Cyclometalated iridium(III) complexes induce immunogenic cell death in HepG2 cells via paraptosis.

{"full_text": " Bioorganic Chemistry 140 (2023) 106837\n\n\n Contents lists available at ScienceDirect\n\n\n Bioorganic Chemistry\n journal homepage: www.elsevier.com/locate/bioorg\n\n\n\n\nCyclometalated iridium(III) complexes induce immunogenic cell death in\nHepG2 cells via paraptosis\nJiaxin Liao a, 1, Yuqing Zhang a, 1, Minying Huang a, 1, Zhijun Liang a, Yao Gong a, Ben Liu a,\nYuling Li a, Jiaxi Chen a, *, Wei Wu a, Zunnan Huang b, *, Jing Sun a, b, *\na\n School of Pharmacy, Guangdong Medical University, Dongguan 523808, China\nb\n Key Laboratory of Computer-Aided Drug Design of Dongguan City, Guangdong Medical University, Dongguan 523808, 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: Immunotherapy has been shown to provide superior antitumor efficacy by activating the innate immune system\nIr(III) complexes to recognize, attack and eliminate tumor cells without seriously harming normal cells. Herein, we designed and\nAnticancer synthesized three new cyclometalated iridium(III) complexes (Ir1, Ir2, Ir3) then evaluated their antitumor ac\u00ad\nCytotoxicity\n tivity. When co-incubated with HepG2 cells, the complex Ir1 localized in the lysosome, where it induced par\u00ad\nParaptosis\n aptosis and endoplasmic reticulum stress (ER stress). Notably, Ir1 also induced immunogenic cell death (ICD),\nICD inducer\nDC promoted dendritic cell maturation that enhanced effector T cell chemotaxis to tumor tissues, down-regulated\nImmunity proportions of immunosuppressive regulatory T cells within tumor tissues and triggered activation of anti\u00ad\nHepG2 cells tumor immunity throughout the body. To date, Ir1 is the first reported iridium(III) complex-based paraptosis\nDAMPs inducer to successfully induce tumor cell ICD. Furthermore, Ir1 induced ICD of HepG2 cells without affecting cell\nER stress cycle or reactive oxygen species levels.\n\n\n\n\n1. Introduction exposing calreticulin (CRT) molecules on tumor cell surfaces while also\n triggering extranuclear release of high-mobility group box 1 protein\n In the field of cancer therapy, the harnessing of the immune system (HMGB-1) [7]. Notably, ICD was defined by the Nomenclature Com\u00ad\nto combat cancer is increasingly being evaluated as a promising mittee on Cell Death (NCCD) in 2020 as a stress-induced regulatory cell\nemerging antitumor strategy [1]. The most well-known antitumor im\u00ad death (RCD)-related process that, under certain circumstances, can\nmune system-harnessing agents include immune checkpoint inhibitors cause the body\u2019s inflammatory response to induce adaptive cytotoxic T\n(ICIs) that target host immune cell surface molecules CTLA-4, PD-1 and lymphocyte (CTL)-mediated immunity and long-term immunological\nPD-L1. ICIs act by blocking immune checkpoint functions and by stim\u00ad memory. Interestingly, it has been demonstrated that certain drugs can\nulating tumor-specific T-cell immune responses that convert immune kill tumor cells, while also invoking ICD by activating an immune\nsystem effector cells into tumor-killing machines [2,3]. While ICI ther\u00ad response within the tumor microenvironment. Based on this premise,\napy has been shown to effectively combat multiple cancers, treatment researchers are increasingly developing and evaluating new chemo\u00ad\nefficacy varies substantially according to tumor type and can even vary therapeutic agents for use in combination with tumor immunotherapies,\nbetween patients with the same type of malignancy [4,5]. As a result, with ICDs performing both functions[8\u201310]. Nevertheless, in clinical\ntraditional anticancer treatments, such as surgery, radiation and practice only a small number of ICD-inducing chemotherapeutic agents\nchemotherapy, continue to be viewed as clinical best practices in some have been identified, including adriamycin[11,12], mitoxantrone[13]\ncountries. and cyclophosphamide[14], as well as the aforementioned agents\n Early researchers in the field of ICI therapy, Casares and colleagues, doxorubicin and oxaliplatin.\ndemonstrated that the anthracycline doxorubicin supported the gener\u00ad As non-platinum metal complexes, iridium(III) complexes, have\nation of an immune response within tumors in the absence of any increasingly attracted the attention of researchers, due to their excellent\nadjuvant [6]. More recently, oxaliplatin was found to promote ICD by antitumor activities and remarkable luminescent features[15,16].\n\n\n\n * Corresponding authors at: School of Pharmacy, Guangdong Medical University, Dongguan 523808, China (J. Sun).\n E-mail addresses: jiaxi@gdmu.edu.cn (J. Chen), zn_huang@gdmu.edu.cn (Z. Huang), sunjing@gdmu.edu.cn (J. Sun).\n 1\n Contributed equally to this work.\n\nhttps://doi.org/10.1016/j.bioorg.2023.106837\nReceived 10 July 2023; Received in revised form 25 August 2023; Accepted 3 September 2023\nAvailable online 7 September 2023\n0045-2068/\u00a9 2023 Elsevier Inc. All rights reserved.\n\fJ. Liao et al. Bioorganic Chemistry 140 (2023) 106837\n\n\nEncouragingly, results of early studies revealed that Ir(III) complexes synthetic routes to generate the three complexes, as presented in Scheme\ntargeted the endoplasmic reticulum (ER) to kill tumor cells via S2. Results of elemental analysis, ESI-MS and 1H NMR of ligand L and\napoptosis, while also acting as immunomodulating adjuvants to promote the three complexes are presented in Fig. S1-S8; All compounds (L, Ir1-\ntumor cell killing via ICD[17]. Similarly, results of the current study Ir3) are >95% pure by HPLC analysis (Fig. S9-S12). UV\u2013Vis absorption\nsuggest that Ir(III) complexes target the ER to induce an obvious ICD spectra of Ir1-Ir3 in PBS, CH3CN and CH2Cl2 are shown in Fig. S13. The\neffect. However, our results also indicated that tumor cell ICD was main absorption band observed within the absorbance