Cyclometalated Ru(II)-isoquinoline complexes overcome cisplatin resistance of A549/DDP cells by downregulation of Nrf2 via Akt/GSK-3β/Fyn pathway.

{"full_text": " Bioorganic Chemistry 119 (2022) 105516\n\n\n Contents lists available at ScienceDirect\n\n\n Bioorganic Chemistry\n journal homepage: www.elsevier.com/locate/bioorg\n\n\n\n\nCyclometalated Ru(II)-isoquinoline complexes overcome cisplatin\nresistance of A549/DDP cells by downregulation of Nrf2 via Akt/GSK-3\u03b2/\nFyn pathway\nLanmei Chen a, b, 1, Jie Wang a, 1, Xianhong Cai a, Suxiang Chen c, Jingjing Zhang d, e, Baojun Li a,\nWeigang Chen a, Xinhua Guo a, Hui Luo a, b, e, *, Jincan Chen a, b, e, *\na\n Guangdong Key Laboratory for Research and Development of Natural Drugs, The Marine Biomedical Research Institute, School of Pharmacy, Guangdong Medical\nUniversity, Zhanjiang 524023, China\nb\n Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang), Zhanjiang, Guangdong 524023, China\nc\n Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, Western Australia 6150, Australia\nd\n Affiliated Hospital of Guangdong Medical University & Key Laboratory of Zebrafish Model for Development and Disease of Guangdong Medical University, Zhanjiang\n524001, China\ne\n The Marine Biomedical Research Institute of Guangdong Zhanjiang, Zhanjiang, Guangdong 524023, China\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: Both ruthenium (Ru) and isoquinoline (IQ) compounds are regarded as potential anticancer drug candidates.\nCyclometalated Ru(II)-isoquinoline complexes Here, we report the synthesis and characterization of three novel cyclometalated Ru(II)-isoquinoline complexes:\nNrf2 RuIQ-3, RuIQ-4, and RuIQ-5, and evaluation of their in vitro cytotoxicities against a panel of cell lines including\nCisplatin resistance\n A549/DDP, a cisplatin-resistant human lung cancer cell line. A549/DDP 3D multicellular tumor spheroids\nAkt/GSK-3\u03b2/Fyn\n (MCTSs) were also used to detect the drug resistance reversal effect of Ru(II)-IQ complexes. Our results indicated\n that the cytotoxic activities against cancer cells of Ru(II)-IQ complexes, especially RuIQ-5, were superior\n compared with cisplatin. In addition, RuIQ-5 exhibited low toxicity towards both normal HBE cells in vitro and\n zebrafish embryos in vivo. Further investigation on cellular mechanism of action indicated that after absorption\n by A549/DDP cells, RuIQ-5 was mainly distributed in the nucleus, which is different from cisplatin. Besides,\n RuIQ-5 could induce apoptosis through mitochondrial dysfunction, reactive oxygen species (ROS) accumulation,\n ROS-mediated DNA damage, and cycle arrest at both S and G2/M phases. Moreover, RuIQ-5 could inhibit the\n overexpression of Nrf2 through regulation of Akt/GSK-3\u03b2/Fyn signaling pathway and hindering the nuclear\n translocation of Nrf2. Based on these findings, we firmly believe that the studied Ru(II)-IQ complexes hold great\n promise as anticancer therapeutics with high effectiveness and low toxicity.\n\n\n\n\n1. Introduction Therefore, the clinical application of cisplatin has been greatly limited\n [2,3]. Cisplatin resistance in lung cancer is closely associated with\n Lung cancer is the most frequent cause of cancer-related deaths aberrant activation of the nuclear factor erythroid 2-related factor 2\nworldwide. Non-small cell lung cancer (NSCLC) is a highly metastatic (Nrf2) [4].\nand aggressive subtype, accounting for about 80% of all lung cancer Nrf2 is a nuclear factor that coordinates the expression and induction\ncases [1]. Chemotherapy is an important part of comprehensive treat\u00ad of a battery of cytoprotective proteins-encoding genes [5]. Under\nment for lung cancer patients. Cisplatin (DDP) is one of the common normal conditions, Nrf2 acts as a transcription factor that plays a key\nchemotherapeutic agents for the treatment of NSCLC, however, the ef\u00ad role in the regulation of normal cells in generating protective responses\nficacy and the spectrum of anti-tumor activities of cisplatin is signifi\u00ad and resisting adverse effects of external stress stimuli. However, upon\ncantly compromised due to drug resistance and induced side effects. stimulation by internal and external free radicals or chemicals, Nrf2 is\n\n\n * Corresponding authors at: Guangdong Key Laboratory for Research and Development of Natural Drugs, The Marine Biomedical Research Institute, School of\nPharmacy, Guangdong Medical University, Zhanjiang 524023, China.\n E-mail addresses: luohui@gdmu.edu.cn (H. Luo), jincanchen@126.com (J. Chen).\n 1\n L. Chen and J. Wang contribute equally to this work.\n\nhttps://doi.org/10.1016/j.bioorg.2021.105516\nReceived 7 June 2021; Received in revised form 18 November 2021; Accepted 21 November 2021\nAvailable online 25 November 2021\n0045-2068/\u00a9 2021 Elsevier Inc. All rights reserved.\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\noverexpressed, leading to enhanced expression of protective genes IQ)]+ (bpy = 2,2\u2032 -bipyridine, RuIQ-3), [Ru(bpy)2(1-(4-F-Ph)-7-OCH3-\nincluding those encoding phase II detoxification enzymes, antioxidant IQ)]+ (RuIQ-4) and [Ru(bpy)2(1-(4-F-Ph)-6,7-(OCH3)2-IQ)]+ (RuIQ-5)\nenzymes, and multidrug resistance protein (MRP) in response to stimuli, (see Fig. 1), and evaluated their anticancer activity on different human\nand thus promoting tumor drug resistance [6,7]. Therefore, Nrf2 has carcinoma cell lines. RuIQ-5 was then identified as the best performing\nbecome an attractive therapeutic target for cisplatin resistance. Recent complex among them. Further examination of IC50 value for RuIQ3-\nstudies revealed that the function of Nrf2 can be regulated by the Akt/ RuIQ-5 in 3D multicellular tumor spheroids (MCTSs) confirmed its su\u00ad\nGSK-3\u03b2/Fyn pathway via controlling Fyn-mediated export and degra\u00ad perior anticancer efficacy in vitro. It is worth mentioning that RuIQ-5\ndation of nuclear Nrf2 [8\u201310]. Inhibition of the Nrf2 signaling pathway displayed selective cytotoxicity to cancer cells that it exhibited only low\ncan reverse the drug resistance of lung cancer cells, thus enhancing the toxicity on both normal HBE cells in vitro and zebrafish embryos in vivo.\nefficacy of chemotherapy [11\u201314]. Recent studies also demonstrated Subsequent investigation on the mechanism of tumor inhibition effect\nthat inhibition of the Nrf2 pathway could block drug resistance in both showed that RuIQ-5 could induce ROS-mediated apoptosis of A549/\nNSCLC cells and mice bearing NSCLC xenografts [15,16]. DDP cells through Nrf2 inhibition. The study also revealed that RuIQ-5\n Isoquinoline (IQ) and its derivatives are among the alkaloids with could trigger mitochondrial dysfunction and DNA damage, while it\nnitrogen heterocyclic structure which exist widely in nature and possess inhibited protein kinase B (PKB or Akt) and Glycogen synthase kinase\na variety of physiological activities, such as anticancer, analgesic, and (GSK-3\u03b2) signaling pathway. Taken together, our results indicated that\nanti-inflammatory [17]. In terms of anticancer activity, isoquinoline Ru(II)-isoquinoline complexes tested in this work could be developed as\nalkaloids can inhibit the proliferation, migration and invasion of tumor Nrf2 inhibitors that reverse the drug resistance of A549/DDP cells,\nby many ways, such as cell cycle arrest, induction of apoptosis, and demonstrating the promise and potential of Ru(II)-isoquinoline com\u00ad\ninhibition of the activities of cyclooxygenase-2 (COX-2), telomerase, and plexes as effective and safe NSCLC therapeutics.