Amitriptyline down-regulates coenzyme Q⁠10 biosynthesis in lung cancer cells

UNCORRECTED PROOF European Journal of Pharmacology xxx (2017)xxx-xxx Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com Amitriptyline down-regulates coenzyme Q⁠10 biosynthesis in lung cancer cells Tamara Ortiz⁠a⁠, ⁠b,Marina Villanueva-Paz⁠c,Eduardo Daz-Parrado⁠a,Matilde Illanes⁠a,Ana Fern ndez-Rodr guez⁠a, Jos A. Snchez-Alc zar⁠c,Manuel de Miguel⁠a⁠, ⁠ aDpto. Citología e HistologíaNormal yPatológica, Facultad de Medicina, Universidad de Sevilla, Avda. Sánchez Pizjuáns/n, 41009 Sevilla, Spain bEscuela de Nutrición y Dietética, Facultad de Ciencias de la Salud, Universidad del Desarrollo, Concepción, Chile cCentro Andaluz de Biologíadel Desarrollo (CABD), Universidad Pablo de Olavide-CSIC, Centro de InvestigaciónBiomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Carretera de Utrera, 41013 Sevilla, Spain ARTICLE INFO Keywords: Amitriptyline Coenzyme Q⁠10 Oxidative stress Antioxidants Oxidative therapy ABSTRACT Amitriptyline, atricyclic antidepressant, has been proposed as an antitumoral drug in oxidative therapy. Its pro-apoptotic effects, mediated by high reactive oxygen species generation, have been already described. In this study we analysed the effect of amitriptyline on the biosynthesis of coenzyme Q⁠10 (CoQ), an essential component for electron transport and a potent membrane antioxidant involved in redox signaling. We treated H460 cells, anon-small-cell lung cancer cell line, with amitriptyline and we analysed CoQ levels by HPLC and CoQ biosynthesis rate, as well as the enzymes involved in CoQ biosynthesis by real-time PCR and Western blot. Amitriptyline treatment induced adose-dependent decrease in CoQ levels in tumor cells. CoQ decreased levels were associated with down-regulation of the expression of COQ4 gene, as well as decreased Coq4 and Coq6 protein levels. Our Vndings suggest that the effect of amitriptyline on CoQ biosynthesis highlights the potential of this drug for antitumoral oxidative therapy. 1. Introduction It has been observed that most cancer cells have altered mitochondria and increased reactive oxygen species steady state levels, making them more vulnerable to reactive oxygen species-mediated damage and resulting in increased cell death (Szatrowski and Nathan, 1991; Burdon, 1995; Pelicano et al., 2004; Tomasetti et al., 2015). Thus, manipulating reactive oxygen species levels by redox modulation could exert a remarkable antitumoral effect and potentially induce programmed cell death without causing significant toxicity to normal cells (Trachootham et al., 2009; Zhang et al., 2015). This strategy is called oxidation therapy" and it is achieved through delivering cytotoxic reactive oxygen species to solid tumors, or alternatively inhibiting the antioxidative enzyme system (Fang et al., 2009; Pathania et al., 2014; Fang et al., 2008). Recent studies have shown that tricyclic antidepressants (TCAs), such as imipramine, desipramine, nortriptyline or amitriptyline, induce cell death through mechanisms involving neurotrophin receptor (Pula et al., 2013), (JNK)/c-Jun pathway (Jahchan et al., 2013), Fas death receptor (Yuan et al., 2015); or mechanisms involving mitochondria-mediated cell death (Yuan et al., 2015; Zhang et al., 2013). Our group has proposed amitriptyline as an anticancer agent for oxidation therapy targeted to mitochondrial as it increases oxidative stress by generating high amounts of reactive oxygen species and decreasing antioxidant activity in several human cancer cell lines, leading to cell death through caspase-3-dependent apoptosis (Cordero et al., 2010). Reactive oxygen species increase seems to be due to an alteration of complex I+III of the mitochondrial respiratory chain (Cordero et al., 2009, 2010; Villanueva-Paz et al., 2016). It is noteworthy that amitriptyline decreases the amount or activity of several antioxidants