wavelength\naccompanied by Ir(III) complex targeting of mitochondria and lyso\u00ad range of 320\u2013380 nm corresponds to the internal electron transition of\nsomes with unknown antitumor effects, which is of great significance, the ligands, while the weak absorption band within the absorbance\nsince Ir(III) complexes that target lysosomes to induce ICD and chemo\u00ad wavelength range of 395\u2013420 nm belongs to the metal-to-ligand charge\ntherapeutic agents that induce both cancer cell paraptosis and ICD have transfer transition[18]. When excited by light of wavelength 405 nm, all\nnot yet been reported. three complexes emitted light in PBS, CH3CN, and CH2Cl2 at 298 K\n In this work, three cyclometalated Ir(III) complexes, Ir1, Ir2 and Ir3, (Fig. S14). Measurements of oil\u2013water partition coefficients for Ir1-Ir3\nwere designed, synthesized and evaluated to serve as antitumor agents using the shaker flask method provided logPo/w values for the com\u00ad\n(Fig. 1). Thereafter, Ir1 lysosome-specific targeting, cellular uptake and plexes that were ranked in decreasing order as Ir1 (1.71) > Ir3 (1.45) >\ncytotoxic effects on a non-tumor cell-derived cell line and various types Ir2 (0.32).\nof cancer cells were studied, with results showing that the Ir1 anticancer\nmechanism of action involved caspase-independent paraptosis. More\u00ad 2.2. Antitumor activities and cellular locations of complexes in vitro\nover, results obtained from additional studies conducted using HepG2\ncells indicated that Ir1 induced ER stress and also triggered ICD by Five human cancer cell lines and five murine cancer cell lines were\nuncovering CRT on tumor cell surfaces while also promoting extracel\u00ad evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium\nlular HMGB-1 release and substantial ATP secretion. Furthermore, in\u00ad bromide (MTT) assays in order to assess in vitro effects of ligand L, the\njection of Ir1-treated homologous tumor cells into C57BL/6 mice three complexes and the cisplatin (CDDP) on cell viability (Table S1).\nsignificantly prevented growth of transplanted tumor in vivo. Based on MTT assay results, all three complexes exhibited excellent\n antitumor activity against cancer line cells. Notably, Ir2 (IC50: 1.6 ~ 3.3\n2. Results and discussion \u03bcM) and Ir3 (IC50: 1.8 ~ 6.2 \u03bcM) exhibited greater cytotoxicity towards\n all tumor cell types as compared to corresponding effects of Ir1 (IC50:\n2.1. Synthesis of Ir(III) complexes and characterization of their 4.2 ~ 37.4 \u03bcM). However, Ir1 (IC50: 24.4 \u00b1 0.2 \u03bcM) was less toxic to\nphotophysical properties non-tumorigenic L02 cells than Ir2 (IC50: 2.1 \u00b1 0.2 \u03bcM) and Ir3 (IC50:\n 4.3 \u00b1 0.4 \u03bcM). This result prompted us to select Ir1 for further investi\u00ad\n Ligand L (2-(quinoxalin-6-yl)-1H-imidazo[4,5-f][1,10]phenanthro\u00ad gation, since Ir1 would likely be less cytotoxic to normal cells than to\nline) was prepared as follows: 1,10-phenanthroline-5,6-dione, glacial tumor cells (e.g. HepG2 cells) and thus would provide superior selec\u00ad\nacetic acid and ammonium acetate were added to a reaction flask then tivity for HepG2 cells vs. non-tumorigenic cells in in vitro assays.\nthe mixture was refluxed until all solutes were dissolved. Thereafter, the Cyclometallic Ir(III) complexes have rich optical properties that\nreaction was initiated by adding quinoxalin-6-carboxaldehyde to the allow their intracellular distributions to be tracked using fluorescence\nsolution then the mixture was refluxed until the solid in the flask was microscopy[19]. As shown in Fig. S15A and Fig. S15B, Ir1 completely\ncompletely dissolved. Next, the pH of the solution was adjusted to 7, entered the cytoplasm during the 6-h co-incubation with HepG2 cells.\nresulting in precipitation of a solid product that was subsequently Meanwhile, co-staining of Ir1-treated cells with mitochondrial MTR\nwashed with water and ethanol then dried to generate L. Thereafter, probe (MitoTracker\u00ae Green) and lysosomal LTR probe (LysoTracker\u00ae\ncomplexes Ir1-Ir3 were synthesized by reacting two equivalents of L Green), revealed Ir1-associated red fluorescence that overlapped\nwith one equivalent of Ir(III) chloro-bridged dimer using similar negligibly with the green fluorescence emitted by the mitochondrial\n\n\n\n\n Fig. 1. Molecular structure of complexes Ir1-Ir3.\n\n 2\n\fJ. Liao et al. Bioorganic Chemistry 140 (2023) 106837\n\n\nprobe (PCC = 0.368) and overlapped significantly with the green fluo\u00ad Table S2).\nrescence emitted by the lysosomal probe (PCC = 0.842). Thus, after Unlike apoptosis, paraptosis-induced cell death is characterized by\nentering HepG2 cells, Ir1 mainly entered the cytoplasm then entered the production of sizable cytoplasmic vacuoles[22]. After cells were\nlysosomes. Investigation of this process using a cellular uptake assay treated with Ir1, the internal cellular structure was examined via light\nrevealed that the amount of Ir1 entering cells was reduced at low tem\u00ad microscopy and transmission electron microscopy (TEM). As shown in\nperature (4 \u25e6 C) or in the presence of the metabolic inhibitor carbonyl Fig. 3A and Fig. 3B, cytoplasmic vacuoles appeared, of which some\ncyanide 3-chlorophenylhydrazone (CCCP), but was not affected by the contained large amounts of unfolded proteins and others contained\npresence of chloroquine, an endocytosis inhibitor (Fig. S15C). Taken debris resembling broken mitochondrial ridges that were accompanied\ntogether, these results indicate that Ir1 entered cells via an energy- by enlarged ER and mitochondrial structures. Previous studies have\ndependent mechanism. identified certain ICD inducers that can cause mitochondrial swelling\n when treating Hepatocellular carcinoma[23,24]. Taken together, these\n2.3. Investigation of the cell death mechanism of Ir1 results suggest that Ir1 promoted paraptosis in HepG2 cells.