Induction of ferroptosis of iridium(III) complexes localizing at the mitochondria and lysosome by photodynamic therapy.

{"full_text": " Journal of Inorganic Biochemistry 264 (2025) 112808\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\nInduction of ferroptosis of iridium(III) complexes localizing at the\nmitochondria and lysosome by photodynamic therapy\nYajie Niu a , Shuanghui Tang a , Jiongbang Li a , Chunxia Huang a , Yan Yang b,* , Lin Zhou a,\nYunjun Liu a,*, Xiandong Zeng a,*\na\n School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China\nb\n Department of Pharmacy, The Affiliated Guangdong Second Provincial General Hospital of Jinan University, Guangzhou 510317, 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, [Ir(ppy)2(DMHBT)](PF6) (ppy = deprotonated 1-phenylpyridine, DMHBT = 10,12-dimethylpter-\nIridium (III) metal complexes idino[6,7-f][1,10]phenanthroline-11,13-(10,12H)-dione, 8a), [Ir(bzq)2(DMHBT)](PF6) (bzq = deprotonated\nPhotosensitizer benzo[h]quinoline, 8b) and [Ir(piq)2(DMHBT)](PF6) (piq = deprotonated 1-phenylisoquinoline, 8c) were syn-\nApoptosis\n thesized and characterized by HRMS, 13C NMR and 1H NMR. In vitro cytotoxicity experiments showed that 8a,\nFerroptosis\n 8b, 8c show moderate cytotoxicity against B16 cells, while the cytotoxicity of the complexes 8a, 8b and 8c to-\nImmunogenic cell death\n ward B16 cells was greatly improved upon light irradiation, which can be used as photosensitizers to exert\n anticancer efficacy in photodynamic therapy (PDT). After being taken up by cells, 8a, 8b, 8c were localized in the\n mitochondria, resulting in a large amount of Ca2+ in-flux, a burst release of ROS, a sustained opening of mito-\n chondrial permeability transition pore, and a decrease of the mitochondrial membrane potential, which led to\n mitochondrial dysfunction and further activation of caspase 3 and Bcl-2 family proteins to induce apoptosis.\n Overloaded ROS reacted with polyunsaturated fatty acids on the cell membrane, and initiated lipid peroxidation,\n inhibited the x\u2212c -system-glutathione (GSH)-glutathione peroxidase 4 (GPX4) antioxidant defense system, and\n upregulated the expression of the damage-associated molecules, HMGB1, CRT, and HSP70. The presence of Fer-1\n was effective on increasing the cell survival, which demonstrates that the complexes possess the potential to\n induce ferroptosis and immunogenic cell death. In addition, 8a, 8b and 8c induced autophagy by inhibiting the\n AKT/PI3K/mTOR signaling pathway, downregulating p62 and promoting Beclin-1 expression upon light\n irradiation.\n\n\n\n\n Abbreviations: 8a, [Ir(ppy)2(DMHBT)](PF6); 8b, [Ir(bzq)2(DMHBT)](PF6); 8c, [Ir(piq)2(DMHBT)](PF6) 8a(light), 8b(light) and 8c(light) stands for the treatment of\nthe cells with complexes 8a, 8b and 8c for 4 h, then irradiation for 30 min (LED lamp, 460 nm, 7.03 J/cm2).; A549, human lung carcinoma; B16, mouse melanoma\ncells; AKT, protein kinase B; Bak, Bcl-2, homologous antagonist/killer; Bax, Bcl-2 associated x protein; Bcl-2, B-cell lymphoma-2; Beclin1, recombinant Beclin 1; bzq,\ndeprotonated benzo[h]quinoline; C11-BODIPY581/591, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bromo-3a,4a-diaza-s-indene-3-undecanedioic acid; calcein AM,\ncalcein acetoxymethyl ester; caspase 3, cysteinyl aspartate specific proteinase 3; CRT, calreticulin protein; DAMPs, damage-associated molecular patterns; DAPI, 4\u2032,6-\ndiamidino-2-phenylindole; DHR123, dihydrorhodamine 123; DCFH-DA, 2\u2032,7\u2032-dichlorodihydro-fluorescein diacetate; DHE, dihydroethidium; DMHBT, 10,12-dime-\nthylpteridino[6,7-f][1,10]phenanthroline-11,13-(10,12H)-dione; DMSO, dimethylsulfoxide; DPBF, 1,3-diphenylisobenzofuran; Fer-1, ferrostatin-1; GPX4, gluta-\nthione peroxidase 4; GSH, glutathione; HMGB1, high mobility group box-1; HOMO, highest occupied molecular orbital; HPF, hydroxyphenyl fluorescein; HPLC, high\nperformance liquid chromatography; HRMS, high resolution mass spectrometry; HSP70, heat shock protein 70; IC50, half-maximum inhibitory concentration; ICD,\nimmunogenic cell death; JC-1, 5,5\u2032,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-tetraethylbenzenecarboxamidinylcarbocyanine iodide; LC3, autophagy marker light chain 3; LDH, lactate\ndehydrogenase; LO2, human normal hepatocytes; logP, lipid-water partition coefficient; LUMO, lowest unoccupied molecular orbital; MDA, malondialdehyde; MDIP,\n2-(7-methoxybenzo[d][1,3]dioxol-5-yl)-1H-imidazo[4,5-f][1,10]phenanthroline; m-TOR, mammalian target of rapamycin; MTT, 3-(4,5-dimethylthiazole-2-yl)-\ndiphenyltetrazolium bromide; MMP, mitochondrial membrane potential; MPTP, mitochondrial membrane permeability transition pore; NMR, nuclear magnetic\nresonance; p62, polyubiquitin-binding protein; PARP, poly ADP-ribose polymerase; PBS, phosphate buffer saline; PDT, photodynamic therapy; PI3K, phosphati-\ndylinositol 3-kinase; piq, deprotonated 1-phenylisoquinoline; p-mTOR, phosphorylated mammalian target of rapamycin; PPD, pteridino[6,7-f][1,10]phenanthroline-\n11,13-diamine; ppy, deprotonated 1-phenylpyridine; PVDF, polyvinylidene difluoride; ROS, reactive oxygen species..\n * Corresponding authors.\n E-mail addresses: yany@gd2h.org.cn (Y. Yang), lyjche@gdpu.edu.cn (Y. Liu), zengxiandong@gdpu.edu.cn (X. Zeng).