Bisindolylmethane (BIM) is an important alkaloid derived from cruciferous plants that exhibits anti-cancer biological activity. Molecules with a BIM skeleton have long been widely recognized by medicinal chemists. Unfortunately, due to limitations in synthetic methods, research on this skeleton has mainly focused on symmetrical 3,3'-bisindolylmethane derivatives (3,3'-BIMs), wherein the two indole rings possess identical structures. In contrast, research on unsymmetrical 3,3'-BIMs has progressed slowly. Meanwhile, trifluoromethyl is a widely used "star group" in drug design due to its ability to improve pharmacological and pharmaceutical properties. To discover novel BIMs with significant anti-cancer potential and to study the variety of their structures, we designed and synthesized a series of unsymmetrical trifluoromethyl-containing BIMs based on previously reported SN1-type dehydrative nucleophilic substitution by our group. These novel BIMs inhibit various cancer cells, especially lung cancer cells A549. Among them, the compound 5b effectively induces endoplasmic reticulum stress (ERS) and leads to apoptosis, demonstrating outstanding anti-non-small cell lung cancer (NSCLC) effects in vitro and in vivo, and was significantly more effective than the BIM group. The IC50 values of BIM and 5b against A549 cells are 54.76 ± 4.7 μM and 3.88 ± 0.1 μM, respectively. Specifically, anti-tumor activity assays in vivo found the inhibitory rates of 34.80 % for BIM and 80.32 % for 5b. Hematoxylin-eosin (H&E) results showed that 5b does not cause chronic organ damage. Clearly, 5b greatly enhances anti-cancer efficacy, effectively restraining cell colonies and proliferation in the S phase. Additionally, 5b increases the content of reactive oxygen species (ROS), causing a decline in the mitochondrial membrane potential and promoting Ca2+ release. Further studies on the mechanism revealed that 5b induces ERS by activating the PERK-elF2α-CHOP signaling pathway, which could further regulate caspase and Bcl-2 family proteins, leading to apoptosis. These results demonstrate that 5b may be potent anticancer candidates for A549 tumor.

In this study, we designed and synthesized a new ligand, FMPIP (2-(3-fluoro-2-methylphenyl)-1H-imidazo[4,5-f][1,10]phenanthroline), and its three cyclometalated iridium(III) complexes: [Ir(ppy)2(FMPIP)](PF6) (Ir3a, ppy = 2-phenylpyridine), [Ir(bzq)2(FMPIP)](PF6) (Ir3b, bzq = benzo[h]quinoline), and [Ir(piq)2(FMPIP)](PF6) (Ir3c, piq = 1-phenylisoquinoline). We evaluated antiproliferative activity of Ir3a, Ir3b and Ir3c against hepatocellular carcinoma cell lines (Huh7, HepG2, SK-Hep1, HCCLM3) using 3-(4,5-dimethylthiazole-2-yl)-diphenyltetrazolium bromide (MTT) assay. Ir3a-Ir3c exhibited negligible cytotoxicity toward all tested cell lines, each with half-maximal inhibitory concentration (IC50) values > 100 μM, which was caused by poor solubility of Ir3a, Ir3b and Ir3c in phosphate-buffered saline (PBS). To overcome this limitation, we encapsulated Ir3a-Ir3c into liposomes to produce Ir3alp, Ir3blp and Ir3clp. Ir3alp, Ir3blp and Ir3clp significantly enhanced antiproliferative potency (IC50 < 5.0 μM). To further enhance anticancer efficacy, we used three kinds of targeted functional liposome to encapsulate Ir3a to form Ir3aT1lp, Ir3aT2lp and Ir3aT3lp. Against Huh7 cells, Ir3aT1lp, Ir3aT2lp and Ir3aT3lp showed IC50 values of 1.34 ± 0.04 μM, 1.26 ± 0.01 μM, and 1.23 ± 0.07 μM, respectively. Mechanistic studies revealed that Ir3alp, Ir3aT3lp, Ir3blp and Ir3clp trigger intracellular reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and apoptosis, we further observed some markers consistent with ferroptosis. Ir3aT3lp displayed potent antitumor efficacy with a tumor growth inhibition rate of 64.90%, simultaneously, they induce an increment of CD8+ T cells. These results show that Ir3alp, Ir3aT3lp, Ir3blp and Ir3clp induced ferroptosis to stimulate immune response to elevate the CD8+ T cells to exert antitumor efficiency.

