate/jinorgbio\n\n\n\n\nMitochondria-targeted cyclometalated iridium (III) complexes: Dual\ninduction of A549 cells apoptosis and autophagy\nLanmei Chen a, b, c, 1, Hong Tang a, b, c, 1, Weigang Chen a, Jie Wang a, Shenting Zhang a, b, c,\nJie Gao a, b, Yu Chen a, Xufeng Zhu b, c, *, Zunnan Huang a, b, c, *, Jincan Chen a, b, c, *\na\n Key Laboratory of Computer-Aided Drug Design of Dongguan City, Guangdong Key Laboratory for Research and Development of Natural Drugs, School of Pharmacy,\nGuangdong Medical University, Dongguan, Guangdong 523808, PR China\nb\n The Marine Biomedical Research Institute, Guangdong Medical University, Zhanjiang, Guangdong 524023, PR China\nc\n The Marine Biomedical Research Institute of Guangdong Zhanjiang, Zhanjiang, Guangdong 524023, PR China\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: In this study, we synthesized 4 cyclometalated iridium complexes using N-(1,10-phenanthrolin-5-yl)picolinamide\nIridium complexes (PPA) as the main ligand, denoted as [Ir(ppy)2PPA]PF6 (ppy = 2-phenylpyridine, Ir1), [Ir(bzq)2PPA]PF6 (bzq =\nMitochondria benzo[h]quinoline, Ir2), [Ir(dfppy)2PPA]PF6 (dfppy = 2-(3,5-difluorophenyl)pyridine, Ir3), and [Ir(thpy)2PPA]\nA549 cells\n PF6 (thpy = 2-(thiophene-2-yl)pyridine, Ir4). Compared to cisplatin and oxaliplatin, all four complexes exhibited\nApoptosis\nAutophagy\n significant anti-tumor activity. Among them, Ir2 demonstrated higher cytotoxicity against A549 cells, with an\n IC50 value of 1.6 \u00b1 0.2 \u03bcM. The experimental results indicated that Ir2 primarily localized in the mitochondria,\n inducing a large amount of reactive oxygen species (ROS) generation, that decreased in mitochondrial membrane\n potential (MMP), reduced ATP production, and further impaired mitochondrial function, leading to cytochrome c\n release. Additionally, Ir2 caused cell cycle arrest at the S phase and induced apoptosis through the AKT-mediated\n signaling pathway. Further investigations revealed that Ir2 could simultaneously induce both apoptosis and\n autophagy in A549 cells, with the latter acting as a non-protective mechanism that promoted cell death. More\n importantly, Ir2 exhibited low toxicity to both normal LO2 cells in vitro and zebrafish embryos in vivo.\n Consequently, these newly developed Ir(III) complexes show great potential in the development of novel and\n low-toxicity anticancer agents.\n\n\n\n\n1. Introduction exceeds the sum of breast cancer, prostate cancer, and colorectal cancer\n [4]. The treatment of NSCLC faces significant challenges, as many late-\n Cancer is recognized as one of the leading causes of death worldwide stage lung cancer patients are unable to tolerate the side effects of\n[1]. Among these cancers, lung cancer accounts for 11.6% of the total chemotherapy [5]. Therefore, there is an urgent need to explore a\ndiagnosed cases of cancer globally [2]. Lung cancer is classified into treatment approach that is both low in side effects and highly efficient.\nsmall-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), Some metal-based complexes, such as iridium, ruthenium, rhenium,\nwith the latter comprising nearly 85% of cases [3]. According to relevant gold, and platinum, exhibit certain roles in cancer treatment. These\nliterature, the number of cancer-related deaths caused by lung cancer metal complexes target the cell nucleus, mitochondria, and endoplasmic\n\n\n Abbreviations: PPA, N-(1,10-phenanthrolin-5-yl)picolinamide; PBS, phosphate buffered saline; ppy, 2-phenylpyridine; bzq, benzo[h]quinoline; dfppy, 2-(3,5-\ndifluorophenyl)pyridine; thpy, 2-(thiophen-2-yl)pyridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ICP-MS, Inductively Coupled Plasma\nMass Spectrometry; ROS, reactive oxygen species; ESI-MS, electrospray ionization mass spectrometry; FBS, fetal bovine serum; MMP, mitochondrial membrane\npotential; JC-1, 5,5\u20326,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-tetraethylimidacarbocyanine iodide; DCFH-DA, 2,7-dichlorodi-hydroflfluorescein diacetate; DMEM, dulbecco's modified\neagle medium; ATP, Adenosine Triphosphate; SCLC, small-cell lung cancer; NSCLC, non-small-cell lung cancer; PA, Pyridinic acid; NO, nitric oxide; DMSO, dimethyl\nsulphoxide; HOBT, hydroxybenzotriazole; DIEA, N, N-diisopropylethylamine; EDCI, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; LTR, Lyso Tracker Red; MTR,\nMito Tracker Red; CDKs, cyclin-dependent kinases; MDC, Monodansylcadaverine; AO, Anthracene Orange; AVOAs, acidic vacuolar organelles; TEM, transmission\nelectron microscopy; PS, Phosphatidylserine; UV\u2013vis, ultraviolet visible; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis..\n * Corresponding authors.\n E-mail addresses: xufeng910730@126.com (X. Zhu), zn_huang@gdmu.edu.cn (Z. Huang), chenjc@gdmu.edu.cn (J. Chen).\n 1\n These authors contributed equally to this work.\n\nhttps://doi.org/10.1016/j.jinorgbio.2023.112397\nReceived 11 August 2023; Received in revised form 1 October 2023; Accepted 6 October 2023\nAvailable online 14 October 2023\n0162-0134/\u00a9 2023 Elsevier Inc. All rights reserved.