\ntopoisomerase [18]. In recent years, many studies have shown that\nisoquinoline alkaloids can reverse multidrug resistance (MDR) of tumor 2. Results and discussion\ncells. For instance, Li et al. reported that isoquinoline derivative HZ08\ncan block Adriamycin (ADM) resistance of MCF-7/ADM cells through 2.1. Synthesis and characterization\ninhibition of drug efflux protein, P-glycoprotein (P-gp) [19,20]. Lei et al.\nreported that isoquinoline alkaloids extracted from Rhizoma Corydalis The synthetic routes of precursor cis-[Ru(bpy)2Cl2] (L = bpy, phen),\ncan inhibit the transcription of multidrug resistance gene 1 (MDR1) and ligands 1-(4-F-Ph)-IQ, 1-(4-F-Ph)-7-OCH3-IQ, and 1-(4-F-Ph)-6,7-\nmultidrug resistance associated protein 1 (MRP1), and thus reduce the (OCH3)2-IQ, and the cyclometalated Ru(II)-IQ complexes RuIQ-3,\nproduction of P-gp (coded by MDR1) and MRP1 (coded by MRP1), RuIQ-4, and RuIQ-5 are depicted in Scheme 1. Firstly, the ligands 1-(4-\nleading to reversal of the drug resistance of MCF-7/ADR [21]. Zeng et al. F-Ph)-IQ, 1-(4-F-Ph)-7-OCH3-IQ, and 1-(4-F-Ph)-6,7-(OCH3)2-IQ were\nsummarized twenty-six types of isoquinoline alkaloids and found that synthesized according to the previously reported method [36]. Sec\u00ad\nsome of the alkaloids displayed more potent anti-MDR effects than the ondly, the precursor cis-[Ru(bpy)2Cl2]\u22c52H2O was obtained through\npositive control, against the tested cancer cells [22]. In addition, Liang reflowing RuCl3\u22c5nH2O,LiCl and bpy in N,N-Dimethylformamide (DMF)\net al. showed that the antitumor activities of the metal (Zn2+, Ni2+, solution at 140 \u25e6 C overnight [37,38]. Finally, complexes RuIQ-3, RuIQ-\nCu2+)-isoquinoline derivatives coordination complexes are significantly 4, and RuIQ-5 were obtained by reacting cis-[Ru(bpy)2Cl2] with 1-(4-F-\nhigher than that of the isoquinoline ligands alone, indicating that iso\u00ad Ph)-IQ, 1-(4-F-Ph)-7-OCH3-IQ, or 1-(4-F-Ph)-6,7-(OCH3)2-IQ in ethyl\nquinoline derivatives are ideal metal ligands [23]. alcohol at 80 \u25e6 C overnight under the protection of argon. Then, com\u00ad\n Ruthenium (Ru) complexes have attracted a great deal of interest as plexes RuIQ-3, RuIQ-4, and RuIQ-5 were characterized by electrospray\nanticancer drug candidates because of their low toxicity, diverse ionization mass spectrometry (ESI-MS, Fig. S1\u2013S3 in supplementary\nmechanisms of action, and non-cross resistance in contrast to the con\u00ad materials), and proton nuclear magnetic resonance (1H NMR and 13C\nventional platinum (Pt)-based agents. In recent decades, an increasing NMR, Fig. S4\u2013S9 in supplementary materials). The counter anion of all\nnumber of reports have supported that Ru complexes have emerged as these complexes is PF6-.\nthe most outstanding candidates for cancer treatment [24]. Ru com\u00ad\npounds have shown selective bioactivity and the capability of over\u00ad 2.2. Stability and UV\u2013visible spectral studies\ncoming the resistance that Pt-based therapeutics face, making them\ncompetitive oncotherapeutic candidates in rational drug design ap\u00ad Stability of antineoplastic drugs is essential to ensure their efficacy\nproaches [25]. So far, four Ru complexes including imidazolium(imid\u00ad and safety. The stability of the synthesized cyclometalated Ru(II)-IQ\nazole)-(dimethylsulfoxide) tetrachlororuthenate(III) (NAMI-A) [26,27], complexes in aqueous solution was tested by UV\u2013vis spectroscopy. As\nindazolium trans-tetrachlorobis(1H-indazole) ruthenium(III)] (KP1019) shown in Fig. S10-S12 in supplementary materials, the Ru(II)-IQ com\u00ad\n[28,29], KP133929 [30] and Ru(II)-based therapeutics TLD1433 [31] plexes were basically stable in phosphate buffer saline (PBS) within 48 h\nhave entered clinical trials. In addition, Ru(II) compound DW1/2, a that the UV absorption peak showed no obvious change. The UV\u2013visible\nprotein kinase inhibitor, is currently in preclinical development stage spectral characteristics of the complexes are similar to those of other\n[25,32]. Chao et al. found that tridentate ring cyclometalated Ru com\u00ad isoquinoline Ru complexes [39]. RuIQ-3, RuIQ-4, and RuIQ-5 showed\nplex [Ru(pbpy)(adtpy)]+ exhibits effective inhibitory activity against intense absorption bands in the range of 260\u2013300 nm, which was\nA549 human NSCLC cells and cisplatin resistant A549/DDP cells with attributed mainly to intra-ligand \u03c0-\u03c0* transitions. The relatively weak\nhalf-maximal inhibitory concentration (IC50) values of 0.55 \u03bcM and bands at 330\u2013400 nm were most likely attributed to \u03c0-\u03c0* transitions. The\n0.57 \u03bcM, respectively [33]. Another cyclometalated Ru complex, [Ru relatively broad and weak absorption bands at 425\u2013590 nm were\n(phen)(bzq)(tbtfpip)]+ has displayed 178-fold higher anti-tumor activ\u00ad assigned to the metal-to-ligand charge transfer (MLCT) absorption.\nity against A549/DDP cells than cisplatin. Further studies showed that\nthere was no significant difference in absorption rates of [Ru(phen)(bzq) 2.3. Cytotoxicity on 2D cancer cell cultures\n(tbtfpip)]+ between A549 and A549/DDP cells [34]. After absorption by\ncells, this complex was mainly concentrated in mitochondria and could The in vitro cytotoxicities of Ru(II)-IQ complexes RuIQ-3, RuIQ-4,\ndownregulate the expression level of thioredoxin reductase (TrxR). This and RuIQ-5 against four selected human cancer cell lines (lung adeno\u00ad\ndistinct mechanism of action might be the main reason for the reversal of carcinoma A549, cisplatin resistant A549/DDP, hepatocellular carci\u00ad\nA549/DDP resistance [34,35]. noma HepG2, and breast cancer MCF-7) and one human normal\n Encouraged by these findings, we synthesized three novel Ru(II) bronchial epithelial cell line HBE were determined by 3-(4,5-dime\u00ad\ncomplexes with isoquinoline (IQ) as main ligand: [Ru(bpy)2(1-(4-F-Ph)- thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after co-\n\n 2\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\n\n\n Fig. 1. The chemical structure of ligand bpy, 1-(4-F-Ph)-IQ and Ru(II) complexes RuIQ-3, RuIQ-4, and RuIQ-5.\n\n\nincubation of cells with Ru(II)-IQ complexes for 48 h. The chemother\u00ad (RuIQ-3, RuIQ-4, RuIQ-5) resulted in marked reduction in the viability\napeutic agent cisplatin was chosen as a positive control. As shown in of A549 and A549/DDP cells in a concentration- and time-dependent\nTable 1, the IC50 values of the ligands 1-(4-F-Ph)-IQ, 1-(4-F-Ph)-7-OCH3- manner. Since RuIQ-5 was identified as the most efficacious candidate\nIQ, 1-(4-F-Ph)-6,7-(OCH3)2-IQ and the precursor cis-[Ru(bpy)2Cl2] among the three Ru(II)-IQ complexes, it was thereby chosen for further\nagainst the tested cancer cell lines all exceeded 150 \u03bcM, and therefore, investigations.\nthese ligands and precursor were deemed ineffective when used alone. To verify the anti-proliferation ability of the complex RuIQ-5 on\nIn contrast, the cyclometalated Ru(II)-IQ complexes exhibited greatly A549 and A549/DDP cells, wound-healing assay was performed. As\nenhanced cytotoxicities and were more potent than cisplatin against the shown in Fig. 2, after treatment of cells with 2.0 \u03bcM of RuIQ-5 for 24 h,\ncancer cell lines. The IC50 values of the Ru(II)-IQ complexes (RuIQ-3: the wound closure ratio decreased by 29% (A549) and 22% (A549/\n4.5 \u03bcM, RuIQ-4: 3.8 \u03bcM, RuIQ-5: 2.2 \u03bcM) against A549 cells were much DDP), respectively, indicating that RuIQ-5 could effectively inhibit the\nlower than those of cisplatin (15.6 \u03bcM) and other structurally similar migration of both A549 and A549/DDP cells even at a low dosage.