Antiproliferative Activity of Gold(I) N-Heterocyclic Carbene and Triphenylphosphine Complexes with Ibuprofen Derivatives as Effective Enzyme Inhibitors

A series of gold(I) complexes of ligand ibuprofen-alkynyl (but-3-yn-1-yl 2-(4-isobutylphenyl)propanoate, LE) with N-heterocyclic carbene (LC: 1,3-dimethylimidazol-2-ylidene) and triphenylphosphine (PPh₃) ligands with formula (LE)Au (LC) (complex 1 ) and (LE)Au (PPh₃) (complex 2 ) were synthesized and fully characterized by spectroscopic methods. In order to reveal the cytotoxicity mechanism, the interaction of complex 1 or 2 with cysteine (HCys) has been studied by experimental and density functional theory (DFT) methods. The compounds were investigated for their anticancer activity against MCF-7, MDAMB 231 breast cancer cells, HT-29 colon cancer cells and MCF-10A non-tumor breast cell line. The results were compared with cisplatin and auranofin as reference drugs. The complex 2 showed more cytotoxic activity than complex 1 . The complex 2 was 4.2, 3.7, and 1.7 fold more active than cisplatin against HT-29, MDA-MB-231, MCF-7 cancer cell lines, respectively. The inhibition of thioredoxin reductase of complexes 1 and 2 including cytosolic (TrxR1) and mitochondrial (TrxR2) thioredoxin reductase and also the inhibition of glutathione reductase (GR) were studied in detail. Moreover, the cellular uptake and reactive oxygen species (ROS) generation of compounds were investigated. Based on the DFT calculations a relationship between the σ-donor ability of the isolated ligands and cytotoxicity is suggested.     

A Gold(I) Complex Containing an Oleanolic Acid Derivative as a Potential Anti-Ovarian-Cancer Agent by Inhibiting TrxR and Activating ROS-Mediated ERS

Many cancer cells critically rely on antioxidant systems for cell survival and are vulnerable to further oxidative impairment triggered by agents generating reactive oxygen species (ROS). Therefore, the classical design and development of inhibitors that target antioxidant defense enzymes such as thioredoxin reductase (TrxR) can be a promising anticancer strategy. Herein, it is shown that a gold(I) complex containing an oleanolic acid derivative ( 4 b ) induces apoptosis of ovarian cancer A2780 cells by activating endoplasmic reticulum stress (ERS). It can inhibit TrxR enzyme activity to elevate ROS, mediate ERS and mitochondrial dysfunction, and finally leads to cell cycle arrest and apoptosis of A2780 cells. Notably, this complex inhibits A2780 xenograft tumor growth accompanied by increased ERS level and decreased TrxR activity in tumor tissues.     

Selective Activation of a Prodrug by Thioredoxin Reductase Providing a Strategy to Target Cancer Cells

Elevated reactive oxygen species and antioxidant defense systems have been recognized as one of the hallmarks of cancer cells. As a major regulator of the cellular redox homeostasis, the selenoprotein thioredoxin reductase (TrxR) is increasingly considered as a promising target for anticancer drug development. The current approach to inhibit TrxR predominantly relies on the modification of the selenocysteine residue in the C‐terminal active site of the enzyme, in which it is hard to avoid the off‐target effects. By conjugating the anticancer drug gemcitabine with a 1,2‐dithiolane scaffold, an unprecedented prodrug strategy is disclosed that achieves a specific release of gemcitabine by TrxR in cells. As overexpression of TrxR is frequently found in different types of tumors, the TrxR‐dependent prodrugs are promising for further development as cancer chemotherapeutic agents.     

Unveiling the Potential of Innovative Gold(I) and Silver(I) Selenourea Complexes as Anticancer Agents Targeting TrxR and Cellular Redox Homeostasis

A series of NHC-based selenourea Ag(I) and Au(I) complexes were evaluated for their anticancer potential in vitro, on 2D and 3D human cancer cell systems. All NHC-based selenourea complexes possess an outstanding cytotoxic potency, which was comparable or even better than that of the reference metallodrug auranofin, and were also able to overcome both platinum-based and multi-drug resistances. Intriguingly, their cytotoxic potency did not correlate with solution stability, partition coefficient or cellular uptake. On the other hand, mechanistic studies in cancer cells revealed their ability to strongly and selectively inhibit the redox-regulating enzyme Thioredoxin Reductase (TrxR), being even more effective than auranofin, a well-known TrxR inhibitor, without affecting other redox enzymes such as Glutathione Reductase (GR). The inhibition of TrxR in H157 human cancer cells caused, in turn, the disruption of cellular thiol-redox homeostasis and of mitochondria pathophysiology, ultimately leading to cancer cell death through apoptosis.     

