Cyclometalated iridium(III) complexes as anti-breast cancer and anti-metastasis agents via STAT3 inhibition.

{"full_text": " Journal of Inorganic Biochemistry 251 (2024) 112427\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\nCyclometalated iridium(III) complexes as anti-breast cancer and\nanti-metastasis agents via STAT3 inhibition\nYan Su a, b, 1, Jin Yang b, 1, Meng-Meng Wang b, Hong-Bao Fang b, Hong-Ke Liu b,\nZheng-Hong Yu a, *, Zhi Su b, *\na\n Department of Rheumatology and Immunology, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210002, China\nb\n Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023,\nChina\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: Breast cancer is the most commonly diagnosed cancer and second\u2011leading cause of cancer deaths in women.\nIridium(III) complex Signal transducer and activator of transcription 3 (STAT3) plays a critical role in promoting breast cancer cell\nSTAT3 inhibition proliferation, invasion, angiogenesis, and metastasis, and the high expression of STAT3 is related to the occur\u00ad\nAnti-cancer\n rence and poor chemotherapy sensitivity of breast cancer. Iridium(III) complexes Ir-PTS-1\u2013 4 containing a\nAnti-metastasis\n pterostilbene-derived ligand were synthesized to inhibit the STAT3 pathway in breast cancer. Ir-PTS-4 inhibited\nAutophagy\nCell cycle arrest the proliferation of breast cancer cells by suppressing the expression of phosphorylated STAT3 and STAT3-\n related cyclin D1, arresting cell cycle in the S-phase, inducing DNA damage and reactive oxygen species\n (ROS) generation, eventually leading to autophagic cell death. The cell metastasis and invasion were also\n inhibited after Ir-PTS-4 treatment. Besides, Ir-PTS-4 exhibited excellent anti-proliferation activity in 3D multi\u00ad\n cellular tumor spheroids, showing potential for the treatment of solid tumors. This work presents the rational\n design of metal-based anticancer agents to block the STAT3 pathway for simultaneously inhibiting breast cancer\n proliferation and metastasis.\n\n\n\n\n1. Introduction potential as anticancer agents [7\u20139]. By interfering with STAT3, several\n metal complexes have been shown to suppress tumor development and\n Breast cancer (BC) is responsible for the majority of cancer-related metastasis. Leung et al. reported a series of cyclometalated rhodium(III)\ndeaths in women. Patients with primary breast cancer have a 5-year complexes as direct inhibitors of STAT dimerization [10]. Ma et al.\nsurvival rate of 99%; however, the development of metastases reduces designed a benzofuran-conjugated iridium(III) as an inhibitor of STAT3\nsurvival rates to 28% [1]. 20\u201330% of breast cancer cases metastasize to activity in prostate cancer cells [11]. Rimoldi et al. synthesized Pt(II)\ndistant organs, accounting for 90% of breast cancer-related fatalities [2]. complexes bearing 1,2,5-oxadiazole ligands which exhibited strong\nGreat efforts are being made to develop targeted medicines to treat interaction with STAT3, leading to inhibition of STAT3 signaling\ndistinct types of BC [3]. Signal transducer and activator of transcription pathway [12]. Gou et al. designed and prepared a series of Pt(II) com\u00ad\n3 (STAT3), a member of the cytoplasmic transcription factor family, plexes by conjugating four non-steroidal anti-inflammatory drugs\ntransduces extracellular growth factor and cytokine signals and modu\u00ad (NSAIDs) derivatives with Pt(II) agents to inhibit metastasis and inva\u00ad\nlates the levels of genes related to cell cycle, angiogenesis, anti- sion of A2780 cells by suppressing the COX-2/JAK2/STAT3 axis [13].\napoptosis, and invasion/migration [4,5]. Abnormal STAT3 signaling is Recently, Wang et al. reported two pterostilbene (PTS) modified Pt(IV)\nusually associated with tumor development and targeting the STAT3 complexes SPP and DPP to combat BC via inhibiting the JAK-STAT3\nprotein may be a possible anti-tumor and anti-metastatic treatment pathway and regulating the tumor microenvironment [14]. The devel\u00ad\nstrategy [6]. opment of iridium complexes as potent anticancer agents has received\n Metal complexes have stimulated researchers' interest because of increasing attention in recent years. On one hand, iridium complexes are\ntheir unique photochemical and photophysical properties and their widely used in biological imaging and biosensors because of their rich\n\n\n * Corresponding authors.\n E-mail addresses: 13327800182@189.cn (Z.-H. Yu), zhisu@njnu.edu.cn (Z. Su).\n 1\n Authors contributed equally.\n\nhttps://doi.org/10.1016/j.jinorgbio.2023.112427\nReceived 17 October 2023; Received in revised form 6 November 2023; Accepted 11 November 2023\nAvailable online 14 November 2023\n0162-0134/\u00a9 2023 Published by Elsevier Inc.\n\fY. Su et al. Journal of Inorganic Biochemistry 251 (2024) 112427\n\n\nphotophysical properties, including high quantum yield, large Stokes ligand (E)-2-(4-(3,5-dimethoxystyryl) phenoxy) acetic acid and 4-ami\u00ad\nshift, long fluorescence lifetime, good light stability, and strong cell nomethyl-4-methyl-2,2-bipyridyl were prepared according to previ\u00ad\npenetration. On the other hand, diverse mechanisms of iridium com\u00ad ously reported methods [21,22]. To a suspension of (E)-2-(4-(3,5-\nplexes are exerted to inhibit the activities of cancer cells, including DNA dimethoxystyryl) phenoxy) acetic acid (0.314 g, 1 mmol), EDCI (0.230\nbinding [15\u201317], protein kinase activity inhibition [18,19] and so on. g, 1.2 mmol), HOBt (0.108 g, 0.8 mmol) in DMF, 4-aminomethyl-4-\nUntil now, only one iridium-based STAT3 inhibitor has been reported, methyl-2,2-bipyridyl (0.239 g, 1.2 mmol) and 0.4 mL trimethylamine\ntherefore, it is still urgently necessary to develop novel multifunctional were added in a 50 mL Schlenk tube. The reaction mixture was stirred\nanti-tumor agents that can both eradicate cancer cells and prevent under argon atmosphere at ambient temperature for 48 h, then treated\nmetastasis. with ice-cold water and a deep rose white precipitate was filtered. The\n As PTS has been previously reported as a STAT3 inhibitor [20], we obtained solid was dissolved in CH2Cl2 and purified by column chro\u00ad\nhypothesized that the combination of the PTS-modified ligand with matography on silica gel eluted with CH2Cl2/CH3OH. 1H NMR (400\ncyclometalated iridium(III) complex into a single molecular entity could MHz, Chloroform\u2011d) \u03b4 (ppm):\u03b4 8.64 (dd, J = 5.0, 0.9 Hz, 1H), 8.55 (dd, J\npotentially generate a metal-based inhibitor of STAT3 for the treatment = 5.0, 0.8 Hz, 1H), 8.32 (dd, J = 1.7, 0.8 Hz, 1H), 8.24 (dt, J = 1.7, 0.9\nof breast cancer. In this study, we designed and synthesized four PTS- Hz, 1H), 7.51\u20137.45 (m\uff0c2H), 7.25 (dd, J = 5.0, 1.8 Hz, 1H), 7.18\u20136.89\nderived ligand-conjugated cyclometalated iridium(III) complexes (Ir- (m, 6H), 6.67 (d, J = 2.3 Hz\uff0c2H), 6.41 (t, J = 2.3 Hz, 1H), 4.72\u20134.60\nPTS-1\u2013 4). Ir-PTS-4 inhibited the proliferation of breast cancer cells by (m, 4H), 3.85 (s, 6H), 2.46 (s, 3H)\u3002.