Iridium(III) complexes become a research hotspot due to their unique chemical properties and biological activities. Herein, we synthesized two iridium(III) complexes: [Ir(bzq)2(BDIP)]PF6 (bzq = benzo[h]quinoline, BDIP = 2-(6-bromobenzo[d][1,3]dioxol-5-yl)-1H-imidazo[4,5-f][1,10]phenanthroline, Ir10a) and [Ir(piq)2(BDIP)]PF6 (piq = 1-phenylisoquinoline, Ir10b). The antitumor activities and mechanisms of the complexes and liposome-loaded complexes Ir10alip and Ir10blip were studied. Through the cellular uptake, we found that complexes and liposomes enter cells, but the less amount of Ir10a and Ir10b in the cells was observed. Ir10a, Ir10b, Ir10alip and Ir10blip inhibit the formation of cell colonies and cell invasion, cause a decrease in the mitochondrial membrane potential, and induce a release of cytochrome C. Ir10a, Ir10b, Ir10alip and Ir10blip cause apoptosis through p38-MAPK pathway. We also found that the expression of CRT, HSP70, and HMGB1 increased, proving that Ir10a, Ir10b, Ir10alip and Ir10blip caused immunogenic cell death, which further activates immune response to increase CD8+ T cells. The decrease of GSH, the increase of MDA, as well as the decreased expression of GPX4 and ferritin, provide strong evidence for ferroptosis. We further investigated the in vivo antitumor activities of Ir10b and Ir10blip through xenograft tumor experiments. The tumor inhibition rates of Ir10b and Ir10blip reached 33.02% and 67.92%, respectively, which proved that Ir10blip had a better antitumor effect in vivo. The above results demonstrate that Ir10a, Ir10b, Ir10alip and Ir10blip are expected to provide an efficient and low-toxicity treatment option for patients with cervical cancer, and this work plays an important role in the future development of antitumor drugs.

In this article, using NNIP (2-(2-nitronaphthalen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) as a ligand to synthesise and characterise a new iridium(III) complex, [Ir(piq)(2)(NNIP)]PF6 (Ir1, where piq = 1-phenylisoquinoline) and to explore its anticancer activity as a photosensitiser against HeLa cancer cells and the corresponding mechanisms of inducing cancer cell death. The cytotoxicity of Ir1 against HeLa, B16 and normal NIH3T3 cells was assessed using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Unexpectedly, Ir1 initially shows no cytotoxicity against those cells (half maximal inhibitory concentration, IC50 > 200 mu M) in the dark. However, upon white light irradiation, Ir1 significantly increased cytotoxicity, especially on HeLa cancer cells with a low IC50 value of 3.1 +/- 0.3 mu M. The anticancer mechanism was explored through various techniques, including cellular uptake, mitochondrial co-localisation, ROS production, mitochondrial permeability transition pore opening and the change in the mitochondrial membrane potential. Subsequently, lipid peroxidation was investigated with a C11-BODIPY581/591 probe to affirm the occurrence of ferroptosis. Additionally, metabolic impacts were probed by conducting lactate dehydrogenase release and adenosine 5 '-triphosphate (ATP) quantification assays. Apoptosis, pyroptosis and immunogenic cell death were also explored. The light-activated antitumour in vivo revealed that Ir1 can effectively inhibit the tumour growth with an inhibitory rate of 53.2%. These findings demonstrate that Ir1 induces cancer cell demise by a mitochondrial apoptotic pathway mediated by ROS, ferroptosis and pyroptosis.

