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We introduce ruthenosomes, a fusion of liposomal and reactive oxygen species (ROS)-generating properties meticulously engineered as potent ferroptosis inducers (FINs), marking a significant advancement in metallodrug design for cancer therapy. Formed through the self-assembly of oleate-conjugated ruthenium complexes, these ruthenosomes exhibit exceptional cellular uptake, selectively accumulating in mitochondria and causing substantial disruption. This targeted mitochondrial damage significantly elevates ROS levels, triggering autophagy and selectively activating ferritinophagy. Together, these processes sensitize cancer cells to ferroptosis. In vivo, ruthenosomes effectively suppress colorectal tumor growth, underscoring their therapeutic potential. Our study pioneers a design strategy that transforms ruthenium complexes into liposome-like structures capable of inducing ferroptosis independent of light activation. By leveraging ruthenosomes as multifunctional nanocarriers, this research offers a versatile and powerful platform for ROS-mediated, ferroptosis-driven cancer cell eradication. Show less

Title: New rhodium(III)-triphenylphosphine complexes with 5-halogenate-8-hydroxyquinoline as ligands: synthesis, characterization, cytotoxicity, and mechanism of action. Abstract: The incorporation of triphenylphosphine (PPh3) can enhance the antiproliferative activity of complexes. Herein, four Rh(III) complexes GUPT1-GUPT4 were synthesized. GUPT4 exhibited stronger anticancer activity than HGU, cisplatin, and GUPT1-GUPT3 against human non-small cell lung A549 and its cisplatin-resistant A549 cell line (CR-A549), with IC50 values of 6.73 ± 0.41 and 5.11 ± 0.16 μM, respectively. The antiproliferative activity of the four RhIII complexes increased with different 5-substituted ligands in the following order: H (GUPT1) < Br (GUPT2) < Cl (GUPT3) < F (GUPT4). GUPT3 and GUPT4 induce CR-A549 mitochondrial autophagy and ATP blockade, leading to apoptosis. In addition, the inhibition rate of GUPT4 on A549 was 39.1 %, showing potential antitumor efficacy. Thus, GUPT3 and GUPT4 can be considered as promising non-Pt drug candidates for lung cancer treatment. Show less

Title: Mitochondrial-targeted iridium(III) complexes suppress tumor growth through inducting immunogenic cell death to activate immune response. Abstract: 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. Show less

Background/objectives

Breast cancer (BC) remains one of the most prevalent and deadly cancers worldwide, with limited access to advanced treatments in developing regions. There is a critical need for novel therapies with unique mechanisms of action, especially to overcome resistance to conventional platinum-based drugs. This study investigates the anticancer potential of the ruthenium complex Bis(quinolin-8-olato)bis(triphenylphosphine)ruthenium(II) (Ru(quin)₂) in ER-positive (T47D) and triple-negative (MDA-MB-231) BC cell lines.

Results

Ru(quin)₂ demonstrated dose-dependent cytotoxicity, with IC50 values of 48.3 μM in T47D cells and 45.5 μM in MDA-MB-231 cells. Its cytotoxic effects are primarily driven by apoptosis, as shown by increased BAX expression, enhanced caspase-3 activity, reduced Aurora B kinase levels, and elevated histone release. Ru(quin)₂ also induced autophagy, evidenced by LC3-I to LC3-II conversion and reduced SQSTM1, partially mediated through MAPK signaling. Furthermore, Ru(quin)₂ induced G0/G1 cell cycle arrest by downregulating cyclin D1, CDK4, and CDK6, alongside upregulation of the CDK inhibitor p21.

Conclusions

Ru(quin)₂ emerges as a potent candidate for BC treatment, with multiple mechanisms of action involving apoptosis, autophagy, and cell cycle arrest. Further studies are warranted to elucidate its detailed molecular mechanisms and evaluate its therapeutic potential in vivo, moving toward clinical applications for both ER-positive and triple-negative BC management. Show less

In recent years, photodynamic therapy (PDT) has emerged as a promising alternative to classical chemotherapy for treating cancer. PDT is based on a nontoxic prodrug called photosensitizer (PS) activated by light at the desired location. Upon irradiation, the PS reacts with the oxygen present in the tumor, producing cytotoxic reactive oxygen species (ROS). Compounds with highly conjugated π-bond systems, such as porphyrins and chlorins, have proven to be excellent light scavengers, and introducing a metal atom in their structure improved the generation of ROS. In this work, a series of tetrapyrrole-ruthenium(II) complexes derived from protoporphyrin IX and the commercial drug verteporfin were designed as photosensitizers for PDT. The complexes were almost nontoxic on human gastric cancer cells under dark conditions, revealing remarkable cytotoxicity upon irradiation with light. The ruthenium atom in the central cavity of the chlorin ligand allowed combined mechanisms in photodynamic therapy, as both singlet oxygen and superoxide radicals were detected. Additionally, one complex produced large amounts of singlet oxygen under hypoxic conditions. Biological assays demonstrated that the ruthenium derivatives caused cell death through a caspase 3 mediated apoptotic pathway and via CHOP, an endoplasmic reticulum stress-inducible transcription factor involved in apoptosis and growth arrest. Show less

Title: Induction of ferroptosis of iridium(III) complexes localizing at the mitochondria and lysosome by photodynamic therapy. Abstract: 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. Show less

