A Rhenium Isonitrile Complex Induces Unfolded Protein Response-Mediated Apoptosis in Cancer Cells.

A Journal of Accepted Article Title:A Rhenium Isonitrile Complex Induces Unfolded Protein Response-Mediated Apoptosis in Cancer Cells Authors:A. Paden King, Sierra C. Marker, Robert V. Swanda, Joshua J. Woods, Shu-Bing Qian, and Justin J. Wilson This manuscript has been accepted after peer review and appears as an Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article. To be cited as: Chem. Eur. J. 10.1002/chem.201902223 Link to VoR: http://dx.doi.org/10.1002/chem.201902223 Supported by 10.1002/chem.201902223 COMMUNICATION A Rhenium Isonitrile Complex Induces Unfolded Protein Response-Mediated Apoptosis in Cancer Cells A. Paden King,‡[a] Sierra C. Marker, ‡[a] Robert V. Swanda,[b] Joshua J. Woods,[c] Shu-Bing Qian,[b] Justin J. Wilson[a]* Abstract: Complexes of the element Re have recently been shown to scaffolds may give rise to promising drug candidates.[6–11] In this possess promising anticancer activity via mechanisms of action that context, our group has been exploring the anticancer activity of are distinct from the conventional metal-based drug cisplatin. In this polypyridyl rhenium(I) tricarbonyl complexes.[12–15] Certain study, we report our investigations on the anticancer activity of the members of this class of compounds exhibit potent cytotoxic complex [Re(CO)(dmphen)(p-tol-ICN)]+ (TRIP) where dmphen = 2,9- activity that can be leveraged for their use as anticancer 3 dimethyl-1,10-phenanthroline and p-tol-ICN = 4-methylphenyl agents.[16–23] Here, we describe our investigation of a new isonitrile. TRIP was synthesized via literature methods and rhenium(I) tricarbonyl complex bearing a chelating polypyridyl exhaustively characterized. This compound exhibits potent in vitro ligand and an axial isonitrile ligand as a potent anticancer agent. anticancer activity in a wide variety of cell lines. Flow cytometry and Our efforts to understand the mechanism of action of this immunostaining experiments indicate TRIP induces intrinsic tricarbonyl rhenium isonitrile polypyridyl (TRIP) complex have apoptosis. Comprehensive biological mechanistic studies revealed that it is an effective ER stress-inducing agent with demonstrate this compound triggers the accumulation of misfolded significant antiproliferative activity. proteins, which causes endoplasmic reticulum (ER) stress, the unfolded protein response, and apoptotic cell death. Furthermore, TRIP was synthesized by treating the previously reported TRIP induces hyperphosphorylation of eIF2α, translation inhibition, complex [Re(CO) (dmphen)OTf] with excess 4-methylphenyl 3 mitochondrial fission, and induction of proapoptotic ATF4 and CHOP. isonitrile in tetrahydrofuran (Figure 1). TRIP was fully These results establish TRIP as a promising anticancer agent based characterized using 1H NMR and IR spectroscopy, HR-MS, and on its potent cytotoxic activity and ability to induce ER stress. X-ray diffraction (Figures S1 and S2, Tables S1 and S2). The purity of the complex was verified via elemental analysis and HPLC (Figure S3, Table S3). The water-soluble complex is The endoplasmic reticulum (ER) is a major regulator of luminescent upon irradiation with UVA and blue light and exhibits cancer cell proliferation, metastasis, angiogenesis, and a luminescence quantum yield of 3% and a lifetime of 1.05 µs in chemotherapy resistance.[1] Cancer cells often exhibit higher aqueous, air-equilibrated phosphate buffer (Figures S4–S6). The rates of protein synthesis than non-cancer cells, which raises their complex is stable indefinitely as a solid and in aqueous solution ER protein load and leads to higher basal levels of ER stress.[2] for over one week (Figures S7 and S8). TRIP is also stable in the To handle this ER stress, cancer cells often employ the unfolded presence of millimolar concentrations of glutathione (Figure S9). protein response (UPR). The UPR is typically cytoprotective, and Based on TRIP’s favorable physical properties and high stability, its increased activation in cancer cells can cause them to be more we evaluated its potential as an anticancer agent in vitro. virulent and more resistant to chemotherapy.[3] However, acute inductions of high levels of ER stress can shift the UPR to activate apoptosis.