The synthesis of the ligand 2-(2-methyl-4-hydroxyl)phenyl-1H-imidazo[4,5-f][1,10]phenanthroline (MHIP) and its corresponding new iridium(III) complexes [Ir(ppy)2(MHIP)]PF6 (ppy = 2-phenylpyridine, 9a), [Ir(bzq)2(MHIP)]PF6 (bzq = benzo[h]quinolone, 9b) and [Ir(piq)2(MHIP)]PF6 (piq = 1-phenylisoquinoline, 9c) was reported. The antiproliferative activity of compounds 9a-9c on HepG2, B16, and A549 cancer cells as well as on normal NIH 3T3 cells was tested using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that the three complexes showed moderate cytotoxicity in A549 and B16 cells. However, after further irradiation, the cytotoxicity was greatly enhanced; especially, 9a, 9b and 9c displayed significant cytotoxicity toward B16 cells with a low IC50 value of 3.1 ± 0.3 μM for 9a, 4.9 ± 0.8 μM for 9b, and 0.4 ± 0.1 μM for 9c. The effects of 9a-9c on the invasive ability of B16 cells were explored via colony formation and scratch experiments. Results demonstrated that the complexes could efficiently block cell proliferation and migration. The co-localization assay found that 9a-9c accumulated in the mitochondria and led to the apoptosis of B16 cells by decreasing mitochondrial membrane potential, altering the structure of microtubule proteins, damaging the structure of cellular DNA, and changing the expression of related proteins. The decrease in glutathione (GSH) concentration, the increase in malondialdehyde (MDA), the downregulation of GPX4, and C11-BODIPY staining results confirmed that 9a, 9b and 9c led to ferroptosis. In addition, we explored the relevant signaling pathways through an RNA sequencing assay and speculated on the possible anticancer mechanisms. Together, the results of this study indicate that the synthesized new iridium(III) complexes 9a-9c can induce cell death via ROS-mediated mitochondrial dysfunction, apoptosis and ferroptosis.

In this paper, we synthesised an organic small molecule IPD (4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-N,N-diphenylaniline) and its three new iridium(III) metal complexes [Ir(ppy)2(IPD)(PF6)] (Ir2a, ppy = 2-phenylpyridine), [Ir(piq)2(IPD)(PF6)] (Ir2b, piq = 1-phenylisoquinoline) and [Ir(bzq)2(IPD)(PF6)] (Ir2c, bzq = benzo[h]quinoline). After completing the synthesis and purification of the complexes, we used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method to investigate the in vitro cytotoxicity of the complexes on cancer A549, SK-hep1 and normal NIH 3T3 cells. In the dark, the complexes have no cytotoxic activity; however, upon irradiation, Ir2a and Ir2b show a high anticancer effect on inhibiting cancer proliferation of A549 and SK-hep1 cells. We explored the ability of Ir2a and Ir2b to inhibit cell proliferation and invasion by scratch and cell colony formation assays. We also detected cell cycle block, gamma-H2AX, intracellular reactive oxygen levels, co-localisation and mitochondrial membrane potential, and found that Ir2a and Ir2b, located at the mitochondria, increase intracellular ROS levels, cause a decrease in mitochondrial membrane potential and induce mitochondrial dysfunction. Additionally, Ir2a and Ir2b cause apoptosis and autophagy. RNA-sequence assays suggest that Ir2a upregulates 101 genes and downregulates 143 genes. Additionally, Ir2a and Ir2b cause immunogenic cell death.