such as catalase and superoxide dismutase (Cordero et al., 2010; Moreno-Fernandez et al., 2008), and reduces levels of coenzyme Q⁠10 (CoQ) (Bentinger et al., 2007). CoQ or ubiquinone is alipid-soluble component of virtually all cell membranes and is both an essential component for electron transport in oxidative phosphorylation of mitochondria (Battino et al., 1990), and apowerful antioxidant (Turunen Corresponding author. Email address: [email protected] (M. de Miguel) http://dx.doi.org/10.1016/j.ejphar.2017.01.017 Received 19 July 2016; Received in revised form 11 January 2017; Accepted 13 January 2017 Available online xxx 0014-2999/© 2016 Published by Elsevier Ltd. Full length article UNCORRECTED PROOF T. Ortiz et al. European Journal of Pharmacology xxx (2017)xxx-xxx et al., 2004). Paradoxically, CoQ is also involved in superoxide production by the respiratory chain (James, Smith, and Murphy, 2004). Antioxidant and prooxidant mechanisms of CoQ are shown in Fig. 1. On the other hand, it is described that deVciency of CoQ decreases the activity of enzymes of the mitochondrial respiratory chain, the mitochondrial membrane potential, the expression of the mitochondrial proteins involved in oxidative phosphorylation and cell growth rates; and this deVciency also increases cell death, the production of reactive oxygen species, mitochondrial permeabilization and triggers mitochondria elimination by mitophagy (Rodriguez-Hernandez et al., 2009; Cotan et al., 2011). Given the critical role of CoQ in mitochondrial function, it has been suggested that CoQ levels could be auseful biological marker of mitochondrial function (Haas et al., 2008). In vivo studies have shown that treatment with amitriptyline induces CoQ deVciency and oxidative stress in some organs and peripheral blood cells (Bautista-FerruVno et al., 2011; Moreno-Fernandez et al., 2012). Therefore, alterations in the amount of CoQ are somehow involved in the increase in oxidative stress induced by amitriptyline, although no study has investigated the effect of amitriptyline in the biosynthesis of CoQ at the molecular level. The present study aimed to assess the changes provoked by amitriptyline in gene expression and protein levels of the enzymes involved in CoQ biosynthesis, as well as CoQ biosynthesis rate, in ahuman lung cancer cell line. 2. Materials and methods 2.1. Reagents Amitriptyline and trypsin were purchased from Sigma Chemical Co. (St Louis, Missouri, USA). Hoechst 3342 and MitoSOX Red were purchased from Invitrogen/Molecular Probes (Eugene, Oregon, USA). Anti-COQ proteins and Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and from Research Diagnostic Inc. (Flanders, New Jersey, USA), respectively. 2.2. Cell culture H460 cells (from human non-small cell lung carcinoma: NSCLC) were purchased from the American Type Culture Collection (Manassas, Virginia, USA) and cultured in RPMI-1640 medium supplemented with L-glutamine, antibiotic/antimycotic solution (Sigma Chemical Co., St. Louis, MO, USA), and with 10% fetal bovine serum (FBS). Cells were incubated at 37 Cin a5% CO⁠2 atmosphere. 2.3. Dose-dependent toxicity, and viability assays H460 cells were cultured with different concentration of amitriptyline (0 M, 20 M, 50 M, and 100 M) for 6h, 12 h, and 24 h. After incubation, cell viability was analysed by trypan blue assay and an automated cell counter (TC10 )according to the manufacturer s instructions (Bio-Rad Laboratories, Inc.). The rest of the studies were performed after treating H460 cells with 50 Mamitriptyline for 24 h. To study the putative restore effects of external CoQ on cell viability of amitriptyline-treated cells, H460 cultures were supplemented with 50 Mamitriptyline and 25 MCoQ for 24 h. 2.4. Biochemical determination of coenzyme Q⁠10 levels Lipid extraction from H460 cells after treatment with several doses of amitriptyline (20 M, 50 M, and 100 M) for 