Iridium(III) complexes inhibit the proliferation and migration of BEL-7402 cells through the PI3K/AKT/mTOR signaling pathway.

{"full_text": " Journal of Inorganic Biochemistry 241 (2023) 112145\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\nIridium(III) complexes inhibit the proliferation and migration of BEL-7402\ncells through the PI3K/AKT/mTOR signaling pathway\nJing Chen a, Haimei Liu a, Yichuan Chen a, Huiyan Hu a, Chunxia Huang a, Yi Wang a,\nLijuan 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, Guangdong Provincial Engineering Center of Topical Precise Drug Delivery System, Guangdong\nPharmaceutical 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: Iridium(III) complexes are largely studied as anti-cancer complexes due to their excellent anti-cancer activity. In\nIridium(III) complex this article, two new iridium(III) complexes [Ir(piq)2(THPIP)]PF6 (THPIP = 2,4-di-tert-butyl-6-(1H-imidazo[4,5-\nApoptosis f][1,10]phenanthrolin-2-yl)phenol, piq = deprotonated 1-phenylisoquinoline) (Ir1) and [Ir(bzq)2(THPIP)]PF6\nAutophagy\n (bzq = deprotonated benzo[h]quinolone) (Ir2) were synthesized. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte\u00ad\nROS\n trazolium bromide (MTT) assays showed that complex Ir1 exhibits moderate activity (IC50 = 29.9 \u00b1 4.6 \u03bcM) and\nImmunogenic cell death\n Ir2 shows high cytotoxicity (IC50 = 9.8 \u00b1 1.8 \u03bcM) against BEL-7402 cells. Further studies on the mechanism\n showed that Ir1 and Ir2 induced apoptosis by changing the mitochondrial membrane potential, Ca2+ release,\n ROS accumulation, and cell cycle arrest at the S phase. The complexes can effectively inhibit cell colony for\u00ad\n mation and migration. The expression of B-cell lymphoma-2 (Bcl-2) family proteins, PI3K (phosphatidylinositol\n 3-kinase), AKT (protein kinase B), mTOR (mammalian target of rapamycin), and p-mTOR was studied by\n immunoblotting. Complexes Ir1 and Ir2 downregulated the expression of anti-apoptotic protein Bcl-2 and\n increased the expression of autophagy-related proteins of Beclin-1 and LC3-II. Further experiments showed that\n the complexes inhibited the production of glutathione (GSH) and increased the amounts of malondialdehyde\n (MDA). Fluorescence of HMGB1 was significantly increased. We also investigated the effect of the complexes on\n the expression of genes using RNA-sequence analysis, we further calculated the lowest binding energies between\n the complexes and proteins using molecular docking. Taken together, the above results indicated that complexes\n Ir1 and Ir2 induce apoptosis in BEL-7402 cells through a ROS-mediated mitochondrial dysfunction and inhi\u00ad\n bition of the PI3K/AKT/mTOR signaling pathway.\n\n\n\n\n Abbreviations: A549, human lung cancer; AKT, protein kinase B; BAX, Bcl-2-Associated X; BCA, Bicinchoninic acid; Bcl-2, B-cell lymphoma-2; BEL-7402, (human\nhepatocellular carcinoma); BH3, The Bcl-2 Homology Domain 3; Bzq, deprotonated benzo[h]quinolone; CCCP, Carbonyl cyanide 3-chlorophenylhydrazone; CRT,\ncalmodulin; DAMPs, damage-associated molecular patterns; DAPI, 4\u2032 ,6-diamidino-2-phenylin-dole; DCF, 2\u2032 ,7\u2032 -Dichlorofluorescein; DCFH-DA, 2\u2032 ,7\u2032 dichloro\u00ad\nfluorescein diacetate; DMEM, dulbecco\u2019s modified eagle medium; DMSO, Dimethyl sulfoxide; ECL, enhanced chemiluminescence; FAK, Focal adhesion kinase; FBS,\nFetal bovine serum; Fluo-3 AM, Fluo-3-pentaacetoxy-methyl ester; GSH, glutathione; HeLa, human cervical carcinoma; HepG2, human hepatocellular carcinoma;\nHMGB1, high mobility group box 1; HSP, heat shock protein; IC50, half maximal inhibitory concentration; ICD, immunogenic cell death; JC-1, 5,5\u2032 ,6,6\u2032 -tetrachloro-\n1,1\u2032 ,3,3\u2032 -tetraethyl-benzimidazolylcarbocyanine iodide; LC3, microtubule-associated protein 1 light chain 3 alpha; LGA, Lamarckian genetic algorithm; LC3-I, a\ncytosolic form of LC3; LC3-II, LC3-phosphatidylethanolamine conjugate; LO2, liver cell line; MDA, malondialdehyde; MDC, monodansylcadaverine; MMP, mito\u00ad\nchondrial membrane potential; M-TOR, mammalian target of rapamycin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly ADP-\nribose polymerase; PBS, phosphate buffer saline; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; Piq, deprotonated 1-phenylisoquinoline; PVDF, poly\u00ad\nvinylidene fluoride; RIPA, Radio-Immunoprecipitation Assay; RNA-seq, RNA sequencing; ROS, Reactive Oxygen Species; RPMI, Roswell Park Memorial Institute;\nSDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SOD, superoxide dismutase; THPIP, 2,4-di-tert-butyl-6-(1H-imidazo[4,5-f][1,10]phenan\u00ad\nthrolin-2-yl)phenol; TMS, tetramethylsilane; VEGF, vascular endothlial growth factor..\n * Corresponding author.\n E-mail address: lyjche@gdpu.edu.cn (Y. Liu).\n\nhttps://doi.org/10.1016/j.jinorgbio.2023.112145\nReceived 23 November 2022; Received in revised form 11 January 2023; Accepted 20 January 2023\nAvailable online 23 January 2023\n0162-0134/\u00a9 2023 Elsevier Inc. All rights reserved.\n\fJ. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112145\n\n\n\n\n Scheme 1. Synthetic route of THPIP, complexes Ir1, Ir2 and structures of Ru1, Ru2 and Ru3.\n\n\n1. Introduction intracellular redox imbalance and alteration of mitochondrial mem\u00ad\n brane potential leading to necrosis and apoptosis of tumor cells by Ir(III)\n Hepatocellular carcinoma is one of the deadliest forms of cancer and complexes [16]. In addition, autophagy and apoptosis are two important\nthe second cancer death worldwide, with approximately 800,000 new physiological activities that control cell survival and death, both of\npatients each year, making it a global health challenge [1\u20133]. Although which are anti-cancer pathways [17]. It was reported that two Ir(III)-\ntherapies such as surgery, chemotherapy, and immunotherapy have \u03b2-caroline complexes were identified as potent inducers of autophagy\nbeen used in the clinical management of hepatocellular carcinoma, the [18]. Our previous work reported that Ir(III) complexes can inhibit cell\nprognosis for patients with hepatocellular carcinoma remains unfavor\u00ad proliferation by inducing apoptosis and autophagy [19].