Mitochondria-Accumulating Rhenium(I) Tricarbonyl Complexes Induce

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Biological and Medical Applications of Materials and Interfaces

Mitochondria-Accumulating Rhenium(I) Tricarbonyl Complexes Induce Cell Death via Irreversible Oxidative Stress and Glutathione Metabolism Disturbance Fang-Xin Wang, Jin-Hao Liang, Hang Zhang, Ze-Hua Wang, Qin Wan, Cai-Ping Tan, Liang-Nian Ji, and Zongwan Mao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01057 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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ACS Applied Materials & Interfaces

Mitochondria-Accumulating Rhenium(I) Tricarbonyl Complexes Induce Cell Death via Irreversible Oxidative Stress and Glutathione Metabolism Disturbance Fang-Xin Wang, Jin-Hao Liang, Hang Zhang, Ze-Hua Wang, Qin Wan, Cai-Ping Tan,* LiangNian Ji and Zong-Wan Mao* MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun YatSen University, Guangzhou 510275, P. R. China KEYWORDS: rhenium(I) complexes, apoptosis, necroptosis, mitochondrial dysfunction, oxidative stress, glutathione metabolism

ABSTRACT Mitochondria play a critical role in tumorigenesis. Targeting mitochondria and disturbing related events has been emerging as a promising way for chemotherapy. In this work, two binuclear rhenium(I) tricarbonyl complexes of the general formula [Re2(CO)6(dip)2L](PF6)2 (dip = 4,7-diphenyl-1,10-phenanthroline; L = 4,4’-azopyridine (ReN) or 4,4’-dithiodipyridine (ReS)) were synthesized and characterized. ReN and ReS can react with glutathione. They exhibit good in vitro anticancer activity against cancer cell lines screened. Besides, they can target mitochondria, cause oxidative stress, and disturb glutathione metabolism. Both ReN and ReS can induce necroptosis and caspase-dependent apoptosis simultaneously. We also demonstrate that ReN and ReS can inhibit tumor growth in nude mice bearing carcinoma xenografts. Our study

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shows the potential of Re(I) complexes as chemotherapeutic agents to kill cancer cells via a mitochondria-to-cellular redox strategy.

1 Introduction Compared with normal cells, aberrant cancer cells evade growth suppressors and possess a much higher replication rate.1 In order to maintain robust proliferation, biosynthesis and energy production by mitochondria are often upregulated in cancer cells, which is regarded as metabolic reprogramming.1-2 This alteration is responsible for elevated reactive oxygen species (ROS), as well as excessive reductant species as counterparts.3 Cancer cells adapt to microenvironmental changes by regulating related metabolites and enzymes. There are increasing studies demonstrating that targeting mitochondria and interfering redox homeostasis is a potentially effective method for chemotherapy.4-6 Since the discovery of cisplatin (CDDP), metallodrugs have been playing an important role in chemotherapy. In clinics, approximately 50% of chemotherapeutic treatments involve Pt-based drugs.7 However, Pt-based drugs can induce inevitable side effects and drug resistance.8-9 Rhenium complexes have attracted attention of researchers, because of their intrinsic properties advantageous for chemotherapy. Firstly, the cell death mechanisms induced by Re complexes are diverse, such as apoptosis, paraptosis and necroptosis.10-12 This may overcome some limitations associated with existing drugs. Secondly, they are applicable for photodynamic therapy (PDT) and photoactivatable therapy through CO-releasing mechanisms, which can be activated by proper irradiation in tumor regions.13-14 Besides, Re tricarbonyl complexes are used for bioimaging through fluorescence or vibration, which are helpful to visualize cellular distribution, and the mechanism of action.15-16 Except “cold” Re complexes, “hot” radioactive 186/188Re complexes and


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analogues 99mTc complexes show good biosafety, and have already been applied in clinics.17 Some Re complexes that can interact with biomolecules, such as DNA and proteins. Che and co-workers reported that Re complexes with pyridine-triazine ligand can bind to DNA minor groove with a preference for AT-rich sequences.18 Metzler-Nolte and co-workers reported that Re complexes with imine ligands can act as potential inhibitors of GSK-3β kinase.19 Our group conjugated Re complexes with small enzyme inhibitors, such as Vorinostat (SAHA, histone deacetylase inhibitor) and dichloroacetate (DCA, pyruvate dehydrogenase kinase inhibitor), to improve the therapeutic effects.10,

20-23

However, in most cases, the anticancer mechanisms of rhenium complexes are

unclear, which includes the specific cell death modes, the effects on various intracellular homeostasis (e.g., redox and metabolism), the impacts on cell signaling pathways, and their specific biomolecular targets. Understanding how the rhenium complexes work is beneficial for further clinical application. In this work, two binuclear Re(I) tricarbonyl complexes of the general formula [Re2(CO)6(dip)2L](PF6)2 (dip = 4,7-diphenyl-1,10-phenanthroline ; L = 4,4’-azopyridine (ReN) or 4,4’-dithiodipyridine (ReS)) were synthesized and characterized. The highly lipophilic rhenium moiety is expected to improve cellular uptake efficacy and mitochondria-targeting property, and the azo/disulfide bond in the bridge linkage group can react with reductant.24 The anticancer activities were investigated against several cancer cell lines in vitro and a nude mouse xenograft model in vivo. The anticancer efficacies including mitochondrial dysfunction, oxidative stress, glutathione metabolism disturbance, and cell death mode have been studied. In all, our study reveals that ReN and ReS can target mitochondria, influence redox homeostasis, and show their potential as chemotherapeutic agents.