Title: Functional Upgrading of an Organo-Ir(III) Complex to an Organo-Ir(III) Prodrug as a DNA Damage-Responsive Autophagic Inducer for Hypoxic Lung Cancer Therapy. Abstract: The efficiency of nitrogen mustards (NMs), among the first chemotherapeutic agents against cancer, is limited by their monotonous mechanism of action (MoA). And tumor hypoxia is a significant obstacle in the attenuation of the chemotherapeutic efficacy. To repurpose the drug and combat hypoxia, herein, we constructed an organo-Ir(III) prodrug, IrCpNM, with the composition of a reactive oxygen species (ROS)-inducing moiety (Ir-arene fragment)-a hypoxic responsive moiety (azo linker)-a DNA-alkylating moiety (nitrogen mustard), and realized DNA damage response (DDR)-mediated autophagy for hypoxic lung cancer therapy for the first time. Prodrug IrCpNM could upregulate the level of catalase (CAT) to catalyze the decomposition of excessive H2O2 to O2 and downregulate the expression of the hypoxia-inducible factor (HIF-1α) to relieve hypoxia. Subsequently, IrCpNM initiates the quadruple synergetic actions under hypoxia, as simultaneous ROS promotion and glutathione (GSH) depletion to enhance the redox disbalance and severe oxidative and cross-linking DNA damages to trigger the occurrence of DDR-mediated autophagy via the ATM/Chk2 cascade and the PIK3CA/PI3K-AKT1-mTOR-RPS6KB1 signaling pathway. In vitro and in vivo experiments have confirmed the greatly antiproliferative capacity of IrCpNM against the hypoxic solid tumor. This work demonstrated the effectiveness of the DNA damage-responsive organometallic prodrug strategy with the microenvironment targeting system and the rebirth of traditional chemotherapeutic agents with a new anticancer mechanism. Show less

Title: White light increases anticancer effectiveness of iridium(III) complexes toward lung cancer A549 cells. Abstract: Anticancer activity has been extensively studies. In this article, three ligands 2-(6-bromobenzo[d][1,3]dioxol-5-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (BDIP), 2-(7-methoxybenzo[d][1,3]dioxol-5-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (MDIP), 2-(6-nitrobenzo[d][1,3]dioxol-5-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (NDIP) and their iridium(III) complexes: [Ir(ppy)2(BDIP)](PF6) (ppy = deprotonated 2-phenylpyridine, 3a), [Ir(ppy)2(MDIP)](PF6) (3b) and [Ir(ppy)2(NDIP)](PF6) (3c) were synthesized. The cytotoxicity of 3a, 3b, 3c against Huh7, A549, BEL-7402, HepG2, HeLa, and non-cancer NIH3T3 was tested using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The results obtained from the MTT test stated clearly that these complexes demonstrated moderate or non-cytotoxicity toward Huh7, BEL-7402, HepG2 and HeLa except A549 cells. To improve the anticancer efficacy, we used white light to irradiate the mixture of cells and complexes for 30 min, the anticancer activity of the complexes was greatly enhanced. Particularly, 3a and 3b exhibited heightened capability to inhibit A549 cells proliferation with IC50 (half maximal inhibitory concentration) values of 0.7 ± 0.3 μM and 1.8 ± 0.1 μM, respectively. Cellular uptake has shown that 3a and 3b can be accumulated in the cytoplasm. Wound healing and colony forming showed that 3a and 3b significantly hinder the cell migration and growth in the S phase. The complexes open mitochondrial permeability transition pore (MPTP) channel and cause the decrease of membrane potential, release of cytochrome C, activation of caspase 3, and finally lead to apoptosis. In addition, 3a and 3b cause autophagy, increase the lipid peroxidation and lead to ferroptosis. Also, 3a and 3b increase the expression of calreticulin (CRT), high mobility group box 1 (HMGB1), heat shock protein 70 (HSP70), thereby inducing immunogenic cell death. Show less

Colon cancer is one of the most commonly diagnosed cancers and is recognized as the most aggressive tumor of the digestive system. Aberrant activation of signal transducer and activator of transcription 3 (STAT3) is associated with proliferation, metastasis and immunosuppression of the tumor cells. Here, to inhibit the STAT3 pathway and suppress metastasis in colon cancer cells, the half-sandwich iridium complex Ir-ART containing an artesunate-derived ligand was synthesized. The complex showed remarkable antiproliferative activity against human colon cancer HCT-116 cells and exhibited a concentration-dependent reduction in STAT3 protein expression. Mechanism study demonstrates that Ir-ART is located mainly in the nucleus and mitochondria, causing γ-H2AX and cyclin B1 reduction and reactive oxygen species accumulation and mitochondrial membrane potential loss, ultimately leading to autophagic cell death. The migration of cancer cells was also inhibited via metalloproteinase 9 downregulation. Furthermore, Ir-ART could initiate antitumor immune responses by eliciting immunogenic cell death and downregulating immunosuppressive cytokine cyclooxygenase-2. Taken together, Ir-ART is expected to be further applied to chemotherapy and immunotherapy for colon cancer. Show less

The development of anticancer drugs to treat triple-negative breast cancer (TNBC) is an ongoing challenge. Immunogenic cell death (ICD) has garnered considerable interest worldwide as a promising synergistic modality for cancer chemoimmunotherapy. However, only few drugs or treatment modalities can trigger an ICD response and none of them exert a considerable clinical effect against TNBC. Therefore, new agents with potentially effective chemoimmunotherapeutic response are required. In this study, five new cyclometalated Ir(III) complexes containing isoquinoline alkaloid CˆN ligands were designed and synthesized. Among them, Ir-1 exhibited the highest in vitro cytotoxicity. Mechanistically, Ir-1 could trigger autophagy-dependent ferroptosis and a subsequent ferroptosis-dependent ICD response as well as indoleamine 2,3-dioxygenase (IDO) inhibition via reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress in MDA-MB-231 cells. When immunocompetent BALB/c mice were vaccinated with Ir-1-treated dying TNBC cells, antitumor CD8⁺ T-cell response and Foxp3⁺ T-cell depletion were induced, resulting in long-lasting antitumor immunity in TNBC cells. Moreover, combination therapy with Ir-1 and anti-PD1 could substantially augment in vivo therapeutic effects. Based on these results, Ir-1 is a promising candidate for chemoimmunotherapy against TNBC and its effects are mediated synergistically via ICD induction and IDO blockage. Show less