A Multitarget Gold(I) Complex Induces Cytotoxicity Related to Aneuploidy in HCT‐116 Colorectal Carcinoma Cells

A novel alkynyl phosphane gold(I) complex (trimethylphosphane)(3‐(1,3‐dimethylxanthine‐7‐yl)prop‐1‐yn‐1‐yl)gold(I) 1 displayed mutiple biological activites including selective proliferation inhibitory, anti‐metastatic, and anti‐angiogenic effects. The complex also induced effects related to aneuploidy in HCT‐116 colon carcinoma cells, which might be mainly ascribed to the dysfunction of mitochondrial bioenergetics and downregulation of glycolysis. Induction of aneuploidy beyond a critical level can provide an effective strategy to target cancer, in particular colorectal tumours with a low tolerance of aneuploidy, and could be of relevance for 1 and other metallodrugs.     

Mitochondria-targeted chemotherapeutics: the rational design of gold(I) N-heterocyclic carbene complexes that are selectively toxic to cancer cells and target protein selenols in preference to thiols

A family of lipophilic, cationic Au(I) complexes of N-heterocyclic carbenes (NHCs) have been designed as new mitochondria-targeted antitumor agents that combine both selective mitochondrial accumulation and selective thioredoxin reductase inhibition properties within a single molecule. Two-step ligand exchange reactions with cysteine (Cys) and selenocysteine (Sec) occur with release of the NHC ligands. At physiological pH the rate constants for the reactions with Sec are 20- to 80-fold higher than those with Cys. The complexes are selectively toxic to two highly tumorigenic breast cancer cell lines and not to normal breast cells, and the degree of selectivity and potency are optimized by modification of the substituent on the simple imidazolium salt precursor. The lead compound is shown to accumulate in mitochondria of cancer cells, to cause cell death through a mitochondrial apoptotic pathway and to inhibit the activity of thioredoxin reductase (TrxR) but not the closely related and Se-free enzyme glutathione reductase.     

Redirecting Cisplatin and Doxorubicin to Mitochondria Affords Highly Effective PlatinumIV Prodrug Against Triple Negative Breast Cancer

A mitochondria targeting dual-action platinum^IV prodrug exhibits high anticancer activity in triple negative breast cancer cells. The complex intervenes in several cellular processes including DNA damage, perturbation of mitochondrial bioenergetics and induction of necrosis to kill cancer cells.     

Thioredoxin Reductase Activity may be More Important than GSH Level in Protecting Human Lens Epithelial Cells against UVA Light

This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L‐buthionine‐(S,R)‐sulfoximine (BSO) or 1‐chloro‐2,4‐dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O₂, 3% and 20%, were employed during a 1 h exposure of the cells to 25 J cm^?2 of UVA radiation (338–400 nm wavelength, peak at 365 nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O₂ compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA‐induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA‐induced cell damage.     

A Chemical–Biological Evaluation of Rhodium(I) N‐Heterocyclic Carbene Complexes as Prospective Anticancer Drugs

Rhodium(I) complexes bearing N‐heterocyclic carbene (NHC) ligands have been widely used in catalytic chemistry, but there are very few reports of biological properties of these organometallics. A series of Rh^I‐NHC derivatives with 1,5‐cyclooctadiene and CO as secondary ligands were synthesized, characterized, and biologically investigated as prospective antitumor drug candidates. Pronounced antiproliferative effects were noted for all complexes, along with moderate inhibitory activity of thioredoxin reductase (TrxR) and efficient binding to biomolecules (DNA, albumin). Biodistribution studies showed that the presence of albumin lowered the cellular uptake and confirmed the transport of rhodium into the nuclei. Changes in the mitochondrial membrane potential (MMP) were observed as well as DNA fragmentation in wild‐type and daunorubicin‐ or vincristine‐resistant Nalm‐6 leukemia cells. Overall, these studies indicated that Rh^I‐NHC fragments could be used as partial structures of new antitumor agents, in particular in those drugs designed to address resistant malignant tissues.     