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2 Results and Discussions 2.1 Synthesis and Characterization ReN and ReS were synthesized by a general method as described in Scheme S1.20 Briefly, the precursor [Re(dip)(CO)3(CH3CN)](PF6) and the bridging ligand (4,4’-azopyridine or 4,4’dithiodipyridine) were refluxed at a 2:1 molar ratio in tetrahydrofuran (THF) overnight in the dark under an inert atmosphere with nitrogen. The product was evaporated to remove the solvent, and then purified by silica flash column chromatography with CH2Cl2/CH3OH as eluent. The products were recrystallized in CH3CN/ethyl ether. ReN and ReS were characterized by ESI-MS, 1H NMR spectroscopy,

13C

NMR spectroscopy, and elemental analysis (Figure S1‒S8). The structure of

ReN (CCDC NO. 1828899) was also characterized by single crystal X-ray diffraction method (Figure 1B, Table S1 and S2). The purity was further proved by powder X-ray diffraction (Figure S9). ReN is in a trans isomerism, with Re atoms coordinate in a distorted octahedral geometry and CO ligands arrange in a facial fashion.


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Figure 1. A) Chemical structures of ReN and ReS. B) X-ray crystal structure of ReN. The thermal ellipsoids are drawn at a 30% probability level (symmetry code; A = 2-x, -y, 1-z). Hydrogen atoms, counter ion (PF6‒) and solvent molecule (CH3CN) are omitted for clarity. 2.2 Photophysical Property Most Re(I) tricarbonyl complexes possess long-lived phosphorescence, large Stokes shifts and high quantum yields, which is advantageous for intracellular sensing and bioimaging.25-26 ReN and ReS possess intense absorption bands at around 260‒320 nm that can be assigned to intraligand (π→π*) transitions, as well as relatively weak bands at 320‒450 nm that attribute to metal to ligand charge-transfer (MLCT) process (Figure 2A). They exhibit yellow to red emission in CH2Cl2 upon excitation at 405 nm (Figure 2B). Compared with ReN, ReS displays a higher emission quantum yield and a longer lifetime (Table S3). Besides, ReS possesses a better emission capability in vivo after the intratumoral injection (Figure 2C).

Figure 2. A) UV-vis absorption spectra and B) normalized fluorescence emission spectra of ReN and ReS at 10 μM in CH2Cl2 at 25 °C. The excitation wavelength is 405 nm. C) Fluorescence emission of ReN and ReS in vitro and in vivo (5 min and 1 h after injection, 1 mg/kg). ReN and


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ReS were dissolved in PET diluent (6% polyethylene glycol 400, 3% ethanol and 1% Tween 80 in PBS). The excitation wavelength is 430 nm, and the maximum emission wavelength is 600 nm. 2.3 Reaction with Glutathione (GSH) GSH is the most abundant reductant with a concentration of 2‒10 mM in cells.27 Azo bond and disulfide bond have been applied for designing prodrugs and hypoxia probes.24,

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After the

addition of GSH, the mass-to-charge peak shifts from 694.75 to 695.90 for ReN, and from 712.15 to713.45 for ReS (Figure S10 and S11). Besides, the alteration of chemical shifts in 1H NMR also proves the redox reaction (Figure S12 and S13). Azo bond is reduced to NH−NH, and disulfide bond breaks to form thiol. This redox reaction can also be detected by photophysical behaviors. As Figure 3A shows, the absorption spectra of ReN and ReS change within 20 min after the addition of GSH (20 μM). The half-life is less than 1 min for both Re complexes, which indicates the reaction processes rapidly (Figure S14). The emission intensities of ReN and ReS increase by 7.4-fold and 1.3-fold, respectively (Figure 3B). The fluorescence lifetimes vary from 93 ns to 121 ns for ReN, and from 108 ns to 123 ns for ReS (Figure 3C).


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Figure 3. A) The absorption spectra of ReN and ReS (20 μM) after reaction with GSH (20 μM). B) The fluorescence emission spectra and C) the fluorescence lifetimes of ReN and ReS (20 μM) with or without GSH (1 mM) in H2O. The excitation wavelength is 405 nm. 2.4 Cytotoxicity in Vitro As reported in literature, the cytotoxicity of Re complexes can be tuned by ligands and chemical structures, which varies from 0.1 μM to over 100 μM.29 Herein, the cytotoxicity of ReN and ReS was evaluated in A549 (human lung adenocarcinoma epithelial), A549R (cisplatin-resistant A549), HeLa (human cervical cancer), HepG2 (human hepatocellular liver carcinoma), MCF-7 (human breast adenocarcinoma), U2SO (human osteosarcoma), PC3 (human prostate cancer) cells and LO2 (human normal liver) cells (Table 1). The IC50 values of ReS against the cancer cell lines tested fall between 0.8 and 2.1 μM, which are lower than those obtained for CDDP. ReN is more toxic towards most of the cancer cell lines than cisplatin except for PC3 and HepG2. ReN and ReS possess higher anticancer activities than most reported Re-based anticancer agents.29 Notably, they can also kill A549R cells effectively, which indicates they may overcome CDDP-induced resistance. Besides, ReN and ReS can also inhibit colony formation in vitro (Figure 4). Table 1. IC50 values (μM) of tested complexes towards different cell lines in vitro.a Complex Cell line

