Light activation of iridium(III) complexes driving ROS production and DNA damage enhances anticancer activity in A549 cells.

{"full_text": " Journal of Inorganic Biochemistry 236 (2022) 111977\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\nLight activation of iridium(III) complexes driving ROS production and DNA\ndamage enhances anticancer activity in A549 cells\nWenlong Li a, Chuanling Shi a, Xiaoyun Wu a, Yuanyuan Zhang a, Haimei Liu a, Xiuzhen Wang a, *,\nChunxia Huang a, Lijuan Liang a, Yunjun Liu a, b, **\na\n School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China\nb\n Guangdong Provincial Key Laboratory of Advanced Drug Delivery Systems and Guangdong Provincial Engineering Center of Topical Precise Drug Delivery System,\nGuangdong Pharmaceutical University, Guangzhou 510006, 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: The work aimed to synthesize and characterize two iridium(III) complexes [Ir(ppy)2(IPPH)](PF6) (Ir1, IPPH =\nIridium(III) complexes (2S,3R,5S,6R)-2-(2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenoxy)-6-(hydroxymethyl)tetrahydro-2H-\nPhotoactivation pyran-3,4,5-triol, ppy = 2-phenylpyridine), [Ir(piq)2(IPPH)](PF6) (Ir2, piq = 1-phenylisoquinoline). The cyto\u00ad\nApoptosis\n toxicity of the complexes against BEL-7402, A549, HCT-116, B16 cancer cells and normal LO2 was evaluated\nImmunogenic cell death\n through 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) method. The complexes show no\nMitochondria\n cytotoxic activity (IC50 > 100 \u03bcM) against these cancer cells, while their cytotoxicity can significantly be elevated\n upon illumination. The IC50 values range from 0.2 \u00b1 0.05 to 35.5 \u00b1 3.5 \u03bcM. The cellular uptake, endoplasmic\n reticulum and mitochondria localization, reactive oxygen species, the change of mitochondrial membrane po\u00ad\n tential, \u03b3-H2AX levels, cycle arrest, apoptosis and the expression of B-cell lymphoma-2 were investigated. The\n calreticulin (CRT), heat shock protein 70 (HSP70), high mobility group box 1 (HMGB1) were explored. This\n study demonstrates that photoactivatable complexes induce cell death in A549 through ROS-mediated endo\u00ad\n plasmic reticulum stress-mitochondrial pathway, DNA damage pathways, immunogenic cell death (ICD), acti\u00ad\n vation of PI3K/AKT signaling pathway and inhibit the cell growth at S phase.\n\n\n\n\n1. Introduction related death is not the tumor itself but the metastasis from the tumor\n [2]. Since the discovery of cisplatin by Rosenberg in the 1960s, the ef\u00ad\n Cancer remains one of the most challenging and dangerous health ficacy of platinum drugs in cancer chemotherapy has been limited by\nburden for human beings worldwide [1]. The main cause of cancer- side effects such as drug resistance, neurotoxicity, nephrotoxicity,\n\n\n Abbreviations: A549, human non-small cell lung cancer cells; ATP, adenosine triphosphate; AKT, protein kinase B; B16, mouse melanoma cells; Bax, Bcl-2\nassociated x protein; BCA, bicinchoninic acid; Bcl-2, B-cell lymphoma-2; BEL-7402, human hepatocarcinoma cells; Caspase-3, cysteinyl aspartate specific proteinase-\n3; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CRT, calreticulin; DAPI, 4\u2032 ,6-Diamidino-2-phenylindole; DCF, 2\u2032 ,7\u2019-Dichlorofluorescein; DCHF, 2\u2032 ,7\u2019-\nDichlorodihydrofluorescein; DCHF-DA, 2\u2032 ,7\u2019-Dichlorodihydrofluorescein diacetate; DMEM, Dulbecco's Modified Eagle Medium; DMSO, dimethylsulfoxide; EDTA,\nethylene diamine tetraacetic acid; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HCT-116, human colorectal carcinoma cells; HeLa, human cervical\ncancer cells; HepG2, human-hepatocarcinoma cells; HMGB1, high mobility group box1; HSP70, heat shock protein 70; ICD, immunogenic cell death; IPPH,\n(2S,3R,5S,6R)-2-(2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol; JC-1, 5,5\u2032 -6,6\u2032 -tetrachloro-1,1\u2032 -3,3\u2032 -\ntetrethylbenzimidalylcarbocyanine iodide; LED, light emitting diode; LO2, human normal liver cells; MDA, malondialdehyde; MMP, mitochondrial membrane po\u00ad\ntential; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide; NBCS, Newborn calf serum; PARP, poly ADP-ribose polymerase; PBS, phosphate buffer\nsolution; PDT, photodynamic therapy; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; piq, 1-phenylisoquinoline; PMSF, phenylmethylsulfonyl fluoride;\nHppy, 2-phenylpyridine; PVDF, polyvinylidene difluoride; RIPA, 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate; RLU, Relative\nluminometer units; RNase, ribonuclease; ROS, reactive oxygen species; RPMI 1640, Roswell Park Memorial Institute 1640; SDS-PAGE, sodium dodecyl sulfate\npolyacrylamide gel electrophoresis; TBA, thiobarbituric acid; TBST 20, mM Tris\u2013HCl, 150 mM NaCl, 0.2% Tween 20; TMS, tetramethylsilane; Tris, Tris(hydrox\u00ad\nymethyl)aminomethane; Tween 20, polysorbate 20.\n * Corresponding author.\n ** Corresponding author at: School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China.\n E-mail addresses: wxzqq1234@gdpu.edu.cn (X. Wang), lyjche@gdpu.edu.cn (Y. Liu).\n\nhttps://doi.org/10.1016/j.jinorgbio.2022.111977\nReceived 25 July 2022; Received in revised form 10 August 2022; Accepted 19 August 2022\nAvailable online 23 August 2022\n0162-0134/\u00a9 2022 Elsevier Inc. All rights reserved.\n\f W. Li et al.\n2\n\n\n\n\n Journal of Inorganic Biochemistry 236 (2022) 111977\n Scheme 1. Synthetic route for ligand and its complexes Ir1 and Ir2\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\nhepatotoxicity and selectivity of treatment [3\u20136]. Unfortunately, the and 200 \u25e6 C, respectively and the quoted m/z values are for the major\nresistance and toxic side effects associated with chemotherapy, radio\u00ad peaks in the isotope distribution.