A Rhein‐Based Rh(III) Arene Complex with Anti‐tumor Cell Proliferative Activity Inhibits RNA Demethylase FTO

{"full_text": " For submission: https://mc.manuscriptcentral.com/cjoc\n \u4e2d \u56fd \u5316 \u5b66 - An International Journal\n For published articles: https://onlinelibrary.wiley.com/journal/16147065\n\n\n\nCite this paper: Chin. J. Chem. 2022, 40, 1156\u20121164. DOI: 10.1002/cjoc.202100901\n\n\n\n\nA Rhein-Based Rh(III) Arene Complex with Anti-tumor Cell\nProliferative Activity Inhibits RNA Demethylase FTO\nLu Liu,\u2021,a,b Yaqiong Kong,\u2021,a Liang He,c Xiuxiu Wang,d Meng-Meng Wang,a Hongjiao Xu,b Cai-Guang Yang,b,e\nZhi Su,*,a Jing Zhao,*,d Zong-Wan Mao,*,c Yue Huang,*,b,e and Hong-Ke Liu*,a\na\n School of Chemistry and Materials Science, Nanjing Normal University, Nanjing, Jiangsu 210023, China\nb\n State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China\nc\n School of Chemistry, Sun Yat-Sen University, Guangzhou, Guangdong 510275, China\nd\n School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210023, China\ne\n School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences,\nHangzhou, Zhejiang 310024, China\n\n\n\nComprehensive Summary\n Metallodrugs with fine-tuned coordination between metals and bioactive ligands can achieve cytotoxic effects in cancer therapy and\n have been considered as a new approach for drug design. However, it has yet to be elucidated whether these metallodrugs target epi-\n transcriptomic proteins for gene expression regulation. This report describes a rhein-based Rh(III)-arene complex, Rh1, that exhibited\n promising antiproliferative effects in several tumor cell lines. Rh1 induced cell death through the autophagy, cell cycle arrest, and ac-\n 6 6\n cumulation of intracellular reactive oxygen species (ROS). In addition, Rh1 upregulated the global N -methyladenosine (m A) levels in\n A549 cells in the fat mass- and obesity-associated protein (FTO)-dependent manner. Collectively, the metal-based FTO inhibitor Rh1\n 6\n effectively suppressed tumor cell proliferation and modulated the abundance of cellular m A, highlighting the potential of met-\n al-based agents to target and regulate epitranscriptomics for tumor suppression.\n\n\n\n\nKeywords\n Rhodium | mRNA |Antiproliferation | FTO | m6A\n\n\n\n*E-mail: liuhongke@njnu.edu.cn, huangyue@simm.ac.cn, View HTML Article Supporting Information\ncesmzw@mail.sysu.edu.cn, jingzhao@nju.edu.cn, zhisu@njnu.edu.cn\n\u2021\n These authors contributed equally to this work.\n\n\n\n1156 \u00a9 2022 SIOC, CAS, Shanghai, & WILEY-VCH GmbH Chin. J. Chem. 2022, 40, 1156\u20141164\n\f 16147065, 2022, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cjoc.202100901 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nRh(III) Arene Complex Inhibits RNA Demethylase FTO Chin. J. Chem.\n\nBackground and Originality Content Furthermore, some metal complexes escaped and migrated to\n other organelles once stimulated by photoirradiation to produce\n [20]\n Coordination complexes incorporating metal ions with biolog- ROS. Therefore, such targeted luminescent metal complexes\nically active ligands have been broadly used as diagnostic tools, provide a structural foundation for the further design and optimi-\n [1] zation of metallodrugs.\nanticancer drugs, and bioimaging agents. Cisplatin, as a land-\nmark discovery of metal complexes, has been used clinically for In this study, rhein, a reported inhibitor for the fat mass- and\n [2] [21]\ntreating numerous human cancers. However, drug resistance obesity-associated protein (FTO), which was identified as a\nand undesirable side effects of cisplatin have led to other plati- critical demethylases involved in regulating cellular mRNA stability\n 6 [22]\nnum-containing anticancer drugs or combination therapies of by erasing m A from mRNA, was taken as the ligand and used\ncisplatin with other drugs being considered as novel strategies for to develop the first example of a rhein-based Rh(III) arene com-\n [3]\nchemotherapy. In recent years, coordination complexes with plex that maintained the inhibitory activity for FTO demethylation.\nseveral transition metals have attracted increasing attention be- The complex, termed Rh1, exhibited FTO-dependent antiprolifera-\n 6 6\ncause of their variable oxidation states, higher selectivity for can- tive activity by upregulating the global N -methyladenosine (m A)\n [4]\ncer cells, and iron mimicking properties. The ruthenium (Ru) levels in A549 cells. In addition, Rh1 induced cell death through\ncomplexes KP1019 and NAMI-A progressed to clinical trials, but autophagy, cell cycle arrest, and the generation of intracellular\n [5]\nthese were terminated for various reasons. Diverse approaches ROS. These findings offer a new insight of metal-based complexes\nsuch as novel scaffold incorporation, light activation, catalytic targeting the FTO demethylase for tumor suppression.