\nable [4]. The drug resistance and severe side effects of platinum drugs Ruthenium(II) complexes (Ru1, Ru2, Ru3, Scheme 1) with THPIP\nhave limited their clinical use, which prompts researchers to develop (2,4-di-tert-butyl-6-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)\nother metal compounds such as iridium(III) complexes as potential phenol) as ligand showed high inhibitory activity against A549 lung\ntumor imaging and therapeutic agents [5\u201310]. Cao et al. reported a se\u00ad cancer cell and moderate cytotoxic activity toward BEL-7402 cells [20].\nries of Ir(III) complexes with bipyridyl ligands that significantly reduced To explore the difference in the anticancer effect of the classical Werner\nmitochondrial membrane potential (MMP), oxygen consumption rate, Ru(II) complexes and organometallic Ir(III) complexes, in this paper, we\nand ATP production [11]. Thomas et al. synthesized a series of guani\u00ad used iridium(III) in place of Ru(II), and synthesized two new organo\u00ad\ndino and thiourea iridium compounds with high cytotoxicity based on metallic iridium(III) complexes: [Ir(piq)2(THPIP)(PF6) (piq = deproto\u00ad\nthe 2-aminobenzimidazole unit, offering new potential therapeutic av\u00ad nated 1-phenylisoquinoline) (Ir1) and [Ir(bzq)2(THPIP)]PF6 (bzq =\nenues for ovarian cancer [12]. Chen et al. reported four iridium(III) deprotonated benzo[h]quinolone) (Ir2) (Scheme 1), these complexes\ncomplexes to induce both autophagy and apoptosis and eventually lead were characterized by fluorescence, UV\u2013Vis, HRMS, 1H NMR and 13C\nto A549 cells death [13]. NMR spectra. Their anticancer activity was examined by cytotoxicity in\n It has been suggested that anticancer drugs may damage mitochon\u00ad vitro, apoptosis, mitochondrial localization, mitochondrial membrane\ndria by increasing the permeability of the outer mitochondrial mem\u00ad potential, intracellular Ca2+ levels, reactive oxygen species (ROS),\nbrane, which in turn affects the mitochondrial membrane potential cellular uptake, cell cloning, cell migration, cell cycle arrest and\n(\u0394\u03a8m) [14]. And the defective mitochondrial function is involved in the immunoblotting.\ninduction of cancer cell death [15]. Huang et al. reported a light-induced\n\n\n 2\n\fJ. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112145\n\n\n2. Experimental H, 3.78, N, 7.77%. HRMS (CH3CN): Calcd for C53H44N6OPF6Ir: m/z =\n 973.3210 [(M-PF6)+] (Fig. S2a, supporting information). Found: m/z =\n2.1. Materials and methods 973.2503 [(M-PF6)+]. 1H NMR (DMSO\u2011d6, 500 MHz, Fig. S2b, sup\u00ad\n porting information): \u03b4 9.02 (d, 4H, J = 8.0 Hz), 8.41 (d, 2H, J = 8.0 Hz),\n All reagents and solvents are commercially purchased, and they were 8.27 (s, 1H), 8.00 (d, 1H, J = 7.5 Hz), 7.90\u20137.85 (m, 5H), 7.46 (d, 2H, J\nused without further purification. IrCl3\u22c53H2O was obtained from = 6.5 Hz), 7.38 (d, 2H, J = 6.5 Hz), 7.23 (s, 1H), 7.17 (t, 2H, J = 7.5 Hz),\nKunming Boren Precious Metals Co., Ltd. 1-phenylisoquinoline (piq) and 6.96 (t, 2H, J = 7.5 Hz), 6.33 (d, 2H, J = 7.0 Hz), 1.48 (s, 9H), 1.35 (s,\nbenzo[h]quinolone (bzq) were purchased from Beijing HWRK Chem Co., 9H). 13C NMR (DMSO\u2011d6, 125 MHz, Fig. S2c, supporting information):\nLtd. 2-hydroxy-3,5-di-tert-butylbenzaldehyde was purchased from 174.08, 167.13, 158.53, 158.31, 156.63, 156.47, 152.73, 152.66,\nAcros Organics, Inc. A549 (human lung cancer), HepG2 (human hepa\u00ad 151.10, 150.67, 149.58, 148.44, 142.41, 142.31, 139.69, 139.50,\ntocellular carcinoma), BEL-7402 (human hepatocellular carcinoma), 135.76, 134.21, 131.74, 131.52, 130.54, 129.72, 128.71, 126.22,\nHeLa (human cervical carcinoma), and human normal liver LO2 cells 124.76, 123.39, 123.02, 122.56, 122.29, 36.86, 23.06.\nwere obtained from the cell center of Sun Yat-Sen University (Guangz\u00ad\nhou, China). Fetal bovine serum (FBS) and Duchenne Modified Eagle 2.3. Cell culture\nMedium (DMEM) were obtained from Gibco. Fluorescent dyes and\nrelated assay kits were purchased from Beyotime Biotechnology (Shang The cell lines used in this paper were all cultured in a medium\nHai). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide containing 10% fetal bovine serum, HepG2, A549, HeLa and LO2 were\n(MTT) was purchased from Biosharp Biotechnology Company (Beijing, cultured in DMEM high glucose medium, and BEL-7402 cells were\nChina). 1,10-phenthroline and DMSO were purchased in the Guangzhou cultured in RPMI-1640 medium. The cells used for drug administration\nchemical reagent factory (Guangzhou). 4\u2032 ,6-diamidino-2-phenylin-dole and cell experiments were cultured in 6-well, 12-well, or 96-well cell\n(DAPI) was obtained from Equation Biotechnology Co., Ltd. (Beijing, culture plates.\nChina). NMR spectra were performed with DMF-d7 as solvent and tet\u00ad\nramethylsilane (TMS) as an internal standard at AVANCE III 500 MHz\n 2.4. Studies on cytotoxicity in vitro\nnuclear magnetic resonance spectrometer (Bruker Bio-Spin,\nSwitzerland) at room temperature. HRMS was detected through direct\n The viability of cells treated by complexes Ir1 and Ir2 was evaluated\ninjection in the Xevo G2-XS QT mass analyzer (Waters, USA). UV\u2013Visible\n by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\nand emission spectra were measured in the UV-2550 ultraviolet spec\u00ad\n (MTT) method [22]. The cells were plated in 96-well microplates (1 \u00d7\ntrophotometer and RF-5301PC fluorescence spectrophotometer (Shi\u00ad\n 104 cells per well) and cultured overnight in a 5% CO2 incubator at\nmadzu, Japan). The absorbance of microplates was recorded in a\n 37 \u25e6 C. Next day, when the cell density increased to about 50% ~ 60%,\nMultiskan\u2122 FC Microplate Photometer (Thermo Fisher Scientific, USA).\n the cells were treated with different concentrations of the complexes\nThe apoptosis and cell cycle distribution were performed using FACS\n (1.56, 3.125, 6.25, 12.5, 25, 50 and 100 \u03bcM) for 48 h (the complexes\nCalibur flow cytometer (BD, USA).\n were dissolved in DMSO and the final concentration of DMSO is less than\n 0.05%). Then MTT (9:1, v/v) was added into every well and incubated at\n2.2. Synthesis of complexes\n 37 \u25e6 C for 4 h. The purple formazan product was dissolved with 100 \u03bcL\n dimethyl sulfoxide. Finally, a microplate reader was used to detect the\n2.2.1. Synthesis of [Ir(piq)2(THPIP)]PF6 (Ir1)\n absorbance value at a wavelength of 490 nm. The cell viability was\n A mixture of THPIP (0.212 g, 0.5 mmol) [20] and cis-[Ir\n calculated according to the following equation:\n(piq)2Cl]2\u2022H2O (0.338 g, 0.25 mmol) [21] was dissolved in 42 mL of (( )/ )\ndichloromethane and methanol (V:V = 2:1) and refluxed at 40 \u25e6 C for 6 h Viability% = Asample \u2212 Amedium control (Acell control \u2212 Amedium control ) \u00d7 100\nunder argon to produce a reddish-brown solution. After cooled to room\n Whereas Asample, Amedium and Ablank are the absorbance values of\ntemperature, an excess of ammonium hexafluorophosphate solution was\n sample, medium and control.