\n\nhttps://doi.org/10.1016/j.jinorgbio.2024.112808\nReceived 14 October 2024; Received in revised form 12 November 2024; Accepted 6 December 2024\nAvailable online 9 December 2024\n0162-0134/\u00a9 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n1. Introduction complex was entrapped into the liposome and exhibits very high cyto-\n toxic efficiency (IC50 = 4.1 \u00b1 0.2 \u03bcM) [11]. Chao et al. reported that the\n Melanoma is a highly metastatic tumor that can spread rapidly functionalized Ir(III) complexes selectively localize at the mitochondria\nthroughout the body, making it difficult for radiotherapy and chemo- and produce 1O2 and O\u2022- 2 upon two-photon irradiation to efficiently\ntherapy to effectively counteract the development of highly diffuse prevent the tumor growth [12]. Additionally, after irradiation, [Ir\nmetastases [1,2]. Photodynamic therapy (PDT) is a cancer treatment (C\u2227N)2(dppz)]+ caused leakage of lysosomal content into the cytoplasm\nmodality based on the interaction between photosensitizers, light, and and induced oncosis-like cell death in HeLa cells [13]. Most these\nmolecular oxygen, and the use of photoexcitation of photosensitizers to compounds are type II photosensitizers, namely, the photosensitizers\nproduce cytotoxic oxygen-related substances that promote tumor cell transfer its energy to molecular oxygen (3O2), resulting in population of\ndeath through necrosis or apoptosis [3,4]. Generally, the photosensi- singlet oxygen (1O2), 1O2 is reactive species able to kill cells [14]. As a\ntizers need a long triplet excited state lifetime to prompt electron photosensitizer in photodynamic therapy (PDT), we also uncovered that\ntransfer to produce oxygen radicals (type I pathway) or energy transfer the iridium(III) complexes can transfer 3O2 into 1O2 to promote cancer\nto generate singlet oxygen (1O2, type II pathway) [5\u20137]. In recent years, cell apoptosis in our lab [15\u201317].\nphotodynamic therapy (PDT) has been indicated as an effective means to To gain more insight into the anticancer activity, in this article, we\nimprove melanoma treatment [8,9]. It has been shown that PDT can designed and synthesized [Ir(ppy)2(DMHBT)](PF6) (ppy: 1-phenylpyri-\nachieve long-term tumor control through immune induction of tumor dine, DMHBT: 10,12-dimethylpteridino[6,7-f][1,10]phenanthroline-\ncells and enhancement of the ferroptosis pathway, respectively [9,10]. 11,13-(10,12H)-dione, 8a), [Ir(bzq)2(DMHBT)](PF6) (bzq: benzo[h]\nLiu et al. found that [Ir(bzq)2(PPD)](PF6) (PPD = pteridino[6,7-f][1,10] quinoline, 8b) and [Ir(piq)2(DMHBT)](PF6) (piq: 1-phenylisoquinoline,\nphenanthroline-11,13-diamine) shows no cytotoxic activity (IC50 > 100 8c). 8a and 8b display moderate or low cytotoxicity toward B16 and\n\u03bcM), upon light irradiation, the IC50 value is 18.0 \u00b1 1.6 \u03bcM, while the A549 cells. To improve the anticancer efficiency, the co-incubation of\n\n\n\n\n Scheme 1. Synthetic route of DMHBT, 8a, 8b and 8c.\n\n\nTable 1\nIC50 (\u03bcM) values of 8a, 8b and 8c toward the selected cancer cells for 48 h.\n complex B16 SI A549 SI LO2\n\n IC50dark IC50light IC50dark IC50light IC50dark IC50light\n\n 8a 25.2 \u00b1 2.3 8.2 \u00b1 0.2 \u200b 49.0 \u00b1 8.9 37.6 \u00b1 2.7 \u200b > 100 > 100\n 8b 11.4 \u00b1 1.2 6.5 \u00b1 0.2 5.3 37.6 \u00b1 4.6 16.8 \u00b1 3.1 2.0 47.1 \u00b1 2.8 34.2 \u00b1 5.4\n 8c 1.6 \u00b1 0.1 1.1 \u00b1 0.1 5.4 1.4 \u00b1 0.2 1.0 \u00b1 0.1 5.9 13.3 \u00b1 1.9 5.9 \u00b1 0.9\n cisplatin 18.3 \u00b1 1.4 \u2014 \u200b 6.3 \u00b1 0.5 \u2014 \u200b 18.2 \u00b1 2.2 \u2014\n\nSelectivity Index (SI): IC50 value of LO2 versus cancer cells upon light irradiation.\n\n 2\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n\n\nFig. 1. (a and b) The change in the absorbance of DPBF at 412 nm during 3 min light irradiation, [DPBF] = 20 \u03bcM, [Ir complexes] = 10 \u03bcM, (c and d) superoxide\nanion assay using DHR123 as a probe, [DHR123] = 10 \u03bcM, [Ir complexes] = 10 \u03bcM, (e and f) Energy gap between LUMO and HOMO orbitals or between S1 and T1.\n\n\n\n\n 3\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n\n\n Fig. 1. (continued).\n\n\ncancer cells with 8a, 8b and 8c for 4 h, the cells were irradiated for 30 2.1.2. Synthesis of [Ir(bzq)2(DMHBT)]PF6 (8b)\nmin (LED lamp, 460 nm, 7.03 J/cm2), we unexpectedly found that the The synthesis of 8b was similar to 8a, with the substitution of cis-[Ir\nanticancer efficiency of 8a [8a(light)], 8b [(8b(light)] and 8c [(8c (bzq)2Cl]2 (0.25 mmol, 0.30 g) [19] for cis-[Ir(ppy)2Cl]2. Yield: 87 %.\n(light)] on B16 cells was greatly enhanced. Thereafter, we explored the HRMS (CH3CN, Fig. S4, SI): Calcd for C44H28IrN8O2PF6: m/z = 893.2874\nanticancer effect and mechanism through apoptosis, ROS, mitochondrial ([M-PF6]+), found: m/z = 893.2935. 1H NMR (DMSO\u2011d6, 500 MHz,\nmembrane potential, autophagy and so on. The results demonstrate that Fig. S5, SI): \u03b4 9.72 (d, 1H, J = 8.0 Hz), 9.47 (d, 1H, J = 8.0 Hz), 8.54 (d,\n8a(light), 8b(light) and 8c(light) trigger cell demise by apoptosis, 2H, J = 8.0 Hz), 8.32 (dd, 1H, J = 1.0, J = 5.0 Hz), 8.27 (dd, 1H, J = 1.0,\nautophagy and ferroptosis. J = 5.0 Hz), 8.12\u20138.06 (m, 4H), 7.99 (d, 2H, J = 8.0 Hz), 7.89 (d, 2H, J =\n 8.5 Hz), 7.59 (d, 2H, J = 8.0 Hz), 7.49\u20137.45 (m, 2H), 7.23 (t, 2H, J = 7.5\n2. Experimental Hz), 6.30 (d, 2H, J = 7.5 Hz), 3.86 (s, 3H), 3.47 (s, 3H). 13C NMR\n (DMSO\u2011d6, 125 MHz, Fig. S6, SI): 172.51, 162.79, 159.57, 156.72,\n2.1. Synthesis and characterization 153.57, 152.24, 151.09, 150.41, 149.79, 148.40, 148.17, 146.79,\n 141.19, 140.77, 138.10, 136.56, 134.85, 134.64, 134.23, 130.80,\n2.1.1. Synthesis of [Ir(ppy)2(DMHBT)]PF6 (8a) 130.25, 130.22, 129.96, 129.53, 129.06, 128.98, 128.77, 127.17,\n Under argon, DMHBT (0.17 g, 0.5 mmol) [18] and Cis-[Ir(ppy)2Cl]2 124.71, 123.23, 123.19, 120.99, 36.25, 31.24.\n(0.27 g, 0.25 mmo1) [19] were reacted in 15 mL of methanol and 30 mL\ndichloromethane at a constant temperature of 40 \u25e6 C for 6 h, then 1.0 g of 2.1.3. Synthesis of [Ir(piq)2(DMHBT)]PF6 (8c)\nammonium hexafluorophosphate was added after the solution was The synthesis and purification of 8c was the same as 8a, with the\ncooled to room temperature and continuously stirred for 1.5 h. After substitution of cis-[Ir(piq)2Cl]2 (0.25 mmol, 0.32 g) [19] for cis-[Ir\nremoving the solvent in the filtrate, a yellow-brown solid was gained. (ppy)2Cl]2. Yield: 78 %. HRMS (CH3CN, Fig. S7, SI): Calcd. for\nNeutral alumina column chromatography was used to purify the crude C48H32IrN8O2PF6: m/z = 945.2280 ([M-PF6]+), found: m/z = 945.2111.