\nmechanisms of action, and pH-dependent modulation of reactivi-\n [6]\nty have been applied for the design of metallodrugs. The half-\n Results and Discussion\nsandwich arene complexes coordinated with metal centers such\nas rhodium (Rh) and iridium (Ir) and ancillary ligands play syner- Chemical synthesis of the metal complexes Rh1 and Ru1\ngistic roles in fine-tuning the in vitro and in vivo anticancer prop-\n [7]\nerties of the compounds. However, further work is required to The half-sandwich organometallic Rh(III) complex Rh1 and\nunderstand the mechanisms of the anticancer activity. Ru(II) complex Ru1 were synthesized by refluxing the ligand\n 5 X X\n Early mechanistic studies demonstrated that the half-sand- Rhein-L with the dimer *(\u03b7 -Cp )RhCl2]2 (Cp = pentamethylcyclo-\n X 6\nwich metal-arene complexes interacted with the N7 of guanine, pentadienyl) (RhCp ) and *(\u03b7 -p-cymene)RuCl2]2 (Rucym), respec-\nsimilar to the interaction of cisplatin with DNA, and this was be- tively (Schemes 1 & S1). The ligand Rhein-L and complexes of Rh1\n [8-9] and Ru1 were fully characterized with proton nuclear magnetic\nlieved to be one of the main causes of the anticancer activity. 1\nRecent studies revealed that enzymes were the potential binding resonance ( H NMR) spectroscopy and electrospray ionization\n [10]\ntargets for the metal-arene complexes. Several classes of pro- mass spectrometry (ESI-MS) (Figures S1\u2014S3).\nteins with different roles, including kinases and DNA-repair pro- Photophysical properties of Rhein-L, Rh1, and Ru1 were stud-\nteins have been characterized as the targets of metallodrugs in ied in DMSO/H2O solution (V/V, 1 : 99) at room temperature, and\n [11]\nvitro and in vivo. Using an integrated proteomics-based target- the three samples displayed similar UV absorption and fluores-\nresponse profiling approach, a series of Ru(II)-arene (RAPTA) cence emission. The ligand Rhein-L exhibited maximum UV ab-\ncompounds were determined to inhibit thioredoxin reductase and sorption at 438 nm and a photoluminescence emission band at\n [12]\ncathepsin B, or mainly target the cytoskeletal protein plectin to 610 nm with excitation at 450 nm in the DMSO/H2O solution at\n [13]\naffect the motility of cancer cells. In addition, metal-based in- room temperature (Figure S4). The complexes Rh1 and Ru1 dis-\nhibitors have been developed to selectively target the histone played two obvious UV absorptions around 300 nm and 425 nm\ndeacetylases (HDACs) to modulate the epigenetic status and (Figure S5); the high-energy bands (\uf03c 320 nm) could be assigned\n [14]\nachieve the anticancer effect. to the ligand-centred (LC) transitions, while the relatively low-\n The rich photophysical properties and unique optical parame- energy band at ~425 nm could be ascribed to the contribution\nters of the metal complexes mean cellular imaging can be used to from both ligand-to-ligand charge transfer (LLCT) transitions and\nelucidate the various biological processes and in vivo trafficking of metal-to-ligand charge transfer (MLCT).\n [15]\nthese drugs. Subsequently, organelle-targeting strategies have\nbeen applied to improve the anticancer therapeutic effects by Rh1 exhibits enhanced cytotoxic activity versus its dimer and\ndelivering the metallodrugs to specific cell compartments such as ligand in tumor cell lines\n [16]\nthe nucleus, mitochondria, and lysosomes. For example, some In vitro cytotoxicity of Rh1 and Ru1, their dimers RhCpX and\nIr(III) complexes were reported to accumulate in lysosomes, lead- Rucym, respectively, and the ligand Rhein-L against tumor cells\ning to lysosomal damage and induction of autophagy or apopto- was assessed using the cell lines human lung carcinoma A549,\n [17]\nsis. Other metal complexes tended to locate in mitochondria cisplatin-resistant A549 (A549R), human breast cancer MCF-7,\nand induced caspase-dependent apoptosis by mitochondrial dam- epithelial ovarian carcinoma A2780, and acute promyelocytic\nage, cellular ATP depletion, mitochondrial respiratory inhibition, leukemia NB4. Cisplatin was set as a positive control. Rh1 exhibit-\n [18-19]\nand elevated production of reactive oxygen species (ROS). ed marked cytotoxicity towards the tested tumor cell lines and\n\nScheme 1 Chemical structures of Rhein-L, RhCpx dimer, Rh1 (RhCpX-Rhein), Ru-cym dimer, and Ru1 (Ru-cym-Rhein)\n\n\n\n\nChin. J. Chem. 2022, 40, 1156\u20141164 \u00a9 2022 SIOC, CAS, Shanghai, & WILEY-VCH GmbH www.cjc.wiley-vch.de 1157\n\f 16147065, 2022, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cjoc.202100901 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nConcise Report Liu et al.\n\n\n\nshowed preferred antiproliferative activity in A549 and NB4 cells,\nwith estimated IC50 values of 3.0 \u03bcmol/L and 2.3 \u03bcmol/L, respec-\ntively (Table S1). Rh1 also demonstrated the potency to overcome\nthe cisplatin resistance in A549R cells, with IC50 values of 3.0\n\u03bcmol/L and 8.8 \u03bcmol/L for A549 and A549R cells, respectively. In\ncontrast, the cytotoxic/antiproliferative effects of the ligand\nRhein-L were almost abolished in A549R cells (IC50 of 3.9 \u03bcmol/L\nfor A549 and 35.0 \u03bcmol/L for A549R, respectively). Ru1 exhibited\nonly moderate activity against tumor cells, with IC50 values greater\nthan 15 \u03bcmol/L for all tested cell lines. Compared with the RhCpX\ndimer, Rhein-L, and Ru1 complex, Rh1 displayed the best efficacy\nfor inhibiting the viability of A549 cells (Figure 1A). Furthermore,\nRh1 significantly inhibited colony formation in A549 cells (Figure\n1B). Based on these findings, Rh1 was selected for further inves-\ntigation.