Title: Anticancer activity of 8-hydroxyquinoline-triphenylphosphine rhodium(III) complexes targeting mitophagy pathways. Abstract: Metallodrugs exhibiting distinct mechanisms of action compared with cisplatin hold promise for overcoming cisplatin resistance and improving the efficacy of anticancer drugs. In this study, a new series of rhodium (Rh)(III) complexes containing tris(triphenylphosphine)rhodium(I) chloride [(TPP)3RhCl] (TPP = triphenylphosphine, TPP=O = triphenylphosphine oxide) and 8-hydroxyquinoline derivatives (H-XR1-H-XR4), namely [Rh(XR1)2(TPP)Cl]·(TPP=O) (Yulin Normal University-1a [YNU-1a]), [Rh(XR2)2(TPP)Cl] (YNU-1b), [Rh(XR3)2(TPP)Cl] (YNU-1c), and [Rh(XR4)2(TPP)Cl] (YNU-1d), was synthesized and characterized via X-ray diffraction, mass spectrometry and IR. The cytotoxicity of the compounds YNU-1a-YNU-1d in Hep-G2 and HCC1806 human cancer cell lines and normal HL-7702 cell line was evaluated. YNU-1c exhibited cytotoxicity and selectivity in HCC1806 cells (IC50 = 0.13 ± 0.06 μM, selectivity factor (SF) = 384.6). The compounds YNU-1b and YNU-1c, which were selected for mechanistic studies, induced the activation of apoptotic pathways and mitophagy. In addition, these compounds released cytochrome c, cleaved caspase-3/pro-caspase-3 and downregulated the levels of mitochondrial respiratory chain complexes I/IV (M1 and M4) and ATP. The compound YNU-1c, which was selected for in vivo experiments, exhibited tumor growth inhibition (58.9 %). Importantly, hematoxylin and eosin staining and TUNEL revealed that HCC1806 tumor tissues exhibited significant apoptotic characteristics. YNU-1a-YNU-1d compounds are promising drug candidates that can be used to overcome cisplatin resistance. Show less

Lipid-mediated phase separation is crucial for the formation of lipophilic spontaneous domain to regulate lipid metabolism and homeostasis, furtherly contributing to multiple cell death pathways. Herein, a series of Ru(II) lipid-mimics based on short chains or midchain lipids are developed. Among them, Ru-LipM with two dodecyl chains significantly induces natural lipid phase separation via hydrocarbon chain-melting phase transitions. Accompanied by the aggregation of Ru-LipM-labeled lipophilic membrane-less compartments, most polyunsaturated lipids are increased and the autophagic flux is retarded with the adaptor protein sequestosome 1 (p62). Upon low-dose irradiation, Ru-LipM further drives ferritinophagy, providing an additional source of labile iron and rendering cells more sensitive to ferroptosis. Meanwhile, the peroxidation of polyunsaturated lipids occurs due to the deactivation of glutathione peroxidase 4 (GPX4) and the overexpression of acyl-CoA synthetase long-chain family member 4 (ACSL4), leading to the immunogenic ferroptosis. Ultimately, both innate and adaptive immunity are invigorated, indicating the tremendous antitumor capability of Ru-LipM in vivo. This study presents an unprecedented discovery of small molecules capable of inducing and monitoring lipid phase separation, thereby eliciting robust immune responses in living cells. It provides a biosimulation strategy for constructing efficient metal-based immune activators. Show less

Title: Design, synthesis and biological evaluation of liposome entrapped iridium(III) complexes toward SGC-7901 cells. Abstract: In this study, two new iridium(III) polypyridyl complexes [Ir(bzq)2(DIPH)](PF6) (bzq = deprotonated benzo[h]quinoline, DIPH = 4-(2,5-dibromo-4-(1H-imidazo[4,5-f][1,10]phenanthrolim-2-yl)-4-hydroxybutan-2-one) (Ir1) and [Ir(piq)2(DIPH)](PF6) (piq = deprotonated 1-phenylisoquinoline) (Ir2) were synthesized and characterized by elemental analysis, HRMS, 1H and 13C NMR. The cytotoxic activity of Ir1, Ir2, Ir1lipo and Ir2lipo against cancer cells SGC-7901, HepG2, A549, HeLa, B16 and normal NIH3T3 cells in vitro was evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) method. Ir1 and Ir2 showed no cytotoxic activity, but their liposome-entrapped Ir1 (Ir1lipo) and Ir2 (Ir2lipo) showed significant cellular activity, especially sensitive to SGC-7901 with IC50 values of 4.7 ± 0.2 and 12.4 ± 0.5 μM, respectively. The cellular uptake, endoplasmic reticulum (ER) localization, autophagy, tubulin polymerization, glutathione (GSH), malondialdehyde (MDA) and release of cytochrome c were investigated to explore the mechanisms of apoptosis. The calreticulin (CRT), heat shock protein 70 (HSP70), high mobility group box 1 (HMGB1) were also explored. Western blotting showed that Ir1lipo and Ir2lipo inhibited PI3K (phosphoinositide-3 kinase), AKT (protein kinase B), p-AKT and activated Bcl-2 (B-cell lymphoma-2) protein and apoptosis-regulated factor caspase 3 (cysteinyl aspartate specific proteinase-3) and cleaving PARP (poly ADP-ribose polymerase). The results demonstrated that Ir1lipo and Ir2lipo induce cell apoptosis through targeting the endoplasmic reticulum (ER), cause oxidative stress damage, inhibiting PI3K/AKT signaling pathway, immunogenic cell death (ICD) and inhibit the cell growth at G2/M phase. Show less