In this study, a new ligand 2-(4-(1H-imidazol-1-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline (IPIP) was synthesized and reacted with cis-[Ru(phen)₂Cl₂]·2H2O and cis-[Ru(dip)₂Cl₂]·2H2O to form two new Ru(II) complexes [Ru(phen)2(IPIP)](PF6)2 (Ru1a) and [Ru(dip)2(IPIP)](PF6)2 (Ru1b). The cytotoxicity of Ru1a and Ru1b against both cancer and normal cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The results revealed that Ru1a exhibits moderate and Ru1b shows no cytotoxic activity in the dark. However, after 1 h of irradiation, Ru1a exhibited significant cytotoxicity against HCT116 cells, while Ru1b still showed no cytotoxic activity toward the selected cancer cells. Further investigation via cell uptake, mitochondrial localization, mitochondrial membrane potential, cytochrome C release, and western blotting techniques confirmed that Ru1a induced apoptosis in HCT116 cells via the mitochondrial pathway. RNA-sequence assay showcases that Ru1a caused cell death through ferroptosis. The levels of lipid peroxidation product of malondialdehyde (MDA) were elevated, while the expression of GPX4 was reduced. These findings affirmed that Ru1a promotes cell death through ferroptosis. Antitumor in vivo confirmed that Ru1a exerts anticancer activity in the tumor microenvironment (TME) by inducing immunogenic cell death and activating immune responses to enhance CD8+ T cells, thereby directly killing colorectal cancer cells.

Herein, we reported the synthesis and characterization of two new iridium(III) complexes [Ir(ppy)2(fpp)](PF6) (Ir1a, ppy = 2-phenylpyridine, fpp = 2-(2,2-difluorobenzo[1,3]dioxol-5-yl-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(bzq)2(fpp)](PF6) (Ir1b, bzq = benzo[h]quinoline) through high resolution mass spectrometry (HRMS), 1H NMR and 13C NMR. The cytotoxicity in vitro of Ir1a and Ir1b on normal NIH3T3 cells and cancer SGC-7901, A549, SK-Hep1 cells was tested using MTT (3-(4,5-dimethylthiazole-2-yl)-diphenyltetrazolium bromide) method. Ir1a exhibits high cytotoxicity on SGC-7901 cells (IC50 = 2.7 ± 0.7 µM), whereas Ir1b shows moderate cytotoxicity toward the selected cancer cells. The ROS content was investigated using a fluorescence probe of 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA), the results show that Ir1a and Ir1b elevate ROS content. The co-localization and the change of mitochondrial membrane potential were explored. Apoptotic studies using Annex V/PI double staining method demonstrate that Ir1a and Ir1b can efficiently cause apoptosis. Ir1a and Ir1b inhibit the cell proliferation at the G2/M period. Additionally, lipid peroxidation and downregulation of ferritin protein suggest that Ir1a and Ir1b can trigger ferroptosis.

In this work, we have carefully designed and synthesized two Ru(II) metal complexes: [Ru(phen)2(HMPIP)] (PF6)2 (6a, where phen = 1,10-phenanthroline, HMPIP = 2-(2-hydroxy-3-methylphenyl-1H-imidazo[4,5-f][1,10] phenanthroline) and [Ru(bpy)2(HMPIP)](PF6)2 (6b, where bpy = 2,2 '-bipyridine). Using 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) to explore the cytotoxicity of 6a and 6b towards HepG2, B16, A549, SGC-7901, HCT116 and non-cancer LO2. The complexes exhibited cytotoxicity activity against HepG2 cells. The capacity of 6a and 6b to impede the proliferation and dissemination of cancer cells was evaluated by conducting proliferation and migration experiments and 3D model. The anticancer mechanism was investigated in detail. The utilization of cycle blocking assays revealed that 6a and 6b induced a G0/G1 phase arrest in HepG2 cells. The cellular uptake experiments show that the complexes enter the cell nuclei, then escape from the cell nuclei into the cytoplasm, finally accumulate in the mitochondria. Apoptosis assays and the examination of proteins indicated that the complexes were capable of efficiently inducing apoptosis in HepG2 cells. Additionally, the potential induction of autophagy-mediated cell death was explored. The observed reduction in glutathione (GSH) levels and glutathione peroxidase 4 (GPX4) expression suggested a disruption of redox homeostasis within cancer cells, an increment in malondialdehyde (MDA) amount, together with BODIPY staining experiment, confirm that 6a and 6b can induce ferroptosis. Interestingly, in a nude mouse model, 6a showed a significant suppression of tumor growth with an inhibition rate of 63.4 %, without causing any weight loss of mice. The studies on the mechanism show that 6a causes immune cell death, increase the amount of TNF-alpha and IFN-gamma, reduce IL-10 content, which further activates immune response to increase CD8+ T cells to prevent tumor growth. Therefore, 6a inhibits the tumor growth through stimulating the immune response to increase CD8+ T cells. In addition, the experiments in vitro show that the complexes through inhibition of PI3K/AKT/mTOR signaling pathway and intrinsic mitochondria pathway to cause cell apoptosis. These results demonstrate that Ru (II) complexes may be potent anticancer candidates for HepG2 tumor.

