Sunitinib-induced oxidative imbalance and retinotoxic effects in rats

Depósito de Investigación de la Universidad de Sevilla https://idus.us.es/ This is an Accepted Manuscript of an article published by ELSEVIER in LIFE SCIENCES, Vol. 257, on 2020, available at: https://doi.org/10.1016/j.lfs.2020.118072 Copyright 2020 Elsevier. En idUS Licencia Creative Commons CC BY-NC-ND Journal Pre-proof Sunitinib-induced oxidative imbalance and retinotoxic effects in rats Álvaro Santana-Garrido, Claudia Reyes-Goya, Helder André, Óscar Aramburu, Alfonso Mate, Carmen M. Vázquez PII: S0024-3205(20)30823-7 DOI: https://doi.org/10.1016/j.lfs.2020.118072 Reference: LFS 118072 To appear in: Life Sciences Received date: 15 June 2020 Revised date: 5 July 2020 Accepted date: 6 July 2020 Please cite this article as: Á. Santana-Garrido, C. Reyes-Goya, H. André, et al., Sunitinibinduced oxidative imbalance and retinotoxic effects in rats, Life Sciences (2020), https://doi.org/10.1016/j.lfs.2020.118072 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier. Journal Pre-proof 1 Sunitinib-induced oxidative imbalance and retinotoxic effects in rats Álvaro Santana-Garridoa,b, Claudia Reyes-Goyaa, Helder Andréc, Óscar Aramburud, Alfonso Matea,b,*, Carmen M. Vázqueza,b a Departamento de Fisiología, Facultad de Farmacia, Universidad de Sevilla. CL Profesor García González 2, 41012 Sevilla, Spain. b Epidemiología Clínica y Riesgo Cardiovascular, Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocío – Consejo Superior de Investigaciones Científicas – Universidad de Sevilla. Avda. Manuel Siurot s/n, 41013 Sevilla, Spain. c Department of Clinical Neuroscience, St. Erik Eye Hospital, Karolinska Institutet. 11282 Stockholm, Sweden d Servicio de Medicina Interna, Hospital Universitario Virgen Macarena, E-41009 Sevilla, Spain *Corresponding author. Departamento de Fisiología, Facultad de Farmacia, Universidad de Sevilla, CL Profesor García González 2, 41012 Sevilla, Spain. Phone number: +34 954 556 518. E-mail: [email protected]s Keywords: Sunitinib; NADPH oxidase; ocular side effects; oxidative stress; retina. Journal Pre-proof Journal Pre-proof 2 Abstract Aims: Sunitinib (Su), a tyrosine kinase inhibitor, is one of the most commonly used antiangiogenic drugs. Some studies have described retinal detachment and photoreceptor damage following systemic exposure to Su, despite beneficial effects achieved with local treatment of ocular pathologies. The aim of this study was to explore the role of NADPH oxidase system and oxidative stress in eyes from Su-treated animals. Main methods: Male Wistar rats were administered 25 mg Su/kg body weight/day incorporated in the chow for 3 weeks. Upon treatment completion, NADPH oxidase activity and ROS levels were measured in ocular tissue by chemiluminiscence and dihydroethidium (DHE) staining, respectively. The expression of NADPH oxidase isoforms (NOX1, NOX2 and NOX4), antioxidant enzymes and endothelial/inducible nitric oxidase isoforms (eNOS/iNOS) in the eyecup and/or retina were measured via immunofluorescence, immunoblotting and RTqPCR. Key findings: NADPH oxidase activity/expression increased in eyecup and retinas from Sutreated rats. Immunohistofluorescence studies in retinal layer confirmed a higher signal of NADPH oxidase isoforms after Su treatment. Treated animals also presented with reductions in NO levels and eNOS expression, whereas iNOS was upregulated. Finally, a significant depletion of antioxidant enzyme glutathione peroxidase was measured in eyecups of rats following Su exposure, and the opposite pattern was seen for glutathione reductase and superoxide dismutase. Significance: This study demonstrates that Su treatment is associated with NADPH oxidasederived oxidative stress in the eye. Long-term treatment of Su should be properly monitored to avoid retinotoxic effects that might result in ocular pathologies and sight-threatening conditions. Journal Pre-proof Journal Pre-proof 3 Abbreviation list AMD: age-related macular degeneration CNV: corneal neovascularization DAPI: 4′,6-diamidino-2-phenylindole DBP: diastolic blood pressure DHE: dihydroethidium DPI: diphenyleneiodonium GAPDH: glyceraldehyde-3-phosphate dehydrogenase GCL: ganglion cell layer GSH: reduced glutathione GSH-Px: glutathione peroxidase GSH-Red: glutathione reductase INL: inner nuclear layer IPL: inner plexiform layer L-NAME: N-nitro-L-arginine methyl ester NADPH: nicotinamide adenine dinucleotide phosphate, reduced form NO: nitric oxide NOS: nitric oxide synthase, endothelial (eNOS) and inducible (iNOS) isoforms O2.