Title: Selectively attacking tumor cells of Ru/Ir-arene complexes based on meclofenamic acid Abstract: Breast cancer (BC) is one of the most common malignant tumors and often accompanied by inflammatory processes. Inflammation is an essential component of the tumor microenvironment, which might influence tumor proliferation and metastasis. Herein, three metal-arene complexes MA-bip-Ru, MA-bpy-Ir, and MA-bpy-Ru were prepared by tethering the non-steroidal anti-inflammatory drug meclofenamic acid (MA). Among them, MA-bip-Ru and MA-bpy-Ir showed lower cytotoxicity towards cancer cells, but MA-bpy-Ru showed significantly high selectivity and cytotoxicity towards MCF-7 cells through the autophagic pathway and exhibited no toxicity against normal HLF cells, showing potential for selective treatment of tumor cells. MA-bpy-Ru could also effectively destroy the 3D multicellular tumor spheroids, demonstrating its potential for clinical application. Besides, MA-bip-Ru, MA-bpy-Ir, and MA-bpy-Ru exhibited anti-inflammatory properties superior to MA, notably downregulating the expression of cyclooxygenase-2 (COX-2) and inhibiting the secretion of prostaglandin E2 in vitro. These findings demonstrated that MA-bpy-Ru was capable of intervening in inflammatory processes and showed the potential of MA-bpy-Ru to act as a selective anticancer agent, thus presenting a new mechanism of action for metal-arene complexes. Show less

Title: Iridium(III) complexes inhibit the proliferation and migration of BEL-7402 cells through the PI3K/AKT/mTOR signaling pathway. Abstract: Iridium(III) complexes are largely studied as anti-cancer complexes due to their excellent anti-cancer activity. In this article, two new iridium(III) complexes [Ir(piq)2(THPIP)]PF6 (THPIP = 2,4-di-tert-butyl-6-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenol, piq = deprotonated 1-phenylisoquinoline) (Ir1) and [Ir(bzq)2(THPIP)]PF6 (bzq = deprotonated benzo[h]quinolone) (Ir2) were synthesized. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that complex Ir1 exhibits moderate activity (IC50 = 29.9 ± 4.6 μM) and Ir2 shows high cytotoxicity (IC50 = 9.8 ± 1.8 μM) against BEL-7402 cells. Further studies on the mechanism showed that Ir1 and Ir2 induced apoptosis by changing the mitochondrial membrane potential, Ca2+ release, ROS accumulation, and cell cycle arrest at the S phase. The complexes can effectively inhibit cell colony formation and migration. The expression of B-cell lymphoma-2 (Bcl-2) family proteins, PI3K (phosphatidylinositol 3-kinase), AKT (protein kinase B), mTOR (mammalian target of rapamycin), and p-mTOR was studied by immunoblotting. Complexes Ir1 and Ir2 downregulated the expression of anti-apoptotic protein Bcl-2 and increased the expression of autophagy-related proteins of Beclin-1 and LC3-II. Further experiments showed that the complexes inhibited the production of glutathione (GSH) and increased the amounts of malondialdehyde (MDA). Fluorescence of HMGB1 was significantly increased. We also investigated the effect of the complexes on the expression of genes using RNA-sequence analysis, we further calculated the lowest binding energies between the complexes and proteins using molecular docking. Taken together, the above results indicated that complexes Ir1 and Ir2 induce apoptosis in BEL-7402 cells through a ROS-mediated mitochondrial dysfunction and inhibition of the PI3K/AKT/mTOR signaling pathway. Show less

Title: Rhodium(III)-Picolinamide Complexes Act as Anticancer and Antimetastasis Agents via Inducing Apoptosis and Autophagy. Abstract: As a continuation of our endeavors in discovering metal-based drugs with cytotoxic and antimetastatic activities, herein, we reported the syntheses of 11 new rhodium(III)-picolinamide complexes and the exploration of their potential anticancer activities. These Rh(III) complexes showed high antiproliferative activity against the tested cancer cell lines in vitro. The mechanism study indicated that Rh1 ([Rh(3a)(CH3CN)Cl2]) and Rh2 ([Rh(3b)(CH3CN)Cl2]) inhibited cell proliferation by multiple modes of action via cell cycle arrest, apoptosis, and autophagy and inhibited cell metastasis via FAK-regulated integrin β1-mediated suppression of EGFR expression. Furthermore, Rh1 and Rh2 significantly inhibited bladder cancer growth and breast cancer metastasis in a xenograft model. These rhodium(III) complexes could be developed as potential anticancer agents with antitumor growth and antimetastasis activity. Show less

