White light increases anticancer effectiveness of iridium(III) complexes toward lung cancer A549 cells.

{"full_text": " Journal of Inorganic Biochemistry 259 (2024) 112652\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\nWhite light increases anticancer effectiveness of iridium(III) complexes\ntoward lung cancer A549 cells\nGechang Li a, 1 , Jing Chen a, 1 , Yufeng Xie a , Yan Yang b, * , Yajie Niu a , Xiaolan Chen a ,\nXiandong Zeng a , Lin Zhou a , Yunjun Liu a, *\na\n School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China\nb\n Department of Pharmacy, Guangdong Second Provincial General Hospital, 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: Anticancer activity has been extensively studies. In this article, three ligands 2-(6-bromobenzo[d][1,3]dioxol-5-\nIridium(III) complexes yl)-1H-imidazo[4,5-f][1,10]phenanthroline (BDIP), 2-(7-methoxybenzo[d][1,3]dioxol-5-yl)-1H-imidazo[4,5-f]\nPhotodynamic therapy (PDT) [1,10]phenanthroline (MDIP), 2-(6-nitrobenzo[d][1,3]dioxol-5-yl)-1H-imidazo[4,5-f][1,10]phenanthroline\nApoptosis\n (NDIP) and their iridium(III) complexes: [Ir(ppy)2(BDIP)](PF6) (ppy = deprotonated 2-phenylpyridine, 3a), [Ir\nAutophagy\n (ppy)2(MDIP)](PF6) (3b) and [Ir(ppy)2(NDIP)](PF6) (3c) were synthesized. The cytotoxicity of 3a, 3b, 3c against\nFerroptosis\n Huh7, A549, BEL-7402, HepG2, HeLa, and non-cancer NIH3T3 was tested using 3-(4,5-dimethylthiazole-2-yl)-\n 2,5-diphenyl tetrazolium bromide (MTT) method. The results obtained from the MTT test stated clearly that\n these complexes demonstrated moderate or non-cytotoxicity toward Huh7, BEL-7402, HepG2 and HeLa except\n A549 cells. To improve the anticancer efficacy, we used white light to irradiate the mixture of cells and com\u00ad\n plexes for 30 min, the anticancer activity of the complexes was greatly enhanced. Particularly, 3a and 3b\n exhibited heightened capability to inhibit A549 cells proliferation with IC50 (half maximal inhibitory concen\u00ad\n tration) values of 0.7 \u00b1 0.3 \u03bcM and 1.8 \u00b1 0.1 \u03bcM, respectively. Cellular uptake has shown that 3a and 3b can be\n accumulated in the cytoplasm. Wound healing and colony forming showed that 3a and 3b significantly hinder\n the cell migration and growth in the S phase. The complexes open mitochondrial permeability transition pore\n (MPTP) channel and cause the decrease of membrane potential, release of cytochrome C, activation of caspase 3,\n and finally lead to apoptosis. In addition, 3a and 3b cause autophagy, increase the lipid peroxidation and lead to\n ferroptosis. Also, 3a and 3b increase the expression of calreticulin (CRT), high mobility group box 1 (HMGB1),\n heat shock protein 70 (HSP70), thereby inducing immunogenic cell death.\n\n\n\n\n1. Introduction recognized as the most prevalent form of cancer worldwide for several\n decades [4]. Considerable advancements have been made in compre\u00ad\n Cancer continues to proliferate and escalate in incidence in the 20st hending biochemical mechanisms and formulating therapeutic drugs\ncentury [1]. Surgical intervention, radiation therapy, and chemotherapy through extensive investigations into the reactivity and interactions\nare employed as conventional approaches for cancer treatment. between transition metal complexes and biomolecules like DNA and\nChemotherapy involves administering cytotoxic drugs that target both proteins [5,6]. Cisplatin, a platinum-based complex, has been widely\nresting and dividing cells. The objective of cancer chemotherapy is to used in cancer treatment since its approval for clinical use. It is notable\nimpede the multiplication, invasion, metastasis, and fatality caused by that carboplatin and oxaliplatin are also extensively employed in anti\u00ad\ncancer cells [2]. Despite the progress made in therapeutic approaches cancer therapy [7]. Despite the current achievements of platinum-based\nsuch as targeted therapies that focus on oncogenes, for example tyrosine medications, they come with a lot of notable constraints. Firstly, their\nkinase inhibitors, and immunotherapies like immune checkpoint in\u00ad efficacy is confined to a specific spectrum of cancers. Secondly, certain\nhibitors, chemotherapy continues to be the primary choice of treatment tumors may acquire or possess inherent resistance toward these medi\u00ad\nfor various types of cancer [3]. Lung cancer has been consistently cations. Lastly, they frequently trigger severe adverse effects including\n\n\n * Corresponding authors.\n E-mail addresses: yany@gd2h.org.cn (Y. Yang), lyjche@gdpu.edu.cn (Y. Liu).\n 1\n These authors contribute equally.\n\nhttps://doi.org/10.1016/j.jinorgbio.2024.112652\nReceived 14 April 2024; Received in revised form 29 May 2024; Accepted 25 June 2024\nAvailable online 26 June 2024\n0162-0134/\u00a9 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\nqueasiness, suppression of bone marrow function, and toxicity in the 2.2. Synthesis of ligands and complexes\nkidneys. In recent times, the demand for metal-based compounds in\ncancer treatment has been on the rise. Photodynamic therapy (PDT) is a 2.2.1. Preparation of BDIP, MDIP and NDIP\nminimally invasive procedure that has gained clinical approval and 2-(6-nitrobenzo[d][1,3]dioxol-5-yl)-1H-imidazo[4,5-f][1,10]phe\u00ad\ndemonstrates selective cytotoxic activity against malignant cells. This nanthroline (NDIP) was synthesized according to the literatures [24]. 2-\ntechnique involves using a photosensitizer followed by exposure to light (6-bromobenzo[d][1,3]dioxol-5-yl)-1H-imidazo[4,5-f][1,10]phenan\u00ad\nat a wavelength corresponding to the sensitizer\u2019s absorbance band. In throline (BDIP) and 2-(7-methoxybenzo[d][1,3]dioxol-5-yl)-1H-imi\u00ad\nthe existence of oxygen, this gives rise to a sequence of occurrences that dazo[4,5-f][1,10]phenanthroline (MDIP) were prepared as following:\nlead to prompt demise of tumor cells, impairment to the microvascular 1,10-phenanthroline-5,6-dione (0.42 g, 2.0 mmol), 6-bromobenzo[d]\nsystem, and stimulation of localized inflammatory reactions [8]. With [1,3]dioxole-5-carbaldehyde or 5-methoxybenzo[d][1,3]dioxole-5-car\u00ad\nthe advent of recent technological progressions, photodynamic therapy baldehyde (2.0 mmol, 0.36 g), and NH4Ac (40.0 mmol, 3.08 g) and\n(PDT) exhibits immense promise in becoming widely adopted for cancer glacial CH3COOH were refluxed at 130 \u25e6 C for 2 h, thereafter, the solu\u00ad\ntreatment. tion was neutralized with concentrated NH3\u22c5H2O and a yellow precipi\u00ad\n Iridium(III) polypyridine complexes have been utilized in cancer tate washed with ice water was gained.