Phosphorescent rhenium(I) complexes conjugated with artesunate: Mitochondrial targeting and apoptosis-ferroptosis dual induction.

{"full_text": " Journal of Inorganic Biochemistry 223 (2021) 111537\n\n\n Contents lists available at ScienceDirect\n\n\n Journal of Inorganic Biochemistry\n journal homepage: www.elsevier.com/locate/jinorgbio\n\n\n\n\nPhosphorescent rhenium(I) complexes conjugated with artesunate:\nMitochondrial targeting and apoptosis-ferroptosis dual induction\nRui-Rong Ye *, 1, Bi-Chun Chen 1, Jun-Jian Lu , Xiu-Rong Ma , Rong-Tao Li *\nFaculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, PR 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: Cell death is essential for cancer, which can be induced through multiple mechanisms. Ferroptosis, a newly\nRhenium(I) complex emerging form of non-apoptotic cell death, involves the generation of iron-dependent reactive oxygen species\nArtesunate (ROS). In this study, we designed and synthesized two artesunate (ART) conjugated phosphorescent rhenium(I)\nMitochondria\n complexes (Re(I)-ART conjugates), [Re(N^N)(CO)3(PyCH2OART)](PF6) (Re-ART-1 and Re-ART-2)\nAnticancer activity\nApoptosis\n (Py = pyridine, N^N = 1,10-phenanthroline (phen, in Re-ART-1) and 4,7-diphenyl-1,10-phenanthroline (DIP, in\nFerroptosis Re-ART-2)) that can specifically locate in the mitochondria of human cervical carcinoma (HeLa). Mechanism\n studies show that Re-ART-1 and Re-ART-2 exhibit high cytotoxicity against cancer cells lines and can induce\n both apoptosis and ferroptosis in HeLa cells through mitochondrial damage, caspase cascade, glutathione (GSH)\n depletion, glutathione peroxidase 4 (GPX4) inactivation and lipid peroxidation accumulation. As a result, this\n work presents the rational design of Re(I)-ART conjugates as a promising strategy to induce both apoptosis and\n ferroptosis and improve therapeutic efficiency of cancer treatment.\n\n\n\n\n1. Introduction rhenium complexes designed by Massi et al. can induce cell cycle arrest\n by inhibiting the phosphorylation of Aurora-A kinase [12]. Falasca et al.\n Rhenium(I) organometallic complexes with rich photophysical and reported that rhenium complexes can block the spread of cancer by\nphotochemical properties have been developed as luminescent probes inhibiting the signal cascade induced by fibroblast growth factor re\u00ad\n[1,2]. 186/188Re is widely used as radio-pharmaceuticals [3]. Recently, ceptor [13], and sarcoma. Zobi et al. synthesized a Re(I) tricarbonyl\nrhenium(I) tricarbonyl complexes, one class of the rhenium(I) organo\u00ad complex with selective cytotoxicity towards HCT-116 cells [14]. We\nmetallic complexes, have been extensively reported as anticancer agents previously designed and synthesized a rhenium(I)-based histone\n[4\u20137]. For instance, Mao and Tan et al. [8] reported that rhenium(I) deacetylase inhibitor, which could target mitochondria and suppress\ntricarbonyl complexes could accumulate in mitochondria and subse\u00ad histone deacetylases activity [15]. We also explored the anticancer\nquently cause cell death by exerting irreversible oxidative stress and mechanisms of mononuclear and dinuclear rhenium(I) complexes,\nglutathione (GSH) metabolism disorder. Wilson et al. reported a which were closely associated with organelle-specific localization [16].\nrhenium isonitrile complex that can induce apoptosis mediated by In summary, the possibility of oncotherapy and superior luminescent\nunfolded protein response, which also exhibits in vivo and in vitro properties of rhenium(I) tricarbonyl complexes make them meaningful\nanticancer activity and resistance to ovarian cancer cells [9\u201311]. The for future anti-cancer drug research.\n\n\n\n Abbreviations: ART, Artesunate; ROS, Reactive oxygen species; GSH, Glutathione; GSSH, Oxidized glutathione; RCD, Regulated cell death; GPX4, Glutathione\nperoxidase IV; MDA, Malondialdehyde; DMEM, Dulbecco\u2019s Modified Eagle Medium; FBS, Fetal bovine serum; PBS, Phosphate-buffered saline; DCC, Dicyclohex\u00ad\nylcarbodiimide; DMAP, 4-N,N-dimethylaminopyridine; PLE, Porcine liver esterase; HPLC, High performance liquid chromatography; ICP-MS, Inductively coupled\nplasma mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTDR, MitoTracker Deep Red FM; LTDR, LysoTracker Deep Red\nFM; CCCP, Cyanide 3-chlorophenylhydrazone; MMP, Mitochondrial membrane potential; Rh123, Rhodamine 123; H2DCFDA, 2\u2032 ,7\u2032 -dichlorodihydrouorescein diac\u00ad\netate; DCF, 2\u2032 ,7\u2032 -dichlorodihydrouorescein; Hoechst 33342, 2\u2032 -(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5\u2032 -bi-1H-benzimidazole trihydrochloride; ATP, Aden\u00ad\nosine triphosphate; FITC, Fluorescein Isothiocyanate; PI, propidium iodide; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma-2; PARP, Poly (ADP-ribose)\npolymerase; DFO, Deferoxamine; Fer-1, Ferrostatin-1; BODIPY, 4,4-difluoro-3a,4a-diaza-s-indacene.