\ntherapy and surgery make cancer treatment troublesome [7]. Photody\u00ad\nnamic therapy (PDT) has attracted great attention for its high efficiency,\nlow side effects and overcoming drug resistance compared to traditional 2.2. Synthesis of complexes\ncancer treatment strategies [8\u201310]. In PDT, the photosensitizer is acti\u00ad\nvated by light and the excited triplet state of the photosensitizer converts 2.2.1. Preparation of ligand IPPH\nthe ground state 3O2 into the active singlet oxygen (1O2) which kills A mixture of 1,10-phenanthroline-5,6-dione (0.315 g, 1.5 mmol),\ncancer cells (type II reactions). The Radical species such as superoxide helicine (0.426 g, 1.5 mmol), NH4Ac (2.31 g, 30 mnol) was dissolved in\nion (O\u22122 ) and hydroxyl radical (\u22c5OH) were formed by electron transfer ethanol (20 mL), then the mixture was refluxed at 78 \u25e6 C for 3 h, after\nreactions (type I reactions) [11\u201313]. cooling to room temperature, pale yellow precipitate was collected. The\n In our early work, we found that a range of iridium(III) complexes precipitate was washed three times with ice water, the yellow powder\nhave unique anticancer mechanisms, such as mitochondrial dysfunction, was obtained. Yield: 79%. Anal. Cacld for C25H22N4O6: C, 63.29, H,\nDNA binding, ROS elevation, immunogenic cell death, endoplasmic re\u00ad 4.67, N, 11.81%. Found: C, 63.53, H, 4.34, N, 11.98%. ESI-MS (CH3OH):\nticulum stress, the release of cytochrome c [14\u201318]. Encouragingly, m/z = 473.20 ([M-1]).\niridium(III) complexes are extensively investigated as showing great\npotential for photocatalytic cancer therapy in the field of photodynamic 2.2.2. Synthesis of complex [Ir(ppy)2(IPPH)]PF6 (Ir1)\ntherapy (PDT) agent [19\u201333]. Glucose is essential to mammalian cells A mixture of cis-[Ir(ppy)2Cl]2 (0.28 g, 0.25 mmol) [40] and IPPH\nbecause it is not only a precursor of glycoproteins, triglycerides, and (0.237 g, 0.5 mmol) was dissolved in 42 mL of dichloromethane and\nglycogen but also a crucial energy source through the generation of methanol (v/v, 2:1). The mixture was refluxed at 40 \u25e6 C for 6 h under the\nadenosine triphosphate (ATP) [34\u201338], moreover, the iridium com\u00ad protection of argon. Upon cooling, saturated aqueous NH4PF6 was added\nplexes containing glucose can increase cell uptake and show high anti\u00ad to the reaction solution with stirring at room temperature for 2 h to\ncancer activity [39]. To obtain much information on the anticancer obtain yellow precipitate. The crude product was initially separated\nactivity, in this work, two new pending glucose iridium(III) complexes using silica gel column with dichloromethane-acetone (v/v, 1:3) as\n[Ir(ppy)2(IPPH)](PF6) (Ir1, ppy = 2-phenylpyridine, IPPH = eluent, the yellow band was collected. The solvent was removed under\n(2S,3R,5S,6R)-2-(2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phe\u00ad reduced pressure and the yellow powder was obtained. Yield: 83%. 1H\nnoxy)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol) and [Ir NMR (Acetone\u2011d6, 500 MHz): \u03b4 9.25 (dd, 2H, J = 1.0, J = 8.5 Hz),\n(piq)2(IPPH)](PF6) (Ir2, piq = 1-phenylisoquinoline, Scheme 1) were 8.48\u20138.45 (m, 1H), 8.37 (d, 2H, J = 5.0 Hz), 8.24 (d, 2H, J = 8.5 Hz),\ndesigned, synthesized and characterized by HRMS, 1H NMR and 13C 8.07\u20138.02 (m, 2H), 7.93 (d, 2H, J = 8.0 Hz), 7.89 (t, 2H, J = 8.0 Hz),\nNMR. Ir1 and Ir2 showed no activity against selected BEL-7402 (human 7.73 (d, 2H, J = 6.0 Hz), 7.58\u20137.55 (m, 1H), 7.49\u20137.45 (m, 1H),\nhepatocarcinoma cells), A549 (human lung cancer cells), HCT116 7.39\u20137.34 (m, 1H), 7.08 (t, 2H, J = 6.0 Hz), 7.00\u20136.96 (m, 4H), 6.46 (d,\n(human colon cells), B16 (mouse melanoma cells) and normal LO2 2H, J = 7.5 Hz), 5.26 (d, 1H, J = 8.0 Hz), 4.77 (s, 1H), 4.56 (s, 1H),\n(human normal liver cells). However, the complexes were irradiated 4.07\u20133.99 (m, 2H), 3.95\u20133.81 (m, 3H), 3.77\u20133.68 (m, 2H), 3.59 (t, 1H, J\nwith a 65w LED lamp (white light, 5.2 J cm\u2212 2, \u03bb = 450\u2013465 nm) for 40 = 8.0 Hz). 13C NMR (Acetone\u2011d6, 125 MHz): 168.59, 156.72, 150.19,\nmin. Ir1 and Ir2 were highly photoactive in subduing cancer cells, 145.02, 139.30, 132.74, 132.54, 131.11, 130.65, 130.52, 127.68,\nespecially on A549 cells. In addition, cytotoxicity, endoplasmic reticu\u00ad 127.58, 125.67, 124.72, 124.68, 124.23, 123.29, 120.58, 120.25,\nlum and mitochondria localization, apoptosis, intracellular ROS levels, 119.65, 119.61, 104.73, 78.57, 77.39, 75.23, 71.55, 68.85, 62.36.\nthe change of mitochondrial membrane potential, immunogenic cell HRMS (CH3CN): C47H38N6O6IrPF6: m/z = 975.2463 [(M-PF6)+].\ndeath, the expression of B-cell lymphoma-2 (Bcl-2) family proteins were\ninvestigated in detail. 2.2.3. Synthesis of complex [Ir(piq)2(IPPH)]PF6 (Ir2)\n The complex [Ir(piq)2(IPPH)]PF6 (Ir2) was synthesized in an iden\u00ad\n2. Experimental tical method that described for Ir1, with cis-[Ir(piq)2Cl]2 [40] in place of\n cis-[Ir(ppy)2Cl]2. Yield: 72%. 1H NMR (Acetone\u2011d6, 500 MHz): \u03b4 9.24\n2.1. Materials and methods (dd, 2H, J = 1.0, J = 8.5 Hz), 9.11 (d, 2H, J = 8.5 Hz), 8.47 (d, 3H, J =\n 8.0 Hz), 8.26\u20138.24 (m, 2H), 8.04\u20137.99 (m, 4H), 7.91 (t, 2H, J = 6.5 Hz),\n All chemical reagents were purchased and used directly without 7.86 (t, 2H, J = 7.0 Hz), 7.75 (t, 1H, J = 7.5 Hz), 7.69 (t, 1H, J = 8.0 Hz)\nfurther purification. IrCl3\u22c53H2O was obtained from the Kunming Boren 7.61 (d, 2H, J = 6.5 Hz), 7.58\u20137.52 (m, 1H), 7.40 (d, 2H, J = 6.5 Hz),\nPrecious Metals Co., Ltd. 1,10-phenanthroline was obtained from the 7.38\u20137.29 (m, 1H), 7.19 (t, 2H, J = 7.5 Hz), 6.96 (t, 2H, J = 7.5 Hz), 6.48\nGuangzhou Chemical Reagent Factory. Fetal Bovine Serum (FBS) and (d, 2H, J = 7.0 Hz), 5.26 (dd, 1H, J = 2.5, J = 8.0 Hz), 4.76 (s, 1H), 4.55\nNewborn Calf Serum (NBCS) were purchased from Gibco company. (s, 1H), 4.07\u20134.00 (m, 2H), 3.95\u20133.81 (m, 3H), 3.76\u20133.67 (m, 2H), 3.58\nDulbecco's Modified Eagle Medium (DMEM) and Roswell Park Memorial (t, 1H, J = 8.0 Hz). 13C NMR (Acetone\u2011d6, 125 MHz): 169.59, 156.73,\nInstitute (RPMI) 1640 were purchased from Thermo Fisher Scientific 146.57, 141.79, 137.89, 133.03, 132.70, 131.50, 131.32, 130.63,\n(Waltham, USA). The tumor cell lines BEL-7402, A549, HCT116, B16 129.84, 128.39, 127.49, 126.96, 124.71, 123.08, 122.64, 120.24,\ncancer cells and normal LO2 were gained from the Cell Center of Sun 119.65, 104.76, 78.59, 77.39, 75.19, 71.54, 68.84, 62.36. HRMS\nYat-Sen University (Guangzhou, China). 3-(4,5-dimethyl-tiazol-2-ly)- (CH3CN): C55H42N6O6IrPF6: m/z = 1075.2966 [(M-PF6)+].\n2,5-diphenyl-tetrazoliumbromide (MTT) was purchased from Shanghai\nBeyotime Institute of Biotechnology (Shanghai, China). 2\u2032 ,7\u2032 -dichlor\u00ad\nodihydrofluorescein diacetate (DCFH-DA) and 5,5\u2032 -6,6\u2032 -tetrachloro-1,1\u2032 - 2.3. pKa values determination\n3,3\u2032 -tetrethylbenzimidalylcarbocyanine iodide (JC-1) were purchased\nfrom Roche Diagnostics (Indianapolis, IN, USA). 