[4] The higher basal ER stress levels of cancer cells makes them more susceptible than normal cells to apoptosis induction via overactivation of the UPR. Thus, the development of new chemotherapeutic agents that target the ER is a promising strategy for the treatment of cancer.[5] Recently, several transition metal complexes bearing polypyridyl ligands have been discovered to induce anticancer activity via ER stress and the UPR, suggesting that the exploration of these non-traditional [a] A. P. King, S. C. Marker, J. J. Woods, Prof. J. J. Wilson Department of Chemistry and Chemical Biology, Cornell University Ithaca, NY 14853 (USA) E-mail: jjw275@cornell.edu [b] R. V. Swanda, Prof. S.-B. Qian Division of Nutritional Sciences, Cornell University Figure 1. Diagram of TRIP (left) and its X-ray crystal structure (right). Ellipsoids Ithaca, NY 14853 (USA) are drawn at 50% probability. Hydrogen atoms and the counterion are omitted [c] J. J. Woods Robert F. Smith School for Chemical and Biomolecular Engineering for clarity. Cornell University Ithaca, NY 14853 (USA) The cytotoxicity of TRIP was investigated in a panel of cancer and non-cancer cell lines to determine its potential as a ‡Denotes equal contribution therapeutic agent. For comparison, we also evaluated the Supporting information for this article is given via a link at the end of activities of the established metal-based anticancer drug cisplatin the document tpircsunaM detpeccA Chemistry - A European Journal This article is protected by copyright. All rights reserved. 10.1002/chem.201902223 COMMUNICATION and another potent rhenium anticancer agent that we have Given the promising activity of TRIP in a variety of cancer previously investigated in our lab, [Re(CO) (dmphen)(OH )]+ cell lines and its ability to induce intrinsic apoptosis, we explored 3 2 (Neo-Re).[12,15] The concentrations of these complexes required its intracellular localization and early cellular effects. The to reduce cell viability to 50% of the control (IC ) are shown in localization of TRIP was probed by measuring the colocalization 50 Table 1. In comparison to cisplatin and Neo-Re, TRIP has of TRIP luminescence with organelle-specific fluorescent small comparable or greater toxicity in all cancer cell lines tested molecules or fusion proteins. Partial colocalization was observed (Figures S10–S21). Based on its promising anticancer activity, with the LysoTracker Red dye and GalT-dsRed fusion protein, but we submitted TRIP for screening in the National Cancer Institute the majority of TRIP luminescence was cytosolic (Figure S32). (NCI)-60 cell line panel (Figure S22).[24] The results indicate that While performing these colocalization studies, we observed that TRIP is most potent in melanoma and breast cancer cells lines the mitochondrial morphology was noticeably altered in TRIP- and least effective in lung and renal cancer cell lines. The activity treated cells. The mitochondria were significantly rounded and of TRIP in this cell line panel was compared to drugs in the NCI punctate after TRIP treatment, in contrast to the tubular, database via the COMPARE algorithm, which compares the elongated morphology within untreated cells. Time-lapse toxicity profiles of drugs to reveal correlations in their activity.[25] microscopy experiments revealed that TRIP induces these Highest correlations were observed for DNA-binding agents changes after only 30 min of treatment in HeLa cells (Figures 2 chromomycin A3 and actinomycin D and the translation inhibitors and S33, Videos 1–6). Although TRIP-treated mitochondria were pyllanthoside, bruceantin, and didemnin B (Table S4). Notably, visually different, mitochondrial polarization experiments with the the spectrum of activity of TRIP was not correlated to any of the ratiometric sensor JC-1 indicated that the mitochondria remained platinum-based drugs, and it exhibits only a moderate correlation functional (Figures S34 and S35), demonstrating that the (PCC = 0.403) to Neo-Re. The high correlations to established observed changes might be controlled mitochondrial fission rather transcription and translation inhibitors indicates that TRIP may act than fragmentation. These morphology changes were curtailed in similarly. the presence of Mdivi-1, which inhibits dynamin-related protein 1 (Drp1), an essential mediator of fission, confirming that this Table 1. IC50 values of TRIP, Neo-Re, and cisplatin in cancer and non- process is due to mitochondrial fission (Figure 2).