\n * Corresponding authors.\n E-mail addresses: yerr@mail2.sysu.edu.cn (R.-R. Ye), rongtaolikm@163.com (R.-T. Li).\n 1\n These authors contributed equally.\n\nhttps://doi.org/10.1016/j.jinorgbio.2021.111537\nReceived 16 February 2021; Received in revised form 24 June 2021; Accepted 5 July 2021\nAvailable online 9 July 2021\n0162-0134/\u00a9 2021 Elsevier Inc. All rights reserved.\n\fR.-R. Ye et al. Journal of Inorganic Biochemistry 223 (2021) 111537\n\n\n Drugs with a single anticancer mechanism are usually not effective in Herein, two ART conjugated phosphorescent rhenium(I) complexes\ntreating tumors and are prone to drug resistance attributed to the pol\u00ad (Re(I)-ART conjugates), [Re(N^N)(CO)3(PyCH2OART)](PF6) (Re-ART-1\nygenicity and complexity of cancer [17]. It has been reported that the and Re-ART-2) (Scheme 1; Py = pyridine, N^N = 1,10-phenanthroline\ndevelopment of anticancer agents with multiple action mechanisms can (phen, in Re-ART-1) and 4,7-diphenyl-1,10-phenanthroline (DIP, in Re-\nconquer these side effects and drug resistance [18]. Currently, the ART-2)) were designed and synthesized. The corresponding reference\nconjugation of organic molecules with anticancer potential and metal complexes [Re(phen)(CO)3(PyCH2OH)](PF6) (Re-1) and [Re(DIP)\ncomplexes has been demonstrated to be a promising strategy for (CO)3(PyCH2OH)](PF6) (Re-2) that lack the ART group were also\ndeveloping anticancer agents with multiple action mechanisms. More\u00ad included. As the multi-faceted regulators of cell death, mitochondria are\nover, metal complexes generally show superior biological activity than an important target for the development of anti-tumor drugs [38]. It has\norganic ligands, thus combining two of them may exert a synergistic been reported that phosphorescent rhenium(I) complexes with high\neffect [19,20]. Chen et al. reported two copper(\u0406\u0406) complexes with iso\u00ad lipophilicity can preferentially accumulate in mitochondria [15,16,39].\nquinoline derivatives as ligands to induce apoptosis and autophagy in According to our previous research findings, the introduction of an ART\nhuman lung carcinoma (A549) [21]. Mao and Tan et al. prepared group can enhance the lipophilicity of cyclometalated iridium(III)\nrhenium(I) complexes mixed with clinical iron chelator deferasirox to complexes [24]. In this work, the ART group is expected to improve the\nsimultaneously disrupt mitochondrial metabolism and iron homeostasis lipophilicity of rhenium(I) complexes and further potentiate the mito\u00ad\n[22]. Artesunate (ART), a chemical derivative of artemisinin, exerts chondrial targeting ability as well as synergistic anti-tumor activity of Re\ndiverse antitumor effects, such as antiproliferative, cell cycle regulation, (I)-ART conjugates. In this study, we describe the synthesis, character\u00ad\nand pro-apoptosis [23]. Recently, we reported a series of cyclometalated ization, photophysical properties, and anticancer activity of Re(I)-ART\niridium(III) complexes incorporating ART, which exhibited synergistic conjugates as well as the anticancer mechanisms of Re-ART-1 and Re-\nanti-hepatocellular cancer activity [24]. ART-2. Based on our findings, Re(I)-ART conjugates localize in mito\u00ad\n Apoptosis is the classic cell death pathway that causes most of chondria and induce mitochondrial dysfunction, thereby causing human\nregulated cell death (RCD) in mammalian cells [25,26]. In addition, cervical carcinoma (HeLa) cell death through a dual-mode of apoptosis\nseveral regulated non-apoptotic cell death pathways have been recog\u00ad and ferroptosis.\nnized, such as autophagy, necroptosis, ferroptosis, etc. [27]. Ferroptosis,\nwhich plays a key role in a variety of organic diseases, especially cancer, 2. Results and discussion\nis induced by the cumulation of lipid reactive oxygen species (ROS)\nmediated by the incapacitation of cellular GSH-dependent antioxidant 2.1. Synthesis, photophysical characterization\ndefenses [28]. As the main source of ROS, mitochondria play a role in\nthis type of RCD [29\u201331]. In addition, malondialdehyde (MDA), a lipid The reference complex Re-1 was synthesized according to the\nperoxidation marker, reflects the lipid peroxidation status [32]. The reference [40], and Re-2 was obtained by a synthesis method similar to\nconsumption of GSH and the suppression of glutathione peroxidase 4 that of Re-1. The Re(I)-ART conjugates, Re-ART-1 and Re-ART-2, were\n(GPX4) are implicated in ferroptosis [33,34]. Thus, MDA level, cellular obtained through the condensation reaction of Re-1 or Re-2 with ART in\nGSH and GPX4 expression are the hallmarks of ferroptosis formation. anhydrous CH2Cl2 at room temperature using dicyclohexylcarbodiimide\nUntil now, only a few metal-based complexes have been reported to (DCC) as the dehydrating agent and 4-N,N-dimethylaminopyridine\ncause cancer cell death through ferroptosis, including cisplatin [25], (DMAP) as the catalyst (Scheme S1). The products were purified with\niridium(III) complex [35], and iron(III) complex [36]. Moreover, it is silica column chromatography (100:1 CH2Cl2:CH3OH) and character\u00ad\nalso pertinent to note that ART has been reported to have a ferroptosis- ized using ESI-MS, 1H NMR (Figs. S1-S6), and elemental analysis.