\nsuppressing the expression of phosphorylated STAT3 and STAT3-related Synthesis of the complexes Ir-PTS-1\u2013 4: [Ir(ppy)2Cl]2, [Ir(thpy)2Cl]2,\ncyclin D1, arresting the cell cycle in the S-phase, inducing DNA damage [Ir(dzq)2Cl]2, and [Ir(pq)2Cl]2 were prepared according to previously\nand mitochondrial damage, eventually leading to autophagy. The cell reported methods. [23,24] A mixture of N^N-PTS (0.25 mmol, 2 equiv)\nmetastasis and invasion were also inhibited after Ir-PTS-4 treatment. and iridium(III) chloro-bridged dimer (0.125 mmol, 1 equiv) was pre\u00ad\nBesides, Ir-PTS-4 exhibited excellent anti-proliferation activity in 3D pared in 45 mL CH2Cl2/CH3OH (2:1, v/v) solvent by refluxing for 6 h\nmulticellular tumor spheroids, suggesting potential for the treatment of under argon in the dark. After the mixture was cooled to room tem\u00ad\nsolid tumors. This work demonstrated the rational design of metal perature, a 6-fold excess of NH4PF6 was added and stirred for another 2\niridium-based anticancer agents to block the STAT3 pathway for h. The desired product was achieved after purification via column\nsimultaneously inhibiting tumor proliferation and metastasis in breast chromatography on silica gel eluted using CH2Cl2/CH3OH.\ncancer treatment. Complex Ir-PTS-1: 1H NMR (400 MHz, DMSO\u2011d6) \u03b4 (ppm):\u03b4\n 8.84\u20138.62 (m, 3H), 8.26 (t, J = 8.2 Hz, 2H), 7.91 (dd, J = 10.4, 6.5 Hz,\n2. Experimental section 4H), 7.74 (dd, J = 6.0, 2.8 Hz, 1H), 7.69 (t, J = 5.2 Hz, 1H), 7.61 (d, J =\n 5.9 Hz, 2H), 7.50 (dt, J = 12.0, 4.8 Hz, 3H), 7.23\u20137.10 (m, 4H),\n2.1. Materials and instrumentation 7.06\u20136.96 (m, 3H), 6.95\u20136.85 (m, 3H), 6.70 (d, J = 2.1 Hz, 1H),\n 6.54\u20136.44 (m, 1H), 6.41\u20136.30 (m, 2H), 6.24\u20136.15 (m, 2H), 4.73\u20134.48\n All the reagents were purchased from commercial sources and used (m, 4H), 3.77 (d, J = 1.3 Hz, 3H), 2.53 (d, J = 4.6 Hz, 3H). ESI-MS\nwithout further purification unless stated otherwise. All the buffer (CH3OH) calcd for:[Ir-PTS-1-PF\u22126 ]+ m/z = 996.18, found m/z =\ncomponents were of biological grade. Iridium (III) chloride hydrate 996.40. Elemental Analysis Calcd for C52H45F6IrN5O4P: C, 54.73; H,\n(IrCl3) was purchased from J&K Scientific. 1-hydroxybenzotriazole 3.97; N, 6.14. Found: C, 54.98; H, 3.69; N, 6.04.\n(HOBt), 2-phenylpyridine (ppy), 2-(thiophen-2-yl)pyridine (thpy), Complex Ir-PTS-2: 1H NMR (400 MHz, DMSO\u2011d6) \u03b4 (ppm):\u03b4 8.80 (dt,\nbenzo[h]quinoline (dzq), 2-phenylquinoline (pq), NH4PF6, ethyl- J = 15.1, 6.0 Hz, 1H), 8.69\u20138.62 (m, 2H), 7.84\u20137.68 (m, 6H), 7.66\u20137.62\nbromacetat, pterostilbene (PTS), N-(3-Dimethylaminopropyl)-3-ethyl\u00ad (m, 3H), 7.59\u20137.49 (m, 5H), 7.22\u20137.12 (m, 2H), 7.02\u20136.88 (m, 5H), 6.71\ncarbodiimide hydrochloride (EDCI) and 3-(4,5-dimethylthiazol-2-yl)- (d, J = 2.3 Hz, 1H), 6.54\u20136.45 (m, 1H), 6.37 (d, J = 2.3 Hz, 1H), 6.17\n2,5-diphenyltetrazolium bromide (MTT) were purchased from (dd, J = 4.8, 2.5 Hz, 2H), 4.66 (s, 1H), 4.63\u20134.52 (m, 3H), 3.78 (s, 3H),\nHEOWNS. Cisplatin was purchased from Energy Chemical. Dimethyl 3.60 (s, 3H), 2.54 (d, J = 5.0 Hz, 3H). ESI-MS (CH3OH) calcd for:[Ir-\nsulfoxide (DMSO) was purchased from Sigma Aldrich. Mito-tracker PTS-2-PF\u22126 ]+ m/z = 1008.22, found m/z = 1008.20. Elemental Analysis\nGreen, Lyso-tracker Green, ER-tracker Green, Hoechst 33342, ROS Calcd for C48H41F6IrN5O4PS2: C, 49.99; H, 3.58; N, 6.07. Found: C,\nassay kit and JC-1 assay kit were purchased from KeyGen Co. (China). 50.05; H, 3.62; N, 5.98.\nAntibodies for western blot, such as STAT3, p-STAT3, LC3, GAPDH, Complex Ir-PTS-3: 1H NMR (400 MHz, DMSO\u2011d6) \u03b4 (ppm):\u03b4\nRIP3, Bax, Bcl-2, Cytochrome c and \u03b3H2AX were purchased from Abcam 8.81\u20138.67 (m, 4H), 8.57 (ddt, J = 8.2, 6.2, 1.4 Hz, 2H), 8.08\u20138.05 (m,\nCo. (China). All the compounds tested were dissolved in DMSO just 2H), 7.97 (ddd, J = 8.9, 3.8, 2.5 Hz, 2H), 7.91\u20137.86 (m, 2H), 7.72 (dd, J\nbefore the experiments and the final DMSO concentration was <1% (v/ = 5.7, 3.0 Hz, 1H), 7.67\u20137.57 (m, 4H), 7.56\u20137.48 (m, 3H), 7.44\u20137.36 (m,\nv). 2H), 7.18\u20137.14 (m, 3H), 6.99\u20136.95 (m, 1H), 6.89\u20136.86 (m, 1H), 6.68 (d,\n The 1H NMR spectra were recorded on a Bruker AVANCE 400 J = 2.3 Hz, 1H), 6.47 (d, J = 2.7 Hz, 1H), 6.35 (d, J = 2.3 Hz, 1H),\nspectrometer at ambient temperature. UV\u2013vis spectra were recorded on 6.23\u20136.19 (m, 2H), 4.61 (s, 1H), 4.56 (d, J = 7.7 Hz, 3H), 3.76 (s, 3H),\na LAMBDA 365 UV\u2013vis spectrophotometer. The Electrospray ionization 3.58 (s, 3H). ESI-MS (CH3OH) calcd for:[Ir-PTS-3-PF\u22126 ]+ m/z = 1044.31,\nmass spectra (ESI-MS) were recorded on a LCQ system (Thermo Scien\u00ad found m/z = 1044.30. Elemental Analysis Calcd for C56H45F6IrN5O4P:\ntific). MTT data were recorded on a microplate reader (LabServ K3, C, 56.56; H, 3.81; N, 5.89. Found: C, 56.58; H, 3.89; N, 5.78.\nThermo Fisher Scientific, USA). Iridium contents in the samples were Complex Ir-PTS-4: 1H NMR (400 MHz, DMSO\u2011d6) \u03b4 (ppm):\u03b4 8.72 (dt,\ndetermined by ICP-MS (X Series 2, Thermo Fisher, USA). Cell imaging J = 16.7, 6.0 Hz, 1H), 8.59\u20138.52 (m, 4H), 8.27 (t, J = 7.7 Hz, 4H), 7.98\nexperiments were carried out on a confocal microscope (A1, Nikon, (dd, J = 5.8, 2.8 Hz, 1H), 7.93 (dq, J = 8.5, 2.3 Hz, 3H), 7.54\u20137.47 (m,\nJapan). Flow cytometric analysis was done using a flow cytometer (BD 2H), 7.46\u20137.39 (m, 3H), 7.27 (d, J = 8.8 Hz, 1H), 7.18\u20137.07 (m, 6H),\nFACSVerse, USA). Western blotting experiments were conducted on 7.06\u20136.99 (m, 1H), 6.96\u20136.91 (m, 1H), 6.90\u20136.85 (m, 1H), 6.81 (tdt, J =\nMini-Protean Tetra System (BIO RAD, USA). The western blotting signal 6.3, 3.2, 1.3 Hz, 2H), 6.70 (d, J = 2.3 Hz, 1H), 6.48 (d, J = 2.5 Hz, 1H),\nwas enhanced by Tanon High-sig ECL Western Blotting Substrate and 6.42\u20136.31 (m, 4H), 4.67\u20134.55 (m, 2H), 4.49\u20134.40 (m, 2H), 3.78 (s, 3H),\nvisualized by Tanon 5200 Multi. 3.60 (s, 3H), 2.38 (s, 3H). ESI-MS (CH3OH) calcd for:[Ir-PTS-4-PF\u22126 ]+ m/\n z = 1096.30, found m/z = 1096.40. Elemental Analysis Calcd for\n2.2. Synthesis and characterization C60H49F6IrN5O4P: C, 58.06; H, 3.98; N, 5.64. Found: C, 58.11; H, 4.02;\n N, 5.59.