\nadded into the reddish-brown solution and stirred for 30 min. The ob\u00ad\n The IC50 values were obtained by plotting the percentage of cell\ntained precipitate was washed thoroughly with dichloromethane. The\n viability versus concentration on a logarithmic graph and reading off the\ncrude product was purified by column chromatography. Neutral\n concentration at which 50% of cells remained viable relative to the\nalumina (100\u2013200 mesh, pH range 7 \u00b1 0.5) was loaded on the column,\n control. Each experiment was repeated at least three times to obtain\nand a mixture of dichloromethane and acetone (V:V = 3:1) was used as\n mean values.\neluent. The pure Ir1 product was obtained by evaporation under\nreduced pressure. Yield: 71.7%. Anal Calcd for C57H48N6OPF6Ir: C,\n58.50, H, 4.13, N, 7.18%. Found: C, 58.31, H, 4.24, N, 7.36%. HRMS 2.5. Apoptosis assay by flow cytometry\n(CH3CN): Calcd for C57H48N6OPF6Ir: m/z = 1025.3523 [(M-PF6)+].\nFound: m/z = 1025.3558 [(M-PF6)+] (Fig. S1a, supporting informa\u00ad Flow cytometry is an effective method to distinguish between non-\ntion). 1H NMR (DMF-d7, 500 MHz, Fig. S1b, supporting information): \u03b4 apoptotic cells and apoptotic cells. BEL-7402 cells in a logarithmic\n9.14 (d, 4H, J = 8.5 Hz), 8.50 (d, 3H, J = 8.0 Hz), 8.07 (d, 2H, J = 8.0 growth phase were treated with IC50 concentration of the complexes for\nHz), 8.03 (s, 1H), 7.97\u20137.88 (m, 7H), 7.63 (d, 2H, J = 6.5 Hz), 7.54 (d, 48 h, the culture medium was discarded, the cells were collected by\n2H, J = 6.5 Hz), 7.34 (s, 1H), 7.20 (t, 2H, J = 7.0 Hz), 6.98 (t, 2H, J = centrifugation and stained with Annexin V-FITC and incubated at 37 \u25e6 C\n7.5 Hz), 6.49 (d, 2H, J = 7.5 Hz), 1.54 (s, 9H), 1.39 (s, 9H). 13C NMR for 15 min, finally, the cells were detected using flow cytometry.\n(DMF-d7, 125 MHz, Fig. S1c, supporting information): 173.49, 170.15,\n156.65, 147.53, 144.74, 142.82, 138.64, 137.09, 133.78, 133.49, 2.6. Western blot analysis\n132.34, 131.99, 130.77, 129.36, 128.27, 127.68, 127.38, 123.73,\n123.69, 123.36, 33.00, 21.89. BEL-7402 cells were cultured in 6-well culture plates at a density of 4\n \u00d7 105 cells/well. A blank control group was set, and BEL-7402 cells were\n2.2.2. Synthesis of [Ir(bzq)2(THPIP)]PF6 (Ir2) treated with IC50 concentration of Ir1 and Ir2 for 24 h, then washed with\n The complex Ir2 was synthesized in an identical method described ice-cold PBS, and lysed with the RIPA buffer on ice for 20 min. Next, the\nfor complex Ir1, with cis-[Ir(bzq)2Cl]2\u22c5H2O [21] in place of cis-[Ir solution was centrifuged at 4 \u25e6 C for 15 min. After centrifugation, the\n(piq)2Cl]2\u22c5H2O. The yellow product was obtained. Yield: 64.4%. Anal concentration of total protein was measured using the BCA protein assay\nCalcd for C53H44N6OPF6Ir: C, 56.93, H, 3.97, N, 7.52%. Found: C, 56.80, kit. The electrophoresis process was performed on SDS-PAGE with equal\n\n 3\n\fJ. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112145\n\n\nproteins. Then, the separated proteins were absorbed into PVDF mem\u00ad mitochondrial membrane potential, intracellular Ca2+ levels, intracel\u00ad\nbranes. Blocked for 70 min at room temperature and incubated with the lular ROS content, autophagy and molecular mocking studies can be\nprimary antibody overnight at 4 \u25e6 C. Incubated with dilutions of found in the supporting information.\nrespective secondary antibodies coupled with horseradish peroxidase on\nice for 70 min. Finally, the immune complexes were detected using the 2.11. Data analysis\nECL method.\n All data were expressed as mean \u00b1 SD. Statistical significance was\n2.7. Glutathione (GSH) measurement evaluated using t-tests. Differences were considered significant when the\n *P value was less than 0.05.\n BEL-7402 cells (4 \u00d7 105 cells per well) were seeded in six-well plates\novernight. After treatment with IC50 concentration of Ir1 and Ir2 for 24 3. Results and discussion\nh, the cells were trypsinized and washed twice with PBS. Glutathione\nassays were performed using the GSH Assay Kit (Beyotime Biotech\u00ad 3.1. Synthesis and characterization\nnology, China) according to the manufacturer\u2019s protocol, the absor\u00ad\nbance at 412 nm was measured, and then the GSH content was The synthetic route for ligand and its two iridium(III) complexes is\ncalculated. shown in Scheme 1. The ligand THPIP was prepared according to the\n previously reported methods [20]. The complexes [Ir(piq)2(THPIP)]PF6\n2.8. Malondialdehyde (MDA) measurement (Ir1) and [Ir(bzq)2(THPIP)]PF6 (Ir2) were synthesized by the direct\n reaction with THPIP and precursors in dichloromethane and methanol.\n BEL-7402 cells treated with IC50 of Ir1 and Ir2 for 24 h were washed The complexes were characterized by elemental analysis, HRMS, 1H\ntwice with PBS and then the cells were lysed with a mixture of RIPA (50 NMR, and 13C NMR. In the HRMS spectra, the determined molecular\nmM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) weights are consistent with the expected values. In the 1H NMR spectra,\nand PMSF (phenylmethanesulfonylfluoride). Then the cells were the peaks of 8.03 (s, 1H) and 7.34 (s, 1H) for Ir1, 8.27 (s, 1H) and 7.23\ncentrifuged at 12,000 \u00d7g for 10 min at 4 \u25e6 C to remove insoluble cell (s, 1H) for Ir2 are attributed to the hydrogen atoms at the position of b\ndebris. Total protein was determined using the Pierce BCA Protein Assay and a, respectively. However, the peak for the proton on nitrogen atom\nKit (Beyotime Biotechnology, China). To detect lipid peroxidation, the of the imidazole ring was not observed, which may be caused by metal\nlysates obtained from the above operations were assayed by the MDA coordination inducing electron deficiency in the ligand, therefore, the\nAssay Kit (Beyotime Biotechnology, China). Through calculating the NH proton of the imidazole ring is very active and easy to be exchanged\nprotein content and the MDA content in the solution, the amount of between the two imidazole nitrogen atoms in solution. In addition, the\nMDA in the original sample can be obtained. peaks for the proton in the phenol hydroxyl (-OH) are not observed.