\n\fL. Chen et al. Journal of Inorganic Biochemistry 249 (2023) 112397\n\n\nreticulum [6\u201310]. As is well known, the exceptional properties of mol\u00ad design and synthesize four cyclometalated iridium complexes, [Ir\necules originate from their unique structural characteristics [11]. (ppy)2PPA]PF6 (Ir1), [Ir(bzq)2PPA]PF6 (Ir2), [Ir(dfppy)2PPA]PF6 (Ir3),\nCompared to organic compounds, metal-based complexes possess and [Ir(thpy)2PPA]PF6 (Ir4), and their structures were characterized. To\ngreater flexibility in structure, making it easier to introduce other mo\u00ad elucidate their antitumor mechanism, we investigated the cellular up\u00ad\nlecular functional groups on ligands for specific structural modifications take, subcellular localization, impact on mitochondria and reactive ox\u00ad\nin different substrate binding environments. Additionally, metal-based ygen species, anticancer activities, and in vivo safety of these iridium\ncomplexes are relatively stable, allowing for efficient drug effects complexes. The results demonstrated that all synthesized iridium com\u00ad\nwithin the body [12]. Compared to platinum-based complexes, iridium plexes Ir1-Ir4 exhibited significant in vitro activity against tested tumor\ncomplexes exhibit higher tumor cell selectivity with lesser toxicity to cell lines. ICP-MS and laser scanning confocal microscope analysis\nnormal cells [13,14]. In recent years, cyclometalated iridium(III) com\u00ad revealed that all 4 complexes were localized in the mitochondria.\nplexes have aroused significant interest among researchers due to their Moreover, complex Ir2 induced mitochondrial dysfunction, cytochrome\nexcellent photophysical, photochemical properties, and antitumor c release, cell cycle arrest at the S phase, and further triggered apoptosis\nmechanisms [15\u201319]. and autophagy. Importantly, the 4 complexes showed certain safe con\u00ad\n Previously, our group synthesized a series of metal-based ruthenium centrations in vivo, as the zebrafish embryos exhibited a hatching rate\nand iridium complexes, which primarily targeted the cell mitochondria and survival rate over 80% at 96 h when the concentrations of Ir1-Ir4\n[19\u201321]. Mitochondria, as the energy supplier of cells, are double- were below this threshold.\nmembraned organelles in the cytoplasm of eukaryotic cells, serving as\nthe main oxygen-consuming powerhouses [22]. Mitochondria play 2. Results and discussion\ncrucial roles in various physiological processes, apart from providing\nenergy to cells. They are involved in processes such as cell differentia\u00ad 2.1. Synthesis and characterization\ntion, cell signaling, and cell apoptosis, and possess the ability to regulate\ncell growth and the cell cycle. Damage or defects in mitochondria can The synthetic routes for Ir1-Ir4 are depicted in Scheme S1 (Sup\u00ad\nlead to diseases, such as neurodegenerative disorders, metabolic dis\u00ad porting information text). Starting with 2-pyridinic acid and 1,10-phe\u00ad\neases, and cancer [23]. Therefore, mitochondria are considered poten\u00ad nanthrolin-5-amine as raw materials, hydroxybenzotriazole (HOBT),\ntial targets for many chemotherapy drugs. N, N-diisopropylethylenediamine (DIEA), and 1-ethyl-(3-dimethylami\u00ad\n Pyridinic acid (PA) is a natural catabolic product of L-tryptophan nopropyl)carbodiimide hydrochloride (EDCI) were added. The reac\u00ad\nmetabolism synthesized by pyridoxine decarboxylase from 2-amino-3- tion was refluxed at 298 K for 24 h, resulting in the formation of the\ncarboxy aldehyde [24]. PA exhibits various biological activities, such main ligand PPA. The iridium complexes precursor, Ir(C^N)2Cl2, was\nas activating murine macrophages, inducing nitric oxide synthase and obtained by reacting hydrated iridium trichloride, IrCl3\u22c53H2O, with the\nnitric oxide (NO) production, inducing interleukin-8 (IL-8) production, corresponding C^N ligands (ppy, dfppy, bzq, and thpy). The iridium\nand demonstrating anticancer effects [25,26]. PA or its derivatives can complexes Ir1-Ir4 were directly synthesized by connecting the iridium\ndirectly coordinate with metal centers, and such complexes possess precursors with the main ligand according to the reported literature\ncertain antitumor, antimicrobial, or topoisomerase inhibitory activities. methods [30]. The chemical structures of iridium complexes Ir1-Ir4 are\nFor instance, Liu et al. linked PA derivatives with metal iridium, shown in Fig. 1A.