\n Synthesis of the ligand (N^N-PTS): pterostilbene (PTS)-derived\n\n 2\n\fY. Su et al. Journal of Inorganic Biochemistry 251 (2024) 112427\n\n\n2.3. UV\u2013vis spectroscopy and fluorescence measurements medium was removed and replaced with fresh medium/DMSO (v/v,\n 99:1) containing complex Ir-PTS-4 (4 \u03bcM). After incubation for 6 h, the\n A LAMBDA 365 UV\u2013vis spectrophotometer and a FS5 Spectrofluo\u00ad cells were harvested with trypsin, washed with PBS and counted. Cell\nrometer were used with 1 cm path-length quartz cuvettes (3 mL). The pellets were lysed in RIPA Lysis Buffer (Beyotime Biotechnology, China)\nstock solution of Ir-PTS-1, Ir-PTS-2, Ir-PTS-3 and Ir-PTS-4 in DMSO and the nucleus and mitochondria fractions were extracted using the\nwere prepared before measurements, and then diluted suitably with Mitochondria/Nuclei Isolation Kit (KeyGEN, China) according to the\ndistilled water to the required concentration. The absorption spectra manufacturer's instructions. These fractions in the stock buffer were\nwere recorded at ambient temperature unless otherwise stated. Fluo\u00ad then digested with concentrated nitric acid (100 \u03bcL) at 95 \u25e6 C for 2 h,\nrescence spectra were obtained by recording the emission spectra (from hydrogen peroxide (30%, 50 \u03bcL) at 95 \u25e6 C for 1.5 h, and concentrated\n450 to 800 nm) at ambient temperature (\u03bbex = 405 nm). hydrochloric acid (50 \u03bcL) at 95 \u25e6 C for 1.5 h to give fully homogenized\n solutions. Finally, the solutions were diluted with water to a final vol\u00ad\n2.4. Cell culture ume of 2 mL for the measurement and iridium contents in the samples\n were determined by ICP-MS (X Series 2, Thermo Fisher, USA). The\n MCF-7 and 4 T1 were obtained from the Experimental Animal Center average of three parallel experimental data was reported.\nof Sun Yat-Sen University (Guangzhou, China). MCF-10 A was pur\u00ad\nchased from Procell (CL-0525). MCF-7, 4 T1 and MCF-10 A were 2.9. Detection of intracellular Ca2+\ncultured in DMEM (Dulbecco's modified Eagle's medium, Gibco, USA)\nwith 37 \u25e6 C and 5% CO2 supplemented with 10% inactivated fetal bovine The MCF-7 cells were seeded into a 35 mm dish and incubated at\nserum (FBS) and 1% penicillin-streptomycin (Gibco, USA). 37 \u25e6 C for 24 h. After that, the cells were then incubated with fresh\n DMEM containing Ir-PTS-4 at different concentrations. After removing\n2.5. Cellular cytotoxicity assay the culture medium, cells were stained with Fluo-4 AM fluorescent\n probes. Finally, intracellular calcium ions were detected using confocal\n The cytotoxicity of the tested compounds towards cell lines was microscopy.\ndetermined by MTT assay. Cells seeded in 96-well plates were incubated\nin a 5% CO2 atmosphere in 100 \u03bcL of complete medium at 37 \u25e6 C for 12 h. 2.10. Lysosomal damage assay\nThen, 100 \u03bcL of freshly prepared culture medium containing drugs at\ndifferent concentrations was added, and the mixture was incubated for MCF-7 cells were seeded into a 35 mm dish and cultured for 24 h.\nan additional 48 h. 20 \u03bcL of MTT solution (5 mg/mL) was then added to After that, the cells were treated with Ir-PTS-4 for 24 h, and washed with\neach well, and the plates were incubated for an additional 4 h. Finally, PBS three times. Before being imaged, the cells were incubated with AO\nthe medium was removed, and DMSO (150 \u03bcL) was added. The absor\u00ad (5 \u03bcM) for another 0.5 h, The excitation wavelength of AO was 488 nm,\nbance at 570 nm was measured using a microplate reader (LabServ K3, and the capture emission region was 515\u2013545 nm for the green channel\nThermo Fisher Scientific, USA). Each well was triplicated to gain the and 610\u2013640 nm for the red channel.\nmean values. IC50 values quoted are mean \u00b1 standard deviation (S.D.).\n 2.11. Intracellular ROS level analysis\n2.6. Assessment of lipophilicity\n 2\u2032,7\u2032-dichlorofluoresecein diacetate (H2DCHF-DA) was used to mea\u00ad\n Briefly, Ir-PTS-1, Ir-PTS-2, Ir-PTS-3 and Ir-PTS-4 were added to the sure the reactive oxygen species (ROS) accumulation. For the flow\nmixture of octanol/NaCl aqueous solution (1:1, v/v), respectively. The cytometry analysis of cellular ROS, MCF-7 cells in the logarithmic phase\nmixture was shaken at 25 \u25e6 C, 600 rpm for 48 h to achieve partitioning were harvested and placed (3 \u00d7 105 cells/well) into a 6-well plate at the\nequilibrium between octanol and aqueous NaCl. The oil phase and the incubator for 12 h, the cell was treated for 24 h with Ir-PTS-4. The cells\nwater phase were then separated by centrifugation (3000 rpm, 10 min) were dyed with H2DCHF-DA for 30 min, then the cell samples were\nand collected. The concentration of the solute was determined by UV\u2013vis analyzed after washing with culture medium without FBS and analyzed\nabsorbance. The lipo-hydro partition coefficient log Po/w (Po/w = Co/Cw using a BD FACSVerse flow cytometer with excitation at 488 nm and\n= Ao/Aw, A stands for absorbance) were calculated. emission at 530 \u00b1 30 nm. Data were analyzed using FlowJo 7.6.1 soft\u00ad\n ware. 10, 000 cells were acquired for each sample.\n2.7. Intracellular localization study For the confocal microscopy analysis of cellular ROS, MCF-7 cells\n were seeded into 35 mm confocal dishes (JET BIOFIL, Canada). After\n MCF-7 cells were seeded into 35 mm confocal dishes for 12 h at 37 \u25e6 C being cultured overnight, the cells were treated with the complex for 24\nand then incubated for 2 h after adding Ir-PTS-4 (4 \u03bcM). Upon h, after being stained with H2DCHF-DA probe and washed twice with\ncompletion, cells were washed twice with PBS and incubated with Mito- serum-free medium, the cells were immediately observed by confocal\nTracker Green, Lyso-Tracker Green, ER-Tracker Green, or Hoechst microscopy (A1, Nikon, Japan) with excitation at 488 nm and emission\n33342 fluorescent probes at 37 \u25e6 C, respectively. Cells were finally at 530 \u00b1 20 nm.\nwashed with PBS and imaged by confocal microscopy under a 100 \u00d7 oil-\nimmersion objective lens. The excitation wavelength for Ir-PTS-4 and 2.12. Mitochondrial membrane potential (MMP) detection\nHoechst 33342 were set at 405 nm and 352 nm, respectively. And the\nemission wavelength for Ir-PTS-4 and Hoechst 33342 were set at 600 \u00b1 The mitochondrial membrane potential was determined by using JC-\n20 nm and 460 \u00b1 20 nm, respectively. The excitation wavelength for 1 dye. For the flow cytometry analysis of MMP, MCF-7 cells were seeded\nMito-tracker Green, Lyso-tracker Green and ER-tracker Green were set at (3 \u00d7 105 cells/well) in a 6-well cell-culture plate for 12 h. The cells were\n488 nm. The emission wavelength for Mito-tracker Green, Lyso-tracker treated with Ir-PTS-4 for 24 h in an incubator at 37 \u25e6 C. Then the cells\nGreen and ER-tracker Green were set at 520 \u00b1 20 nm. were harvested with 0.25% trypsin, and washed with PBS three times.