\ninducing effect [37]. The electronic absorption and emission spectra of Re-ART-1 and Re-\n\n PF6\n\n PF6\n CO CO\n N N\n OC OC\n Re Re\n OC N OC N\n N N\n\n\n\n\n O O\n O O O O O O\n O O O\n O\n O O O O\n H H Re-ART-1 H H Re-ART-2\n\n PF6\n\n PF6\n CO CO\n N N\n OC OC\n Re Re\n OC N OC N\n N N\n\n\n\n\n HO HO\n\n Re-1 Re-2\n Scheme 1. Chemical structures of rhenium(I) complexes with (Re-ART-1 and Re-ART-2) or without (Re-1 and Re-2) ART.\n\n 2\n\fR.-R. Ye et al. Journal of Inorganic Biochemistry 223 (2021) 111537\n\n\nART-2 are shown in Fig. S7A. Re(I) complexes display two main bands at most potent among all tested compounds. Coupling with ART can\n250\u2013450 nm in the case of the intraligand and metal-to-ligand charge- significantly improve the cytotoxicity of rhenium(I) complexes,\ntransfer transitions. After excitation at 405 nm, Re-ART-1 and Re-ART- whereby Re-ART-1 and Re-ART-2 showed about 4.5- to 12.2-fold\n2 emitted yellow light with a maximum wavelength of around 580 nm higher cytotoxicity than Re-1 and Re-2 against the tested human can\u00ad\n(Fig. S7B). The photophysical data of Re-ART-1 and Re-ART-2 are cer cell lines. In addition, Re-ART-1 and Re-ART-2 display approxi\u00ad\nsummarized in Table S1. mately 3.9- to 57.6-fold greater ability to kill A549R cells than cisplatin,\n We further explored the stability of Re-ART-1\u20132 in PBS by fluores\u00ad indicating that they can conquer the resistance of cisplatin. Further\u00ad\ncence spectroscopy and high performance liquid chromatography more, the cytotoxicity of Re-ART-1 and Re-ART-2 against LO2 cells was\n(HPLC). As shown in Fig. S8 and Fig. S9, no significant changes in the determined to be approximately 2.0- to 8.6-fold lower than that of\nemission intensities and HPLC signals of Re-ART-1\u20132 are observed, HepG2 cells, revealing their selectivity to cancer cells.\nindicating the stability in PBS. Then, the responses of Re-ART-1\u20132 to\u00ad\nwards esterase were monitored by fluorescence spectra. Porcine liver 2.3. Cellular localization and uptake mechanisms\nesterase (PLE) was used as a model to investigate the hydrolytic process\nof ester bonds in Re-ART-1\u20132. As shown in Fig. S10, the emission Since the anticancer mechanism of metal-based complexes is closely\nspectra of Re-ART-1 display increased intensities upon treatment with related to their cellular localization [16], the cellular uptake and\nPLE, while the spectra of Re-ART-2 show decreased intensities. These intracellular distribution of Re-ART-1 and Re-ART-2 were investigated\nresults indicate that Re-ART-1\u20132 can undergo hydrolysis in the presence by confocal microscopy. As shown in Fig. S11, both compounds can\nof esterase. effectively penetrate HeLa cells and exhibit blatant organelle staining.\n Further co-localization study demonstrates that the fluorescence of Re-\n ART-1 and Re-ART-2 overlays with that of MitoTracker Deep Red FM\n2.2. Lipophilicity and in vitro cytotoxicity (MTDR) (Fig. 1A), however, almost no overlap was observed with the\n fluorescence of LysoTracker Deep Red FM (LTDR) (Fig. 1B). The Pear\u00ad\n The cellular uptake, distribution and cytotoxicity of metal-based son\u2019s correlation coefficients of Re-ART-1 and Re-ART-2 with MTDR\ncomplexes are strongly affected by their lipophilicity (log Po/w) [41]. are 0.83 and 0.91, respectively. These data display that Re(I)-ART\nIt has been reported that a molecule with a log Po/w value between conjugates are primarily distributed in mitochondria.\n0 and + 5 and a cation number more than 0 shows a high probability to The pathways for small molecules to penetrate cells mainly include\nlocalize in mitochondria [42]. Therefore, we used the shake flask energy-dependent and energy-independent [43]. To evaluate the entry\nmethod to determine the lipophilicity of Re(I) complexes. The log Po/w of the complexes into cells, we studied the cellular uptake mechanisms\nvalues are listed in the following order: Re-1 (0.66) < Re-ART-1 of Re-ART-1 and Re-ART-2. HeLa cells were pre-treated under different\n(1.08) < Re-2 (1.54) < Re-ART-2 (1.96), which reveals that the conditions and then incubated with Re(I)-ART conjugates (10 \u03bcM) for\nconjugation of ART can enhance the lipophilicity of Re(I) complexes. 