Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus
2018; Springer Nature; Volume: 37; Issue: 13 Linguagem: Inglês
10.15252/embj.201798354
ISSN1460-2075
AutoresLígia C. Gomes‐da‐Silva, Liwei Zhao, Lucillia Bezu, Heng Zhou, Allan Sauvat, Peng Liu, Sylvère Durand, Marion Leduc, Sylvie Souquère, Friedemann Loos, Laura Mondragón, Baldur Sveinbjørnsson, Øystein Rekdal, Gaëlle Boncompain, Franck Perez, Luı́s G. Arnaut, Oliver Kepp, Guido Kroemer,
Tópico(s)Porphyrin Metabolism and Disorders
ResumoArticle28 May 2018free access Transparent process Photodynamic therapy with redaporfin targets the endoplasmic reticulum and Golgi apparatus Lígia C Gomes-da-Silva Chemistry Department, University of Coimbra, Coimbra, Portugal Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Liwei Zhao Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Lucillia Bezu Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Heng Zhou Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Allan Sauvat Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Peng Liu orcid.org/0000-0002-1682-9222 Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Sylvère Durand Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Marion Leduc Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Sylvie Souquere Gustave Roussy Comprehensive Cancer Center, Villejuif, France CNRS, UMR9196, Villejuif, France Search for more papers by this author Friedemann Loos orcid.org/0000-0002-5976-5978 Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Laura Mondragón Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Baldur Sveinbjørnsson Lytix Biopharma AS, Oslo, Norway Institute of Medical Biology, University of Tromsø, Tromsø, Norway Search for more papers by this author Øystein Rekdal Lytix Biopharma AS, Oslo, Norway Institute of Medical Biology, University of Tromsø, Tromsø, Norway Search for more papers by this author Gaelle Boncompain Department of Subcellular Structure and Cellular Dynamics, CNRS, Institut Curie, PSL Research University, Paris, France Search for more papers by this author Franck Perez orcid.org/0000-0002-9129-9401 Department of Subcellular Structure and Cellular Dynamics, CNRS, Institut Curie, PSL Research University, Paris, France Search for more papers by this author Luis G Arnaut Chemistry Department, University of Coimbra, Coimbra, Portugal Search for more papers by this author Oliver Kepp Corresponding Author [email protected] orcid.org/0000-0002-6081-9558 Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Guido Kroemer Corresponding Author [email protected] orcid.org/0000-0002-9334-4405 Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Sorbonne Paris Cité, Université Paris Descartes, Paris, France Université Pierre et Marie Curie, Paris, France Pôle de Biologie, Hôpital Européen Georges Pompidou, APsupp-HP, Paris, France Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Lígia C Gomes-da-Silva Chemistry Department, University of Coimbra, Coimbra, Portugal Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Liwei Zhao Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Lucillia Bezu Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Heng Zhou Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Allan Sauvat Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Peng Liu orcid.org/0000-0002-1682-9222 Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Sylvère Durand Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Marion Leduc Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Sylvie Souquere Gustave Roussy Comprehensive Cancer Center, Villejuif, France CNRS, UMR9196, Villejuif, France Search for more papers by this author Friedemann Loos orcid.org/0000-0002-5976-5978 Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Laura Mondragón Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Baldur Sveinbjørnsson Lytix Biopharma AS, Oslo, Norway Institute of Medical Biology, University of Tromsø, Tromsø, Norway Search for more papers by this author Øystein Rekdal Lytix Biopharma AS, Oslo, Norway Institute of Medical Biology, University of Tromsø, Tromsø, Norway Search for more papers by this author Gaelle Boncompain Department of Subcellular Structure and Cellular Dynamics, CNRS, Institut Curie, PSL Research University, Paris, France