Inhibition of thioredoxin and thioredoxin reductase by 4-hydroxy-2-nonenal in vitro and in vivo

Lipid peroxidation is a cellular process that takes place under physiological conditions and particularly after oxidative stress. 4-Hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, is known to exert a multitude of biological effects and has high reactivity to various cellular components, including DNA and protein. The thioredoxin system, composed of the selenoenzyme thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, plays a key role in redox regulation and is involved in many signaling pathways. The selenocysteine (Sec) and cysteine (Cys) residues (Cys-496/Sec-497) in the active site of TrxR and a pair of Cys residues (Cys-32/Cys-35) in Trx are sensitive to various alkylating reagents. Herein, we report a mechanistic study on the inhibition of rat TrxR by HNE. The inhibition occurs with TrxR only in its reduced form and persists after removal of HNE. Inhibition of TrxR by HNE added to cultured HeLa cells is also observed. In addition, HNE inactivates reduced Escherichia coli Trx irreversibly. We proved that the redox residues (Cys-496/Sec-497 in TrxR and Cys-32/Cys-35 in Trx) were primary targets for HNE modification. The covalent adducts formed between HNE and Trx were also confirmed by mass spectrum. Because the thioredoxin system is one of the core regulation enzymes of cells' function, inhibition of both TrxR and Trx by HNE provides a possibly novel mechanism for explanation of its cytotoxic effect and signaling activity, as well as the further damage indirectly caused under oxidative stress conditions.     

Combinatorial Therapeutic Effect of Inhibitors of Aldehyde Dehydrogenase and Mitochondrial Complex I,and the Chemotherapeutic Drug,Temozolomide against Glioblastoma Tumorspheres

Resident cancer cells with stem cell-like features induce drug tolerance, facilitating survival of glioblastoma (GBM). We previously showed that strategies targeting tumor bioenergetics present a novel emerging avenue for treatment of GBM. The objective of this study was to enhance the therapeutic effects of dual inhibition of tumor bioenergetics by combination of gossypol, an aldehyde dehydrogenase inhibitor, and phenformin, a biguanide compound that depletes oxidative phosphorylation, with the chemotherapeutic drug, temozolomide (TMZ), to block proliferation, stemness, and invasiveness of GBM tumorspheres (TSs). Combination therapy with gossypol, phenformin, and TMZ induced a significant reduction in ATP levels, cell viability, stemness, and invasiveness compared to TMZ monotherapy and dual therapy with gossypol and phenformin. Analysis of differentially expressed genes revealed up-regulation of genes involved in programmed cell death, autophagy, and protein metabolism and down-regulation of those associated with cell metabolism, cycle, and adhesion. Combination of TMZ with dual inhibitors of tumor bioenergetics may, therefore, present an effective strategy against GBM by enhancing therapeutic effects through multiple mechanisms of action.     

Cyclometalated Ruthenium(II) Anthraquinone Complexes Exhibit Strong Anticancer Activity in Hypoxic Tumor Cells

Hypoxia is the critical feature of the tumor microenvironment that is known to lead to resistance to many chemotherapeutic drugs. Six novel ruthenium(II) anthraquinone complexes were designed and synthesized; they exhibit similar or superior cytotoxicity compared to cisplatin in hypoxic HeLa, A549, and multidrug‐resistant (A549R) tumor cell lines. Their anticancer activities are related to their lipophilicity and cellular uptake; therefore, these physicochemical properties of the complexes can be changed by modifying the ligands to obtain better anticancer candidates. Complex 1 , the most potent member of the series, is highly active against hypoxic HeLa cancer cells (IC₅₀=0.53 μM ). This complex likely has 46‐fold better activity than cisplatin (IC₅₀=24.62 μM ) in HeLa cells. This complex tends to accumulate in the mitochondria and the nucleus of hypoxic HeLa cells. Further mechanistic studies show that complex 1 induced cell apoptosis during hypoxia through multiple pathways, including those of DNA damage, mitochondrial dysfunction, and the inhibition of DNA replication and HIF‐1α expression, making it an outstanding candidate for further in vivo studies.     

Fluorescent Coumarin–Artemisinin Conjugates as Mitochondria‐Targeting Theranostic Probes for Enhanced Anticancer Activities

Mitochondria‐targeting theranostic probes that enable the simultaneously reporting of and triggering of mitochondrial dysfunctions in cancer cells are highly attractive for cancer diagnosis and therapy. Three fluorescent mitochondria‐targeting theranostic probes have been developed by linking a mitochondrial dye, coumarin‐3‐carboximide, with a widely used traditional Chinese medicine, artemisinin, to kill cancer cells. Fluorescence images showed that the designed coumarin–artemisinin conjugates localized mainly in mitochondria, leading to enhanced anticancer activities over artemisinin. High cytotoxicity against cancer cells correlated with the strong ability to accumulate in mitochondria, which could efficiently increase the intracellular reactive oxygen species level and induce cell apoptosis. This study highlights the potential of using mitochondria‐targeting fluorophores to selectively trigger and directly visualize subcellular drug delivery in living cells.     