\ntherapy due to their exceptional anti-tumor activity and precise organ\u00ad BDIP: Yield: 75%. Analytical calcd for C20H11N4BrO2: C, 57.30; H,\nelle targeting [9\u201311]. Recently, iridium(III) metal complexes have sur\u00ad 2.64; N, 13.36%. Found: C, 57.08; H, 2.89%; N, 13.55%. HRMS\nfaced as promising contenders for innovative metallodrugs with (CH3CN): m/z = 419.0133. 1HNMR (DMSO\u2011d6, 500 MHz): \u03b4 9.05 (dd, J\npotential anticancer properties due to their capacity to exhibit excep\u00ad = 1.5, J = 4.0 Hz, 2H), 8.86 (dd, J = 2.0, J = 8.5 Hz), 7.84\u20137.81 (m, 2H),\ntional anticancer efficacy through various mechanisms, such as hin\u00ad 7.48 (s, 1H), 7.41 (s, 1H), 6.22 (s, 2H).\ndering protein activity related to biological processes [12,13], MDIP: Yield: 79%. Analytical calcd for C21H14N4O3: C, 68.10; H,\ncatalyzing redox reactions within cells [14,15], and interacting with 3.81; N, 15.13%. Found: C, 68.35; H, 3.66%; N, 15.25%. HRMS\nDNA [16]. (CH3CN): m/z = 386.0879. 1HNMR (CD3OD, 500 MHz): 8.85\u20138.83 (m,\n In our prior research, we discovered that a lot of iridium(III) com\u00ad 2H), 8.55 (s, 2H), 7.57\u20137.55 (m, 2H), 7.19 (s, 1H), 7.02 (s, 1H), 5.96 (s,\nplexes exhibit distinctive mechanism against cancer, including disrup\u00ad 2H), 3.91 (s, 3H).\ntion of mitochondrial function, interaction with DNA, enhancement of\nreactive oxygen species (ROS), evoking immunogenic cell death, trig\u00ad 2.2.2. [Ir(ppy)2(BDIP)](PF6) (3a)\ngering endoplasmic reticulum stress, and discharge of cytochrome c The precursor cis-[Ir(ppy)2Cl]2 [25] was synthesized using\n[17\u201321]. More notably, we have discovered the iridium(III) complex, IrCl3\u22c53H2O and 2-phenylpyridine as starting materials. Cis-[Ir(ppy)2Cl]2\nwhich functions as a photosensitizer in photodynamic therapy (PDT), (0.5 mmol, 0.536 g) and BDIP (1.0 mmol, 0.419 g) were dissolved in 60\n3\n O2 (trilinear state) can be transferred into 1O2 (singlet oxygen), thereby mL of dichloromethane and methanol (v/v, 2:1) and refluxed for 6 h,\ninducing cancer cell apoptosis [22,23]. then about 1.5 g NH4PF6 powder was added to the above solution and\n In this study, we designed three ligands and their iridium(III) metal stirred continuously for 1 h. The filtrate was extracted, the solvent was\ncomplexes [Ir(ppy)2(BDIP)](PF6) (BDIP = 2-(6-bromobenzo[d][1,3] removed, the yellow powder was gained. The crude product underwent\ndioxol-5-yl)-1H-imidazo[4,5-f][1,10]phenanthroline, ppy = deproto\u00ad purification via alumina column chromatography, and dichloromethane\nnated 2-phenylpyridine) (3a), [Ir(ppy)2(MDIP)](PF6) (MDIP = 2-(7- and methanol were used as eluents, and the orange yellow strip was\nmethoxybenzo[d][1,3]dioxol-5-yl)-1H-imidazo[4,5-f][1,10]phenan\u00ad collected, the yellow powder was obtained. Yield: 80%. HRMS (CH3CN,\nthroline) (3b) and [Ir(ppy)2(NDIP)](PF6) (NDIP = 2-(6-nitrobenzo[d] Fig. S1, SI): C42H27BrN6O2PF6Ir: m/z = 919.0811 [(M-PF6)+]. 1H NMR\n[1,3]dioxol-5-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (3c). These (500 MHz, DMSO\u2011d6, Fig. S2, SI): \u03b4 9.00 (dd, J = 1.5, J = 8.0 Hz, 2H),\ncomplexes were characterized through HRMS and NMR spectra. The 8.26 (d, J = 8.5 Hz, 2H), 7.98\u20137.95 (m, 4H), 7.90\u20137.85 (m, 4H), 7.48 (d,\nanticancer effectiveness of the complexes against BEL-7402, HepG2, J = 7.0 Hz, 2H), 7.41 (s, 1H), 7.33 (s, 1H), 7.05 (t, J = 7.5 Hz, 2H), 7.00\nHeLa, A549, Huh 7 and non-cancer NIH3T3 cells was investigated in (t, J = 7.5 Hz, 2H), 6.95 (t, J = 7.5 Hz, 2H), 6.32 (d, J = 8.5 Hz, 2H), 6.14\ndetail. (s, 2H)\u22c513C NMR (125 MHz, DMSO\u2011d6, Fig. S3, SI): \u03b4 172.67, 167.49,\n 151.83, 149.30, 148.17, 147.26, 146.24, 144.55, 143.16, 138.94,\n2. Experimental 131.97, 131.77, 130.63, 126.76, 126.72, 125.45, 124.24, 122.59,\n 120.36, 113.43, 112.87, 111.85, 102.53.\n2.1. Materials and methods\n 2.2.3. [Ir(ppy)2(MDIP)](PF6) (3b)\n IrCl3\u22c53H2O was procured from Kunming Boren Precious Metals Co., This complex was synthesized using the same procedure as 3a, with\nLTD, 2-phenylpyridine, 6-bromobenzo[d][1,3]dioxole-5-carbaldehyde, MDIP instead of BDIP. Yield: 85%. HRMS (CH3CN, Fig. S4, SI):\n5-methoxybenzo[d][1,3]dioxole-5-carbaldehyde and 6-nitrobenzo[d] C43H30N6O3PF6Ir: m/z = 871.2011 [(M-PF6)+]. 1H NMR (500 MHz,\n[1,3]dioxole-5-carbaldehyde were sourced from HERK Chemical Co., DMSO\u2011d6, Fig. S5, SI): \u03b4 9.14 (dd, J = 1.0, J = 8.0 Hz, 2H), 8.28 (d, J =\nLTD (Beijing). Additionally, Guangzhou Chemical Reagent Factory 8.5 Hz, 2H), 8.15 (d, J = 5.5 Hz, 2H), 8.09\u20138.06 (m, 2H), 7.96 (d, J = 7.5\nprovided the 1,10-phenanthroline. The Fetal Bovine Serum (FBS) and Hz, 2H), 7.89 (t, J = 7.5 Hz, 2H), 7.64 (d, J = 5.0 Hz, 1H), 7.53\u20137.51 (m,\nDulbecco\u2019s Modified Eagle Medium (DMEM) were procured from Gibco. 3H), 7.07 (t, J = 7.5 Hz, 2H), 7.02\u20136.95 (m, 4H), 6.31 (d, J = 7.5 Hz,\nBEL-7402 (human hepatocellular), HepG2 (human hepatocellular), 2H), 6.16 (s, 2H), 4.00 (s, 3H). 13C NMR (125 MHz, DMSO\u2011d6, Fig. S6,\nHeLa (human cervical), A549 (human lung cancer), Huh7 (human liver SI): \u03b4 168.83, 152.55, 150.95, 150.79, 149.44, 145.93, 145.50, 145.43,\ncancer) and NIH3T3 (mouse embryonic fibroblasts) cells were obtained 140.52, 138.14, 133.98, 133.11, 132.12, 128.37, 126.93, 125.69,\nfrom the Cell Center of Sun Yat-Sen University (Guangzhou, China). 124.18, 121.86, 108.67, 103.71, 102.44, 58.48.\n The HRMS analysis was performed on Xevo G2-XS QT mass analyzer\n(Waters, USA) by directly injecting the sample. The NMR spectra were 2.2.4. [Ir(ppy)2(NDIP)]PF6 (3c)\nobtained on a Varian-500 spectrometer (500 MHz), DMSO\u2011d6 and tet\u00ad [Ir(ppy)2(NDIP)](PF6) (3c) was prepared using the same procedure\nramethylsilane (TMS) were used as solvent and internal standard. UV- as 3a, with NDIP instead of ligand BDIP. Yield: 84%. HRMS (CH3CN,\n2700 ultraviolet spectrophotometer (Shimadzu, Japan) and F-2500 Fig. S7, SI): C42H27IrN7O4: m/z = 886.2736 [(M-PF6)+]. 1H NMR (500\nfluorescence spectrophotometer (Hitachi, Japan) were utilized to MHz, DMSO\u2011d6, Fig. S8, SI): \u03b4 8.95 (d, J = 8.0 Hz, 2H), 8.26 (dd, J = 1.5,\ndetermine the UV\u2013Vis and fluorescence spectra. J = 8.5 Hz, 2H), 8.02 (d, J = 4.0 Hz, 2H), 7.96\u20137.85 (m, 6H), 7.61 (s,\n 1H), 7.58 (s, 1H), 7.50 (d, J = 6.0 Hz, 2H), 7.06 (t, J = 6.5 Hz, 2H), 7.00\n\n 2\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\n(t, J = 6.0 Hz, 2H), 6.95 (t, J = 7.0 Hz, 2H), 6.32\u20136.29 (m, 4H). 