Search for more papers by this author Franck Perez orcid.org/0000-0002-9129-9401 Department of Subcellular Structure and Cellular Dynamics, CNRS, Institut Curie, PSL Research University, Paris, France Search for more papers by this author Luis G Arnaut Chemistry Department, University of Coimbra, Coimbra, Portugal Search for more papers by this author Oliver Kepp Corresponding Author [email protected] orcid.org/0000-0002-6081-9558 Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Search for more papers by this author Guido Kroemer Corresponding Author [email protected] orcid.org/0000-0002-9334-4405 Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Sorbonne Paris Cité, Université Paris Descartes, Paris, France Université Pierre et Marie Curie, Paris, France Pôle de Biologie, Hôpital Européen Georges Pompidou, APsupp-HP, Paris, France Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Author Information Lígia C Gomes-da-Silva1,2,3,4, Liwei Zhao2,3,4, Lucillia Bezu2,3,4, Heng Zhou2,3,4, Allan Sauvat2,3,4, Peng Liu2,3,4, Sylvère Durand3,4, Marion Leduc2,3,4, Sylvie Souquere6,7, Friedemann Loos2,3,4, Laura Mondragón2,3,4, Baldur Sveinbjørnsson8,9, Øystein Rekdal8,9, Gaelle Boncompain10, Franck Perez10, Luis G Arnaut1, Oliver Kepp *,2,3,4 and Guido Kroemer *,2,3,4,5,11,12,13 1Chemistry Department, University of Coimbra, Coimbra, Portugal 2Faculty of Medicine, University of Paris Sud, Kremlin-Bicêtre, France 3Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France 4Institut National de la Santé et de la Recherche Médicale UMR1138, Equipe 11 labellisée Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France 5Sorbonne Paris Cité, Université Paris Descartes, Paris, France 6Gustave Roussy Comprehensive Cancer Center, Villejuif, France 7CNRS, UMR9196, Villejuif, France 8Lytix Biopharma AS, Oslo, Norway 9Institute of Medical Biology, University of Tromsø, Tromsø, Norway 10Department of Subcellular Structure and Cellular Dynamics, CNRS, Institut Curie, PSL Research University, Paris, France 11Université Pierre et Marie Curie, Paris, France 12Pôle de Biologie, Hôpital Européen Georges Pompidou, APsupp-HP, Paris, France 13Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden *Corresponding author. Tel: +33 1 42 11 45 16; E-mail: [email protected] *Corresponding author. Tel: +33 1 44 27 76 67; E-mail: [email protected] EMBO J (2018)37:e98354https://doi.org/10.15252/embj.201798354 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA-dependent secretory pathway. This led to a general inhibition of protein secretion by PDT-treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT. Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function. Synopsis Redaporfin, a latest generation light-sensitizing agent employed in photodynamic anti-tumor therapy, acts via selective damage of endoplasmic reticulum (ER) and Golgi apparatus (GA), leading to reactive oxygen species-mediated ER stress, compromised protein secretion, and eventually cancer cell death. Redaporfin enriches in the ER and GA in human osteosarcoma cells. Light activated redaporfin causes reactive oxygen species-dependent perturbation of the ER and GA, coupled to irreversible inhibition of GA function, leading to reduced cytokine secretion. Dissipation of the GA by brefeldin A or golgicide A can reduce the redaporfin-mediated phototoxicity. Localized production of reactive oxygen species at the GA is sufficient to kill cancer cells. Introduction Photodynamic therapy (PDT) is a novel antineoplastic treatment modality. At the physicochemical level, the principle of PDT resides in the administration of a photosensitizer that itself is rather inert, yet acquires cytotoxic potential upon absorption of photons that cause the generation of reactive oxygen species (ROS), hence damaging cellular structures. At the practical level, the photosensitizing drug is injected systemically or intratumorally. After a defined time, named the drug-to-light interval (DLI), light with appropriate wavelength is applied to the targeted area. High DLI facilitates optimal redistribution of the compound into organellar, cellular, or subcellular compartments, whereas low DLI is mainly used to target the tumor vasculature. Photodynamic therapy allows