\n After the incubation of 0.5 mL JC-1 working solution for 30 min, the cell\n2.8. Intracellular distribution study samples were washed with 1 \u00d7 incubation buffer and analyzed by using\n a BD FACSVerse flow cytometer.\n The cellular distribution of Ir-PTS-4 in MCF-7 cells was determined For the confocal microscopy analysis of MMP, MCF-7 cells were\nby measuring the iridium contents. Briefly, cells were seeded and seeded into 35 mm confocal dishes (JET BIOFIL, Canada). After cultured\nincubated overnight under standard growth conditions. The culture overnight, the cells were treated with the complex for 24 h at 37 \u25e6 C.\n\n 3\n\fY. Su et al. Journal of Inorganic Biochemistry 251 (2024) 112427\n\n\nAfter being stained with JC-1 working solution for 30 min, the cells were system (Calcein AM: \u03bbex = 488 nm and \u03bbem range 500\u2013550 nm, PI: \u03bbex =\nwashed with incubation buffer and then immediately observed by 561 nm and \u03bbex range 570\u2013620 nm).\nconfocal microscopy (A1, Nikon, Japan).\n 2.18. Statistical analysis\n2.13. Cell cycle analysis\n All biological experiments were performed in triplicate and each\n The cell cycle analysis was conducted as the manufacturer's protocol. experiment was repeated at least twice. The quantitative data are pre\u00ad\nMCF-7 cells were cultured in 6-well plates and incubated with Ir-PTS-4 sented as means \u00b1 standard deviations (SD). Statistical significance was\nfor 24 h. Cells were collected and fixed with 70% ethanol. After storage performed using an unpaired two-tailed Student's t-test. Statistical sig\u00ad\nat 4 \u25e6 C overnight, cells were centrifuged and washed three times with nificance was set at *P < 0.05, and extreme significance was set at **P <\ncold PBS and resuspended in a 500 \u03bcL PBS buffer containing PI and 0.01, and ***P < 0.001.\nRNase A (9:1) for 30 min in the dark. The samples were analyzed by a BD\nFACSVerse flow cytometer and the data were processed using FlowJo 3. Results and discussion\n7.6.1 software.\n 3.1. Synthesis and characterization\n2.14. Wound healing assay\n The modified N^N-PTS ligand was synthesized from 4-aminomethyl-\n MCF-7 cells were seeded in 6-well plates at 10 \u00d7 104 cells per well. 4-methyl-2,2-bipyridyl with pterostilbene (PTS)-derived ligand (E)-2-(4-\nThe cells were allowed to adhere to the surface, and once they reached (3,5-dimethoxystyryl) phenoxy) acetic acid [25,26]. Complexes [Ir\n80% confluence, the monolayer was wounded using a 200-\u03bcl pipette tip (C\u2013N)2(N^N-PTS)](PF6) [C-N = 2-phenylpyridine (ppy, Ir-PTS-1), 2-(2-\nto streak each hole vertically. The cells were washed with PBS three thienyl)pyridine (thpy, Ir-PTS-2), benzo[h]quinoline (dzq, Ir-PTS-3) 2-\ntimes and incubated in serum-free DMEM for another 24 h. Images of the phenylquinoline (pq, Ir-PTS-4)] containing N^N-PTS as ligand were\nscratches were captured with a microscope camera. Data were collected prepared according to literature methods (Fig. 1, Scheme S1) [26]. The\nfrom three independent experiments. The percentage of wound healing structures of Ir-PTS-1\u2013 4 were fully characterized by 1H nuclear mag\u00ad\nwas calculated using the following formula: [1- (empty area 12 (24) h/ netic resonance (NMR) spectroscopy and electrospray ionization-mass\nempty area 0 h)] \u00d7 100. spectrometry (ESI-MS) (Figs. S1-S9). The photophysical properties of\n complexes were first studied (Fig. S10). Complexes Ir-PTS-1\u2013 4 exhibit\n2.15. Transwell migration assay similar absorption spectra in the range of 250\u2013550 nm. Moreover, the\n emission intensity of Ir-PTS-4 was about 6-fold stronger than that of Ir-\n Transwell chambers with 8-\u03bcm pores (Corning, NY) were used to PTS-1/2, which was excited at 405 nm. This finding indicated that Ir-\nevaluate the migration of MCF-7 cells. The upper chamber was filled PTS-4 showed better photophysical properties and could be favorable\nwith 5 \u00d7 104 cells from each sample in serum-free medium for migration for cell imaging. Lipophilicity (log Po/w) has a significant impact on drug\nanalysis. After incubating the cells at 37 \u25e6 C for 24 h, the cells migrating cellular uptake, distribution, and cytotoxicity [27]. The shaking flask\nto the bottom of the filter were incubated with 0.5% crystal violet at method was used to determine the lipophilicity of four complexes. The\nroom temperature for 20 min. For quantitative measurement, five areas log Po/w values for complexes Ir-PTS-1\u2013 4 were determined to be 1.48,\n(at \u00d7100 magnification) were randomly selected under an optical mi\u00ad 1.06, 1.49, and 1.32, respectively (Table S1). This implied that the four\ncroscope, and the cells were counted. complexes exhibited higher lipophilicity, which could help them\n permeate the lipid bilayer of the cell membrane.\n2.16. Western blot analysis\n 3.2. In vitro anticancer activity\n MCF-7 cells were seeded in 10 cm dishes for 12 h, then treated with\nthe Ir-PTS-4 (4 \u03bcM) for 24 h. After that, the harvested cells were lysed by The cytotoxicity of complexes Ir-PTS-1\u2013 4 was tested against breast\nRIPA lysis buffer. The protein concentration was quantified using the cancer cell lines (MCF-7 and 4 T1) and normal breast cell line (MCF-10\nBCA Quantitation Kit. Equal amounts of cellular lysate (20 \u03bcg) were A) by a MTT assay [28], with ligand PTS, N^N-PTS and cisplatin as the\nseparated on SDS polyacrylamide gel electrophoresis and then trans\u00ad reference. The half-maximal inhibitory concentrations (IC50 values)\nferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, obtained after 48 h of drug treatment are listed in Table 1. The ligand\nUSA). Membranes were blocked in QuickBlock\u2122 Blocking Buffer, and PTS and N^N-PTS showed almost no toxicity against the tested cell lines.\nthen incubated overnight with the primary antibodies at 4 \u25e6 C. After a Ir-PTS-1\u2013 4 exhibited strong anti-proliferative activity, with the\nsubsequent washing step, the membrane was incubated with the following order: Ir-PTS-4 > Ir-PTS-1 > Ir-PTS-2 > Ir-PTS-3. Ir-PTS-4,\nappropriate horseradish peroxidase conjugated secondary antibody.\nImages were captured using Tanon High-sig ECL Western Blotting\nSubstrate and Tanon 5200 Multi, and analyzed manually with Image J\nsoftware.\n\n2.17. 