1H and 13C NMR The pKa values of the complexes were assayed by potentiometric\nspectra were recorded on a Varian-500 spectrometer with DMSO\u2011d6 as titration method using a pH meter (Basic pH Meter PB-10, Sartorius)\nsolvent and tetramethylsilane (TMS) as an internal standard at 500 MHz calibrated with standard buffers of pH 4.01, 6.86 and 9.18. The com\u00ad\nat room temperature. Electrospray ionization mass spectra (ESI\u2013MS) plexes were dissolved in a mixture of acetonitrile and water (v/v, 3:7).\nwere recorded on an LCQ system (Finnigan MAT, USA) using acetoni\u00ad The pH value of the above mixture was adjusted to 2 using HCl solution\ntrile as the mobile phase. The spray voltage, tube lens offset, capillary (100 mM) under constant ionic strength (150 mM, KCl). Then KOH so\u00ad\nvoltage, and capillary temperature were set at 4.50 kV, 30.00 V, 23.00 V lution (60 mM) was added until the pH value reaches 12.\n\n 3\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\nTable 1 measured at 490 nm by a Microplate Reader (Multiskan FC, Thermo\nIC50 values (\u03bcM) of the ligand and its complexes toward the selected cancer cells Scientific, Shanghai, China). The obtained results were analyzed with\nfor 48 h. the IBM SPSS Statistics (version 20.0) to obtain the mean IC50 values in\n Complexes BEL-7402 A549 HCT116 B16 LO2 the independent three experiments.\n IPPH > 100 79.6 \u00b1 4.1 > 100 > 100 > 100\n Note: In the following experiments, the location and cellular uptake\n IPPH (light) > 100 > 100 28.6 \u00b1 3.8 > 100 > 100 were performed without irradiation, in the other cell experiments, the\n Ir1 >100 >100 >100 >100 >100 cells were treated with IC50 concentration of the complexes for 4 h, then\n Ir1(light) 30.8 \u00b1 0.8 16.8 \u00b1 2.1 18.0 \u00b1 1.3 35.5 \u00b1 3.5 >100 the cells were irradiated with white light (65 w LED lamp, light dose of\n Ir2 >100 >100 >100 >100 >100\n 5.2 J cm\u2212 2, \u03bb = 450\u2013465 nm) for 40 min.\n Ir2 (light) 5.0 \u00b1 0.4 0.2 \u00b1 0.05 0.8 \u00b1 0.1 32.4 \u00b1 0.9 26.1 \u00b1 3.6\n Cisplatin 15.4 \u00b1 4.1 6.5 \u00b1 0.5 15.6 \u00b1 0.4 20.3 \u00b1 1.0 18.7 \u00b1 0.7\n 2.6. Cellular uptake\nData for cisplatin toward A549, BEL-7402 and LO2 from ref. [52], HCT116 from\nref. [53].\n A549 cells (1 \u00d7 105 cells per well) were inoculated into 12-well\n plates and grown to 50% of the density. After adding 10.0 \u03bcM concen\u00ad\n2.4. Cell culture tration of Ir1 and Ir2, the cells were incubated at 37 \u25e6 C and 5% CO2 for 4\n h. Then, removing the culture medium, the nuclei were stained with\n A549, HCT116, B16 and normal LO2 were cultured in Dulbecco's 4\u2032 ,6-diamidino-2-phenylindole (DAPI) at 37 \u25e6 C for 20 min. The cells\nModified Eagle Medium (DMEM). BEL-7402 cells were cultured in the were washed thrice with cold phosphate buffer solution (PBS) and\nRoswell Park Memorial Institute (RPMI) 1640. The medium for the imaged under the ImageXpress R Micro XLS System (MD company,\nculture of cell lines was supplemented with 10% (v/v) fetal bovine USA).\nserum (FBS, Gibco, USA) and 1% (v/v) the penicillin-streptomycin so\u00ad\nlution. All cells were cultured at 37 \u25e6 C in a humidified atmosphere\n 2.7. Wound healing assay\nconsisting of 5% CO2 and 95% air.\n A wound healing test was used to examine the effects on the\n2.5. Cytotoxicity assay in vitro migration of A549 cells. The cells were seeded in a 6-well plate (5.0 \u00d7\n 105 per well) and incubated for 24 h. The cells were wounded with a\n The cytotoxic activity of Ir1 and Ir2 in vitro against the selected sterile pipette tip and washed three times with PBS to remove any re\u00ad\ncancer cell lines was assayed by 3-(4,5-dimethylthiazole-2-yl)-2,5- sidual cell fragments from the scratch. Then, A549 cells were cultured\nbiphenyl tetrazolium bromide (MTT) method [41]. The cells were with a fresh medium containing 1% FBS. After the addition of the\nseeded in a 96-well microplate (Costar, Corning Corp, New York) at a complexes, 40 min of irradiation to the cells and continued to incubate\ndensity of 1.0 \u00d7 104 cells per well and further grown overnight at 37 \u25e6 C for 24 h. Finally, images were taken at 0 and 24 h under an inverted\nin a 5% CO2 incubator. Next, different concentration (0\u2013100 \u03bcM) of the microscope (Olympus Co., Tokyo, Japan). The wound width was\ncomplexes dissolved in DMSO (final concentration of DMSO of 0.05%, v/ analyzed using Image J software to quantify the speed of wound healing.\nv) were added to the wells. Light activation of complexes was performed\nwith visible light and the cells were irradiated with a 65 w LED lamp 2.8. Measurement of apoptosis by flow cytometry\n(white light, 5.2 J cm\u2212 2). The distance between the lamp and the 96-well\nplate was 20 cm and the temperature was maintained at room temper\u00ad Apoptosis was assessed using Annexin V- FITC apoptosis detection\nature for 40 min during the irradiation period. After incubation at 37 \u25e6 C kit according to the manufacturer's instructions. A549 cells were\nin 5% CO2 for 48 h, replacing the medium in the wells with 90 \u03bcL of exposed to the IC50 concentrations of Ir1 and Ir2 and incubated at 37 \u25e6 C\nmedium and 10 \u03bcL of MTT dye solution (5 mg/mL) and the incubation in 5% CO2 for 24 h. The cells were irradiated for 40 min. Then the cells\nwas allowed to continue for another 4 h. The formed formazan crystals were collected and treated with trypsin-EDTA solution. Eventually, the\nwere dissolved in 100 \u03bcL DMSO according to MTT Kit. Absorbance was cells were washed twice with cold PBS and stained with Annexin V-FITC\n\n\n\n\n Fig. 1. The cellular uptake assay of A549 cells was incubated with Ir1 (10 \u03bcM), Ir2 (10 \u03bcM) for 4 h.\n\n 4\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\n\n\nFig. 2. (a) The wound healing studies after A549 cells were treated with IC50 concentration of Ir1, Ir2 for 24 h upon irradiation. (b) The wound closure ratio values\nwere analyzed with Image J.