13C NMR of 3a or 3b for 6 h upon light or without light, were then washed three\n(125 MHz, DMSO\u2011d6, Fig. S9, SI): \u03b4 172.51, 167.42, 151.45, 150.54, times and stained with a C11-BODIPY fluorescent probe for 30 min and\n149.42, 147.23, 144.52, 143.82, 143.72, 139.01, 132.09, 131.73, photographed. Treatment of A549 cells with IC50 concentrations of 3a or\n130.66, 126.70, 125.48, 124.27, 122.67, 120.38, 109.99, 105.46, 3b upon irradiation or not for 26 h, flow cytometry was applied to\n103.85. determine the red and green fluorescence intensity.\n Note: The procedures for pKa values determination, lipid-water\npartition coefficient, cell culture, detection of singlet oxygen produc\u00ad 2.5. Western blotting assay\ntion, cellular uptake, scratch wound healing and colony-forming assay,\nmitochondrial and lysosomes localization, mitochondrial permeability Treatment of A549 cells with 3a, 3b upon irradiation or not for 26 h,\ntransition pore (MPTP), membrane potential determination (MMP), the concentration of protein in the supernatant was obtained according\ncytochrome C, cell cycle distribution, apoptosis, autophagy, and mea\u00ad to BCA (bicinchoninic acid) assay. Amount of the proteins in the per lane\nsurement of CRT, HSP70, HMGB1, ATP (adenosine triphosphate) can be is equal in the sodium dodecyl sulfate-polyacrylamide gel electropho\u00ad\ndiscovered in the supporting materials. resis. After gel electrophoresis, the gels were transferred to poly\u00ad\n vinylidene difluoride membranes (Millipore) and blocked with 5% non-\n2.3. Cytotoxicity assay in vitro fat milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH\n 8.0) buffer for 3 h. After washing four times with TBST buffer, the\n The complexes were dissolved in DMSO, the final concentrations of membranes were incubated with primary antibody solution at 4 \u25e6 C\nDMSO are <0.05%. The cell viability in the presence of 3a, 3b, 3a (light) overnight, washing three times again, the secondary antibodies were\nand 3b (light) was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5- then conjugated with horseradish peroxidase (1:5000 dilution) for 70\ndiphenyltetrazolium (MTT) [26], cisplatin was applied as a positive min at room temperature. Finally, the blots were treated with the\ncontrol. The cancer cells were seeded into 96-well microplates and Amersham ECL Plus western blotting detection reagents.\nincubated in a 5% CO2 incubator at 37 \u25e6 C for 24 h, then different con\u00ad\ncentrations of complexes (100 \u2192 1.56 \u03bcM) were added to the wells for 2.6. Data analysis\n48 h, while in the light groups, after a 4 h interaction of 3a, 3b and 3c\nwith cancer cells, then irradiated for 30 min and continuously cultured The mean \u00b1 SD was used to express all data, and t-tests were\nfor 48 h, MTT dye solution (20 \u03bcL, 5 mg/mL) was added to each well. employed to evaluate statistical significance. Significance was deter\u00ad\nAfter 4 h, 100 \u03bcL dimethylformamide (50%) and 20% sodium dodecyl mined at a level of *P < 0.05, **P < 0.01, ***P < 0.001.\nsulfate (20%) was applied to dissolve MTT formazan product, the optical\ndensity was measured at 490 nm. Each experiment was performed in 3. Results and discussion\ntriplicate. The cell viability was gained based on the equation:\n 3.1. Syntheses and characterization\n\n\n (( )/ )\nCell viability% = Asample \u2212 Amedium control (Acell control \u2212 Amedium control ) \u00d7100\n\n\n\n The general procedure for 3a, 3b, 3c is illustrated in Scheme 1, li\u00ad\n Whereas Asample, Amedium and Ablank are the absorbance values of gands BDIP, MDIP and NDIP were synthesized through condensation of\nsample, medium and control. 1,10-phenanthroline-5,6-dione with 6-bromobenzo[d][1,3]dioxole-5-\n carbaldehyde, 5-methoxybenzo[d][1,3]dioxole-5-carbaldehyde and 6-\n2.4. Ferroptosis studies nitrobenzo[d][1,3]dioxole-5-carbaldehyde. Subsequently, the synthe\u00ad\n sis of 3a, 3b and 3c was performed via the reaction with [Ir(ppy)2Cl]2\n The glutathione (GSH) and glutathione disulfide (GSSG) Assay Kit and BDIP, MDIP, NDIP in a dichloromethane-methanol under argon.\nwas applied to determine the amount of reduced GSH. Seeded in a 6-well With dichloromethane-methanol as eluent, neutral alumina column\nplate and cultured for 24 h, A549 cells were dealt with IC50 concentra\u00ad chromatography was applied to purify 3a-3c. In the HRMS spectra, [M-\ntions of 3a and 3b for 26 h, while in the light-treated groups, A549 cells PF6]+ signal was observed which corresponded to the expected molec\u00ad\nwere incubated with IC50 concentrations of 3a and 3b for 4 h, then ular weight. Additionally, in the 1HNMR spectra, the signals for the\nirradiation for 30 min and continuously incubated for 26 h. Thereafter, protons in the imidazole ring were not found, the signals at 6.14, 6.16\nA549 cells were trypsinized, washed three times with PBS (phosphate ppm are assigned to the protons (-CH2) in the dioxolane of 3a, 3b,\nbuffer saline). Glutathione content was determined via measuring whereas the peak of 4.00 ppm for 3b is attributed to the protons\nabsorbance at 412 nm. (-OCH3). In the 13CNMR spectra, the signals at 102.53, 102.44 and\n In the assay of malondialdehyde (MDA) content, A549 cells, treated 103.85 ppm are assigned to the carbon atoms in the dioxolane ring of 3a,\nwith IC50 concentrations of 3a and 3b for 26 h upon irradiation or not, 3b, 3c, the peak at 58.48 ppm is attributed to the carbon at -OCH3. The\nwere washed three times with PBS and then lysed using a mixture of purity of 3a, 3b and 3c was detected by high performance liquid chro\u00ad\nRIPA (50 mM Tris, pH = 7.4, 150 mM sodium chloride, 1% NP-40, and matography (HPLC), methanol was used as mobile phase. As observed\n0.5% sodium deoxycholate) and PMSF (phenylmethanesulfonyl fluo\u00ad from Fig. S10 (SI), what a main peak was uncovered revealed that pure\nride). Insoluble cell debris was removed by centrifuging at 15,000g at compounds were gained with a purity value of 96.23, 98.86 and 97.47%\n4 \u25e6 C for 10 min. Total protein was determined using pierce bicinchoninic for 3a, 3b and 3c.