\n 1\nproduct (CH2Cl2 and CH3COCH3 (5:1, v/v) as eluent), the bright yellow H NMR (DMSO\u2011d6, 500 MHz, Fig. S8, SI): \u03b4 9.75 (t, 1H, J = 5.0 Hz),\nband was gathered, the solvent was removed to give a bright yellow 9.52\u20139.50 (m, 1H), 9.02 (d, 2H, J = 9.0 Hz), 8.41 (d, 2H, J = 8.0 Hz),\nproduct. Yield: 80 %. HRMS (CH3CN, Fig. S1, SI): Calcd for C40H28Ir- 8.20 (d, 2H, J = 4.5 Hz), 8.15 (d, 2H, J = 4.0 Hz), 8.08\u20138.03 (m, 2H),\nN8O2PF6: m/z = 845.1964 ([M-PF6]+), found: m/z = 845.2012. 1H NMR 7.91\u20137.87 (m, 4H), 7.52\u20137.45 (m, 4H), 7.19 (t, 2H, J = 7.5 Hz),\n(DMSO\u2011d6, 500 MHz, Fig. S2, SI): \u03b4 9.73 (d, 1H, J = 8.0 Hz), 9.49 (d, 1H, 7.00\u20136.97 (m, 2H), 6.27 (t, 2H, J = 6.5 Hz), 3.86 (s, 3H), 3.48 (s, 3H).\n 13\nJ = 8.0 Hz), 8.34 (d, 1H, J = 4.5 Hz), 8.29 (d, 3H, J = 7 Hz), 8.23\u20138.16 C NMR (DMSO\u2011d6, 125 MHz, Fig. S9, SI): 168.26, 162.78, 159.54,\n(m, 2H), 7.97 (d, 2H, J = 8.0 Hz), 7.90 (t, 2H, J = 7.5 Hz), 7.61 (t, 2H, J 153.38, 153.35, 153.00, 151.66, 151.09, 149.80, 148.20, 147.79,\n= 5.5 Hz), 7.08 (t, 2H, J = 7.5 Hz), 7.04\u20137.00 (m, 2H), 6.97 (t, 2H, J = 145.84, 141.66, 141.61, 141.19, 137.05, 136.63, 134.86, 134.72,\n7.0 Hz), 6.28 (d, 2H, J = 7.5 Hz), 3.86 (s, 3H), 3.47 (s, 3H). 13C NMR 132.57, 132.12, 132.08, 131.16, 131.08, 130.83, 130.36, 129.87,\n(DMSO\u2011d6, 125 MHz, Fig. S3, SI): 178.30, 172.51, 167.25, 162.80, 129.63, 129.11, 128.82, 128.15, 126.94, 126.00, 122.98, 122.63, 36.26,\n159.55, 153.03, 151.70, 151.08, 150.00, 148.16, 147.95, 144.51, 31.24.\n141.14, 139.30, 136.55, 134.81, 134.61, 131.66, 130.78, 130.26,\n129.54, 129.09, 128.80, 125.60, 124.33, 122.99, 36.26, 31.25.\n\n\n\n 4\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n\n\nFig. 2. (a) The co-location of 8a, 8b and 8c in the mitochondria after an exposure of B16 cells to IC50 concentration for 4 h. (b) Intracellular Ca2+ content, (c) ROS\nlevels, (d) superoxide anion assay using DHE as a probe upon light irradiation, DHE fluorescence intensity, [DHE] = 5 \u03bcM, [Ir complexes] = 2 \u00d7 IC50 \u03bcM, (e) hydroxyl\nfree radical assay with HPF as a probe upon irradiation, HPF fluorescence intensity, [HPF] = 10 \u03bcM, [Ir complexes] = 2 x IC50 \u03bcM, (f) MPTP assay, (g) change of the\nmitochondrial membrane potential and (h) the ratio of red/green in B16 cells treated with IC50 concentration of 8a(light), 8b(light) and 8c(light) for 24 h. (For\ninterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)\n\n\n\n\n 5\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n\n\n Fig. 2. (continued).\n\n\n2.2. Determination of purity after 4 h, discarding the culture medium and 100 \u03bcL of DMSO was used\n to dissolve residual MTT, recording the absorbance at 490 nm.\n The purity of 8a, 8b and 8c was determined at 25 \u25e6 C via HPLC. The\nmobile phases A and B were H2O and CH3OH with a flow rate of 3 mL/\n 2.4. Intracellular lipid peroxidation\nmin. The purity of 8a, 8b and 8c was determined by different elution\nprograms of H2O:CH3OH of 7:93 for 8a, 5:95 for 8b, and 10:90 for 8c,\n C11-BODIPY581/591 is a fluoroboron fluorescent derivative with good\nwith a detected wavelength of 210 nm.\n photostability and low fluorescence artifacts for further determination\n of intracellular lipid peroxidation. In the qualitative experiments, B16\n cells (4 \u00d7 105 per well) were inoculated into 6-well plates for 24 h and\n2.3. In vitro toxicity assay\n exposed to 8a (8.2 \u03bcM), 8b (6.5 \u03bcM) and 8c (1.1 \u03bcM) for 4 h and irra-\n diated for 30 min (LED lamp, 460 nm, 7.03 J/cm2), and progressively\n The in vitro cytotoxicity of 8a, 8b and 8c on cancer cells was explored\n cultured for 24 h. The cells were washed and dyed with C11-BODIPY581/\nusing 3-(4,5-dimethylthiazole-2-yl)-diphenyltetrazolium bromide 591\n (2.5 \u03bcM) for 30 min at 37 \u25e6 C, ultimately, the cells were subsequently\n(MTT) [20]. The cells were homogeneously incubated in 96-well plates\n observed under a microscope.\n(2.5 x 105 cells per well) for 24 h and treated with a series of concen-\ntration gradient of 8a, 8b and 8c (1.56, 3.125, 6.25, 12.5, 25.0, 50.0,\n100 \u03bcM) for 4 h, then the cells were irradiated for 30 min (LED lamp, 2.5. Ferrostatin-1 (Fer-1) assay\n460 nm, 7.03 J/cm2) and continuously cultured for 24 h. The culture\nmedium was discarded, and the cells were treated with MTT (9:1, v/v), Cultured in 96-well plates (1 \u00d7 104 cells per well) for 24 h, B16 cells\n\n 6\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n\n\nFig. 3. Cell cycle distribution was detected after an exposure of B16 cells (a) to IC50 concentration of 8a (b), 8b (c), 8c (d), 8a(light, e), 8b(light, f) and 8c(light, g) for\n24 h.\n\n\nwere treated via the following eight groups: (I) blank group, (II) blank ppm for 8a, 172.51 ppm for 8b, 168.26 ppm for 8c are assigned to the\ngroup + Fer-1, (III) 8a(light), (IV) 8a(light) + Fer-1, (V) 8b(light), (VI) carboxyl groups, while 36.26 and 31.25 ppm for 8a, 36.25 and 31.24\n8b(light) + Fer-1, (VII) 8c(light), and (VIII) 8c(light) + Fer-1 for 4 h, the ppm for 8b and 36.26 and 31.24 ppm for 8c are allocated to the two\ncells were irradiated for 30 min (LED lamp, 460 nm, 7.03 J/cm2), and carbon atoms in the two -CH3 groups.\ncontinuously cultured for 24 h, then MTT was added into the cells and 8a, 8b and 8c display two or three peaks, 268 nm (\u03b5 = 48,900), 366\nthe cell viability was measured. nm (\u03b5 = 20,500) and 400 nm (\u03b5 = 12,500) for 8a, 262 nm (\u03b5 = 32,396),\n 359 nm (\u03b5 = 16,957) and 401 nm (\u03b5 = 12,504) for 8b, 289 nm (\u03b5 =\n2.6. Western blot analysis 31,066) and 384 nm (\u03b5 = 18,705) for 8c, respectively (Fig. S10, SI). 8a,\n 8b and 8c emit weak green fluorescence, with a maximum locating at\n Equal amounts of protein samples of the same concentration were 604, 603 and 614 nm (Fig. S11, SI).\nloaded into each lane of a sodium dodecyl sulfate-polyacrylamide gel The stability of 8a, 8b and 8c was measured and found that the peak\nand electrophoresed for 3 h (50 V, 400 mA). Gel proteins were trans- shapes during 72 h have no change, indicating that 8a, 8b and 8c are\nferred semi-dry onto PVDF membranes in an all-purpose protein transfer stable in PBS solution (Fig. S10, SI).