In this work, we describe a novel ruthenium-xanthoxylin complex, [Ru(phen)₂(xant)](PF₆) (RXC), that can eliminate colorectal cancer (CRC) stem cells by targeting the chaperone Hsp90. RXC exhibits potent cytotoxicity in cancer cell lines and primary cancer cells, causing apoptosis in HCT116 CRC cells, as observed by cell morphology, YO-PRO-1/PI staining, internucleosomal DNA fragmentation, mitochondrial depolarization, and PARP cleavage (Asp214). Additionally, RXC can downregulate the HSP90AA1 and HSP90B1 genes and the expression of HSP90 protein, as well as the expression levels of its downstream/client elements Akt1, Akt (pS473), mTOR (pS2448), 4EBP1 (pT36/pT45), GSK-3β (pS9), and NF-κB p65 (pS529), implying that these molecular chaperones can be molecular targets for RXC. Moreover, this compound inhibited clonogenic survival, the percentage of the CRC stem cell subpopulation, and colonosphere formation, indicating that RXC can eliminate CRC stem cells. RXC reduced cell migration and invasion, decreased vimentin and increased E-cadherin expression, and induced an autophagic process that appeared to be cytoprotective, as autophagy inhibitors enhanced RXC-induced cell death. In vivo studies showed that RXC inhibits tumor progression and experimental metastasis in mice with CRC HCT116 cell xenografts. Taken together, these results highlight the potential of the ruthenium complex RXC in CRC therapy with the ability to eliminate CRC stem cells by targeting the chaperone Hsp90. Show less

[Ru(5-FU)(PPh₃)₂(bipy)]PF₆ (Ru/5-FU) is a novel ruthenium complex with 5-fluorouracil with promising potential against colorectal cancer (CRC). In the present study, we investigated the molecular mechanism of Ru/5-FU action in HCT116 CRC cells. Ru/5-FU exhibited potent cytotoxicity on a panel of cancer cell lines and on primary cancer cells and induced apoptosis in HCT116 CRC cells. Ru/5-FU reduced AKT1 gene transcripts, as well as the expression of Akt1 and Akt (pS473) and downstream Akt proteins mTOR (pS2448), S6 (pS235/pS236), 4EBP1 (pT36/pT45), GSK-3β (pS9) and NF-κB p65 (pS529), but not Akt upstream proteins Hsp90 and PI3K p85/p55 (pT458/pT199), indicating an inhibitory action of Akt/mTOR signaling. Ru/5-FU increased LC3B expression and reduced p62/SQSTM1 levels, indicating autophagy induction. Curiously, the autophagy inhibitors 3-methyladenine and chloroquine increased Ru/5-FU-induced cell death, indicating an induction of cytoprotective autophagy by this compound. Ru/5-FU also reduced clonogenic survival, as well as the percentage of CD133+ cells and colonosphere formation, indicating that Ru/5-FU can suppress stem cells in HCT116 cells. Ru/5-FU inhibited cell migration and invasion in wound healing assays and Transwell cell invasion assays, along with a reduction in vimentin expression and an increase in E-cadherin levels, indicating that Ru/5-FU can interfere with epithelial-mesenchymal transition. Ru/5-FU also inhibited in vivo HCT116 cell development and experimental lung metastases in mouse xenograft models. Altogether, these results indicate that Ru/5-FU is an anti-CRC chemotherapy drug candidate with the ability to suppress stemness in CRC cells by inhibiting Akt/mTOR signaling. Show less

Title: Mitochondria-targeted cyclometalated iridium (III) complexes: Dual induction of A549 cells apoptosis and autophagy. Abstract: In this study, we synthesized 4 cyclometalated iridium complexes using N-(1,10-phenanthrolin-5-yl)picolinamide (PPA) as the main ligand, denoted as [Ir(ppy)2PPA]PF6 (ppy = 2-phenylpyridine, Ir1), [Ir(bzq)2PPA]PF6 (bzq = benzo[h]quinoline, Ir2), [Ir(dfppy)2PPA]PF6 (dfppy = 2-(3,5-difluorophenyl)pyridine, Ir3), and [Ir(thpy)2PPA]PF6 (thpy = 2-(thiophene-2-yl)pyridine, Ir4). Compared to cisplatin and oxaliplatin, all four complexes exhibited significant anti-tumor activity. Among them, Ir2 demonstrated higher cytotoxicity against A549 cells, with an IC50 value of 1.6 ± 0.2 μM. The experimental results indicated that Ir2 primarily localized in the mitochondria, inducing a large amount of reactive oxygen species (ROS) generation, that decreased in mitochondrial membrane potential (MMP), reduced ATP production, and further impaired mitochondrial function, leading to cytochrome c release. Additionally, Ir2 caused cell cycle arrest at the S phase and induced apoptosis through the AKT-mediated signaling pathway. Further investigations revealed that Ir2 could simultaneously induce both apoptosis and autophagy in A549 cells, with the latter acting as a non-protective mechanism that promoted cell death. More importantly, Ir2 exhibited low toxicity to both normal LO2 cells in vitro and zebrafish embryos in vivo. Consequently, these newly developed Ir(III) complexes show great potential in the development of novel and low-toxicity anticancer agents. Show less

Title: Luminescent 11-{Naphthalen-1-yl}dipyrido[3,2-a:2',3'-c]phenazine-Based Ru(II)/Ir(III)/Re(I) Complexes for HCT-116 Colorectal Cancer Stem Cell Therapy. Abstract: Due to a number of unpleasant considerations, marketed drugs have steadily lost their importance in the treatment of cancer. In order to find a viable cancer cell diagnostic agent, we therefore focused on metal complexes that displayed target adequacy, permeability to cancer cells, high standard water solubility, cytoselectivity, and luminescent behavior. In this aspect, luminescent 11-{naphthalen-1-yl} dipyrido [3,2-a:2',3'-c] phenazine based Ru(II)/Ir(III)/Re(I) complexes have been prepared for HCT-116 colorectal cancer stem cell therapy. Our study successfully established the possible cytotoxicity of IrL complex at different doses on HCT-116 colorectal cancer stem cells (CRCSCs). Additionally, an immunochemistry analysis of the complex IrL showed that the molecule was subcellularly localized in the nucleus and other regions of the cytoplasm, where it caused nuclear DNA damage and mitochondrial dysfunction. The level of BAX and Bcl-2 was further quantified by qRT-PCR. The expression of proapoptotic BAX showed increased expression in the complex IrL-treated cell compared to the control, indicating the potential of complex IrL for apoptotic induction. Upon further validation, complex IrL was developed as an inhibitor of autophagy for the eradication of cancer stem cells. Show less