\n Reactive oxygen species (ROS) are intracellular intermediates pro\u00ad\n Medication-induced apoptosis is often accompanied by the trig\u00ad duced by the mitochondrial respiratory chain during normal aerobic\ngering of a series of biochemical processes[20,21]. Ir1-induced nuclear metabolism, as well as in cells treated with most known chemothera\u00ad\nmorphological changes did not exhibit typical apoptosis features, peutic agents, which exert their antitumor effects by promoting intra\u00ad\nincluding fragmented nuclei, plasma membrane blebbing and apoptotic cellular ROS production causes excessive tumor cell oxidative damage\nbodies (Fig. 2A). In order to rule out apoptosis as an Ir1 cell death and functional abnormalities[25\u201327]. In this study, the fluorescent\nmechanism, Annexin V staining assays were conducted to detect cellular probe 2\u2032,7\u2032-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to\nphosphatidylserine externalization, a recognized marker of early detect ROS levels in Ir1-treated cells. Surprisingly, green fluorescence\napoptosis. As compared to results obtained for control cells, only a was very weak in Ir1-treated cells, while flow cytometric quantification\nnegligible change in the proportion of membrane-linked protein V- of intracellular ROS levels revealed that with increasing Ir1 concentra\u00ad\npositive cells was observed after cells were treated with Ir1 (Fig. 2B). In tion, intracellular ROS intensity decreased considerably to levels that\naddition, due to the fact that caspase activation is considered a key were even lower than corresponding control group levels (Fig. S17). In\nbiochemical indicator of apoptosis, caspase-3 and poly(ADP-ribose) general, moderate intracellular amounts of ROS are required by normal\npolymerase-1 (PARP) activation in cells treated with CDDP or Ir1 cells to sustain cellular processes, such as tissue homeostasis, cell dif\u00ad\nwere assessed via protein blot analysis. The results of this analysis ferentiation and proliferation and regulation of intracellular and extra\u00ad\nrevealed activation of both caspase-1 and PARP enzymes by CDDP, cellular signaling[28]. However, excessive inhibition of ROS production\nwhile no alterations in activation states of these enzymes in Ir1-treated leads to disruption of intracellular phosphorylation, dephosphorylation\ncells were observed (Fig. 2C), thus demonstrating that Ir1-induced cell and disruption of redox homeostasis that cause intracellular dysfunction\ndeath did not occur via a conventional apoptotic process. Moreover, Ir1 and eventual cell death. Meanwhile, studies have shown that production\ntreatment exerted no cell cycle-associated effects (Fig. S16 and of giant mitochondria is induced when cells are exposed to various types\n\n\n\n\nFig. 2. (A) Confocal microscopy analysis of morphological alterations of HepG2 cells treated with CDDP (20 \u03bcM) and different concentration of Ir1 (5, 10 and 20 \u03bcM)\nfor 24 h. Cells were stained with DAPI. (B) Flow cytometric quantification of annexin V single labeled HepG2 cells were treated with Ir1 and CDDP for 24 h. (C)\nWestern blot analysis of PARP, Caspase-3 and Cleaved Caspase-3 proteins.\n\n 3\n\fJ. Liao et al. Bioorganic Chemistry 140 (2023) 106837\n\n\n of antitumor immunity[39,40]. In general, cell surface membrane CRT\n expression[41], HMGB-1 translocation outside the nucleus[42] and\n extracellular adenosine triphosphate (ATP) release are major ICD fea\u00ad\n tures that can be used to distinguish ICD from other tumor cell-killing\n mechanisms[43].\n It has been reported that CRT protein, a highly conserved calcium-\n binding protein located primarily in the ER that maintains Ca2+ ho\u00ad\n meostasis, can be transferred from the ER lumen to the cell membrane\n surface during early stages of ICD development[44,45]. Here, cell sur\u00ad\n face CRT protein was detected after cells were treated with different Ir1\n concentrations. As shown in Fig. 4C, green fluorescence observed at the\n cell surface gradually increased with increasing Ir1 concentration,\n which indicated that Ir1 was able to induce CRT transfer to the cell\n membrane surface, while no fluorescence was observed in the vehicle-\n treated group. Due to the fact that tumor cells in late-stage ICD can\n promote DCs maturation by releasing HMGB-1 and by stimulating DCs\n presentation of tumor antigens to T cells[46], HMGB-1 protein content\n in extracellular fluid was monitored here using ELISAs (Fig. 4D). From\n the results, it can be seen that 10 \u03bcM Ir1 treatment of HepG2 cells\n triggered release from cells of a peak amount of HMGB-1, while the\n amount of HMGB-1 released from cells treated with 20 \u03bcM Ir1 was much\n lower. Due to the fact that ATP is a direct energy source for cells, release\n of ATP from cells in an advanced ICD state can activate and recruit in\u00ad\n flammatory pathway-associated antigen-presenting cells (APCs) to\n enhance immune system activity[47]. In this study, we used a chem\u00ad\n iluminescence kit to detect ATP content of extracellular fluid (Fig. 4E)\n and found that incubation of cells with 10 \u03bcM Ir1 was associated with\n greatest extracellular ATP content. Collectively, the abovementioned\n results indicate that 10 \u03bcM Ir1 treatment was associated with greatest\n ICD induction.\n At the same time, ICD induction experiments were performed using\nFig. 3. (A) Light microscopy images of HepG2 cells treated with 10 \u03bcM Ir1. (B)\n cells of the Hepa1-6 cell line, which are derived from murine hepato\u00ad\nThe representative TEM images explaining the morphological features of\n cellular carcinoma cells. As compared to CDDP-treated Hepa1-6 cells,\nHepG2 cells treated with Ir1 (10 \u03bcM) for 12 h and 24 h.