\nsystem for 20 min, and the PVDF membranes loaded with protein bands The purity of 8a, 8b and 8c was examined by HPLC (Fig. S12, SI),\nwere closed with 0.5 % skimmed milk for 1 h. The membrane was only one main peak was found during 30 min, indicating that 8a, 8b and\nincubated with the primary antibody at 4 \u25e6 C in a refrigerator overnight. 8c are pure (purity >95 %).\nNext day, the primary antibody was removed, the cells were incubated\nwith the second antibody for 1 h. Finally, the protein bands were 3.2. Lipid-water partition coefficient (logP) and in vitro cytotoxicity\nobserved in FluorChem E. studies\n\n2.7. Data analysis Lipid-water partition coefficient (logP) is used to determine the\n concentration of a compound in the lipid phase and the aqueous phase\n All data were expressed using mean \u00b1 SD. And t-test was used to according to the literature [21]. The logP values are \u2212 0.19, 0.32 and \u2212\nevaluate the statistical significance. *P < 0.05 indicates a significant 0.49 for 8a, 8b and 8c, suggesting that 8a, 8b and 8c can enter cells.\ndifference. Based on the principle that succinate dehydrogenase in the mitochon-\n dria of living cells can reduce exogenous 3-(4,5-dimethylthiazole-2-yl)-\n3. Results and discussion diphenyltetrazolium bromide (MTT) to blue-violet crystalline formazan,\n whereas dead cells do not have such a function, we have used the MTT\n3.1. Synthesis and characterization colorimetric assay to examine the cytotoxicity of 8a, 8b, and 8c on B16\n and A549 cancer cells. As listed in the Table 1, 8a and 8b showed\n The ligand DMHBT was prepared with 1,10-phenanthroline-5,6- moderate or low cytotoxic effects on B16 and A549 in the dark, while 8c\ndione and 5,6-diamino-1,3-dimethyluridine in ethanol and refluxed for showed strong anti-tumor activity on B16 and A549 cells. To enhance\n8 h. The corresponding iridium(III) complexes 8a, 8b and 8c were ob- anticancer activity, after the treatment of cancer cells with the com-\ntained by the reaction of DMHBT with [Ir(ppy)2Cl]2, [Ir(bzq)2Cl]2 and plexes for 4 h and irradiation (LED lamp, 460 nm, 7.03 J/cm2) for 30\n[Ir(piq)2Cl]2 in CH3OH/CH2Cl2 (Scheme 1). In the 1H NMR spectra, 3.86 min, the anticancer efficiency was greatly enhanced, particularly, 8a, 8b\n(s,3H) and 3.47 (s, 3H) for 8a and 8b, 3.86 (s, 3H) and 3.48 (s, 3H) for 8c and 8c show high anticancer efficiency on B16 cells with low IC50 values\nare attributed to the protons in the two methyl groups (-CH3). 178.30 of 8.2 \u00b1 0.2, 6.5 \u00b1 0.2 and 1.1 \u00b1 0.1 \u03bcM, but these complexes show low\n\n 7\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n\n\n Fig. 4. The intracellular CRT (a), HSP70 (b) and HMGB1 (c) assays in B16 cells treated with IC50 concentrations of 8a(light), 8b(light) and 8c(light) for 24 h.\n\n\n\n\n 8\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n\n\n Fig. 4. (continued).\n\n\nselectivity index (SI) values (Table 1). The cytotoxic activity of 8a(light), calculated based on the following Eq. [30]:\n8b(light) and 8c(light) is higher than cisplatin, but lower than [Ir ( / ) ( / )\n(ppy)2(MDIP)](PF6) (IC50 = 0.7 \u00b1 0.3 \u03bcM) [16] and organo- \u03a6sample = \u03a6Ref \u00d7 Ksample KRef \u00d7 FRef Fsample .\niridium\u2013albumin bioconjugate Ir(III) complex Ir-HSA (human serum K is the slope, F is calibration factor of the absorbance, F = 1\u201310-OD\nalbumin) (IC50 = 1.1 \u00b1 0.3 \u03bcM) [22]. (OD is the absorbance of the photosensitizer at the light source wave-\n Comparing the cytotoxicity of 8a, 8b and 8c on B16 and A549 cells, length). The quantum yield \u03a6 was calculated to be 0.44, 0.55 and 0.61\nB16 cells were selected for the subsequent series of experiments. for 8a, 8b and 8c, respectively. These complexes show moderate quan-\n Photodynamic therapy (PDT) has been widely applied in cancer tum yield. Therefore, the complexes exhibit moderate\ntreatment due to its unique spatial selectivity and low toxicity, and it has photocytotoxicity.\nbeen reported that singlet oxygen (1O2) is the key to the death. The We also measured the generation of superoxide anion (O\u2022\u2013 2 ) using\nphotosensitizer is irradiated by a light source in the ground state to dihydrorhodamine 123 (DHR123) as a probe. The fluorescence intensity\nbecome an excited state, and in the excited state photosensitizer trans- increased by 22.04, 28.18 and 19.75 times for 8a, 8b and 8c (Fig. 1c and\nfers energy to the ground state oxygen to generate 1O2, which is capable d), which confirmed that 8a, 8b and 8c may produce O\u2022\u2013 2 . The quantum\nof damaging cells and causing cell death [23\u201327]. Light irradiation en- yield \u03a6 was measured to be 0.012 for 8a, 0.036 for 8b and 0.024 for 8c\nhances cytotoxic activity of 8a, 8b and 8c on B16 and A549 cells, this is ([Ru(bpy)3]2+ in PBS, 0.042) [31]. As a result, upon light irradiation, 8a,\nclosely to the generation of singlet oxygen (1O2) or superoxide anion 8b and 8c not only generate 1O2, but also produce O\u2022\u2013\n 1 2 , they may be used\n(O\u2022\u2013\n 2 ). Firstly, we investigated O2 and quantum yield \u03a6. 1,3-Diphenyli- as type I and II photosensitizer in photodynamic therapy.\nsobenzofuran (DPBF) is a singlet oxygen capture agent that can indi- The cytotoxic activity of 8a, 8b and 8c on B16 and A549 cells can be\nrectly detect 1O2. As shown in the Fig. 1a and b, during 5 min, a decrease explained according to the energy gap (\u0394E) between HOMO and LUMO\nof 48.7 % for 8a, 46.2 % for 8b, 50.3 % for 8c was discovered in the orbitals. Generally, the low \u0394E value corresponds to a high cytotoxicity.