\nnon-cyclometalated Ru(II) complexes [39]. It\u2019s worth noting that the\nselectivity index (SI) of the cyclometalated Ru(II)-IQ complexes (RuIQ- 2.4. Cytotoxicity on 3D multicellular tumor spheroids (MCTSs)\n3, RuIQ-4, RuIQ-5) is about 5\u20138 times the SI of cisplatin. Moreover,\nRuIQ-3, RuIQ-4, and RuIQ-5 showed potent inhibitory activities 3D multicellular tumor spheroid (MCTS) model has received\nagainst cisplatin-resistant A549/DDP as well, with their resistant factor increasing interest in recent years, since it can realistically mimics the\n(RF) values close to 1.0, which were lower than 3.13 in the positive extracellular matrix (ECM) and the in vivo situations and therefore have\ncontrol (cisplatin) group. The higher SI and lower RF values of the Ru been widely applied in the drug discovery process [42]. In particular,\n(II)-IQ complexes suggests that they possess higher efficacy and research based on MCTSs can gain insights into the metabolic and pro\u00ad\nimproved safety profile as potential cancer therapeutics, which was also liferative gradients in tumors which contribute to the therapeutic chal\u00ad\nsupported by previous reports [40,41]. In order to confirm the cytotoxic lenges of cancers, such as altered responsiveness of chronically hypoxic\neffect of the Ru(II)-IQ complexes on lung cancer cells, MTT assay was tumor cells, and resistance to multiple apoptosis-inducing agents.\nperformed with a concentration gradient of each complex and a time MCTSs with diameters of approximately 400 \u03bcm resemble solid tumors\ngradient for the A549 and A549/DDP cells (Fig. S13 in supplementary in terms of pathophysiological conditions, such as specific hypoxic areas\ninformation). As shown in Fig. S13, treatment of Ru(II)-IQ complexes in the center of a tumor, and gradient proliferation rates of tumor cells\n\n 3\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\n\n\n complex R1 R2\n\n RuIQ-3 H H\n\n RuIQ-4 OCH3 H\n\n RuIQ-5 OCH3 OCH3\n\nScheme 1. The synthetic routes of precursor cis-[Ru(bpy)2Cl2], ligands 1-(4-F-Ph)-IQ, 1-(4-F-Ph)-7-OCH3-IQ, and 1-(4-F-Ph)-6,7-(OCH3)2-IQ, and the cyclometalated\nRu(II)-IQ complexes RuIQ-3, RuIQ-4 and RuIQ-5.\n\n\n[43,44]. Therefore, we tested the cytotoxicities of the Ru(II)-IQ com\u00ad 14 days. The size of the RuIQ-5 treated MCTSs were apparently smaller\nplexes (RuIQ-3, RuIQ-4, RuIQ-5) against A549 and A549/DDP MCTSs than the cisplatin treated and untreated MCTSs, which means the pro\u00ad\nwith diameters of approximately 400 \u03bcm [42]. As shown in Fig. 3(A), the liferation of A549 and A549/DDP cells was significantly inhibited by\nIC50 value of cisplatin against A549 MCTSs was 62 \u03bcM and it even RuIQ-5. Moreover, RuIQ-5 inhibited the growth of MCTSs in a clear-cut\nexceeded 200 \u03bcM in A549/DDP MCTSs. In contrast, the IC50 values of dose dependent manner (Fig. S14), thus confirming the antitumor ac\u00ad\nthe Ru(II)-IQ complexes against A549 MCTSs (RuIQ-3: 4.9 \u03bcM, RuIQ-4: tivity of RuIQ-5.\n4.2 \u03bcM, RuIQ-5: 2.8 \u03bcM) and A549/DDP MCTSs (RuIQ-3: 5.2 \u03bcM, RuIQ- To visualize the antitumor effectiveness of RuIQ-5, MCTSs were\n4: 4.3 \u03bcM, RuIQ-5: 2.9 \u03bcM) were much lower, indicating that the treated with 1.0, 2.0, and 4.0 \u03bcM of RuIQ-5 respectively, after which the\ncyclometalated Ru(II)-IQ complexes can efficiently inhibit tumor growth spheroids were stained with calcein AM (staining the living cells) and\nand overcome drug resistance. propidium iodide (PI, staining the dead cells), respectively. As shown in\n Meanwhile, we monitored the growth rate of MCTSs by adminis\u00ad Fig. 3(B), A549 or A549/DDP cells in untreated MCTSs emitted steady\ntering different concentrations of RuIQ-5 (Fig. S14). MCTSs were green fluorescence, indicating that the cells were living. In contrast,\ntreated with RuIQ-5 (1.0, 2.0, 4.0 \u03bcM) for 72 h, then 50% of the plated when MCTSs were treated with 1.0, 2.0, and 4.0 \u03bcM of RuIQ-5 the green\nmedium was replaced with fresh medium every two days. In this way, fluorescence was significantly weakened and the bright-red fluorescence\nloss or disruption of the MCTSs could be avoided [40]. As shown in gradually increase, indicating that cells had been damaged. In addition,\nFig. S14, the growth of MCTSs was significantly delayed by RuIQ-5 after the red fluorescence was dependent on the dose of RuIQ-5. These results\n\n\n 4\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\nTable 1 less than 20% at 72 h, and all the embryos died at 96 h. These results\nIC50 values (\u03bcM) of Ru(II)-IQ complexes (RuIQ-3, RuIQ-4, RuIQ-5) against the indicated that RuIQ-5 (less than 13.6 \u03bcg/mL) possessed little in vivo\nselected human cancer cell lines.a toxicity in zebrafish embryos, which demonstrated an acceptable safety\n Complex IC50 (\u03bcM) RFb SIc profile. Therefore, it is expected that RuIQ-5 could be developed as a\n A549 A549/ HepG2 MCF-7 HBE\n low-toxicity agent against lung cancer. Hence, further studies (which\n DDP were discussed in later sections) were performed to elucidate mecha\u00ad\n nism underlying the antitumor effect and the reversal of cisplatin\n cis-[Ru >200 >200 >200 >200 \u2013 \u2013 \u2013\n (bpy)2Cl2] resistance of RuIQ-5 in A549/DDP cells.\n 1-(4-F-Ph)- 179.6 >200 196.4 >200 >200 \u2013 \u2013\n IQ \u00b1 6.3 \u00b1 6.9\n 1-(4-F-Ph)- 168.7 >200 192.2 191.6 >200 \u2013 \u2013 2.6. Lipophilicity and cellular uptake\n 7-OCH3- \u00b1 6.4 \u00b1 5.1 \u00b1 6.3\n IQ To evaluate the ability of Ru(II)-IQ complexes entering cells, their\n 1-(4-F-Ph)- 152.5 185.8 182.6 \u2013 \u2013\n lipophilicity, expressed as oil\u2013water partition coefficient (logPo/w), was\n >200 \uff1e200\n 6,7- \u00b1 5.8 \u00b1 5.4 \u00b1 5.1\n (OCH3)2- determined by the shake-flask method using inductively coupled plasma\n IQ mass spectrometry (ICP-MS). As shown in Fig. 5A, the logPo/w values\n RuIQ-3 4.5 \u00b1 4.9 \u00b1 8.4 \u00b1 7.9 \u00b1 25.2 1.09 5.14 were in the following order: RuIQ-5 (1.25) > RuIQ-4 (1.10) > RuIQ-3\n 0.5 0.5 0.6 0.7 \u00b1 1.6\n (0.65) > cisplatin (-4.53), which was in accordance with the order of\n RuIQ-4 3.8 \u00b1 4.1 \u00b1 7.8 \u00b1 7.2 \u00b1 23.8 1.08 5.80\n 0.4 0.3 0.5 0.5 \u00b1 2.1 cellular metal content (Fig. 5B), confirming that a higher lipophilicity of\n RuIQ-5 2.2 \u00b1 2.3 \u00b1 7.2 \u00b1 6.8 \u00b1 18.3 1.04 7.96 Ru(II)-IQ complexes enhanced their tumor penetration and cellular\n 0.3 0.2 0.4 0.5 \u00b1 0.1 uptake. It\u2019s worth noting that there was no significant difference in the\n Cisplatin 15.6 \u00b1 48.8 18.4 \u00b1 21.5 \u00b1 19.8 3.13 1.27 uptake of Ru(II)-IQ complexes between A549 and A549/DDP cell lines,\n 1.1 \u00b1 2.1 1.5 1.3 \u00b1 1.2\n in contrast, the uptake of cisplatin by A549/DDP was significantly lower\n a\n Cell viability was determined by MTT assay after ligands or complexes than A549, implying that the ability of the Ru(II)-IQ complexes (RuIQ-3,\ntreatment with cells for 48 h. RuIQ-4, RuIQ-5) to reverse drug resistance may be related to their\n b\n RF (resistant factor) = IC50 (A549/DDP)/IC50 (A549). efficient penetration into target tumor cells [48]. Besides, the subcel\u00ad\n c\n SI (selectivity index) = IC50 (HBE)/IC50(A549/DDP).