Source: https://pubs.rsc.org/en/content/articlehtml/2019/mt/c9mt00051h
Timestamp: 2019-04-26 16:37:30+00:00

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The ruthenium complex sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1339/IT-139) showed preclinical activity in a variety of in vivo tumor models including a highly predictive colon cancer model. The compound has entered clinical trials, where patients experienced disease stabilization accompanied by mild side effects. KP1339, a GRP78 inhibitor, disrupts endoplasmic reticulum (ER) homeostasis leading to cell death. The PERK/eIF2α-branch of the ER plays an essential role in the cascade of events triggering immunogenic cell death (ICD). ICD makes dying cancer cells ‘visible’ to the immune system, initiating a prolonged immune response against the tumor. As some metal-based chemotherapeutics such as oxaliplatin are able to induce ICD, we investigate whether KP1339 could also trigger induction of the ICD signature. For this, we employ a three-dimensional colon cancer spheroid model and show for the first time that the treatment with KP1339, a ruthenium-based complex, triggers an ICD signature hallmarked by phosphorylation of PERK and eIF2α, exposure of calreticulin on the cell membrane, release of high mobility group box 1 and secretion of ATP.
The impact of metal-based compounds on the immunological aspects of cancer is now emerging as a critical component of their therapeutic potential. It has been demonstrated based on preclinical and clinical data, that the immunomodulatory effects of platinum complexes provide a promising platform for their combination with immunotherapies. However, further studies are necessary to elucidate the complex interaction between cancer and the immune system and the role of metal-based drugs in modulating their intricate interplay. Our work highlights the importance of studying the immunological aspects of metal-based compounds, and emphasizes the potential of ruthenium-derived complexes in this emerging field.
Therefore, it is of major therapeutic relevance to explore the immunogenic potential of both clinically applied anti-cancer drugs and currently developed compounds. In this context, it is of utmost interest to characterize the molecular mechanisms of how cells die upon treatment as this subsequently influences the onset of ICD. Chemotherapy-induced ICD depends on the emission of key immunomodulatory damage-associated molecular patterns (DAMPs), such as: pre-apoptotic calreticulin (CRT) surface-exposure, extracellular adenosine triphosphate (ATP) and high mobility group box 1 (HMGB-1).
The endoplasmic reticulum (ER) possesses three different transmembrane receptors: IRE1α, ATF6 and PERK. Normally, these proteins are bound by the ER chaperone BiP. Once unfolded proteins accumulate, BiP releases ATF6, PERK and IRE1α, allowing their activation of the unfolded protein response (UPR). Under prolonged ERS conditions UPR is activated as an attempt to restore homeostasis by shutting down protein synthesis and arresting the cell cycle. If this condition persists, the PERK branch of the UPR via ATF4 induces the activation of CHOP, leading to apoptosis.
We hypothesized that KP1339 could induce the hallmarks of ICD, making it the first recognized ruthenium-based complex that is likely able to trigger ICD in vitro. In order to support our hypothesis, 3D tumor spheroids derived from three different colon adenocarcinoma cell lines (HCT-15, HCT-116 and HT-29) were treated with KP1339, oxaliplatin or cisplatin. In response to all treatments, tumor spheroids underwent cell death as indicated by flow cytometry studies with annexin-V fluorescein isothiocyanate and propidium iodide (PI) (SF1, ESI†).
Fig. 1 Representative immunoblot images from a capillary Western blot system. The total load of PERK, eIF2α, and GAPDH, respectively, of HCT-116 cells from spheroids is shown. For PERK and eIF2α the phosphorylation status is also analyzed, whereby phosphorylation is only observed upon treatment with KP1339 and oxaliplatin for 24 h. *GAPDH: loading control for pPERK, tPERK, p-eIF-2α. **GAPDH: loading control for t-eIF2α.
The next step was to investigate whether CRT translocates from the cytoplasm to the cell membrane upon treatment. To demonstrate this, we performed flow cytometry studies, where spheroids were treated for 24 hours with oxaliplatin, cisplatin, KP1339 or were left untreated, followed by staining with an anti-CRT antibody and propidium iodide (live/dead staining). In all cell lines, viable (propidium iodide negative) cells isolated from spheroids treated with KP1339 and oxaliplatin showed increased expression of CRT on the cellular membrane compared to cisplatin treated and untreated controls (Fig. 2 and SF3, ESI†). Additionally, cryosections of spheroids treated under the same conditions as described above were subjected to immunofluorescence staining followed by confocal microscopy. Cells were stained with a cell membrane marker (wheat germ agglutinin), anti-CRT antibody and DAPI (for nuclei) under non-cell-permeabilizing conditions. This approach confirmed the results obtained by flow cytometry, with CRT co-localizing with the cell membrane in KP1339 and oxaliplatin treated spheroids (Fig. 3 and SF4–SF6, ESI†).