\nabsorbance of DPBF at 417 nm. Owing to the moderate change in the As depicted in Fig. 1e, in the LUMO orbitals, the electronic clouds\nabsorbance of DPBF, which indicated that 8a, 8b and 8c show a mod- distribute in the main ligand DMHBT, while the electronic cloud focus\nerate ability to generation of 1O2. While in our previous work [28], we on the ancillary ligands in the HOMO orbitals, the \u0394E values for 8a, 8b\ndiscovered that the iridium(III) complex [Ir(piq)2(DBDIP)](PF6) (DBDIP and 8c are 2.5565, 2.4545, 2.2983 eV, hence, the \u0394E values follow the\n= 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1H-imidazo[4,5-f][1,10] sequence of 8a > 8b > 8c, furthermore, the energy gaps between S1 and\nphenanthroline) containing dioxane ring shows very significant photo- T1 (Fig. 1f) are the same order as the \u0394E values, which indicates that the\ntoxicity, before and after illumination, the change in the absorbance of cytotoxicity follows the order of 8a < 8b < 8c, this is line with the\nDPBF reached 95.29 %, the IC50 values change from >100 to 1.7 \u00b1 0.1 cytotoxicity obtained from MTT experiments.\n\u03bcM and > 100 to 0.31 \u00b1 0.1 \u03bcM toward BEL-7402 and A549 cells,\nrespectively. Hence, the photocytotoxicity of 8a, 8b and 8c is lower than 3.3. Cellular uptake studies\nthat of [Ir(piq)2(DBDIP)](PF6).\n The quantum yield \u03a6 ([Ru(bpy)3]2+, 0.81, methanol) [29] was The iridium(III) metal complexes emit green fluorescence, and we\n\n 9\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n\n\nFig. 5. (a) Co-location of 8a, 8b and 8c in the lysosomes after the treatment of B16 cells for 4 h, (b) the proteins expression induced by a treatment of B16 cells with\nIC50 concentration of 8a(light), 8b(light) and 8c(light) for 24 h, (c) autophagy assay of B16 cells treated with 2 \u00d7 IC50 concentration of 8a(light), 8b(light) and 8c\n(light) for 8 h.\n\n\nutilized this property to observe the cellular uptake. The cell nuclei were to determine whether the complexes locate at the mitochondria. As\ndyed with DAPI, after the treatment of B16 cells with 2 \u00d7 IC50 concen- depicted in Fig. 2a, the overlap of the fluorescence from the mitochon-\ntration of 8a, 8b and 8c for 20 h, we observed the green fluorescence in dria (red) and the green fluorescence emitted by the complexes affirmed\nthe cells. The overlap of green fluorescence with the blue fluorescence that 8a, 8b and 8c distributed in the mitochondria.\nobtained from DAPI-stained cell nuclei indicated that 8a, 8b and 8c were Mitochondria are the core of energy metabolism and the hub of\nable to enter the cells and accumulate in the cell nuclei (Fig. S13, SI). calcium (Ca2+) signaling, although Ca2+ is a positive effector of energy\n production, overloaded Ca2+ leads to mitochondrial dysfunction and\n thus induces cell death [32]. Further studies have confirmed that Ca2+\n3.4. Co-localization at the lysosome, mitochondria, ROS and Ca2+\n endocytosis generates ROS, and overloaded Ca2+ generates excess ROS\ncontent detection\n which in turn causes the mitochondrial membrane permeability transi-\n tion pore (MPTP) to remain open and the mitochondrial membrane\n The mitochondrial pathway is one of the most important mecha-\n potential to decrease, thereby causing mitochondrial damage and ulti-\nnisms that cause apoptosis to occur, and ROS production is inextricably\n mately inducing the onset of apoptosis [33,34]. As observed from\nlinked to mitochondria. To determine the distribution of 8a, 8b and 8c\n Fig. 2b, after B16 cells were treated with 8a, 8b, 8c and irradiated for 30\ninside the living cells, MitoTracker dyes as fluorescence probe was used\n\n 10\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n\n\nFig. 6. (a) Apoptosis assay of B16 cells (I) treated with IC50 concentration of 8a (II), 8b (III), 8c (IV), 8a(light, V), 8b(light, VI) and 8c(light, VII) for 24 h, (b and c) the\nexpression of apoptotic-related proteins in the dark or light irradiation.\n\n\nmin (LED lamp, 460 nm, 7.03 J/cm2), the intracellular Ca2+ concen- Fig. 2c, in the dark, 8a, 8b and 8c caused a slight change in the green\ntration increased, which was manifested by the significantly enhanced fluorescence intensity. However, upon light irradiation, the green fluo-\ngreen fluorescence in the drug group, we observed an enhancement of rescence increased by 30.6 times for ROSUP (positive), 176.3 times for\nthe fluorescence in 8a, 8b and 8c-treated groups by 2.02, 1.55 and 1.66 8a(light), 211.9 times for 8b(light) and 12.3 times for 8c(light), indi-\ntimes than that in the control, respectively. Next, we examined the ROS cating that 8a, 8b and 8c can efficiently elevate the intracellular ROS\nconcentration (2\u2032,7\u2032-dichlorodihydro-fluorescein diacetate (DCFH-DA) levels upon irradiation.\nas fluorescence probe). To eliminate the impact of the green fluores- To further confirm the kinds of ROS, we used dihydroethidium\ncence emitted by the complexes, the complexes were used as references. (DHE) as probe to examine the superoxide anion, See from Fig. 2d, the\nUnder normal conditions, mitochondria produce appropriate amounts of red fluorescence intensity elevated by 2.3 times for 8a(light), 2.4 times\nreactive oxygen species (ROS) as second messengers to regulate the for 8b(light) and 2.7 times for 8c(light) in comparison to the control. The\nphysiological processes, when ROS content continues to increase beyond results suggest an ability of 8a, 8b, 8c to enhance superoxide anion.\nthe intracellular antioxidant system, the high ROS level leads to mito- Additionally, we also examined the hydroxyl free radical content with\nchondrial damage, autophagy, and apoptosis [35\u201338]. As shown in hydroxyphenyl fluorescein (HPF) as a fluorescence probe. The green\n\n\n 11\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n\n\nFig. 7. (a, b) C11-BODIPY581/591 was used as a fluorescence dye to determine the red and green fluorescence, (c) cell viability assay in the presence of Fer-1, (d)\nGSH content, (e) MDA content, (f) release of LDH after B16 cells were incubated with IC50 concentrations of 8a, 8b and 8c for 24 h upon light irradiation.\n\n\n\n\n Fig. 8. Molecular docking studies of 8a, 8b and 8c with PDB ID: 1BNA.