\n lular distribution of RuIQ-5 in A549 and A549/DDP cells was further\n determined by ICP-MS with cisplatin as a control (Fig. 5C). As shown in\nrevealed that RuIQ5 could inhibit the growth of MCTSs and induce Fig. 5C, RuIQ-5 complexes were predominantly accumulated in the\ntumor cell death. nucleus and only a small fraction of the complexes were found in\n mitochondria and cytoplasm, implying that RuIQ-5 might affect\n2.5. Fish embryo acute toxicology test (FET). genome replication and gene expression. In contrast, cisplatin was\n mainly distributed in the cytoplasm of A549/DDP cells instead of the\n Embryo-larval zebrafish has served as a useful model for toxicology nucleus (Fig. 5C). These results suggest that RuIQ-5 is a highly cell-\nand drug development studies. More than just an alternative to mouse, permeable, nucleus-targeting Ru(II) complex, which is similar to other\nzebrafish model has emerged as a complement to mammals in toxico\u00ad cyclometalated Ru(II) complexes reported earlier such as [Ru(bpy)\nlogical studies that the toxicological results can predict the effects in (phpy)(dppz)]+ [49], [Ru(dmb)2(1-Ph-\u03b2C)](PF6) [50], [Ru(bpy)2(1-Ph-\nmammals [45\u201347]. In this work, zebrafish embryos were used as a \u03b2C)](PF6) [50], [Ru(bpy)2(1-Ph-IQ)]+ [39], and [Ru(phen)2(1-Ph-IQ)]+\nvertebrate model for the in vivo toxicity evaluation of the newly syn\u00ad [39].\nthesized Ru(II)-IQ complex RuIQ-5. In general, drug molecules cross cell membrane and reach cytoplasm\n As shown in Fig. 4A, after exposure to RuIQ-5 at concentrations less through adenosine-5\u2032 -triphosphate (ATP)-dependent active transport or\nthan 13.6 \u03bcg/mL (16.0 \u03bcM) for 72 h, all zebrafish embryos grew into nonenergy-dependent passive diffusion [51]. To evaluate the internali\u00ad\njuveniles, with an acceptable cumulative hatch rate of 80% (Fig. 4B). zation pathway of RuIQ-5 complexes, A549 and A549/DDP cells were\nAnd, the lethality rate was lower than 20% even after 96 h (Fig. 4C). treated with RuIQ-5 at low temperature (4 \u25e6 C) or pretreated with so\u00ad\nHowever, when the concentration of RuIQ-5 increased to 13.6 \u03bcg/mL, dium azide (NaN3) in combination with 2-Deoxy-D-glucose (2-DOG), a\nsome zebrafish embryos developed abnormally with a noticeable spinal commonly used strategy to block ATP-dependent active transportation.\ncurvature (Fig. 4A). When the RuIQ-5 concentration was increased to As shown in Fig. 5D, both low temperature and NaN3/2-DOG could\n27.2 \u03bcg/mL (32.0 \u03bcM), the hatch rate of zebrafish embryo decreased to strongly inhibit the cellular uptake of RuIQ-5, confirming that RuIQ-5\n\n\n\n\nFig. 2. Wound healing assay of A549 and A549/DDP cells treated with or without RuIQ-5 for 24 h. (A) Typical images were taken at 0 and 24 h. (B) Quantification of\nmicroscope images. Data are quoted as mean \u00b1 SD of three replicates. \u00d7 \u00b1 s, n = 3. ###P < 0.001 vs A549 control, ***P < 0.001 vs A549/DDP control.\n\n 5\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\n\n\nFig. 3. Cytotoxicity assay of 3D MCTSs model. (A) IC50 values of cisplatin and Ru(II)-IQ complexes (RuIQ-3, RuIQ-4, RuIQ-5) against A549 and A549/DDP MCTSs\nafter 72 h drug exposures. (B) Calcein AM and PI dual-staining of MCTSs treated by indicated concentrations of RuIQ-5 for 72 h. \u00d7 \u00b1 s, n = 3. ###P < 0.001 vs A549\ncontrol, ***P < 0.001 vs A549/DDP control.\n\n\nenters A549 and A549/DDP cells in an energy-dependent manner. electrophoresis (SCGE), also known as comet assay, is an efficient and\nEndocytosis is the most common energy-dependent pathway. Therefore, rapid method for detecting DNA damage that the damage degree is\nwe further investigated the internalization pathway of RuIQ-5 with two directly reflected by the length of comet tails [56,57]. To determine\nendocytosis inhibitors nystatin (inhibitor of clathrin-mediated endocy\u00ad whether RuIQ-5 complexes can induce DNA damage or not, comet assay\ntosis) and sucrose (inhibitor of lipid raft-mediated endocytosis) [52]. was performed. As shown in Fig. 7A, both A549 and A549/DDP cells in\nSimilarly, cellular uptake level of RuIQ-5 was also reduced in both the the control did not display comet tail artifact, indicating that the DNA in\nnystatin and sucrose treatment groups. These results suggested that untreated cells was intact. After incubation with 1.0 \u03bcM of RuIQ-5 for\nRuIQ-5 complexes were actively transported into A549 and A549/DDP 24 h, both A549 and A549/DDP cells exhibited legible comet tails,\ncells mainly through endocytosis. indicating that mild DNA damage has occurred. When the concentration\n of RuIQ-5 was increased to 4 \u03bcM, long and thick comet tails could be\n2.7. RuIQ-5 triggers inhibition of DNA replication and DNA damage observed, suggesting that severe DNA damage has occurred. The length\n of comet tails was then quantified by Image J software and shown in\n Since RuIQ-5 accumulated mostly in the cell nucleus, we tested the Fig. 7B. These results suggested that RuIQ-5 could induce DNA damage\ninhibitory effect of RuIQ-5 on DNA replication by carrying out an in a dose-dependent manner, which might be responsible for the inhi\u00ad\nantiproliferation assay [49]. 5-ethynyl-2\u2032 -deoxyuridine (EdU) is a bition of DNA amplification.\nthymidine analogue that can be integrated into DNA as a red fluorescent DNA damage can directly affect DNA replication, transcription, and\nmarker in active cell proliferation, thereby it was used in this assay [53]. lead to cell cycle arrest [58]. To investigate the effects of RuIQ-5 on cell\nAs shown in Fig. 6, a large amount of red fluorescence was observed in cycle distribution, A549 and A549/DDP cells were treated with RuIQ-5\nthe nucleus of A549 and A549/DDP cells in the control groups, indi\u00ad (1.0, 2.0, 4.0 \u03bcM) for 24 h and analyzed by flow cytometry. The results in\ncating the newly replicated DNA. In contrast, the amount of newly Fig. S15 showed that treatment of A549 and A549/DDP cells with RuIQ-\nreplicated DNA was decreased significantly in the cells treated with 5 significantly increased the percentages of cells in both the S and G2/M\ndifferent concentrations of complex RuIQ-5 (1.0, 2.0, 4.0 \u03bcM). When the phases in a concentration-dependent manner. For instance, the per\u00ad\nconcentration of RuIQ-5 reached 4.0 \u03bcM, the synthesis of DNA was centage of A549/DDP cells in G2/M phase was 6.15% before treatment,\nalmost completely inhibited. These results suggested that complex and increased to 10.83% and 18.67% when treated with 1.0 and 4.0 \u03bcM\nRuIQ-5 could effectively inhibit the DNA amplification in A549 and of RuIQ-5, respectively. These findings denoted that, like other cyclo\u00ad\nA549/DDP cells. metalated Ru(II)-IQ complexes [39], RuIQ-5 could induce cell cycle\n DNA damage can lead to inhibition of its replication, failure to repair arrest at S and G2/M phases in A549 and A549/DDP cells.\ndamaged DNA may induce cell death. Therefore, DNA damage is\nconsidered to be a marker of apoptosis [54,55]. Single cell gel\n\n 6\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\n\n\nFig. 4. Toxicity assessment of RuIQ-5 in developing zebrafish embryos. (A) Toxicity of RuIQ-5 to zebrafish embryos at various concentrations (1.7, 3.4, 6.8, 13.6,\n27.2 \u03bcg/mL) within 96 h on a 4 \u00d7 objective lens in the microscope. (B) Cumulative hatch rate of zebrafish embryos in the presence/absence of RuIQ-5 at various\nconcentrations (0, 1.7, 3.4, 6.8, 13.6, 27.2 \u03bcg/mL) every 24 h. (C) Lethality rate of zebrafish embryos in the presence/absence of RuIQ-5 at different concentrations\n(0, 1.7, 3.4, 6.8, 13.6, 27.2 \u03bcg/mL) every 24 h.\n\n\n2.8. RuIQ-5 induces apoptosis in A549 and A549/DDP cells 2.9. RuIQ-5 induces mitochondrial dysfunction\n\n Cell cycle arrest and DNA damage may result in cell division Mitochondrial dysfunction is involved in apoptotic cell death [49].