Tissue ReN

ReS

CDDP

U2SO

Bone

1.4 ± 0.1

1.1 ± 0.1

10.7 ± 0.9

HeLa

Cervix

1.6 ± 0.1

1.8 ± 0.1

9.2 ± 0.1

A549

Lung

1.1 ± 0.1

0.8 ± 0.1

9.6 ± 0.8


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A549R

Lung

8.3 ± 0.1

2.1 ± 0.1

51.3 ± 1.9

MCF-7

Breast

11.8 ± 1.3

1.5 ± 0.1

14.2 ± 0.8

PC3

Prostate

> 25

1.1 ± 0.1

10.5 ± 0.3

HepG2

Liver

> 25

1.6 ± 0.1

9.0 ± 0.1

LO2

Liver

7.5 ± 0.1

2.0 ± 0.1

15.7 ± 0.6

IC50 value is the drug concentration that is necessary for 50% inhibition of cell viability. Data

are presented as means ± standard deviations obtained in at least three independent experiments.

Figure 4. Colony formation inhibition in HeLa cells. Cells were treated with ReN, ReS and CDDP at indicated concentrations for 24 h, and then incubated with fresh media for another 7 d. NC represents untreated cells. 2.5 Induction of Mitochondrial Dysfunction Mitochondria are pivotal organelles for energy production, biosynthesis, metabolism, and cell death.5 As ReN and ReS can emit phosphorescence, it is easy to track their cellular distribution. As Figure 5A shows, the fluorescence of ReN and ReS overlay with that of MitoTracker Deep


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Red (MTDR) with high Pearson’s colocalization coefficiencies (PCC= 0.82 for ReN, and PCC = 0.75 for ReS). ICP-MS measurement also shows that ReN and ReS can accumulate in mitochondria (Figure 5B). The tubular mitochondria become swollen and globular after treated with ReN and ReS for 2 h (Figure 5C). The mitochondrial membrane potential (MMP) also decreases in a dose-dependent way (Figure S15). At a concentration of 8 μM, the percentage of cells with depolarized mitochondria increases from 5.7 ± 0.3% (NC) to 54.2 ± 2.1% (ReN) and 77.4 ± 2.5% (ReS). These results indicate that ReN and ReS can target and depolarize mitochondria. There are thousands of copies of the mitochondrial DNA (mtDNA) in the mitochondrial genome.30 Compared with nuclear DNA, mtDNA mutates at a higher rate in various cancers to alter mitochondrial metabolism, enhance tumorigenesis, and adapt to microenvironment.31 Herein, the damage of mtDNA was detected by real-time quantitative polymerase chain reaction (real-time qPCR). After treated with ReN and ReS at 8 μM for 6 h, the relative copy number of mtDNA decreases to 10.7 ± 6.6% and 10.8 ± 0.9%, respectively. It is important to monitor the mitochondria status and the respiration-related activity.32 The mitochondrial respiration profile of HeLa cells is shown in Figure 5E. Cells treated with ReN and ReS display a dose-dependent decrease on oxygen consumption rate (OCR). ATP production and basal/maximal/non-mitochondrial respiration are diminished (Figure S16). Besides, the proton leak shows a dose-dependent increase, which impacts on mitochondrial coupling efficiency and ROS. The expression of 13 genes encoded in mtDNA is down-regulated after treatment, and these genes are the essential subunits of oxidative phosphorylation system (Figure 5F). Among them, COX1, COX2 and COX3 (cytochrome c oxidase, Complex IV) are the most impacted genes. Complex IV is the final enzyme in the electron transport chain of respiration that is closely related


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to MMP maintenance and ATP synthesis.33 These results elucidate that ReN and ReS can impair mitochondrial bioenergetics status and cellular respiration.

Figure 5. A) Representative colocalization graphs of ReN and ReS with MTDR in HeLa cells. Cells were labeled with MTDR (150 nM, 30 min) and incubated with ReN and ReS at 8 μM for 1 h. Re complexes are excited at 405 nm, and MTDR is excited at 633 nm. Emission is collected at 550‒700 nm for Re complexes, and 650‒750 for MTDR. Scale bars: 20 μm. B) The contents of Re element in mitochondria and cytosol measured by ICP-MS. Cells were treated with ReN and ReS at 10 μM for 1 h. C) Mitochondrial morphological changes in HeLa cells. Cells were labeled with MTDR (150 nM, 30 min) and incubated with ReN and ReS at 8 μM for 2 h. Scale bar: 10 μm. D) Relative copy number of mtDNA in HeLa cells. Cells are incubated with ReN and ReS at 8 μM for 6 h. **p < 0.01. E) Mitochondrial respiration OCR profile of HeLa cells. Cells were treated with ReN or ReS at indicated concentrations for 6 h. Cells were treated with oligomycin (oligo, 1 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.125 μM), and the combination of rotenone (rot, 0.5 μM) and antimycin A (AA, 0.5 μM) by sequence. F) The impact