6h, 12 h, and 24 h was performed as described earlier (Santos-Ocana et Fig. 1. Antioxidant and prooxidant mechanisms of Coenzyme Q(CoQ). Mitochondrial Respiratory Chain: Mitochondrial electron transport chain transfers electrons from NADH and FADH2 to molecular oxygen to form H⁠2Othrough multiprotein complexes, named complex IIV, and of aseries of carriers such as Wavoproteins, iron-sulfur proteins, CoQ and cytochromes. Antioxidant Activity: The antioxidant capacity of CoQ depends on its redox forms: fully reduced (Ubiquinol, UQH⁠2), semireduced (Ubisemiquinone, UQH ⁠-)and oxidized (Ubiquinone, UQ). Antioxidants such as -tocopherol ( -TOH) can donate ahydrogen ion to asuperoxide radical (O2 -) to form tocopheroxyl radical (TO ), which in turn can react with UQH⁠2 for recycle -TOH and produce UQH ⁠-.At the same time, UQH⁠2 reacts with lipid-derived free radicals such as alkyl radical (L ), formed by the attack of areactive species (hydroxyl radical: HO )to ahydrogen atom (H) from unsaturated fatty acids of phospholipids (LH); and with peroxyl radical (LOO ), formed by the spontaneous union of Lwith oxygen molecules. Finally, UQH ⁠− reacts with radicals to generate more UQ and to inhibit the chain reaction of lipid peroxidation. Prooxidant activity: Ubisemiquinone spontaneously oxidizes in the presence of oxygen. The superoxide formed produces hydrogen peroxide (H⁠2O⁠2). O2 −can react with UQH⁠2 or UQ to form further ubisemiquinone radicals, starting achain reaction of autoxidation. However, this reaction occurs when there is adisorder of the phospholipid bilayer. 2 UNCORRECTED PROOF T. Ortiz et al. European Journal of Pharmacology xxx (2017)xxx-xxx al., 2002). The cell samples were lysed with 1% SDS and vortexed for 1min. Amixture of ethanol:isopropanol (95:5) was added and samples were vortexed for 1min. To recover CoQ, 5ml of hexane was added and samples were centrifuged at 1000gfor 5min at 4C. The upper phases from three extractions were recovered and dried on arotatory evaporator. Lipid extract was suspended in 1ml of ethanol, dried in aspeed vac and kept at −20 C. Samples were suspended in the suitable volume of ethanol prior to high-performance liquid chromatography (HPLC) injection. Lipid components were separated by aProminence Shimadzu HPLC system (Shimadzu ScientiVc Instruments Columbia, MD, USA) equipped with aShimadzu Shim-pack XR-ODS column. Coenzyme Q⁠9 (CoQ⁠9)was used as an internal standard. CoQ levels were analysed using an ultraviolet SPD-20A detector. 2.5. CoQ biosynthesis rate Human H460 cells were cultured in 10 ml of PBS (Invitrogen) containing 1gof glucose/100 ml, 10% FBS, penicillin (100 units/ml), and streptomycin (100 mg/ml), with and without 50 Mamitriptyline for 24 h. Then, 0.5 mCi of [3 H]mevalonate (3.25 Ci/mmol, Perkin-Elmer Life Sciences, Waltham, MA, USA) was added, and incubation continued for an additional 24 h. Finally, the cells were harvested by trypsinization and stored at −20 Cfor later analysis. Lipids were extracted from cells with chloroform/methanol/water (1:1:0.3) at 37 Cfor 1 h with magnetic stirring). The extracts thus obtained were adjusted to achieve a Vnal chloroform/methanol/ water ratio of 3:2:1, and complete phase separation was then accomplished by centrifugation. The lower chloroform phase was removed and evaporated to dryness under aWow of nitrogen, and the residue was subsequently redissolved in chloroform. These solutions were placed onto asilica column (50 mg/1.5 ml; Extract-Clean; Alltech, DeerVeld, IL, USA) and thereafter eluted with 6ml of chloroform. After evaporation of the solvents, the neutral lipids were dissolved in chloroform/methanol (2:1). For analysis of CoQ, the samples were analysed by thin layer chromatography. The lipid extracts were supplemented with 5 g of cold CoQ and then loaded onto silica gel plates (Silica gel 60, Merck KGaA, Darmstadt, Germany). The plates were developed with hexane/ethyl ether 80:20 and bands corresponding to these metabolites were dyed with iodine vapors. The band corresponding to CoQ was scrapped oX from the silica gel plates and transferred to liquid scintillation vials. Radioactivity was then measured by liquid scintillation in aBeckman Coulter LS6500 liquid scintillator. 