\nblockage and apoptosis [59]. Apoptosis often causes cell morphological Apoptosis-inducing factors released by mitochondrion, such as cyto\u00ad\nchanges, such as nuclear condensation and fragmentation, and forma\u00ad chrome c play an important role in the initiation of apoptosis [63\u201367].\ntion of apoptotic bodies [60]. A549 or A549/DDP cells were treated with The decline of mitochondrial membrane potential (MMP) is an early\nRuIQ-5 (1.0, 2.0, 4.0 \u03bcM) for 24 h, and the RuIQ-5-induced morpho\u00ad marker of apoptosis. We used 5,5\u2032 ,6,6\u2032 -Tetrachloro-1,1\u2032 ,3,3\u2032 -tetrae\u00ad\nlogical changes of cells were observed by Hoechst33342 fluorescent thylbenzimidazolylcarbocyanine iodide (JC-1) dye to detect the decline\nstaining. As shown in Fig. S16A and 16B, after staining with Hoechst of MMP in A549 and A549/DDP cells after RuIQ-5 treatment. As shown\n33342, nuclear condensation and apoptotic bodies were observed in the in Fig. 9A, after treatment of cells with RuIQ-5 (1.0, 2.0, 4.0 \u03bcM) for 24\nRuIQ-5-treated cells. Moreover, cell apoptosis was evaluated by acri\u00ad h, the green fluorescence was significantly enhanced while the intensity\ndine orange/ethidium bromide (AO/EB) dual-staining. As shown in of red fluorescence was weakened, indicating a decrease in MMP. The\nFig. S16C, in the control groups, A549 and A549/DDP cells with normal changes of MMP were quantitatively analyzed by flow cytometry, as\nmorphology were labeled homogeneously with green fluorescence. In shown in Fig. 9B and Fig. S17, the MMP decline in A549 and A549/DDP\ncontrast, clearly morphological features of apoptosis and apoptotic cells cells was dependent on the concentration of RuIQ-5.\nshowing orange or red fluorescence were observed in the RuIQ-5 In addition, western blot analysis was performed to confirm the\ntreatment groups. These preliminary results indicated that RuIQ-5 could mitochondrial pathways in apoptosis induced by RuIQ-5 treatment. The\ninduce apoptosis in both A549 and A549/DDP cells. results in Fig. 9C indicated that the ratios of Bcl-2/Bax and Bcl-xl/Bad\n In order to further quantitatively analyze the cell death-inducing were decreased, the apoptosis inducing factor, cytochrome c was\nability of RuIQ-5, annexin V-fluorescein isothiocyanate (FITC)/PI released into the cytosol. The increase of cytosolic cytochrome c was\nstaining was performed and analyzed using flow cytometry. Fig. 8A and dependent on the dosage of RuIQ-5 (Fig. 9C). These results further\n8B showed that pre-incubation of A549 or A549/DDP cells with complex confirmed that RuIQ-5 could induce mitochondrial dysfunction, which\nRuIQ-5 of different concentrations (1.0, 2.0, 4.0 \u03bcM) for 24 h enhanced contributes to apoptosis in A549 and A549/DDP cells.\nthe percentage of apoptotic cells. Specifically, the proportion of the\nRuIQ-5 treated cells in early apoptosis and late apoptosis/necrosis was\nincreased significantly in a dose-dependent manner. Western blot was 2.10. RuIQ-5 stimulates intracellular ROS accumulation\nused to detect apoptosis-related proteins to confirm the occurrence of\napoptosis. As shown in Fig. 8C, RuIQ-5 significantly increased the It has been demonstrated that ROS can induce apoptosis via a variety\nexpression level of cleaved caspase-3, -8, -9, and poly (ADP-ribose) po\u00ad of mechanisms. For example, increased ROS can activate the intrinsic\nlymerase (PARP) in both A549 and A549/DDP cells in a concentration- pathway of apoptosis by stimulating the depolarization of MMP [68].\ndependent manner. These results undoubtedly demonstrated that RuIQ- However, the majority of cancer cells can remain viable in the presence\n5 could induce apoptosis in A549 and A549/DDP cells [61,62]. of intrinsic oxidative stress, thereby enabling them to avoid apoptosis\n and become resistant to many chemotherapeutic drugs [69,70]. In order\n to elucidate the underlying mechanism of RuIQ-5-induced apoptosis in\n A549 and A549/DDP cells, we investigated their cellular ROS levels\n using 7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) fluorescent\n\n 7\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\n\n\nFig. 5. Lipophilicity and cellular uptake of cisplatin and Ru(II)-IQ complexes (RuIQ-3, RuIQ-4, RuIQ-5) in A549 and A549/DDP cells. (A) LogPo/w values of cisplatin\nand Ru(II)-IQ complexes. (B) Cellular Ruthenium or platinum contents determined in A549, A549/DDP cells after 24 h incubation with cisplatin, RuIQ-3, RuIQ-4, or\nRuIQ-5 at 2.0 \u00b5M, respectively. \u00d7 \u00b1 s, n = 3. ***P < 0.01 vs cisplatin treatment group of A549. (C) Subcellular distribution of ruthenium or platinum contents in\nA549 and A549/DDP cells after incubation with cisplatin or RuIQ-5 at 2.0 \u00b5M for 24 h. x \u00b1 s, n = 3. #P < 0.05, ##P < 0.01 vs A549 nucleus, *P < 0.01, ***P < 0.001\nvs A549/DDP nucleus. (D) Intracellular uptake of RuIQ-5 in A549 and A549/DDP cells under different endocytosis-inhibited conditions at 2.0 \u03bcM. \u00d7 \u00b1 s, n = 3. #P\n< 0.05, ##P < 0.01 vs A549 control, **P < 0.01, ***P < 0.001 vs A549/DDP control.\n\n\n\n\nFig. 6. Anti-proliferation assay of A549 and A549/DDP cells treated by EdU after 1.0, 2.0 4.0 \u03bcM of RuIQ-5 treatment for 12 h. (A) Anti-proliferation assay in A549\ncells. (B) Anti-proliferation assay in A549/DDP cells.\n\n\nprobe. It was observed that, prior to RuIQ-5 treatment, the intensity of obtained by flow cytometry (Fig. 10A). The results in Fig. 10A and B\nDCFH-DA fluorescence in A549/DDP cells was stronger than that of revealed that the increase in the cellular ROS levels as a result of RuIQ-5\nA549 cells, which might be associated with the drug resistance in A549/ treatment was dependent on the dose (1.0, 2.0, 4.0 \u03bcM).\nDDP cells [70]. Furthermore, as shown in Fig. S18, compared with the To further explore the role of ROS in triggering apoptosis, two ROS\ncontrol group, both A549 and A549/DDP cells exhibited obvious green scavengers, NAC and GSH, were used. As shown in Fig. 10C, the pro\u00ad\nfluorescence when treated with RuIQ-5 at 1.0, 2.0, 4.0 \u03bcM for 12 h, duction of cellular ROS was significantly reduced in the RuIQ-5/NAC\nindicating an increase in the cellular ROS level. A similar result was also and RuIQ-5/GSH treatment groups, in contrast to the cells treated by\n\n 8\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\n\n\nFig. 7. DNA damage was examined by comet assay. (A) A549 and A549/DDP cells were treated with various concentrations (1.0, 2.0, 4.0 \u03bcM) of RuIQ-5 for 24 h,\nand DNA fragmentation was examined by comet assay. (B) Quantification of DNA tails in the comet assay. The length of DNA tails in microscopy images was\nquantified by Image J. \u00d7 \u00b1 s, n = 3. ##P < 0.01, ###P < 0.001 vs A549 control, **P < 0.01, ***P < 0.001 vs A549/DDP control.\n\n\nRuIQ-5 alone. Moreover, NAC or GSH pretreatment led to increased RuIQ-5 treatment led to downregulation of Nrf2, which could improve\nA549 and A549/DDP cell viability as well (Fig. 10D). Altogether, these the sensitivity of A549/DDP cells to antineoplastic drugs by reversal of\nresults revealed that activation of ROS generation by RuIQ-5 played an drug resistance in cells.\nimportant role in inducing apoptosis in A549 and A549/DDP cells.