Broken electron transfer pathway in enzyme: Gold clusters inhibiting TrxR1/Trx via cell studies and theory simulations

Thioredoxin reductase 1 (TrxR1) is over activity in tumor cell to maintain their redox balance. Although gold clusters have great potential in antitumor drug as they could well inhibit TrxR1, the molecular mechanism has not been disclosed yet. In this work, we revealed gold clusters can well inhibit the activity of TrxR1 in lung tumor cells and further disclosed the inhibition mechanism by using computational simulation methods. We firstly inferred the binding sites of gold in the hydrophobic cavities on TrxR1. The simulation results show that the gold ion (released from Au cluster) interact with –SH of Cys189 in TrxR1, this greatly increase the distance between the C-terminal redox center of TrxR1 and the Trx redox center, thereby destroy the electron transfer pathway between them. Our electron transfer destroying mechanism is different from the previous hypothesis that gold binds to the Sec498 of TrxR1 which has never been proved by experimental and theory studies. This work provides a new understanding of the gold clusters to inhibit TrxR1 activity.     

Imine‐N‐Heterocyclic Carbenes as Versatile Ligands in Ruthenium(II) p‐Cymene Anticancer Complexes: A Structure–Activity Relationship Study

A family of novel imine‐N‐heterocyclic carbene ruthenium(II) complexes of the general formula [(η⁶‐p‐cymene)Ru(C^N)Cl]PF₆⁻ (where C^N is an imine‐N‐heterocyclic carbene chelating ligand with varying substituents) have been prepared and characterized. In this imine‐N‐heterocyclic carbene chelating ligand framework, there are three potential sites that can be modified, which distinguishes this class of ligand and provides a body of flexibilities and opportunities to tune the cytotoxicity of these ruthenium(II) complexes. The influence of substituent effects of three tunable domains on the anticancer activity and catalytic ability in converting coenzyme NADH to NAD⁺ is investigated. This family of complexes displays an exceedingly distinct anticancer activity against A549 cancer cells, despite their close structural similarity. Complex 9 shows the highest anticancer activity in this series against A549 cancer cells (IC₅₀=14.36 μm ), with an approximately 1.5‐fold better activity than the clinical platinum drug cisplatin (IC₅₀=21.30 μm ) in A549 cancer cells. Mechanistic studies reveal that complex 9 mediates cell death mainly through cell stress, including cell cycle arrest, inducing apoptosis, increasing intracellular reactive oxygen species (ROS) levels, and depolarization of the mitochondrial membrane potential (MMP). Furthermore, lysosomal damage is also detected by confocal microscopy.     

Mechanism‐Guided Design and Synthesis of a Mitochondria‐Targeting Artemisinin Analogue with Enhanced Anticancer Activity

Understanding the mechanism of action (MOA) of bioactive natural products will guide endeavor to improve their cellular activities. Artemisinin and its derivatives inhibit cancer cell proliferation, yet with much lower efficiencies than their roles in killing malaria parasites. To improve their efficacies on cancer cells, we studied the MOA of artemisinin using chemical proteomics and found that free heme could directly activate artemisinin. We then designed and synthesized a derivative, ART‐TPP, which is capable of targeting the drug to mitochondria where free heme is synthesized. Remarkably, ART‐TPP exerted more potent inhibition than its parent compound to cancer cells. A clickable probe ART‐TPP‐Alk was also employed to confirm that the attachment of the TPP group could label more mitochondrial proteins than that for the ART derivative without TPP (AP1). This work shows the importance of MOA study, which enables us to optimize the design of natural drug analogues to improve their biological activities.     

Mitochondria‐Targeted Cancer Therapy Using a Light‐Up Probe with Aggregation‐Induced‐Emission Characteristics

Subcellular organelle‐specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria‐targeting probe (AIE‐mito‐TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation‐induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE‐mito‐TPP probe can selectively accumulate in cancer‐cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE‐mito‐TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light‐up probe provides a unique strategy for potential image‐guided therapy of cancer cells.     

Necroptosis Induced by Ruthenium(II) Complexes as Dual Catalytic Inhibitors of Topoisomerase I/II

Inducing necroptosis in cancer cells is an effective approach to circumvent drug‐resistance. Metal‐based triggers have, however, rarely been reported. Ruthenium(II) complexes containing 1,1‐(pyrazin‐2‐yl)pyreno[4,5‐e][1,2,4]triazine were developed with a series of different ancillary ligands ( Ru1 ‐ 7 ). The combination of the main ligand with bipyridyl and phenylpyridyl ligands endows Ru7 with superior nucleus‐targeting properties. As a rare dual catalytic inhibitor, Ru7 effectively inhibits the endogenous activities of topoisomerase (topo) I and II and kills cancer cells by necroptosis. The cell signaling pathway from topo inhibition to necroptosis was elucidated. Furthermore, Ru7 displays significant antitumor activity against drug‐resistant cancer cells in vivo. To the best of our knowledge, Ru7 is the first Ru‐based necroptosis‐inducing chemotherapeutic agent.