3D tumor spheroids viability assays\n\n The multicellular tumor spheroids (MCTSs) were prepared with\nMCF-7 cells by seeding 2500 cells/well in an Ultra-Low Attachment 96-\nwell plate (Corning). The spheroids were incubated for 1 day. The\ndiameter of the MCF-7 MCTSs proved to be around 500 \u03bcm after 1 day.\nThe spheroid growth was monitored using a live-cell phase-contrast\nmicroscope (Axio Observer, Zeiss). After 5 d incubation, the spheroids\nwere washed twice with PBS, stained with Calcein AM/PI following the\nmanufacturer's instructions (Beyotime, China), and fixed in 4% para\u00ad\nformaldehyde. Spheroids were placed in a glass-bottom dish and imaged\nat different depths (z-stacking) with a confocal scanning microscopy Fig. 1. Chemical structures of complexes Ir-PTS-1\u2013 4.\n\n 4\n\fY. Su et al. Journal of Inorganic Biochemistry 251 (2024) 112427\n\n\nTable 1 cellular localization of the Ir-PTS-4 in MCF-7 cells was investigated by\nIC50 values (\u03bcM) of tested compounds towards different cell lines. Data are co-localization staining with commercial dyes Lyso-Tracker Green,\npresented as means \u00b1 standard deviations obtained in at least three independent Mito-Tracker Green, endoplasmic reticulum (ER)-Tracker Green and\nexperiments and the treatment period was 48 h. Hoechst 33342. The Pearson's correlation coefficients (PCCs) of complex\n Compounds MCF-7 4 T1 MCF-10 A Ir-PTS-4 were 0.55, 0.53, 0.54 and 0.08 with ER-Tracker Green, Mito-\n PTS >100 >100 >100 Tracker Green, Lyso-Tracker Green and Hoechst 33342, respectively\n N^N-PTS >100 >100 >100 (Fig. 3a). These results revealed that Ir-PTS-4 localized in the ER,\n Ir-PTS-1 4.1 \u00b1 0.1 3.3 \u00b1 0.2 3.7 \u00b1 0.3 mitochondria and lysosome of MCF-7 cells. To further assess the intra\u00ad\n Ir-PTS-2 4.6 \u00b1 0.3 5.6 \u00b1 0.1 5.4 \u00b1 0.4 cellular distribution of Ir-PTS-4, the iridium content of MCF-7 cells was\n Ir-PTS-3 4.7 \u00b1 0.1 5.1 \u00b1 0.6 11.5 \u00b1 0.1\n Ir-PTS-4 1.9 \u00b1 0.1 3.6 \u00b1 0.1 6.5 \u00b1 0.1\n quantified by inductively coupled plasma-mass spectrometry (ICP-MS).\n Cisplatin 4.3 \u00b1 0.1 9.6 \u00b1 0.2 10.8 \u00b1 1.2 As shown in Fig. S12, there were about 65% and 32% of iridium were\n localized in the cytosol and mitochondria in the Ir-PTS-4-treated MCF-7\n cells, respectively. These data displayed that Ir-PTS-4 are primarily\nwith an IC50 value of 1.9 \u03bcM to MCF-7 cells, is the most potent among all distributed in mitochondria, lysosome and ER.\ncompounds. Notably, the toxicity of Ir-PTS-4 to normal cells was\nreduced by about 3.4-fold, showing better cell selectivity compared with 3.5. Induction of Ca2+ overload and lysosomal membrane\nIr-PTS-1\u2013 3 and cisplatin. Based on the cytotoxic profile, we chose Ir- permeabilization\nPTS-4 as the targeting compound for further mechanism investigation.\n According to previous studies, the ER is highly sensitive to Ca2+\n3.3. Cellular accumulation concentration turbulence [30\u201332]. Fluo-4 AM can be hydro-lysed by the\n esterase enzymes to Fluo-4 which can bind to free Ca2+, and turn fluo\u00ad\n Due to the excellent photophysical properties of Ir(III) complexes, rescent [33]. Therefore, in order to depict the efflux of Ca2+ induced by\nthe accumulation of Ir-PTS-4 in MCF-7 cells was detected by confocal Ir-PTS-4 via ER stress, Fluo-4 AM was used as an \u201cOFF-ON\u201d probe for\nlaser scanning microscopy (CLSM) and flow cytometer. As shown in intracellular Ca2+. As shown in Fig. 3b, the fluorescence intensity was\nFig. 2, an increased red fluorescence intensity in MCF-7 cells was significantly increased in a dose-dependent manner after treatment with\nobserved, suggesting the uptake of Ir-PTS-4 was highly dependent on Ir-PTS-4, which suggested an increased intracellular Ca2+ level. While\nthe concentration and the incubation time. We further examined the MCF-7 cells in the control group did not show this phenomenon, sug\u00ad\ncellular uptake mechanisms of Ir-PTS-4 by assessing the influence of gesting that Ir-PTS-4 could induce the efflux of Ca2+ from the ER.\nincubation temperature (37 \u25e6 C and 4 \u25e6 C), metabolic inhibitor (2-deoxy- Due to the accumulation of complex Ir-PTS-4 in the lysosome, we\nD-glucose and oligomycin) and endocytic inhibitor (NH4Cl) on the further detected whether Ir-PTS-4 could induce lysosomal membrane\ncellular uptake level of Ir-PTS-4. As shown in Fig. S11, compared with permeabilization (LMP) by acridine orange (AO) staining. As shown in\nthat at 37 \u25e6 C, the fluorescence intensity of Ir-PTS-4 in MCF-7 cells was Fig. 3c, MCF-7 cells incubated with Ir-PTS-4 showed obvious green\nmarkedly reduced on incubation at 4 \u25e6 C or pretreatment with metabolic fluorescence of lysosomes as the control, whereas the red fluorescence\ninhibitor. Meanwhile, the cellular uptake efficiency is barely affected by intensity in the Ir-PTS-4-treated MCF-7 cells was significantly attenu\u00ad\npretreated with NH4Cl. These results demonstrated that Ir-PTS-4 ated, suggesting the occurrence of LMP.\npenetrated the cell membrane primarily through an energy-dependent\nmechanism instead of endocytic pathways. 3.6. Mitochondrial dysfunction\n\n3.4. Subcellular localization Since Ir-PTS-4 could accumulate in the mitochondria of MCF-7 cells,\n the impacts on mitochondrial functionality were then studied. Mito\u00ad\n Studies on cellular localization of phosphorescent metal complexes chondria is the main organelle for reactive oxygen species (ROS) pro\u00ad\nmay offer more clues for the study of anticancer mechanisms [29]. The duction and ROS elevation could cause mitochondrial damage [34].\n\n\n\n\nFig. 2. (a) Temporal profiles of confocal imaging and (b) quantification analysis, and (c) dose-dependent profiles of flow cytometry results and (d) quantification\nanalysis of cellular uptake of Ir-PTS-4 in MCF-7 cells. Scale bar: 20 \u03bcm.\n\n 5\n\fY. Su et al. Journal of Inorganic Biochemistry 251 (2024) 112427\n\n\n\n\nFig. 3. (a) Subcellular distribution confocal study of Ir-PTS-4 in MCF-7 cells by co-localization imaging. The cells were co-stained with Mito-Tracker Green (200 nM)\nor Lyso-Tracker Green (50 nM), or ER-Tracker Green (1 \u03bcM), or Hoechst 33342 (10 \u03bcg/mL). (b) Confocal images of intracellular Ca2+ assays with Fluo-4 AM in MCF-7\ncells for 24 h. (c) AO staining for LMP analysis in MCF-7 cells. (d-e) Flow cytometry quantification of the ROS generation and (f-g) flow cytometry results of a JC-1\nassay in MCF-7 cells after treatment with Ir-PTS-4, respectively, for 24 h. Scale bar: 20 \u03bcm. (For interpretation of the references to colour in this figure legend, the\nreader is referred to the web version of this article.)