\n Similar results can be found in the literature [23]. In the 13C NMR\n2.9. Immunofluorescence analysis spectra, the peaks of 33.00 ppm for Ir1, 36.86 ppm for Ir2 are assigned\n the tert-butyl carbon atoms (-C(CH3)3), while the peaks of 21.89 ppm for\n BEL-7402 cells (4 \u00d7 105 cells/well) were seeded in 12-plate over\u00ad Ir1 and 23.06 ppm for Ir2 are attributed to the methyl carbon atoms\nnight, the cells were treated with IC50 concentration of Ir1 and Ir2 for (-CH3).\n24 h, the cells were fixed in 75% ethanol for 30 min. Then the cells were The stability of the complexes Ir1 (30 \u03bcM) and Ir2 (9.8 \u03bcM) in PBS\nblocked with an immunostaining blocking solution at 4 \u25e6 C for 1 h. solution was investigated by UV\u2013Vis spectra, as shown in Fig. S3a and\nSubsequently, the cells were washed three times with immunostaining 3b (supporting information), no change in the peak shape at 0 and 24 h\nsolution and incubated with CRT antibody, HMGB1 antibody, and was found, indicating that the complexes are stable. Owing to low sol\u00ad\nHSP70 antibody for 12 h. After washing with immunostaining solution ubility, some precipitates were observed at 24 h, therefore, the absor\u00ad\nand incubation with secondary antibody for 1 h in the dark, then the bance decreases. In addition, we also determined the UV\u2013Vis spectra of\ncells were stained with Hoechst for 30 min, washed with cold PBS, and ligand, [Ir(piq)2Cl]\u22c5H2O and [Ir(bzq)2Cl]\u22c5H2O (Fig. S3c, supporting in\u00ad\nimmediately photographed under Image Xpress Micro XLS. formation), the peak shapes are different with the complexes Ir1 and\n Ir2, which further suggests that the complexes are stable. The lumi\u00ad\n2.10. RNA sequence analysis nescence of complexes Ir1 (30 \u03bcM, \u03bbex = 357 nm) and Ir2 (9.8 \u03bcM, \u03bbex =\n 354 nm) in PBS solution are obtained at room temperature, the\n BEL-7402 cells were inoculated in 6-well culture plates (5 \u00d7 105 maximum for Ir1 and Ir2 appears at 611 nm (Fig. S3d, supporting in\u00ad\ncells) and incubated in a 37 \u25e6 C incubator for 24 h. The BEL-7402 cells formation). The luminescence quantum yield (\u03a6) of the complexes was\nwere exposed to 2 \u00d7 IC50 concentration of Ir1 for 24 h. Adherent cells determined according to the literature [24], [Ru(bpy)3]2+ (\u03a6 = 0.04,\nwere washed twice using pre-chilled PBS. The appropriate amount of methanol) was used as a reference. The luminescence quantum yields for\nRNA extraction solution (Servicebio, China) was added, and cells were Ir1 and Ir2 were determined to be 0.30, and 0.04, respectively. The\nwell blown using a pipette to lyse them adequately, followed by isolation time-resolved fluorescence decay profiles for complexes were deter\u00ad\nof total RNA using Trizol Reagent (Invitrogen Life Technologies), after mined using time-correlated single-photon counting (TCSPC) spectros\u00ad\nwhich the concentration, quality and integrity were determined using a copy. The average fluorescence lifetimes of complexes Ir1 and Ir2 are\nNanoDrop spectrophotometer (Thermo Scientific). Sequencing libraries 189.5 and 16.1 ns, respectively.\nwere generated using the TruSeq RNA Sample Preparation Kit (Illumina, The purity of the complexes was determined by HPLC using meth\u00ad\nSan Diego, CA, USA). To select cDNA fragments with a length of pref\u00ad anol and water (Vmethanol:VH2O = 60:40 for Ir1, 80:20 for Ir2) as mobile\nerably 200 bp, library fragments were purified using the AMpure XP phase, during a period of 30 min, only a peak was observed, indicating\nsystem (Beckman Coulter, Beverly, CA, USA). The library fragments that the complexes are pure (Fig. S3e) (supporting information), the\nwere then quantified using Agilent high-sensitivity DNA analysis on a values of the purity for Ir1 and Ir2 are 97.41 and 97.00%, respectively.\nBioAnalyst 2100 system (Agilent, Santa Clara, CA, USA). Finally, the\nsequencing library was sequenced on a Hiseq platform (Illumina) by 3.2. Cellular uptake studies\nShanghai Personal Biotechnology Cp. Ltd. (China).\n The experimental procedures for luminescence quantum yields, cell It is very important to study whether the complexes can effectively\nuptake analysis, wound scratch, colony formation, Cell cycle distribu\u00ad enter the cells. Researchers have proposed several pathways for the\ntion, location assays of Ir1 and Ir2 at the mitochondria, the change of complexes to enter the cells including passive diffusion through the\n\n 4\n\fJ. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112145\n\n\nTable 1 3.3. Cytotoxic activity assay of the complexes toward cancer cells\nIC50 (\u03bcM) values of Ir1, Ir2, THPIP and cisplatin toward the selected cancer and\nnormal cells for 48 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\n Complex BEL-7402 A549 HepG2 HeLa LO2 (MTT) method was used to evaluate the cytotoxic activity of the com\u00ad\n THPIP 37.4 \u00b1 1.4 38.1 \u00b1 1.4 >100 27.2 \u00b1 1.0 45.4 \u00b1 2.3\n plexes Ir1, and Ir2 against cancer cells BEL-7402, HepG2, A549, HeLa,\n Ir1 29.9 \u00b1 4.6 >100 >100 >100 >100 and liver cell line (LO2), the culture medium was used as a blank control.\n Ir2 9.8 \u00b1 1.8 >100 >100 25.7 \u00b1 0.8 >100 As depicted in Table 1, complex Ir1 only shows moderate cytotoxic\n Cisplatin 15.4 \u00b1 4.1 6.5 \u00b1 0.5 9.1 \u00b1 0.9 5.7 \u00b1 1.7 18.5 \u00b1 0.7 activity against BEL-7402 cells, no cytotoxicity toward HepG2, HeLa and\nData for cisplatin from Ref [27]. A549 cells. While complex Ir2 exhibits high cytotoxic activity against\n BEL-7402, no cytotoxic activity toward HepG2 and A549 cells, which is\nmembrane, transport proteins, and endocytosis [25]. To confirm related to the uptaken amount of the complexes by different cancer cells.