\nresulting in complexes with significant antitumor activity, exhibiting a The main ligand, PPA, and complexes Ir1-Ir4 were characterized by\n5-fold increase in IC50 value against A549 cells compared to cisplatin elemental analysis, ESI-MS (Fig. S1-S4), and 1H NMR (Fig. S5-S9). In the\n[27]. Chew-Hee Ng et al. designed and synthesized metal-based zinc-PA 1\n H NMR spectrum of PPA, a distinct peak at 11.12 ppm corresponding to\ncomplexes with topoisomerase I inhibitory activity [28]. Amanda Lee E. the amide bond proton (N\u2013H, N15) was observed (Fig. S5), indicating\nManicum et al. connected pyridinic acid and its fluorinated derivatives the successful condensation of the amide bond. In the ESI-MS spectra of\nwith metal rhenium, synthesizing complexes that demonstrated prom\u00ad Ir1-Ir4, ion peaks at 801.12 (Ir1), 849.11 (Ir2), 871.20 (Ir3), and 811.12\nising cytotoxicity against HeLa and A549 cells [29]. (Ir4) [M-PF6]+ were detected, matching the theoretical values, con\u00ad\n Based on this, in this study, we modified PA and obtained N-(1,10- firming the successful synthesis of the corresponding iridium complexes\nphenanthrolin-5-yl)picolinamide (PPA) through condensation reaction (Fig. S1-S4). Furthermore, upon coordination with iridium metal ions,\nwith 5-amino-1,10-phenanthroline, and used it as the main ligand to the 1H NMR signals of the CH protons (Ar\u2013H, C7, C8) adjacent to the N\n atoms in the main ligand PPA were affected by changes in the electronic\n cloud density upon ligand coordination with the metal atom. During the\nTable 1\n formation of Ir1-Ir4, the 1H NMR signals of PPA's CH protons (Ar\u2013H, C7,\nIC50 values of tested compounds towards different cell lines.\n C8) experienced a noticeable shift from 9.16 ppm and 9.09 ppm towards\n Complex IC50a (\u03bcM) higher magnetic fields. As a result, the corresponding signals around\n A549 HeLa HepG2 MCF-7 LO2 SIb 9.16 ppm and 9.09 ppm were not observed in the 1H NMR spectra of Ir1-\n 60.6 \u00b1 71.1 \u00b1 68.2 \u00b1 75.5 \u00b1 80.5 \u00b1 Ir4. Additionally, after coordination with iridium metal ions, the other\n PPA 1.33\n 0.2 0.5 0.7 0.3 0.9 proton signals in the PPA moiety did not show significant changes. For\n Ir1\n 3.3 \u00b1 3.6 \u00b1\n 4.9 \u00b1 0.6\n 3.7 \u00b1 13.7 \u00b1\n 4.15 instance, the signal of the CH proton (Ar\u2013H, C20) adjacent to the N\n 0.5 0.4 0.6 0.9 atom remained nearly the same in the 1H NMR spectra of Ir1-Ir4, located\n 1.6 \u00b1 1.8 \u00b1 4.1 \u00b1 12.2 \u00b1\n Ir2\n 0.2 0.3\n 3.4 \u00b1 0.6\n 0.4 0.6\n 7.63 around 8.84 ppm. Moreover, in the 1H NMR spectra of Ir1-Ir4, clear\n 5.2 \u00b1 6.3 \u00b1 5.9 \u00b1 16.9 \u00b1 signals at 11.46 ppm (Ir1), 11.47 ppm (Ir2), 11.48 ppm (Ir3), and 11.47\n Ir3 7.8 \u00b1 0.2 3.25\n 0.2 0.4 0.7 0.7 ppm (Ir4) corresponding to the amide bond protons were observed\n 4.3 \u00b1 5.1 \u00b1 6.6 \u00b1 16.4 \u00b1 (Fig. S6-S9), further confirming the successful coordination of the main\n Ir4 4.7 \u00b1 0.5 3.81\n 0.4 0.2 0.6 0.9\n ligand.\n 18.6 \u00b1 21.8 \u00b1 20.4 \u00b1 13.5 \u00b1 17.8 \u00b1\n Cisplatin\n 1.6 2.1 1.5 1.1 1.2\n 0.96 Fig. 1B displays the UV\u2013Vis absorption spectra of Ir1-Ir4 in CH3CN\n 13.5 \u00b1 11.8 \u00b1 17.9 \u00b1 13.1 \u00b1 15.7 \u00b1 solution at 298 K. Three distinct absorption bands are observed within\n Oxaliplatin 1.16\n 0.9 1.2 1.3 1.1 0.9 the range of 200\u2013600 nm. Ir1-Ir4 exhibit prominent absorptions at 265\n a\n IC50 values are drug concentrations necessary for 50% inhibition of cell nm, 261 nm, 250 nm, and 275 nm, while the absorption peaks at 331 nm\nviability. Data are presented as mean \u00b1 standard deviation and cell viability was and 419 nm are relatively broad [31].\nassessed after 48 h incubation. The absorption spectra in the CH2Cl2 solution and PBS buffer (pH =\n b\n SI (selectivity index) = IC50 (LO2)/IC50 (A549). 7.4) also exhibited similar results (Fig. 1C, D). Furthermore, in the\n\n 3\n\fL. Chen et al. Journal of Inorganic Biochemistry 249 (2023) 112397\n\n\n\n\nFig. 2. Cellular uptake and distribution of Ir1-Ir4 in A549 cells. (A) Cellular Ir contents were determined in A549 cells after 24 h incubated with 1, 2, and 4 \u03bcM Ir1-\nIr4, respectively. (B) Cellular Ir contents were determined in A549 cells incubated with 2 \u03bcM Ir2 in 3 h. (C) LogPO/W values of Ir1-Ir4. (D) Subcellular distribution of Ir\ncontent in A549 cell after incubated with 1, 2, 4 \u03bcM Ir1-Ir4 for 24 h CLSM images of A549 cells co-labeled with Ir complexes (2 \u03bcM, 3 h), and (E) Lyso Tracker Red\n(LTR; 50 nM, 30 min). \u03bbex = 561 nm, \u03bbem = 617 \u00b1 36 nm (LTR), (F) Mito Tracker Red (MTR; 150 nM, 30 min). \u03bbex = 561 nm, \u03bbem = 617 \u00b1 36 nm (MTR) and \u03bbex =\n405 nm, \u03bbem = 560 \u00b1 40 nm (Ir complexes); All images share the same scale bar: 10 \u03bcm. (For interpretation of the references to colour in this figure legend, the reader\nis referred to the web version of this article.)