\n\n\n(2 \u03bcL) and propidium iodide (PI, 2 \u03bcL) for 20 min in the dark. The mL) at 37 \u25e6 C for 0.5 h. Subsequently, the cells were rinsed with cold PBS\napoptosis rates were immediately determined by flow cytometry and photographed under ImageXpress Micro XLS system as soon as\n(Beckman Instruments, NJ). possible. To investigate the effect of complexes on mitochondrial\n membrane potential (MMP), A549 cells were treated with the IC50\n2.9. Cell cycle arrest determination concentration of Ir1 and Ir2 for 24 h. Four hours after the addition of the\n complexes, the cells were irradiated for 40 min. The cells were stained\n A549 cells were cultured in a 6-well plate (4 \u00d7 105 cells per well) with 5,5\u2032 -6,6\u2032 -tetrachloro-1,1\u2032 -3,3\u2032 -tetrethylbenzimidalylcarbocyanine\novernight and incubated with IC50 concentration of Ir1 and Ir2. After 4 iodide (JC-1) at 37 \u25e6 C for 30 min after trypsin and collection. After\nh, the cells were irradiated for 40 min. Then the cells were harvested by centrifugation to remove the supernatant, the cell pellet was suspended\ntrypsinization and centrifuged 5 min at 1000 g. The supernatant was in PBS. The ratio of red/green fluorescence intensity was measured by\ncarefully aspirated, washed with cold PBS and fixed with 75% ethanol at FACS Calibur flow cytometry (Beckman Instruments, NJ).\n4 \u25e6 C overnight. According to guidance of the manufacturer's in\u00ad\nstructions, the cells were washed thrice with PBS and resuspended in 2.12. Determination of reactive oxygen species (ROS)\n200 \u03bcL of assay buffer with 0.1% Triton X-100, 4 \u03bcL of PI (propidium\niodide, 0.02 mg/mL) and 4 \u03bcL of RNase (ribonuclease, 0.2 mg/mL), and A549 cells were placed into a 12-well plate and incubated overnight.\nkept at room temperature for 30 min in the dark. The cells were analyzed After discarding the medium, fresh DMEM containing IC50 concentra\u00ad\nby flow cytometry (Beckman Instruments, NJ). tion of Ir1 and Ir2 was added and incubated for 4 h at 37 \u25e6 C in 5% CO2,\n the cells were irradiated (white light) for 40 min. After 24 h, the cells\n2.10. Endoplasmic reticulum localization were stained with 10 \u03bcM of 2\u2032 ,7\u2019-Dichlorodihydrofluorescein diacetate\n (DCHF-DA) in the dark. The cell nuclei were stained with 4\u2032 ,6-Dia\u00ad\n The A549 cells were seeded in a 12-well plate at a density of 1.0 \u00d7 midino-2-phenylindole (DAPI) for 20 min and observed under the\n105 cells per well. After incubation for 24 h, the cells were treated with ImageXpress R Micro XLS System (MD company, USA).\nIC50 concentration of Ir1 and Ir2 for 4 h. The cells were washed twice\nwith cold PBS and then incubated with ER Tracker Red (1 \u03bcM) at 37 \u25e6 C 2.13. Lipid peroxidation assay\nfor 20 min. After that, the cells were washed twice with cold PBS and\nobserved under ImageXpress R Micro XLS System (MD company, USA). The intracellular malondialdehyde (MDA) levels were measured\n using the thiobarbituric acid (Beyotime, Shanghai, China) method.\n2.11. Mitochondrial localization and membrane potential determination Briefly, A549 cells were cultured in a 6-well plate (4 \u00d7 105 cells per well)\n(MMP) for 24 h. After the density reached 90%, the IC50 concentration of the\n complexes were added to the cells for 4 h, then the cells were irradiated\n The A549 cells were incubated with IC50 concentration of Ir1 and Ir2 for 40 min, after 24 h, the cells were harvested by trypsinization, pre\u00ad\nfor 4 h and further co-incubated with MitoTracker \u00ae Deep Red FM (1 \u03bcg/ pared in lysis buffer and centrifuged at 12,000 g for 15 min at 4 \u25e6 C. The\n\n 5\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\n\n\nFig. 3. Cell cycle distribution of A549 cells (a) were treated with IC50 concentration of Ir1 (b) and Ir2 (c) (without irradiation) or Ir1 (d) and Ir2 (e) (irradiation for\n40 min).\n\n\nsupernatants were reacted with TBA (thiobarbituric acid) and the re\u00ad incubated with a fluorescein isothiocyanate (FITC)-conjugated second\u00ad\naction products were measured spectrophotometrically at 530 nm. The ary antibody (Beyotime, Shanghai, China; 1:200) for 80 min at 4 \u25e6 C. The\nconcentration of MDA was calculated by a calibration curve using nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) and im\u00ad\n1,1,3,3-tetrathoxypropane propane as a standard and expressed as \u03bcg/g ages were acquired under Micro XLS System (MD company, USA).\nprotein.\n 2.16. Western blotting assay\n2.14. Measurement of CRT, HSP70, HMGB1\n A549 cells were seeded into a 6-well plate at a density of 2.0 \u00d7 106\n A549 cells were seeded in a 12-well plate at the density of 1.0 \u00d7 105 cells per well for 24 h and treated with IC50 concentrations of the Ir1 and\ncells/well and incubated overnight. Then, the cells were treated with Ir2 for 4 h, the cells were irradiated for 40 min. After 24 h, the cells were\nIC50 concentration of Ir1 and Ir2 for 4 h, the cells were irradiated for 40 washed three times with cold PBS and lysed in lysis buffer (phosphatase\nmin. After 24 h, the cells were fixed with 75% alcohol, incubated with an and protease inhibitor mixture) on ice and centrifuged at 12,000 rpm for\nimmunostaining blocker for 1 h. Then the cells were incubated with 15 min at 4 \u25e6 C. The protein content of the sample was detected using a\nrabbit antibodies (CRT, HSP70 and HMGB1) at 1:70 dilutions at 4 \u25e6 C BCA assay. Samples with equal protein concentrations were separated\novernight. Next, the plate was washed with immunol staining wash by 10 or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis\nbuffer three times and incubated with FITC-labeled Goat Anti-Rabbit IgG (SDS-PAGE). Separated proteins were transferred from the gels to pol\u00ad\nin the dark for 1 h. After that, the cells were washed twice, then stained yvinylidene difluoride (PVDF) membranes. The membranes were\nwith DAPI for 20 min, and the images of CRT, HSP70 and HMGB 1 blocked with 5% skim milk in TBST (20 mM Tris\u2013HCl, 150 mM NaCl,\nexpression were obtained under the ImageXpress Micro XLS System (MD 0.2% Tween 20, pH 8.0) buffer for 1 h at room temperature and incu\u00ad\ncompany, USA). bated with primary antibodies in diluent overnight at 4 \u25e6 C. The poly\u00ad\n vinylidene difluoride (PVDF) membranes were washed four times with\n2.15. Immunofluorescence staining of \u03b3-H2AX TBST and incubated with secondary antibodies conjugated with horse\u00ad\n radish peroxidase (1:5000 dilution) for 70 min. Finally, the membranes\n Immunofluorescence staining for \u03b3-H2AX was measured using the were washed three times with TBST and developed with BeyoECL Star\nDNA damage assay kit by \u03b3-H2AX immunofluorescence (Beyotime, solution, followed by imaging with FluorChem instrument (Pro\u00ad\nShanghai, China). A549 cells were treated with IC50 concentration of Ir1 teinSimple, CA, USA). The protein expression level was analyzed using\nand Ir2 for 4 h in a 12-well plate (1.0 \u00d7 105 cells/well), the cells were the Image J software.