\nwhether the complexes can enter the cells, the cellular uptake was Interestingly, the complexes reveal no cytotoxic efficacy toward normal\ninvestigated under Image Xpress Micro XLS. After the treatment of BEL- liver LO2 cells. Comparing the IC50 value, Ir2 shows higher cytotoxic\n7402 cells with IC50 concentration of Ir1 and Ir2 for 24 h, as shown in effect on BEL-7402 cells than that of cisplatin, but lower than those of\nFig. S4a (supporting information), the complexes emit weak green iridium(III) complexes [Ir(bzq)2(FTTP)](PF6) (FTTP = 2-(3-fluo\u00ad\nfluorescence, the cell nuclei were stained blue with DAPI, the overlap of ronaphthalen-2-yloxy)-1,4,8,9-tetraazatriphenylene, IC50 = 1.6 \u00b1 0.09\ngreen and blue fluorescence indicated that the complexes may enter the \u03bcM) and [Ir(piq)2(FTTP)](PF6) (IC50 = 1.1 \u00b1 0.02 \u03bcM) [26]. In our\ncell and distribute in the cytoplasm. We also quantitatively determined previous work [20], we synthesized three Ru(II) complexes Ru1, Ru2\nthe cell uptake using flow cytometry, as shown in Fig. S4b (supporting and Ru3 containing THPIP ligand (Scheme 1), the anticancer activity of\ninformation), BEL-7402 cells were treated with IC50 concentration of Ir1 the Ru(II) complexes against BEL-7402 cells is lower than that of Ir(III)\nor Ir2 for 24 h, the green fluorescence intensity increased by 3.08 and complex Ir2, but their cytotoxicity is higher than Ir1 against BEL-7402\n4.70 times for Ir1 and Ir2 compared with that in the control, while the cells, moreover, the anticancer efficacy of complexes Ru1 (IC50 = 8.6 \u00b1\ntreatment of A549 cells with IC50 concentration of Ir1 and Ir2 for 24 h, 1.1 \u03bcM), Ru2 (IC50 = 13.1 \u00b1 1.1 \u03bcM) and Ru3 (IC50 = 7.3 \u00b1 1.4 \u03bcM) is\nthe green fluorescence intensity only increased by 1.27 and 1.16 times, higher than Ir(III) complexes toward A549 cells. Although Ru(II) and Ir\nrespectively. Hence, the cellular uptaken amounts of the complexes by (III) complexes containing the same main ligand THPIP, different metal\nBEL-7402 cells are more than those by A549 cells. ion and different ancillary ligand result in different anticancer effect on\n the same cancer cells. Additionally, we also determined the IC50 values\n of the complexes against A549 and HepG2 cells upon irradiation (65 w\n LED lamp, white light, \u03bb = 450\u2013465 nm, 5.2 J cm\u2212 2). After the addition\n\n\n\n\nFig. 1. (a) The apoptosis was performed after BEL-7402 cells (I) were incubated with IC50 concentration of Ir1 (II), and Ir2 (III) for 48 h by flow cytometry. (b) The\nexpression of caspase 3 and Bcl-2 family proteins in BEL-7402 cells treated with IC50 concentration of the complexes for 24 h, \u03b2-actin was used as an internal control.\n(c) The gray values of PARP, Cleaved PARP, Caspase 3, Bax and Bcl-2 proteins.\n\n 5\n\fJ. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112145\n\n\nof the complexes, 30 min of irradiation to the cells and continued to The mitochondrial pathway is precisely regulated by signaling of mul\u00ad\nincubate for 48 h, the IC50 values of the complexes Ir1 and Ir2 toward tiple physiological activities, and Bcl-2 family proteins and caspase\nA549 and HepG2 cells are more than 100 \u03bcM, which indicates that cascade reaction [35,36]. Therefore, the changes in the expression levels\nirradiation has no influence on the cytotoxic activity. To further inves\u00ad of the corresponding proteins can directly reflect the extent of apoptosis\ntigate the antiproliferative mechanism of Ir1 and Ir2, BEL-7402 cells [37]. The results were presented in Fig. 1b and c, the expression of\nwere chosen for the following cell experiments. apoptosis-related proteins caspase 3 and Bcl-2 was significantly\n decreased in the Ir1- and Ir2-treated groups compared with that in the\n3.4. Effects of Ir1 and Ir2 on the inhibition of migration and colony- control group. Under normal conditions, caspase 3 exists in the form of\nforming assay the zymogen, and once the cells are stimulated to produce apoptotic\n signals, it is shear-activated by caspase 8/9/10 to play a pro-apoptotic\n Cell migration is the most frequently used in vitro biological method role [38]. The reduction of the expression of caspase 3 indicated that\nto test the drug efficacy. As depicted in Fig. S5a and b (supporting in\u00ad it was sheared intracellularly, which implied the smooth transmission of\nformation), the wound width of the blank group was significantly apoptotic signals. BAX is a core member of the Bcl-2 family of proteins,\nreduced. After the cells were treated with IC50 concentration of the which regulates the balance between cell survival and death [39]. The\ncomplexes Ir1 and Ir2 for 24 h, the distance between the wound edges expression of the anti-apoptotic protein BAX was increased compared to\nincreased compared with the control group, which indicated that com\u00ad the blank group. PARP is the main substrate for activation of caspase-3\nplexes effectively inhibit the migration of BEL-7402 cells. The colony and cleaved PARP is an apoptosis-specific biomarker [40]. Thus, inhi\u00ad\nformation is used to test the proliferative capacity of cancer cells [28]. bition of PARP expression can block the DNA repair process and accel\u00ad\nAs demonstrated in Fig. S5c and d (supporting information), the number erate apoptosis. Western blotting assay showed that the expression level\nof cells was significantly reduced compared to the control group after an of PARP was decreased, which further confirmed that Ir1 and Ir2 can\nexposure of BEL-7402 cells to IC50 concentration of the complexes for activate the pro-apoptotic protein BAX through targeting Bcl-2 family\n24 h. Focal adhesion kinase (FAK) is overexpressed in some human tu\u00ad proteins and cascade enzyme proteins, while inhibiting the activation\nmors such as liver and breast cancers and it can play an important role in level of Bcl-2, promoting caspase 3 shearing to deliver and amplify\ntumor progression [29,30]. Reduced FAK expression was associated apoptotic signals and eventually inhibiting the repair of damaged DNA\nwith loss of adhesion, reduced migration, and induction of apoptosis to lead to apoptosis of BEL-7402 cells.\n[31]. As shown in Fig. S5e and f (supporting information), the com\u00ad\nplexes downregulated the expression of FAK protein compared with that 3.7. Complex-mitochondrial co-localization and membrane potential\nin the control group. All the above results demonstrated that Ir1 and Ir2 detection\nsignificantly inhibit the migration of BEL-7402 cells.