\n\n\nIntracellular ROS levels were determined by flow cytometry using a ROS 75.6%. CLSM analysis was also performed to detect the MMP in MCF-7\nprobe, 2\u2032,7\u2032-dichlorofluorescein diacetate (H2DCF-DA). H2DCF-DA will cells. Ir-PTS-4-treated MCF-7 cells emitted strong green fluorescence\nbe oxidized to highly fluorescent 2\u2032,7\u2032-dichlorofluorescein (DCF) by a (Fig. S13b), thus suggesting that Ir-PTS-4 has a strong ability to impair\nwide range of cellular ROS [35]. As shown in Fig. 3d-e, after treatment the MMP. These results indicated that Ir-PTS-4 could induce ROS\nwith Ir-PTS-4 for 24 h, a dramatic dose-dependent increase in DCF elevation and loss of MMP.\nfluorescence intensity was observed in MCF-7 cells, indicating the gen\u00ad\neration of ROS. An approximately 3.5-fold increase in DCF fluorescence\n 3.7. Cell death mechanism study\nintensity was observed in cells treated with Ir-PTS-4 (4 \u03bcM). Meanwhile,\nCLSM images indicated a concentration-dependent elevation of ROS\n Many studies have shown that damage to mitochondrial and lyso\u00ad\ngeneration in MCF-7 cells was observed after Ir-PTS-4 the treatment\n somal can lead to autophagy [37,38]. LC3 (microtubule-associated with\n(Fig. S13a).\n protein light chain 3) is a common molecular marker of autophagy [39].\n The effects on the mitochondrial membrane potential (MMP) were\n During autophagy, LC3-I (cytoplasmic type I) is hydrolyzed by enzymes\nevaluated using the JC-1 assay kit [36]. The changes in MMP were re\u00ad\n to cut off a polypeptide and produce LC3-II (type II LC3) located in the\nflected by the increase of JC-1 monomers (green fluorescence) and\n membrane of autophagic cells. The marker of autophagy is the conver\u00ad\ndecrease of JC-1 aggregate (red fluorescence). As shown in Fig. 3e-f, in\n sion of LC3 protein from type I to type II [40]. From the results of the\ncomparison with the control, a significant dose-dependent loss of MMP s\n western blot experiment, the ratio of LC3-II to LC3-I increased signifi\u00ad\nwas observed in Ir-PTS-4-treated MCF-7 cells, dropping from 12.2% to\n cantly after treatment with Ir-PTS-4, compared to the control (Fig. 4a\n\n 6\n\fY. Su et al. Journal of Inorganic Biochemistry 251 (2024) 112427\n\n\nand c). All these data collectively suggest that Ir-PTS-4 can induce anticancer mechanism for Ir-PTS-4 towards MCF-7 cells involved STAT3\nautophagy in MCF-7 cells. inhibition and cell cycle arrest.\n We further investigated the apoptosis of MCF-7 cells induced by Ir-\nPTS-4 by flow cytometry after annexin V-FITC and propidium iodide 3.9. Inhibition of cell migration and invasion\n(PI) double staining. As shown in Fig. S14, flow cytometry analysis\nindicated that there is no distinct dose-dependent increase of the Migration is a key step in cancer progression, and emerging evidence\napoptotic MCF-7 cells. The expressions of apoptosis-related proteins Bax indicated that aberrantly elevated STAT3 is one of the key driving fac\u00ad\n(proapoptotic protein), Bcl-2 (anti-apoptotic protein), cytochrome c and tors of cancer metastasis and invasiveness [43]. Hence, the anti-\nnecrosis marker RIP3 (receptor-interacting kinase protein 3) were not migration activity of Ir-PTS-4 was tested by a wound healing assay\nchanged in Ir-PTS-4-treated MCF-7 cells as compared with that of the and a transwell assay. As shown in Fig. 5a and c, the wound closure ratio\ncontrol. These data suggested that the cell death mediated by Ir-PTS-4 of Ir-PTS-4-treated MCF-7 cells was only 11.3%, in contrast to 75.9% of\noccurred via a non-apoptotic/necrotic pathway but autophagic the control group after 24 h incubation. As shown in Fig. 5b, the number\npathways. of cells migrating through pores was significantly decreased by Ir-PTS-4\n in a dose-dependent manner in MCF-7 cells. Migration cells treated with\n Ir-PTS-4 (2 \u03bcM) were observed in only 14.7% of the chamber compared\n3.8. Inhibition of STAT3 and cell cycle arrest to the control group (Fig. 5d). These results suggested that Ir-PTS-4\n could effectively inhibit cell migration and invasion.\n Recent clinical and preclinical findings suggest overexpressed and\nconstitutively activated STAT3 is involved in the progression, prolifer\u00ad 3.10. Biological evaluation on 3D multicellular tumor spheroids\nation, and metastasis of breast cancer [41], the mechanism of action of\nIr-PTS-4 on STAT3 signaling was further explored in MCF-7 cells by 3D multicellular tumor spheroids (MCTSs) with the tumor micro\u00ad\nimmunoblotting. As shown in Fig. 4b-c, Ir-PTS-4 reduced the expression environments of intercellular and cell-extra-cellular matrix interactions\nof phosphorylated STAT3 (p-STAT3) in a dose-dependent manner rela\u00ad have been used to mimic the solid tumor in vitro [44]. As shown in\ntive to the control, while the expression of STAT3 was only slightly Fig. 6a, the control spheroids grew to a diameter of around 600 \u03bcm after\naffected. The results indicated that Ir-PTS-4 selectively decreased the 5 days of incubation, which demonstrated the great proliferation ca\u00ad\nlevel of p-STAT3, which was not due to a constitutional decrease in total pacity of MCF-7 spheroids. The diameter shrank to only 350 \u03bcm after 5\nSTAT3 expression. days of incubation with Ir-PTS-4 (10 \u03bcM). In addition, a Calcein AM/PI\n It has been reported that STAT3 can upregulate cyclin D1 to promote double staining was examined to further illustrate the anti-proliferation\nthe proliferation of breast cancer cells, indicating a potential involve\u00ad performance. Calcein AM only enters into live cells and emits green\nment of STAT3 in the cell cycle [42]. The cyclin D1 expression and the fluorescence, whereas PI only enters into dead cells with red fluores\u00ad\ncell cycle distribution of MCF-7 cells were analyzed after exposure to Ir- cence under irradiation. As shown in Fig. 6b, the untreated control\nPTS-4 for 24 h. As shown in Fig. 4b-d, Ir-PTS-4 induced a significant MCTSs exhibited a bright green fluorescence signal indicating that the\ndownregulation of cyclin D1 and arrested cell cycle in the S phase cells were alive. However, the MCTSs treated with Ir-PTS-4 showed\n(24.8%) as compared with the control (14.5%). Cell cycle arrest could weak green fluorescence as well as strong red fluorescence, indicating\ninduce cellular DNA damage. Then, the expression of \u03b3-H2AX in MCF-7 the superior capacity of Ir-PTS-4 to inhibit cell growth and induce cell\ncells was determined by western blotting, which is a biomarker of DNA death of 3D MCTSs.