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of ReN and ReS on the transcription of mitochondrial genes. HeLa cells were incubated with Re complexes (8 μM) for 6 h. 2.6 Induction of Oxidative Stress Cancer cells are frequently under persistent oxidative stress.34 Excessive ROS can accelerate tumorigenesis and alter metabolic pathways, while lethal level of ROS can trigger cell death.35 The impact of ReN and ReS on primary ROS was detected by 2’,7’-dichlorofluorescin-diacetate (DCFH-DA) staining. ReN and ReS can induce a 3.8-fold and 4.1-fold ROS increase after incubation for 2 h (Figure 6A), respectively. Superoxide (O2•-) is also increasing after treatment (Figure 6B). These excessive primary ROS can result in secondary ROS, such as lipid peroxidation.36 Malondialdehyde (MDA), a reactive metabolite that forms covalent adducts with biomacromolecules, can reflect lipid peroxidation status and oxidative stress.37 A concentrationdependent increase of MDA is observed in Re-treated cells (Figure 6C). The content of MDA increases by approximately 4.5-fold in cells treated with ReN or ReS at 8 µM for 12 h. There are some enzymes in cells to regulate the excess ROS, such as superoxide dismutase (SOD) and catalase (CAT).38 SOD can catalyze the dismutation of superoxide radical into oxygen or hydrogen peroxide.39 Hydrogen peroxide is also highly reactive and can be degraded by CAT.40 The catalytic ability of SOD decreases after treatment with ReN and ReS for 12 h (Figure 6D). This can explain the result of increased superoxide. Besides, the CAT activities in Re-treated cells are not altered significantly (Figure 6E). The lethal level of ROS cannot be scavenged effectively, which causes an irreversible damage to cells (Figure S17). The process of oxidative stress induced by Re complexes is shown in Figure 6F.


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Figure 6. A) ROS elevation in HeLa cells treated with ReN or ReS for 2 h detected by flow cytometry. MFI represents mean fluorescence intensity of DCF. The excitation wavelength is 488 nm. The emission is collected at 500‒550 nm. B) Superoxide in HeLa cells induced by ReN and ReS for 2 h. Cells were stained with MitoSOX (5 μM) for 30 min. MitoSOX is excited at 488 nm, and the emission wavelength is 550‒600 nm. Scale bar: 20 μm. C) Lipid oxidation level in HeLa cells. Cells were treated with CDDP, ReN and ReS at the indicated concentrations for 12 h. D) SOD activity in HeLa cells. Cells were treated with CDDP, ReN and ReS for 12 h. Unit (U) of SOD activity presents the enzyme activity when the inhibition ratio of the xanthine-based coupled reaction is 50%. E) CAT activity in HeLa cells. Cells were treated with CDDP, ReN and ReS for 12 h. U presents the amount that catalyzes the conversion of 1 μM of hydrogen peroxide per minute in pH 7.0 at 25 °C. *p < 0.05 and **p < 0.01. F) The process of irreversible oxidative stress induced by Re complexes. 2.7 Disturbance of Glutathione Metabolism Redox homeostasis is fundamental to maintain cellular function and survival.41 Many redox enzymes and species are overexpressed in cancer cells to sustain antioxidant defense.42 The


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chemotherapy that is against antioxidant pathways shows considerable potential.42 Herein, metabolomics was used to analyze potential drug-related metabolites and pathways. HeLa cells were treated with ReN and ReS at 8 µM for 6 h, and the metabolites were detected by gas chromatography–time-of-flight mass spectrometry (GC–TOF–MS) after derivative reaction. 520 peaks were detected and 219 of them were identified as metabolites (Figure S18). In the PCA model (Figure 7A) and the OPLS-DA model (Figure 7B), the untreated group is well separated from the Re-treated groups. The contents of most differential metabolites decrease after Retreatment (Figure S20), which indicates the anticancer mechanism of ReN and ReS may be similar. 26 and 19 differential metabolites of ReN- and ReS-treated groups are identified after filter (p < 0.05 and VIP > 1, Table S4). Through pathway enrichment and KEGG pathway analysis, the most vital metabolism pathways affected by Re complexes are the amino acid-related pathways (aminoacyl-tRNA biosynthesis and cyanoamino acid metabolism) and glutathione metabolism. Aminoacyl-tRNA biosynthesis is affected by amino acids and ATP.43 Essential amino acids (threonine, valine, and isoleucine) and non-essential amino acids (glycine, alanine, proline, serine and aspartic acid) decrease significantly (Figure 7D). ATP level is also declined because of mitochondrial damage (Figure S16). L-lactate, D-glucose, D-fructose 2,6-bisphosphate in glycolysis and L-malic acid in tricarboxylic acid cycle (TAC) are diminished, which reflects the attenuation of energy production. Cyanoamino acid metabolism is also influenced by the alteration of essential and non-essential amino acids. The most down-regulated metabolite is oxoproline that is derived from GSH by γ-glutamyl cyclotransferase.27 The glutathione metabolism is mainly affected by GSH and oxoproline, both in concentration and conversion.27


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Figure 7. A) PCA score plot and B) OPLS-DA score plot of metabolome profiling. HeLa cell were treated with ReN and ReS at 8 µM for 6 h. C) Bubble plot of KEGG pathway analysis of HeLa cells treated with ReN. D) Relative content of important differential metabolites. The relative content is normalized by the peak area of adonitol (internal standard). The alteration of glutathione metabolism is further studied by detecting the redox-related enzymes and species.44 Oxidation of GSH to form GSSG can be carried out by the direct interaction with radicals, or by glutathione peroxidases (GPx) during the reduction of hydrogen


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peroxide.44 As Figure 8A shows, the contents of GSH and GSSG are down-regulated after treatment by ReN and ReS. The ratios of GSH/GSSG are also declined. NADPH acts as reducing equivalents for redox process.45 NADPH-dependent reduction of GSSG can be catalyzed by glutathione reductase (GR).45 As shown in Figure 8B, the levels of NADPH and NADP+ increase after treatment by ReN and ReS for 12 h. Besides, the enzyme activity of GPx increases, whereas enzyme activity of GR decreases after treatment by ReN and ReS for 12 h (Figure 8C and 8D). These results show how Re complexes influence metabolism by redox-related species and enzymes.