\n\n\nfluorescence intensity increased by 2.44, 2.67 and 2.76 times for 8a cleaved into monomers, and the fluorescence changes from red to green\n(light), 8b(light) and 8c(light) in comparison to the control (Fig. 2e), [39]. As shown in Fig. 2g, there was a significant decrease in red fluo-\nindicating that 8a, 8b and 8c can elevate the intracellular hydroxyl free rescence in the 8a(light), 8b(light) and 8c(light) groups compared to the\nradical content upon light irradiation. Taken together, we conclude that control. On the contrary, the green fluorescence was significantly\n8a, 8b and 8c can generate O\u2022\u2013 2 and \u22c5OH upon light irradiation. enhanced, which can be further illustrated by the red/green ratio. The\n The complexes act on the mitochondria and cause an increase of ratio of red/green fluorescence decreased (Fig. 2h), which affirmed that\nROS, which will trigger an open of mitochondrial permeability transi- 8a(light), 8b(light) and 8c(light) can trigger a decline of MMP.\ntion pore (MPTP). To prove the envision, we applied calcein AM as a\nprobe to examine MPTP. Calcein AM emits green fluorescence, after 3.5. Cell cycle distribution\nMPTP opens, CoCl2 binds with calcein AM to quench the green fluo-\nrescence, which was confirmed by the significant reduction of green Cell proliferation is an important way to maintain biological growth\nfluorescence in the positive, 8a(light), 8b(light) and 8c(light)-treated and development, but it is also a major mechanism for cancer devel-\ngroups in comparison to the control (Fig. 2f), suggesting that 8a opment [40\u201342]. The infinite proliferation of tumor cells makes cancer\n(light), 8b(light) and 8c(light) were able to induce the sustained opening an extremely destructive and incurable disease, therefore, hindering the\nof MPTP. development of the cell cycle has become a main tactics in the cancer\n The opening of MPTP will lead to the decrease of mitochondrial therapy, inhibiting the proliferation of tumor cells stops the develop-\nmembrane potential (MMP). To affirm this hypothesis, JC-1 (5,5\u2032,6,6\u2032- ment of malignant tumors. In this regard, we carried out experiments to\ntetrachloro-1,1\u2032,3,3\u2032-tetraethylbenzenecarboxamidinylcarbocyanine io- explore the effects of 8a, 8b and 8c on the cell cycle of B16 with or\ndide) was used to examine the change of mitochondrial membrane po- without irradiation. Comparing with the blank group (a), the distribu-\ntential (MMP). It is well known that JC-1 is freely transmitting through tion of B16 cells in G0/G1 phase after the action of 8a (b), 8b (c) and 8c\nthe cell, when the mitochondria is depolarized, the JC-1 polymer is (d) was slightly increased by 4.00, 6.91 and 2.01 %. However, upon\n\n 12\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n\n\n Fig. 9. Mechanism of the complexes inducting cell death.\n\n\nirradiation, in comparison to the control, the distribution of cells in G0/ 8b and 8c was found to be able to overlap with the red (lysosome),\nG1 phase increased by 9.98, 10.81 and 11.23 % for 8a(light, e), 8b(light, indicating that 8a, 8b and 8c were able to enter the lysosome (Fig. 5a).\nf) and 8c(light, g) (Fig. 3). Obviously, upon irradiation, the complexes We further explored the effect of 8a, 8b and 8c on the autophagy upon\nshow higher efficiency on preventing cell proliferation at the G0/G1 irradiation (LED lamp, 460 nm, 7.03 J/cm2). AKT/PI3K/mTOR\nphase than those without irradiation. signaling pathway is the main pathway that initiates and mediates the\n occurrence of autophagy in cells, when this signaling pathway is acti-\n3.6. Cell death studies vated, the autophagy is inhibited, inversely, when this pathway is\n inhibited, the expression of AKT, PI3K, and mTOR proteins decreases,\n3.6.1. Cellular immunogenicity assay and the autophagy will be activated [47]. It has been demonstrated that\n Immunogenic cell death (ICD) is a form of regulatory cell death that inhibition of melanoma growth can be achieved by inhibiting this\nactivates tumor-specific immune responses. In certain photodynamic signaling pathway [48]. The polyubiquitin-binding protein p62 binds\ntherapy and radiotherapy treatments, signals released by inducing the autophagy marker light chain 3 (LC3) for transport to autophago-\napoptosis in tumor cells that can serve as an immune response are somes, where it is degraded by autophagy [49]. Beclin-1 is essential for\ndamage-associated molecular patterns (DAMPs) [43], which can release the formation and maturation of autophagosomes [50]. 8a(light), 8b\ncalreticulin protein (CRT), heat shock proteins (HSP) and high mobility (light) and 8c(light) reduced AKT, PI3K, mTOR, p-mTOR, p62 expres-\ngroup box-1 (HMGB1). Therefore, inducing tumor cells to undergo sion and upregulated the expression of Beclin-1 (Fig. 5b), which indi-\nimmunogenic cell death to release damage-associated molecular pat- cated that 8a(light), 8b(light) and 8c(light) can induce cellular\nterns has become a key manner in antitumor therapy [44]. As depicted autophagy by inhibiting the AKT/PI3K/mTOR signaling pathway.\nin Fig. 4a-c, upon irradiation, the green fluorescence increases, indi- Additionally, we also investigated the autophagy using mono-\ncating interaction of 8a(light), 8b(light) and 8c(light) on B16 cells dansylcadaverine (MDC) as green fluorescence probe. As depicted in\ncauses an enhancement of CRT (a), HSP70 (b) and HMGB1 (c) proteins. Fig. 5c, treatment of B16 cells with 2 \u00d7 IC50 concentration of 8a(light),\nThe results demonstrate that 8a, 8b and 8c can induce immunogenic cell 8b(light) and 8c(light) for 8 h, the autophagic vesicles were uncovered,\ndeath in B16 cells upon irradiation. indicating that 8a, 8b and 8c can cause autophagy upon light irradiation.\n\n\n3.6.2. Autophagy studies 3.6.3. Apoptosis and mechanism studies\n Autophagy is an important strategy in tumor therapy, which is Apoptosis is the autonomous death of cells under gene regulation,\ncrucial to maintain cellular homeostasis in response to different forms of which is a way for the body to remove senescent and damaged cells.