2.6. Western blotting for Coq proteins H460 cells were treated with 50 Mamitriptyline for 24 h. Whole cellular lysates were prepared in abuXer, gentle shaking, composed of 0.9% NaCl, 20 mM Tris-HCl, pH 7.6, 0.1% triton X-100, 1mM phenylmethyl-sulfonylWuoride, and 0.01% leupeptine. Electrophoresis was carried out in a10 15% acrylamide SDS/PAGE. Proteins were transferred to Immobilon membranes (Amersham Pharmacia Biotech, Madrid, Spain). Mouse anti-Coq proteins and mouse antiglyceraldehyde 3-phos- phate dehydrogenase antibodies were used to detect proteins by Western blotting. The proteins were electrophoresed, transferred to nitrocellulose membranes, and, after blocking overnight at 4C, incubated with the respective antibody solution at 1:1000 dilutions. Then, membranes were probed with their respective secondary antibody labelled with Wuorescence (1:2500). Immunolabelled proteins were detected by using a chemiluminiscence method (Bio-Rad Laboratories Inc., Hercules, California, USA). The protein concentration was determined by the Bradford method (Bradford, 1976). 2.7. Quantitative real-time RT-PCR Total cellular RNA was puriVed from cultured H460 cells after treatment with 50 Mamitriptyline for 24 husing the TRIsure method (Bioline, London, UK), according to the manufacturer's instructions. RNA concentration was determined spectrophotometrically. In order to avoid genomic DNA contamination, one microgram of total RNA from each sample was incubated in gDNA Wipeout BuXer (Quantitect Reverse Transcription Kit, Qiagen. Hilden, Germany) at 42 Cfor 5min. RNA samples were subsequently retrotranscribed to cDNA using QuantiTect Reverse Transcription Kit (Qiagen. Hilden, Germany). Quantitative RT-PCRs were performed in aMiniopticon unit (Bio-Rad, Hercules, CA) making use of the SensiMix One-Step qRT-PCR Kit (Bioline, London, UK), in accordance to the comparative 2⁠CT method described by Livak and Schmittgen (Livak and Schmittgen, 2001). The thermal cycling conditions used were: denaturation at 95 Cfor 20 s, alignment at 54 C for 20 sand elongation at 72 Cfor 20 s, for 40 cycles. Beta-actin was used as an internal control in each reaction. Primers utilized are listed in Table 1. All reactions were performed in duplicate. Reaction mixtures, without RNA, were used as negative controls in each run. Table 1 Primers used for qRT-PCR analysis, with their annealing temperatures and size of PCR products. Gene Primer sequence PCR product (bp) Annealing temp. beta-actin F: 5 -CCAGATCATGTTTGAGACC-3 275 55 R: 5 -ATGTCACGCACGATTTCCC-3 COQ2 F: 5 -AGAACAGCCAATCGTCCAATAGC-3 220 55 R: 5 -CCCAATTAAATGTCAAGCCCAAGG-3 COQ4 F: 5 -CCCCAGACACCCGAGCAC-3 154 55 R: 5 -ACAGCCTCAAACCATTTCACCAC-3 COQ6 F: 5 -CTGCTGTTGTGGCTACTCTGC-3 128 55 R: 5 -GGACCAAACCAAGGAACTCAAGG-3 COQ7 F: 5 -GGACGCTGATGGAGGAGGAC-3 144 55 R: 5 -TGCTCTTCAGGACGGCATAGG-3 3 UNCORRECTED PROOF T. Ortiz et al. European Journal of Pharmacology xxx (2017)xxx-xxx 2.8. Mitochondrial reactive oxygen species production Mitochondrial reactive oxygen species generation was assessed by MitoSOX , a red mitochondrial superoxide indicator. Approximately 110⁠6 H460 cells treated with 50 Mfor 24 hwere incubated with 1 M MitoSOX for 30 min at 37 C, washed twice with PBS and resuspended in 500 lof PBS and analysed by Wow cytometry. H460 cultures were also treated with 25 MCoQ in order to investigate the effect of supplemented CoQ on amitriptyline-induced reactive oxygen species production. 