1 h. Compared to that at 37 \u25e6 C, the suppressed cellular luminescence was\n As rhenium is an exogenous element, the quantitative measurement observed when treated with Re-ART-1 and Re-ART-2 at 4 \u25e6 C or pre-\nof the cellular uptake levels of Re(I) complexes was studied by induc\u00ad treated with the metabolic inhibitor cyanide 3-chlorophenylhydrazone\ntively coupled plasma mass spectrometry (ICP-MS) measurement. Upon (CCCP) (Fig. 2). However, the cellular luminescence was barely influ\u00ad\nincubation with 10 \u03bcM Re(I) complexes on HeLa cells for 1 h, the enced for pre-treated HeLa cells with endocytic inhibitor chloroquine.\nintracellular rhenium contents of the compounds are in the following This result means that Re(I)-ART conjugates are taken up through the\norder: Re-ART-2 (2013.4 ng/106 cells) > Re-2 (1043.2 ng/106 energy-dependent mechanism instead of endocytic pathways.\ncells) > Re-ART-1 (847.3 ng/106 cells) > Re-1 (278.7 ng/106 cells).\nThus, the cellular uptake efficiency of Re(I) complexes is well correlated\n 2.4. Mitochondrial damage\nwith their lipophilicities.\n The antiproliferative effects of Re-ART-1 and Re-ART-2 were tested\n Mitochondrial integrity can be reflected by the mitochondrial\nagainst HeLa, A549, A549R (cisplatin-resistant A549), HepG2 (human\n membrane potential (MMP) [44]. As Re-ART-1 and Re-ART-2 mainly\nhepatocellular liver carcinoma) and LO2 (human normal liver) cells. For\n accumulate in mitochondria, their impact on MMP was examined by\ncomparison, Re-1, Re-2, ART, the mixtures of Re-1/2 with ART (Re-\n confocal microscopy using rhodamine 123 (Rh123) staining. Rh123 is\n1 + ART, Re-2 + ART) and cisplatin were included. According to the\n an MMP-dependent fluorescent probe, a decrease in fluorescence in\u00ad\nIC50 values in Table 1, the antiproliferative effects of the compounds are\n tensity of Rh123 can indicate the loss of MMP [45]. After treating HeLa\nas follows: ART < Re-1 \u2248 Re-1 + ART < cisplatin < Re-ART-1 < Re-\n cells with different doses of Re-ART-1 or Re-ART-2 for 6 h, the fluo\u00ad\n2 \u2248 Re-2 + ART < Re-ART-2, which are unanimous with their lip\u00ad\n rescence intensity of Rh123 decreased in a concentration-dependent\nophilicity. Re-ART-2, with IC50 values ranging from 0.8\u20131.5 \u03bcM, is the\n manner (Fig. 3), indicating the loss of MMP.\n Mitochondria are the main organelles for cellular energy production,\nTable 1 as a result of the loss of MMP, cellular adenosine triphosphate (ATP)\nIC50 values of tested compounds towards different cell linesa. levels should be disturbed [46]. The impact of Re(I)-ART conjugates on\n Compound IC50 (\u03bcM) cellular ATP levels was measured, using Re-1\u20132 as controls. As shown in\n HeLa A549 A549R HepG2 LO2 Fig. 4, the intracellular ATP levels decreased in a concentration-\n dependent manner after treating HeLa cells with Re(I) complexes for\n Re-ART-1 5.2 \u00b1 0.5 13.2 \u00b1 1.7 21.9 \u00b1 2.5 11.4 \u00b1 1.2 22.4 \u00b1 3.0\n Re-ART-2 0.8 \u00b1 0.1 1.1 \u00b1 0.2 1.5 \u00b1 0.2 1.0 \u00b1 0.1 8.6 \u00b1 1.1 6 h, while Re-ART-1\u20132 performed better than Re-1\u20132 at the same\n Re-1 63.8 \u00b1 0.7 79.8 \u00b1 4.2 > 100 91.9 \u00b1 2.5 > 100 concentration. Notably, compared with the control, treatment of HeLa\n Re-2 4.8 \u00b1 0.2 5.1 \u00b1 0.3 6.7 \u00b1 0.5 5.5 \u00b1 1.1 8.8 \u00b1 0.8 cells with 12.5 \u03bcM Re-ART-1 or Re-ART-2 reduced the intracellular ATP\n Re- 59.1 \u00b1 1.7 62.4 \u00b1 5.8 87.8 \u00b1 5.2 85.4 \u00b1 1.8 85.3 \u00b1 2.1 levels to 40.5% (Re-ART-1) and 19.9% (Re-ART-2), respectively. The\n 1 + ART\n Re- 4.9 \u00b1 0.6 4.6 \u00b1 0.3 5.4 \u00b1 1.0 5.4 \u00b1 0.4 6.0 \u00b1 1.6\n results imply that Re(I)-ART conjugates can cause the dysfunction of\n 2 + ART mitochondria.\n ART > 100 > 100 > 100 > 100 > 100\n cisplatin 20.0 \u00b1 2.1 18.2 \u00b1 1.5 86.5 \u00b1 9.0 22.4 \u00b1 2.0 30.5 \u00b1 2.8 2.5. Elevation of intracellular ROS levels\n a\n IC50 values are drug concentrations necessary for 50% inhibition of cell\nviability. The data are presented as mean \u00b1 standard deviation (SD) and cell In addition to energy production, mitochondria are also the main site\nviability is assessed after 48 h incubation. of ROS production [47]. It has been reported that the loss of MMP is\n\n 3\n\fR.-R. Ye et al. Journal of Inorganic Biochemistry 223 (2021) 111537\n\n\n\n\nFig. 1. The colocalization assay of Re(I)-ART conjugates (10 \u03bcM) with MTDR (150 nM) (A) or LTDR (50 nM) (B) (Re(I)-ART conjugates: \u03bbex = 405 nm,\n\u03bbem = 560 \u00b1 20 nm; MTDR and LTDR: \u03bbex = 633 nm, \u03bbem = 665 \u00b1 20 nm). Scale bar: 20 \u03bcm.\n\n\n\n\nFig. 2. Cellular uptake mechanisms of Re(I)-ART conjugates. HeLa cells were incubated with Re(I)-ART conjugates (10 \u03bcM) under different temperatures (37 \u25e6 C and\n4 \u25e6 C), pre-treated with CCCP (30 \u03bcM) or chloroquine (50 \u03bcM) (\u03bbex = 405 nm, \u03bbem = 560 \u00b1 20 nm). Scale bar: 20 \u03bcm.\n\n\n\n\n Fig. 3. Re(I)-ART-induced the loss of MMP analyzed by confocal microscopy with Rh123 staining (\u03bbex = 488 nm, \u03bbem = 530 \u00b1 20 nm). Scale bar: 20 \u03bcm.