to limit the overall side effects and to control the spatial and temporary extension of the treatment with unprecedented precision. It is minimally invasive with superior functional outcome as compared to surgery. Both in preclinical models and in the clinical context, PDT-elicited anti-tumor immunity has been observed (Agostinis et al, 2011; Dabrowski & Arnaut, 2015). The first-in-class photosensitizer approved for clinical use in cancer treatment is porfimer. This hematoporphyrin derivative presented promising results in patients, despite limitations including low molar absorption, poor tissue penetration of light at 630 nm, and a rather long-lasting photosensitivity, obliging patients to stay in the dark (Allison & Sibata, 2010). Some of these limitations were partially overcome by the second generation of photosensitizers including temoporfin, silicon phthalocyanine-4, talaporfin, and verteporfin, which are endowed with intense absorption bands at longer wavelengths and shorter periods of photosensitivity (Allison & Sibata, 2010). The latest generation photosensitizers include the bacteriochlorins, padeliporfin, and redaporfin (Redp), which are currently under clinical evaluation. Major advantages include a high ROS quantum yield and absorption in high spectral regions (740–780 nm) that allow to treat relatively deep lesions. Fast clearance of padeliporfin limits PDT protocols to targeting of the tumor vasculature. In contrast, redaporfin is endowed with higher stability that is compatible with both long and short DLI (Arnaut et al, 2014). Redaporfin-based PDT was shown to be effective in the treatment of immunocompetent mice bearing subcutaneous tumors with up to 85% of cure rate. Recently, phase I/II clinical trials have successfully been concluded in advanced head and neck cancer patients (NCT02070432). The anticancer effect of redaporfin-based PDT was partially ascribed to the host immune system as the treatment efficacy dropped dramatically in immunodeficient mice (Rocha et al, 2015; Pucelik et al, 2016). Anti-tumor immunity activated by redaporfin-PDT inhibits the growth of distant (and non-irradiated) lesions and protects cured mice from rechallenge with live cancer cells (Rocha et al, 2015; Pucelik et al, 2016). This immunostimulatory effect of redaporfin is reminiscent of that induced by hypericin (Hyp). Indeed, PDT with hypericin has been the first PDT to be shown to induce immunogenic cell death (ICD; Garg et al, 2012). Photodynamic therapy with hypericin triggers ICD through a pathway that involves endoplasmic reticulum (ER) stress (Garg et al, 2012), as indicated by the phosphorylation of eukaryotic translation initiation factor 2-alpha (eIF2α) by the ER stress kinase eIF2α kinase 3 (EIF2AK3, best known as PERK) and the exposure of calreticulin at the cell surface (Galluzzi et al, 2012; Garg et al, 2012, 2016; Rufo et al, 2017). Indeed, the induction of ER stress appears to be required for the induction of ICD, as this has been shown for chemotherapy and radiotherapy as well (Obeid et al, 2007; Panaretakis et al, 2009; Kepp et al, 2014; Garg et al, 2015; Galluzzi et al, 2017). Captivated by the aforementioned premises, we decided to investigate the cellular and molecular mechanisms through which redaporfin kills cancer cells. Here, we show that redaporfin specifically accumulated in the endoplasmic reticulum (ER) and, in addition, in the Golgi apparatus (GA). Light-activated redaporfin causes selective damage to these subcellular compartments, elicits ER stress, and irreversibly compromises GA-dependent secretion. Unexpectedly, the ER/GA compartment targeted by redaporfin appears to operate upstream of the mitochondrial pathway of apoptosis to cause cell killing. Results Phototoxic effects of redaporfin on Golgi apparatus and endoplasmic reticulum To determine the subcellular tropism of redaporfin, we loaded this agent into cells for 20 h (Appendix Fig S1A–D) and then visualized its distribution based on its fluorescent properties (excitation at 510 nm, emission of 750 nm; Arnaut et al, 2014). This 20-h preincubation period with redaporfin effectively sensitizes the cells to PDT-induced killing (Appendix Fig S1G and H). Fluorescence microscopy imaging revealed that