2.9. Statistical analysis All results are expressed as means values S.D. Each experiment was repeated three times. The unpaired Student's t-test was used to evaluate the significance of differences between groups (treatments and control). The level of significance was set at Pvalue of less than 0.05. 3. Results 3.1. Dose-dependent toxicity of amitriptyline and reactive oxygen species production The toxicity of amitriptyline was studied on cultured H460 cells by analysing cell viability. Fig. 2shows the percentages of living cells after administration of the drug at different concentrations (20 M, 50 M, and 100 M), and at several times (6 h, 12 h, and 24 h). The values obtained from controls at time 0 h (T⁠0)were very similar among groups with different drug concentrations (data not shown). After 12 hand 24 hof incubations, the average number of cells was notably decreased in the presence of amitriptyline at concentration of 50 M(61% and 36%, respectively) and 100 M(28% and 26%, respectively) as compared with control cultures (97% and 98%, respectively). In consequence of these results, the condition selected for the rest of the experiments was 50 Mamitriptyline for 24 hbecause it was the lowest dose with high effect on cell viability. Interestingly, when cells were treated for 24 hwith 50 Mamitriptyline supplemented with 25 MCoQ, the percentage of cell viability increased significantly from 55% (amitriptyline alone) to 79% (Fig. 3A). On the other hand, when we analysed reactive oxygen species production in H460 cells treated with Fig. 2. Effects of amitriptyline on cell survival. Amitriptyline dose-dependent toxicity in H460 cells treated for 6h, 12 h, and 24 h. H460 cells were treated with amitriptyline 20, 50, and 100 M, and cell counting was determined as shown in Section 2.3 of Material and Methods.Results are expressed as mean S.D. of three independent experiments. *P<0.05 between control and treated cells. Fig. 3. Effect of supplementary CoQ on survival percentages and reactive oxygen species production in H460 cells treated with 50 Mamitriptyline for 24 h. A: Cell viability for cells treated with amitriptyline and supplemented with 25 MCoQ. B: Mitochondrial reactive oxygen species generation assessed by MitoSOX in tumor cells in the presence of amitriptyline and supplementary CoQ. Results are expressed as mean S.D. of three independent experiments. *P<0.05 between control and treated cells; ** P<0.05 between amitriptyline treated cells and those complemented with CoQ. 50 Mamitriptyline we found asignificant increase in reactive oxygen species levels (83.92 a.u.; control: 58.36 a.u.) that was restored to normal values in the presence of supplementary CoQ (56.87 a.u.) (Fig. 3B). Amultivariate analysis showed that cells treated with 50 M amitriptyline for 24 hhad astrong positive association between cell death and reactive oxygen species production in H460 cells (Coef.=0.893), with statistical significance. 3.2. Biochemical analysis of coenzyme Q⁠10 levels We determined the levels of CoQ by HPLC in H460 cells after treatment with 20 M, 50 M, and 100 Mamitriptyline for 6h, 12 h, and 24 h. We found that amitriptyline treatment induced asignificant dose-dependent decrease of CoQ levels (Fig. 4A). The data, expressed as pmol CoQ/mg protein, were 224.67 1.34 for control cells and 130.47 10.63 for H460 cells treated with 50 Mamitriptyline for 24 h. To elucidate whether CoQ levels down-regulation was due to a decrease in CoQ biosynthesis rate, we examined the incorporation of the radiolabelled CoQ precursor [3H]mevalonate in 50 Mamitriptyline-treated cells for 24 h. We observed that amitriptyline induced asignificant decrease of CoQ biosynthesis (Fig. 4B). Multivariate analysis showed that higher amount of CoQ was associated with adecrease in cell viability and in reactive oxygen species levels (Coef.=−0.5964 and Coef.=−0.8027, respectively); all variables being statistically significant. 