\n\n\n\n\n Figure 2 Rh1 accumulates in the lysosome and induces an autophagy\n phenotype in A549 cells. (A) Intracellular colocalization of Rh1 with\n LysoTracker Red (LTR) and Hoechst 33342. The excitation wavelengths for\n Rh1, LTR, and Hoechst 33342 were 405 nm, 357 nm, and 633 nm, respec-\n tively, and the emitted fluorescence was collected at 600 \u00b1 20 nm, 660 \u00b1\n 20 nm, and 460 \u00b1 20 nm, respectively. Scale bar, 10 \u00b5m. (B) LC3 protein\n expression in A549 cells after Rh1 treatment. LC3 expression was analyzed\nFigure 1 Rh1 exhibits an antiproliferative effect in A549 cells. (A) IC50 by western blotting (left panel) and quantified with Image J software (right\nvalues of Rh1 dimer (RhCpX), Rhein-L, Rh1, and cisplatin for inhibiting cell panel). (C) Immunofluorescence imaging of LC3 in A549 cells pre-treated\nviability of A549 cells. (B) Effects of Rh1 on colony formation of A549 cells. with 10 \u03bcmol/L Rh1 for 12 h. Scale bar, 20 \u03bcm. ****, p < 0.0001.\n\n\nRh1 accumulates in the lysosomes of A549 cells and leads to Rh1 induces the intracellular ROS generation and cell cycle\nautophagic cell death arrest\n To understand the subcellular accumulation of Rh1, immuno- The excessive generation of ROS is an important mechanism\nfluorescence staining was performed on A549 cells after incuba- for the metal-based anticancer agents and has also been reported\n [25]\ntion with Rh1. Fluorescence microscopy examination of the cells to induce autophagy. The generation of ROS in A549 cells was\nrevealed that Rh1 exhibited a green fluorescence that merged detected by using the fluorescent probe DCFH-DA (2\u2019,7\u2019-dichloro-\n [26]\nwith the lysosome dye LysoTracker Red (LTR), suggesting that Rh1 fluorescin diacetate). The number of fluorescence dots present\npredominantly localized in the lysosomes (Figure 2A). Autophagy with DCFH-DA probing was significantly increased in the A549\nis an evolutionarily conserved stress-responsive process that cul- cells exposed to Rh1 treatment (Figure 3A). Moreover, the fluo-\nminates with the lysosomal degradation of redundant or poten- rescence intensity was enhanced in a time-dependent manner\n [23]\ntially dangerous cytosolic entities. The accumulation of LC3 (Figure 3B). Thus, the Rh1 complex effectively induced the gener-\npuncta and the transformation from LC3-I to LC3-II were used as ation of intracellular ROS.\n [24]\ntypical biomarkers to monitor autophagic responses in the cells. Increasing evidence suggests that autophagy and the cell cycle\n [27]\nWestern blotting showed that endogenous LC3-II increased dose are coordinated and reciprocally regulated. The effect of Rh1\ndependently upon Rh1 treatment over 24 h in A549 cells (Figure treatment on cell cycle distribution in A549 cells was examined by\n2B), indicating that Rh1 induced an autophagy phenotype. Im- propidium iodide (PI)/RNase staining and subsequent flow cyto-\nmuno\ufb02uorescence assessment of LC3-II demonstrated that metry analysis. Rh1 could induce G0/G1 cell cycle arrest in a\nLC3-positive puncta were significantly elevated upon treatment dose-dependent manner (Figures 3C and S6) and the proportion\nwith Rh1, manifesting the emergence of autophagosome vacuoles of A549 cells in G0/G1 phase increased from 57.16% to 77.46%\n(Figure 2C). These observations suggested that treatment with after Rh1 treatment for 24 h. Collectively, these data demon-\nRh1 caused autophagic cell death in A549 cells. strated that Rh1 accumulates in lysosomes, induces ROS produc-\n\n1158 www.cjc.wiley-vch.de \u00a9 2022 SIOC, CAS, Shanghai, & WILEY-VCH GmbH Chin. J. Chem. 2022, 40, 1156\u20141164\n\f 16147065, 2022, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cjoc.202100901 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nRh(III) Arene Complex Inhibits RNA Demethylase FTO Chin. J. Chem.\n\n\n\n\n Figure 4 Effects of Rhein-L and Rh1 on FTO demethylation of m6A in vitro.\n (A and B) Detection of Rhein-L (A) and Rh1 (B) inhibition of FTO demethyl-\n ation on ssDNA using the restriction enzyme digestion assay. In PAGE im-\n age (left panel), the upper band is 39-nt DNA with dm6A incorporation,\n and the lower 22- and 17-nt bands represent the demethylated products\n after DpnII digestion. IC50 values of the inhibition curve were drawn using\n Image J quantification (right panel). Meclofenamic acid (MA) was used as a\n positive control of FTO inhibitor at 50 \u03bcmol/L.\n\nFigure 3 Rh1 induces excessive ROS production and cell-cycle arrest in as an oncogene in A549 cells, which was consistent with the re-\n [28-29]\nA549 cells. (A) Fluorescence microscopy images of intracellular ROS pro- ported results. Rh1 markedly suppressed the proliferation of\nduction in A549 cells treated with 10 \u03bcmol/L Rh1. Scale bar, 200 \u03bcm. (B) A549 cells but exhibited a much milder effect on A549 shFTO cells.\nTime-dependent ROS production measured with a fluorescence spec- The IC50 value of Rh1 for inhibiting the proliferation of A549 shNC\ntrometer. (C) Effects of Rh1 treatment on cell-cycle distribution using flow (negative control) cells was 3.3 \u03bcmol/L, while the IC50 values for\ncytometry. Rh1 on A549 shFTO-1 and shFTO-2 cells were 10.1 \u03bcmol/L and\n 10.4 \u03bcmol/L, respectively (Figure 5G). In addition, A549 shFTO\ntion, arrests the cell cycle at G0/G1 phase, and leads to cell death cells showed significant dose-dependent drug sensitivity against\nby autophagy in A549 cells. Rh1 (Figure 5H), suggesting that the inhibitory effect of Rh1 on\n the proliferation of cells was dependent on FTO protein.