\n 3. Conclusions\n2.11. Akt/GSK-3\u03b2/Fyn signaling pathway was involved in RuIQ-5-\ninduced apoptosis In this study, three Ru(II)-IQ complexes [Ru(bpy)2(1-(4-F-Ph)-IQ)]+\n (RuIQ-3), [Ru(bpy)2(1-(4-F-Ph)-7-OCH3-IQ)]+ (RuIQ-4), and [Ru\n Nrf2, a key transcriptional regulator of antioxidant and anti- (bpy)2(1-(4-F-Ph)-6,7-(OCH3)2-IQ)]+ (RuIQ-5) were synthesized, char\u00ad\ninflammatory enzymes, is abundantly expressed in cancer cells [71]. acterized and evaluated biologically. Among them, RuIQ-5 exhibited\nIn NSCLC patients, Nrf2 is usually deregulated at both the transcrip\u00ad the highest cellular uptake and thus displayed the most potent anti-\ntional level and protein level [72]. It has been shown that GSK-3\u03b2, a proliferation activities against both A549 and A549/DDP cells. Studies\nsubtype of GSK3, can regulate Fyn-mediated export and degradation of on the intracellular distribution showed that RuIQ-5 was mainly\nNrf2 in the nucleus [8\u201310]. GSK-3\u03b2 phosphorylates Fyn, which subse\u00ad distributed in the nucleus other than cytoplasm upon absorption by\nquently phosphorylates and activates Nrf2, leading to the promotion of A549/DDP cells. Cytotoxicity assays based on 3D MCTSs tumor spheroid\napoptosis by enhanced Nrf2 degradation; however, activated Akt can models further demonstrated the ability of Ru(II)-IQ complexes to\nphosphorylate the Ser9 residue of GSK-3\u03b2, thereby inhibiting GSK-3\u03b2 overcome multicellular drug resistance. Moreover, RuIQ-5 exhibited\nactivity and protecting cells from apoptosis [73,74]. The reversal of low toxicity both towards normal HBE cells in vitro and zebrafish em\u00ad\ntumor drug resistance by RuIQ-5 may be related to the Akt/GSK3\u03b2/Fyn bryos in vivo, displaying a promising safety profile. Further, molecular\nsignaling pathway. Hence, we evaluated the effect of RuIQ-5 on the mechanism studies revealed that RuIQ-5 could induce ROS-mediated\nexpression levels of Akt and GSK3\u03b2 in both A549 and A549/DDP cells. apoptosis in A549 and A549/DDP cells through mitochondrial\nAs shown in Fig. 11A, the expression of p-Akt, p-GSK3\u03b2, and Nrf2 in dysfunction, DNA damage, and cell cycle arrest in S and G2/M phases\nA549/DDP cells was higher than that in A549 cells, while p-Fyn level in (Fig. 12). Studies also showed that RuIQ-5 could reverse the drug\nA549/DDP cells was lower than that in A549 cells. After treatment with resistance of A549/DDP cells through regulation of Akt/GSK3\u03b2/Fyn\nRuIQ5, in A549 cells, p-Akt and total Nrf2 decreased, but GSK3\u03b2 and p- signaling pathway (Fig. 12). Based on these findings, we believe that Ru\nFyn did not change much. However, in A549/DDP cells, RuIQ5 treat\u00ad (II)-isoquinoline complexes could be developed as efficient Nrf2 in\u00ad\nment downregulated the expression of p-Akt and p-GSK3\u03b2, while GSK3\u03b2 hibitors and overcome the cisplatin resistance in NSCLC.\nand p-Fyn levels were increased . In addition, RuIQ5 treatment resulted\nin a decrease in the levels of nucleus Nrf2 and its downstream resistance- 4. Experimental section\nassociated proteins MRP1 and HO-1 in A549/DDP cells in a\nconcentration-dependent manner (Fig. 11B). The above results indi\u00ad 4.1. Materials and general methods\ncated that RuIQ5 could reverse the drug resistance in A549/DDP cells\nthrough regulation of Akt/GSK3\u03b2/Fyn pathway. All reagents and solvents were purchased commercially and used\n The localization of Nrf2 is essential for its biological function [4,6]. without further purification. DMSO, 3-(4,5-dimethylthiazol-2-yl)-2,5-\nThe effects of RuIQ-5 on the distribution of Nrf2 in cells were observed diphenyltetrazolium bromide (MTT), Hoechst 33342, phosphate buff\u00ad\nby immunofluorescence staining, with PI3K inhibitor LY294002 used as ered saline (PBS), 5,5\u2032 6,6\u2032 -tetrachloro-1,1\u2032 ,3,3\u2032 -tetraethylimidacarbo\u00ad\na positive control. We found that Nrf2 was mainly expressed in the cyanine iodide (JC-1), 2,7-Dichlorodi-hydrofluorescein diacetate\ncytoplasm of A549 cells, while Nrf2 fluorescence was significantly (DCFH-DA), Annexin V-FITC apoptosis detection kit, QuantiPro\u2122 BCA\nenhanced in A549/DDP cells, with increased expression in the nucleus assay kit, and ECL\u2122 Start Western blotting detection reagent were\n(Fig. 11C and 11D). RuIQ-5 treatment reduced the fluorescence in\u00ad purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell mitochondria\ntensity of Nrf2 in A549/DDP cells and hindered its nuclear expression isolation kit, BeyoClick EdU-594 cell proliferation assay kit, calcein AM\n(Fig. 11D). However, in A549 cells, the ruthenium complex RuIQ-5 had (excitation at 488 nm, emission at 515 nm), propidium iodide (PI)\nno significant effect on the nuclear translocation of Nrf2. (excitation at 535 nm, emission at 617 nm), and CellTiter-Lumi\u2122 Plus\n Taken together, regulation of Akt/GSK3\u03b2/Fyn signaling pathway by luminescent cell viability assay kit were purchased from Beyotime\n\n 9\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\n\n\nFig. 8. Ru(II)-IQ complex RuIQ-5 induced A549 and A549/DDP cells apoptosis. (A) A549 and A549/DDP cells apoptosis was detected by annexin V/PI assay after\nco-incubation with 1.0, 2.0, 4.0 \u03bcM of RuIQ-5 for 24 h. (B) Populations for A549, A549/DDP cells in four stages. \u00d7 \u00b1 s, n = 3. ##P < 0.01, ###P < 0.001 vs A549\ncontrol, **P < 0.01, ***P < 0.001 vs A549/DDP control. (C) The expression levels of caspase-3, PARP and cleaved caspase-3/8/9 and PARP were evaluated in a\nconcentration-dependent manner with RuIQ-5 (1.0 2.0 4.0 \u03bcM) treatment for 24 h. GAPDH was used as internal control.\n\n\n(Shanghai, China). Ruthenium standard solution was purchased from 4.2. Synthesis and characterization\nAladdin Chemistry Co. Ltd (Shanghai, China). Primary and secondary\nantibodies were purchased from Cell Signaling Technology, Inc. Com\u00ad 1-(4-F-Ph)-IQ, 1-(4-F-Ph)-7-OCH3-IQ, 1-(4-F-Ph)-6,7-(OCH3)2-IQ\netAssay\u00ae reagent kit was purchased from Trevigen (Gaithersburg, MD, [36] and cis-[Ru(bpy)2Cl2]\u22c52H2O [36] were prepared as previous liter\u00ad\nUSA). Dulbecco\u2019s Modified Eagle Medium (DMEM) and fetal bovine ature. And these complexes were characterized by Elemental micro\u00ad\nserum (FBS) were obtained from HyClone. analyses, ESI-MS, 1H NMR and UV\u2013Vis spectra.\n Protein bands were visualized using ChemiDoc\u2122 XRS + Imaging\nSystem (Bio-Rad, USA). Elemental microanalyses (C, H, and N) were 4.2.1. Synthesis of [Ru(bpy)2(1-(4-F-Ph)-IQ)](PF6) (RuIQ-3)\nperformed using a Perkin\u2013Elmer 240Q elemental analyzer. Electrospray A mixture of cis-[Ru(bpy)2Cl2]\u22c52H2O (0.052 g, 0.1 mM), 1-(4-F-Ph)-\nionization mass spectrometry (ESI-MS) was recorded on Agilent LC- IQ (0.0223 g, 0.1 mM), Ag(CF3SO3) (0.064 g, 0.25 mM) and 10%\nMS6430B Spectrometer. 1H NMR spectra were recorded on a Bruker methanol solution of (CH3)4NOH(0.012 g, 0.13 mol) in anhydrous\nAVANCE 400 spectrometer (400 MHz) at room temperature. UV\u2013visible ethanol (10 mL) was refluxed under argon at 80 \u25e6 C for 12 h. After the\n(UV\u2013Vis) and emission spectra were measured on Perkin\u2013Elmer reaction, AgCl was removed by filtration, the red precipitate (0.04 g)\nLambda-850 spectrophotometer at 25 \u25e6 C. Flow cytometry was per\u00ad was then obtained by a dropwise addition of saturated aqueous KPF6\nformed using an EPICS\u00ae XL-MCL flow cytometer (BECKMAN COULTER, solution. Finally, the purple-red precipitate was dried under vacuum and\nUSA). Fluorescence observation was performed by Ti-E inverted mi\u00ad purified by column chromatography on neutral alumina with a mixture\ncroscope (Nikon, Japan). Microplate was read by Infinite M200 Pro of CH3CN-toluene (1:3, v/v) as eluent. Yield: 72 %. Anal. calc. for\nmultimode microplate reader (Tecan, Switzerland). C35H25F7N5PRu: C, 53.83; H, 3.23; N, 8.97; found: C, 53.80; H, 3.25; N,\n\n\n 10\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\n\n\nFig. 9. RuIQ-5 induced changes of mitochondrial membrane potential (MMP) in A549 and A549/DDP cells. (A) Fluorescence microscope analysis of cellular MMP\nlevel by JC-1 staining after 1.0, 2.0 and 4.0 \u03bcM of RuIQ-5 treatment for 24 h. (B) Flow cytometry analysis of cellular MMP level after 1.0, 2.0 and 4.0 \u03bcM of RuIQ-5\ntreatment for 24 h. (C) The expression levels of Bcl-2 family proteins and cytochrome c in cytosol were evaluated in a concentration-dependent manner with RuIQ-5\ntreatment for 24 h. GAPDH was used as internal control.