\nstress in vivo and ex vivo. While macroautophagy is a lysosome- Cancer is a disease of cell-autonomous proliferation that fails to respond\ndependent degradation pathway involving the degradation of cyto- to appropriate stimuli to evade apoptosis, resulting in unlimited prolif-\nplasmic proteins and organelles [45,46]. Lysosomes contain a variety of eration of cancer cells [51]. To explore the apoptotic efficiency of the\nhydrolytic enzymes, which are involved in a variety of biochemical complexes, Annexin V/PI were used as a double staining probe. In the\nfunctions, and can break down biomolecules by releasing hydrolytic dark conditions, 8a (II), 8b (III) and 8c (IV) cause an early apoptosis with\nenzymes to remove digested remnants in the organelles while supplying an increase of 8.37 %, 14.19 % and 7.48 %, respectively. However, upon\nenergy to the cells. Hence, we firstly investigated whether the complexes irradiation, 8a(light) (V), 8b(light) (VI) and 8c(light) (VII) increased by\nenter the lysosomes. After B16 cells were subjected to 8a (8.2 \u03bcM), 8b 31.7 %, 37.3 % and 9.8 % in the cells at the early apoptosis (Fig. 6a).\n(6.5 \u03bcM) and 8c (1.1 \u03bcM) for 4 h, the green fluorescence emitted by 8a, These results revealed that 8a, 8b and 8c can cause apoptosis with or not\n\n\n 13\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\nirradiation, moreover, 8a, 8b and 8c show higher apoptotic efficiency SI). The DNA-binding affinities (Fig. S14b, SI) were measured to be 1.74\nupon irradiation than those in the dark. The above results also demon- (\u00b1 0.42) \u00d7 105 for 8a, 1.80 (\u00b1 0.51) \u00d7 105 for 8b, 2.47 (\u00b1 0.60) \u00d7 105 for\nstrate that 8a and 8b may be as effective photosensitizers to cause 8c. The DNA-binding affinities are comparable with Ru(II) complex\napoptosis. \u0394-[Ru(bpy)2(dmppd)]2+ (3.1 \u00b1 0.3) \u00d7 105 M\u2212 1 [18] and [Co\n Apoptosis includes intrinsic and extrinsic pathways, which is basi- (bpy)2(DMHBT)]3+ (6.3 \u00b1 0.1) \u00d7 105 M\u2212 1 [57]. Therefore, the com-\ncally characterized by the fact that pro-apoptotic proteins (e.g., Bax, plexes exhibit high DNA-binding ability. The DNA-binding affinities\nBak) and anti-apoptotic proteins (Bcl-2) of the Bcl-2 family promote the follow the sequence of 8c > 8b > 8a, this is well line with those of\nrelease of cytochrome c from mitochondria, which activates the down- cytotoxicity. The binding site and binding energy with double strand\nstream cascade reaction of caspases to cleave a variety of proteins, ul- DNA (PDB ID: 1BNA) were calculated through molecular docking\ntimately leading to the occurrence of apoptosis. Activation of caspase 3 (Autodock 4.2). 8a, 8b and 8c interact with DNA through intercalation at\nand cleavage of PARP proteins, which are hallmarks of apoptosis, are the minor groove accompanied by hydrogen bond of DA18:H3 for 8a,\nused to identify the cell death pathways involved [52]. The expression DG16:H22 for 8b and 8c (Fig. 8), respectively. The binding energy of 8a,\nlevels of apoptotic proteins were examined by western blot, and the 8b, 8c with DNA is \u2212 36.4, \u2212 37.2 and \u2212 40.5 KJ\u22c5mol\u2212 1. The results from\nresults were shown in Fig. 6b and c, we discovered that 8a, 8b and 8c can the binding energy show that DNA-binding affinity is 8a < 8b < 8c, this\neffectively downregulate the expression of PARP, caspase 3 and Bcl-2, is consistent with the sequence from electronic absorption titration.\nupregulate the expression of Bax upon light irradiation or not, further- Consequently, we conclude that the complexes target the DNA and\nmore, 8a(light), 8b(light) and 8c(light) cause higher efficiency on the induce cancer cell demise.\nprotein expression than 8a, 8b and 8c.\n 4. Conclusions\n3.6.4. Ferroptosis studies\n Among the many different forms of regulated cell death, ferroptosis Three new iridium(III) metal complexes with DMHBT as ligand were\nis a ROS-dependent regulated cellular necrosis, which is mainly caused synthesized. Single state oxygen and superoxide anion assays showed\nby intracellular oxidative membrane damage due to iron accumulation that 8a, 8b and 8c can increase 1O2 and O\u2022\u2013 2 as photosensitizers for the\nand lipid peroxidation. Imbalance in intracellular redox regulation, ROS treatment of cancer in photodynamic therapy. In vitro cytotoxicity ex-\noverload, and massive production of lipid peroxides play an important periments revealed that 8a, 8b and 8c exhibited moderate or low cyto-\nrole in promoting ferroptosis [53\u201355]. To study lipid peroxidation toxicity against various tumor cell lines, whereas the in vitro toxicity of\ncaused by 8a(light), 8b(light) and 8c(light), 4,4-difluoro-5-methyl-4- the complexes was greatly improved upon light irradiation, with the\nbora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C11-BODIPY581/591) strong ability to kill B16 cells. The cellular uptake demonstrated that the\nwas emerged as a probe [56]. Treatment of B16 cells with IC50 con- complexes enter the cells and accumulate in the cell nuclei.Co-\ncentrations of 8a(light), 8b(light), and 8c(light) resulted in a significant localization experiments revealed that the complexes were able to\ndecrease in the red fluorescence (non-oxidized) and an increase in the localize at the mitochondria. Since apoptosis via the intrinsic pathway is\ngreen fluorescence (oxidized) (Fig. 7a). In addition, Fig. 7b shows a mediated by oxidatively damaged mitochondria, we found that the\ndecrease in the red/green fluorescence ratio. These results confirmed complexes led to a massive inward flow of Ca2+ and a great enhance-\nthat 8a(light), 8b(light) and 8c(light) can cause lipid peroxidation. ment of ROS, which in turn led to the sustained opening of the MPTP and\n The ability to enhance intracellular iron accumulation by increasing the decrease of MMP, resulting in mitochondrial dysfunction and\niron uptake, limiting iron