\nirradiated for 40 min. After 24 h, the cells were washed three times with\ncold PBS and blocked at 4 \u25e6 C for 1 h using QuickBlock\u2122 Blocking Buffer 2.17. Data analysis\nfor Immunol Staining. Then the cells were incubated for 1 h at room\ntemperature with an anti-\u03b3-H2AX primary antibody (rabbit monoclonal; The results were expressed as mean \u00b1 SD. Statistical significance was\nBeyotime, Shanghai, China; 1:200). A549 cells were washed with evaluated through a one-way analysis of variance. The *P values <0.05\nImmunol Staining Wash Buffer containing 0.5% Triton X-100 and was considered statistically significant.\n\n 6\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\n\n\nFig. 4. Apoptosis assay of A549 cells (I) exposure to IC50 concentration of Ir1 (II), Ir2 (III) (without irradiation) or Ir1 (IV) and Ir2 (V) upon irradiation for 40 min.\n\n\n3. Results and discussion dissolved in methanol and diluted with water. The \u03a6 values were\n calculated according to the following equation:\n3.1. Synthesis and characterization ( / )\n \u03a6c = \u03a6r \u00d7 Ic Ar ns 2 Ir Ac nr 2\n The ligand IPPH was prepared through condensation of 1,10-phe\u00ad Where I is integrated emission intensity, A is absorbance intensity, n\nnanthroline-5,6-dione with helicine in the ethanol. The complexes Ir1 is refractive index of solvent. The subscript r stands for reference, c\nand Ir2 were synthesized by the direct reaction with [Ir(ppy)2Cl2]2 or [Ir stands for the complexes.\n(piq)2Cl2]2 and IPPH in a mixture of dichloromethane and methanol The luminescence quantum yields for Ir1 and Ir2 were determined to\nunder argon. The crude product was purified on a silica gel (100\u2013200 be 0.13 and 0.05.\nmesh) column and dichloromethane\u2013acetone (1:3, v/v) as eluent. The The stability of the complexes in PBS solution at room temperature\ncomplexes were characterized by HRMS, 1H NMR and 13C NMR. In the was detected at 0 and 24 h, as shown in Fig. S3 (supporting information),\nspectra of HRMS, the signal of [M-PF6]+ was observed, the determined no change in the shape of the peaks was observed, indicating that the\nmolecular weights are consistent with the expected values. The purity of complexes are stable in PBS solution.\nthe complexes was detected using methanol and water (Vmethanol:Vwater\n= 97:3 for Ir1, 95:5 for Ir2) as mobile phase by HPLC. As shown in\n 3.2. pKa values determination\nFig. S1 (supporting information), during the period of 30 min, only a\npeak for Ir1 and Ir2 was discovered, indicating that the complexes are\n The dissociation constant (pKa) of the complexes has a considerable\npure.\n influence on the physicochemical and pharmacokinetic properties and\n The UV\u2013Vis spectra of 20.0 \u03bcM Ir1 and Ir2 in ethanol were shown in\n can be used to determine whether it can cross the blood-brain barrier\nFig. S2a (supporting information), the maximum absorbance of Ir1 and\n [43]. Compounds with minimally one charge with a pKa > 4 for acids\nIr2 appeared at 277 (\u03b5 = 57,595), 289 (\u03b5 = 67,630) nm, respectively.\n and correspondingly a pKa < 10 for bases can cross the cell membrane by\nThe complexes can luminescence in ethanol at room temperature, with a\n passive diffusion [44]. The pH values for Ir1 and Ir2 are 6.61 and 6.29,\nmaximum for Ir1 and Ir2 (Fig. S2b, supporting information) appearing\n respectively. The pKa values were obtained by a graph of pH versus\nat 559 nm (\u03bbex = 275 nm) and 589 nm (\u03bbex = 290 nm), respectively. The\n volume of KOH. As shown in Fig. S4 (supporting information), the pKa\nluminescence of the complexes in PBS solution was also detected, the\n values are 7.13 for Ir1, 7.02 for Ir2, indicating that the complexes can\ncomplexes emit weak green fluorescence (Fig. S2c, supporting\n cross the cell membrane to enter to the cells.\ninformation).\n The luminescence quantum yield (\u03a6) of the complexes was deter\u00ad\nmined under argon according to the literature [42] using [Ru(bpy)3]2+ 3.3. Irradiation increases the cytotoxicity\n(\u03a6 = 0.04, methanol) as a refence, the complexes (10 \u03bcM) were\n The cytotoxicity of IPPH, Ir1 and Ir2 toward BEL-7402, A549, HCT-\n\n 7\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\n\n\nFig. 5. Location assay (a) endoplasmic reticulum and (b) mitochondria of A549 cells incubated with IC50 concentration of Ir1 and Ir2 for 4 h and stained with ER-\nTracker Red or Mito-Tracker Red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)\n\n\n116, B16 cancer cells and normal LO2 was determined by MTT assay. the presence of white light, Ir2 exhibits very high anticancer effect on\nThe obtained IC50 values are listed in Table 1. As expected, IPPH shows A549 cells with a very low IC50 value (IC50 = 0.2 \u00b1 0.05 \u03bcM). Therefore,\nno or low cytotoxic activity. Unexpectedly, complexes Ir1 and Ir2 show Ir2 exhibits higher photosensitizer efficacy toward A549 cells than\nweakly cytotoxic activity against BEL-7402, A549, HCT-116, B16 cancer WSIr7, [Ir(ppy)2(ipbc)](PF6) (ipbc = 4\u2032 -(1H-imidazo[4,5-f][1,10]phe\u00ad\ncells and normal LO2 cells (IC50 > 100 \u03bcM). To enhance anticancer nanthrolin-2-yl)-(1,10-biphenyl)-4-carbaladehyde, IC50 = 48.5 \u00b1 3.8\nactivity of the complexes, the cells were treated with different concen\u00ad \u03bcM, light) [46] and [Ir(ppy)2(FBPIP)[(PF6) (FBPIP = 2-(4-formyl)ben\u00ad\ntration of the complexes for 4 h, then the cells were irradiated with white zeno[4,5-f][1,10]phenanthroline, IC50 = 22.9 \u00b1 1.2 \u03bcM) [47]. Similar\nlight (65 w LED lamp, light dose of 5.2 J cm\u2212 2, \u03bb = 450\u2013465 nm) for 40 results were observed for the cytotoxic activity of other families of\nmin, the complexes showed remarkable activity against all the cancer iridium(III) complexes [31,32]. In general, upon irradiation, the com\u00ad\ncells. In particular, Ir2 exhibits very high ability to kill A549 and plexes can increase intracellular ROS (e.g. superoxide, hydroxyl radicals\nHCT116 cells with a low IC50 value of 0.2 \u00b1 0.05 and 0.8 \u00b1 0.1 \u03bcM. or peroxides) and 1O2. Both ROS and 1O2 rapidly interact with adjacent\nComparing the IC50 values, Ir2 shows higher cytotoxic activity than Ir1 biomolecules disrupting normal cell functions that finally drives to cell\nand cisplatin under the identical conditions. Hence, irradiation can in\u00ad death [48,49]. According to the literature [50,51], we infer iridium(III)\ncrease the cytotoxic activity of the complexes toward cancer cells. Gupta complexes Ir1 and Ir2 can effectively sensitize ground-state oxygen\net al. synthesized a Ir(III) dipyrrinato complex with sugar moiety (3O2) to produce cytotoxic singlet oxygen (1O2), making them highly\n(WSIr7), the IC50 values of the complex against A549 cells are 84.3 \u00b1 1.9 effective photosensitizers for use in photodynamic therapy (PDT).