\n Hepa1-6 cells treated with 15 \u03bcM Ir1 exhibited significant ICD charac\u00ad\n teristics, including significantly increased cell surface CRT over-\nof free radicals[29]. In turn, free radicals alter mitochondrial membrane\n expression, extranuclear release of HMGB-1 and extracellular ATP\nbiochemical properties, resulting in reduced oxygen consumption,\n release. In addition, results of Fluo-3 AM calcium probe-based assays\nphosphorylation capacity and intracellular ROS levels. As shown in\n and protein immunoblotting assays indicated that ER stress occurred in\nFig. 3B, intracellular mitochondria of Ir1-treated cells appeared swollen\n Hepa1-6 cells after treatment with 15 \u03bcM Ir1 (Fig. 5A-Fig. 5E).\nand damaged, with broken membrane ridges observed. This result\n Expression of CD86 and CD80 molecules on cell surfaces of bone\nindicated that observed reduced intracellular ROS levels after Ir1\n marrow-derived dendritic cells (BMDCs) are used to confirm DC matu\u00ad\ntreatment may have been caused by giant mitochondria production, as\n rity. In this study, bone marrow cells were extracted using the Lutz\nconsistent with reported effects of paraptotic agents[30].\n method then relevant growth-stimulating factors were added to stimu\u00ad\n late differentiation of BMDCs into DC cells, which was confirmed via\n2.4. In vitro ICD studies flow cytometric analysis of cell surface CD80 and CD86 levels. There\u00ad\n after, Hepa1-6 cells and BDMCs were incubated with various Ir1 con\u00ad\n Cells undergoing paraptosis exhibit calcium level imbalances and centrations followed by flow cytometric assessments of CD80 and CD86\nROS-related mitochondrial dysfunction[31], as well as disruption of key levels. The results of these experiments revealed that Hepa1-6 cells\nER functions associated with Ca2+ storage, protein folding and post- pretreated with 5 \u03bcM Ir1 exhibited greater cell surface expression of\ntranslational modification[32,33]. Importantly, ER stress occurs when CD80 as compared to untreated control cells. Meanwhile, BDMCs\ncells are damaged or endostasis becomes imbalanced[34]. To assess treated with 15 \u03bcM Ir expressed both CD86 and CD80 on cell surfaces in\ncellular ER stress, Ir1-treated and untreated cells were stained with large amounts. However, cell surface amounts of CD80 decreased when\nFluo-3 AM, a probe commonly used to monitor intracellular Ca2+ levels cells were treated with 30 \u03bcM Ir1, indicating that CD80 expression was\n[35]. Staining results revealed that in Ir1-treated cells, cellular fluo\u00ad weakened at higher Ir1 concentrations. Taken together, these results\nrescence was mainly concentrated in the ER as an indication that Ca2+ suggest that excessive Ir1 not only led to rapid death of Hepa1-6 cells,\nhad been released from the ER into the cytoplasm, which is a sign of but also attenuated the ICD effect (Fig. 5F).\nsevere ER stress (Fig. 4A). Meanwhile, C/EBP-homologous protein\n(Chop) and eukaryotic initiation factor 2\u03b1 (eIF2\u03b1) levels were monitored 2.5. In vivo antitumor immunity\nin cells treated with various Ir1 concentrations via western blotting. As\nseen in Fig. 4B, over-expression of Chop and phosphorylation of eIF2\u03b1 To investigate in vivo antitumor activity of Ir1, we chose C57BL/6\n(p-eIF2\u03b1), which are both signatures of ER stress[36\u201338], were observed mice with natural immunity rather than immunodeficient nude mice.\nafter Ir1 treatment. Hepa1-6 cells treated with 15 \u03bcM Ir1 or 20 \u03bcM CDDP for 24 h served as\n Cells undergoing ICD can activate tumor-specific immune responses \u201cvaccines\u201d that were injected subcutaneously into mice, while mice in\nby releasing molecules known as damage-associated molecular patterns the control group were injected with Hepa1-6 cells treated for 24 h with\n(DAMPs). DAMPs support long-term efficacy of anticancer drugs via two PBS solution. On the seventh day after vaccination (day 0), small pieces\nmechanisms: through direct killing of cancer cells and through induction of tumor tissue were transplanted into the right leg of each mouse then\n\n 4\n\fJ. Liao et al. Bioorganic Chemistry 140 (2023) 106837\n\n\n\n\nFig. 4. (A) Confocal images of intracellular Ca2+ in HepG2 cells treated with Ir1 (5, 10 and 20 \u03bcM) for 12 h, stained with Fluo-4 AM. (B) Western blot analysis of\neIF2\u03b1, p-eIF2\u03b1 and Chop proteins. (C) Confocal images of CRT on the surface of HepG2 cells treated with Ir1 in different concentrations. (D) ELISA analysis of HMGB-\n1 in HepG2 cell supernatant treated with Ir1 in different concentrations. (E) Analysis of ATP levels in cell supernatant of different concentrations of Ir1-treated\nHepG2 cell. (***p < 0.001, **p < 0.01, *p < 0.05).\n\n\non day 16, six mice from each group were euthanized then spleen and thereafter. On day 30, after the remaining mice were photographed from\ntumor tissues were harvested (Fig. 6A). Notably, the mean tumor vol\u00ad the back and side (Fig. S18), they were euthanized then their tumor\nume of the CDDP group was lower than that of the control group, sug\u00ad masses were removed. As shown in Fig. 6B and Fig. 6C, the Ir1 group\ngesting that CDDP may have triggered an immune effect that inhibited mean tumor mass did not increase and even decreased after treatment,\ntumor growth. Meanwhile, the mean tumor volume of the Ir1 group while tumors grew rapidly along inguinal sides of mice in the control\nincreased slowly for 10 days after tumor transplantation then plateaued group. By contrast, tumors in the CDDP group were slightly smaller than\n\n\n 5\n\fJ. Liao et al. Bioorganic Chemistry 140 (2023) 106837\n\n\n\n\nFig. 5. (A) Confocal images of intracellular Ca2+ in Hepal-6 cells treated with Ir1 (5, 15 and 30 \u03bcM) for 12 h, stained with Fluo-4 AM. (B) Western blot analysis of\neIF2\u03b1, p-eIF2\u03b1 and Chop proteins. (C) Confocal images of CRT on surface of Hepal-6 cells treated with Ir1 (15 \u03bcM) and CDDP (20 \u03bcM) Hepa1-6 cells. (D) ELISA\nanalysis of HMGB-1 in cell supernatant of Ir1-treated (15 \u03bcM) and CDDP-treated (20 \u03bcM) Hepa1-6 cells. (E) Analysis of ATP levels in cell supernatant of Ir1-treated\n(15 \u03bcM) and CDDP-treated (20 \u03bcM) Hepa1-6 cells. (F) Flow cytometric quantification of BMDC maturation in Hepa1-6 cells after co-culture with Ir1 (5, 15 and 30\n\u03bcM) for 24 h. (***p < 0.001).