4 UNCORRECTED PROOF T. Ortiz et al. European Journal of Pharmacology xxx (2017)xxx-xxx Fig. 4. Effects of amitriptyline on CoQ levels and biosynthesis rate. A: H460 cells were treated with amitriptyline at different doses (20 M, 50 M, and 100 M) for 6h, 12 h, and 24 h; and CoQ levels were determined by HPLC as described in Section 2.4 of Material and Methods.B: For the analysis of CoQ biosynthesis rate, control and 50 Mamitriptyline-treated cells were incubated with [3H]mevalonate for 24 h, and radiolabelled CoQ was analysed as described in Section 2.5 of Material and Methods.Results are expressed as mean S.D. of three independent experiments. *P<0.05 between control and treated cells. 3.3. Amitriptyline modulates COQ gene expression In order to underline the effect of amitriptyline on CoQ synthesis in H460 cells, we also determined the expression levels of key genes and proteins involved in CoQ synthesis. Expression levels of COQ2, COQ4,COQ6 and COQ7 genes were analysed at mRNA level by real time quantitative RT-PCR and at protein level by Western blot. The relative number of gene copies within the total RNA isolated from amitriptyline-treated cells was compared to the number in their non-treated counterparts. Amitriptyline induced significant down-regulation of mRNA levels of COQ4 gene and up-regulation of COQ2 and COQ7 (Fig. 5). Accordingly, Western blotting analysis revealed adecrease in the expression levels of Coq4 and Coq6 translated proteins and an increase in the expression levels of Coq7 (Fig. 6), indicating that the decrease of CoQ induced by amitriptyline can be caused by both transcription and translational alterations of key components of the CoQ biosynthesis pathway. Fig. 5. qRT-PCR based analysis of the effect of amitriptyline treatment on coenzyme-Q genes COQ2,COQ4,COQ6 and COQ7 in H460 cell cultures treated with 50 Mamitriptyline for 24 h. Graphs show the speciVc mRNA relative amount using appropriate primers (Table 1) and normalized to the beta-actin mRNA expression. *P<0.05. 4. Discussion Mitochondria have recently emerged as novel targets for cancer therapy due to its important roles in cellular function such as programmed cell death regulation and reactive oxygen species generation (He et al., 2015c; Weinberg and Chandel, 2015). Increased generation of reactive oxygen species and an altered redox status have been observed in cancer cells, and recent studies suggest that these biochemical properties can be used for therapeutic beneVts (Trachootham et al., 2009; Fang et al., 2007; Chandra et al., 2000). Oxidative therapy is presented as an antitumoral strategy based on oxidative susceptibility of most cancer cells due to their intrinsic high oxidative stress, close to the lethal threshold. The proposed mechanism consists in the use of pro-oxidant agents that generate high levels of reactive oxygen species or inhibit reactive oxygen species elimination by diminishing the antioxidant system of tumor cells, leading to oxidative damage and cell death through the activation of the caspase cascade (Chandra et al., 2000; Fang et al., 2009; Tomasetti et al., 2015; Conklin, 2004). In recent studies, we have shown that amitriptyline, atricyclic antidepressant commonly prescribed for depression and therapeutic treatments of several neuropathic and inWammatory illnesses, increases reactive oxygen species levels and induces apoptosis through mitochondrial dysfunction, inhibiting mitochondrial complex III activity (Cordero et al., 2009; Bentinger et al., 2007; Villanueva-Paz et al., 2016); and it has been propose to be considered as aputative new anticancer drug (Cordero et al., 2010; Villanueva-Paz et al., 2016). Amitriptyline induces high reactive oxygen species generation as aresult of mitochondrial dysfunction, provoking ahigher level of apoptosis of tumor cells than common chemotherapeutic drugs (Cordero et al., 2010), and reduces important antioxidants of the cell-defense machinery, dramatically limiting