A new ligand, 2-(2-hydroxyl-4-methyl)phenyl-1H-imidazo[4,5-f][1,10]phenanthroline (IPMP), and [Ir(ppy)2(IPMP)]PF6 (7a), [Ir(bzq)2(IPMP)]PF6 (7b), and [Ir(piq)2(IPMP)]PF6 (7c) have been prepared and characterized by HRMS, NMR spectra. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that 7b exhibited excellent activity (IC50 = 4.5 ± 0.4 μM), while 7a and 7c showed good cytotoxicity (IC50 = 8.5 ± 0.9 μM and 8.9 ± 2.2 μM) against non-small cell lung cancer A549 cells. The experiments of cellular uptake and mitochondrial localization demonstrate that these new iridium(III) complexes are readily taken up by A549 cells and accumulate in the mitochondria and damage the structure of the mitochondria, which results in the loss of mitochondrial membrane potential (MMP), elevated lipid peroxidation, as well as DNA damage, the inhibition of microtubule polymerization, hindrance of the cell cycle in the G0/G1 phase, and release of cytochrome c, collectively leading to apoptosis. Furthermore, upregulation of Beclin-1, overexpression of NF-κB and downregulation of GPX4 protein were observed, which resulted in the activation of autophagy, pyroptosis and ferroptosis, respectively. In the C57BL/6 mouse model, the 7b demonstrated promising in vivo antitumor efficacy, with a tumor inhibitory rate of 66.9 %. Additionally, the complexes induce an immunogenic cell death to activate immune response, further enhance CD8+ T cells and efficiently inhibit tumor growth. Collectively, we consider that the complexes may be utilized as potential candidate agents for the treatment of A549 cancer.

In this study, [Ir(ppy)2(DMHBT)](PF6) (ppy = deprotonated 1-phenylpyridine, DMHBT = 10,12-dimethylpteridino[6,7-f][1,10]phenanthroline-11,13-(10,12H)-dione, 8a), [Ir(bzq)2(DMHBT)](PF6) (bzq = deprotonated benzo[h]quinoline, 8b) and [Ir(piq)2(DMHBT)](PF6) (piq = deprotonated 1-phenylisoquinoline, 8c) were synthesized and characterized by HRMS, 13C NMR and 1H NMR. In vitro cytotoxicity experiments showed that 8a, 8b, 8c show moderate cytotoxicity against B16 cells, while the cytotoxicity of the complexes 8a, 8b and 8c toward B16 cells was greatly improved upon light irradiation, which can be used as photosensitizers to exert anticancer efficacy in photodynamic therapy (PDT). After being taken up by cells, 8a, 8b, 8c were localized in the mitochondria, resulting in a large amount of Ca2+ in-flux, a burst release of ROS, a sustained opening of mitochondrial permeability transition pore, and a decrease of the mitochondrial membrane potential, which led to mitochondrial dysfunction and further activation of caspase 3 and Bcl-2 family proteins to induce apoptosis. Overloaded ROS reacted with polyunsaturated fatty acids on the cell membrane, and initiated lipid peroxidation, inhibited the xc--system-glutathione (GSH)-glutathione peroxidase 4 (GPX4) antioxidant defense system, and upregulated the expression of the damage-associated molecules, HMGB1, CRT, and HSP70. The presence of Fer-1 was effective on increasing the cell survival, which demonstrates that the complexes possess the potential to induce ferroptosis and immunogenic cell death. In addition, 8a, 8b and 8c induced autophagy by inhibiting the AKT/PI3K/mTOR signaling pathway, downregulating p62 and promoting Beclin-1 expression upon light irradiation.