\nIdentification of the inhibition activity of Rh1 on FTO de- Further studies were performed to exclude the effects of Rh1\nmethylation on methylation of histone and genomic DNA. Rh1 only minimally\n Rhein was previously shown to be the first FTO inhibitor.\n [7] altered major histone methylations (Figure S8A) and genomic\nTherefore, the rhein-based complex Rh1 was examined to deter- 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)\nmine whether the complex retained the inhibitory activity on FTO modifications in A549 cells (Figure S8B). Collectively, Rh1 might\ndemethylation. The demethylation activity of FTO was completely achieve antitumor effects through targeting the FTO-mediated\n x\nabolished in the presence of 50 \u03bcmol/L RhCp dimer and Rh1, regulatory pathway of RNA demethylation rather than the epige-\nwhile Ru-cym dimer and Ru1 treatment displayed no inhibition netic histone or DNA demethylation.\nactivity under the same conditions (Figure S7A). The IC50 values of\n X\nRhein-L, Rh1, and the dimer RhCp , respectively, on FTO demeth- Conclusions\nylation were quantified to further determine the inhibitory effects\nof the compounds. The IC50 values of Rhein-L and Rh1 for the As a result of the clinical success of cisplatin in the early 1960s,\ninhibition of FTO demethylation were 20.9 \u03bcmol/L and 9.9 \u03bcmol/L, considerable progress has been made with metal complexes for\n X\nrespectively (Figures 4A and 4B). Meanwhile, the RhCp dimer biological application in the past half century. However, drug re-\nexhibited enhanced FTO inhibition activities compared with sistance and undesirable side-effects of cisplatin are major obsta-\nRhein-L or Rh1, with an IC50 value of 2.3 \u03bcmol/L (Figure S7). These cles in current clinical chemotherapy. To address these problems,\n x\nresults demonstrate that both RhCp dimer and Rhein-L contrib- novel elements such as new metal centers, ancillary ligands, and\nute to the inhibitory activity of FTO, and Ru1 does not show an flexible scaffolds have been applied to the design of metal com-\ninhibitory effect that may be attributed to Ru-cym dimer. plexes to improve the biological activities. Furthermore, metal\n substitution or novel scaffold of complexes may facilitate the clin-\nRh1 exhibits FTO-dependent antiproliferative activity via the\n ical introduction of the next generation of platinum or other met-\nFTO/m6A axis in A549 cells al-based anticancer agents. For the half-sandwich metal-arene\n 6\n To investigate whether Rh1 could alter the level of m A modi- complex, the arene moiety favors structural diversity and hydro-\n 6\nfication in A549 cells, the abundance of m A was investigated in phobicity, which not only provides additional functionalities but\nvitro by dot blot assay under dose-dependent conditions. As ex- also facilitates cellular uptake by ameliorating lipophilicity. In the\n 6\npected, the m A abundance in total RNA and mRNA markedly current study, a rhein-based Rh(III) arene complex, Rh1, was syn-\nincreased after Rh1 treatment (Figures 5A and 5B). To further thesized and found to significantly repress the proliferation of\ndetermine whether the inhibitory effect of Rh1 on cell prolifera- A549 cells, including those that were resistant to cisplatin (Figure\ntion relied on FTO, stable FTO knockdown (shFTO) A549 cells were 1 and Table S1). Similar to the classical metal complexes, Rh1 ex-\ngenerated with lentivirus-mediated shRNA. The efficacy of erted antiproliferative effects on A549 cells by accumulating in\nknockdown was confirmed by the expression levels of FTO protein lysosomes and inducing autophagy, arresting the cell cycle at\nand mRNA (Figures 5C and 5D). MTT and colony formation assays G0/G1 phase, and significantly increasing the level of intracellular\nrevealed that knockdown of FTO in A549 cells significantly sup- ROS (Figures 2 and 3). Rhein was originally used as a laxative and\npressed cell proliferation (Figures 5E and 5F), indicating FTO stomach drug and was reported as the first FTO inhibitor, so the\n\nChin. J. Chem. 2022, 40, 1156\u20141164 \u00a9 2022 SIOC, CAS, Shanghai, & WILEY-VCH GmbH www.cjc.wiley-vch.de 1159\n\f 16147065, 2022, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cjoc.202100901 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nConcise Report Liu et al.\n\n\n\n\nFigure 5 Rh1 exhibits FTO-dependent antiproliferative activity in A549 cells. (A and B) Dot blot assay determination of m6A abundance in total RNA (A)\nand mRNA (B) in A549 cells treated with Rh1 for 48 h. MB (methylene blue) represents loading control of RNA samples. (C and D) The FTO protein (C) and\nmRNA (D) levels in A549 cells infected with lentivirus carrying indicated shRNAs. shNC, control cells. shFTO-1 or shFTO-2, FTO-knockdown cells. (E) Effects\nof shFTO on A549 cell viability. (F) Effects of shFTO on colony formation of A549 cells. (G) IC 50 values of Rh1 for inhibiting cell viability in shNC and shFTO\nA549 cells. (H) Effects of indicated dosage of Rh1 on cell viability in shNC and shFTO A549 cells. ****, p < 0.0001; ***, p < 0.001.