[26] Because cancer cell lines. mitochondrial fission is often associated with autophagy,[27] we examined the expression of LC3, an autophagosome marker,[28] Compound IC50 (µM) in A2780 cells upon treatment with TRIP. After 24 h, a large increase in LC3II expression relative to LC3I was observed in A2780 A2780 HeLa A549 HEK293 (ovarian CP70 (cervical (lung (kidney) cells treated with TRIP (Figure S36). Based on these results, it is cancer) (cisplatin- cancer) cancer) clear that TRIP induces both autophagy and apoptosis. Because resistant TRIP does not depolarize the mitochondria or cause release of ovarian cytochrome c on short time scales, we hypothesized that a cancer) different organelle, such as the ER, may be the key target of this TRIP 1.7 ± 0.7 1.9 ± 1 1.4 ± 0.2 1.4 ± 0.6 1.9 ± 0.2 compound. Neo-Re 5.7 ± 0.6 6.0 ± 0.2 4.4 ± 1.3 7.7 ± 2.4 9.0 ± 0.3 Cisplatin 1.3 ± 0.1 12 ± 3 6.6 ± 0.7 5.6 ± 0.5 1.7 ± 0.2 To determine the type of cell death induced by TRIP, the cytotoxicity of this compound in A2780 cells was evaluated in the presence of inhibitors of various established cell death pathways. Inhibitors of necroptosis, paraptosis, and ferroptosis did not alter TRIP’s activity, but the pan-caspase inhibitor Z-VAD-FMK significantly decreased TRIP’s cytotoxicity (Figures S23–S27). Because the activation of caspases is often critical for the execution of apoptosis, this result indicates that TRIP may be inducing apoptosis. To confirm that TRIP induces caspase- dependent apoptosis, we first performed western blots to detect apoptosis markers caspase 3 and cleaved PARP (Figure S28). We further verified this cell death pathway by performing the annexin V assay, which selectivity stains apoptotic cells (Figures S29 and S30). To determine whether TRIP induced apoptosis by the intrinsic pathway, the release of cytochrome c from the mitochondria was tracked using flow cytometry (Figure S31). Cytochrome c release occurs on the same time scale as Figure 2. HeLa cells stained with MitoTracker Red and Hoechst dye treated apoptosis induction by TRIP, indicating that TRIP induces intrinsic with TRIP (5 μM) for 0 and 30 min (top panels). HeLa cells stained with apoptosis. tpircsunaM detpeccA Chemistry - A European Journal This article is protected by copyright. All rights reserved. 10.1002/chem.201902223 COMMUNICATION MitoTracker Red and Hoechst dye cotreated with TRIP (5 μM) and Mdivi-1 (50 μM) for 0 and 30 min (bottom panels). Scale bar = 10 μm. Because of the potential connections between mitochondrial fission, autophagy, and ER stress, we explored the effects of the ER stress modulator salubrinal on the cytotoxicity of TRIP in A2780 cells.[29] Salubrinal operates by inhibiting dephosphorylation of the master regulatory protein eukaryotic initiation factor 2α (eIF2α), an integral component of the UPR.[29– 32] The presence of salubrinal increases the activity of TRIP by a factor of 4 (Figure 3A). Based on this synergy, we explored the possibility that TRIP was acting to cause phosphorylation of eIF2α. Western blot analysis of A2780 cells treated with TRIP confirms the induction of eIF2α phosphorylation as little as 2 h after exposure (Figures 3B and S37), indicating that this process is one of the first cellular responses. Next, we explored the downstream effects of eIF2α phosphorylation. The most immediate and pronounced effect of eIF2α phosphorylation is the inhibition of translation.[33] To probe whether the levels of phosphorylation induced by TRIP were sufficient to inhibit protein translation, we measured endogenous global translation levels Figure 3. (A) Dose-response curve of A2780 cells treated with TRIP in the using the puromycin incorporation assay.