\n\n\nfluorescence spectroscopic measurements, complexes Ir1-Ir4 showed 2.2. In vitro antiproliferative activity\ndistinct emission peaks upon excitation at 405 nm, while emission peaks\nwere negligible when excited at 488 nm (Fig. S10), consistent with the To evaluate the in vitro cytotoxicity of the synthesized iridium\nconfocal microscopy analysis results (Fig. S11). As shown in Fig. 1E, F, complexes, we tested them against four tumor cell lines (A549, Hela,\nG, the emission wavelength of Ir3 under 405 nm excitation was 520 \u00b1 HepG2, and MCF-7) and one normal cell line (LO2). Cisplatin and oxa\u00ad\n20 nm, while both Ir1 and Ir2 exhibited red-shifted emission peaks at liplatin were used as control compounds. The MTT assay was employed\n560 \u00b1 20 nm. Notably, compared to Ir1-Ir3, complex Ir4 demonstrated for screening, and the results are shown in Table 1. All iridium com\u00ad\nthe largest redshift in emission wavelength, located at 600 \u00b1 20 nm. plexes exhibited significant toxicity against the selected tumor cell lines,\nFinally, we assessed the stability of complexes Ir1-Ir4. As shown in with the cytotoxicity ranking as follows: Ir2 > Ir1 > Ir4 > Ir3. Particu\u00ad\nFig. S12, after incubation in PBS buffer for 72 h, the UV\u2013Vis spectra of larly, Ir2 demonstrated the highest cytotoxicity against A549 cells, with\nthe complexes remained almost unchanged, indicating good stability of an IC50 value of 1.6 \u00b1 0.2 \u03bcM, which was 11-fold higher than that of\nthese complexes in PBS buffer. cisplatin and 8-fold higher than that of oxaliplatin. Notably, the\n\n\n 4\n\fL. Chen et al. Journal of Inorganic Biochemistry 249 (2023) 112397\n\n\n\n\nFig. 3. (A) The ATP levels in A549 cell after incubation with 1, 2, 4 \u03bcM Ir2 for 12 h. CLSM images of (B) JC-1 stained and (C) ROS stained A549 cells after treatment\nwith Ir2 (1,2,4 \u03bcM) for 12 h. scale bar: 100 \u03bcm. (D) Western blotting analysis of Cyto-c (Cyt.)and Cyto-c (Mit.) protein in A549 cells treated with Ir2 (1,2,4 \u03bcM) for 12\nh. GAPDH was used as an internal control. DCF, \u03bbex = 488 nm, \u03bbem = 525 \u00b1 25 nm; JC-1 aggregate, \u03bbex = 585 nm, \u03bbem = 595 \u00b1 20 nm; JC-1 monomer, \u03bbex = 488 nm,\n\u03bbem = 525 \u00b1 25 nm. Scale bar: 100 \u03bcm. The data are presented as mean \u00b1 standard deviation (SD) with ***P < 0.001.\n\n\nantiproliferative activity of the 4 complexes against the normal cell line increased with longer incubation time, indicating an accumulation of\nLO2 was lower compared to the other tumor cells, with a selectivity iridium within the cells.\nindex of 4.15, 7.63, 3.25, and 3.81, all higher than those of cisplatin The lipophilic (log P value) is used to evaluate pharmacokinetic\n(0.96) and oxaliplatin (1.16), indicating Ir1-Ir4 have certain selectivity parameters related to drug action, such as lipophilicity, cellular uptake,\ntowards human cancer cells. metabolism, and bioavailability [32]. In order to assess the lipophilicity\n of the iridium complexes, we determined the water-octanol partition\n2.3. Cellular uptake and subcellular localization coefficient (P) using a conventional shake-flask technique. Log P values\n \u2264 \u2212 4 indicate poor lipid bilayer permeability, while values \u22654 indicate\n The cellular uptake level of complexes is correlated with their poor water solubility [33]. As shown in Fig. 2C, the oil-water distribu\u00ad\ncytotoxicity, generally, the more uptake, the stronger the cytotoxicity tion coefficients of the four iridium complexes were all above 0.5, falling\n[31]. To elucidate the relationship between cellular uptake and cyto\u00ad within the ideal lipophilicity range. The Log P values of Ir1-Ir4 were\ntoxicity, we employed ICP-MS to measure the intracellular iridium 0.89, 1.02, 0.91, and 0.93, respectively, with Ir2 having the highest\ncontent. As shown in Fig. 2A, after incubating the iridium complexes lipophilicity of 1.02. Previous studies have shown that there is a rela\u00ad\nwith A549 cells for 24 h, the intracellular iridium content increased in a tionship between the lipophilicity of metal-based complexes and their\nconcentration-dependent manner. Among them, Ir2 exhibited the cytotoxicity, where higher lipophilicity tends to result in stronger\nhighest uptake among the four complexes, with a content of 522 \u00b1 12 ng cytotoxicity [34,35]. For some ruthenium [36] and platinum [37]\nof metal iridium per 106 cells at a concentration of 4 \u03bcM, followed by Ir1 complexes, increased lipophilicity can enhance cellular uptake, thereby\nand Ir4 with 415 \u00b1 12 ng, while Ir3 complex showed the least cellular leading to enhanced cytotoxicity. Among the four complexes synthe\u00ad\nuptake, with metal iridium content of 263 \u00b1 14 ng. These are consistent sized in this study, based on the high cellular uptake and lipophilicity of\nwith the results of cellular cytotoxicity in vitro. Furthermore, we Ir2, it exhibited the best cellular activity.\nobserved that the intracellular iridium content of Ir2 increased in a time- Cell organelles are \u201cminiature organs\u201d within the cytoplasm with\ndependent manner within 3 h of incubation with A549 cells (Fig. 2B). specific shapes, structures, and functions. Different organelles have\nSimilar results were observed by using confocal microscopy (Fig. S13), specific functions and work together harmoniously to carry out essential\nwhere the yellow fluorescence intensity of Ir2 in cells gradually physiological processes within the cell. Many antitumor agents can\n\n\n 5\n\fL. Chen et al. Journal of Inorganic Biochemistry 249 (2023) 112397\n\n\n\n\nFig. 4. (A) Distribution of cell cycle of A549 cells incubated with Ir2 (1,2,4 \u03bcM) for 12 h, the obtained data from flow cytometry was dealt with FlowJo. (B)The\nhistogram of the cell cycle of A549 cells was treated with Ir2 (1,2,4 \u03bcM) for 12 h. (C) Western blotting analysis of CDK2, Cyclin A protein in A549 cells treated with\nIr2 (1,2,4 \u03bcM) for 12 h. GAPDH was used as an internal control. (D) A549 cells were treated with various concentrations (1,2,4 \u03bcM) of Ir2 for 24 h, and DNA\nfragmentation was examined by comet assay. (E) Quantification of DNA tails in the comet assay. The length of DNA tails in microscopy images was quantified by\nImage J. The data are presented as mean \u00b1 standard deviation (SD) with ***P < 0.001.