\n\n\nclosely related to the increase of intracellular ROS [48]. ART [49] or Re-ART-2 was detected by staining with 2\u2032 ,7\u2032 -dichlorodihydrouorescein\nrhenium(I) complexes [15,16] can induce the elevation of ROS levels. diacetate (H2DCFDA). H2DCFDA is non-fluorescent and can be oxidized\nHerein, the intracellular ROS-accumulating capability of Re-ART-1 and by intracellular ROS to highly fluorescent 2\u2032 ,7\u2032 -dichlorofluorescein\n\n\n 4\n\fR.-R. Ye et al. Journal of Inorganic Biochemistry 223 (2021) 111537\n\n\n testing apoptosis [53]. As a member of the cysteine protease family,\n caspase-3 facilitates the cleavage of PARP in the execution phase of\n apoptosis [54]. As shown in Fig. 8, Re-ART-1 and Re-ART-2 markedly\n up-regulated the expression of Bax and down-regulated the expression of\n Bcl-2. Moreover, Re-ART-1 and Re-ART-2 also induced the cleavage of\n caspase-3 and PARP in a dose-dependent manner. These data indicate\n that Re(I)-ART conjugates induce the apoptosis of HeLa cells through a\n caspase-dependent pathway.\n\n\nFig. 4. ATP levels in HeLa cells treated with different concentrations of Re(I) 2.7. Induction of ferroptosis\ncomplexes (* compared with control, * P < 0.05, ** P < 0.01, *** P < 0.001).\n The abnormal increase of intracellular ROS is closely related to\n(DCF) [50]. As shown in Fig. 5, treating HeLa cells with Re-ART-1 or Re- apoptosis and ferroptosis [55,56]. Ferroptosis, which is controlled by\nART-2 caused a concentration-dependent increase in DCF fluorescence iron-dependent lipid hydroperoxides accumulation [27], can be inhibi\u00ad\nintensity, indicating the elevation of ROS. ted by antioxidant ferrostatin-1 (Fer-1) or iron chelators (e.g., deferox\u00ad\n amine (DFO)) [57]. Re-ART-1 and Re-ART-2 were proved their ability\n to induce apoptosis. To examine whether the occurrence of ferroptosis\n2.6. Induction of apoptosis was involved in HeLa cell death induced by Re(I)-ART conjugates, we\n investigated the effects of Re-ART-1 and Re-ART-2 each concomitantly\n Mitochondria are important players in controlling the intrinsic incubated with DFO and Fer-1 on the viability of HeLa cells, using Re-\npathway of apoptosis [51]. Mitochondrial damage and ROS accumula\u00ad 1\u20132 as controls. As shown in Fig. 9A, B and Fig. S12, compared with cells\ntion are two important phenomena of apoptosis, we tested the ability of treated with Re(I) complexes alone, cells treated with DFO and Fer-1\nRe(I)-ART conjugates to induce apoptosis. As shown in Fig. 6, HeLa cells exhibited an increase in cell viability, except for Re-1. As for DFO, the\ntreated with Re-ART-1 and Re-ART-2 exhibited typical changes in viability of HeLa cells increased 1.7-fold for Re-2, 1.8-fold for Re-ART-\napoptotic morphology (such as cell shrinkage and nuclear fragmenta\u00ad 1, and 2.5-fold for Re-ART-2. As for Fer-1, the viability of HeLa cells\ntion) in a concentration-dependent manner. Moreover, annexin V-FITC/ increased 1.7-fold for Re-2, 1.9-fold for Re-ART-1, and 2.6-fold for Re-\nPI (FITC: fluorescein isothiocyanate; PI: propidium iodide) double ART-2. The results partially indicate that Re-2 and Re-ART-1\u20132 can\nstaining analysis demonstrated that HeLa cells treated with Re-ART-1 or induce ferroptosis, whereby Re-ART-1\u20132 exhibit a better ferroptosis\nRe-ART-2 for 24 h increased the percentage of apoptotic cells in a inducing ability than Re-1\u20132.\nconcentration-dependent manner (Fig. 7). Specifically, treatment with GPX4 is important in preventing lipid peroxidation, which is inac\u00ad\nRe-ART-1 (20.8 \u03bcM) or Re-ART-2 (3.2 \u03bcM) significantly increased the tivated by the depletion of GSH, thus inducing ferroptosis [34,58,59].\npercentage of apoptotic cells from 2.75% (control) to 29.30% (Re-ART- Accordingly, we further measured the depletion of GSH in HeLa cells. As\n1) and 32.22% (Re-ART-2), respectively. Overall, the results indicate shown in Fig. 9C, HeLa cells treated with Re-ART-1 and Re-ART-2\nthat Re(I)-ART conjugates can induce HeLa cell apoptosis. significantly reduced the GSH levels in a concentration-dependent\n To further investigate the apoptotic mechanism induced by Re(I)- manner. The influence of Re-ART-1 and Re-ART-2 on the expression\nART conjugates, we examined the changes in Bcl-2-associated X pro\u00ad of GPX4 was also examined. As shown in Fig. 9D, HeLa cells treated with\ntein (Bax), B-cell lymphoma-2 (Bcl-2), caspase-3, and Poly (ADP-ribose) Re-ART-1 and Re-ART-2 remarkably decreased the levels of GPX4\npolymerase (PARP) expression. The Bcl-2 family contains both pro- expression in a concentration-dependent manner. It has been reported\napoptotic protein Bax and anti-apoptotic protein Bcl-2. During the suppression of GPX4 activity can lead to intracellular lipid peroxi\u00ad\napoptosis, Bax undergoes translocation from the cytoplasm to mito\u00ad dation accumulation, which is an important indicator of ferroptosis\nchondria, changes its conformation, triggers the emancipation of cyto\u00ad [60]. Therefore, the influence of Re-ART-1 and Re-ART-2 on lipid\nchrome C, and participates in the caspase activation pathway associated peroxidation was also assessed by measuring the levels of MDA. As\nwith mitochondria [52]. PARP is an important biochemical marker for shown in Fig. 9E, treated HeLa cells with Re-ART-1 or Re-ART-2\n\n\n\n\nFig. 5. Re(I)-ART-induced the elevation of intracellular ROS levels examined by confocal microscopy with H2DCFDA staining (\u03bbex = 488 nm, \u03bbem = 530 \u00b1 20 nm).