-: superoxide anion ONL: outer nuclear layer OPL: outer plexiform layer OS: outer segments OXI: oxypurinol ROT: rotenone SBP: systolic blood pressure SOD: superoxide dismutase Su: Sunitinib TKI: tyrosine kinase inhibitor Journal Pre-proof Journal Pre-proof 4 1. Introduction Sunitinib (Su) is a tyrosine kinase inhibitor (TKI) commonly used as an angiogenesis inhibitor. Its mechanistic targets include the inhibition of vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs), and the stem cell factor receptor (KIT) [1]. Both the US Food and Drug Administration (FDA) and the European Commission approved Su treatment in several conditions including advanced renal carcinoma [2], gastrointestinal stromal tumour (GIST, a rare tumour of the stomach, bowel, or oesophagus), and advanced pancreatic neuroendocrine tumours [3]. Moreover, Su is recommended as an alternative to imatinib mesylate when the treatment with the latter fail to control the cancer growth or when the patient is not eligible for treatment with imatinib [4]. Several evidences have demonstrated the effectiveness of local administration of Su in different ocular pathologies with neovascularization events [5–7]. Thus, subconjunctival treatment with Su is more effective for inhibition of corneal neovascularization than that with bevacizumab [8]. In addition, Su showed a strong anti-fibroblastic effect on conjunctival wound healing in a rabbit model with experimental trabeculectomy [9]. In the same way, adjuvant Su treatment showed positive effects on uveal melanoma patients [10]. However, negative effects of systemic treatment with Su, such as retinal detachment or damage to photoreceptor layer, have also been reported [11,12]. It is well known that chronic Su exposure can lead to side effects including cardiotoxicity and arterial hypertension, amongst others [13]. Although several hypotheses have been proposed [13,14], the precise mechanisms involved in the pathogenesis of these toxic effects are still unclear. Previous evidence suggested that Su can promote the activation of the endothelin (ET) system, which leads to increased blood pressure [15]. Moreover, ET activates vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the most important source of Journal Pre-proof Journal Pre-proof 5 superoxide anion (O2.-, a potent prooxidant) in hypertension-related organ damage [16–18]; this, in turn, would produce an increase in oxidative stress at the vascular level. Recent experiments in our laboratory support the role of NADPH oxidase in the development of ocular diseases (mainly at the retinal level) in hypertensive, N-nitro-L-arginine methyl ester (LNAME)-treated rats (Santana et al., unpublished observations). The fact that the eye is a highly vascularized organ suggests that ocular microcirculation might constitute an additional target of Su-associated side effects not only because of increased risk for hypertension development but also due to deleterious direct effect of this drug. The aim of the present study was to explore the effects of systemic treatment with Su on the retina, in order to explore possible mechanisms involved in the development of ocular pathologies following Su treatment. To this purpose, the experiments described here include determinations of NADPH oxidase activity and O2.- production; protein and mRNA location/expression of NOX1, NOX2, NOX4 and p22phox isoforms/subunits of NADPH oxidase; protein expression of antioxidant enzymes (namely, glutathione peroxidase, (GSH-Px1/2), glutathione reductase (GSH-Red), and superoxide dismutase (SOD-1)); nitric oxide (NO) relative concentration, as well as protein expression of both endothelial (eNOS) and inducible (iNOS) isoforms of nitric oxide synthase. All measurements were carried out in ocular tissue (eyecup, retina) from Su-administered animals and were compared with control samples from nontreated Wistar rats processed in parallel. 