Fig. 2 Representative flow cytometry histogram showing increased CRT expression on the surface of (A) cells from HCT-116 spheroids treated with KP1339 and oxaliplatin, compared to untreated (control) and cisplatin-treated spheroids, after 24 h of exposure. (B) Analysis of calreticulin membrane exposure of treated spheroids after drug exposure for 24 h. Mean + STD (ns: not significant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; Tukey's range test).
Fig. 3 Representative immunofluorescence analysis (confocal microscopy) of paraformaldehyde-fixed (PFA) HCT-116 spheroids. Exposure of calreticulin on the cell membrane is observed after 24 h treatment with KP1339 (100 μM) and oxaliplatin (20 μM), as indicated by co-localization of calreticulin and membrane associated wheat germ agglutinin (WGA).
We next evaluated HMGB-1 release from cells treated with KP1339. This protein is present in the nucleus, then translocates to the cytoplasm and is eventually released to the extracellular space upon treatment with ICD inducers.37 We measured HMGB-1 content in supernatants originated from treated and untreated spheroids by means of ELISA. Indeed, upon treatment with oxaliplatin, cisplatin and KP1339 all three cell lines showed a pronounced release of HMGB-1 from the corresponding tumor spheroids (SF7, ESI†).
We also assessed HMGB-1 localization by using immunofluorescence and super-resolution confocal microscopy. Cryosections of KP1339 treated spheroids showed an enhanced signal of this protein in the cytoplasm, confirming the findings obtained by ELISA (Fig. 4, SF8 and SF9, ESI†).
Fig. 4 Representative immunofluorescence analysis (structure illumination microscopy) of paraformaldehyde-fixed (PFA) spheroids. Release of HMGB-1 (red and range indicator) into the cytoplasm of HCT-116 spheroids is observed after 24 h treatment with KP1339 (100 μM).
In the context of verifying ATP release, we first investigated whether KP1339 is able to induce autophagy. It has been previously shown that ATP release during chemotherapy-driven ICD relies on activation of the autophagic machinery.32 Therefore, we probed the expression of some key autophagy markers such as Beclin-1 and LC3A/B-II. Upregulation of Beclin-1 and LC3A/B-II in spheroids treated with KP1339 was clearly observed as early as 24 hours after drug treatment. In contrast, cisplatin treated spheroids did not show any pronounced effect, confirming the findings of other studies where cisplatin failed to induce autophagy (SF2, ESI†). The supernatants of treated and untreated spheroids were collected to determine ATP levels by using a luciferase based assay. All drugs induced an increase in ATP release with KP1339-treated cells exhibiting a pronounced release (Fig. 5).
Fig. 5 Analysis of ATP levels in supernatants from treated spheroids after drug exposure for 24 h. Mean + STD (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; Tukey's range test).
The production of reactive oxygen species (ROS) was subsequently assessed. To demonstrate the oxidative stress response, ROS production was tracked by using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) as an indicator. All drug treatments led to ROS production, with KP1339 exhibiting the most marked effect amongst all treatments (SF10, ESI†).
In type I ICD, ERS is induced as a collateral effect of the actual mode of action of a compound. Oxaliplatin induces type I ICD due to DNA damage resulting in ERS. Type II ICD inducers, on the other hand, trigger a focused ERS and are known to more efficiently elicit danger signals, e.g., photodynamic therapy with hypericin and oncolytic viruses. There is only a limited number of type II ICD inducers reported in the literature, and for a better understanding of this specific type of ICD, more inducers are required for further studies.38 Based on our findings, KP1339 may qualify as a type II ICD inducer due to its main mode of action that relies on ERS16 (SF2, ESI†) and oxidative stress (SF10 and SF11, ESI†). Most of the chemotherapeutic agents currently used in the clinic, are unable to trigger ICD, often because they do not induce ERS and the consequent CRT exposure.
In accordance with other studies, we confirm here that oxaliplatin induces all the key hallmarks of ICD, while cisplatin failed to stimulate CRT translocation to the cell membrane despite inducing other DAMPs.15,17,20,37 Chemically closely related drugs often exhibit an unexpected degree of heterogeneity in their capacity to trigger ICD, e.g., cisplatin and oxaliplatin.29 Failure to elicit one of the key events in the course of ICD is sufficient to impair the immunogenicity of cell death.21,36 To our knowledge, this is the first study employing a 3D model, where a ruthenium-based complex is shown to induce the ICD signature in vitro.
Many chemotherapeutic agents are particularly effective when administered in combination regimens. Immunological effects may explain the success of such combinations, e.g., 5-fluorouracil (with folinic acid) and oxaliplatin (FOLFOX), where the individual agents can target malignant cells through their different modes of action, but can also exert a combined long lasting immunostimulatory effect.
We would like to thank Prof. Andreas Wanninger and his team (Institute of Zoology, University of Vienna) as well as John Soltys (Department of Chemical Catalysis, University of Vienna), Dr Wolfgang Kandioller (Institute of Inorganic Chemistry, University of Vienna), Christina Puri and Oliver Bergner (Boehringer Ingelheim RCV, Vienna) for helpful discussions and for supporting this work.
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