Title: Asymmetrically Coordinated Heterodimetallic Ir-Ru System: Synthesis, Computational, and Anticancer Aspects. Abstract: Herein, we present an unprecedented formation of a heterodinuclear complex [{(ppy)2IrIII}(μ-phpy){RuII(tpy)}](ClO4)2 {[1](ClO4)2} using terpyridyl/phenylpyridine as ancillary ligands and asymmetric phpy as a bridging ligand. The asymmetric binding mode (N∧N-∩-N∧N∧C-) of the phpy ligand in {[1](ClO4)2} is confirmed by 1H, 13C, 1H-1H correlated spectroscopy (COSY), high-resolution mass spectrum (HRMS), single-crystal X-ray crystallography techniques, and solution conductivity measurements. Theoretical investigation suggests that the highest occupied molecular orbital (HOMO) and the least unoccupied molecular orbital (LUMO) of [1]2+ are located on iridium/ppy and phpy, respectively. The complex displays a broad low energy charge transfer (CT) band within 450-575 nm. The time-dependent density functional theory (TDDFT) analysis suggests this as a mixture of metal-to-ligand charge transfer (MLCT) and ligand-to-ligand charge transfer (LLCT), where both ruthenium, iridium, and ligands are involved. Complex {[1](ClO4)2} exhibits RuIIIrIII/RuIIIIrIII- and RuIIIIrIII/RuIIIIrIV-based oxidative couples at 0.83 and 1.39 V, respectively. The complex shows anticancer activity and selectivity toward human breast cancer cells (IC50; MCF-7: 9.3 ± 1.2 μM, and MDA-MB-231: 8.6 ± 1.2 μM) over normal breast cells (MCF 10A: IC50 ≈ 21 ± 1.3 μM). The Western blot analysis and fluorescence microscopy images suggest that combined apoptosis and autophagy are responsible for cancer cell death. Show less

Title: Targeted liposomes encapsulated iridium(III) compound greatly enhance anticancer efficacy and induce cell death via ferroptosis on HepG2 cells. Abstract: In this study, ligands 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline (PIP), 2-(2-nitrophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline (NPIP), 2-(2-nitronaphthalen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (NNIP) and their iridium(III) metal compounds [Ir(ppy)2(PIP)](PF6) (ppy = 2-phenylpyridine, 1a), [Ir(ppy)2(NPIP)](PF6) (1b), [Ir(ppy)2(NNIP)](PF6) (1c) were designed and synthesized. The anti-cancer activities of 1a, 1b and 1c on BEL-7402, HepG2, SK-Hep1 and non-cancer LO2 were detected using MTT method. 1a shows moderate, 1b and 1c display low or no anti-cancer activities. To elevate the anti-cancer effectiveness, encapsulating the compounds 1a, 1b and 1c into the ordinary or targeted liposomes to produce 1alip, 1blip, 1clip, or targeted 1aTlip, 1bTlip and 1cTlip. The IC50 values of 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip against HepG2 cells are 7.9 ± 0.1, 8.6 ± 0.2, 16.9 ± 0.5, 5.9 ± 0.2, 7.3 ± 0.1 and 9.7 ± 0.7 μM, respectively. Specifically, the anti-tumor activity assays in vivo found that the inhibitory rates are 23.24 % for 1a, 61.27 % for 1alip, 76.06 % for 1aTlip. It is obvious that the targeted liposomes entrapped iridium(III) compound greatly enhance anti-cancer efficacy. Additionally, 1alip, 1blip and 1clip or targeted 1aTlip, 1bTlip and 1cTlip can effectively restrain the cell colony and proliferation in the G0/G1 period. 1alip, 1blip, 1clip, 1aTlip, 1bTlip and 1cTlip can increase reactive oxygen species (ROS) concentration, arouse a decline in the mitochondrial membrane potential and promote Ca2+ release. RNA-sequence was applied to examine the signaling pathways. Taken together, the liposomes or targeted liposomes encapsulated compounds trigger cell death by way of apoptosis, autophagy, ferroptosis, disruption of mitochondrial function and PI3K/AKT/mTOR signaling pathways. Show less

Title: Cyclometalated iridium(III) complexes as anti-breast cancer and anti-metastasis agents via STAT3 inhibition. Abstract: Breast cancer is the most commonly diagnosed cancer and second‑leading cause of cancer deaths in women. Signal transducer and activator of transcription 3 (STAT3) plays a critical role in promoting breast cancer cell proliferation, invasion, angiogenesis, and metastasis, and the high expression of STAT3 is related to the occurrence and poor chemotherapy sensitivity of breast cancer. Iridium(III) complexes Ir-PTS-1- 4 containing a pterostilbene-derived ligand were synthesized to inhibit the STAT3 pathway in breast cancer. Ir-PTS-4 inhibited the proliferation of breast cancer cells by suppressing the expression of phosphorylated STAT3 and STAT3-related cyclin D1, arresting cell cycle in the S-phase, inducing DNA damage and reactive oxygen species (ROS) generation, eventually leading to autophagic cell death. The cell metastasis and invasion were also inhibited after Ir-PTS-4 treatment. Besides, Ir-PTS-4 exhibited excellent anti-proliferation activity in 3D multicellular tumor spheroids, showing potential for the treatment of solid tumors. This work presents the rational design of metal-based anticancer agents to block the STAT3 pathway for simultaneously inhibiting breast cancer proliferation and metastasis. Show less