\nand 17.8 \u00b1 0.5 \u03bcM in the dark and in the presence of light, respectively. Notably, Ir1 and Ir2 displayed strong PDT efficacy under irradiation and\nThe phototoxicity index (PI) shows that the photosensitizer (WSIr7) is Ir2 can be used as photosensitizer for the treatment of cancer. Based on\nabout 4 times more active under light than under the dark conditions these results, A549 cells were chosen for the undergoing experiments.\n[45]. While Ir2 has no cytotoxic activity in the dark (IC50 > 100 \u03bcM), in\n\n 8\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\n\n\nFig. 6. (a) Intracellular ROS assay of A549 cells exposure to ROSUP, IC50 concentration of Ir1 or Ir2 in the absence or presence of irradiation. (b) Assay of DCF\nfluorescence intensity after 24 h exposure of A549 cells (I) to ROSUP (II), IC50 concentration of Ir1 (III, V) or Ir2 (IV, VI) in the absence or presence of irradiation.\n\n\n\n 9\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\n increase the anticancer efficacy.\n\n 3.7. Analysis of inducing apoptosis\n\n Apoptosis, the programmed cell death, is finely regulated at the gene\n levels resulting in the efficient removal of damaged cells [56]. There\u00ad\n fore, we further investigated the effect of the complexes on apoptosis in\n the presence or absence of irradiation. As shown in Fig. 4, in the control\n (I), the percentage of early apoptosis was 2.19%. without irradiation, Ir1\n (II) and Ir2 (III) cause a weak apoptotic effect. After irradiation for 40\n min, the early apoptotic rate reached 14.9% and 15.3% after treatment\n with IC50 concentration of Ir1 (IV) and Ir2 (V), the apoptotic percentage\n increases by 12.71% and 13.11% for Ir1 and Ir2. This is consistent with\n the cell cytotoxic activity. These results reveal that the complexes can\n effectively induce apoptosis upon irradiation, and they may become\n promising PDT agents for the treatment of cancer.\nFig. 7. The determination of the concentration of MDA in A549 treated with\nIC50 concentration of Ir1 and Ir2 upon irradiation or not for 40 min. 3.8. Subcellular localization of the complexes\n\n The endoplasmic reticulum (ER) is a specialized organelle that reg\u00ad\n3.4. Analysis of the uptake of complexes\n ulates protein homeostasis that orchestrates and monitors the synthesis,\n folding, assembly, trafficking and degradation of all proteins destined\n The cellular uptake was monitored under the ImageXpress High\n [57]. Homeostasis imbalance in ER results in ER stress which further\nContent Screening System. A549 cells were treated with Ir1 (10 \u03bcM) and\n induces apoptosis [58]. Most of the iridium(III) complexes containing N-\nIr2 (10 \u03bcM) for 4 h and stained by DAPI (\u03bbex = 340 nm, \u03bbem = 488 nm),\n based ligands, such as polypyridyl, N-heterocyclic carbene (NHC),\nas the fluorescence probe. As shown in Fig. 1, Ir1 and Ir2 emit green\n phenanthroline, can accumulate in the ER membrane and mitochondria\nfluorescence due to autofluorescence properties, and the cell nuclei were\n [30,59]. The location of the complexes at the endoplasmic reticulum and\nstained blue. The merge of the green and blue fluorescence indicates that\n mitochondria after a 4 h exposure of A549 cells to IC50 concentration of\nthe complexes successfully crossed the cell membrane and entered the\n Ir1 and Ir2 was further investigated under the ImageXpress Micro XLS\ncell and mainly distribute in the cytoplasm and few amount of the\n System. As displayed in Fig. 5a, the green fluorescence of the complexes\ncomplexes distribute in the cell nuclei.\n significantly overlaps with the red fluorescence of ER-Tracker Red (\u03bbex\n = 587 nm, \u03bbem = 615 nm), suggesting that the complexes localized at the\n3.5. Wound healing assays endoplasmic reticulum. The Pearson's colocalization coefficients (PCC)\n were calculated by analyzing the red and green fluorescence intensity\n The tendency of tumor cells to metastasize and spread is a major (Image pro plus 6.0 software) in 50 cells according to literature [60].\ncause of cancer treatment failure [54]. A wound healing assay is a The PCC values are 0.94 for 1 and 0.95 for 2. To explore whether the\nstandard technique in vitro to evaluate cell migration capacity [55]. As complexes located at the mitochondria, A549 were treated with IC50\nshown in Fig. 2a and b, compared with the control group, Ir1 and Ir2 concentration of Ir1 and Ir2 for 4 h, the mitochondria were stained red\ncould attenuate the healing of wounded cell monolayers to a certain with Mito-Tracker Red (\u03bbex = 579 nm, \u03bbem = 599 nm). The complexes\nextent, but still could not stop the tendency of A549 cell migration. emit weak green fluorescence, the merge of the red and green fluores\u00ad\nHowever, after irradiation, Ir1 (light) and Ir2 (light) can effectively cence indicates that the complexes were localized at the mitochondria\ninhibit the cell migration, and the width of the wound's edge was almost (Fig. 5b). The PCC values are 0.98 for 1 and 0.94 for 2. The Pearson's\nunchanged. Wound area analysis showed that a significant reduction in colocalization coefficients for endoplasmic reticulum and mitochondria\nthe percent of wound closure was observed in A549 cells exposed to indicate an existence of positive correlation. Hence, according to the\nphotoactivated Ir1 and Ir2 compared to control cells. While Ir1 and Ir2 PCC values, we consider that the complexes accumulate both in the\nshow weak ability to prevent the cell migration without irradiation. endoplasmic reticulum and mitochondria.\nTherefore, the above results indicated that the complexes effectively\nhindered the migration of A549 cells upon irridiation and follow the 3.9. Cellular reactive oxidative species (ROS) production\norder of Ir2 (light) > Ir1 (light) > Ir2 > Ir1.