Figure 8. A) Concentrations of GSH and GSSG in HeLa cells. Cells were treated with ReN and ReS at 8 µM for 12 h. B) Content of NADP+ and NADPH in HeLa cells. Cells were treated with ReN and ReS at 8 µM for 12 h. C) GPx activity in HeLa cells after treated with ReN and ReS at 8 µM for 12 h. U present that 1 µM NAPDH is catalyzed by GPx to form NADP+ per minute under 25 °C and pH 8.0, with the existence of GSH, GR and Cum-OOH. D) GR activity in HeLa cells


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after treated with ReN and ReS at 8 µM for 12 h. U presents that 1 µM GSSG can be catalyzed by GR to GSH per minute under 25 °C and pH 7.5. *p < 0.05 and **p < 0.01. E) The disturbance of glutathione metabolism induced by Re complexes. 2.8 Induction of Apoptosis and Necroptosis It has been reported that Re complexes can induce apoptosis, paraptosis and necroptosis, or cell death belonging to none of these common pathways.10-12, 20 After Re-treatment, many vacuoles in cytosol appear in HeLa cells, and cell membrane shrinks and bubbles (Figure S21). Furthermore, increased cellular debris in Re-treated cells is detected by propidium iodide (PI) staining. After incubation for 24 h at 8 µM, the percentage of cell debris in sub-G1 phase increases from 1.2 ± 0.1% to 55.7 ± 3.2% and 19.8 ± 1.0% for ReN and ReS, respectively (Figure S22). Cells in different status were also detected by annexin V/PI staining. As Figure 9A shows, the treatment of Re complexes leads to a dose-dependent increase in the percentage of cells in apoptotic and necrotic phase, as well as non-viable phase. At a concentration of 8 µM, ReN and ReS induce the increase in percentage of apoptotic and necrotic cells from 13.0 ± 0.8% to 57.9 ± 2.0% and 36.5 ± 1.0%, respectively. The percentages of non-viable cells increase from 0.7 ± 0.1% to 16.7 ± 0.7% (ReN) and 47.3 ± 2.8% (ReS). The intracellular Ca2+ overload associated with cell death was detected by Fluo-4 AM.46 As Figure 9B shows, the emission intensity of Fluo-4 AM increases in a dose-dependent manner in Re-treated cells. Caspases are important regulators of apoptosis.47 Compared with untreated cells, activation of caspase 3/7 is increased with the drug dose (Figure 9C). ReN and ReS at 8 µM can remarkably increase the caspase-3/7 activity by 9.8-fold and 4.6-fold, respectively. However, although necrosis and necroptosis are caspase-independent, they allow the cells bypass caspase activation.48 Thus, we used Nec-1 (necrostatin-1, a necroptosis inhibitor) and z-


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VAD-fmk (a pan-caspase inhibitor) to further verify cell death modes.49-50 As shown in Figure 9D, the cell viability increases after pretreatment with Nec-1. Besides, the cell viabilities can further be elevated by the combined pretreatment of Nec-1 and z-VAD-fmk (Figure 9E). This result further proves that Re(I) complexes induce apoptosis and necroptosis simultaneously. Additionally, pretreatment with 3-MA (3-methyladenine, an autophagy inhibitor) and cycloheximide (a paraptosis inhibitor) shows no obvious effects on Re-induced cell death (Figure S23 and S24), which excludes the possibility of autophagy and paraptosis.51-52

Figure 9. A) Cell apoptosis detected by flow cytometric quantification with annexin V/PI staining. HeLa cells were treated with ReN and ReS for 24 h at the indicated concentrations. λex = 488 nm. λem = 500‒550 nm (annexin V). λem = 600‒650 nm (PI). B) Ca2+ level in HeLa cells after incubated ACS Paragon Plus Environment

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with ReN and ReS for 6 h. λex = 488 nm. λem = 500‒550 nm. C) Activation of caspase 3/7 in HeLa cells. The cells were incubated with CDDP, ReN and ReS at the indicated concentrations for 6 h. D) Cell viability after pre-treatment by necroptosis inhibitor. The cells were pretreated with or without Nec-1 (20 μM) for 1 h, and then incubated with ReN and ReS at indicated concentrations for 24 h. E) Cell viability after pre-treatment by necroptosis inhibitor and apoptosis inhibitor. The cells were pretreated with z-VAD-fmk (20 μM) or z-VAD-fmk (20 μM) and Nec-1 (20 μM) together. ReN and ReS were incubated with cells for 24 h. *p < 0.05, **p < 0.01. 2.9 Inhibition of Xenograph Growth in Vivo The antitumor activity of ReN and ReS were assessed in a mouse xenograph model in vivo. When solid tumors grew to 200‒250 mm3, nude mice (n=7) were separated evenly into 4 groups.53 The intratumoral injections (5 mg/kg) were performed at day 0 and day 9. The graph of tumors at day 19 is shown in Figure 10A. ReN and ReS can inhibit tumor growth with a ratio of 44 ± 15% and 50 ± 16% respectively, which is less effective than CDDP (30 ± 14%) (Figure 10B). Compared with CDDP (MW = 300.05), binuclear ReN (MW = 1681.4) and ReS (MW = 1717.5) are in low concentrations in mole fraction. The side effects of different complexes were evaluated by body weight, survival rate and hematoxylin and eosin (H&E) staining of organs. The average body weights of mice treated with PET diluent, ReN, ReS and CDDP are 20.3 ± 1.0 g, 18.3 ± 2.1 g and 19.6 ± 1.0 g and 16.4 ± 2.6 g, respectively. As Figure 10E shows, there is no obvious pathological change in the organs from each group, which indicates ReN and ReS possess no severe side effects.