\nacid (BCA) protein assay kit. The lysates obtained were detected with the The UV\u2013Vis spectra of complexes 3a-3c (10.0 \u03bcM) in PBS were\nMDA assay kit. The content of MDA can be obtained by calculating the determined (Fig. S11a, SI). The maximum absorbance wavelengths\ncontent of protein and MDA in the solution. appear at 273 nm (\u03b5 = 62,150), 258 nm (\u03b5 = 41,100) and 270 nm (\u03b5 =\n Lipid peroxidation was detected by 4,4-difluoro-5-(4-phenyl-1,3- 77,290) for 3a, 3b and 3c, respectively. The complexes emit weak green\nbutadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11- fluorescence (Fig. S11b, SI), appearing at 628 nm (\u03bbex = 273 nm), 627\nBODIPY) fluorescent probe. A549 cells, treated with IC50 concentrations nm (\u03bbex = 258 nm), 628 nm (\u03bbex = 270 nm).\n\n 3\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\n\n\n H\n\n\n\n\n H\n\n\n\n\n H\n\n\n\n\n Scheme 1. Synthetic route of BDIP, MDIP, NDIP, 3a, 3b and 3c.\n\n\n The stability of 3a, 3b and 3c in PBS solution was determined by Notably, all pKa values for 3a-3c exceed 4, indicating its potential ability\nusing UV\u2013Vis spectra. As depicted in Fig. S12 (SI), at 0 and 24 h, the to traverse cell membranes and serve as a potent drug candidate.\npeak shape has no change, indicating that the complexes are stable. Additionally, we determined the lipid-water partition coefficient\n (logP) using shake flash method [28]. The logP values for 3a, 3b and 3c\n were measured to be 0.4987, 0.1956 and 0.7833, respectively, indi\u00ad\n3.2. pKa values determination cating that the complexes can enter the cancer cells.\n\n Substances that have pKa values higher than 4 for acids and lower\n 3.3. Irradiation increases the cytotoxicity\nthan 10 for bases predominantly charged at physiological pH can cross\nthe blood-brain barrier. The pKa value of a drug directly affects its lip\u00ad\n In a cytotoxicity study, the average IC50 concentrations values of 3a,\nophilicity, solubility, protein binding, and permeability. Consequently,\n 3b and 3c against BEL-7402, HepG2, HeLa, A549, Huh7 and non-cancer\nit has a significant influence on crucial pharmacokinetic characteristics\n NIH3T3 cells were determined applying 3-(4,5-dimethylthiazole-2-yl)-\nsuch as absorption, distribution, metabolism, and excretion [27]. The\n 2,5-diphenyl tetrazolium bromide (MTT) [26]. The obtained results are\ntitration curve of 3a-3c is depicted in Fig. S13 (SI), the pH values of 3a-\n presented in Table 1, 3a exhibits moderate anti-tumor activity against\n3c were measured as 6.59, 6.77, and 6.40, respectively, while the pKa\n BEL-7402, HepG2, HeLa and Huh7 cells. Surprisingly, 3a shows high\nvalues were determined to be 6.43, 6.85, and 6.05 for 3a, 3b and 3c.\n anticancer efficiency toward A549 cells with a low IC50 value of 4.0 \u00b1\n 0.6 \u03bcM, while 3b and 3c display no or low cytotoxic activity on the above\nTable 1 cells. To enhance the anti-tumor activity, after the cells were incubated\nIC50 values (\u03bcM) of 3a, 3b and 3c treating cancer cells for 48 h. with 3a, 3b or 3c for 4 h, then the cells was subjected to LED lamp (white\n Complex BEL- HepG2 HeLa A549 Huh7 NIH3T3 light, 65 W, \u03bb = 460 nm, 7.03 J/cm2) exposure of 30 min, 3a-3c dem\u00ad\n 7402 onstrates significant anti-tumor effects, especially, 3a and 3b possess\n 17.1 \u00b1 9.3 \u00b1 19.1 \u00b1 4.0 \u00b1 16.6 \u00b1 16.3 \u00b1 strong antiproliferative ability to prevent A549 cell proliferation with a\n 3a\n 0.4 1.7 1.3 0.6*** 1.6 1.7 low IC50 value of 0.7 \u00b1 0.3 and 1.8 \u00b1 0.1 \u03bcM. Their anticancer activity\n 3a 6.4 \u00b1 2.8 \u00b1 19.7 \u00b1 0.7 \u00b1 8.8 \u00b1\n 7.3 \u00b1 0.4 on A549 cells is higher than that of cisplatin. The complexes show\n (light) 0.4 0.06 0.8 0.3** 1.3\n 26.8 \u00b1 26.8 \u00b1 moderate cytotoxic effect on non-cancer NIH3T3 cells. Among the three\n 3b > 100 > 200 > 100 > 200\n 5.7** 2.1 complexes, upon irradiation, 3a as Br atom as substitute shows the\n 3b 43.2 \u00b1\n > 100 > 100\n 1.8 \u00b1 30.0 \u00b1 11.9 \u00b1 highest cytotoxic effect on all the selected cancer cells, this may be\n (light) 5.7 0.1** 1.6 1.0 caused by the strongest ability to produce ROS.\n 34.3 \u00b1 17.5 \u00b1\n 3c > 200 > 200 > 200 > 100\n 3.4 0.7\n To investigate the reason for different cytotoxicity of 3a, 3b and 3c\n 7.3 \u00b1 34.3 \u00b1 29.2 \u00b1 13.9 \u00b1 toward cancer cells under white light, we determined the singlet oxygen\n 3c (light) > 100 > 200\n 0.9 1.8* 3.9 0.6 (1O2) and quantum yield \u03a6.\n Cisplatin\n 14.8 \u00b1 9.4 \u00b1 5.7 \u00b1\n 6.6 \u00b1 0.7 nd nd When combined with 1O2, 1,3-diphenylisobenzofuran (DPBF) is\n 3.4 1.5 1.1\n irreversibly oxidated to form 1,2-dibenzoylbenzene, the absorbance\nData for cisplatin from Ref [20, 21], *p < 0.05, **p < 0.01, ***p < 0.001. deceases. As shown in Fig. S14 (SI), the absorbance of DPBF at 411 nm\n\n 4\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\nreduced by 76.0% for 3a, 66.3% for 3b and 48.6% for 3c, respectively, blue. The overlapping signals from green and blue fluorescence indicate\nhence, 3a as Br atom as substitute exhibits the strongest ability to pro\u00ad that both 3a and 3b possess a strong capability to efficiently enter cells\nduce singlet oxygen among these complexes. These results indicating and mainly distribute within their cytoplasm (Fig. 2a).\nthat the complexes can produce singlet oxygen which further leads to To quantify the cellular uptake, cancer A549 cells were cultured with\ncancer cell death. 3a or 3b (IC50 concentration) for 26 h, the results obtained are presented\n In addition, the quantum yield \u03a6 were calculated from the following in Fig. 2b. Compared to that in the control group, the treated groups\nEq. [29]: exposed to 3a, 3b, 3a (light) and 3b (light) exhibited a different degree\n ( / ) ( / ) increase in green fluorescence intensity, further indicating their excel\u00ad\n\u03a6sample = \u03a6Ref \u00d7 Ksample KRef \u00d7 FRef Fsample\n lent ability to penetrate cells. These findings suggest that both 3a and 3b\n K is the slope, F is calibration factor of the absorbance, F = 1\u201310-OD possess remarkable cell penetration capabilities as well as cytoplasmic\n(OD is the absorbance of the photosensitizer at the light source wave\u00ad distribution.