\n\nthose of the control group, but were significantly larger than those of the CDDP group (4.01 \u00b1 0.64%). It is noteworthy that the proportion of\nIr1 group. In addition, Ir1 treatment did not lead to significant mortality immunosuppressive regulatory T cells (Treg cells) in spleens of mice was\nor remarkable changes in body weight among the experiment (Fig. 6D). elevated in the CDDP group (1.34 \u00b1 0.32%) as compared to the corre\u00ad\nFurthermore, H&E staining of paraffin-embedded sections of heart, sponding proportions of the other two groups (Control: 1.13 \u00b1 0.01%,\nliver, spleen, lung and kidney tissues revealed no obvious lesions in Ir1: 0.54 \u00b1 0.01%). Proportions of Treg cells, a subset of T cells that can\ntissues of Ir1 group mice. Taken together, these results indicate that Ir1 significantly inhibit immune system activities, are significantly elevated\nwas not toxic to surrounding organs (Fig. 6E). in cancer patients[48,49] and appear to help tumor cells block normal\n In order to assess Ir1 effects on in vivo immune system function, immune surveillance to thereby enable immune escape[50,51]. In the\nproportions of DC cells and T cells belonging to different T cell subsets in CDDP group, the proportion of splenic Treg cells was significantly greater\nspleen and tumor tissues after 16 days of tumor transplantation was than that of the control group, suggesting that immunosuppression may\nexamined via flow cytometric analysis. The results indicated that the occur via multiple mechanisms. For example, immunosuppression\nproportion of splenic DCs (CD11c+ cells) was significantly greater in the observed in the CDDP group was not solely due to CDDP toxic effects on\nIr1 group (5.56 \u00b1 1.25%) than in the control group (3.10 \u00b1 0.79%), bone marrow cells, but may have also been caused by activities of CD8+\nwhile a slight increase in splenic DC proportion was observed in the T cells. CD8+ T cells can act as cytotoxic T lymphocytes in vivo by\n\n 6\n\fJ. Liao et al. Bioorganic Chemistry 140 (2023) 106837\n\n\n\n\nFig. 6. (A) Schematic diagram of the ICD vaccine experiment. (B) The tumor volume growth curves of the mice in each group. (C) Photographs of tumors removed\nfrom the mice after treatment (30 days). (D) The weight of the mice throughout the follow-up period. (E) Histological examination of the main organs of the mice\n(30 days).\n\n\nrecognizing tumor antigens to thereby become activated tumor-killing indicating that treatment of tumor cells with Ir1 enhanced immune\neffector cells via secretion of perforin, TNF-\u03b1 and other immune fac\u00ad system antitumor activity in vivo (Fig. S19 and Fig. 7A1-7F1).\ntors[52,53]. In fact, the proportion of Treg cells has been reported to be In tumor tissues, proportions of DCs, CD3+ T cells, CD4+ T cells and\nnegatively correlated with the proportion of CD8+ CTL cells, such that CD8+ T cells in the Ir1 group (25.61 \u00b1 3.36%, 86.99 \u00b1 2.98%, 22.19 \u00b1\nthe CD8+ T cell/Treg cell ratio has been commonly used as a cancer 1.59%, 11.29 \u00b1 2.37%, respectively) were greater than corresponding\npatient clinical prognostic indicator[54]. Here the splenic CD8+ T cell/ control group proportions (8.38 \u00b1 1.11%, 57.07 \u00b1 11.50%, 3.47 \u00b1\nTreg cell ratio of the Ir1 group (33.89%) was considerably higher than 2.42%, 2.73 \u00b1 1.29%, respectively) and CDDP group proportions (10.85\nthat of the control group (10.79%) and CDDP group (15.54%), thus \u00b1 1.24%, 68.67 \u00b1 11.10%, 6.69 \u00b1 4.01%, 6.42 \u00b1 2.91%, respectively)\n\n\n 7\n\fJ. Liao et al. Bioorganic Chemistry 140 (2023) 106837\n\n\n\n\nFig. 7. Quantitative analysis by flow cytometry of the percentage of different cells in the spleen (A1-E1) and tumor (A2-E2). (A1, A2) DC, (B1, B2) CD3+ T cell, (C1,\nC2) CD3+CD4+ T cell, (D1, D2) CD3+CD8+ T cell, (E1, E2) CD4+Foxp3 cell, (F1, F2) CD8+/Foxp3 ratio. (***p < 0.001, **p < 0.01, *p < 0.05).\n\n\n(Fig. S20 and Fig. 7A2-7F2). Of these cell types, DCs and CD4+ T cells 3. Conclusion\nwere present in significantly greater proportions in the Ir1 group than in\nthe other groups, suggesting that Ir1-induced ICD effects could effec\u00ad In this study, three new cyclometalated Ir(III) complexes were syn\u00ad\ntively enable these types of cells to recognize, chemotactically attract thesized, characterized and assessed for antitumor activities, which and\nAPCs, and enhance antigen presentation functions in response to were shown via MTT assays to be excellent for all three Ir(III) complexes.\nimmunologically recognized tumor antigens. The greater proportion of However, as compared to Ir2 and Ir3, Ir1 exhibited less cytotoxicity\nCD8+ T cells in tumor tissues of the Ir1 group vs. corresponding pro\u00ad toward LO2 cells and was more selectively cytotoxic against cancer cells.