\n\n\ntarget specific subcellular organelles, accumulate at specific sites, (LTDR, 100 nM) were relatively low, at 0.38, 0.31, 0.34, and 0.27,\nenhance cytotoxic potency, and diminish toxic side effects [38,39]. In\u00ad respectively. These results indicate that Ir1-Ir4 mainly accumulate and\nvestigations have revealed that organometallic compounds can target localize in mitochondria, potentially inducing cell death through\norganelles (e.g., mitochondria, lysosomes, and the nucleus), leading to mitochondrial-mediated pathways. According to previous reports, a\ncellular dysfunction and subsequent organelle damage [40\u201342]. We series of cyclometalated iridium complexes with the same main ligand\nused ICP-MS to detect the distribution of iridium complexes in subcel\u00ad and only different ancillary ligands should have similar anticancer\nlular compartments. As shown in Fig. 2D, after incubating iridium mechanisms [43]. Since Ir1-Ir4 share the same main ligand (PPA) and\ncomplexes with A549 cells for 24 h, the content of metal iridium in exhibit similar activities against the selected tumor cell lines (Table 1),\nmitochondria increased in a concentration-dependent manner, with Ir2 all of them localize to mitochondria, in this work, we selected the most\nreaching 344 \u00b1 6.5 ng at a concentration of 4 \u03bcM, and the distribution of active compound, Ir2, and A549 cells for further investigation into the\nIr4 in mitochondria was the second highest, with an iridium content of anticancer mechanisms.\n290 \u00b1 12.3 ng. Owing to the intrinsic phosphorescence of Ir1-Ir4, their\nlocalization within cells can thus be investigated by confocal laser 2.4. Mitochondrial dysfunction\nscanning microscopy (CLSM), as shown in Fig. 2E, F. A549 cells were co-\nincubated with iridium complexes for 3 h, followed by incubation with Based on the above results, the iridium complexes Ir1-Ir4 were co-\nspecific fluorescent probes for mitochondria and lysosomes for 30 min. localized with mitochondria. Since mitochondria play a crucial role in\nThe Pearson coefficients for the co-localization of Ir1-Ir4 with mito\u00ad cellular energy production, a decrease in mitochondrial membrane po\u00ad\nchondria were 0.87, 0.93, 0.81, and 0.89, respectively, consistent with tential (MMP) can interfere with ATP production in the cell [44]. To\nthe ICP-MS data in Fig. 2D. Meanwhile, the co-localization coefficients verify this, the effect of Ir2 on intracellular ATP levels was measured\nof the complexes with the lysosome-specific dye LysoTracker Deep Red (Fig. 3A). After incubation of A549 cells with 1, 2, and 4 \u03bcM of Ir2 for 12\n\n 6\n\fL. Chen et al. Journal of Inorganic Biochemistry 249 (2023) 112397\n\n\n\n\nFig. 5. (A) CLSM images of MDC stained and (B) AO stained A549 cells after treatment with Ir2 (1,2,4 \u03bcM) for 12 h. MDC (\u03bbex = 488 nm, \u03bbem = 518 \u00b1 20 nm), AO\n(\u03bbex = 488 nm, \u03bbem = 510 \u00b1 20 nm (green); \u03bbem = 625 \u00b1 20 nm (red). scale bar: 50 \u03bcm. (C) TEM images of A549 cells co-labeled with Ir2 (2 \u03bcM, 12 h). (D) Western\nblotting analysis of LC3 protein in A549 cells treated with Ir2 (2 \u03bcM) and Rapamycin (1 \u03bcM) for 12 h. GAPDH was used as an internal control. (E) The histogram of\napoptosis inhibitors Z-VAD-fmk (20 \u03bcM) and autophagy inhibitors CQ (2.5 \u03bcM) on cell viability. The data are presented as mean \u00b1 standard deviation (SD) with **P\n< 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)\n\n\nh, intracellular ATP levels significantly decreased in a dose- and time- release of cytochrome c from mitochondria, which in turn induces cell\ndependent manner. Subsequently, the cell staining was performed apoptosis. Western blot analysis revealed an increase in cytochrome c\nusing the dye 5,5\u2032,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-tetraethylbenzimidazo\u00ad level in the cytoplasm and a decrease in mitochondria (Fig. 3D). This\nlylcarbocyanine iodide (JC-1), and changes in MMP were detected using further confirms that the Ir2 complex causes mitochondrial functional\nfluorescence microscopy. JC-1 is a dual-emission dye that exhibits damage.