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Figure 10. Inhibition of xenograph in nude mice in vivo. A) Tumors at day 19 after the experiment. B) Tumor volume, C) body weight and D) survival rate of nude mice after treatment with PET diluent, CDDP ReN and ReS. CDDP was dissolved in PBS. E) H & E staining of organs. Scale bars: 100 μm.

3 Conclusion In this work, two mitochondria-accumulating binuclear Re(I) tricarbonyl complexes ReN and ReS were synthesized and characterized. ReN and ReS mainly accumulate in mitochondria and cause oxidative stress, mitochondrial dysfunction and slow down bioenergetic rate. Besides, glutathione metabolism and redox homeostasis are disturbed by the impacted redox-related enzymes and species. ReN and ReS induce necroptosis and caspase-dependent apoptosis simultaneously, and inhibit tumor growth in vivo. In conclusion, our work shows that rhenium


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complexes can exert their anticancer effects by irreversible oxidative stress and cellular redox imbalance. This study gives us inspiration to design novel Re complexes in order to optimize the kinetics of redox modulation, as well as anticancer capacity in the near future.

4 Experiment Section 4.1 Materials and Measurements Re(CO)5Cl, CF3SO3Ag, NH4PF6, 4,4’-azopyridine, 4,4’-dithiodipyridine, dip, CDDP, MTT, JC-1, cycloheximide, crystal violet, DCFH-DA and 3-MA were purchased from Sigma Aldrich, USA. Nec-1 was purchased from MedChemExpress, China. MTDR was purchased from Life Technologies, USA. Fluo-4 AM and z-VAD-fmk were purchased from Beyotime, China. Primers were synthesized by Sangon, China. A549, A549R, HeLa, HepG2, MCF-7, U2SO, PC3 and LO2 cells were obtained from Experimental Animal Centre of Sun Yat-Sen University, China. Female BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Co Ltd, China. ReN and ReS were dissolved in DMSO just before the experiments, and the concentration of DMSO in biological experiments was 1% (v/v). All studies involving animals were approved by the university animal care and use committee. 1H

NMR spectra and

13C

NMR spectra were recorded on a Bruker Avance 400 spectrometer,

Germany. ESI-MS spectra were recorded on a Shimadazu LCMS-2020 spectrometer, Japan. Elemental analysis was carried out using an Elemental Vario EL CHNS analyser, Germany. UVvis spectra were recorded on a Varian Cary 300 spectrophotometer, USA. Fluorescence emission measurements were conducted on an FLS 980 combined fluorescence lifetime and steady state spectrometer, UK. [Ru(bpy)3]Cl2 (bpy = 2,2’-bipyridine) was used as the reference for


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fluorescence quantum yield. Cells were monitored by an LSM 710 confocal laser scanning fluorescence microscopy, Germany. Absorption and luminescence were measured by an Infinite M200pro microplate reader, Switzerland. Flow cytometry was conducted on a BD FACSCalibur, USA. OCR was detected by a Seahorse XFe24 analyser, USA. Realtime qPCR was performed in a LightCycler 480 II, Switzerland. Re element was detected by X Series 2 ICP-MS, USA. Untargeted metabolome profiling was tested by an Agilent 7890 gas chromatograph system coupled with a Pegasus 4D time-of-flight mass spectrometer, USA. Bioimaging in vivo was performed by Kodak In-Vivo Imaging Systems, USA. 4.2 Synthetic Procedure of Re Precursor Re(dip)(CO)3Cl was prepared according to a reported procedure.10 Briefly, Re(CO)5Cl (1.4 mmol) and dip (1.5 mmol) was refluxed in toluene (70 mL) for over 4 h. The flask was placed on ice overnight. The precipitant was collected and washed with hexane. Then it was converted to [Re(dip)(CO)3(CH3CN)](PF6) according to a standard method with some modifications.20 Re(dip)(CO)3Cl (1.0 mmol) and AgCF3SO3 (1.0 mmol) were refluxed in CH3CN (200 mL) for 24 h in the dark under an inert atmosphere of N2. The white precipitate was removed, and NH4PF6 aqueous solution (over 10-fold) was added into the solution. The yellow precipitate was collected by centrifugation and washed by double distilled water. 4.3 Characterization of Re(I) Complexes ReN Yield: 0.131 g (78%). ESI-MS: m/z [M−2PF6]2+ 694.75, [Re(dip)(CO)3+L]+ 786.55. 1H NMR (400 MHz, d6-DMSO) δ 9.79 (d, J = 5.4 Hz, 4H), 8.83‒8.80 (m, 4H), 8.19 (d, J = 5.4 Hz, 4H), 8.12 (s, 4H), 7.65 (s, 20H), 7.62‒7.60 (m, 4H).

