Original Article 5-Fluorouracil-Induced Neurotoxicity in Rat Cerebellum Granule Cells Involves Oxidative Stress and Activation of Caspase-3 Pathway
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Int J Clin Exp Med 2019;12(3):2334-2343 www.ijcem.com /ISSN:1940-5901/IJCEM0077214 Original Article 5-fluorouracil-induced neurotoxicity in rat cerebellum granule cells involves oxidative stress and activation of caspase-3 pathway Yang Zhang1*, Nuoya Yin2,3*, Shengxian Liang2,3, Su Shen1, Dandan Li1, Francesco Faiola2,3 1Department of Pharmacy, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; 2State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China; 3College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China. *Equal contributors. Received April 2, 2018; Accepted October 29, 2018; Epub March 15, 2019; Published March 30, 2019 Abstract: 5-Fluorouracil (5-FU) is a widely used anticancer drug that acts by blocking DNA replication. Although the side effects of 5-FU are well documented, the mechanism of 5-FU-induced neurotoxicity remains unclear. The current study was performed to investigate the toxicity of 5-FU to rat cerebellum granule cells (CGCs) and to eluci- date the corresponding molecular mechanisms. We demonstrated that 5-FU exhibited significant cellular toxicity to CGCs, in a dose-dependent manner, and that it altered intracellular Ca2+ levels. The accumulation of intracellular re- active oxygen species in CGCs revealed that 5-FU also induced oxidative stress. Moreover, 5-FU-treated cells showed elevated caspase-3 activity. Furthermore, intraperitoneally administered 5-FU caused slight degenerative changes in the rat cerebellum granular layer. Taken together, these findings revealed that 5-FU substantially impaired the survival of CGCs by inducing oxidative stress and activating the caspase-3 apoptotic pathway. Keywords: 5-fluorouracil, neurotoxicity, cerebellum granule cells, oxidative stress, caspase Introduction have been observed for almost all classes of anticancer drugs, including alkylating agents 5-Fluorouracil (5-FU) is a commonly used che- [6], antimetabolites [7], and even antihormonal motherapeutic agent that blocks DNA synthe- agents [8]. Typical side effects of 5-FU include sis and replication via inhibition of thymidylate myelosuppression, nausea, vomiting, diarrhea, synthase and incorporation of 5-FU metabo- and stomatitis [9]. Previous studies have shown lites into RNA and DNA [1]. Presently, 5-FU is that 5-FU has the potential to induce toxicity in still a component of most of the currently various tissues. Cardiotoxicity is a well-defined applied regimens to treat solid cancers of the side effect of 5-FU, which often occurs as myo- gastrointestinal tract, breast, head and neck, cardial ischemia, and to a lesser extent, as car- and pancreas [2]. Derivatives of 5-FU, including diac arrhythmias, hyper- and hypotension, left carmofur, tegafur, and capecitabine (prodrugs ventricular dysfunction, and cardiac arrest which are metabolized to 5-FU, their only active mediated by multifactorial pathophysiological product in vivo), are also often used clinically as mechanisms [10]. Additionally, 5-FU was dem- anticancer drugs [3-5]. onstrated to be genotoxic, as indicated by chro- mosomal damage in animals treated with 5-FU Most anticancer agents that kill cancer cells [11]. McQuade et al. reported that 5-FU induced also have a low therapeutic index, thus affect- gastrointestinal dysfunction and enteric neuro- ing a diverse range of normal cell types. This toxicity in vivo [12]. Furthermore, 5-FU was also leads to a myriad of adverse side effects on found to cause hepatic damage from overpro- multiple organ systems and may severely limit duction of free radicals and inflammatory medi- their activity. Such effects on normal tissues ators [13]. Neurotoxicity induced by 5-fluorouracil 5-Fluorouracil not only causes adverse effects at 1×106 cells/mL in 6-well plates (Corning Inc., in primary organs where it accumulates, but Corning, NY, USA), which had been coated with also in secondary locations such as the central poly-L-lysine (0.01%; Sigma-Aldrich, St. Louis, nervous system (CNS) upon systemic circula- MO, USA). Subsequently, CGCs were cultured tion [14]. Mustafa et al. have shown earlier that in Dulbecco’s modified Eagle’s medium/F12 5-FU readily crosses the blood-brain barrier (HyClone, Logan, UT, USA) supplemented with (BBB) and affects spatial working memory and 5% horse serum (GIBCO), 5% fetal bovine serum newborn neurons in the adult rat hippocampus. (GIBCO), 25 mM KCl (Sigma-Aldrich), 1% Moreover, there is some evidence that 5-FU L-glutamine (HyClone), 100 U/mL penicillin, and can cross the BBB by simple diffusion and 100 μg/mL streptomycin (GIBCO), under stan- cause neurotoxicity including nystagmus, at- dard culture conditions (37°C, 5% CO2). After axia, dysarthria, and epilepsy [15, 16]. 