\nlength). [Ru(bpy)3]2+ was used as a reference (\u03a6 = 0.81, methanol) Colony formation is an effective method for assessing the complexes\n[30]. The quantum yields \u03a6 for 3a, 3b and 3c were determined to be to prevent the cell proliferative capacity, and this can be quantified by\n0.814, 0.679 and 0.382, respectively. The quantum yield of 3a is the calculating the colony formation rate. Following exposure to IC50 con\u00ad\nlargest among the three complexes, hence, 3a exhibits higher anticancer centrations of 3a and 3b for 7 days (Fig. 2c), the number of A549 cells\nactivity than 3b and 3c. decreased by 33.3% and 36.3% compared to that in the control. How\u00ad\n We also applied Gaussian 09 package [31] at the level of B3LYP ever, under irradiation, there was a significant reduction in cell numbers\ndensity functional in conjunction with the 6-31G(d) basis sets for C, H, by 81.5% for 3a and 80.9% for 3b, respectively (Fig. S15a, SI). These\nN, O, Br, atoms, LanL2DZ for Ir atom to calculate the energy of the results affirm that both 3a and 3b effectively suppress A549 cell\nlowest unoccupied orbital (LUMO) and the highest occupied orbital proliferation.\n(HOMO) to verify the cytotoxic order of 3a > 3b. In general, small \u0394E Tumor metastasis is a complex process involving a series of changes,\nvalue between LUMO and HOMO will result in easy deformation of the including cell migration, invasion, tumor growth, and angiogenesis.\nelectron cloud corresponding to high anticancer activity. As depicted in Observing from Fig. 2d, after exposure of A549 cells to 3a or 3b for 26 h,\nFig. 1, the \u0394E values are 2.6207 eV for 3a, 2.7712 eV for 3b, respec\u00ad the number of cells decreased by 10.8% and 23.4%. On the other hand,\ntively. The \u0394E value of 3a is smaller than that of 3b, indicating that the upon irradiation, the number of cells decreased by 28.5% for 3a and\ncytotoxic activity of 3a is higher than those of 3b, which is consistent 35.1% for 3b (Fig. S15b, SI). The findings hint that 3a and 3b effectively\nwith the cytotoxic activity of these complexes. Similar results were inhibit A549 cell migration.\nfound in the other iridium(III) complexes [32,33]. The focal adhesion kinase (FAK) is a highly phosphorylated kinase\n Owing to 3c exhibiting low cytotoxicity on A549 cells, in the during cell adhesion, playing a pivotal role in tumor invasion and\nfollowing cell experiments, 3a and 3b were used to explore the anti\u00ad metastasis [35,36]. In patients, elevated FAK expression is associated\ncancer efficiency on A549 cells. with poor prognosis and resistance to certain major treatments [37]. As\n depicted in Fig. 2e, both 3a and 3b downregulated the expression of FAK\n protein. These findings once again substantiate that both 3a and 3b\n3.4. Cellular uptake, cell colonies and wound healing studies possess significant capability to inhibit A549 cell migration.\n\n Understanding the mechanisms underlying the interaction between 3.5. Cell cycle arrest analysis\ncomplexes and cells, as well as their impact on cellular uptake, is crucial\nfor exploring the biomedical applications of complexes, particularly in If there is DNA damage, the cell cycle will stop at appropriate\ndrug delivery. Various factors, such as disparities in complex physical checkpoint, and cell cycle arrest gives cell additional time to repair the\nproperties or alterations in cell membrane characteristics, can influence damage, thus reducing the mutations and preventing the development\nthe process of interaction and uptake by cells [34]. Treatment of A549 of tumors [38]. To explore the complexes to block the cell growth at G0/\ncells with 3a and 3b (IC50 concentrations) for 6 h, we employed image G1, S or G2/M phase, the cell cycle distribution was studied. See from\nXpress Micro XLS to investigate cell uptake and determine whether the Fig. 3a, the percentage of cells in the control was 67.58% in the G0/G1\ncomplexes could penetrate in the cells. Due to inherent properties of the stage, 28.51% in the S stage, and 5.90% in the G2/M stage. In the 3a, 3b,\ncomplex itself, both 3a and 3b were able to emit green fluorescence 3a (light), 3b (light) treatment groups, an increase in the cell at the S\nwhile 4\u2032,6-diamidino-2-phenylindole (DAPI) staining rendered nuclei phase of 12.86% for 3a, 14.47% for 3b, 18.29% for 3a (light) and\n 23.57% for 3b (light) was found, which suggests that 3a and 3b block the\n cell growth at S phase. Upon irradiation, 3a (light) and 3b (light) show\n higher efficiency than 3a and 3b without light.\n The regulation of the cell cycle involves cyclins, serine or threonine\n cyclin-dependent kinases (CDKs) [39]. Cyclin B1 and cyclin A2 pre\u00ad\n dominantly expressed in the S and G2/M phases. Since the formation of\n a complex between cyclin proteins and CDK proteins is necessary for\n regulating the cell cycle, we also analyzed CDK2 and CDK4 proteins. As\n depicted in Fig. 3b, the expression of cyclin B1, cyclin A2, CDK2, and\n CDK4 proteins were validly inhibited. These findings suggest that 3a and\n 3b primarily induce cell stasis in the S phase through reduced expression\n of cyclin B1, cyclin A2, CDK2, and CDK4. Taken together, these results\n unveil that 3a and 3b block proliferation in A549 at the S phase.\n\n 3.6. Localization, mitochondrial permeability transition pore and\n membrane potential determination\n\n In addition to biological energy functions, mitochondria play a\n crucial role in regulating cell death, maintaining ion homeostasis,\n Fig. 1. The energy of LUMO and HOMO of 3a and 3b. oxidizing carbohydrates and fatty acids, as well as participating in\n\n 5\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\n\n\nFig. 2. (a) Cellular uptake of a 6 h exposure of A549 cells to IC50 concentrations of 3a, 3b, 3a (light) and 3b (light). (b) Ratio of green fluorescence, (c) colony\nformation, (d) wound healing assay, (e) expression of FAK protein while A549 cells were exposed to IC50 concentrations of 3a, 3b, 3a (light) and 3b (light) for 26 h.