[34] As early as 2 h post presence of 25 μM salubrinal (blue) or absence of salubrinal (red). (B) Western incubation, A2780 cells treated with TRIP incorporated blot of untreated (–), cisplatin (C, 10 μM), TRIP (5 μM), or bortezomib (B, 25 substantially less puromycin compared to the untreated controls, nM) for 24 h in A2780 cells. (C) Western blot of A2780 cells incubated with TRIP indicating much lower rates of translation (Figure 3C). The role of (5 μM) over 0, 0.5, 1, 1.5, and 2 h with puromycin (10 min, left blot) and A2780 eIF2α in these processes was confirmed by testing TRIP in a cells untreated (–), cisplatin (C, 10 μM), TRIP (5 μM), or bortezomib (B, 25 nM) mutant MEF cell line incapable of eIF2α phosphorylation. The treated for 24 h with puromycin (10 min, right blot). (D) Confocal microscopy mutant cells showed no changes in translation levels after TRIP images of HeLa cells treated with ThT (5 μM) at 0 and 30 min in the absence treatment (Figures S38 and S39). (top panels) and the presence (bottom panels) of TRIP (5 μM) at 0 and 30 min. Hyperphosphorylation of eIF2α can lead to apoptosis via Scale bar = 50 μm. upregulation of the stress-related transcription factors ATF4 and CHOP.[35] We measured the upregulation of these proteins in response to TRIP treatment and found that both ATF4 and CHOP were upregulated (Figure 3B), linking the observed eIF2α phosphorylation and apoptosis. Phosphorylation of eIF2α also results in cell cycle arrest in the G1 phase.[36] Cells treated with TRIP showed an 18% increase in the population of cells in the G1 phase and a corresponding decrease in the number of cells in the S phase as opposed to untreated cells (Figure S40). Thus, the ability of TRIP to stall cells in the G1 phase is fully consistent with its induction of eIF2α phosphorylation. These results indicate that TRIP induces ER stress, triggering eIF2α phosphorylation and the resulting downstream effects, culminating in cellular apoptosis. Phosphorylation of eIF2α often occurs due to the accumulation of misfolded proteins. To determine whether the observed phosphorylation was due to protein misfolding, the extent of misfolded protein accumulation induced by TRIP was evaluated using the dye Thioflavin T, (ThT) which fluoresces in the presence of protein aggregates.[37] The fluorescence intensity of ThT increased significantly in HeLa cells treated with TRIP in comparison to untreated cells within 30 min (Figures 3D, S41 and S42, Videos 7 and 8). Given the observation of fast protein aggregation upon treatment with TRIP, the induction of protein misfolding is most likely the cause of the ER stress and activation Figure 4. Proposed mechanism of ER-stress and apoptosis induction by TRIP. of the UPR. A summary of our current understanding of TRIP’s mechanism of ER stress induction and the subsequent cellular response is shown in Figure 4. TRIP induces ER stress in less than 30 min after exposure due to the accumulation of misfolded tpircsunaM detpeccA Chemistry - A European Journal This article is protected by copyright. All rights reserved. 10.1002/chem.201902223 COMMUNICATION proteins. Misfolded protein accumulation leads to the respectively. Ms. Sam Davalos is thanked for assistance in phosphorylation of eIF2α, which initiates autophagy, shuts down preparing the Table of Contents Figure. global protein translation, and upregulates ATF4. Prolonged eIF2α phosphorylation and upregulation of ATF4 leads to Keywords: bioinorganic chemistry • cancer • endoplasmic expression of the proapoptotic protein CHOP, which induces reticulum stress • metallodrug • translation inhibition mitochondrial membrane depolarization and release of cytochrome c. Cytochrome c release then results in caspase [1] H. Urra, E. Dufey, T. Avril, E. Chevet, C. Hetz, Trends in Cancer activation and initiation of apoptosis. Although we have 2016, 2, 252–262. investigated potential causes of eIF2α phosphorylation, including proteasome inhibition, HSP90 inhibition, and reactive oxygen [2] Y.-P. Vandewynckel, D. Laukens, A. Geerts, E. Bogaerts, A. Paridaens, X. Verhelst, S. Janssens, F. Heindryckx, H. Van species generation, we found no evidence that TRIP triggers Vlierberghe, Anticancer Res. 2013, 33, 4683–4694. protein misfolding via these pathways (Figures S43–S47). Recently, a range of diverse metal complexes have been shown [3] M. J. Mann, L. M. Hendershot, Cancer Biol. Ther. 2006, 5, 736–740. to induce ER stress.[7-11,38-47] The major mechanism of action proposed for these agents is through the production of ROS. Only [4] R. Sano, J. C. Reed, Biochim. Biophys. Acta - Mol. Cell Res. 2013, 1833, 3460–3470. a few studies have discovered metal complexes that induce ER stress in the absence of ROS generation.