redaporfin strongly colocalized with the GA and ER. Redaporfin-emitted fluorescence spatially co-distributed with the GA-specific marker GALT1, stably expressed as a green fluorescent protein (GFP) fusion, and with the ER-specific marker CALR-GFP. Only minimal colocalization was observed with mitochondria visualized with MitoTracker Green or with lysosomes stained with quinacrine (Figs 1A and B, and EV1C and D; Appendix Fig S1E and F). Next, we asked whether dispersion of the GA would affect the subcellular localization of redaporfin. For this, we used brefeldin A (BFA), a natural antiviral compound synthesized by Eupenicillium brefeldianum that interrupts protein transport from the ER to the GA by abolishing the association of COP-I protein with the Golgi membrane (Duden et al, 1991). Alternatively, we used golgicide A (GCA), a pharmacological inhibitor of the cis-Golgi ArfGEF GBF1 that arrests the secretion of soluble and membrane-anchored proteins (Saenz et al, 2009). Dispersion of the GA with BFA or GCA led to a redistribution of the redaporfin fluorescence toward a diffuse cytoplasmic pattern, while induction of GA fragmentation with nocodazole (Cole et al, 1996) caused redaporfin to relocalize into multiple cytoplasmic dots (Appendix Fig S1I and J). Subcellular fractionation confirmed the ER/GA tropism of redaporfin. Purified ER micelles and Golgi stacks (but not mitochondria contained in heavy membrane fractions) from redaporfin-pretreated cells (Fig 1F–H) exhibited a distinct high-pressure liquid chromatography (HPLC) peak of UV absorbance (Fig 1C–E) that corresponded in its spectrum to that of redaporfin (Fig 1I and J). F2BOH, a molecule chemically related to redaporfin where the peripheral sulfonamide (SO2NHCH3) groups were replaced by sulfonic acid (SO3H) groups (Fig EV1A and B), exhibited a different subcellular distribution without any ER/GA tropisms but instead a preferentially lysosomal localization, as determined by fluorescence microscopy (Fig EV1C–H). F2BOH is hydrophilic, with an n-octanol:water partition coefficient POW = 0.04, whereas redaporfin is lipophilic, POW = 80 (Arnaut et al, 2014). Figure 1. Redaporfin accumulates at sites of the ER and Golgi apparatus A, B. Co-occurrence of redaporfin (5 μM) with markers of the GA, ER, and mitochondria in U2OS cells (A) and their Pearson correlation coefficient (PCC) (B). Bars indicate means ± SD of triplicates of one representative experiment out of two repeats. Asterisks indicate significant differences with respect to untreated cells, ***P < 0.001 (one-way ANOVA). Scale bar: 10 μm. C–H. Detection of redaporfin (5 μM) by HPLC-UV in ER and Golgi fractions from U2OS cells incubated with redaporfin (C–E). Fractions were tested for purity by immunoblotting using CALR, GALT1, and TOMM20 antibodies (F–H). I, J. Absorption spectra of redaporfin from the Golgi fraction and the pure standard. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. F2BOH preferentially accumulates at acidic vesicles A, B. Molecular structures of the halogenated bacteriochlorins, redaporfin (LUZ11) (A), and F2BOH (LUZ10) (B). C–F. Representative images of U2OS cells showing the co-occurrence of redaporfin (C, D) and F2BOH (E, F) with acidic vesicles stained with quinacrine and the respective Pearson correlation coefficients. Scale bar: 10 μm. G, H. Representative images of U2OS-GALT1-GFP cells showing the lack of co-occurrence of F2BOH with the GA (G) and the corresponding Pearson correlation coefficient (H). Scale bar: 10 μm. Data information: Data are indicated as means ± SD of triplicates of one representative experiment out of 2–4 repeats. Asterisks indicate significant differences with respect to untreated cells. ***P < 0.001 (one-way ANOVA). Download figure Download PowerPoint Next, we used transmission electron microscopy to examine the ultrastructural effects induced by redaporfin treatment followed by light illumination. While the non-exposed, inactive drug failed to affect the structural morphology of any organelle, its exposure to light (i.e., PDT) resulted in marked alterations of the ER/GA compartment such as dilation and vacuolization of the ER as well as disorganization of Golgi cisterns (Fig 2A). Cells expressing the GA marker GALT1-GFP