\n\n\nvarious catabolic and anabolic pathways [40]. Studies have demon\u00ad disruption on the green fluorescence. It is well known that the red\nstrated that organometallic complexes possess the ability to specifically fluorescence stands for high mitochondrial membrane potential (MMP),\ntarget organelles such as mitochondria and lysosomes, leading to green fluorescence indicates a low MMP. As depicted in Fig. 4e, bright\ncellular dysfunction and subsequent organelle damage [41]. The local\u00ad red (monomer) and poor green fluorescence (aggregates) was discov\u00ad\nization of these complexes at the mitochondria and lysosomes was ered corresponding to high MMP (control), while in the cisplatin (pos\u00ad\ndetermined using Mito Tracker Deep Red and Lyso-Tracker Red probes. itive), 3a, 3b, 3a (light) and 3b (light)-treated groups, we uncovered a\nObserve from Fig. 4a and b, 3a and b emitted green fluorescence, lyso\u00ad reduced red and an increasing green fluorescence corresponding to low\nsomes and mitochondria were dyed red, merge of red and green indi\u00ad MMP. The ratio of red/green fluorescence was measured and depicted in\ncated that 3a, 3b successfully entered A549 cells and acted on the Fig. 4f, the results further affirm that 3a and 3b can effectively cause a\nmitochondria and lysosomes. Pearson\u2019s colocalization coefficients (PCC) reduction in MMP. In summary, these findings suggest that 3a and 3b\nwere calculated based on the red and green fluorescence in 50 cells by interact on the mitochondria, lead to mitochondrial damage and ulti\u00ad\nImage pro plus 6.0 software according to literature [42]. For lysosomes, mately cause apoptosis.\nthe PCC values are 0.9882 for 3a, 0.9880 for 3b, 0.9976 for 3a (light),\nand 0.9989 for 3b (light). For mitochondria, the PCC values are 0.9808 3.7. Studies of intracellular cytochrome c level\nfor 3a, 0.9951 for 3b, 0.9934 for 3a (light), and 0.9976 for 3b (light),\nthese results demonstrate that the positive correlations exist. In addition to its involvement in electron transfer, cytochrome c also\n Interaction of mitochondria will lead to an open of mitochondrial plays an important role in apoptosis [45] and is essential for caspase-\npermeability transition pore (MPTP), the MPTP opening has been linked mediated apoptosis [46]. Therefore, it is imperative to detect intracel\u00ad\nto a lot of types of neuronal demise, apoptosis, ischemia-induced cell lular cytochrome c level. As depicted in Fig. S16a (SI), poor green\ndeath, excitotoxicity-related damage, and neurotoxicity [43]. Normally, fluorescence was seen in the blank. However, after treatment with 3a,\nMPTP is turned off and CoCl2 cannot enter mitochondria. On the con\u00ad 3b, 3a (light) and 3b (light) (IC50 concentration) for 6 h, we observe\ntrary, after the complexes act on the mitochondria, which induces a obvious green fluorescence, which hinted that cytochrome c was liber\u00ad\nlarge amount of extracellular Ca2+ to enter cells, resulting in mito\u00ad ated from the mitochondria. Subsequently, a high-content analysis sys\u00ad\nchondrial calcium overload and passive MPTP opening. Observe from tem was used to quantitatively detect the content of cytochrome c, as\nFig. 4c and d, a bright green fluorescence was uncovered (control). shown in Fig. S16b (SI), green fluorescence enhances compared with\nHowever, the green fluorescence reduced (lonomycin, positive control; that in the control. These findings demonstrated that iridium(III) metal\n3a, 3b, 3a (light) and 3b (light)-treated groups). This is because CoCl2 complexes lead to mitochondrial dysfunction, finally prompt the liber\u00ad\ncan enter the mitochondria to bind calcein AM and further lead to ation of cytochrome c from the mitochondria.\nfluorescence quenching after an open of mitochondrial permeability\ntransition pore. Hence, a decrease in the green fluorescence indicated 3.8. Apoptosis and apoptotic mechanism studies\nthat 3a and 3b may lead to an open of mitochondrial permeability\ntransition pore. Programmed cell death, also known as apoptosis, is a natural process\n Mitochondrial membrane potential serves as a sensitive indicator of that eliminates senescent cells. Many treatments for cancer activate\nmitochondrial damage, primarily occurring as an early event in the pathways that induce apoptosis and lead to the elimination of malignant\nintrinsic pathway of mitochondrial cell death. It plays a crucial role in cells. However, when the signals that control apoptosis are disrupted,\nmaintaining physiological ATP production through the electron trans\u00ad particularly when the anti-apoptotic system is triggered, it enables\nport chain, while significant loss of mitochondrial membrane potential cancer cells to evade this natural process of cell death. As a consequence,\nleads to energy depletion and subsequent cell demise [44]. To access the uncontrolled cell division occurs, leading to tumor survival and resis\u00ad\nimpact of 3a and 3b on mitochondrial damage, we employed 5,5\u2032-6,6\u2032- tance to treatment, ultimately resulting in the recurrence of cancer [47].\ntetrachloro-1,1\u2032-3,3\u2032-tetramethylbenzomethylene carbon cyanide iodide See from Fig. 5a, the early apoptotic rate was 2.21% (control). After\n(JC-1) as fluorescent probes to assess alterations in mitochondrial treating A549 cells with IC50 concentration of 3a and 3b for 26 h, the\nmembrane potential using the complexes as references to rule out early apoptosis rates in the 3a and 3b-treated groups only increased by\n\n 6\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\n\n\nFig. 3. (a) The distribution of A549 (I) cell cycle and (b) the expression of CDK2, CDK4, cyclin A2, and cyclin B1 after a 26 h of treatment with 3a (4.0 \u03bcM, II), 3b\n(26.8 \u03bcM, III), 3a (light) (0.7 \u03bcM, IV) and 3b (light) (1.8 \u03bcM, V).\n\n\n4.89% and 3.19%, respectively. However, upon irradiation, the early growth and homeostasis in both normal and malignant tissues within\napoptosis rate caused by 3a (light) and 3b (light) was increased by multicellular organisms [53]. The B-cell lymphoma/leukemia-2 (Bcl-2)\n14.19% and 7.89%, respectively. The results showed that 3a and 3b protein family, consisting of pro-apoptotic and anti-apoptotic proteins,\ncould effectively induce the early apoptosis of A549 cells upon plays an important role in apoptosis regulation, especially through\nirradiation. intrinsic pathways, as they are upstream of irreversible cell damage and\n Pharmaceuticals or therapeutic approaches that can normalize primarily act on mitochondria, in which Bcl-2-associated X (Bax) also\napoptosis signaling pathways hold the potential to eradicate cancer has pro-apoptotic effects [54]. p38 mitogen-activated protein kinase\ncells, as they heavily depend on these abnormalities for survival [48]. (p38 MAPK) is often activated by a lot of environmental stress stimuli\nThe PI3K/AKT/mTOR (PI3K: phosphoinositide 3 kinase; AKT: Protein and cytokines that cause inflammation. Excessive inflammation can lead\nKinase B; mTOR: mammalian target of rapamycin) signaling pathway to many diseases, including cancer [55]. Vascular endothelial growth\nplays a crucial role in promoting malignant processes and conferring factor (VEGF) is an important regulator of angiogenesis and can be\ndrug resistance among individuals diagnosed with solid tumors [49]. involved in regulating the apoptosis of vascular endothelial cells [56],\nThe growth and progression of colorectal cancer (CRC) are significantly playing a key role in the malignant behavior of breast cancer cells [57].