\n\n [34]\ninhibitory activity of Rh1 on FTO was then determined, indicating has yet to be elucidated. The current study successfully synthe-\nthat Rh1 may possess different mechanisms of action. As ex- sized a rhein-derived metal complex Rh1 with FTO inhibitory ac-\npected, Rh1 maintained the inhibitory activity towards FTO de- tivity. Knocking down FTO expression with lentivirus revealed that\nmethylase. In addition, the RhCpX dimer exhibited the best inhib- FTO downregulation repressed the proliferation and colony for-\nitory activity (Figures 4 and S7), which indicated the FTO inhibitory mation of A549 cells, which was similar to the effects of Rh1\n 6\ncapability of Rh1 may arise from the synergy of the ligand Rhein-L treatment. In addition, Rh1 significantly increased the m A abun-\nand RhCpX dimers. However, the Rucym dimer did not show the dance and exhibited FTO-dependent antiproliferative activity in\ninhibitory activity on FTO (the data are not shown) and inhibitory A549 cells. Collectively, Rh1 was shown to suppress the prolifera-\n 6\nmechanism for the RhCpX dimer was unclear. tion of A549 cells by targeting the FTO/m A axis. Although more\n Antiproliferative effects on A549 cells by accumulating in ly- systematic studies are warranted to verify the involvement of Rh1\nsosomes and inducing autophagy, arresting the cell cycle at G0/G1 in FTO-regulated targets or pathways, this study has provided\nphase, and significantly increasing the level of intracellular ROS evidence that metal complexes targeting epitranscriptomics hold\n(Figures 2 and 3). Rhein was originally used as a laxative and potential for tumor suppression.\nstomach drug and was reported as the first FTO inhibitor, so the\ninhibitory activity of Rh1 on FTO was then determined, indicating\nthat Rh1 may possess different mechanisms of action. As ex-\n Experimental\npected, Rh1 maintained the inhibitory activity towards FTO de-\n X Materials\nmethylase. In addition, the RhCp dimer exhibited the best inhib-\nitory activity (Figures 4 and S7), which indicated the FTO inhibitory The m6A-containing ssDNA substrate was synthesized in Gen-\ncapability of Rh1 may arise from the synergy of the ligand Rhein-L eray Company. The biological reagents for enzymatic assay such as\n X\nand RhCp dimers. However, the Ru-cym dimer did not show the Tris, \u03b1-ketoglutarate, (NH4)2Fe(SO4)2 and L-ascorbic acid were\ninhibitory activity on FTO (the data are not shown) and inhibitory purchased from Shanghai Bioengineering Company. The compo-\n X\nmechanism for the RhCp dimer was unclear. nents for cell cultures such as Dulbecco's Modified Eagle Medium\n Several specific or non-specific small-molecule inhibitors of (DMEM), RPMI1640 medium, Fetal Bovine Serum (FBS), penicillin/\nFTO have been identified and these can be classified into streptomycin and trypsin/EDTA solution were purchased from\n2-oxoglutarate (2OG)-competitive, substrate competitive, dual Gibco. The sodium of meclofenamic acid (MA) was obtained from\n2OG- and substrate-competitive, and unvalidated mechanisms TCI.\n [30]\ndepending on their mode of action. More recently, several\nsmall molecules, such as FB23-2, CS1, CS2, and Dac51, have been Instruments\ndeveloped that exhibit clinical potential for cancer therapy by ESI-MS was recorded by the Thermo LCQFLEET electrospray\n [31-33] 1\ntargeting FTO. However, to our knowledge, no metal complex mass spectrometer. H NMR was determined by Bruck AVANCE\nwith FTO inhibitory activity has been characterized to date. The 400 MHz spectrometer, and the UV-visible spectra were collected\ncatalytic nanoparticles OsSx-PEG NPs were recently shown to by Varian Cary 50 Probe UV-vis spectrometer. The fluorescence\nincrease the methylation level of mRNA, but the cellular target spectrum was performed by Perkin-Elmer LS55 fluorescence\n\n1160 www.cjc.wiley-vch.de \u00a9 2022 SIOC, CAS, Shanghai, & WILEY-VCH GmbH Chin. J. Chem. 2022, 40, 1156\u20141164\n\f 16147065, 2022, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cjoc.202100901 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nRh(III) Arene Complex Inhibits RNA Demethylase FTO Chin. J. Chem.\n\nspectrometer, and the digital circular second spectrometer was instructions and the absorbance was recorded at 450 nm. Each\nusing Chriascan instrument produced by Applied Photophysics well was in triplicate. The DMSO control was viewed as 100% and\nCompany in the United Kingdom. The multifunctional enzyme the IC50 value was calculated with nonlinear regression analysis\nlabeling instrument model was recorded by Tecan Infinite M1000 using equation in GraphPad Prism 8.\nPro, flow cytometry was SORP FACSARIA II, and the fluorescent\n Colony formation assay\nmicroscope model was Axio Observer. CHI 660E electrochemical\nwork station was used with a conventional three-electrode sys- A549 and A549R cells were seeded at a density of 1000\ntem. cells/well into a six-well plate and incubated at 37 \u00b0C for 12 d. The\n culture medium was refreshed every 3 d. Cells were fixed with 4%\nSynthesis of ligand Rhein-L paraformaldehyde for 15 min and then stained with 1% crystal\n The modified ligand (Rhein-L, 4, 5-dihydroxy-N-((4'-methyl-[2, violet for 10 min.