exhibited a reduction in the fluorescent signal after PDT (Fig 2B and C), and similarly, endogenous GALT1 detected by immunofluorescence staining diminished post-PDT (Fig 2D and E). The same effect was observed with hypericin, a photosensitizer that targets the ER and GA (Ritz et al, 2008; Appendix Fig S2A and B). In contrast, F2BOH (which targets lysosomes) did not cause any reduction in endogenous GALT1 visualized by immunofluorescence staining (Appendix Fig S2C and D). Quantitative immunoblots confirmed that PDT with redaporfin induced a reduction in the abundance of several GA proteins such as Golgi brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1), golgin subfamily A member 2 (GOLGA2), and galactosyltransferase 1 (GALT1), as well as that of two ER proteins, eukaryotic translation initiation factor 2-alpha (eIF2α) kinase 3 (EIF2AK3) and protein disulfide-isomerase A3 (PDIA3). In contrast, no major decrease in mitochondrial import receptor subunit TOM20 homolog (TOMM20) or in the cytoskeleton protein β-actin was observed (Fig 2F–I). Figure 2. Ultrastructural changes of ER and Golgi after redaporfin-PDT A. Representative images of U2OS cells analyzed 6 h after redaporfin-PDT by transmission electron microscopy. Additional negative controls included cells incubated with redaporfin (5 μM) without photoactivation or cells submitted to light irradiation in the absence of redaporfin. Nuc marks nucleus, ER marks endoplasmic reticulum, GA marks Golgi apparatus, and Mito marks mitochondria. Scale bar: 1 μm B–E. Representative images of U2OS-GALT1-GFP (B) and U2OS stained for GALT1 (D) 6 h after PDT with redaporfin (5 μM). Quantitative analysis represents the average area of GALT1+ Golgi structures per cell (C, E). Scale bars: 10 μm. F–I. Six hours after treatment of U2OS cells with PDT with redaporfin (5 μM), protein was collected and tested by immunoblotting for different GA-, ER-, or mitochondria-specific proteins. Representative immunoblots (F, G) and densitometry data (H, I) are depicted. Data information: Ctr represents untreated cells and Redp* indicates irradiated cells. Bars indicate means ± SEM of three independent experiments. Asterisks indicate significant differences with respect to untreated cells, *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). Download figure Download PowerPoint Redaporfin-based PDT also induced signs of ER stress including phosphorylation of eIF2α detectable by immunoblot (Fig 3A and B) and immunofluorescence staining (Fig EV2A and B) as early as 1 h after the treatment. Systematic analysis of cells in which each of the four eIF2α kinases (EIF2AK1 to 4) was deleted by CRISPR/Cas9 technology (Fig 3C and D) revealed that the sole kinase responsible for eIF2α phosphorylation induced by redaporfin-based PDT was EIF2AK1 (previously known as heme-responsive inhibitor, HRI; Fig 3E and F), perhaps reflecting the molecular similarities between heme and redaporfin, which both bear porphyrin rings (Fig EV1A). Thus, redaporfin-based PDT elicits eIF2α phosphorylation through a pathway that is different from canonical ER stressors such as thapsigargin (Thaps) and tunicamycin (Tun), yet similar to arsenite (Fig EV2C and D). Knockout of EIF2AK1 (but not that of EIF2AK2, EIF2AK3, or EIF2AK4) sensitized the cells to redaporfin-dependent phototoxicity (Fig 3G–H). Redaporfin-based PDT failed to elicit the activation of ATF4 and DDIT3, two transcription factors downstream of eIF2α phosphorylation (Fig EV2E–G). Nevertheless, biosensor cell lines measuring different arms of the unfolded stress response confirmed the induction of ER stress at the levels of ATF6, as indicated by the significant translocation to the nucleus of ATF6-GFP and the IRE1–XBP1 axis, as indicated by the XBP1-GFP reporter construct which only becomes visible when IRE1 mediates an in-frame splicing (Fig EV2H–K). Knockdown of either ATF6 or IRE1 failed to sensitize the cells to redaporfin-dependent phototoxicity (Fig EV2L and M). Although redaporfin-mediated PDT led to a reduction in protein synthesis, this inhibition was not complete (Fig EV3). Redaporfin-mediated ER damage was also associated with increased cytosolic Ca2+ levels (Appendix Fig S3
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