13C

NMR (400 MHz, d6-DMSO) δ 196.91, 195.87,

192.38, 192.18, 157.61, 156.70, 156.38, 155.16, 155.11, 154.98, 154.49, 152.32, 152.21, 152.05,


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147.16, 147.13, 135.57, 135.47, 130.55, 130.34, 129.70, 128.83, 128.66, 127.88, 127.51, 126.51, 126.38, 119.51, 116.48, 116.43. Elemental analysis for C64H40F12N8O6P2Re2·0.5×H2O, calculated: C 45.53%, H 2.45%, N 6.64%; found: C 45.87%, H 2.48%, N 6.70%. ReS Yield: 0.133 g (78%). ESI-MS: m/z [M−2PF6]2+ 712.15, [Re(dip)(CO)3+L]+ 822.55. 1H NMR (400 MHz, d6-DMSO) δ 9.72 (d, J = 5.4 Hz, 4H), 8.40 (d, J = 6.9 Hz, 4H), 8.16 (d, J = 5.4 Hz, 4H), 8.13 (s, 4H), 7.66 (s, 20H), 7.44 (d, J = 6.9 Hz, 4H).

13C

NMR (400 MHz, d6-DMSO) δ

196.93, 195.84, 192.20, 155.10, 155.03, 154.51, 152.75, 152.13, 151.98, 151.44, 150.03, 147.43, 147.10, 138.88, 135.58, 135.42, 130.35, 129.72, 128.78, 128.68, 128.53, 127.86, 127.45, 126.53, 126.39, 125.43, 124.10, 122.43, 119.87. Elemental analysis for C64H40F12N6O6P2Re2S2·0.5×H2O, calculated: C 44.57%, H 2.40%, N 4.87%, S 3.71%; found: C 44.57%, H 2.48%, N 4.91%, S 3.71%. 4.4 Crystallographic Structure Determination The crystal of ReN was recrystallized in CH3CN/ethyl ether. Single crystal X-ray diffraction measurement was performed on a Bruker Smart 1000 CCD diffractometer, Germany. The crystal structure was solved by direct methods with the program SHELXL. Solvent molecules were removed by Squeeze function in PLATON. The structural plots were drawn by the xp package in SHELXL. PXRD data were obtained by a Bruker D8 ADVANCE X-ray powder diffractometer (Cu-Kα). 4.5 Cell Viability Assay The cytotoxicity of compounds or complexes was determined by MTT assay as previously described.54 To study the impact of the inhibitors, cells were pre-treated with inhibitors at indicated concentrations for 1 h, and then incubated with tested compounds.


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4.6 Cellular Distribution by ICP-MS The detection of Re element by ICP-MS was performed as previously described with some modification.55 After treated with Re complexes, mitochondria and cytosol were isolated by the Mitochondria Isolation Kit (Thermo Scientific, USA) according to the manufacturer’s protocol. The fractions were lysed in radio immunoprecipitation assay (RIPA) buffer. The concentration of protein was measured using the BCA assay. 4.7 Mitochondrial Stress Analysis The mitochondrial stress analysis was measured as previously described by the Seahorse XF Cell Mito Stress Test Kit.56 The optimal concentrations of inhibitors were tested before the formal experiment. HeLa cells (3×104 cells/well) were seeded and incubated for 24 h. Then cells were incubated with ReN or ReS at indicated concentrations for 6 h. OCR was detected by a Seahorse XFe24 analyser, followed by sequential treatment of oligomycin (1 μM), FCCP (0.125 μM), antimycin A (0.5 μM) and rotenone (0.5 μM). 4.8 Real-time qPCR Total RNA from HeLa cells was isolated using TRIzol (Thermo Fisher, USA) according to the manufacturer’s instructions. The cDNA was synthesized using PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Japan). 5 ng cDNA was mixed with 0.5 μM of primer for final concentration. SYBR Green PCR buffer (Thermo Fisher, USA) was used for real-time qPCR. The reaction mixtures were incubated at 50 °C for 2 min and at 95 °C for 10 min, and then followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The primer sequences are used as reported.57


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4.9 Measurement of MDA MDA was detected by the Lipid Peroxidation MDA Assay Kit (S0131, Beyotime, China) according to the manufacturer’s protocol. HeLa cells were treated with CDDP, ReN or ReS at the indicated concentrations for 12 h. Cells were homogenized in RIPA lysis buffer and then centrifuged. The supernatant was collected and used for the analysis of both MDA and protein levels. The absorbance at 532 nm was measured by an Infinite M200pro microplate reader. The content of MDA was normalized to the protein level. 4.10 Measurement of Enzyme Activity The activities of SOD (S0101), CAT (S0051), GPx (S0058) and GR (S0055) were determined by assay kits (Beyotime, China) following the manufacturer’s protocols. HeLa cells were treated with CDDP, ReN or ReS at the indicated concentrations for 12 h. The enzyme activity was normalized to the protein level. Notice: the definition of enzyme activity U is different in each enzyme. According to the manufacturer’s protocol, U of SOD enzyme presents the catalytic activity when the inhibition ratio in this xanthine-based coupled reaction is 50%. If the inhibition ratio is 50%, the SOD enzyme activity is 1 U (50% / (1－50%) units). If the inhibition ratio is 60%, the SOD enzyme activity is 1.5 U (60% / (1 － 60%) units). The absorption was recorded on an Infinite M200pro microplate reader. 4.11 Detection of GSH and GSSG GSH and GSSG was detected by GSH and GSSG Assay Kit (S0053, Beyotime, China) according to the manufacturer’s protocol. HeLa cells were treated with ReN or ReS at the indicated concentrations for 12 h. Cells were harvested and lysed by two successive rounds of freezing