\ninfluenced by the PI3K/AKT/mTOR pathway [50]. Poly ADP-ribose Therefore, western blotting was employed to analyze key proteins\npolymerase 1 (PARP-1) is an enzyme that binds to DNA and plays a associated with the intrinsic apoptotic pathway. Observe from Fig. 5b,\nrole in DNA damage, programmed cell death, and maintaining genetic following a 26 h process with 3a, 3b on irradiation or not, the down-\nintegrity [51]. PARP is indispensable in apoptosis [52]. The activation of regulation of PARP, caspase 3, and VEGFA were observed. Further\u00ad\na cascade of caspase enzymes characterizes the executive stage of more, there was a reduction in the expression levels of PI3K, AKT,\napoptosis. Caspase-3 is believed to initiate proteolysis in the process of mTOR, p-mTOR, and Bcl-2, notably more significant reductions were\napoptosis execution, while also serving as a crucial regulator for cellular observed in the light-treated group. Additionally, we also found that the\n\n 7\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\n\n\nFig. 4. Co-location of the complexes in the lysosomes (a) and mitochondria (b), (c) mitochondrial permeability transition pore (MPTP) open, (d) green fluorescence\nintensity, (e) change of mitochondrial membrane potential, (f) the ratio of red/green fluorescence intensity. (For interpretation of the references to colour in this\nfigure legend, the reader is referred to the web version of this article.)\n\n\n\nexpression levels of p38 MAPK and Bax increased. These findings indi\u00ad ratio was reduced (Fig. 6e), further hinting that 3a, 3b, 3a (light) and 3b\ncate that 3a and 3b cause apoptosis via hindrance of PI3K/AKT/mTOR (light) caused a high degree of lipid peroxidation.\nand intrinsic apoptotic signaling pathways mediated by mitochondria. Ferrostatin-1 (Fer-1), a well-established ferroptosis inhibitor, has\n gained significant popularity for its extensive utilization [65,66]. The\n3.9. Detection of ferroptosis inhibitory effect of Fer-1 on ferroptosis is mainly dependent on the in\u00ad\n hibition of lipid peroxidation [67]. To assess whether increasing lipid\n Ferroptosis is an emerging mechanism of cellular demise character\u00ad peroxidation inducing ferroptosis, we used Fer-1 to evaluate the change\nized by lipid peroxidation that is reliant on iron [58,59]. The primary in the cell viability. As shown in Fig. 6f, after the cells were pretreated\nfeatures of Ferroptosis include the elevation in malondialdehyde (MDA) with Fer-1 and 3a, 3b, 3a (light), 3b (light) for 36 h, the cell viability\nconcentration, reactive oxygen species (ROS) level, and Fe2+ level, along increases compared with those caused by 3a, 3b, 3a (light), 3b (light)\nwith a reduction in glutathione (GSH) content [60]. The GSH (reduced alone. These results suggested that the increasing lipid peroxidation may\nglutathione) and MDA levels were assessed using the GSH and GSSG induce ferroptosis.\nAssay Kit as well as the MDA assay kit (Beyotime Biotechnology, China), Iron is an essential element needed by cells and is thought to be a key\nrespectively. Observe from Fig. 6a and b, we found that the level of GSH factor in ferroptosis. Ferritin acts as an essential iron storage protein in\nreduced, MDA amounts increased after an incubation of A549 cells with cells, forming polymers with crystalline iron nuclei [68]. Ferritin plays\n3a, 3b, 3a (light), 3b (light) for 26 h. The results suggest that 3a, 3b can an important role in preventing cardiac ferroptosis and subsequent heart\nreduce GSH concentration, increase MDA amounts, resulting in a sus\u00ad failure [69]. As shown in Fig. 6g, compared with that in the control, the\ntained oxidative stress response. expression levels of glutathione peroxidase 4 (GPX4) and Ferritin were\n p53 governs the expression of a range of genes responsible for up\u00ad downregulated. In summary, reduction of GSH, increase of MDA,\nholding equilibrium within the body, encompassing those implicated in decrease in the ratio of red/green dyed by C11-BODIPY, enhancement of\nregulating the progression of cell cycles, maintaining redox balance, cell viability in the presence of Fer-1 and downregulation of Ferritin and\nfacilitating DNA replication and mending, orchestrating programmed GPX4 together affirm that the complexes promote A549 cell death by\ncell death, and promoting cellular self-degradation [61\u201363]. Protein inducing the ferroptosis pathway.\nimmunoblotting was applied to measure p53 expression, and the results\nwere depicted in Fig. 6c, we found that the expression of p53 increased, 3.10. Autophagy studies\nwhich further substantiates that 3a, 3b can induce oxidative stress\nthrough p53 pathway and eventually lead to apoptosis. Not only is there significant crosstalk between autophagy and\n The main cause of lipid peroxidation in ferroptosis is the depletion of various cell death modes, but autophagy also modulates mitochondrial\nglutathione peroxidase 4 (GPX4) levels. GPX4, a selenium-dependent recycling, thereby regulating hepatocyte apoptosis through the mito\u00ad\nenzyme that relies on glutathione (GSH), plays a crucial role as a chondrial pathway [70]. Autophagy contributes to the overall degra\u00ad\nphospholipid hydroperoxidase and effectively suppresses the production dation of cytoplasm and mitochondria [71,72]. Activation of the\nof lipid ROS [64]. C11-BODIPY dye can transform its fluorescence from autophagy pathway boosts ferroptosis by facilitating ferritin degrada\u00ad\nred to green after reacting with lipid free radicals produced during lipid tion [73]. To investigate whether the complexes can induce autophagy,\nperoxidation, which is applied to evaluate the level of lipid peroxidation we employed fluorescent eosinophilic dye monodansylcadaverine\nin the ferroptosis. Observe from Fig. 6d, after A549 cells were dealt with (MDC) to detect autophagosome formation. The results are presented in\nIC50 concentration of 3a, 3b, 3a (light), 3b (light), the green fluorescence Fig. 7a, green fluorescence was observed in the 3a, 3b, 3a (light), 3b\nintensity is enhanced, while the red fluorescence intensity is reduced. (light)-treated groups, moreover, we observed a lot of autophagic vac\u00ad\nThe quantitative analysis of red/green ratio showed that the red/green uoles, indicating that complexes 3a, 3b, 3a (light), 3b (light) can validly\n\n 8\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\n\n\n Fig. 4. (continued).