This paper introduces a new ligand, 4,6-dichloro-5-(1H-imidazo [4,5-f]phenanthroline-2-yl)pyrimidin-2-amine (DPPA), and its corresponding new iridium(III) complexes: [Ir(ppy)2(DPPA)](PF6) (2a) (where ppy represents deprotonated 2-phenylpyridine), [Ir(bzq)2(DPPA)](PF6) (2b) (with bzq indicating deprotonated benzo[h]quinoline), and [Ir(piq)2(DPPA)](PF6) (2c) (piq denoting deprotonated 1-phenylisoquinoline). The cytotoxic effects of both DPPA and 2a, 2b, and 2c were evaluated against human lung carcinoma A549, melanoma B16, colorectal cancer HCT116, human hepatocellular carcinoma HepG2 cancer cell lines, as well as the non-cancerous LO2 cell line using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. While DPPA exhibited moderate anticancer activity toward A549, B16, HCT116 and HepG2 cells, complexes 2a, 2b, and 2c displayed remarkable efficacy against A549, B16, and HCT116 cells. The cell colonies and wound healing were investigated. Moreover, various aspects of the anticancer mechanisms were explored. The cell cycle analyses revealed that the complexes block cell proliferation of A549 cells during the S phase. Complex 2c induce an early apoptosis, while 2a and 2b cause a late apoptosis. The interaction of 2a, 2b and 2c with endoplasmic reticulum and mitochondria was identified, leading to elevated ROS and Ca2+ amounts. This resulted in a reduced mitochondrial membrane potential, mitochondrial permeability transition pore opening, and an increase of cytochrome c. Also, ferroptosis was investigated through measurements of intracellular glutathione (GSH), malondialdehyde (MDA), and recombinant glutathione peroxidase (GPX4) protein expression. The pyroptosis was explored via cell morphology, release of lactate dehydrogenase (LDH) and expression of pyroptosis-related proteins. RNA sequencing was applied to examine the signaling pathways. Western blot analyses illuminated that the complexes regulate the expression of Bcl-2 family proteins. Additionally, an in vivo antitumor study demonstrated that complex 2c exhibited a remarkable inhibitory rate of 58.58% in restraining tumor growth. In summary, the findings collectively suggest that the iridium(III) complexes induce cell death via ferroptosis, apoptosis by a ROS-mediated mitochondrial dysfunction pathway and GSDMD-mediated pyroptosis.

In this study, the ligand EIPP (5-ethoxy-2-(1H-imidazo[4,5-f] [1,10] phenanthrolin-2-yl)phenol) and [Ir(ppy)2(EIPP)](PF6)] (5a, ppy = 2-phenylpyridine) and [Ir(piq)2(EIPP)](PF6)] (5b, piq = 1-phenylisoquinoline) were synthesized and they were entrapped into liposomes to produce 5alipo and 5blipo. 5a and 5b were characterized via HRMS, NMR, UV-vis and IR. The cytotoxicity of 5a, 5b, 5alipo and 5blipo on cancer and non-cancer cells was estimated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). MTT assay demonstrated that 5a and 5b did not show any significant cellular activity but their liposome-encapsulated 5alipo and 5blipo had significant toxic effects. The mechanism of 5alipo, 5blipo-inducing apoptosis was explored by studying cellular uptake, mitochondrial localization, mitochondrial membrane potential, cytochrome C, glutathione (GSH), malondialdehyde (MDA) and protein immunoblotting. The results demonstrated that 5alipo and 5blipo caused a release of cytochrome C, downregulated the expression of Bcl-2, upregulated the expression of BAX, activated caspase 3, and downregulated PARP expression. It was shown that 5alipo and 5blipo could inhibit cancer cell proliferation in G2/M phase by regulating p53 and p21 proteins. Additionally, 5alipo and 5blipo induced autophagy through an adjustment from LC3-I to LC3-II and caused ferroptosis. The in vivo antitumor activity of 5alipo was examined in detail.