\nportions of other groups indicates that Ir1-induced ICD significantly Results of mechanistic investigations revealed that Ir1 entered the\nreduced tumor volumes or eradicated tumors by recruiting CD8+ T cells cytoplasm and localized within lysosomes to induce tumor cell death via\nand down-regulating Treg cell activities. Comparisons of Ir1 group Treg a mechanism that primarily involved paraptosis. In addition, Ir1 was\ncell proportions obtained for tumor tissues (0.47 \u00b1 0.08%) revealed found to induce ER stress in HepG2 cells, which further enhanced the\nthey were both lower than that of the control group (1.78 \u00b1 0.77%), and ICD effect (i.e., surface exposure of CD8+ T cells and extracellular\nwere more markedly lower than that of the CDDP group (2.89% \u00b1 release of HMGB1 and ATP). Moreover, Ir1 also promoted DCs matu\u00ad\n0.55%). Therefore, the Ir1-induced ICD effect not only enhanced ration, enhanced chemotaxis of effector T cells to tumor tissue sites,\nchemotactic activities of DCs and T cells in tumor tissues, but also alleviated immunosuppression by down-regulating the proportion of\nreduced both the proportion of Treg cells in the tumor microenvironment Treg cells in tumor tissues, activated host immunity and ultimately\nand the immunosuppressive effect observed in the cellular immunized tumor-vaccinated mice against liver cancer tumors. Inter\u00ad\nmicroenvironment. estingly, Ir1 did not cause an increase in cellular ROS, as has been\n\n 8\n\fJ. Liao et al. Bioorganic Chemistry 140 (2023) 106837\n\n\nreported for other metal complexes with known ICD effects (e.g., oxa\u00ad 4.1.1.4. Synthesis of [Ir(tpy)2L]PF6 (Ir3). Ir3 was synthesized in a\nliplatin). Nonetheless, the unique antitumor activities and properties of method similar to Ir1, except [Ir(tpy)2Cl]2 (0.28 g, 0.25 mmol, 1 equiv)\nthe three Ir(III) complexes designed in this study provide a foundation to was used instead of [Ir(piq)2Cl]2. Yield: 0.16 g (31.3%). Anal. Calcd for\nguide further development of Ir(III) complexes for use in inducing ICD. C45H32F6IrN8P (1022.20): C, 52.89; H, 3.16; N, 10.96. Found: C, 52.76;\n To date, immunoregulatory functions of antitumor chemothera\u00ad H, 3.11; N, 11.02. ESI-MS: m/z = 874.8 [M\u2212 PF-6]+. 1H NMR (400 MHz,\npeutic agents have not been systematically investigated. Here, we found DMSO\u2011d6) \u03b4 9.21 (d, J = 8.0 Hz, 2H), 9.13\u20138.87 (m, 3H), 8.78 (d, J = 8.7\nthat although CDDP did not induce an ICD effect, tumor growth was still Hz, 1H), 8.35 (d, J = 8.8 Hz, 1H), 8.29\u20138.17 (m, 4H), 8.11 (d, J = 5.1 Hz,\ninhibited in CDDP group mice after they received transplanted tumor 2H), 7.87 (t, J = 8.1 Hz, 4H), 7.50 (d, J = 5.6 Hz, 2H), 6.93 (dd, J = 16.8,\ncells. Thus, this result suggests that CDDP treatment induced immuno\u00ad 7.1 Hz, 4H), 6.11 (s, 2H), 2.12 (s, 6H), 1.22 (s, 1H).\nmodulation, an effect that has never been reported before for CDDP.\nHowever, the proportion of Treg cells within tumor tissues of CDDP Author contributions\ngroup mice was markedly greater than that of the control group and thus\nreduced the CD8+ T cell tumor-killing effect, warranting further study. J. X. Liao, Y. Q. Zhang and J. X. Chen designed, synthesized and\nNevertheless, the results presented here highlight the importance of characterized three Ir(III) complexes. J. X. Liao, Z. J. Liang, Y. Gong, Y.\neffects of chemotherapeutic agents on the immune response and thus L. Li, B. Liu and W. Wu conducted the in vitro and in vivo experiments. Z.\nmay broaden the range of applications of these classical agents and open N. Huang and J. Sun performed the analysis of data. M. Y. Huang and J.\nup new avenues for development of chemotherapeutics. Sun wrote the manuscript.\n\n4. Experimental section\n Declaration of Competing Interest\n\n4.1. Synthetic protocol and characterization\n The authors declare that they have no known competing financial\n interests or personal relationships that could have appeared to influence\n4.1.1. Synthesis and characterization\n the work reported in this paper.\n [Ir(piq)2Cl]2[55], [Ir(dfppy)2Cl]2[56], and [Ir(tpy)2Cl]2[57] were\nprepared according to previously reported methods.\n Data availability\n\n4.1.1.1. Synthesis of 2-(quinoxalin-6-yl)-1H-imidazo[4,5-f][1,10]phe\u00ad No data was used for the research described in the article.\nnanthroline (L). A mixture of 1,10-phenanthroline-5,6-dione (0.53 g,\n2.5 mmol) and ammonium acid (3 g, 39 mmol) was dissolved in glacial\n Acknowledgments\nacetic acid (80 mL) and stirred for 15 min at 60 \u25e6 C. After the mixture\ndissolved, quinoxaline-6-formaldehyde (0.40 g, 2.5 mmol) was added\n This work was supported by the Discipline Construction Project of\nand the solution refluxed for another 90 min at 100 \u25e6 C. After the mixture\n Guangdong Medical University (4SG22004G), Dongguan Science and\nwas cooled to room temperature, ammonia was added to adjust the pH\n Technology of Social Development Program (20211800905082), the\nto neutral. Then, the yellow precipitate was precipitated, which was\n Key Scientific Research Projects of Colleges and Universities in Guang\u00ad\npurified with water and absolute ethanol. Yield: 0.60 g (69.0%). Anal.\n dong Province (2020ZDZX2031), and Medical Industry Innovation\nCalcd for C21H12N6 (348.11): C, 72.40; H, 3.47; N, 24.12. Found: C,\n Project of Guangdong Medical University (4SG22305P).\n72.25; H, 3.43; N, 24.10. ESI-MS: m/z = 349.0 [M+H+]+. 1H NMR (400\nMHz, DMSO\u2011d6) \u03b4 9.08\u20138.99 (m, 3H), 8.99\u20138.90 (m, 4H), 8.77 (dd, J =\n Appendix A. Supplementary data\n8.7, 2.0 Hz, 1H), 8.27 (d, J = 8.8 Hz, 1H), 7.83 (dd, J = 8.1, 4.3 Hz, 2H),\n1.88 (s, 1H).\n Supplementary data to this article can be found online at https://doi.\n org/10.1016/j.bioorg.2023.106837.\n4.1.1.2. Synthesis of [Ir(piq)2L]PF6 (Ir1). A mixture of L (0.18 g, 0.50\nmmol, 2 equiv) and [Ir(piq)2Cl]2 (0.32 g, 0.25 mmol, 1 equiv) was\n References\nprepared in 80 mL CH2Cl2/CH3OH (2:1, v/v) by refluxing for 4 h under\nargon. After the solution has been cooled to room temperature, an [1] H.Z. Deng, W.J. Yang, Z.J. Zhou, R. Tian, L.S. Lin, Y. Ma, J.B. Song, X.Y. Chen,\nappropriate amount of NH4PF6 was added and the stirring continued for Targeted scavenging of extracellular ROS relieves suppressive immunogenic cell\n1 h. The pure product was gained after purification by column chro\u00ad death, Nat. Commun. 11 (2020) 4951.\n [2] C. Boutros, A. Tarhini, E. Routier, O. Lambotte, F.L. Ladurie, F. Carbonnel,\nmatography on silica gel diluted with CH2Cl2/CH3OH (60:1, v/v). Yield: H. Izzeddine, A. Marabelle, S. Champiat, A. Berdelou, E. Lanoy, M. Texier,\n0.16 g (29.3%). Anal. Calcd for C51H32F6IrN8P (1094.20): C, 55.99; H, C. Libenciuc, A.M. Eggermont, J.C. Soria, C. Mateus, C. Robert, Safety profiles of\n2.95; N, 10.24. Found: C, 56.10; H, 2.31; 10.10. ESI-MS: m/z = 949.0 anti-CTLA-4 and anti-PD-1 antibodies alone and in combination, Nat. Rev. Clin.