\n Due to the intrinsic fluorescent properties of iridium(III) complexes,\n3.5. Effects of Ir1 and Ir2 on cell cycle arrest their subcellular localization can be readily analyzed using confocal\n microscopy [41]. DAPI nuclear staining and mitochondrial staining\n Loss of cell cycle control is a hallmark of tumorigenesis [32]. The were used to identify subcellular compartments where Ir1 and Ir2\neffect of complexes on cell cycle progression was investigated using flow accumulated. As seen Fig. S7a (supporting information), the cell nuclei\ncytometry. As given in Fig. S6a (supporting information), the treatment were stained blue with DAPI, the mitochondria were stained red with\nof BEL-7402 cells (I) with IC50 concentration of Ir1 (II) and Ir2 (III) Mito Tracker Red, and the complexes emit weak green fluorescence, the\nincreased the S-phase portion in the cells and resulted in a corresponding overlap of red and green fluorescence indicated that Ir1 and Ir2 were\ndecrease in G0/G1 portion compared with that in the control group. At predominantly accumulated in the mitochondria. Pearson\u2019s colocaliza\u00ad\nthe same time, there was only a slight decrease in the G2/M phase. The tion coefficients (PCC) were calculated by analyzing the red and green\nresults showed that the complexes induce cell cycle arrest at the S phase. fluorescence intensity (Image pro plus 6.0 software) in 50 cells accord\u00ad\nProtein p21, a founding member of the cell cycle protein-dependent ing to literature [42]. The PCC values for Ir1, Ir2 are 0.93 and 0.97,\nkinase inhibitors, is an important cell cycle regulator that inhibits a which demonstrated an existence of positive correlation. Mitochondrial\nvariety of cell cycle proteins, and it is also an essential target gene to ion channels, as emerging tumor targets as the regulation of these ion\ncause cell cycle arrest [33]. As shown in Fig. S6b and c (supporting transport proteins, may affect mitochondrial membrane potential\ninformation), the complexes upregulated the expression of p21 protein (MMP) and it is an early apoptotic marker [14,43,44]. The reduction of\ncompared with that in the control. The results suggested that Ir1 and Ir2 MMP is a key factor in the process of apoptosis [45].\ninduce cell cycle arrest in the S phase and increase the expression of p21 To determine whether Ir(III) complex-induced apoptosis occurred\nprotein. accompanied by the mitochondrial damage, we determined the changes\n of MMP in BEL-7402 cells with 5,5\u2032 -6,6\u2032 -tetrachloro-1,1\u2032 -3,3\u2032 -tetre\u00ad\n3.6. Apoptosis detection thylbenzimidalylcarbocyanine iodide (JC-1) as fluorescence probe. JC-1\n emits red fluorescence as an aggregate at high MMP, and JC-1 emits\n Apoptosis is a critical mechanism for maintaining intracellular life- green fluorescence as a monomer corresponding to low MMP [46\u201348].\ndeath balance to prevent cancer and other related diseases [34]. The As shown Fig. S7b (supporting information), after BEL-7402 cells were\nfeatures of apoptotic cell mainly include condensation of chromatin treated with CCCP, IC50 concentration of the complexes Ir1 and Ir2 for\nmaterial, fragmentation of nuclear DNA, cell shrinkage, dynamic 24 h, there was a significant decrease in red fluorescence and an increase\nmembrane blebbing, and loss of adhesion. To further study the ability of in green fluorescence compared with that in the control group. The ratio\nthe complexes causing cell death, the effect of complexes on apoptosis in of red/green fluorescence intensity is shown in Fig. S7c (supporting\nBEL-7402 cells was determined using the Annexin V-FITC/propidium information), the red/green ratio in the CCCP, Ir1 and Ir2-treated\niodide (PI) method. The results are shown in Fig. 1a, the early apoptotic groups reduced compared with that in the control group, further indi\u00ad\nrate in the control group (I) was 0.12%. When BEL-7402 cells were cating a decrease of red and an increase of green fluorescence intensity.\ntreated with IC50 concentration of complexes Ir1 or Ir2 for 48 h, the These results indicated that the complexes co-localize in the mitochon\u00ad\npercentage of early apoptosis was 8.85% for Ir1 (II) and 11.4% for dria, induce a decrease in the mitochondrial membrane potential and\ncomplex Ir2 (III). The results indicated that the complexes can effec\u00ad finally cause mitochondrial damage.\ntively induce early apoptosis of BEL-7402 cells.\n It is well known that apoptosis is mainly achieved through exogenous\ndeath receptor- pathway and/or endogenous mitochondria pathway.\n\n 6\n\fJ. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112145\n\n\n\n\n Fig. 2. Measurements of GSH levels (a) and MDA content (b) after BEL-7402 cells were treated with IC50 concentration of Ir1 and Ir2 for 24 h.\n\n\n\n3.8. Analysis of intracellular Ca2+ levels\n The quantified green fluorescence intensity of DCF is shown in\n Ca2+ release from the mitochondria play a key role in cell physiology Fig. S9b (supporting information), the green fluorescence intensity was\n[49]. To determine the change of intracellular Ca2+ concentration, Fluo- significantly increased by 1.89, 2.75 and 2.45 times for Rosup (positive\n3 AM was used as a fluorescent probe. As shown in Fig. S8a (supporting control), Ir1 and Ir2 compared with that in the control. These results\ninformation), the green fluorescence in the control group was extremely further show that the complexes are capable to increase intracellular\nweak, indicating a low Ca2+ level in the BEL-7402 cells. After the cells ROS levels.\nwere treated with IC50 concentration of the complexes for 24 h, the\ngreen fluorescence significantly increased, indicating an increase of\n 3.10. Determination of the intracellular glutathione (GSH) and\nintracellular Ca2+ content. To exclude the cross-interference of the weak\n malondialdehyde (MDA) levels\ngreen fluorescence caused by the complexes, the green fluorescence\nintensity was determined according to the following equation:\n Glutathione (GSH) depletion is an early observed marker of\n\n\n\nIgreen fluorescence = Igreen fluorescence of the complexes in the cells + Fluo \u2212 3 AM\u2013Igreen fluorescence of the complexes in the cells\n\n\n\n apoptosis and participates in the detoxification and deactivation of\n metal-based anticancer complexes [51\u201353]. To further explore the ef\u00ad\n As shown in Fig. S8b (supporting information), the green fluores\u00ad fects of Ir(III) complex-induced ROS activation on mitochondria and\ncence intensity increased by 1.88 times for Ir1 and 1.67 times for Ir2 apoptosis, the changes of intracellular GSH levels were determined. As\ncompared with that in the control group after BEL-7402 cells were shown in Fig. 2a, comparing to the control group, the intracellular GSH\ntreated with IC50 concentration of Ir1 and Ir2 for 24 h, which further levels decreased by 16.13% and 12.57% after BEL-7402 cells were\nconfirms that the complexes can enhance intracellular Ca2+ treated with IC50 concentration of complexes Ir1 and Ir2 for 24 h,\nconcentration. respectively. Additionally, we also determined the ratio of GSH/GSSG\n (glutathione disulfide), in the control, the ratio of GSH/GSSG is 7.34 \u00b1\n 1.87, after an exposure of BEL-7402 cells to IC50 concentration of Ir1 or\n3.9. Qualitative and quantitative measurement of ROS generation Ir2 for 24 h, the ratios of GSH/GSSG are 3.73 \u00b1 0.86 and 4.31 \u00b1 0.25,\n respectively. Therefore, the complexes can reduce the content of intra\u00ad\n Mitochondrial dysfunction can lead to ROS production and enhance cellular GSH and cause an increase of oxidant stress.