\n2'-bipyridin]-4-yl)methyl)-9,10-dioxo-9,10-dihydroanthracene-2- Western blotting\ncarboxamide) was synthesized from 4-aminomethyl-4-methyl-2,2-\n A549 cells were seeded into 6-well plates and treated with the\nbipyridyl, which was prepared in a slightly modified way according\n [35-36] indicated concentrations of metal complex for 24 h at 37 \u00b0C. Cells\nto literature procedures. Rhein (0.5 mmol), EDCI (0.5 mmol),\n were washed three times with PBS and lysed in RIPA Lysis Buffer\nHOBt (0.4 mmol), 4-aminomethyl-4-methyl-2,2-bipyridyl (2.0\n with a protease inhibitor cocktail (C600387, Sangon Biotech) for\nmmol) and 10 \u03bcL triethylamine were mixed and kept at 0 \u00b0C for 10\n 30 min on ice. The lysed cells were centrifuged at 14 000 r/min for\nh. Yellow precipitation was obtained after water was added, and\n 30 min at 4 \u00b0C, and quantified by a BCA protein assay reagent kit\nfurther purified with the column chromatography (yield: 0.18 g,\n 1 (P0011, Beyotime). The protein samples were separated on 12%\n78%). H NMR (400 Hz, DMSO-d6) \u03b4: 11.93 (s, 2H), 9.66 (t, 1H),\n SDS-PAGE and transferred onto nitrocellulose membranes\n8.63 (d, 1H), 8.52 (d, 1H), 8.38 (s, 1H), 8.28\u20148.22 (m, 2H), 7.85\n (HATF0010, Millipore) for western blotting analysis. The primary\n(dd, 2H), 7.77 (d, 1H), 7.42 (t, 2H), 7.28 (d, 1H), 4.63 (d, 2H), 2.42\n antibodies used were FTO (ab124892, Abcam, 1 : 3000), LC3 rabbit\n(s, 3H).\n monoclonal-antibody (AL221, Beyotime, 1 : 1000), GAPDH mouse\nSynthesis of Rh1 and Ru1 monoclonal-antibody (60004-1-Ig, Proteintech, 1 : 5000). HRP\n 5 X X\n The dimers *(\u03b7 -Cp )RhCl2]2 (Cp = pentamethylcyclopentadi- conjugated Goat Anti-mouse (CW0103, Cwbio) or Anti-rabbit IgG\n 6\nenyl) or *(\u03b7 -p-cymene)RuCl2]2 (0.3 mmol) and Rhein-L (0.2 mmol) (CW0102, Cwbio) was used as secondary antibody.\nwere mixed and stirred in CH3OH at room temperature for 3 h. Fluorescence microscopy\nThe solution was further concentrated by the rotary vacuum For subcellular accumulation analysis, A549 cells were treated\nevaporation, and excessive NH4PF6 was added. The product could with 5.0 \u03bcmol/L Rh1 for 5 h and then co-incubated with 150\nbe filtered after frozen in the refrigerator. nmol/L Lyso-Tracker Red (LTR) or 10 \u03bcg/mL Hoechst 33342 at\n Rh1 (*(\u03b75-CpX)Rh(N^N-Rhein)Cl]PF6) was obtained as a yellow 37 \u00b0C for 15 min, respectively. Cells were washed with pre-chilled\n \u2012 + 1\npowder (Yield: 0.12g, 68%). ESI-MS: m/z 738.10 [Rh1\u2012PF6 ] . H PBS for three times and visualized immediately with the fluores-\nNMR (400 MHz, DMSO-d6) \u03b4: 11.95 (s, 2H), 9.77 (t, 1H), 8.92 (d, cence microscopy. The excitation wavelengths for Rh1, LTR and\n1H), 8.82 (d, 1H), 8.68 (s, 1H), 8.58 (s, 1H), 8.25 (d, 1H), 7.89 (s, Hoechst 33342 were set at 405 nm, 633 nm and 357 nm, respec-\n1H), 7.88\u20147.84 (m, 1H), 7.78 (dd, 2H), 7.72 (d, 1H), 7.44 (d, 1H), tively. And the emission wavelength was 600 \u00b1 20 nm for Rh1, 660\n 13\n4.77 (d, 2H), 2.69 (s, 3H), 1.66 (s, 15H). C NMR (101 MHz, DMSO- \u00b1 20 nm for LTR and 460 \u00b1 20 nm for Hoechst 33342, respectively.\nd6) \u03b4: 191.76, 181.60, 165.24, 161.81, 161.57, 154.17, 153.79, For LC3 immunofluorescence analysis, A549 cells were seeded\n153.47, 152.85, 152.42, 151.79, 141.29, 141.14, 138.07, 134.13, into 35 mm dishes and treated with 10 \u03bcmol/L Rh1 or DMSO for\n133.64, 126.49, 125.13, 124.76, 123.17, 122.40, 119.97, 118.31, 12 h, followed by washing twice with the serum-free medium.\n118.02, 116.50, 97.19, 21.20, 8.84. Then cells were fixed with 3.7% formaldehyde solution for 15 min\n Ru1 (*(\u03b76-p-cymene)Ru(N^N-Rhein)Cl]PF6) was obtained as a and washed with a PBS solution containing 0.2% TritonX-100 for\n 1\nyellow powder (Yeild: 0.11g, 65%). H NMR (400 MHz, DMSO-d6) \u03b4: another 15 min. Liquid was discarded and pre-diluted anti-LC3B\n9.72 (s, 1H), 9.44 (d, 1H), 9.36 (d, 1H), 8.60 (s, 1H), 8.51 (s, 1H), rabbit antibody (ab48394, Abcam) was incubated to cells for 2 h\n8.17 (s, 1H), 7.90 (s, 1H), 7.83\u20147.79 (m, 1H), 7.74 (d, 1H), 7.66 (dd, at room temperature. After incubation and washing, the\n2H), 7.41 (d, 1H), 6.19 (d, 2H), 5.94 (d, 2H), 4.75 (d, 2H), 3.17 (s, FITC-labelled goat anti-rabbit IgG was added at 37 \u00b0C for another 1\n2H), 2.59 (s, 3H), 2.33\u20142.26 (m, 1H), 2.15 (s, 3H), 0.95 (d, 6H). h. The supernatant was removed and the cells were stained with\nCell culture Hoechst 33342 for another 15 min. After washing twice with PBS,\n cells were observed immediately by fluorescence microscopy with\n A549 cells, cisplatin-resistant A549 (A549R) cells, A2780 cells\n the excitation wavelength set at 488 nm for FITC and 357 nm for\nand MCF-7 cells were cultured in DMEM with 10% fetal bovine Hoechst 33342. The emission wavelength was 530 \u00b1 20 nm for\nserum (FBS) (10099141, Gibco) and antibiotics. NB4 cells were\n FITC and 460 \u00b1 20 nm for Hoechst 33342, respectively.\ncultured in RPMI1640 supplemented with 10% FBS and antibiotics.\nAll the cells were maintained in a humidified 5% CO2 atmosphere ROS detection\nat 37 \u00b0C. A549 cells were seeded into 35 mm dishes and treated with\n the Rh1 complexes at required concentrations for different time\nIn vitro cytotoxicity determination\n 3\n points. The detection of ROS was performed in accordance with\n Approximately 5\u00d710 cells/well were seeded into 96-well plate the manufacturer\u2019s instructions (KGAF018, KeyGEN BioTECH).