\nScale bar: 20 \u03bcm.\n\n 5\n\fR.-R. Ye et al. Journal of Inorganic Biochemistry 223 (2021) 111537\n\n\n\n\nFig. 6. Re(I)-ART-induced HeLa cells apoptosis measured by confocal microscopy with Hoechst 33342 staining (\u03bbex = 405 nm, \u03bbem = 460 \u00b1 20 nm). Scale bar: 20 \u03bcm.\n\n\n\n\n Fig. 7. Re(I)-ART-induced HeLa cells apoptosis measured by flow cytometry with Annexin V-FITC/PI staining.\n\n\n exhibited a dose-dependent increase in MDA levels. Particularly, cells\n treated with 3.2 \u03bcM Re-ART-2 showed a more than 8.5-fold increase in\n MDA levels. Lipid peroxidation was further measured using a lipid\n peroxidation-sensitive dye C11-BODIPY (4,4-difluoro-3a,4a-diaza-s-\n indacene) 581/591. The red fluorescence is representative of non-\n oxidized lipids in cells labelled with C11-BODIPY 581/591, while the\n green fluorescence represents oxidized lipids. As shown in Fig. 10, HeLa\n cells in the Re-ART-1\u20132 groups show the strongest green fluorescence in\n confocal images compared to those of cells in the other groups. Flow\n cytometric assay also confirms that Re-ART-1\u20132 induced lipid peroxide\n accumulation (Fig. S13). These results collectively demonstrate that Re\n (I)-ART conjugates inhibit the GPX4 pathway and induce lipid peroxi\u00ad\n dation accumulation, leading to ferroptosis.\n\n 3. Conclusions\n\nFig. 8. Expression of apoptosis-related protein (Bax, Bcl-2, caspase-3 and In summary, we have developed two ART conjugated phosphores\u00ad\nPARP) in HeLa cells treated with Re(I)-ART conjugates. cent rhenium(I) complexes, Re-ART-1 and Re-ART-2, with dual anti\u00ad\n cancer mechanisms. These two complexes display high cytotoxicity\n towards human tumor cells. Cellular uptake experiments reveal that Re-\n ART-1 and Re-ART-2 can effectively target mitochondria. Mechanistic\n\n\n 6\n\fR.-R. Ye et al. Journal of Inorganic Biochemistry 223 (2021) 111537\n\n\n\n\nFig. 9. The ferroptosis induced by Re(I)-ART conjugates in HeLa cells. The effects of Re-ART-1 and Re-ART-2 each concomitantly incubated with DFO (100 \u03bcM) (A)\nand Fer-1 (100 nM) (B) on the viability of HeLa cells. GSH level (C), expression of GPX4 (D) and MDA level (E) in HeLa cells treated with Re-ART-1 and Re-ART-2 (*\ncompared with control, * P < 0.05, ** P < 0.01, *** P < 0.001; # Fer-1 (\u2212 )/DFO (\u2212 ) compared with Fer-1 (+)/DFO (+), # P < 0.05, ## P < 0.01, ### P < 0.001).\n\n\nstudies indicate that Re-ART-1 and Re-ART-2 can induce apoptosis 4.2. Preparation of rhenium(I) complexes\nthrough the depolarization of MMP, consumption of cellular ATP,\nelevation of ROS and caspase cascade. Furthermore, Re-ART-1 and Re- Re(phen)(CO)3Cl [61], Re(DIP)(CO)3Cl [62] and [Re(phen)\nART-2 can inhibit the GPX4 pathway and induce lipid peroxidation (CO)3(PyCH2OH)](PF6) (Re-1) [40] were synthesized following the\naccumulation, thus leading to ferroptosis. Overall, these Re(I)-ART literature procedures.\nconjugates show promising application as synergetic anticancer [Re(DIP)(CO)3(PyCH2OH)](PF6) (Re-2): The preparation of Re-2\nagents, which can induce a dual-killing mode of apoptosis-ferroptosis to was similar to that of Re-1 except that Re(DIP)(CO)3Cl was used instead\nenhance cancer therapy. of Re(phen)(CO)3Cl. Yield: 0.250 g (orange powder), 85%. 1H NMR\n (600 MHz, [D6]DMSO) \u03b4 9.84 (d, J = 5.4 Hz, 2H), 8.54 (d, J = 5.4 Hz,\n4. Experimental section 2H), 8.23 (d, J = 5.4 Hz, 2H), 8.16 (s, 2H), 7.73\u20137.68 (m, 10H), 7.33 (d,\n J = 5.6 Hz, 2H), 5.53 (s, 1H), 4.46 (d, J = 5.4 Hz, 2H). ESI-MS (CH3OH):\n4.1. Materials and instruments m/z 712 [M \u2212 PF6]+. Elemental analysis: calcd (%) for\n C33H23F6N3O4PRe: C, 46.26; H, 2.71; N, 4.90; found: C, 46.56; H, 2.87;\n Re(CO)5Cl (Sigma Aldrich), phen (J&K), DIP (J&K), PyCH2OH N, 4.79.