\ndamage [14]. As shown in Fig. 4b-c, a concentration-dependent increase\nin the expression of \u03b3-H2AX was observed in MCf-7 cells, indicating that\nIr-PTS-4 could induce DNA damage. Thus, we concluded that the\n\n\n\n\nFig. 4. (a and b) Immunoblotting of LC3-I/II, p-STAT3, STAT3, Cyclin D1 and \u03b3H2AX in MCF-7 cells treated with Ir-PTS-4 for 24 h. (c) Quantitative analysis of\nimmunoblotting in a-b. (d) Flow cytometry results of cell cycle distribution in MCF-7 cells after treatment with Ir-PTS-4 for 24 h.\n\n 7\n\fY. Su et al. Journal of Inorganic Biochemistry 251 (2024) 112427\n\n\n\n\nFig. 5. (a) Wound-healing assay of MCF-7 cells after incubation with Ir-PTS-4 and (c) the percent wound closure was calculated. (b) Representative images of MCF-7\ncells were observed by transwell invasion assay and (d) the number of migrated cells was counted. Scale bar: 100 \u03bcm.\n\n\n\n\nFig. 6. (a) Representative images of 3D MCF-7 tumor spheroids after being treated with Ir-PTS-4. (b) Images of MCF-7 3D MCTSs treated with Ir-PTS-4 for 5 days,\nand stained with Calcein AM/PI for the live/dead cells. Scale bar: 100 \u03bcm.\n\n\n\n\n 8\n\fY. Su et al. Journal of Inorganic Biochemistry 251 (2024) 112427\n\n\n4. Conclusion [7] C.P. Tan, Y.Y. Lu, L.N. Ji, Z.W. Mao, Metallomics insights into the programmed cell\n death induced by metal-based anticancer compounds, Metallomics 6 (2014)\n 978\u2013995.\n In conclusion, we have developed four PTS-derived ligand conju\u00ad [8] L. Feng, Y. Geisselbrecht, S. Blanck, A. Wilbuer, G.E. Atilla-Gokcumen,\ngated cyclometalated iridium(III) complexes Ir-PTS-1\u2013 4 to combat P. Filippakopoulos, K. Kraling, M.A. Celik, K. Harms, J. Maksimoska,\nbreast cancer cells via inhibiting STAT3 signaling pathway. All four R. Marmorstein, G. Frenking, S. Knapp, L.O. Essen, E. Meggers, Structurally\n sophisticated octahedral metal complexes as highly selective protein kinase\ncomplexes exhibit higher cytotoxicity towards breast cancer cells, inhibitors, J. Am. Chem. Soc. 133 (2011) 5976\u20135986.\nespecially Ir-PTS-4. Besides, Ir-PTS-4 could increase the intracellular [9] A.G. Quiroga, C. Navarro Ranninger, Contribution to the SAR field of metallated\nROS and reduce the mitochondrial membrane potential, arrest the cell and coordination complexes, Coord. Chem. Rev. 248 (2004) 119\u2013133.\n [10] D.-L. Ma, L.-J. Liu, K.-H. Leung, Y.-T. Chen, H.-J. Zhong, D.S.-H. Chan, H.-M.\ncycle in the S-phase, and induce DNA damage, eventually leading to D. Wang, C.-H. Leung, Antagonizing STAT3 dimerization with a rhodium(III)\nautophagy. The expression level of phosphorylated STAT3 and STAT3- complex, Angew. Chem. Int. Ed. 53 (2014) 9178\u20139182.\nrelated cyclin D1 were obviously down-regulated in MCF-7 cells after [11] T.-S. Kang, W. Wang, H.-J. Zhong, Z.-Z. Dong, Q. Huang, S.W.F. Mok, C.-H. Leung,\n V.K.W. Wong, D.-L. Ma, An anti-prostate cancer benzofuran-conjugated iridium\nbeing treated with Ir-PTS-4. The cell metastasis and invasion were also (III) complex as a dual inhibitor of STAT3 and NF-\u03baB, Cancer Lett. 396 (2017)\ninhibited after Ir-PTS-4 treatment. Ir-PTS-4 also exhibited excellent 76\u201384.\nanti-proliferation activity in 3D multicellular tumor spheroids, sug\u00ad [12] F. Porta, G. Facchetti, N. Ferri, A. Gelain, F. Meneghetti, S. Villa, D. Barlocco,\n D. Masciocchi, A. Asai, N. Miyoshi, S. Marchiano\u0300, B.-M. Kwon, Y. Jin, V. Gandin,\ngesting the potential to inhibit solid tumors in vivo. The combination of C. Marzano, I. Rimoldi, An in vivo active 1,2,5-oxadiazole Pt(II) complex: a\nPTS-derived ligand with iridium-based metal complex presents the promising anticancer agent endowed with STAT3 inhibitory properties, Eur. J.\nrational design of metal-based anticancer agents to block STAT3 Med. Chem. 131 (2017) 196\u2013206.\n [13] J. Zang, B. Zhang, Y. Wang, X. Wang, S. Gou, Design, synthesis and biological\npathway for simultaneously inhibiting tumor proliferation and\n evaluation of antitumor platinum(II) agents conjugated with non-steroidal anti-\nmetastasis. inflammatory drug species, Bioorg. Chem. 120 (2022), 105633.\n [14] L. Cai, Y. Wang, H. Chen, Y. Tan, T. Yang, S. Zhang, Z. Guo, X. Wang, Platinum(IV)\n complexes as inhibitors of STAT3 and regulators of the tumor microenvironment to\nCRediT authorship contribution statement control breast cancer, J. Med. Chem. 66 (2023) 11351\u201311364.\n [15] M. Ali Nazif, J.A. Bangert, I. Ott, R. Gust, R. Stoll, W.S. Sheldrick, Dinuclear\n Yan Su: Investigation, Writing \u2013 original draft. Jin Yang: Investi\u00ad organoiridium(III) mono- and bis-intercalators with rigid bridging ligands:\n synthesis, cytotoxicity and DNA binding, J. Inorg. Biochem. 103 (2009)\ngation. Meng-Meng Wang: Investigation. Hong-Bao Fang: Supervi\u00ad 1405\u20131414.\nsion, Funding acquisition. Hong-Ke Liu: Supervision, Funding [16] Z. Liu, A. Habtemariam, A.M. Pizarro, S.A. Fletcher, A. Kisova, O. Vrana, L. Salassa,\nacquisition. Zheng-Hong Yu: Supervision, Funding acquisition. Zhi Su: P.C. Bruijnincx, G.J. Clarkson, V. Brabec, P.J. Sadler, Organometallic half-\n sandwich iridium anticancer complexes, J. Med. Chem. 54 (2011) 3011\u20133026.\nWriting \u2013 original draft, Supervision, Funding acquisition. [17] J. Ruiz, V. Rodriguez, N. Cutillas, K.G. Samper, M. Capdevila, O. Palacios,\n A. Espinosa, Novel C,N-chelate rhodium(III) and iridium(III) antitumor complexes\n incorporating a lipophilic steroidal conjugate and their interaction with DNA,\nDeclaration of Competing Interest Dalton Trans. 41 (2012) 12847\u201312856.\n [18] A. Wilbuer, D.H. Vlecken, D.J. Schmitz, K. Kraling, K. Harms, C.P. Bagowski,\n E. Meggers, Iridium complex with antiangiogenic properties, Angew. Chem. Int.\n The authors declare that they have no known competing financial Ed. 49 (2010) 3839\u20133842.\ninterests or personal relationships that could have appeared to influence [19] C. Kunick, I. Ott, Metal complexes as protein kinase inhibitors, Angew. Chem. Int.\n Ed. 49 (2010) 5226\u20135227.\nthe work reported in this paper. [20] Z. Ma, X. Zhang, L. Xu, D. Liu, S. Di, W. Li, J. Zhang, H. Zhang, X. Li, J. Han, X. Yan,\n Pterostilbene: mechanisms of its action as oncostatic agent in cell models and in\nData availability vivo studies, Pharmacol. Res. 145 (2019), 104265.\n [21] Y. Li, X. Qiang, Y. Li, X. Yang, L. Luo, G. Xiao, Z. Cao, Z. Tan, Y. Deng,\n Pterostilbene-O-acetamidoalkylbenzylamines derivatives as novel dual inhibitors\n Data will be made available on request. of cholinesterase with anti-beta-amyloid aggregation and antioxidant properties\n for the treatment of Alzheimer's disease, Bioorg. Med. Chem. Lett. 26 (2016)\n 2035\u20132039.