[9,38,39,48] TRIP’s ability to [5] R. Ojha, R. K. Amaravadi, Pharmacol. Res. 2017, 120, 258–266. induce ER stress independent of ROS generation indicates that it operates via a different mechanism than many other metallodrugs [6] J. Pracharova, G. Vigueras, V. Novohradsky, N. Cutillas, C. Janiak, targeting the ER. H. Kostrhunova, J. Kasparkova, J. Ruiz, V. Brabec, Chem. - A Eur. J. 2018, 24, 4607–4619. Collectively, these results establish TRIP as a promising anticancer agent that kills cells by causing the accumulation of [7] R. Cao, J. Jia, X. Ma, M. Zhou, H. Fei, J. Med. Chem. 2013, 56, misfolded proteins. TRIP’s favorable physical and photophysical 3636–3644. properties, as well as its high potency, make it a candidate for future studies and a platform for the design of more potent [8] T. Zou, C.-N. Lok, Y. M. E. Fung, C.-M. Che, Chem. Commun. 2013, 49, 5423–5425. analogues. Our current efforts are directed toward synthesizing a variety of related complexes in order to develop a structure- [9] X. Meng, M. L. Leyva, M. Jenny, I. Gross, S. Benosman, B. Fricker, activity relationship and performing proteomics studies to identify S. Harlepp, P. Hébraud, A. Boos, P. Wlosik, P. Bischoff, C. Sirlin, M. Pfeffer, J.-P. Loeffler, C. Gaiddon, Cancer Res. 2009, 69, 5458– TRIP’s molecular mechanism of action. 5466. Supporting information for this article is given via a link at the end of the document. CCDC 1902045 contain the [10] J. S. Nam, M.-G. Kang, J. Kang, S.-Y. Park, S. J. C. Lee, H.-T. Kim, supplementary crystallographic data for this paper. These data J. K. Seo, O.-H. Kwon, M. H. Lim, H.-W. Rhee, T.-H. Kwon, J. Am. Chem. Soc. 2016, 138, 10968–10977. can be obtained free of charge from The Cambridge Crystallographic Data Centre. [11] K. Suntharalingam, T. C. Johnstone, P. M. Bruno, W. Lin, M. T. Hemann, S. J. Lippard, J. Am. Chem. Soc. 2013, 135, 14060– 14063. Acknowledgements [12] K. M. Knopf, B. L. Murphy, S. N. MacMillan, J. M. Baskin, M. P. Barr, E. Boros, J. J. Wilson, J. Am. Chem. Soc. 2017, 139, 14302– 14314. This research was supported by the College of Arts and Sciences at Cornell University, the Cornell Technology Licensing Office [13] S. C. Marker, S. N. MacMillan, W. R. Zipfel, Z. Li, P. C. Ford, J. J. Cornell Technology Acceleration and Maturation (CTAM) fund, Wilson, Inorg. Chem. 2018, 57, 1311–1331. and by the Office of the Assistant Secretary of Defense for Health Affairs through the Ovarian Cancer Research Program under [14] C. C. Konkankit, B. A. Vaughn, S. N. MacMillan, E. Boros, J. J. Wilson, Inorg. Chem. 2019, 58, 3895-3909. award no. W81XWH-17-1-0097. This work made use of the NMR facility at Cornell University, which is supported, in part, by the [15] C. C. Konkankit, A. P. King, K. M. Knopf, T. L. Southard, J. J. NSF under award number CHE-1531632. A. Paden King and R. Wilson, ACS Med. Chem. Lett. 2019, 10, 822-827. Swanda thank the National Institute of Health, National Institute of General Medical Sciences, for a Chemical Biology Interface [16] C. C. Konkankit, S. C. Marker, K. M. Knopf, J. J. Wilson, Dalton Trans. 2018, 47, 9934–9974. (CBI) Training Grant (grant number T32GM008500). Work in the lab of S.-B. Qian is supported by NIH grants R01GM1222814 and [17] P. V. Simpson, I. Casari, S. Paternoster, B. W. Skelton, M. Falasca, R21CA227917 and by the Howard Hughes Medical Institute M. Massi, Chem. - A Eur. J. 2017, 23, 6518–6521. (award number 55108556). J. J. Woods is supported by the [18] N. Agorastos, L. Borsig, A. Renard, P. Antoni, G. Viola, B. Spingler, NSFGRFP (DGE-1650441). We would like to thank the BRC P. Kurz, R. Alberto, Chem. - A Eur. J. 2007, 13, 3842–3852. imaging facility at Cornell University for help with flow cytometry experiments. We would also like to thank Prof. Warren Zipfel and [19] A. Kurzwernhart, W. Kandioller, C. Bartel, S. Bächler, R. Trondl, G. Prof. Jeremy Baskin for assistance with the fluorescence decay Mühlgassner, M. A. Jakupec, V. B. Arion, D. Marko, B. K. Keppler, et al., Chem. 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Wilson* rhenium tricarbonyl Page No. – Page No. complex kills A Rhenium Isonitrile Complex Induces Unfolded Protein cancer cells Response-Mediated Apoptosis in Cancer Cells by inducing the accumulation of unfolded proteins, leading to activation of the unfolded protein response and apoptosis. [a] A. P. King, S. C. Marker, J. J. Woods, Prof. J. J. Wilson tpircsunaM detpeccA Chemistry - A European Journal This article is protected by copyright. All rights reserved.