\n In PDT, photosensitizers and tumor cells were exposed to trans\u00ad\n3.6. Cell cycle arrest analysis formation and endogenous molecular oxygen was converted to ROS\n 1\n (\u2022OH, O\u2022\u2212 2 , and O2) [61,62]. The elevated cellular ROS levels activate\n To determine whether the complexes regulate the cell cycle, A549 various signaling pathways, causing mitochondrial injury and oxidative\ncells were treated with IC50 concentration of Ir1 and Ir2 for 24 h stained stress, which in turn induces apoptosis [63,64]. The distribution of the\nwith propidium iodide (PI), and cell cycle distribution was analyzed by complexes in the mitochondria and the high phototoxicity induce\nflow cytometry. As shown in Fig. 3, in the control, the percentage of cellular apoptosis promoted us to study intracellular ROS generation.\nA549 cells in the S phase was 24.62%. Treatment of A549 cells with IC50 The generation of reactive oxygen species (ROS) in A549 cells was\nconcentration of Ir1 and Ir2 for 24 h, the percentages in the cells at S measured using 2\u2032 ,7\u2032 -dichlorodihydrofluorescein diacetate (DCHF-DA,\nphase are 25.71% and 30.48%, respectively. The slight changes in in\u00ad \u03bbex = 488 nm, \u03bbem = 525 nm) as fluorescence probe. DCHF-DA pene\u00ad\nhibition of S phase were observed for the complexes without irradiation. trates into the cell membrane to enter the cells and is hydrolyzed by\nHowever, A549 cells exposure to IC50 concentration of Ir1 and Ir2 for 4 intracellular esterase and converted into 2\u2032 ,7\u2032 -dichlorodihydro\u00ad\nh, the cells were irradiated for 40 min, the percentages in the cells at S fluorescein DCFH (non-fluorescent), which is rapidly oxidized by ROS\nphase are 33.03% and 50.77%, an increase of 8.41% for Ir1 and 26.15% into 2\u2032 ,7\u2032 -dichlorofluorescein (DCF). The fluorescence of DCF and the\nfor Ir2 at the S phase compared with that in the control was found, levels of intracellular reactive oxygen species were positively correlated.\naccompanied by a corresponding decrease in the G0/G1 phase. The re\u00ad As shown in Fig. 6a, when A549 cells were treated with IC50 concen\u00ad\nsults further demonstrate that photoactivation of complexes can greatly tration of the complexes Ir1 and Ir2 for 24 h, no obvious change of the\n\n 10\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\n\n\nFig. 8. Assay of mitochondrial membrane potential after A549 cells were exposed to CCCP (positive control) and IC50 concentration of Ir1 and Ir2 in the absence or\npresence of irradiation.\n\n\n\n\n 11\n\f W. Li et al.\n12\n\n\n\n\n Journal of Inorganic Biochemistry 236 (2022) 111977\n Fig. 9. Images of CRT (a), HMGB1 (b) and HSP70 (c) levels after A549 cells incubated with IC50 concentration of Ir1 and Ir2 for 24 h upon irradiation or not.\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\n\n\n Fig. 9. (continued).\n\n\ngreen fluorescence of intracellular DCF was observed compared with and Ir2. However, upon irradiation, DCF fluorescence intensity in\u00ad\nthat in the control. However, under the irradiation of white light, the creases by 18.3 and 20.1 times for Ir1 (V) and Ir2 (VI) compared with\ngreen fluorescence of A549 cells treated with ROSUP (positive control) the control. Consequently, under irradiation, the complexes can greatly\nand IC50 concentration of Ir1 and Ir2 was significantly enhanced, indi\u00ad increase the intracellular ROS levels.\ncating that the complexes can increase intracellular ROS levels upon\nirradiation.\n To quantitatively compare the efficacy of Ir1 and Ir2 on intracellular 3.10. Detection lipid peroxidation levels\nROS levels, DCF green fluorescence intensity was determined by flow\ncytometry. As illustrated in Fig. 6b, in the control (I), the fluorescence The excessive intracellular accumulation of reactive oxygen species\nintensity of DCF was 2.6 \u00d7 104. After A549 cells were exposed to ROSUP contributes to lipid peroxidative damage promoting ferroptosis which is\n(II) and IC50 concentration of Ir1 (III) and Ir2 (IV), the green fluores\u00ad a form of regulated cell death [65,66]. To investigate the effect of the\ncence intensity increases by 16.3, 1.07 and 1.02 times for ROSUP, Ir1 complexes on lipid peroxidation, the amount of lipid peroxidation\n product malondialdehyde (MDA) was measured in A549 cells. As\n\n 13\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\n\n\nFig. 10. (a) Immunofluorescence analysis of phosphorylated \u03b3-H2AX induced by IC50 concentration of Ir1 and Ir2 in the absence or presence of white light pho\u00ad\ntoirradiation. (b) Western blot analysis of \u03b3-H2A after an exposure of A549 cells to IC50 concentration of Ir1, Ir2 in the absence or presence of white light photo\u00ad\nirradiation (450\u2013465 nm, light dose = 5.2 J cm\u2212 2). (c) The gray values of \u03b3-H2AX protein.\n\n\n 14\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\n\n\nFig. 11. (a) Western blot analysis of PARP, PI3K, AKT, caspase 3, Bcl-2 and Bax after an exposure of A549 cells to IC50 concentration of Ir1, Ir2 in the absence or\npresence of irradiation (white light, 450\u2013465 nm, light dose = 5.2 J cm\u2212 2). (b) The quantitative values of the expression of PARP, PI3K, AKT, caspase 3, Bcl-2, Bax,\n\u03b2-actin was used as the internal control.\n\n\n\n\n Fig. 12. The molecular mechanism of the complexes inducing apoptosis in A549 cells.\n\n\ndisplayed in Fig. 7, compared with the control group, after an exposure a decrease in mitochondrial membrane potential, which results in\nof 24 h of A549 cells to IC50 concentration of Ir1 and Ir2, a slight in\u00ad apoptosis [67\u201369]. The effect of the complexes on the mitochondrial\ncrease in the content of malondialdehyde (MDA) was discovered. membrane potential (\u0394\u03a8m) in A549 cells was investigated using dye\nHowever, A549 cells were treated with IC50 concentration of Ir1 and Ir2 5,5\u2032 ,6,6\u2032 -tetrachloro-1,1\u2032 ,3,3\u2032 -tetraethylbenzimidazolylcarbocyanine io\u00ad\nupon irradiation for 40 min, the content of MDA increases by 6.00 times dide (JC-1, green, \u03bbex = 514 nm, \u03bbem = 529 nm, red, \u03bbex = 585 nm, \u03bbem =\nfor Ir1 and 8.32 times for Ir2 compared with that in the control. These 590 nm) as fluorescence probe. JC-1 dye forms aggregates with red\ndata demonstrated that the complexes can induce oxidant stress to fluorescence at the high