2. Materials and Methods 2.1. Animals and experimental design This study was conducted in accordance with the European Union (EU) Directive 2010/63/EU and the National (RD 53/2013) guidelines for the care and use of Laboratory animals, and was approved by the competent Institutional Animal Care and Use Committee (approval reference Journal Pre-proof Journal Pre-proof 6 #08/03/2017/034, issued by Junta de Andalucía, Dirección General de Producción Agrícola y Ganadería). Male Wistar rats aged 10-12 weeks (average body weight = 257 ± 6 g) were obtained from the Centre for Animal Production and Experimentation (Seville, Spain). Rats were divided into two groups of seven animals each: (1) Control group (untreated rats feeding with a standard pellet and free access to tap water) and (2) Sunitinib group (rats treated for 3 weeks with 25 mg/kg body weight/day of Su (Su-11248; Sutent®; Pfizer Inc., New York, USA) incorporated into the standard pellet diet and with free access to tap water). The chosen dosage was in agreement with previous studies with TKI [19], and it was adjusted weekly, to ensure continuous proper dosage, according to the evolution of the animals’ body weight and food intake. Animals were housed in individual cages and maintained under controlled, standard conditions (23 ± 1 ºC, 12h/12h light/dark cycles). 2.2 Blood pressure and heart rate measurements Systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate were measured on a weekly basis by the indirect method of tail-cuff occlusion in conscious animals using a NIPREM 645 pressure recorder (CIBERTEC, Barcelona, Spain). The reported SBP and DBP values are the average of three-to-four successive measurements. Blood pressure and body weight were measured every seven days at the same time. 2.3. Tissue isolation and homogenization After experimental treatment, rats were anesthetized with a mix of ketamine (75 mg/kg i.p.) and diazepam (10 mg/kg i.p.). Both eyes were enucleated as described elsewhere [20] and collected in petri dishes with Krebs solution. Under binocular stereoscopic microscope, both lens and vitreous body were separated through corneal incision. Isolated retinas or whole eyecups, as appropriate, were snap-frozen in liquid nitrogen and stored at -80 ºC until use. When necessary, both retina and eyecup were homogenized in 50 mM phosphate buffer (pH Journal Pre-proof Journal Pre-proof 7 7.4) with protease inhibitors (Roche/Sigma-Aldrich, Madrid, Spain) using a Potter-Elvehjem tissue grinder. These homogenates were immediately centrifuged at 10000 x g for 10 min, and the supernatant was used to determine protein concentration by the Bradford method [21]. 2.4. NADPH oxidase activity measurements Both eyecup and retina homogenates were used to determine NADPH oxidase activity by lucigenin-enhanced chemiluminescence, as previously reported [22]. In order to confirm the source of superoxide anions (O2.-), homogenate samples of both eyecup and retina were preincubated for 5 minutes at 37 ºC with different inhibitors (Sigma-Aldrich, Madrid, Spain) at 0.1 mmol/L: DPI (inhibitor of flavoproteins); oxypurinol (inhibitor of xanthine oxidase) and rotenone (mitochondrial complex I inhibitor). 2.5. Measurement of superoxide anion levels and nitric oxide concentration The fluorescent dye, dihydroethidium (DHE; MedChemExpress, Madrid, Spain; Cat. No. HYD0079) was used to measure in situ O2.- production following a previously established protocol [23]. Paraffin-embedded sections (5 m) were prepared from eyes following an intravitreal injection of 4% paraformaldehyde (PFA) in PBS, then post-fixed in 4% PFA for 24 h. DAPI Fluoromount-G® (SouternBiotech Associates, Inc, Birmingham, AL; Cat. No. 0100-20) was used to mount deparaffinized sections incubated with DHE for 20 min at 37º C. A fluorescence microscope (Olympus DP73, Tokyo, Japan) and Image J program (NIH freeware, version 2.0.0) were used to measure the intensity of the staining. Meanwhile, nitrite and nitrate (NOx) levels in eyecup homogenates were estimated by the Griess method [24]. 