\nAcknowledgments [22] Q. Wu, K.Y. Zhang, P. Dai, H. Zhu, Y. Wang, L. Song, L. Wang, S. Liu, Q. Zhao,\n W. Huang, Bioorthogonal \u201clabeling after recognition\u201d affording an FRET-based\n We appreciate the financial support from the National Natural Sci\u00ad luminescent probe for detecting and imaging Caspase-3 via photoluminescence\n lifetime imaging, J. Am. Chem. Soc. 142 (2020) 1057\u20131064.\nence Foundation of China (NSFC) (Grant No. 21977052, 22077066) and [23] B.B. Chen, N.L. Pan, J.X. Liao, M.Y. Huang, D.C. Jiang, J.J. Wang, H.J. Qiu, J.\nDistinguished Young Scholars of Jiangsu Province (BK20230006). Y. Su X. Chen, L. Li, J. Sun, Cyclometalated iridium(III) complexes as mitochondria-\nwas supported by the Jiangsu Excellent Postdoctoral Program and NSF targeted anticancer and antibacterial agents to induce both autophagy and\n apoptosis, J. Inorg. Biochem. 219 (2021), 111450.\nof Jiangsu Province (BK20231090). [24] C. Fu, Q. Lv, J. Fan, S. Wu, M. Lei, X. Zhang, X. Li, W. Zhou, Y. Yu, W. Ren, C. Zhao,\n G. Liao, Discovery of polypyridyl iridium(III) complexes as potent agents against\n resistant Candida albicans, Eur. J. Med. Chem. 233 (2022), 114250.\nAppendix A. Supplementary data\n [25] B. Wang, T. Liu, Z. Wu, L. Zhang, J. Sun, X. Wang, Synthesis and biological\n evaluation of stilbene derivatives coupled to NO donors as potential antidiabetic\n Supplementary data to this article can be found online at https://doi. agents, J. Enzyme Inhib. Med. Chem. 33 (2018) 416\u2013423.\norg/10.1016/j.jinorgbio.2023.112427. [26] M.-M. Wang, F.-J. Xu, Y. Su, Y. Geng, X.-T. Qian, X.-L. Xue, Y.-Q. Kong, Z.-H. Yu,\n H.-K. Liu, Z. Su, A new strategy to fight metallodrug resistance: mitochondria-\n relevant treatment through mitophagy to inhibit metabolic adaptations of cancer\nReferences cells, Angew. Chem. Int. Ed. 61 (2022), e202203843.\n [27] R.-R. Ye, B.-C. Chen, J.-J. Lu, X.-R. Ma, R.-T. Li, Phosphorescent rhenium(I)\n complexes conjugated with artesunate: mitochondrial targeting and apoptosis-\n [1] R.L. Siegel, K.D. Miller, H.E. Fuchs, A. Jemal, Cancer statistics, 2021, CA Cancer J.\n ferroptosis dual induction, J. Inorg. Biochem. 223 (2021), 111537.\n Clin. 71 (2021) 7\u201333.\n [28] Y. Su, H. Lin, Y. Tu, M.-M. Wang, G.-D. Zhang, J. Yang, H.-K. Liu, Z. Su, Fighting\n [2] C.L. Chaffer, R.A. Weinberg, A perspective on cancer cell metastasis, Science 331\n metallodrug resistance through alteration of drug metabolism and blockage of\n (2011) 1559\u20131564.\n autophagic flux by mitochondria-targeting AIEgens, Chem. Sci. 13 (2022)\n [3] L. Yang, P. Shi, G. Zhao, J. Xu, W. Peng, J. Zhang, G. Zhang, X. Wang, Z. Dong,\n 1428\u20131439.\n F. Chen, H. Cui, Targeting cancer stem cell pathways for cancer therapy, Signal\n [29] R.R. Ye, C.P. Tan, M.H. Chen, L. Hao, L.N. Ji, Z.W. Mao, Mono- and dinuclear\n Transduct. Target. Ther. 5 (2020) 8.\n phosphorescent rhenium(I) complexes: impact of subcellular localization on\n [4] J. Huynh, A. Chand, D. Gough, M. Ernst, Therapeutically exploiting STAT3 activity\n anticancer mechanisms, Chemistry 22 (2016) 7800\u20137809.\n in cancer - using tissue repair as a road map, Nat. Rev. Cancer 19 (2019) 82\u201396.\n [30] L. Xu, P.P. Zhang, X.Q. Fang, Y. Liu, J.Q. Wang, H.Z. Zhou, S.T. Chen, H. Chao,\n [5] K.S. Siveen, S. Sikka, R. Surana, X. Dai, J. Zhang, A.P. Kumar, B.K.H. Tan, G. Sethi,\n A ruthenium(II) complex containing a p-cresol group induces apoptosis in human\n A. Bishayee, Targeting the STAT3 signaling pathway in cancer: role of synthetic\n cervical carcinoma cells through endoplasmic reticulum stress and reactive oxygen\n and natural inhibitors, Biochim. Biophys. Acta 2014 (1845) 136\u2013154.\n species production, J. Inorg. Biochem. 191 (2019) 126\u2013134.\n [6] H. Yu, H. Lee, A. Herrmann, R. Buettner, R. Jove, Revisiting STAT3 signalling in\n [31] R.V. Rao, H.M. Ellerby, D.E. Bredesen, Coupling endoplasmic reticulum stress to\n cancer: new and unexpected biological functions, Nat. Rev. Cancer 14 (2014)\n the cell death program, Cell Death Differ. 11 (2004) 372\u2013380.\n 736\u2013746.\n\n\n 9\n\fY. Su et al. Journal of Inorganic Biochemistry 251 (2024) 112427\n\n[32] Y. Liu, Y. Ye, Proteostasis regulation at the endoplasmic reticulum: a new [39] J. Kim, M. Kundu, B. Viollet, K.L. Guan, AMPK and mTOR regulate autophagy\n perturbation site for targeted cancer therapy, Cell Res. 21 (2011) 867\u2013883. through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2011) 132\u2013141.\n[33] B. Yuan, J. Liu, R. Guan, C. Jin, L. Ji, H. Chao, Endoplasmic reticulum targeted [40] Q.P. Liang, T.Q. Xu, B.L. Liu, X.P. Lei, J.R. Hambrook, D.M. Zhang, G.X. Zhou,\n cyclometalated iridium(iii) complexes as efficient photodynamic therapy Sasanquasaponin IotaIotaIota from Schima crenata Korth induces autophagy\n photosensitizers, Dalton Trans. 48 (2019) 6408\u20136415. through Akt/mTOR/p70S6K pathway and promotes apoptosis in human melanoma\n[34] D. Trachootham, J. Alexandre, P. Huang, Targeting cancer cells by ROS-mediated A375 cells, Phytomedicine 58 (2019), 152769.\n mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov. 8 (2009) [41] G.L. Wong, S.G. Manore, D.L. Doheny, H.-W. Lo, STAT family of transcription\n 579\u2013591. factors in breast cancer: pathogenesis and therapeutic opportunities and\n[35] C.P. LeBel, H. Ischiropoulos, S.C. Bondy, Evaluation of the probe 2\u2032,7\u2032- challenges, Semin. Cancer Biol. 86 (2022) 84\u2013106.\n dichlorofluorescin as an indicator of reactive oxygen species formation and [42] S. Zou, Q. Tong, B. Liu, W. Huang, Y. Tian, X. Fu, Targeting STAT3 in cancer\n oxidative stress, Chem. Res. Toxicol. 5 (1992) 227\u2013231. immunotherapy, Mol. Cancer 19 (2020) 145.\n[36] M. Xia, Y. Zhang, K. Jin, Z. Lu, Z. Zeng, W. Xiong, Communication between [43] S. Li, W. Zhang, Y. Yang, T. Ma, J. Guo, S. Wang, W. Yu, L. Kong, Discovery of oral-\n mitochondria and other organelles: a brand-new perspective on mitochondria in available resveratrol-caffeic acid based hybrids inhibiting acetylated and\n cancer, Cell Biosci. 9 (2019) 27. phosphorylated STAT3 protein, Eur. J. Med. Chem. 124 (2016) 1006\u20131018.\n[37] M. Redmann, M. Dodson, M. Boyer-Guittaut, V. Darley-Usmar, J. Zhang, [44] C.S. Shin, B. Kwak, B. Han, K. Park, Development of an in vitro 3D tumor model to\n Mitophagy mechanisms and role in human diseases, Int. J. Biochem. Cell Biol. 53 study therapeutic efficiency of an anticancer drug, Mol. Pharm. 10 (2013)\n (2014) 127\u2013133. 2167\u20132175.\n[38] T. Kanki, K. Furukawa, S. Yamashita, Mitophagy in yeast: molecular mechanisms\n and physiological role, Biochim. Biophys. Acta 2015 (1853) 2756\u20132765.\n\n\n\n\n 10\n\f", "pages_extracted": 10, "text_length": 69701}