\n(J&K), ART (Alfa Aesar), DCC (Alfa Aesar), DMAP (Alfa Aesar), NH4PF6 Re-ART-1: The synthetic route of Re-ART-1 was shown in Scheme\n(Alfa Aesar), Dulbecco\u2019s Modified Eagle Medium (DMEM, Gibco), fetal S1. Firstly, the mixture of ART (10 equiv.), DCC (1.5 equiv.) and DMAP\nbovine serum (FBS, Gibco), penicillin-streptomycin (Gibco), 3-(4,5- (1.5 equiv.) were stirred in dry CH2Cl2 for 30 min, and then Re-1 (1\ndimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (J&K), equiv.) in dry CH2Cl2 was added drop-wise. After stirring the mixture at\nCCCP (J&K), chloroquine (J&K), Rh123 (J&K), H2DCFDA (J&K), ambient temperature for 24 h, the solvent was evaporated. The crude\nHoechst 33342 (J&K), Fer-1 (J&K), DFO (J&K). Annexin V-FITC product was purified using column chromatography on silica gel by\nApoptosis Detection Kit, GSH and GSSG (oxidized glutathione) Assay Kit elution with CH2Cl2/CH3OH (100:1). Yield: 0.223 g (orange powder),\nand MDA Assay Kit were purchased from Beyotime (Jiangsu, China). 70%. 1H NMR (600 MHz, [D6]DMSO) \u03b4 9.77 (d, J = 5.1 Hz, 2H), 9.05 (d,\nPrimary antibodies against Bax, Bcl-2, caspase-3, PARP and GPX4 were J = 8.3 Hz, 2H), 8.44 (d, J = 6.3 Hz, 2H), 8.32 (s, 2H), 8.28\u20138.24 (m,\npurchased from Cell Signaling Technology. Re-ART-1, Re-ART-2, Re-1, 2H), 7.28 (d, J = 5.8 Hz, 2H), 5.60 (d, J = 9.7 Hz, 1H), 5.52 (s, 1H), 5.03\nRe-2 and ART were dissolved in dimethyl sulfoxide (DMSO) just before (s, 2H), 2.64 (s, 4H), 2.24\u20132.13 (m, 2H), 1.99 (dd, J = 13.9, 2.4 Hz, 1H),\nthe experiments, and the concentration of DMSO in biological experi\u00ad 1.84\u20131.79 (m, 1H), 1.62\u20131.56 (m, 2H), 1.54\u20131.50 (m, 1H), 1.41 (dt,\nments was 1% (v/v). Cisplatin was dissolved in 0.9% sodium chloride J = 13.1, 6.5 Hz, 2H), 1.33\u20131.27 (m, 1H), 1.22 (s, 3H), 1.20\u20131.14 (m,\nsolution just before use. 1H), 0.99\u20130.92 (m, 1H), 0.89 (d, J = 6.2 Hz, 3H), 0.63 (d, J = 7.1 Hz,\n A LCQ DECA XP spectrometer was used for obtaining ESI-MS spectra. 3H). ESI-MS (CH3OH): m/z 926.2315 [M \u2212 PF6]+. Elemental analysis:\nA Bruker Avance 600 spectrometer was used for obtaining 1H NMR calcd (%) for C40H41F6N3O11PRe: C, 44.86; H, 3.86 N, 3.92; found: C,\nspectra. A SpetraMax M2 plate reader was used for determining cell 44.56; H, 3.89; N, 3.79.\nviability. A Nikon A1R/A1 laser-scanning confocal microscope was used Re-ART-2: Complex Re-ART-2 was prepared following a similar\nfor obtaining cell imaging images. A CyFlow Space flow cytometer was procedure to that of Re-ART-1 except that Re-2 was used instead of Re-\nused for performing the flow cytometry analysis. 1. Yield: 0.258 g (brown powder), 75%. 1H NMR (600 MHz, [D6]DMSO)\n \u03b4 9.84 (d, J = 5.3 Hz, 2H), 8.60 (d, J = 5.3 Hz, 2H), 8.22 (t, J = 4.9 Hz,\n 2H), 8.17 (s, 2H), 7.74\u20137.67 (m, 10H), 7.37 (d, J = 5.3 Hz, 2H), 5.61 (d,\n J = 9.8 Hz, 1H), 5.52 (s, 1H), 5.10 (s, 2H), 2.66 (s, 4H), 2.24\u20132.13 (m,\n\n\n 7\n\fR.-R. Ye et al. Journal of Inorganic Biochemistry 223 (2021) 111537\n\n\n 4.5. Cell lines and culture conditions\n\n HeLa, HepG2, A549, A549R and LO2 cells were purchased from\n Nanjing KeyGen Biotechnology Co., Ltd., and cultured in DMEM or\n RPMI 1640 complete medium at 37 \u25e6 C with 5% CO2 atmosphere.\n\n 4.6. Lipophilicity\n\n The lipophilicity of rhenium(I) complexes is presented as log Po/w\n values, which were obtained from a previous study [63].\n\n 4.7. ICP-MS measurement\n\n HeLa cells were seeded in 10 cm tissue culture dishes and incubated\n for 24 h. The medium was removed and replaced with fresh medium\n containing Re(I) complexes (10 \u03bcM). After 1 h incubation, the cells were\n washed with PBS, trypsinized and collected. The cells were counted and\n digested with HNO3 (65%, 0.5 mL). The rhenium content in cells was\n determined by ICP-MS.\n\n 4.8. In vitro cytotoxicity assay\n\n The anticancer activities of rhenium(I) complexes against HeLa,\n A549, A549R, HepG2 and LO2 cells were detected using MTT assay. The\n tested cells were placed in 96-well plates at a density of 5 \u00d7 104/well for\n 24 h at 37 \u25e6 C. After replacing the culture medium with a fresh medium\n containing the Re(I) complexes with/without inhibitors (Fer-1 or DFO)\n at the indicated concentration, the cells were cultured at 37 \u25e6 C for 48 h.\n Afterwards, MTT solution was added and co-incubated at 37 \u25e6 C for an\n additional 4 h. Last, 150 \u03bcL/well DMSO was used to take over the media\n and dissolve the MTT-formazan crystals. A SpetraMax M2 plate reader\n was applied to detect the absorbance of living cells at 570 nm.\n\nFig. 10. Confocal imaging of HeLa cells incubated with Re(I) complexes 4.9. Cellular localization assay\n(4 \u00d7 IC50, 6 h) via C11-BODIPY 581/591 staining (\u03bbex = 488 nm,\n\u03bbem = 510 \u00b1 20 nm (green); \u03bbex = 581 nm, \u03bbem = 591 \u00b1 20 nm (red)). Scale bar: HeLa cells (5 \u00d7 105/well) were seeded in a 35 mm glass-bottom cell\n20 \u03bcm. (For interpretation of the references to colour in this figure legend, the culture dish overnight, then co-cultured with Re(I)-ART conjugates\nreader is referred to the web version of this article.) (10 \u03bcM) and MTDR (150 nM) or LTDR (50 nM) for 1 h. Subsequently,\n cells were immediately monitored by confocal microscopy. Excitation\n2H), 2.01\u20131.96 (m, 1H), 1.83\u20131.78 (m, 1H), 1.59\u20131.54 (m, 2H), 1.49 wavelength of Re(I)-ART conjugates was 405 nm and their emission\n(dd, J = 10.8, 6.7 Hz, 1H), 1.42\u20131.36 (m, 2H), 1.33\u20131.28 (m, 1H), 1.22 wavelength was 560 \u00b1 20 nm. For MTDR and LTDR, the excitation and\n(s, 3H), 1.18\u20131.14 (m, 1H), 1.10 (d, J = 7.0 Hz, 1H), 0.88 (d, J = 6.4 Hz, emission wavelengths were 633 nm and 665 \u00b1 20 nm, respectively.