\nand cultured at 37 \u00b0C for 12 h. For the adherent cell lines, the Briefly, cells were stained with 10 \u03bcmol/L H2DCFDA in the dark for\nmedia were refreshed with indicated concentrations of the tested 30 min, and washed with PBS for three times to discard the dye\ncomplexes, dimers or the ligand. After treatment for 48 h, the cell solutions. The fluorescence intensity was measured immediately\nviability was determined by adding 10 \u03bcL MTT solution for 4 h. by the fluorescence microscopy or fluorescence spectrometer\nThe purple product was dissolved with DMSO reagent and rec- with an excitation wavelength at 488 nm and an emission wave-\norded at 490 nm with a microplate reader (Tecan Infinite M1000 length at 530 \u00b1 30 nm.\nPro). For the suspension NB4 cells, the tested compounds were\nimmediately added when cells were seeded. The CCK-8 kit Cell cycle analysis\n(MA0218, Meilunbio) was used according to the manufacturer\u2019s The cell cycle analysis was conducted as the manufacturer\u2019s\n\n\nChin. J. Chem. 2022, 40, 1156\u20141164 \u00a9 2022 SIOC, CAS, Shanghai, & WILEY-VCH GmbH www.cjc.wiley-vch.de 1161\n\f 16147065, 2022, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cjoc.202100901 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nConcise Report Liu et al.\n\n\n\nprotocol (KGA512, KeyGEN BioTECH). A549 cells were cultured in experiments.\n6-well plates and incubated with Rh1 (0, 0.5, 2.5, 5 \u03bcmol/L, re-\nspectively) for 24 h. Cells were collected and fixed 70% ethanol. Supporting Information\nAfter storage at 4 \u00b0C overnight, cells were centrifuged and washed\nthree times with cold PBS and resuspended in a 500 \u03bcL PBS buffer The supporting information for this article is available on the\ncontaining PI and RNase A (9 : 1) for 30 min in the dark. The sam- WWW under https://doi.org/10.1002/cjoc.202100901.\nples were analyzed by a flow cytometry and the data were pro-\ncessed using the ModFit LT 2.0 software (Verity Software House,\nUSA). Acknowledgement\nProtein expression and purification We thank NSFC (Nos. 22077066, 21771109, 21778033,\n The N-terminal 31 residues truncated FTO gene was sub- 21701195, 21837006, 21977052 and 21907101).\ncloned into pET28a vector, encoding a His-tag fusion FTO protein.\nThe expression and purification of FTO were modified from pre- References\n [37]\nviously reported methods. The protein was purified by Nickle-\naffinity chromatography, followed by a superdex 200 gel filtration [1] Ko, C.-N.; Li, G.; Leung, C.-H.; Ma, D.-L. Dual function luminescent\n(GE Healthcare). Fractions were collected and checked by 12% transition metal complexes for cancer theranostics: The combination\nSDS-PAGE. The proteins were stored at \u201280 \u00b0C for further bioas- of diagnosis and therapy. Coord. Chem. Rev. 2019, 381, 79\u2012103.\nsays. [2] Rosenberg, B.; Van Camp, L.; Krigas, T. Inhibition of Cell Division in\n Escherichia coli by Electrolysis Products from a Platinum Electrode.\nInhibition of FTO demethylation in vitro\n Nature 1965, 205, 698\u2012699.\n The PAGE-based assay for inhibition of FTO demethylation in [3] Peng, K.; Liang, B.-B.; Liu, W.; Mao, Z.-W. What blocks more anti-\n [37-38]\nvitro was performed as previously described. A reaction mix- cancer platinum complexes from experiment to clinic: Major prob-\nture, containing 0.5 \u03bcmol/L FTO, 1 \u03bcmol/L 39-nt ssDNA lems and potential strategies from drug design perspectives. Coord.\n 6\n(5\u2019-ATTGCCATTCTCGATAGG(m A)TCCGGTCAAACCTAGACGAA-3\u2019), Chem. Rev. 2021, 449, 214210.\n300 \u03bcmol/L \u03b1-ketoglutarate, 280 \u03bcmol/L (NH4)2Fe(SO4)2, 2 mmol/L [4] Zhu, X.; Su, Q.; Feng, W.; Li, F. Anti-Stokes shift luminescent materials\nL-ascorbic acid and inhibitors at required concentrations in 50 for bio-applications. Chem. Soc. Rev. 2017, 46, 1025\u20121039.\nmmol/L Tris-HCl, pH 7.5, was incubated at room temperature for 2 [5] Bergamo, A.; Gaiddon, C.; Schellens, J. H.; Beijnen, J. H.; Sava, G. Ap-\nh. The reaction was terminated by heating at 65 \u00b0C for 15 min. The proaching tumour therapy beyond platinum drugs: status of the art\nssDNA was annealed to the complementary strand and then sub- and perspectives of ruthenium drug candidates. J. Inorg. Biochem.\njected to the DpnII enzyme digestion (R0543L, NEB). The digested 2012, 106, 90\u201299.\ndsDNA was checked on 15% native PAGE with GelRed staining to [6] Yousuf, I.; Bashir, M.; Arjmand, F.; Tabassum, S. Advancement of\nestimate the percentage of inhibition. metal compounds as therapeutic and diagnostic metallodrugs: Cur-\n rent frontiers and future perspectives. Coord. Chem. Rev. 2021, 445,\nRNA extraction and m6A dot blot\n 214104.\n A549 cells were incubated with indicated concentrations of [7] Das, U.; Kar, B.; Pete, S.; Paira, P. Ru(ii), Ir(iii), Re(i) and Rh(iii) based\nRh1 for 48 h. Total RNA was extracted with TRIzol reagents complexes as next generation anticancer metallopharmaceuticals.\n(15596018, Thermofisher Scientific) and mRNA was then isolated Dalton Trans. 2021, 50, 11259\u201211290.\nfrom total RNA using Dynabeads mRNA DIRECT kit (61012, Ther- [8] Liu, H.-K.; Sadler, P. J. Metal Complexes as DNA Intercalators. Acc.\nmofisher Scientific) in accordance with the manufacturer\u2019s proto- Chem. Res. 2010, 44, 349\u2012359.\ncols. RNA samples were denatured and then spotted onto an Im- [9] Liu, H.