In this paper, three new iridium(III) complexes: [Ir(piq)2(DFIPP)]PF6 (piq = deprotonated 1-phenylisoquinoline, DFIPP = 3,4-difluoro-2-(1H-imidazo[4,5-f][1,10]phenenthrolin-2-yl)phenol, 3a), [Ir(bzq)2(DFIPP)]PF6 (bzq = deprotonated benzo[h]quinoline, 3b), and [Ir(ppy)2(DFIPP)]PF6 (ppy = deprotonated 1-phenylpyridine, 3c), were synthesized and characterized. The complexes were found to be nontoxic to tumor cells via 3-(4,5-dimethylthiazole-2-yl)-diphenyltetrazolium bromide (MTT) assay. Surprisingly, its liposome-entrapped complexes 3alip, 3blip, and 3clip on B16 cells showed strong cytotoxicity (IC50 = 13.6 ± 2.8, 9.6 ± 1.1, and 18.9 ± 2.1 μM). Entry of 3alip, 3blip, and 3clip into B16 cells decreases mitochondrial membrane potential, regulates Bcl-2 family proteins, releases cytochrome c, triggers caspase family cascade reaction, and induces apoptosis. In addition, we also found that 3alip, 3blip, and 3clip triggered ferroptosis and autophagy. In vivo studies demonstrated that 3blip inhibited melanoma growth in C57 mice with a high inhibitory rate of 83.95%, and no organic damage was found in C57 mice.

Three new dibenzoxanthenes were synthesized and their antitumor activity were investigated. Compounds 3a-3c showed significant cytotoxicity to HeLa cells. The 1,3-diphenylisobenzofuran (DBPF) assay demonstrated that compounds could produce singlet oxygen. Cell cloning and wound healing assays demonstrated that compounds 3a-3c effectively inhibited HeLa cell cloning and migration. After entering the mitochondria, the compounds caused a decrease in mitochondrial membrane potential, an increase in intracellular ROS and Ca2+ levels, and blocked the cell cycle in the G2/M phase. Through protein immunoblotting, the apoptotic mechanism was studied, the results show that 3a-3c regulated Bcl-2 family protein, caused abnormal mitochondrial function, which led to mitochondrial apoptotic pathway. ROS, GPX4, GSH and MDA assay indicated that compounds 3a-3c caused intracellular lipid peroxidation in HeLa cells leading to ferroptosis. GSDME cleavage and elevation of LDH release induced the occurrence of pyroptosis. Therefore, we conclude that the compounds cause cell death through three pathways: apoptosis, ferroptosis and pyroptosis.

Herein, we synthesized and characterized two novel iridium (III) complexes: [Ir(bzq)2(PPD)](PF6) (4a, with bzq = deprotonated benzo[h]quinoline and PPD = pteridino[6,7-f][1,10]phenanthroline-11,13-diamine) and [Ir(piq)2(PPD)](PF6) (4b, with piq = deprotonated 1-phenylisoquinoline). The anticancer efficacy of these complexes, 4a and 4b, was investigated using 3-(4,5-dimethylthiazole)-2,5-diphenltetraazolium bromide (MTT). Complex 4a exhibited no cytotoxic activity, while 4b demonstrated moderate efficacy against SGC-7901, A549, and HepG2 cancer cells. To enhance their anticancer potential, we explored two strategies: (I) light irradiation and (II) encapsulation of the complexes in liposomes, resulting in the formation of 4alip and 4blip. Both strategies significantly increased the ability of 4a, 4b to kill cancer cells. The cellular studies indicated that both the free complexes 4a, 4b and their liposomal forms 4alip and 4blip effectively inhibited cell proliferation. The cell cycle arrest analysis uncovered 4alip and 4blip arresting cell growth in the S period. Additionally, we investigated apoptosis and ferroptosis pathways, observing an increase in malondialdehyde (MDA) levels, a reduction of glutathione (GSH), a down-regulation of GPX4 (glutathione peroxidase) expression, and lipid peroxidation. The effects on mitochondrial membrane potential and intracellular Ca2+concentrations were also examined, revealing that both light-activated and liposomal forms of 4alip and 4blip caused a decline in mitochondrial membrane potential and an enhancement in intracellular Ca2+ levels. In conclusion, these complexes and them encapsulated liposomes induce cell death through apoptosis and ferroptosis.