\nvoltage-dependent accumulation in mitochondria. It can exist as either a\ngreen fluorescence monomer in depolarized mitochondria or a red 2.5. Induction of cell cycle arrest by Ir2 in A549 cells\nfluorescence aggregate in polarized mitochondria, allowing the evalu\u00ad\nation of mitochondrial polarization status through the red/green fluo\u00ad One of the characteristics of tumors is the rapid and excessive growth\nrescence ratio [45]. As shown in Fig. 3B, A549 cells were treated with of malignant cells, primarily caused by dysregulation of cell cycle\nvarying concentrations of Ir2 for 12 h. Compared to the control group, regulation [48,49]. Interfering with cell cycle distribution is regard as\nan increase in Ir2 concentration resulted in observable changes from red one of the most common mechanisms of anti-cancer candidate com\u00ad\nto green fluorescence, indicating that Ir2 could induce a decrease in pounds [50]. Studies have shown that metal-based complexes can\nMMP. inhibit cell division by inducing cell cycle arrest, leading to the sup\u00ad\n The mitochondrial respiratory chain is a primary site for the gener\u00ad pression of tumor cell proliferation [51]. To investigate the effect of Ir2\nation of reactive oxygen species (ROS) in organisms [46]. There are on the cell cycle of A549 cells, flow cytometry analysis was performed.\nreports indicating that the increase of ROS is related to the change of As shown in Fig. 4A and B, compared to the control, the percentage of\nMMP [47]. To investigate the impact of Ir2 on intracellular ROS levels, cells in the S phase increased by 7.69% when the concentration of Ir2\ncell staining with 2\u2032,7\u2032-dichlorodihydrofluorescein diacetate (DCFH-DA) was increased from 1 \u00d7 IC50 to 4.0 \u00d7 IC50. This indicates that Ir2 disrupts\nwas performed. DCFH-DA is non-fluorescent but can transform into the cell cycle, arresting cells in the S phase. It is well known that the\nfluorescent DCF when exposed to intracellular ROS. As shown in Fig. 3C, progression of the cell cycle is regulated by the formation of a series of\ncompared to the control group, Ir2 promoted a concentration-dependent specific cyclin-dependent kinases (CDKs) - cyclin complexes. To explore\nincrease in the green fluorescence signal of DCF, indicating elevated the mechanism by which Ir2 induces cell cycle arrest, we used western\nintracellular ROS levels. Mitochondrial dysfunction can lead to the blotting to analyze the effect of Ir2 on the expression of CDK2 and Cyclin\n\n 7\n\fL. Chen et al. Journal of Inorganic Biochemistry 249 (2023) 112397\n\n\n\n\nFig. 6. (A) CLSM images of Annexin V stained A549 cells after treatment with Ir2 (2 \u03bcM) and cisplatin (25 \u03bcM) for 24 h. (Annexin V: \u03bbex = 488 nm; \u03bbem = 525 \u00b1 25\nnm). scale bar: 50 \u03bcm. (B) Western blotting analysis of apoptosis-related protein in A549 cells treated with Ir2 (1,2,4 \u03bcM) for 24 h. (C) Western blotting analysis of\nAKT and P-AKT protein in A549 cells treated with Ir2 (2 \u03bcM) and 3-MA for 24 h. (D) Western blotting analysis of apoptosis-related protein in A549 cells treated with\nLY294002 and Ir2 (2 \u03bcM) for 12 h. GAPDH was used as an internal control.\n\n\nA regulatory proteins. As shown in Fig. 4C, after treatment with Ir2, the 2.6. Induction of autophagy by Ir2 in A549 cells\nexpression of CDK2 and Cyclin A decreased in A549 cells. In summary,\nIr2 induces cell cycle arrest in the S phase by inhibiting the expression Autophagy serves as an adaptive reaction triggered during periods of\nlevels of CDK2 and Cyclin A. stress to uphold cellular energy balance and eliminate protein aggre\u00ad\n DNA damage can lead to inhibition of its replication, failure to repair gates as well as impaired organelles via the autolysosomal degradation\ndamaged DNA may induce cell death. Therefore, DNA damage is route [55]. Autophagy requires a large number of autophagy-related\nconsidered to be a marker of apoptosis [52,53]. Comet assay provides an proteins (Atgs) and functional complexes [56]. For example, Atg genes\nimage of the changes that have happened in the chromatin organization regulate the formation of autophagosomes by forming Atg12-Atg5 and\nin a single cell, which is regarded as a more effective and simple way to LC-II complexes. LC3 family members play a key role in the maturation\ndetect DNA fragmentation or DNA damage in a cell population [54]. of autophagosomes, which are the core organelles of autophagy. At the\nHence, we used comet assay to evaluate whether Ir2 could induce DNA beginning of autophagy, the C-terminal glycine of LC3-I modifies\ndamage. As shown in Fig. 4D, no tail appeared in the control group after phosphatidyl ethanolamine to form LC3-II, which is rapidly translocated\nelectrophoresis, but after A549 cells treated with Ir2 for 24 h, the tail of to the neonatal autophagosome in a point-like distribution. Therefore,\nthe comet gradually became longer as the concentration of the complex the expression level of LC3-II is commonly considered as a marker for\nincreased, indicating that severe DNA damage has occurred. The length detecting autophagy [57].To investigate whether Ir2 induces autophagy\nof comet tails was then quantified by Image J software and shown in in A549 cells, we used the specific fluorescent dye Mono\u00ad\nFig. 4E, when the concentration of the complex Ir2 reached 4 \u03bcM, the dansylcadaverine (MDC) to detect the formation of autophagic vacuoles.