\ncell mobility by elevating cell membrane Ca2+ levels. The 2\u2032 ,7\u2032 - Lipid oxidation or lipid peroxidation is one of the most common\nDichlorodihydrofluorescein diacetate (DCFH-DA) probe is the most oxidative stress indicators, and it is also considered to be one of the\ncommon and sensitive tool for the detection of intracellular ROS levels pathological factors leading to chronic diseases. The most studied\n[50]. To determine whether complex-induced apoptosis in BEL-7402 marker of lipid peroxidation is malondialdehyde (MDA), which directly\ncells was associated with ROS production, we examined the level of represents oxidative damage to cells [54,55]. It is known that the\nintracellular ROS. As shown in Fig. S9a (supporting information), only treatment of cancer patients depletes the antioxidant SOD levels in\nfaint green fluorescence was found in the control group, while BEL-7402 biological systems, leading to increased lipid peroxidation, which is\nwas treated with IC50 concentration of complexes Ir1 and Ir2 for 24 h, a reflected in the increase of intracellular MDA levels [56]. As shown in\nbright green fluorescence was observed. The increased green fluores\u00ad Fig. 2b, compared to the control group, oxidized lipid products MDA\ncence intensity indicated that the complexes increased intracellular ROS levels increased 3.86-fold for complex Ir1 and 2.00-fold for complex Ir2.\ncontent. To exclude the cross-interference of green fluorescence emitted Together, the above experimental results indicated that Ir1 and Ir2 can\nby the complexes, the green fluorescence intensity was calculated ac\u00ad inhibit the production of GSH and increase the content of lipid peroxides\ncording to the following equation: MDA.\n\n\n\n\nIgreen fluorescence = Igreen fluorescence of the complexes in the cells + DCHF \u2212 DA\u2013Igreen fluorescence of the complexes in the cells\n\n\n\n\n 7\n\fJ. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112145\n\n\n\n\n Fig. 3. CRT (a), HMGB 1 (b) and HSP 70 (c) were measured after the treatment of BEL-7402 with IC50 concentration of Ir1 and Ir2 for 24 h.\n\n\n3.11. Ir1 and Ir2 induced autophagy in BEL-7402 cells Beclin-1 contains a BH3 structural domain that binds to B-cell\n lymphoma-2 (Bcl-2) protein, and the release of Beclin-1 from these\n Autophagy plays a crucial regulatory role in drug-induced apoptosis, proteins can induce autophagy [63]. As represented in Fig. S10b and c\nand dysregulation in autophagy may be one of the major factors (supporting information), after treatment of BEL-7402 cells with IC50\ncontributing to sustained cell proliferation and development of tumors concentration of Ir1 and Ir2 for 24 h, the expression levels of LC3-II and\n[57,58]. Anticancer agents have been shown to induce apoptosis and Beclin-1 proteins were increased, but the expression of p62 protein was\nautophagy in cancer cells through the production of ROS [59]. To decreased compared to the control group. These experimental results\nexplore the effects of the complexes on autophagy, BEL-7402 cells were suggested that the complexes can effectively induce autophagy.\ntreated with IC50 concentration of the complexes for 24 h, the cells were\nstained with monodansylcadaverine (MDC), as shown in Fig. S10a\n 3.12. Determination of CRT, HSP70 and HMGB1\n(supporting information), the increased green fluorescence was discov\u00ad\nered compared with that in the control, indicating that the complexes\n Immunogenic cell death (ICD) is a model for drug-induced tumor cell\ncan induce autophagy.\n death that relies on the production of immune signals, induced by\n One of the most common analytic methods for evaluating autophagy\n various stimuli including damage-associated molecular patterns\nbehavior is to quantify the microtubule-related protein MAP1LC3 (LC3)\n (DAMPs) such as calmodulin (CRT), high mobility group box 1 (HMGB1)\nlevel by immunoblot [60]. LC3 is a marker of autophagosomes, which\n and heat shock protein (HSP)70 [64\u201366]. To evaluate the efficacy of the\ncan be transferred from LC3-I to LC3-II when autophagy occurs [61,62].\n complexes on ICD, fluoresceine isothiocyanate (FITC) was used as\n\n 8\n\fJ. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112145\n\n\n\n\nFig. 4. Bioinformatics identified key pathways and biological processes associated with Ir2 sensitivity in BEL-7402 cells. (a) Heat map, (b) Volcano map, (c and d)\nKyoto encyclopedia of genes and genomes pathway analyses.\n\n\nfluorescence probe. As shown in Fig. 3a, the fluorescence of intracellular the periphery of dead cells [68]. Next, we investigated whether HMGB1\nCRT increased after the treatment of BEL-7402 cells with IC50 concen\u00ad can be released from the cells. As shown in Fig. 3b, a significant increase\ntration of complexes Ir1 and Ir2 for 24 h compared that in the control. in the green fluorescence of HMGB1 was observed in the Ir1 and Ir2-\nThorburn et al. reported that autophagy can control the characteristics treated groups compared with that in the control. In addition, HSP70\nof dead cells by regulating the selective release of HMGB1 [67]. An accumulated significantly on the cell membrane after the treatment of\nimportant feature of ICD is the release of HMGB1 from the nucleus into the cells with Ir1 and Ir2 for 24 h (Fig. 3c) was also discovered. Taken\n\n\n\n\nFig. 5. (a) The expression of PI3K, AKT, mTOR, and p-mTOR was detected after BEL-7402 cells were treated with IC50 concentration of Ir1 and Ir2 for 24 h. (b) Gray\nvalues of proteins.