\n\n\n9.18. ESI-MS (MeCN): m/z = 636 ([M \u2212 PF6]+). 1H NMR (400 MHz, 7.35 (m, 3H), 7.28 (d, J = 8.4 Hz, 1H), 7.04 (dd, J = 8.3, 2.6 Hz, 1H),\nDMSO\u2011d6) \u03b4 8.93 \u2013 8.63 (m, 5H), 8.42 (dd, J = 9.0, 5.6 Hz, 1H), 8.24 \u2013 6.82 \u2013 6.65 (m, 1H), 6.66 \u2013 6.58 (m, 1H), 6.04 (dd, J = 9.3, 2.7 Hz, 1H),\n8.06 (m, 1H), 8.02 \u2013 7.85 (m, 5H), 7.85 \u2013 7.71 (m, 3H), 7.71 \u2013 7.49 (m, 3.92 (s, 3H). 13C NMR (100 MHz, DMSO) \u03b4 164.87, 163.19, 160.66,\n3H), 7.51 \u2013 7.26 (m, 5H), 6.74 (td, J = 8.8, 2.8 Hz, 1H), 6.12 (dd, J = 9.2, 159.56, 157.57, 156.81, 156.67, 154.92, 154.08, 150.57, 149.92,\n2.8 Hz, 1H). 13C NMR (100 MHz, DMSO) \u03b4 166.62, 163.39, 160.86, 148.82, 143.82, 140.46, 137.30, 135.87, 135.04, 134.83, 131.54,\n157.56, 156.82, 156.66, 154.91, 154.13, 150.71, 149.94, 148.86, 131.26, 131.18, 129.80, 128.01, 127.51, 127.17, 127.11, 124.24,\n143.61, 142.23, 137.35, 136.14, 135.93, 135.11, 134.89, 132.24, 124.06, 123.95, 122.72, 120.45, 120.33, 120.20, 108.06, 107.84,\n132.15, 130.78, 129.13, 128.06, 127.52, 127.23, 127.12, 126.02, 105.00, 55.99. UV\u2013Vis (\u03bb/nm, \u03b5/M\u2212 1\u2219cm\u2212 1) (PBS): 296 (36650), 492\n125.98, 124.25, 124.09, 123.98, 120.67, 120.36, 120.22, 108.11, (9300).\n107.89. UV\u2013Vis (\u03bb/nm, \u03b5/M\u2212 1\u2219cm\u2212 1) (PBS): 296 (686000), 492\n(134000). 4.2.3. Synthesis of [Ru(bpy)2(1-(4-F-Ph)-6,7-(OCH3)2-IQ)](PF6) (RuIQ-\n 5)\n4.2.2. Synthesis of [Ru(bpy)2(1-(4-F-Ph)-7-OCH3-IQ)](PF6) (RuIQ-4) RuIQ-5 complex was synthesized in a manner identical to that\n The synthesis route of RuIQ-4 was similar to that of RuIQ-3 except described for RuIQ-3, except that the 1-(4-F-Ph)-IQ was replaced by 1-\nthat the 1-(4-F-Ph)-IQ was replaced by 1-(4-F-Ph)-7-OCH3-IQ. Synthe\u00ad (4-F-Ph)-6,7-(OCH3)2-IQ. Synthesized RuIQ-5 complex was a purple-red\nsized RuIQ-4 complex was a purple-red solid with a weigh of 0.040 g, solid with a weigh of 0.042 g, and its yield was 60%. Anal. calc. for\nand its yield was 60%. Anal. calc. for C36H27F7N5OPRu: C, 53.34; H, C37H29F7N5O2PRu: C, 52.86; H, 3.48; N, 8.33; found: C, 52.81; H, 3.50;\n3.36; N, 8.64; found: C, 53.30; H, 3.35; N, 8.63. ESI-MS (MeCN): m/z = N, 8.30. ESI-MS (MeCN): m/z = 695.90 ([M \u2212 PF6]+). 1H NMR (400\n668.06 ([M \u2212 PF6]+). 1H NMR (400 MHz, DMSO\u2011d6) \u03b4 8.83 \u2013 8.72 (m, MHz, DMSO\u2011d6) \u03b4 8.83 \u2013 8.57 (m, 4H), 8.34 (dt, J = 5.6, 1.2 Hz, 1H),\n1H), 8.68 (dt, J = 16.2, 6.6 Hz, 3H), 8.31 (dd, J = 5.9, 1.4 Hz, 1H), 8.22 \u2013 8.13 \u2013 7.87 (m, 6H), 7.87 \u2013 7.81 (m, 1H), 7.76 \u2013 7.69 (m, 1H), 7.60 \u2013\n8.09 (m, 1H), 8.05 (ddd, J = 15.7, 7.7, 1.6 Hz, 1H), 8.01 \u2013 7.91 (m, 4H), 7.32 (m, 7H), 6.99 (s, 1H), 6.62 (td, J = 8.9, 2.8 Hz, 1H), 6.02 (dd, J =\n7.86 \u2013 7.71 (m, 2H), 7.65 \u2013 7.55 (m, 1H), 7.52 \u2013 7.43 (m, 2H), 7.43 \u2013 9.3, 2.7 Hz, 1H), 3.85 (s, 3H), 3.82 (s, 3H). 13C NMR (100 MHz, DMSO) \u03b4\n\n\n 11\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\n\n\nFig. 10. Detection of ROS level after RuIQ-5 treatment. (A) ROS generation was determined by flow cytometry, A549 and A549/DDP cells stained with DCFH-DA\nafter RuIQ-5 complexes treatment for 12 h. (B) Microplate analysis of cellular ROS level by DCFH-DA staining after 1.0, 2.0 and 4.0 \u03bcM of RuIQ-5 treatment for 12 h.\n\u00d7 \u00b1 s, n = 3. #P < 0.05, ##P < 0.01, ###P < 0.001 vs A549 control, *P < 0.05, **P < 0.01, ***P < 0.001 vs A549/DDP control. (C), (D) Effect of NAC, GSH on ROS\ngeneration and cell viability. \u00d7 \u00b1 s, n = 3. ##P < 0.01, ###P < 0.001 vs A549 treatment group, **P < 0.01, ***P < 0.001 vs A549/DDP treatment group.\n\n\n163.78, 163.13, 160.61, 157.59, 156.99, 156.75, 154.98, 153.94, plates was pre-marked with a horizontal line passing through the bottom\n152.88, 151.17, 150.46, 149.98, 148.82, 143.96, 141.02, 137.17, center prior to cell seeding. Wounds were created perpendicular to the\n135.70, 134.84, 134.66, 133.39, 130.93, 130.84, 127.95, 127.46, lines by 10 \u03bcL tips, and detached cells were removed by washing with\n127.10, 124.22, 124.03, 123.92, 121.95, 120.32, 120.18, 119.61, PBS (pH 7.4). The cells were then treated with or without 1.0, 2.0, 4.0\n107.98, 107.75, 106.63, 104.98, 56.36, 56.14. UV\u2013Vis (\u03bb/nm, \u03bcM of complex RuIQ-5 for 24 h, and the images were recorded at 0 and\n\u03b5/M\u2212 1\u2219cm\u2212 1) (PBS): 296 (32900), 493 (7850). 24 h time point, respectively.\n\n4.3. Cell lines and cell culture conditions 4.7. 3D multicellular tumor spheroids (MCTSs) formation\n\n All human cancer cell lines studied in this work were obtained from MCTSs were cultured using the liquid overlay method [76]. A549 or\nthe American Type Culture Collection (ATCC, Manassas, VA). All cell A549/DDP cells in the exponential growth phase were dissociated by a\nlines were maintained in DMEM culture media supplemented with 10% trypsin/EDTA solution to obtain single-cell suspensions. About 2500\nFBS and incubated at 37 \u25e6 C in a 5% CO2 incubator unless otherwise diluted A549 or A549/DDP cells were transferred to 1% agarose-coated\nnoted. The IC50 values were determined by MTT assay according to our transparent 96-well plates with DMEM (200 mL) containing 10% FBS.\nprevious report [74]. A549 MCTSs and A549/DDP MCTSs formed in 4 days with diameters of\n approximately 400 \u03bcm at 37 \u25e6 C in the presence of 5% CO2. After the\n4.4. Stability assay MCTS formation in 96-well plates, cells were imaged using an inverted\n fluorescence microscope. The diameter of each tumor spheroid was\n The stability of the Ru(II)-IQ complexes were measured by UV/Vis calculated by Image J software.\nspectroscopy according to the methodology described in Zheng et al.\u2019\nwork [75]. The complexes were firstly dissolved in DMSO and then 4.8. Cytotoxicity assay of Ru(II)-IQ complexes towards 3D MCTSs\ndiluted with PBS. The UV/Vis spectra were recorded every 6 or 12 h for\n48 h. MCTSs were generated as mentioned above. On fourth day, 50% of\n the plating medium were carefully replaced by fresh DMEM/FBS me\u00ad\n4.5. Viability assay (MTT assay) dium containing different concentrations of RuIQ-5 (1.0, 2.0, 4.0 \u03bcM).\n In parallel, for the untreated group, 50% of the plating medium was\n The cell viability of A549, A549/DDP, HepG2, MCF-7, HBE cells after replaced by fresh medium without RuIQ-5. The cytotoxicities of the\ntreatment with different concentrations of Ru(II)-IQ complexes RuIQ-3, ruthenium complexes were assessed for A549 and A549/DDP cells on\nRuIQ-4 and RuIQ-5 for 48 h was measured by MTT assay [74]. the basis of ATP concentration using CellTiter-Lumi\u2122 Plus luminescent\n cell viability assay kit (Beyotime). The fluorescence of CellTiter-Lumi\u2122\n4.6. Antimetastatic properties Plus luminescent detection reagent was then measured by a microplate\n analyzer.