\nlength of the comet's tails was about 200 \u03bcm. These results suggested MDC is an acidotropic dye commonly used as a specific marker for\nthat Ir2 could induce DNA damage of A549 cells in a concentration- detecting autophagosome formation [58]. As shown in Fig. 5A, no sig\u00ad\ndependent manner. nificant green fluorescence puncta were observed in the control group,\n while bright green fluorescence was visible in A549 cells treated with Ir2\n for 12 h, and the MDC fluorescence intensity increased with the\n\n\n 8\n\fL. Chen et al. Journal of Inorganic Biochemistry 249 (2023) 112397\n\n\n\n\nFig. 7. Toxicity assessment of Ir2 in developing zebrafish embryos. (A) Toxicity of Ir2 to zebrafish embryos at various concentrations within 96 h on a 4 \u00d7 objective\nlens in the microscope. (B) Cumulative hatching rate and (C) Survival rate of zebrafish embryos in the presence/absence of Ir2 at various concentrations every 24 h.\n\n\nincreasing concentration of Ir2. These results suggest that Ir2 induces 2.7. Induction of apoptosis by Ir2 in A549 cells\nautophagy in A549 cells and promotes the formation of autophagic\nvacuoles. Phosphatidylserine (PS) externalization is commonly regarded as a\n Anthracene Orange (AO) is a specific dye that can penetrate intact hallmark of early apoptosis [62]. Therefore, Annexin V staining was\ncell membranes, emitting deep green fluorescence in the cell nucleus used to assess the externalization of PS as an indicator of apoptosis in\u00ad\nand cytoplasm, while displaying red fluorescence in acidic organelles duction by the complexes by confocal microscopy. As shown in Fig. 6A,\nsuch as acidic vacuolar organelles (AVOAs) and lysosomes [59]. We both cisplatin and iridium complexes exhibited significant apoptosis\nfurther used AO staining to confirm whether Ir2 can induce the forma\u00ad fluorescence signals in cells after 24 h of treatment. Additionally,\ntion of autophagic vacuoles in A549 cells. In Fig. 5B, the control group compared to control cells, most cells treated with cisplatin and Ir2 dis\u00ad\nshowed strong green fluorescence, while the experimental group played morphological features characteristic of apoptosis, such as\nexhibited significantly enhanced red fluorescence with increasing con\u00ad membrane blebbing and cell shrinkage. To further confirm the cell\ncentrations of Ir2, indicating that Ir2 can induce the formation of death, we co-treated cells with the apoptosis inhibitor Z-VAD-fmk in\nautophagic vacuoles in A549 cells in a concentration-dependent combination with Ir2 and measured cell viability using the MTT assay.\nmanner. Additionally, we further observed the formation of autopha\u00ad As shown in Fig. 5E, after 24 h of treatment with Ir2 (2 \u03bcM), cell viability\ngolysosomes using transmission electron microscopy (TEM). As shown in A549 cells was <60% compared to the control group. The apoptosis\nin Fig. 5C, the organelles and nuclear envelope in the control group were inhibitor alone did not significantly affect cell viability. However, co-\nintact, with distinct boundaries between organelles, and no apparent incubation of Z-VAD-fmk with Ir2 noticeably increased cell viability,\nfusion was observed. After treatment with Ir2 for 12 h, A549 cells indicating that the iridium complexes primarily induce cell death\nexhibited fusion between mitochondria and lysosomes, indicating the through both autophagy and apoptosis.\ngeneration of autophagolysosomes. To further elucidate the mechanism of apoptosis induced by the\n Furthermore, we examined the expression levels of autophagy- iridium complexes, we investigated the apoptotic signaling pathways.\nrelated proteins using a western blotting assay. As shown in Fig. 5D, Protein kinase B (AKT) regulates cell survival and proliferation, playing\nafter 12 h of treatment with Ir2, the conversion of LC3-I to LC3-II a crucial role in cell survival and apoptosis by modulating the expression\noccurred, and the expression of LC3-II increased compared to the con\u00ad of Bcl-2/Bax proteins [63,64]. Bcl-2/Bax proteins, located upstream of\ntrol group, consistent with the results of autophagy inducer rapamycin. mitochondria, play a critical role in governing the permeability of the\nDuring autophagy, p62 is a commonly used marker protein for mitochondrial membrane, and their overexpression can control the\nmeasuring autophagic flux [60]. Treatment of A549 cells with Ir2 for 12 activation of downstream Caspase 3 enzyme, thereby mediating cell\nh resulted in a decrease in the expression level of p62 protein, indicating death [65]. As shown in Fig. 6B and C, after 24 h of treatment with Ir2,\nthe formation of autophagosomes (Fig. 5D). Studies have shown that the apoptotic regulatory factor Cleaved caspase 3 and the pro-apoptotic\nautophagy is a double-edged sword: on one hand, it