\n\n 9\n\fJ. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112145\n\n\nTable 2\nDocking information for the 10 proteins with the corresponding Ir1 and Ir2. The values are the lowest binding energies (kcal/mol) between the protins and Ir1 and Ir2.\n Complex FAK p21 PARP Caspase 3 Bax Bcl-2 p62 Beclin-1 mTOR PI3K\n\n Ir1 \u2212 8.4 \u2212 8.8 \u2212 6.7 \u2212 8.9 \u2212 5.3 \u2212 2.9 \u2212 4.5 \u2212 10.9 \u2212 9.4 \u2212 7.1\n Ir2 \u2212 9.3 \u2212 9.5 \u2212 8.7 \u2212 7.7 \u2212 4.5 \u2212 5.6 \u2212 4.6 \u2212 9.4 \u2212 9.6 \u2212 6.6\n\n\n\n\nFig. 6. Molecular docking of the complexes in caspase 3 (PDB: 1GFW) (a, method validation; b, Ir1; c, Ir2), PARP (PDB: 4PJV) (d, method validation; e, Ir1; f, Ir2).\n\n\ntogether, these results suggested that the complexes can induce immu\u00ad pathway is involved in cell metabolism and proliferation, which is key\nnogenic cell death. hallmarks of cancer [71]. The VEGF/PI3K/AKT signaling pathway is\n involved in cell proliferation, differentiation, apoptosis and vascular\n3.13. RNA sequencing assay growth processes [72]. Inhibition of VEGF can reduce tumor angio\u00ad\n genesis and growth by inactivating the PI3K/AKT signaling pathway\n RNA sequencing (RNAseq) can detect different types of cancer and [73]. Western blotting assay was used to detect the expression of PI3K,\nrare diseases and provides researchers insight into developing more AKT, mTOR, etc. As shown in Fig. 5a and b, after a 24 h treatment of\neffective therapies [69]. Shen et al. investigated RNA-seq data of BEL-7402 cells with IC50 concentration of the complexes, Ir1 and Ir2\nautophagy-associated genes and found that enriched words from GO and reduced the phosphorylation level (P-mTOR), down-regulated the\nKEGG enrichment analysis were associated with autophagy and multiple expression of PI3K, AKT, and mTOR compared with those in the control.\ncancers including hepatocellular carcinoma [70]. To investigate in The results provided evidence that the complexes induce apoptosis in\ndepth the role of Ir2 in hepatocellular carcinoma tumorigenesis, we BEL-7402 cell through inhibition of PI3K/AKT/mTOR signaling\nperformed RNA-seq analysis of total RNA after a 24 h treatment of BEL- pathway. Hence, RNA-sequence analysis provides useful help for\n7402 cells with 2 \u00d7 IC50 concentration of Ir2 (Fig. 4a-d). Compared with investigating the apoptotic mechanism.\nthe control group, 47 genes were up-regulated and 193 genes were\ndown-regulated after Ir2 treatment (Fig. 4a and b). After performing 3.15. Molecular docking studies\ndifferential gene expression analysis and downstream gene set enrich\u00ad\nment analysis, we found that the complex Ir2 was mainly associated To confirm the above findings, we performed a molecular docking\nwith Ca2+ ion signaling pathway, PI3K/AKT signaling pathway, VEGF analysis of the compound structures to predict whether Ir1 and Ir2\nsignaling pathway and Focal adhesion (Fig. 4c and d). This was interacted with the proteins including Bcl-2, PI3K, BAX, Caspase 3,\nconsistent with our previous study. The complexes inhibit cell migration PARP, FAK, mTOR, p62, p21 and Beclin-1 (these proteins were inves\u00ad\nand further induce apoptosis through Ca2+ ion signaling pathway, tigated in the autophagy, PI3K/AKT/mTOR signaling pathway, cell\nreducing FAK expression and activating the Focal adhesion signaling cycle arrest and invasion-related protein FAK). We first identify the\npathway. We also observed that the complexes can activate PI3K, AKT, space place of the complexes in the above proteins according to the\nand VEGF signaling pathways, which further demonstrates that the method validation using crystallized and docking ligand, then we\ncomplexes induce apoptosis in BEL-7402 cells through inhibition of calculated the the lowest binding energies to further identify the binding\nPI3K/AKT/mTOR signaling pathway. site of the complexes interacting the proteins. The lowest binding en\u00ad\n ergies of the complexes with the proteins are given in Table 2, the low\n3.14. Effect of Ir1 and Ir2 on the regulation of the PI3K/AKT/mTOR binding energies indicate a large binding affinity between the complexes\npathway and receptors. The binding of the coplexes with five proteins including\n Beclin-1, p21, mTOR, FAK and caspase 3 show relatively low docking\n The results from RNA-sequence analysis indicates that the complexes score binding free energy. The results from the binding free energies\ninduce apoptosis through inhibition of PI3K/AKT/mTOR signaling revealed that complexes exhibited a high affinity of \u2212 10.9 kcal/mol\npathway. The PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase with the Beclin-1 among these proteins. Fig. 6 (caspase: a-c, PARP: d-f)\nB)/mammalian target of rapamycin (mTOR) signaling pathway is shows the optimal docking site view and their activity pockets (other\ninvolved in tumorigenesis and development. The mTOR signaling proteins can be found in the Fig. S11, supporting information). The sites\n\n 10\n\fJ. Chen et al. Journal of Inorganic Biochemistry 241 (2023) 112145\n\n\n\n\n Fig. 7. Apoptotic and autophagy mechanism induced by the complexes Ir1 and Ir2.\n\n\nwhere Ir1 and Ir2 form hydrogen bonds and interact with residues with Data availability\namino acids of different proteins are given in Table S1 (supporting in\u00ad\nformation). Thus, the docking energies were consistent with the western Data will be made available on request.\nblot results, which may further confirm the regulatory effects of PI3K/\nAKT/ mTOR in the Ir1 and Ir2 treatment progression. Acknowledgements\n\n4. Conclusions This work was supported by the National Natural Science Foundation\n of China (No 21877018).\n In summary, we designed, synthesized, and characterized two new Ir\n(III) complexes Ir1 and Ir2. MTT results showed that Ir1 exhibited Appendix A. Supplementary data\nmoderate and Ir2 showed high cytotoxic activity against tumor cells\nBEL-7402. These complexes were able to be taken up by BEL-7402 cells, Supplementary data to this article can be found online at https://doi.\nthe complexes caused a significant decrease in GSH activity, and an org/10.1016/j.jinorgbio.2023.112145.\naccumulation of toxic epoxidized lipid MDA levels, which then led to an\nincrease of reactive oxygen species and intracellular calcium ion levels. 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