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(liquid N2, 5 min) and thawing (37 °C, 5 min). The supernatant was separated by centrifuging at 10,000 × g for 10 min and analysed by assay kit. The absorption was recorded on an Infinite M200pro microplate reader. The concentrations of GSH and GSSG were normalized to the protein level. 4.12 Metabolome Profiling HeLa cells were seeded incubated with ReN or ReS at 8 μM for 6 h. The treatment and the analysis of samples were according to a literature report.58 The cells were collected and quenched by liquid N2 immediately. Samples were extracted with 1 mL CH3OH/CHCl3 (v/v, 3:1), and then added 5 μL adonitol (0.5 mg/mL) as IS. Samples were ultrasound treated for 15 min in ice water, and then centrifuged at 4 °C for 15 min at 12000 rpm. The supernatant was dried completely in a vacuum concentrator. 40 μL methoxy amination hydrochloride (20 mg/mL in pyridine) was added into each sample and incubated for 30 min at 80 °C. 50 μL BSTFA regent (1% TMCS, v/v) was added to react for another 1.5 h at 70 °C. Samples were analyzed in a splitless mode with helium as the carrier gas by GC-TOF-MS with a DB-5 MS capillary column. The front inlet purge flow was 3 mL/min, and the gas flow rate through the column was 1 mL/min. The start temperature was kept at 80 °C for 1 min, then steadily raised to 290 °C in 21 min, then kept at 290 °C for 13 min. The temperature of injection, transfer line, and ion source temperatures were 280, 295, and 220 °C, respectively. The energy was -70 eV in electron impact mode. The data were acquired in full-scan mode with the m/z range of 50−600 at a rate of 10 spectra/s after a solvent delay of 474 s. Metabolite identification and peak integration were performed on Chroma TOF 4.3X software of LECO Corporation and LECO-Fiehn Rtx5 database. SIMCA 15 software and KEGG biochemical pathway database were used for data analysis.


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4.13 Antitumor Evaluation in Vivo The antitumor evaluation in vivo was performed as previously described.54 HeLa xenografts were established by inoculating 2×106 cells via subcutaneous injection into female BALB/c nude mice (4−5 weeks old). The longest diameter (a) and the shortest diameter (b) of the xenograft were measured every two days, and tumour volume (V) was calculated by the formula V = ab2×0.52. 4.14 Preparation of Organs for Pathological Evaluation Organs were collected after the experiment, and fixed with 4% paraformaldehyde at 4 °C. Then they were transferred to 10% formalin neutral buffer solution, and embedded in paraffin. Sections were stained by H&E, and examined under a Zeiss inverted fluorescence microscope, Germany. ASSOCIATED CONTENT Supporting Information. Synthetic scheme; characterization of ReN and ReS; reaction between Re complexes and GSH; cell morphology; cell viability with inhibitor pretreatment; profile of mitochondria respiration; untargeted metabolome profiling; cell cycle analysis; selected bond lengths and bond angles of crystal structure; photophysical data; p value and VIP value of differential metabolites. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Z.W.M.). *E-mail: [email protected] (C.P.T.). Author Contributions


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The chemical synthesis and the characterization were done by J.H.L. and Q.W. The photophysical and biological properties were determined by F.X.W. The biological experiments were done by F.X.W. The in vivo tumor growth inhibition was tested by Z.H.W. and H.Z. The manuscript was written by F.X.W. and revised by C.P.T., Z.W.M. and L.N.J. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This study was supported by the National Natural Science Foundation of China (21778078, 21571196 and 21572282), the 973 program (2015CB856301), the Guangdong Natural Science Foundation (2015A030306023), Innovative Research Team in University of Ministry of Education of China (IRT_17R111) and the Fundamental Research Funds for the Central Universities. We thank Zi-Ming Ye (SYSU) for assistance with single crystal XRD and PXRD, and Dr. Nafees Muhammad (SYSU) for discussion of the reaction between Re complexes and GSH. REFERENCES (1) Hanahan, D.; Weinberg, R. A. Hallmarks of Cancer: The Next Generation. Cell 2011, 144, 646–674. (2) Weinberg, S. E.; Chandel, N. S. Targeting Mitochondria Metabolism for Cancer Therapy. Nat. Chem. Biol. 2015, 11, 9–15. (3) Sabharwal, S. S.; Schumacker, P. T. Mitochondrial ROS in Cancer: Initiators, Amplifiers or an Achilles’ Heel? Nat. Rev. Cancer 2014, 14, 709–721. (4) Chen, Y.; Zhang, H.; Zhou, H. J.; Ji, W.; Min, W. Mitochondrial Redox Signaling and Tumor Progression. Cancers 2016, 8, 40. (5) Fulda, S.; Galluzzi, L.; Kroemer, G. Targeting Mitochondria for Cancer Therapy. Nat. Rev.


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Table of Contents Graphic


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Mitochondria-Accumulating Rhenium(I) Tricarbonyl Complexes Induce

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