efflux and decreasing intracellular iron storage inducing B16 cells to undergo apoptosis. The mechanism studies found\npromoted ferroptosis. Ferritin-1 (Fer-1), as an inhibitor for the ferrop- that 8a(light), 8b(light) and 8c(light) activated Bcl-2 family proteins and\ntosis, was used to examine cell survival. Fig. 7c reveals that the presence caused caspase cascade reaction to induce apoptosis. The complexes can\nof Fer-1 was effective on increasing cell survival in the 8a(light), 8b arrest the cell cycle at the G0/G1 phase, blocking DNA synthesis and\n(light) and 8c(light) groups, thus further confirming the potential of contributing to cell death. We also found that 8a(light), 8b(light) and 8c\nlight-activated 8a, 8b and 8c to induce ferroptosis. (light) inhibit the AKT/PI3K/mTOR signaling pathway, down-regulate\n X\u2212c -system-glutathione (GSH)-glutathione peroxidase 4 (GPX4) is the p62 and promote the expression of Beclin-1, which induced cells to\nmajor antioxidant defense mechanism against the onset of ferroptosis, undergo autophagy. In addition, 8a(light), 8b(light) and 8c(light)\nGSH is an essential cofactor for the proper function of GPX4, and caused a dramatic increase in ROS levels in B16 cells. Further experi-\nincreased GSH expression inhibits ferroptosis. In addition, ferroptosis ments revealed that overloaded ROS reacted with polyunsaturated fatty\ncan be caused by activation of high mobility group protein 1 (HMGB1), acids to initiate lipid peroxidation, inhibited the x\u2212c -system-glutathione\nwhich acts as both a damage-associated molecule and a link between (GSH)-glutathione peroxidase 4 (GPX4) antioxidant defense system, and\nferroptosis and pyroptosis. Fig. 7d shows that 8a(light), 8b(light) and 8c up-regulated the expression of the damage-associated molecule HMGB1\n(light) were able to significantly reduce the GSH content, suggesting that to cause ferroptosis. It also promotes the release of CRT and HSP70,\nthe three complexes were effective on reducing the content of GSH upon which activates tumor-specific immune responses to trigger immuno-\nlight exposure. Additionally, we uncovered that 8a(light), 8b(light) and genic cell death. In summary, 8a, 8b and 8c as photosensitizers target the\n8c(light) increased the content of malondialdehyde (MDA) (a product of DNA and induce cell death via the following five pathways: (I) ROS-\nlipid peroxidation) (Fig. 7e). Fig. 7f shows that the three complexes were mediated mitochondrial dysfunction apoptosis; (II) blocking the cell\nable to up-regulate HMGB1 and inhibit the expression of GPX4 after cycle at G0/G1 phase and inhibiting cell proliferation; (III) inducing\nlight activation, enhanced the release of LDH (Fig. 7 g). Taken together, cellular autophagy; (IV) causing ferroptosis and (V) immunogenic cell\nwe concluded that 8a(light), 8b(light) and 8c(light) were able to inhibit death (Fig. 9). The present work contributes to the further understand-\nthe x\u2212c -system-GSH-GPX4 defense mechanism and induce ferroptosis. ing the anticancer mechanism and provides an help for the design and\n synthesis of photocytotoxic candidates for the treatment of B16 tumors.\n3.7. DNA-binding and molecular docking studies\n Author statement\n The complexes enter cancer cells and accumulate in the cell nuclei,\nwhich stimulates us to consider whether DNA become a drug target. We state that the manuscript has been finished by all authors listed in\nHence, we investigate the DNA-binding of 8a, 8b and 8c with calf the manuscript. The all data are original and real. We agree to be\nthymus (CT-DNA). The absorbance of 8a at 269 nm, 8b at 265 nm and 8c accountable for all aspects of the work to ensure that questions related to\nat 294 nm gradually decreased with an increment of DNA amount with a the accuracy or integrity of any part of the work are appropriately\nhypochmism of 13.6 %, 15.2 % and 15.9 % for 8a, 8b and 8c (Fig. S14a, investigated and resolved.\n\n 14\n\fY. Niu et al. Journal of Inorganic Biochemistry 264 (2025) 112808\n\n\n All authors have read the manuscript and approved the manuscript [11] S. Tian, Q.Y. Nie, H.M. Chen, L.J. Liang, H.Y. Hu, S.H. Tang, J.W. Yang, Y.J. Liu,\n H. Yin, Synthesis, characterization and irradiation enhances anticancer activity of\nto be submitted to JIB.\n liposome-loaded iridium(III) complexes, J. Inorg. Biochem. 256 (2024) 112549.\n [12] T. Feng, Z.X. Tang, J. Karges, J. Sun, K. Xiong, C.Z. Jin, Y. Chen, G. Gasser, L.N. Ji,\nCRediT authorship contribution statement H. Chao, An iridium(III)-based photosensitizer disrupting the mitochondrial\n respiratory chain induces ferritinophagy-mediated immunogenic cell death, Chem.\n Sci. 15 (2024) 6752\u20136762.\n Yajie Niu: Methodology, Investigation. Shuanghui Tang: Software, [13] J. Kasparkova, A. Herna\u0301ndez-Garc\u00eda, H. Kostrhunova, M. Goicur\u00eda,\nData curation. Jiongbang Li: Formal analysis. Chunxia Huang: V. Novohradsky, D. Bautista, L. Markova, M.D. Santana, V. Brabec, J. Ruiz, Novel\n 2-(5-Arylthiophen-2-yl)-benzoazole cyclometalated iridium(III) dppz complexes\nInvestigation. Yan Yang: Writing \u2013 original draft, Conceptualization. exhibit selective phototoxicity in cancer cells by lysosomal damage and oncosis,\nLin Zhou: Software, Formal analysis. Yunjun Liu: Supervision, Funding J. Med. Chem. 67 (2024) 691\u2013708.\nacquisition, Conceptualization. Xiandong Zeng: Writing \u2013 review & [14] J. Karges, Clinical development of metal complexes as photosensitizers for\n photodynamic therapy of cancer, Angew. Chem. Int. Ed. 61 (2022) e202112236.\nediting, Project administration, Investigation. [15] W.Y. Zhang, Q.Y. Yi, Y.J. Wang, F. Du, M. He, B. Tang, D. Wan, Y.J. Liu, H.\n L. Huang, Photoinduced anticancer activity studies of iridium(III) complexes\n targeting mitochondria and tubules, Eur. J. Med. Chem. 151 (2018) 568\u2013584.\n [16] G.C. Li, J. Chen, Y.F. Xie, Y. Yang, Y.J. Niu, X.L. Chen, X.D. Zeng, L. Zhou, Y.J. 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