\n\n\ninduce autophagy. Quantitative analysis of green fluorescence intensity participates in the regulation of multiple signaling pathways and auto\u00ad\nshowed a significant increase in the green fluorescence intensity phagy processes. Specifically, p62 acts as an essential adapter respon\u00ad\n(Fig. 7b). sible for recognizing and delivering specific organelles and protein\n Beclin-1 is a crucial component in the autophagy pathway, involved aggregates to the autophagosome for degradation, known as selective\nin various stages from autophagosome formation to their extension and autophagy [77]. To further affirm the occurrence of autophagy, we\nmaturation [74,75]. Beclin-1 plays a central role in regulating auto\u00ad employed western blot technology to examine the expression of Beclin-1\nphagy and holds potential as a target for tumor therapy through auto\u00ad and p62. Fig. 7c demonstrated an increase and a reduction in the\nphagy modulation [76]. p62 is a multifunctional scaffold protein that expression of Beclin-1 and p62 proteins, respectively. These findings\n\n 9\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\n\n\nFig. 5. (a) The apoptosis in A549 cells (I) and (b) expression of PARP, PI3K, AKT, caspase 3, Bcl-2, p38 MAPK, VEGFA, mTOR, p-mTOR, and Bax after exposure to 3a\n(4.0 \u03bcM, II), 3b (26.8 \u03bcM, III), 3a (light) (0.7 \u03bcM, IV) and 3b (light) (1.8 \u03bcM, V) for 26 h.\n\n\n\n\n 10\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\n\n\nFig. 6. Measurement of GSH (a), and MDA levels (b), (c) Expression of p53 protein, (d) Detection of lipid-peroxidation using C11-BODIPY as fluorescence probe, (e)\nRatio of red/green fluorescence, (f) Cell viability in the absence or presence of Fer-1, (e) Expression of ferritin and GPX4 proteins.\n\n\n\n\nFig. 7. (a) Assay of autophagy with MDC staining, (b) green fluorescence intensity, (c) expression of Beclin-1 and p62 after an exposure of A549 cells to IC50\nconcentration of 3a, 3b, 3a (light) and 3b (light) for 26 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version\nof this article.)\n\n\nfurther support that complexes are ability to initiate autophagy. oncolytic viruses, physical chemotherapy, photodynamic therapy, and\n radiation therapy [78,79]. Releasing molecules during cellular distress\n or demise can serve as immune system stimulants or indicators of po\u00ad\n3.11. Photoactivation of the complexes induces immunogenic cell death\n tential harm. These indicators are commonly referred to as damage-\n(ICD)\n associated molecular patterns (DAMPs) [80]. DAMPs encompass the\n presentation of calreticulin (CRT) and heat-shock proteins (HSP70) on\n Immunogenic cell demise (ICD) is a controlled manner in the cellular\n the surface of cells, extracellular release of adenosine triphosphate\ndemise that has the potential to trigger an adaptive immune response in\n (ATP), high-mobility group box-1 (HMGB1), type I IFNs, and members\na robust immunological setting. It falls under the category of an un\u00ad\n belonging to the interleukin-1 (IL-1) cytokine family [81]. To explore\nspecified phenomenon caused by particular chemotherapy medications,\n\n 11\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\n\n\nFig. 8. ICD assay of CRT (a), HMGB1 (b) and HSP70 (c), the green fluorescence intensity for CRT (d), HMGB1 (e) and HSP70 (f) (***p < 0.001), (g) expression of\nproteins CRT, HMGB1 and HSP70. (For interpretation 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 12\n\fG. Li et al. Journal of Inorganic Biochemistry 259 (2024) 112652\n\n\nthe relationship between the complexes and ICD, we utilized immuno\u00ad Declaration of competing interest\nfluorescence staining to detect changes in CRT, HMGB1, and HSP70\ncontent. As depicted in Fig. 8a-c, treatment of A549 cells with 3a, 3b, 3a The authors declare no competing interest exists.\n(light), 3b (light), green fluorescence increases, indicating 3a, 3b, 3a\n(light), 3b (light) can enhance CRT, HMGB1, and HSP70 content. The Data availability\nquantitative determination of green fluorescence intensity (3a, 3b as\nreference) further confirmed that complexes led to an increase of Data will be made available on request.\nintracellular CRT, HSP70 and HMGB1 content (Fig. 8d-f).\n Next, we assessed the impact of the complexes on the expression Acknowledgements\nlevels of CRT, HSP90, and HMGB1. As depicted in Fig. 8g, we discovered\na significant increase in the expression levels of CRT, HSP70, and We are grateful for National Natural Science Foundation of China\nHMGB1. This further substantiates that the complexes may induce (No 21877018).\nimmunogenic cell demise.\n The luminometer was utilized to measure the content of ATP. As Appendix A. Supplementary data\npresented in Fig. S17 (SI), the ATP content was determined to be 2.56\n\u03bcM in the control. 3a, 3b, 3a (light) and 3b (light) induce an increase of Supplementary data to this article can be found online at https://doi.\n2.00, 1.13, 3.33 and 2.50 \u03bcM in ATP content, respectively. These find\u00ad org/10.1016/j.jinorgbio.2024.112652.\nings suggest that exposure of cells to the complexes significantly elevates\nATP levels in A549 cells, leading to immunogenic cell death. References\n\n4. Conclusions [1] K.A. Robb, A.E. Simon, A. Miles, J. Wardle, Public perceptions of cancer: a\n qualitative study of the balance of positive and negative beliefs, BMJ Open 4\n (2014) e005434.\n We successfully designed and synthesized complexes 3a, 3b and 3c. [2] P.S. Roy, B.J. Saikia, Cancer and cure: a critical analysis, Indian J. Cancer 53\nThe pKa values of 3a-3c are >4, indicating an ability of 3a-3c to enter the (2016) 441\u2013442.\n [3] C.M. Tilsed, S.A. Fisher, A.K. 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