can inhibit cell proteins Bad and Bax were upregulated, while AKT, P-AKT, and the anti-\ndeath, while on the other hand, it can accelerate cell death [61].To apoptotic proteins Bcl-xl and Bcl-2 were downregulated in A549 cells,\nevaluate the relationship between cell autophagy and cell viability, we showing a concentration-dependent change. Based on these findings, we\nused MTT assay to measure the effect of Ir2 on cell viability in the further examined the role of AKT using western blotting assay. As shown\npresence of an autophagy inhibitor. As shown in Fig. 5E, co-incubation in Fig. 6D, pre-treatment with LY294002 (an AKT inhibitor) signifi\u00ad\nof the autophagy inhibitor CQ with Ir2 enhanced cell survival. These cantly reduced the expression of P-AKT and Bcl-2 protein. Therefore, we\nresults suggest that Ir2-induced autophagy in A549 cells is a non- hypothesize that Ir2 primarily induces apoptosis through an AKT-\nprotective mechanism that promotes cell death. mediated signaling pathway.\n\n\n 9\n\fL. Chen et al. Journal of Inorganic Biochemistry 249 (2023) 112397\n\n\n\n\n Fig. 8. The main mechanism of apoptosis and autophagy induced by Ir2 in A549 cells.\n\n\n2.8. Fish embryo acute toxicity test below 8 \u03bcM, indicating a certain level of in vivo safety. Compared to Ir2,\n the safe concentrations for zebrafish embryos of Ir1, Ir3, and Ir4 are 16,\n To further investigate the in vivo toxicity of the complexes, we uti\u00ad 32, and 16 \u03bcM, respectively.\nlized a zebrafish embryo model for in vivo safety assessment. Zebrafish\nembryos share a high degree of homology with mammals [66], undergo 3. Conclusion\nrapid post-fertilization development [67], possess small sizes, and\nexhibit optical transparency, making them widely used as a biological In this study, four metal-based iridium(III) complexes Ir1-Ir4 con\u00ad\nmodel for drug discovery and toxicological evaluation. Zebrafish em\u00ad taining a novel ligand PPA were synthesized and characterized using\nbryos were treated with different concentrations of Ir2, and the cumu\u00ad UV\u2013visible spectroscopy, fluorescence spectroscopy, 1H NMR, elemental\nlative hatching rate and survival rate of embryos were recorded and analysis, and mass spectrometry. In vitro, cell toxicity experiments\nevaluated every 24 h. The survival and hatching status of embryos were demonstrated that complexes Ir1-Ir4 exhibited significant anti-tumor\ncorrelated with the concentration of the complexes. proliferation capabilities, with a certain level of selectivity against\n As shown in Fig. 7, the control group exhibited a hatching rate of normal cells. Cell uptake experiments showed that Ir1-Ir4 could enter\n93% at 48 h, and nearly all embryos hatched into healthy fish at 72 h cells and located in mitochondria. Among them, Ir2 leads to a decrease\nwithout any death or deformities. After adding the Ir2 complex for 96 h, in MMP, a decrease in ATP production, and an increase in ROS pro\u00ad\nonly when the concentration of Ir2 was below 16 \u03bcM, the embryo sur\u00ad duction. Furthermore, the downregulation of cyclin A expression sug\u00ad\nvival rate was higher than 50%, and healthy fish were eventually gested the inhibition of cell proliferation during the S phase. Mechanism\nhatched. However, when the concentration of Ir2 reached 16 \u03bcM, the studies revealed that Ir2 induced cell apoptosis and autophagy by acti\u00ad\nembryo hatching was delayed, and the hatched fish showed morpho\u00ad vating the AKT-mediated signaling pathway and triggering Bcl-2 family\nlogical deformities with a lower survival rate. At a concentration of 32 activation (Fig. 8). Autophagy was identified as a non-protective\n\u03bcM Ir2, embryos had difficulty hatching, and a few hatched fish mechanism that promoted cell death. Additionally, in vivo, toxicity\nexhibited spinal deformities and did not survive. Ir1, Ir3, and Ir4 also experiments indicated that all four complexes exhibited certain safe\nshowed similar results (Fig. S14 - S16). These findings suggest that the concentrations in zebrafish embryos, where concentrations below this\ntoxicity of Ir2 on zebrafish embryos is relatively low at concentrations threshold resulted in embryo hatching and survival rates exceeding 80%\n\n\n 10\n\fL. Chen et al. Journal of Inorganic Biochemistry 249 (2023) 112397\n\n\nat 96 h. In summary, this series of iridium complexes demonstrated [11] R.N. Rao, R.L. Panchangam, V. Manickam, M.M. Balamurali, K. Chanda,\n ChemPlusChem 85 (8) (2020) 1800\u20131812.\nstrong anti-cancer activity and could serve as potential candidates for\n [12] J. Yellol, S.A. Perez, A. Buceta, G. Yellol, A. Donaire, P. Szumlas, P.J. Bednarski,\nanti-tumor metal-based drugs. This work contributes to a better under\u00ad G. Makhloufi, C. Janiak, A. Espinosa, J. Ruiz, J. Med. Chem. 58 (18) (2015)\nstanding of the anti-tumor mechanisms of iridium(III) complexes and 7310\u20137327.\nthe design and synthesis of novel metal-based iridium(III) complexes [13] Y. 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