-K.; Berners-Price, S. J.; Wang, F.; Parkinson, J. A.; Xu, J.; Bella,\nmobilon-Ny + membrane (INYC00010, Millipore). After complete J.; Sadler, P. J. Diversity in Guanine-Selective DNA Binding Modes for\ndrying, RNA samples were crosslinked by an UV irradiation for 3 an Organometallic Ruthenium Arene Complex. Angew. Chem. Int. Ed.\nmin. The membrane was then blocked with 5% non-fat dry milk in 2006, 118, 8333\u20128336.\n1\u00d7PBST buffer for 1 h at room temperature and subsequently [10] Lomzik, M.; Hanif, M.; Budniok, A.; Blauz, A.; Makal, A.; Tchon, D. M.;\n 6\nincubated with rabbit anti-m A antibody (ab151230, Abcam) at Lesniewska, B.; Tong, K. K. H.; Movassaghi, S.; Sohnel, T.; Jamieson, S.\n4 \u00b0C overnight. After three washes with PBST, the membrane was M. F.; Zafar, A.; Reynisson, J.; Rychlik, B.; Hartinger, C. G.; Plazuk, D.\nincubated with anti-rabbit IgG antibody for 1 h at room tempera- Metal-Dependent Cytotoxic and Kinesin Spindle Protein Inhibitory\nture, then washed for another three times in PBST. Finally, the Activity of Ru, Os, Rh, and Ir Half-Sandwich Complexes of Ispinesib-\nmembrane was developed with Amersham ECL Prime Western Derived Ligands. Inorg. Chem. 2020, 59, 14879\u201214890.\nBlotting Detection Reagent (RPN2232, GE Healthcare) and stained [11] Zhang, S.; Zhong, X.; Yuan, H.; Guo, Y.; Song, D.; Qi, F.; Zhu, Z.; Wang,\nwith 0.1% methylene blue (MB) as loading control. X.; Guo, Z. Interfering in apoptosis and DNA repair of cancer cells to\n conquer cisplatin resistance by platinum(iv) prodrugs. Chem. Sci.\nStable cell line generation 2020, 11, 3829\u20123835.\n [31]\n Lentivirus was generated as described previously. Briefly, [12] Casini, A.; Gabbiani, C.; Sorrentino, F.; Rigobello, M. P.; Bindoli, A.;\n1.5 \u00b5g pMD2.G, 0.9 \u00b5g pMDLg/pRRE, 2.1 \u00b5g pRSV-Rev, and 5.4 \u00b5g Geldbach, T. J.; Marrone, A.; Re, N.; Hartinger, C. G.; Dyson, P. J.;\nshRNA plasmids pLKO.1-shFTO or pLKO.1-shNC constructs were Messori, L. Emerging Protein Targets for Anticancer Metallodrugs In-\nco-transfected into HEK293T/17 cells with the Lipo2000 transfec- hibition of Thioredoxin Reductase. J. Inorg. Biochem. 2008, 51, 6773\u2012\ntion reagent (11668500, ThermoFisher) according to the manu- 6881.\nfacturer\u2019s instructions. The target sequences for FTO knockdown [13] Meier, S. M.; Kreutz, D.; Winter, L.; Klose, M. H. M.; Cseh, K.; Weiss,\nwere listed in Table S2. The lentivirus particles were harvested 48 T.; Bileck, A.; Alte, B.; Mader, J. C.; Jana, S.; Chatterjee, A.;\nh and 72 h post transfection, directly pipetted into A549 cells in Bhattacharyya, A.; Hejl, M.; Jakupec, M. A.; Heffeter, P.; Berger, W.;\nthe presence of 6 \u03bcg/mL polybrene (H9268, Sigma-Aldrich). Finally, Hartinger, C. G.; Keppler, B. K.; Wiche, G.; Gerner, C. An Organo-\n5 \u03bcg/mL puromycin (P8833, Sigma-Aldrich) was added into the ruthenium Anticancer Agent Shows Unexpected Target Selectivity\nA549 cells 48 h post incubation to select the positive infected-cells. For Plectin. Angew. Chem. Int. Ed. 2017, 56, 8267\u20128271.\nTo avoid cellular compensation of knockdown efficiency, only [14] Kaur, M.; Loveleen; Kumar, R. Inhibition of histone deacetylases,\nfreshly infected cell lines (fewer than 6 passages) were used in the topoisomerases and epidermal growth factor receptor by metal-\n\n1162 www.cjc.wiley-vch.de \u00a9 2022 SIOC, CAS, Shanghai, & WILEY-VCH GmbH Chin. J. Chem. 2022, 40, 1156\u20141164\n\f 16147065, 2022, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cjoc.202100901 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. 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F.; Feng, Y.\n[26] Cheng, F.; Huang, L.; Wang, H.; Liu, Y.; Kandhadi, J.; Wang, H.; Ji, L.; Q.; Zhang, X. L.; Yang, C. G.; Zhou, X. Fluorescein Derivatives as Bi-\n Liu, H. Photodynamic Therapy Activities of 10-(4-Formylphenyl)-5,15- functional Molecules for the Simultaneous Inhibiting and Labeling of\n bis(pentafluorophenyl)corrole and Its Gallium Complex. Chin. J. Chem. FTO Protein. J. Am. Chem. Soc. 2015, 137, 13736\u201213739.\n [39]\n 2017, 35, 86\u201292.\n[27] Zheng, K.; He, Z.; Kitazato, K.; Wang, Y. Selective Autophagy Regu- [40]\n lates Cell Cycle in Cancer Therapy. Theranostics 2019, 9, 104\u2012125. Manuscript received: December 20, 2021\n[28] Shi, H.; Zhao, J.; Han, L.; Xu, M.; Wang, K.; Shi, J.; Dong, Z. Retro- Manuscript revised: January 17, 2022\n spective study of gene signatures and prognostic value of m6A Manuscript accepted: January 19, 2022\n regulatory factor in non-small cell lung cancer using TCGA database Accepted manuscript online: January 25, 2022\n and the verification of FTO. Aging-US 2020, 12, 17022\u201217037. Version of record online: February 12, 2022\n\n\n\n\nChin. J. Chem. 2022, 40, 1156\u20141164 \u00a9 2022 SIOC, CAS, Shanghai, & WILEY-VCH GmbH www.cjc.wiley-vch.de 1163\n\f16147065, 2022, 10, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cjoc.202100901 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n\n\n\n\n Chin. J. Chem. 2022, 40, 1156\u20141164\n Liu et al.\n\n\n\n\n Second Row: (left) Xiu-Xiu Wang, Jing Zhao, (middle) Yue Huang, Cai-Guang Yang, (right) Meng-Meng Wang, Zhi Su\n First Row: (left to right) Lu Liu, Yaqiong Kong, Hong-Jiao Xu, Liang He, Zong-Wan Mao, Hong-Ke Liu\n\n\n\n\n \u00a9 2022 SIOC, CAS, Shanghai, & WILEY-VCH GmbH\n www.cjc.wiley-vch.de\n Concise Report\n\n\n\n The Authors\n\n\n\n\n 1164\n\f", "pages_extracted": 9, "text_length": 66190}