Three iridium (III) polypyridine complexes [Ir(bzq)₂(maip)](PF₆) (Ir1，bzq = benzo[h]quinoline, maip = 3-aminophenyl-1H-imidazo[4,5-f][1,10]phenanthroline), [Ir(bzq)₂(apip)](PF₆) (Ir2, apip = 2-aminophenyl-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(bzq)₂(paip)](PF₆) (Ir3, paip = 4-aminophenyl-1H-imidazo[4,5-f][1,10]phenanthroline) were synthesized and characterized. The cytotoxic activities of the three complexes against human osteosarcoma HOS, U2OS, MG63 and normal LO2 cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The results showed that Ir1-3 exhibited moderate antitumor activity against HOS with IC₅₀ of 21.8 ± 0. 4 μM,10.5 ± 1.8 μM and 7.4 ± 0.4 μM, respectively. We found that Ir1-3 can effectively inhibit HOS cells growth and blocked the cell cycle at the G0/G1 phase. Further studies revealed that complexes can increase intracellular reactive oxygen species (ROS) and Ca²⁺, which accompanied by mitochondria-mediated intrinsic apoptosis pathway. In addition, autophagy was also investigated. Taken together, the complexes induce HOS apoptosis through a ROS-mediated mitochondrial dysfunction pathway and inhibition of the PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B)/mTOR (mammalian target of rapamycin) signaling pathway. This study provides useful help for understanding the anticancer mechanism of iridium (III) complexes toward osteosarcoma treatment. Show less

Ruthenium(II) arene complexes exhibit promising chemotherapeutic properties. In this study, the effect of the counter anion in Ru(II) complexes was evaluated by analyzing the biological effect of two Ru(II) p-cymene derivatives with the 1,10-phenanthroline-5,6-dione ligand of general-formula [(η⁶-arene)Ru(L)Cl][X] X = CF₃SO₃ (JHOR10) and PF₆ (JHOR11). The biological activity of JHOR10 and JHOR11 was examined in the ovarian carcinoma cell line A2780, colorectal carcinoma cell line HCT116, doxorubicin-resistant HCT116 (HCT116-Dox) and in normal human dermal fibroblasts. Both complexes JHOR10 and JHOR11 displayed an antiproliferative effect on A2780 and HCT116 cell lines, and low cytotoxicity in fibroblasts. Interestingly, JHOR11 also showed antiproliferative activity in the HCT116-Dox cancer cell line, while JHOR10 was inactive. Studies in A2780 cells showed that JHOR11 induced the production of reactive oxygen species (ROS) that trigger autophagy and cellular senescence, but no apoptosis induction. Further analysis showed that JHOR11 presented no tumorigenicity, with no effect in the cellular mobility, as evaluated by thye wound scratch assay, and no anti- or pro-angiogenic effect, as evaluated by the ex-ovo chorioallantoic membrane (CAM) assay. Importantly, JHOR11 presented no toxicity in chicken and zebrafish embryos and reduced in vivo the proliferation of HCT116 injected into zebrafish embryos. These results show that these are suitable complexes for clinical applications with improved tumor cell cytotoxicity and low toxicity, and that counter-anion alteration might be a viable clinical strategy for improving chemotherapy outcomes in multidrug-resistant (MDR) tumors. Show less

In this paper, two new iridium (III) complexes, [Ir(ppy)2(ipbp)](PF6) (Ir1) (ppy = 2-phenylpyridine, ipbp = 3-(1H-imidazo[4,5-f][1,10]phenanthrolin-2yl)-4H-chromen-4-one) and [Ir(bzq)2(ipbp)](PF6) (Ir2) (bzq = benzo[h]quinolone), were synthesized and characterized. The cytotoxicity of the complexes against human colon cancer HCT116 and normal LO2 cells was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The complexes Ir1 and Ir2 show high cytotoxic efficacy toward HCT116 cells with a low IC50 value of 1.75 ± 0.10 and 6.12 ± 0.2 µM. Interestingly, Ir1 only kills cancer cells, not normal LO2 cells (IC50 > 200 µM). The inhibition of cell proliferation and migration were investigated by multiple tumor spheroid (3D) and wound healing experiments. The cellular uptake was explored under a fluorescence microscope. The intracellular reactive oxygen species (ROS), change of mitochondrial membrane potential, glutathione (GSH) and adenine nucleoside triphosphate (ATP) were studied. Apoptosis and cell cycle arrest were performed by flow cytometry. The results show that the complexes induce early apoptosis and inhibit the cell proliferation at the G0/G1 phase. Additionally, the apoptotic mechanism was researched by Western blot analysis. The results obtained demonstrate that the complexes cause apoptosis in HCT116 cells through ROS-mediated mitochondrial dysfunction and the inhibition of PI3K/AKT signaling pathways. Show less