\n Oncol. 13 (2016) 473\u2013486.\n[M\u2212 PF-6]+. 1H NMR (400 MHz, DMSO\u2011d6) \u03b4 9.23 (d, J = 8.5 Hz, 2H),\n [3] E.I. Buchbinder, A. Desai, CTLA-4 and PD-1 pathways: similarities, differences, and\n9.04 (dd, J = 19.0, 5.4 Hz, 5H), 8.82\u20138.78 (m, 1H), 8.40 (d, J = 8.1 Hz, implications of their inhibition, Am. J. Clin. Oncol. 39 (2016) 98\u2013106.\n2H), 8.34 (d, J = 8.8 Hz, 1H), 8.04 (ddd, J = 10.0, 9.2, 3.6 Hz, 6H), [4] A. Rotte, Combination of CTLA-4 and PD-1 blockers for treatment of cancer, J. Exp.\n Clin. Canc. Res. 38 (2019) 255.\n7.90\u20137.83 (m, 4H), 7.41 (dd, J = 13.0, 6.5 Hz, 4H), 7.17 (t, J = 7.6 Hz,\n [5] A. Ribas, J.D. Wolchok, Cancer immunotherapy using checkpoint blockade,\n2H), 6.96 (t, J = 7.4 Hz, 2H), 6.29 (d, J = 7.6 Hz, 2H), 1.20 (s, 1H). Science 359 (2018) 1350\u20131355.\n [6] N. Casares, M.O. Pequignot, A. Tesniere, F. Ghiringhelli, S. Roux, N. Chaput,\n E. Schmitt, A. Hamai, S. Hervas-Stubbs, M. Obeid, F. Coutant, D. Me\u0301tivier,\n4.1.1.3. Synthesis of [Ir(dfppy)2L]PF6 (Ir2). Ir2 was synthesized in a\n E. Pichard, P. Aucouturier, G. Pierron, C. Garrido, L. Zitvogel, G. Kroemer, Caspase-\nmethod similar to Ir1, except [Ir(dfppy)2Cl]2 (0.30 g, 0.25 mmol, 1 dependent immunogenicity of doxorubicin-induced tumor cell death, J. Exp. Med.\nequiv) was used instead of [Ir(piq)2Cl]2. Yield: 0.18 g (33.8%). Anal. 202 (2005) 1691\u20131701.\n [7] A. Tesniere, F. Schlemmer, V. Boige, O. Kepp, I. Martins, F. Ghiringhelli,\nCalcd for C43H24F10IrN8P (1066.13): C, 48.45; H, 2.27; N, 10.51. Found:\n L. Aymeric, M. Michaud, L. Apetoh, L. Barault, J. Mendiboure, J.P. Pignon,\nC, 48.51; H, 2.20; N, 10.30. ESI-MS: m/z = 921.0 [M\u2212 PF-6]+. 1H NMR V. Jooste, P. van Endert, M. Ducreux, L. Zitvogel, F. Piard, G. Kroemer,\n(400 MHz, DMSO\u2011d6) \u03b4 9.24 (d, J = 7.9 Hz, 2H), 9.03 (d, J = 19.1 Hz, Immunogenic death of colon cancer cells treated with oxaliplatin, Oncogene 29\n3H), 8.77 (d, J = 9.8 Hz, 1H), 8.46\u20138.20 (m, 5H), 8.10 (s, 2H), 7.97 (s, (2010) 482\u2013491.\n [8] A. Ahmed, S.W.G. Tait, Targeting immunogenic cell death in cancer, Mol. Oncol.\n2H), 7.58 (s, 2H), 7.03 (d, J = 22.2 Hz, 4H), 5.72 (s, 2H), 1.20 (s, 1H). 14 (2020) 2994\u20133006.\n [9] G. Kroemer, L. Galluzzi, O. Kepp, L. Zitvogel, Immunogenic cell death in cancer\n therapy, Annu. Rev. Immunol. 31 (2013) 51\u201372.\n\n\n 9\n\fJ. Liao et al. Bioorganic Chemistry 140 (2023) 106837\n\n[10] N. Wang, Z. Wang, Z. Xu, X. Chen, G. Zhu, A cisplatin-loaded immuno- [32] I. Cakir, E.A. Nillni, Endoplasmic reticulum stress, the hypothalamus, and energy\n chemotherapeutic nanohybrid bearing immune checkpoint inhibitors for enhanced balance, Trends Endocrinol. Metab. 30 (2019) 163\u2013176.\n cervical cancer therapy, Angew. Chem. 57 (2018) 3426\u20133430. [33] A. Ferna\u0301ndez, R. Ordo\u0301n\u0303ez, R.J. Reiter, J. Gonza\u0301lez-Gallego, J.L. Mauriz, Melatonin\n[11] J.Q. Lu, X.S. Liu, Y.P. Liao, X. Wang, A. Ahmed, W. Jiang, Y. Ji, H. Meng, A.E. Nel, and endoplasmic reticulum stress: relation to autophagy and apoptosis, J. Pineal.\n Breast cancer chemo-immunotherapy through liposomal delivery of an Res. 59 (2015) 292\u2013307.\n immunogenic cell death stimulus plus interference in the IDO-1 pathway, ACS [34] C. Lebeaupin, D. Valle\u0301e, Y. Hazari, C. Hetz, E. Chevet, B. Bailly-Maitre,\n Nano 12 (2018) 11041\u201311061. Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty\n[12] Z. Yu, J.F. Guo, M.Y. Hu, Y.Q. Gao, L. Huang, Icaritin exacerbates mitophagy and liver disease, J. Hepatol. 69 (2018) 927\u2013947.\n synergizes with doxorubicin to induce immunogenic cell death in hepatocellular [35] Q. Liu, H. Ko\u0308rner, H. Wu, W. Wei, Endoplasmic reticulum stress in autoimmune\n carcinoma, ACS Nano 14 (2020) 4816\u20134828. diseases, Immunobiology 225 (2020), 151881.\n[13] W. Wei, H.B. Li, G.A. Zhang, Y. Zhang, K. Wu, R.R. Bao, G.G. Wang, H. Zheng, [36] S.A. Oakes, Endoplasmic reticulum stress signaling in cancer cells, Am. J. Pathol.\n Y. Xia, C.L. Li, Proteasome inhibitors attenuates mitoxantrone-triggered 190 (2020) 934\u2013946.\n immunogenic cell death in prostate cancer cells, Med. Oncol. 37 (2020) 116. [37] S.A. Oakes, F.R. Papa, The role of endoplasmic reticulum stress in human\n[14] M.X. Jiang, J. Zeng, L.Q. Zhao, M.G. Zhang, J.L. Ma, X.W. Guan, W.F. Zhang, pathology, Annu. Rev. Pathol. 10 (2015) 173\u2013194.\n Chemotherapeutic drug-induced immunogenic cell death for nanomedicine-based [38] Z.H. Qi, L.X. Chen, Endoplasmic reticulum stress and autophagy, Adv. Exp. Med.\n cancer chemo-immunotherapy, Nanoscale 13 (2021) 17218\u201317235. Biol. 1206 (2019) 167\u2013177.\n[15] L.C. Lee, K.K. Lo, Luminescent and photofunctional transition metal complexes: [39] M.Q. Zhu, M.D. Yang, J.J. Zhang, Y.Z. Yin, X. Fan, Y. Zhang, S.S. Qin, H. Zhang,\n from molecular design to diagnostic and therapeutic applications, J. Am. Chem. F. Yu, Immunogenic cell death induction by ionizing radiation, Front Immunol. 12\n Soc. 144 (2022) 14420\u201314440. (2021), 705361.\n[16] S. Sen, M. Won, M.S. Levine, Y. Noh, A.C. Sedgwick, J.S. Kim, J.L. Sessler, J. [40] J.Y. Zhou, G.Y. Wang, Y.Z. Chen, H.X. Wang, Y.Q. Hua, Z.D. Cai, Immunogenic cell\n F. Arambula, Metal-based anticancer agents as immunogenic cell death inducers: death in cancer therapy: present and emerging inducers, J. Cell Mol. Med. 23\n the past, present, and future, Chem. Soc. Rev. 51 (2022) 1212\u20131233. (2019) 4854\u20134865.\n[17] L.L. Wang, R.L. Guan, L.N. Xie, X.X. Liao, K. Xiong, T.W. Rees, Y. Chen, L.N. Ji, [41] X.W. Wang, S.W. Wu, F. Liu, D.S. Ke, X.W. Wang, D.L. Pan, W.F. Xu, L. Zhou, W.