mitochondrial membrane potential. Conversely,\npromote ferroptosis of A549 cells upon irradiation. the green fluorescence of the dye forms monomers corresponds to low\n mitochondrial membrane potential. A549 cells were treated with IC50\n concentration of complexes Ir1 and Ir2 for 4 h and irradiated with white\n3.11. Mitochondrial membrane potential assay\n light (5.2 J cm\u2212 2) for 40 min. After 24 h, the cells were stained with JC-\n 1. See from Fig. 8, in the control, JC-1 dye exhibited bright red fluo\u00ad\n Mitochondrial membrane potential (MMP) is an indicator of mito\u00ad\n rescence and weak green fluorescence. Treatment of A549 cells with\nchondrial activity and the oxidative stress caused by the accumulation of\n carbonylcyanide-m-chlorophenylhydrazone (CCCP, positive control)\nROS in cells changes mitochondrial membrane permeability and triggers\n\n 15\n\fW. Li et al. Journal of Inorganic Biochemistry 236 (2022) 111977\n\n\nand IC50 concentration of in the absence or presence of irradiation, the expression of caspase 3, PARP, Bax, Bcl-2, PI3K and AKT. Therefore, we\nred fluorescence intensity decreases and the green fluorescence intensity inferred that the complexes strongly trigger apoptosis upon irradiation\nincreases compared with that in the control. The change from red to by regulating Bcl-2 family proteins and inhibiting the PI3K/AKT\ngreen fluorescence suggests that the complexes can cause a reduction of pathway.\nmitochondrial membrane potential.\n 4. Conclusions\n3.12. Photoactivation of the complexes induces immunogenic cell death\n(ICD) In this article, two new complexes [Ir(ppy)2(IPPH)](PF6) (Ir1) and\n [Ir(piq)2(IPPH)](PF6) (Ir2) were synthesized and characterized by ESI-\n Upon irradiation, photosensitizers acting on the endoplasmic retic\u00ad MS, UV, 1H NMR and 13C NMR. The MTT experimental results show\nulum (ER) can cause robust ROS-based ER stress, thus inducing strong that complexes Ir1 and Ir2 show weakly cytotoxic on the tested tumor\nimmunogenic cell death (ICD) characterized by inducing various dam\u00ad cells. However, upon irradiation with white light for 40 min, the com\u00ad\nage associated molecular patterns (DAMPs) exposure [70]. ICD is plexes exhibit significant phototoxicity, especially toward the A549 cell\ncharacterized by translocation of calreticulin (CRT) from ER to the cell line with an IC50 value of 0.2 \u00b1 0.05 \u03bcM for Ir2. Further studies have\nsurface, secretion of heat shock protein 70 kDa (HSP70), migration of shown that the complexes localize at the endoplasmic reticulum (ER)\nhigh mobility group box 1 (HMGB1) from the nuclei to the cytoplasm and generate ER-stress induces immunogenic cell death, act on the\n[71\u201373]. As shown in Fig. 9, A549 cells were treated with IC50 con\u00ad mitochondria and increased the intracellular ROS, reduced the mito\u00ad\ncentration of Ir1 and Ir2 for 24 h, in the absence of irradiation, no chondrial membrane potential. In addition, the western blotting showed\nobvious change in the green fluorescence was discovered. However, that the complexes increased Bax/Bcl-2 ratio, caspase 3 and decreased\nupon irradiation for 40 min, the green fluorescence intensity enhances PARP (poly ADP-ribose polymerase), PI3K (phosphoinositide-3 kinase)\ncompared with that in the control. The degree of immunogenic cell and AKT (protein kinase B). Meanwhile, the complexes caused DNA\ndeath was reflected by the green fluorescence intensity of the antibody damage (upregulation of \u03b3-H2AX protein expression) and induce cell\n(containing fluorescein isothiocyanate (FITC, \u03bbex = 488 nm, \u03bbem = 518 cycle arrest at S phase. In conclusion, this study demonstrates that\nnm)) labeled CRT (a), HMGB1 (b) and HSP70 (c) proteins. These results photoactivatable iridium(III) complexes induce cell death in A549\nshowed that the complexes can induce immunogenic cell death upon through ROS-mediated endoplasmic reticulum stress-mitochondrial\nirradiation. pathway, DNA damage pathway and ICD (Fig. 12). This work is help\u00ad\n ful for design and synthesis new iridium(III) complexes as potent\n3.13. \u03b3-H2AX fluorescence intensity analysis endoplasmic reticulum and mitochondria-targeted photodynamic ther\u00ad\n apy (PDT) candidates for cancer treatment.\n \u03b3-H2AX is converted from phosphorylation of the Ser-139 residue of\nthe histone variant H2AX, a recognized biomarker for the double-strand Declaration of Competing Interest\nDNA breaks [74,75]. To quantitatively evaluate DNA damage, A549\ncells were treated with IC50 concentration of Ir1, Ir2 in the absence or Authors declare no competing interest exists.\npresence of white light (\u03bb = 450\u2013465 nm, light dose = 5.2 J cm\u2212 2, 40\nmin) for 24 h, and the expression level of \u03b3-H2AX (fluorescence dye Data availability\nFITC, \u03bbex = 488 nm, \u03bbem = 518 nm) was evaluated by immunofluores\u00ad\ncence staining. Fig. 10a illustrated that the control group showed a weak Data will be made available on request.\ngreen fluorescence, IC50 concentration of Ir1 and Ir2 (without irradia\u00ad\ntion) showed relatively strong green fluorescence compared with the\n Acknowledgements\ncontrol. While in the A549 cells treated with IC50 concentration of Ir1\nand Ir2 upon irradiation, bright green fluorescence was observed,\n This work was supported by the National Natural Science Foundation\nindicating that the complexes can increase the content of \u03b3-H2AX. In\n of China (No 21877018) and the Natural Science foundation of\naddition, the expression of \u03b3-H2AX protein was investigated by western\n Guangdong Province (No. 2020A1515010524).\nblot analysis. As shown in Fig. 10b and c, the expression of \u03b3-H2AX\nprotein in A549 cells was increased after treatment with the complexes\n Appendix A. Supplementary data\ncompared with control, these data further confirm that Ir1 and Ir2\nsignificantly cause DNA damage.\n Supplementary data to this article can be found online at https://doi.\n org/10.1016/j.jinorgbio.2022.111977.\n3.14. Expression of Bcl-2 family protein\n\n References\n To verify the effect of white light-activated complexes on the in\u00ad\nduction of apoptosis, the expression of B-cell lymphoma-2 (Bcl-2) family [1] M. Kanapathipillai, A. Brock, D.E. Ingber, Nanoparticle targeting of anti-cancer\nproteins was investigated by Western blot. 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