\n3H), 0.64 (d, J = 7.0 Hz, 3H). ESI-MS (CH3OH): m/z 1078.2942\n[M \u2212 PF6]+. Elemental analysis: calcd (%) for C52H49F6N3O11PRe: C, 4.10. Measurement of MMP\n51.06; H, 4.04; N, 3.44; found: C, 51.17; H, 4.12; N, 3.43.\n HeLa cells (5 \u00d7 105/well) were seeded in a 35 mm glass-bottom cell\n4.3. Stability analysis culture dish overnight. After incubating with Re(I)-ART conjugates for\n 6 h, HeLa cells were rinsed twice with PBS, then stained with Rh123\n The stability of Re-ART-1\u20132 was determined using fluorescence (1 \u03bcg/mL) for 30 min. After washing with PBS twice, cells were observed\nspectra and HPLC. Fluorescence spectra: the stock solution (2 \u00d7 10\u2212 5 M) with confocal microscopy (\u03bbex = 488 nm, \u03bbem = 530 \u00b1 20 nm).\nof complexes in PBS was prepared, and then divided them into two equal\nparts, respectively. The first part of the solution was measured imme\u00ad\n 4.11. Intracellular ATP detection\ndiately. The second part of the solution was kept for 48 h, and then\nmonitored using fluorescence spectrophotometer. HPLC: Re-ART-1\u20132\n The tested cells were placed in 96-well plates at a density of 5 \u00d7 104/\nwere dissolved in acetonitrile and detected with a UV detector set at\n well for 24 h at 37 \u25e6 C. After incubating with Re(I)-ART conjugates for\n254 nm and a flow rate of 1 mL/min and a solvent system of MeOH-H2O\n 6 h, the CellTiter-Glo\u00ae Luminescent Cell Viability Assay kit was used to\n(75: 25).\n measure the cellular ATP level following the manufacturer\u2019s manual.\n4.4. Hydrolysis of Re(I)-ART complexes by PLE in vitro\n 4.12. Measurement of intracellular ROS\n \u2212 5\n Re(I) complexes (2 \u00d7 10 M) in PBS were freshly prepared in quartz\ncuvettes (3 mL), and then PLE (1 \u03bcL) was added. Time-dependent HeLa cells (5 \u00d7 105/well) were seeded in a 35 mm glass-bottom cell\nemission spectra were recorded after reaction for 5 min at 298 K. culture dish overnight. After incubating with Re(I)-ART conjugates for\n 6 h, HeLa cells were rinsed with serum-free DMEM twice, then labelled\n with H2DCFDA (10 \u03bcM). Subsequently, cells were washed and examined\n under confocal microscopy (\u03bbex = 488 nm, \u03bbem = 530 \u00b1 20 nm).\n\n 8\n\fR.-R. Ye et al. Journal of Inorganic Biochemistry 223 (2021) 111537\n\n\n4.13. Hoechst 33342 staining and the data were reported as means \u00b1 SD.\n\n HeLa cells (5 \u00d7 105/well) were seeded in a 35 mm glass-bottom cell\n Declaration of Competing Interest\nculture dish overnight. After incubating with Re(I)-ART conjugates for\n24 h, HeLa cells were fixed with 4% paraformaldehyde. Then, Hoechst\n The authors declare that they have no known competing financial\n33342 (5 \u03bcg/mL) was used to label cells which were observed under\n interests or personal relationships that could have appeared to influence\nconfocal microscopy (\u03bbex = 405 nm, \u03bbem = 460 \u00b1 20 nm).\n the work reported in this paper.\n4.14. Annexin V/PI staining\n Acknowledgements\n 6\n HeLa cells were seeded in 6-well plates at a density of 1 \u00d7 10 /well\nand incubated for 24 h. After incubating with Re(I)-ART conjugates for This work was supported by the National Natural Science Foundation\n24 h, cells were collected and resuspended in 500 \u03bcL of binding buffer of China (21967014, 22007042), Applied Basic Research Projects of\ncontaining 5 \u03bcL annexin V and 10 \u03bcL PI. After 10 min of co-incubation in Yunnan Province (202001AT070036), the Innovative Team of Yunnan\ndark, flow cytometry was used to analyze cells (\u03bbex = 488 nm, Province (2019HC018), High-level Scientific Research Foundation for\n\u03bbem = 530 nm \u00b1 20 nm (annexin V) and 620 \u00b1 20 nm (PI)). Talent Introduction of Kunming University of Science and Technology\n (KKKP201826008).\n4.15. Western blot analysis\n Appendix A. Supplementary data\n Bax, Bcl-2, caspase-3, PARP and GPX4 protein extracts were pre\u00ad\npared from HeLa cells. Cells were seeded in 60 mm plates (2 \u00d7 106/well) Supplementary data to this article can be found online at https://doi.\nand incubated overnight for cell attachment. After treating with org/10.1016/j.jinorgbio.2021.111537.\ndifferent doses of Re(I)-ART conjugates for 24 h, cells were lysed, and\nlysates were centrifuged at 4 \u25e6 C for 20 min. The protein concentration References\nwas determined using the BCA assay kit. 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