\n H. Chao, An ER-targeting iridium(III) complex which induces immunogenic cell D. He, An immunogenic cell death-related classification predicts prognosis and\n death in non-small cell lung cancer, Angew. Chem. 133 (2021) 4707\u20134715. response to immunotherapy in head and neck squamous cell carcinoma, Front\n[18] B.B. Chen, N.L. Pan, J.X. Liao, M.Y. Huang, D.C. Jiang, J.J. Wang, H.J. Qiu, J. Immunol. 12 (2021), 781466.\n X. Chen, L. Li, J. Sun, Cyclometalated iridium(III) complexes as mitochondria- [42] T. Verfaillie, A.V. Vliet, A.D. Garg, M. Dewaele, N. Rubio, S. Gupta, P. Witte,\n targeted anticancer and antibacterial agents to induce both autophagy and A. Samali, P. Agostinis, Pro-apoptotic signaling induced by photo-oxidative ER\n apoptosis, J. Inorg. Biochem. 219 (2021), 111450. stress is amplified by Noxa, not Bim, Biochem. Biophys. Res. Commun. 438 (2013)\n[19] V. Venkatesh, R. Berrocal-Martin, C.J. Wedge, I. Romero-Canelon, C. Sanchez- 500\u2013506.\n Cano, J.I. Song, J.P.C. Coverdale, P. Zhang, G.J. Clarkson, A. Habtemariam, S. [43] R.D.W. Vaes, L.E.L. Hendriks, M. Vooijs, D.D. Ruysscher, Biomarkers of\n W. Magennis, R.J. Deeth, P.J. Sadler, Mitochondria-targeted spin-labelled radiotherapy-induced immunogenic cell death, Cells 10 (2021) 930.\n luminescent iridium anticancer complexes, Chem. Sci. 8 (2017) 8271\u20138278. [44] H. Ruan, B.J. Leibowitz, L. Zhang, J. Yu, Immunogenic cell death in colon cancer\n[20] L.E. Bro\u0308ker, F.A.E. Kruyt, G. Giaccone, Cell death independent of caspases: a prevention and therapy, Mol. Carcinog 59 (2020) 783\u2013793.\n review, Clin. Cancer Res. 11 (2005) 3155\u20133162. [45] L. Minute, A. Teijeira, A.R. Sanchez-Paulete, M.C. Ochoa, M. Alvarez, I. Otano,\n[21] M. Cini, H. Williams, M.W. Fay, M.S. Searle, S. Woodward, T.D. Bradshaw, I. Etxeberrria, E. Bolan\u0303os, A. Azpilikueta, S. Garasa, N. Casares, J.L.P. Gracia, M.\n Enantiopure titanocene complexes\u2013direct evidence for paraptosis in cancer cells, E. Rodriguez-Ruiz, P. Berraondo, I. Melero, Cellular cytotoxicity is a form of\n Metallomics 8 (2016) 286\u2013297. immunogenic cell death, Cancer Res. 80 (2020).\n[22] A. Binoy, D. Nedungadi, N. Katiyar, C. Bose, S.A. Shankarappa, B.G. Nair, [46] Y.H. Li, X.H. Liu, X. Zhang, W. Pan, N. Li, B. Tang, Immunogenic cell death\n N. Mishra, Plumbagin induces paraptosis in cancer cells by disrupting the inducers for enhanced cancer immunotherapy, Chem. Commun. 57 (2021)\n sulfhydryl homeostasis and proteasomal function, Chem. Biol. Interact. 310 12087\u201312097.\n (2019), 108733. [47] G. Kroemer, C. Galassi, L. Zitvogel, L. Galluzzi, Immunogenic cell stress and death,\n[23] M.L. Bian, R. Fan, Z.B. Yang, Y.A. Chen, Z.R. Xu, Y.L. Lu, W.K. Liu, Pt(II)-NHC Nat. Immunol. 23 (2022) 487\u2013500.\n complex induces ROS-ERS-related DAMP balance to harness immunogenic cell [48] T.L. Whiteside, FOXP3+ Treg as a therapeutic target for promoting anti-tumor\n death in hepatocellular carcinoma, J. Med. Chem. 65 (2022) 1848\u20131866. immunity, Expert Opin. Ther. Tar. 22 (2018) 353\u2013363.\n[24] Z.B. Yang, M.L. Bian, L. Lv, X.Y. Chang, Z.F. Wen, F.W. Li, Y.L. Lu, W.K. Liu, [49] A. Tanaka, S. Sakaguchi, Targeting Treg cells in cancer immunotherapy, Eur. J.\n Tumor-targeting NHC-Au(I) complex induces immunogenic cell death in Immunol. 49 (2019) 1140\u20131146.\n hepatocellular carcinoma, J. Med. Chem. 66 (2023) 3934\u20133952. [50] A. Tanaka, S. Sakaguchi, Regulatory T cells in cancer immunotherapy, Cell Res. 27\n[25] Z.R. Xu, J.Q. Xu, S.B. Sun, W. Lin, Y.M. Li, Q.Y. Lu, F.W. Li, Z.B. Yang, Y.L. Lu, W. (2017) 109\u2013118.\n K. Liu, Mecheliolide elicits ROS-mediated ERS driven immunogenic cell death in [51] Y. Ohue, H. Nishikawa, Regulatory T (Treg) cells in cancer: Can Treg cells be a new\n hepatocellular carcinoma, Redox Biol. 54 (2022), 102351. therapeutic target, Cancer Sci. 110 (2019) 2080\u20132089.\n[26] H. Yuan, Z. Han, Y.C. Chen, F. Qi, H.B. Fang, Z.J. Guo, S.R. Zhang, W.J. He, [52] A.M. Leun, D.S. Thommen, T.N. Schumacher, CD8+ T cell states in human cancer:\n Ferroptosis photoinduced by new cyclometalated iridium(III) complexes and its insights from single-cell analysis, Nat. Rev. Cancer 20 (2020) 218\u2013232.\n synergism with apoptosis in tumor cell inhibition, Angew. Chem. 133 (2021) [53] L.M. McLane, M.S. Abdel-Hakeem, E.J. Wherry, CD8 T cell exhaustion during\n 8255\u20138262. chronic viral infection and cancer, Annu. Rev. Immunol. 37 (2019) 457\u2013495.\n[27] N. Diwanji, A. Bergmann, An unexpected friend - ROS in apoptosis-induced [54] W.X. Huff, J.H. Kwon, M. Henriquez, K. Fetcko, M. Dey, The evolving role of CD8\n compensatory proliferation: Implications for regeneration and cancer, Semin. Cell +\n CD28- Immunosenescent T cells in cancer immunology, Int. J. Mol. Sci. 20 (2019)\n Dev. Biol. 80 (2018) 74\u201382. 2810.\n[28] R. Mittler, ROS are good, Trends Plant Sci. 22 (2017) 11\u201319. [55] C.Y. Li, M.X. Yu, Y. Sun, Y.Q. Wu, C.H. Huang, F.Y. Li, A nonemissive iridium(III)\n[29] J.N. Moloney, T.G. Cotter, ROS signalling in the biology of cancer, Semin. Cell Dev. complex that specifically lights-up the nuclei of living cells, J. Am. Chem. Soc. 133\n Biol. 80 (2018) 50\u201364. (2011) 11231\u201311239.\n[30] M.J. Seo, D.M. Lee, I.Y. Kim, D. Lee, M.K. Choi, J.Y. Lee, S.S. Park, S.Y. Jeong, E. [56] Y.M. You, S.Y. Park, Inter-ligand energy transfer and related emission change in the\n K. Choi, K.S. Choi, Gambogic acid triggers vacuolization-associated cell death in cyclometalated heteroleptic iridium complex: facile and efficient color tuning over\n cancer cells via disruption of thiol proteostasis, Cell Death Dis. 10 (2019) 187. the whole visible range by the ancillary ligand structure, J. Am. Chem. Soc. 127\n[31] C.Y. Wu, L.T. Zhou, H.B. Yuan, S.Y. Wu, Interconnections among major forms of (2005) 12438\u201312439.\n regulated cell death, Apoptosis 25 (2020) 616\u2013624. [57] M. Nonoyama, Benzo[h]quinolin-10-yl-N iridium(III) complexes, Chem. Soc. Jpn.\n 47 (1974) 767\u2013768.\n\n\n\n\n 10\n\f", "pages_extracted": 10, "text_length": 60915}