\n The antimetastatic property of complex RuIQ-5 was evaluated by a\nwound-healing assay. A549 and A549/DDP cells were seeded in 6-well 4.9. Staining of MCTSs by calcein AM/PI\nplates (each well contained 2 mL of cell culture media) and allowed to\ngrow and form a confluent monolayer, respectively. Each well of the The cytotoxicity of RuIQ-5 in MCTSs was evaluated by calcein\n\n 12\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\n\n\nFig. 11. Akt/GSK3\u03b2/Fyn signaling pathway involved in RuIQ-5-induced apoptosis. (A) Western blot analysis of Akt/GSK3\u03b2/Fyn signaling pathway related proteins\nin A549 and A549/DDP cells after 24 h exposure of RuIQ-5 at indicated concentrations (1.0, 2.0, 4.0 \u03bcM). (B) Western blot analysis of nucleus Nrf2 and drug-\nresistance related proteins MRP1 and HO-1 in A549 and A549/DDP cells after 24 h exposure of RuIQ-5 at indicated concentrations (1.0, 2.0, 4.0 \u03bcM). (C) and\n(D) Immunofluorescence staining assay of Nrf2 in A549 and A549/DDP cells after 24 h RuIQ-5 exposures.\n\n\nacetoxymethyl ester (AM) and propidium iodide (PI) double staining. respectively. Observations were recorded using a DFC310 FX micro\u00ad\nFirstly, spheroids were treated with 1.0, 2.0, and 4.0 \u00b5M of RuIQ-5 for scope (Leica Microsystems CMS GmbH, Germany) every 24 h until the\n72 h. Treated spheroids were then stained with calcein AM (excitation at test ended. The ethical protocols used for the in vivo zebrafish embryo\n488 nm, emission at 515 nm) and PI (excitation at 535 nm, emission at study were performed in compliance with the ethical regulations of\n617 nm) and subsequently imaged directly using an inverted fluores\u00ad Guangdong Medical University. The experiment was repeated thrice.\ncence microscope.\n 4.11. Measurement of lipophilicity\n4.10. Zebrafish embryotoxicity test\n The distribution coefficient of each Ru(II)-IQ complex, which was\n Zebrafish embryos were provided by the Zebrafish Platform of presented as logPo/w values, was detected by the \u201cshake-flask\u201d method\nAffiliated hospital of Guangdong Medical University. Zebrafish embryos as previously described [50]. Briefly, a suitable quantity of a stock so\u00ad\nwere incubated in 12-well plates with 2 mL of sterile dechlorinated tap lution of a Ru(II)-IQ complex or cisplatin in aqueous NaCl was added to\nwater (SDTW) containing different concentrations (0, 1.7, 3.4, 6.8, 13.6, an equal volume of octanol, and the mixture was shaken at 25 \u25e6 C, 200\n27.2 \u03bcg/mL) of complex RuIQ-5 at 28.0 \u00b1 1.0 \u25e6 C. Twenty zebrafish rpm for 48 h to allow partitioning, followed by centrifugation at 3000\nembryos were assessed in each treatment (concentration) group, rpm for 10 min. The resultant aqueous layer was separated from the\n\n 13\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\n\n\n Fig. 12. Schematic illustration of the mechanism of RuIQ-5 in reversing cisplatin resistance in A549/DDP cells.\n\n\noctanol layer for Ru(II) or Pt analysis. The Ru(II) or Pt content in the BeyoClick EdU-594 cell proliferation assay kit (Beyotime). Firstly, A549\naqueous layer was measured by inductively coupled plasma mass and A549/DDP cells were seeded into 6-well plates and then treated\nspectrometry (ICP-MS) and used to calculate the LogPo/w values ac\u00ad with different concentrations (1.0, 2.0, and 4.0 \u00b5M) of RuIQ5 for 12 h,\ncording to the equation LogPo/w = Log ([Ru]o/[Ru]w). respectively. Then 10 \u03bcM EdU in 200 \u00b5L of DMEM/FBS medium was\n added to each well and the plates were incubated for 2 h. Then the cells\n4.12. Cellular uptake and localization were fixed with 4% paraformaldehyde for 15 min, washed twice with\n 3% bovine serum albumin (BSA), followed by cell permeabilization\n A549 and A549/DDP cells were cultured in 60 mm tissue culture using 0.5% TritonX-100 for 20 min. Subsequently, the cells were washed\ndishes for 12 h, and then treated with 2.0 \u03bcM of RuIQ-5 or cisplatin, with 3% BSA twice, and the Click-it reaction mixture was then added to\nrespectively. Twenty-four hours after treatment, cells were collected. In each well and incubated for 30 min, followed by washing with 3% BSA.\norder to investigate the localization of RuIQ-5 and cisplatin in cells, Cell nucleus staining was performed by adding Hoechst 33342 to each\ndifferent cellular fractions (nuclear, mitochondrial and cytoplasmic well and incubated for 30 min, followed by washing with PBS twice.\nfractions) of A549 and A549/DDP cells were extracted using Cell Finally, the cells were imaged under an inverted fluorescence\nMitochondria Isolation Kit. The dishes were digested with 3 mL of microscope.\nconcentrated nitric acid and 1 mL of perhydrol for 24 h, and then diluted\nto 5 mL with ultrapure water. Finally, the amount of RuIQ-5 complexes 4.16. Comet assay\nor cisplatin uptake by A549 and A549/DDP cells was determined by ICP-\nMS. Single-cell gel electrophoresis (SCGE), also known as comet assay, is\n a reliable method to detect DNA damage which is evidenced by the\n4.13. Studies on the mechanism of cellular uptake comet tails [74]. In this study, comet assay was performed to detect\n RuIQ5-induced DNA damage using the Comet assay reagent kit (Tre\u00ad\n A549 and A549/DDP cells were pretreated with endocytosis in\u00ad vigen) according to the manufacturer\u2019s instructions. DNA of A549 and\nhibitors (sodium azide, NaN3: 2.5 \u03bcM; 2-deoxy-D-glucose, 2-DOG: 10 A549/DDP cells was stained with acridine orange (AO, 20 \u03bcg/mL) and\nmM; sucrose: 20 mM; and nystatin: 2.5 \u03bcg/mL) for 2 h, followed by then photographed by an inverted fluorescence microscope.\ntreatment with 2 \u03bcM of RuIQ-5 for 6 h. The cells were then trypsinized\nand collected, then were determined using ICP-MS. 4.17. Apoptosis assay by AO/EB and Hoechst 33342 staining\n\n4.14. Analysis of cell cycle arrest by flow cytometry A549 and A549/DDP cells were incubated with different concen\u00ad\n trations (1.0, 2.0, and 4.0 \u00b5M) of RuIQ5, respectively. After 24 h, cell\n The cell cycle distribution was investigated by flow cytometry nuclei were counterstained with acridine orange/ethidium bromide\nanalysis as previously described [74,77,78]. A549 and A549/DDP cells (AO/EB) solution (100 \u03bcg/mL AO, 100 \u03bcg/mL EB) or Hoechst 33,342 (5\nwere treated with different concentration doses (1.0, 2.0, 4.0 \u03bcM) of \u03bcg/mL in PBS) for 10 min followed by washing twice with PBS. Cell\nRuIQ-5, respectively. After 24 h incubation, the cells were collected and images were then captured by an inverted fluorescence microscope.\nfixed in 75% ethanol at \u2212 20 \u25e6 C overnight. The cell pellets were then\ncentrifuged and washed with PBS twice, and subsequently stained with 4.18. Apoptosis assay by annexin V/PI double staining assay\nPI in the presence of RNase A (100 \u03bcM) for 30 min at 37 \u25e6 C in the dark.\nAnalysis of samples was performed using a flow cytometer. The Apoptosis assay was performed using AnnexinV-FITC Apoptosis\n Detection Kit as previously described [50]. A549 and A549/DDP cells\n4.15. Cell treatment with EdU were incubated with different concentrations (1.0, 2.0, and 4.0 \u00b5M) of\n RuIQ5, respectively. After 24 h, cells were harvested, washed twice with\n The anti-proliferation activities of RuIQ-5 was measured using a PBS, and re-suspended in 500 \u03bcL of binding buffer. The cell suspensions\n\n 14\n\fL. Chen et al. Bioorganic Chemistry 119 (2022) 105516\n\n\nwere stained with 5 \u03bcL of Annexin V-FITC and 10 \u03bcL of PI at room the Medical Scientific Research Foundation of Guangdong Province of\ntemperature for 15 min in the dark, and subsequently analyzed using a China (A2020414) and the University Student Innovation Experiment\nflow cytometer. Program.\n\n4.19. Measurement of mitochondrial membrane potential Appendix A. 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