Cellular energy metabolism is reprogrammed in cancer to fuel proliferation. In oncological therapy, treatment resistance remains an obstacle and is frequently linked to metabolic perturbations. Identifying metabolic changes as vulnerabilities opens up novel approaches for the prevention or targeting of acquired therapy resistance. Insights into metabolic alterations underlying ruthenium-based chemotherapy resistance remain widely elusive. In this study, colon cancer HCT116 and pancreatic cancer Capan-1 cells were selected for resistance against the clinically evaluated ruthenium complex sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (BOLD-100). Gene expression profiling identified transcriptional deregulation of carbohydrate metabolism as a response to BOLD-100 and in resistance against the drug. Mechanistically, acquired BOLD-100 resistance is linked to elevated glucose uptake and an increased lysosomal compartment, based on a defect in downstream autophagy execution. Congruently, metabolomics suggested stronger glycolytic activity, in agreement with the distinct hypersensitivity of BOLD-100-resistant cells to 2-deoxy-d-glucose (2-DG). In resistant cells, 2-DG induced stronger metabolic perturbations associated with ER stress induction and cytoplasmic lysosome deregulation. The combination with 2-DG enhanced BOLD-100 activity against HCT116 and Capan-1 cells and reverted acquired BOLD-100 resistance by synergistic cell death induction and autophagy disturbance. This newly identified enhanced glycolytic activity as a metabolic vulnerability in BOLD-100 resistance suggests the targeting of glycolysis as a promising strategy to support BOLD-100 anticancer activity. Show less

Title: Lysosome-targeted cyclometalated iridium(III) complexes: JMJD inhibition, dual induction of apoptosis, and autophagy. Abstract: A series of cyclometalated iridium(III) complexes with the formula [Ir(C^N)2 L](PF6) (C^N = 2-phenylpyridine (ppy, in Ir-1), 2-(2-thienyl)pyridine (thpy, in Ir-2), 2-(2,4-difluorophenyl)pyridine (dfppy, in Ir-3), L = 2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)quinolin-8-ol) were designed and synthesized, which utilize 8-hydroxyquinoline derivative as N^N ligands to chelate the cofactor Fe2+ of the Jumonji domain-containing protein (JMJD) histone demethylase. As expected, the results of UV/Vis titration analysis confirm the chelating capabilities of Ir-1-3 for Fe2+, and molecular docking studies also show that Ir-1-3 can interact with the active pocket of JMJD protein, and treatment of cells with Ir-1-3 results in significant upregulation of trimethylated histone 3 lysine 9 (H3K9Me3), indicating the inhibition of JMJD activity. Meanwhile, Ir-1-3 exhibit much higher cytotoxicity against the tested tumor cell lines compared with the clinical chemotherapeutic agent cisplatin. And Ir-1-3 can block the cell cycle at the G2/M phase and inhibit cell migration and colony formation. Further studies show that Ir-1-3 can specifically accumulate in lysosomes, damage the integrity of lysosomes, and induce apoptosis and autophagy. Reduction of mitochondrial membrane potential and elevation of reactive oxygen species also contribute to the antitumor effects of Ir-1-3. Finally, Ir-1 can inhibit tumor growth effectively in vivo and increase the expression of H3K9Me3 in tumor tissues. Our study demonstrates that these iridium(III) complexes are promising anticancer agents with multiple functions, including the inhibition of JMJD and induction of apoptosis and autophagy. Show less

Phase separation of DNA is involved in chromatin packing for the regulation of gene transcription. Visualization and manipulation of DNA phase separation in living cells present great challenges. Herein, we present a Ru(II) complex (Ru1) with high DNA binding affinity and DNA "light-switch" behavior that can induce and monitor DNA phase separation both in vitro and in living cells. Molecular dynamics simulations indicate that the two phen-PPh₃ ligands with positively charged lipophilic triphenylphosphine substituents and flexible long alkyl chains in Ru1 play essential roles in the formation of multivalent binding forces between DNA molecules to induce DNA phase separation. Importantly, the unique environmental sensitive emission property of Ru1 enables direct visualization of the dynamic process of DNA phase separation in living cells by two-photon phosphorescent lifetime imaging. Moreover, Ru1 can change the gene expression pattern by modulating chromatin accessibility as demonstrated by integrating RNA-sequencing and transposase-accessible chromatin with high-throughput sequencing. In all, we present here the first small-molecule-based tracer and modulator of DNA phase separation in living cells and elucidate its impact on the chromatin state and transcriptome. Show less

Ruthenium(II) complexes are currently considered attractive alternatives to the widely used platinum-based drugs. We present herein the synthesis and characterization of half-sandwich ruthenium compounds formulated as [Ru(p-cymene)(L)Cl][CF₃SO₃] (L = 1,1-bis(methylenediphenylphosphano)ethylene, 1; L = 1,1-bis(diphenylphosphano)ethylene, 2), which were characterized by elemental analysis, mass spectrometry, ¹H and ³¹P{¹H} NMR, UV-vis and IR spectroscopy, conductivity measurements and cyclic voltammetry. The molecular structures for both complexes were determined by single-crystal X-ray diffraction. Their cytotoxic activity was evaluated using the MTT assay against human tumor cells, namely ovarian (A2780) and breast (MCF7 and MDA-MB-231). Both complexes were active against breast adenocarcinoma cells, with complex 1 exhibiting a quite remarkable cytotoxicity in the submicromolar range. Interestingly, at concentrations equivalent to the IC₅₀ values in the MCF7 cancer cells, complexes 1 and 2 presented lower cytotoxicity in normal human primary fibroblasts. The antiproliferative effects of 1 and 2 in MCF7 cells might be associated with the induction of reactive oxygen species (ROS), leading to a combined cell death mechanism via apoptosis and autophagy. Despite the fact that in vitro a partial intercalation between complexes and DNA was observed, no MCF7 cell cycle delay or arrest was observed, indicating that DNA might not be a direct target. Complexes 1 and 2 both exhibited a moderate to strong interaction with human serum albumin, suggesting that protein targets may be involved in their mode of action. Their acute toxicity was evaluated in the zebrafish model. Complex 1 (the most toxic of the two) exhibited a lethal toxicity LC₅₀ value about 1 order of magnitude higher than any IC₅₀ concentrations found for the cancer cell models used, highlighting its therapeutic relevance as a drug candidate in cancer chemotherapy. Show less