2.6. RT-PCR RNA was isolated from eyecups following TRIzol® total RNA isolation protocol (Thermo Fisher Scientific, Madrid, Spain). Reverse transcription was performed as previously described [25] Journal Pre-proof Journal Pre-proof 14 NADPH system, which could affect the eye and retina vasculature and lead to the development of ocular diseases [36,57]. When antioxidant enzymes were studied, an increase in protein expression of GSH-Red and SOD-1, together with downregulation of GSH-Px 1/2, was found in eyecup homogenates from Su-treated rats. The reduction in GSH-Px 1/2 expression might contribute to the oxidative imbalance, for GSH-Px enzyme catalyses the reduction of H2O2 to H2O and O2, at the expense of reduced glutathione (GSH-Red). Moreover, the observed increase in GSH-Red might indicate a possible compensation in an attempt to supply GSH, the main component in the antioxidant defence, from oxidized glutathione (GSSG), as it was observed in retinas from hypertensive animals [48]. In addition, an increase in SOD expression might indicate an overactivation of this enzyme secondary to altered O2.- levels. Ocular pathologies such as AMD [58] or chronic glaucoma [59] have been shown to present with oxidative imbalance. In addition, myopic patients seem to have different SOD levels when compared with emmetropic controls [60]. According to Lei et al. [61], it is very complicated to evaluate the implications of oxidative imbalance in the eye, due to the complexity of the biochemical pathways involved. Regarding our results, the changes observed in antioxidant enzyme expression in Su-treated rats might be the result of an increase in the local oxidative stress, with a higher production in O2.- secondary to NADPH oxidase system overexpression and NO metabolism dysfunction. In any case, whether the alterations in oxidative stress observed in Su-exposed animals are significant enough to induce damage in ocular function is currently unknown. Changes in electroretinogram b-wave amplitude have been found in hypertensive rats, which paralleled the increase in superoxide content found in these animals in the ganglion cell layer of the retina [48]. If such (or other) functional alterations are also present in Su-treated animals is an interesting issue that certainly warrants further research. Journal Pre-proof Journal Pre-proof 15 5. Conclusion The present study demonstrates that oral administration of Su for 3 weeks produces retinotoxic effects associated with: i) oxidative damage in ocular tissues (especially in retinal layers), which is accompanied by excess activity/expression of NADPH oxidase system; and ii) modifications of nitric oxide metabolism and antioxidant defence mechanisms that might be key components for the development of retinal diseases. These findings might be linked to hypertensive side effects and/or to negative direct effects of Su. Overall, and despite the accepted beneficial effects of this drug in cancer disease, our findings support the notion that systemic, long-term treatment with TKI should be carefully and continuously evaluated to avoid retinotoxic effects, ocular pathologies, and sight-threatening conditions. Author Contribution Study design and data management: AM, CMV; data acquisition: AS, CR-G; draft/revision of the article: AS, HA, OA, AM, CMV. Funding This study was supported by Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía (2017/00000440; CTS-584). AS is recipient of an FPU predoctoral fellowship from Ministerio de Ciencia, Innovación y Universidades (FPU17/03465). CR-G was supported by Ministerio de Ciencia, Innovación y Universidades, Ayudas para la Promoción de Empleo Joven e Implantación de la Garantía Juvenil en I+D+i 2017-2020 (PEJ2018-004474-A). Acknowledgements We thank technical support from Centro de Innovación, Tecnología e Innovación de la Universidad de Sevilla (CITIUS, Servicio de Biología, Servicio de Microscopía). Journal Pre-proof Journal Pre-proof 16 Conflict of interest statement The authors declare that there are no conflicts of interest. References [1] Q. Jiao, L. Bi, Y. Ren, S. Song, Q. Wang, Y. shan Wang, Advances in studies of tyrosine kinase inhibitors and their acquired resistance, Mol. Cancer. 17 (2018) 1–12. https://doi.org/10.1186/s12943-018-0801-5. [2] R.J. 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