Cyclometalated iridium(iii) compounds have been widely explored due to their outstanding photo-physical properties and multiple anticancer activities. In this paper, three cyclometalated iridium(iii) compounds [Ir(ppy)(2)(DBDIP)]PF6 (5a), [Ir(bzq)(2)(DBDIP)]PF6 (5b), and [Ir(piq)(2)(DBDIP)]PF6 (5c) (ppy: 2-phenylpyridine; bzq: benzo[h]quinoline; piq: 1-phenylisoquinoline, and DBDIP: 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) were synthesized and the mechanism of antitumor activity was investigated. Compounds photoactivated by visible light show strong cytotoxicity against tumor cells, especially toward A549 cells. Biological experiments such as migration, cellular localization, mitochondrial membrane potential and permeability, reactive oxygen species (ROS) and calcium ion level detection were performed, and they demonstrated that the compounds induced the apoptosis of A549 cells through a mitochondrial pathway. At the same time, oxidative stress caused by ROS production increases the release of damage-related molecules and the expression of porogen gasdermin D (GSDMD), and the content of LDH released from damaged cell membranes also increased. Besides, the content of the lipid peroxidation product, malondialdehyde (MDA), increased and the expression of GPX4 decreased. These indicate that the compounds promote cell death by combining ferroptosis and pyroptosis. The results reveal that cyclometalated iridium(iii) compounds 5a-5c may be a potential chemotherapeutic agent for photodynamic therapy of cancers.

This paper unveils a novel perspective on synthesis and characterization of the ligand 5-bromo-2-amino-2'- (phenyl-1H-imidazo[4,5-f][1,10]phenanthroline) (BAPIP), and its iridium(III) complexes [Ir(PPY-)2(BAPIP)] (PF6) (1a, with PPY- as deprotonated 2-phenylpyridine), [Ir(PIQ-)2(BAPIP)](PF6) (1b, piq- denoting deprotonated 1-phenylisoquinoline), and [Ir(BZQ-)2(BAPIP)](PF6) (1c, bzq- signifying deprotonated benzo[h]quinoline). Systematic evaluation of the cytotoxicity of 1a, 1b, and 1c across diverse cell lines encompassing B16, HCT116, HepG2, A549, HeLa, and LO2 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Unexpectedly, compounds 1b and 1c demonstrated no cytotoxicity against the above cell lines. Motivated by the pursuit of heightened anti-proliferative potential, a strategic encapsulation approach yielded liposomes 1alip, 1blip, and 1clip. As expectation, 1alip, 1blip, and 1clip displayed remarkable anti-proliferative efficacy, particularly noteworthy in A549 cells, exhibiting IC50 values of 4.9 +/- 1.0, 5.9 +/- 0.1, and 7.6 +/- 0.2 mu M, respectively. Moreover, our investigation illuminated the mitochondrial accumulation of these liposomal entities, 1alip, 1blip, and 1clip, evoking apoptosis through the mitochondrial dysfunction mediated by reactive oxygen species (ROS). The ferroptosis was confirmed by decrease in glutathione (GSH) concentrations, the downregulation of glutathione peroxidase 4 (GPX4), increase of high mobility group protein 1 (HMGB1), and lipid peroxidation. Simultaneously, pyroptosis as another mode of cell death was undertaken. RNA-sequencing was employed to investigate intricate signalling pathways. In vivo examination provided tangible evidence of 1alip in effectively curbing tumor growth. Collectively, this study provides a multifaceted mode of cellular demise orchestrated by 1a, 1alip, 1blip, and 1clip, involving pathways encompassing apoptosis, ferroptosis, and pyroptosis.