5-Fl- 18-22 h, 10 μM arabinofuranosylcytosine (Flu- uorouracil has also been associated with both ca, St. Louis, MO, USA) was added to inhibit pro- acute and delayed CNS toxicities [17]. Acute liferation of non-neuronal cells. Cells were cul- CNS toxicities, such as pancerebellar syndro- tured for seven additional days until synapses me and subacute encephalopathy with severe formed. cognitive dysfunction, and delayed cerebral de- myelinating syndrome reminiscent of multifocal Evaluation of cell viability leukoencephalopathy, have been increasingly reported following 5-FU therapies [18]. Similar To assess the cytotoxicity of 5-FU on CGCs, neurologic syndromes, such as cerebellum dy- cells were treated with increasing concent- sfunction, encephalopathy, and multifocal infla- rations of 5-FU (0.01-100 μM). After 24 h, cell mmatory leukoencephalopathy, have also been viability was determined by Alamar Blue (AB) observed in cancer patients treated with 5-FU assay. Fluorescence intensity was measured at derivatives [5, 19]. 590 nm upon excitation at 530 nm by using a multi-well fluorometric reader (Thermo Scien- Although the phenomenon of 5-FU-induced tific, Waltham, MA, USA). Dose-response curves neurotoxicity is well recognized, its mecha- were generated to calculate the half maximal nisms remain unclear and need to be elucidat- inhibitory concentration (IC50). ed. Given the large number of individuals treat- ed for cancer, it is imperative to fully uncover Measurement of intracellular free Ca2+ level the molecular mechanisms of 5-FU neurotoxic- ity to develop safer pharmacological strategies Cerebellum granule cells grown on plates were to treat cancer. Cerebellum granule cells (CGCs) loaded with 5 μM Fura-2/AM dye (Sigma-Al- possess similar characteristics as neural tis- drich) for 30 min in PBS without Ca2+ and Mg2+ sues and have been widely used as an in vitro at 37°C and 5% CO2. Cells were then treated model to elucidate the mechanism of action of with three different concentrations of 5-FU a wide range of therapeutic agents [20]. In this (0.1, 1, and 5 μM). The increase in intracellular study, we showed that 5-FU induced apoptosis Ca2+ level was expressed in terms of ratio of in CGCs by stimulating calcium influx, oxidative fluorescence intensity upon excitation at 340 stress, and caspase activation. Furthermore, and 380 nm (F340/F380), which is proportion- the neurotoxicity of 5-FU was demonstrated al to Ca2+ concentration. The F340/F380 ratio by histopathological analysis using Sprague- was measured at an emission wavelength of Dawley rats in an in vivo model. 510 nm every 50 s. The experiments were car- ried out in the dark. Materials and methods Assessment of intracellular reactive oxygen Preparation of primary cell cultures species (ROS) accumulation Primary cultured CGCs were isolated as de- The production of intracellular ROS was mea- scribed previously [21]. Dissociated cerebella sured spectrophotometrically by using the fluo- from 7-day-old Sprague-Dawley rats were cul- rescent probe 2,7-dichlorofluorescindiacetate tured in high-glucose Hank’s balanced salt (DCFH-DA), which passively enters cells and solution (GIBCO, Grand Island, NY, USA) and reacts with ROS to form the highly fluorescent seeded at 5×105 cells/mL in 96-well plates, or compound dichlorofluorescein (DCF). Briefly, 2335 Int J Clin Exp Med 2019;12(3):2334-2343 Neurotoxicity induced by 5-fluorouracil CGCs were incubated with 100 μM DCFH-DA facility in a temperature-, humidity-, and light- (Sigma-Aldrich) for 30 min at 37°C and 5% CO2, controlled environment with a 12 h light/dark before treatment with different concentrations cycle, and were given free access to standard of 5-FU (0.01-5 μM) for 1 h, 4 h and 12 h, laboratory diet and water. The rats were ran- respectively. Hydrogen peroxide (H2O2, 1 mM) domly divided into two groups of six rats each, was used as a positive control. DCF fluores- and received 5-FU (20 mg/kg) in three consec- cence was measured using a microplate spec- utive injections every other day intraperitoneal- trofluorometer (Thermo Scientific) at excitation ly; control rats received equal amounts of 0.9% and emission wavelengths of 485 nm and 530 saline. The body weight of rats was measured nm, respectively. daily. The animals were sacrificed at the end of the experiment on day 7 (rats were first injected Western blot with 5-FU on day 1). After treatment with different concentrations of Histopathological analysis 5-FU for 12 h or 24 h, CGCs were collected and lysed on ice for 30 min in RIPA lysis buffer con- Hematoxylin & eosin (H&E) staining and immu- taining a mixture of protease inhibitors (EDTA- nohistochemistry (IHC) evaluation were per- free Protease Inhibitor Cocktail Tablets; Roche, formed on paraffin-embedded sections of the Basel, Switzerland). CGC samples containing cerebellums that were dissected on day 7. Rats equal amounts of total proteins were analyzed were deeply anaesthetized by intraperitoneal by 10% SDS-PAGE and transferred onto a nitro- injection of ketamine before perfusion fixation. cellulose membrane (Pall, Port Washington, NY, Then, the cerebellum was immediately collect- USA). For immunoblotting, the nitrocellulose ed and cut into 5-μm-thick sections, and sub- membrane was incubated with TBS-T contain- sequently processed with H&E staining.