tumor cell response to reactive oxygen species production; although some studies have reported protective actions and increased antioxidant status by 5 UNCORRECTED PROOF T. Ortiz et al. European Journal of Pharmacology xxx (2017)xxx-xxx Fig. 6. Effects of amitriptyline in the content of CoQ biosynthetic protein in H460 cells treated with 50 Mamitriptyline for 24 h. Western blots were performed as described in Section 2.6 of Material and Methods with antibodies against Coq2, Coq4, Coq6 and Coq7 and GADPH. Protein levels were determined by densitometric analysis of three different Western blots and normalized to GAPDH signal. *P<0.05 between control and treated cells. upregulation of glutathione reductase (GR) and superoxide dismutase (SOD) activity of TCAs (Kolla et al., 2005; Herbet et al., 2013). These discrepancies could be explained by differences in the concentrations of the antidepressants used that could achieve effects sub-cytotoxic (protective) or cytotoxic (deleterious). This Vnding is consistent with previous results found by Yuan et al. in bladder cancer cells (Yuan et al., 2015), where they have shown that other TCAs, such as nortriptyline, also induce caspase-dependent apoptosis by two different pathways: mitochondria-mediated and death receptor-mediated. CoQ is one of the antioxidant that is significantly decreased by amitriptyline treatment, as we have previously demonstrated in both Vbroblasts and tumor cells (Cordero et al., 2009, 2010), although the expression levels of enzymes involved in CoQ biosynthesis were not studied. Furthermore, when CoQ levels were decreased by amitriptyline treatment, CoQ and alpha-tocopherol supplementation ameliorated amitriptyline-induced toxicity in both cultured human primary Vbroblasts and zebraVsh embryos (Cordero et al., 2009). CoQ has been shown to be acomponent of the structure of Complex III, contributing to both its assembly and stability; and it is one of the most important sites of reactive oxygen species production (Quinlan et al., 2013). In mammalian cells, COQ genes involved in the biosynthesis of ubiquinone encode at least nine Coq proteins (Coq1-Coq9), which are mainly located in the matrix side of the inner mitochondrial membrane (Tran and Clarke, 2007), although they are also present in plasma membrane, endomembranes and in serum lipoproteins, where they exert akey component in the antioxidant machinery (Fernandez-Ayala et al., 2005). CoQ is our only lipid soluble antioxidant that is synthesized endogenously. CoQ tissue content is independent on the diet, and it is effective in preventing both lipid peroxidation and protein oxidation as well as defending DNA from oxidative damage, especially important for mitochondrial DNA since it is more susceptible to oxidative damage than nuclear DNA (Bentinger et al., 2007). CoQ is also reported to be an inhibitor of apoptosis by inhibiting mitochondrial depolarization, independently of its free radical scavenging property (Papucci et al., 2003). In the yeast Saccharomyces cerevisiae (representative of eukaryotic cells), CoQ is synthesized by 11 known genes (COQ1-COQ9,YAH1,and ARH1) (Allan et al., 2015). It has been reported that the coordinated function of the eleven polypeptides (Coq1-Coq9, Arh1, and Yah1) is required for the production of CoQ (Nguyen et al., 2014; He et al., 2014). The Coq10 protein is localized to the mitochondria but it is not directly involved in its biosynthesis in S. cerevisiae (Barros et al., 2005). Several evidences suggest that the Coq proteins are associated in amulti-subunit complex in the inner mitochondrial membrane termed "CoQ-synthome" (He et al., 2014; Nguyen et al., 2014; He et al., 2015a; Allan et al., 2015; Kawamukai, 2015). Although the function of some of the Coq proteins remains unknown, such as Coq4, Coq8, and Coq9 (Tran and Clarke, 2007; He et al., 2014; Allan et al., 2015; Kawamukai, 2015), the presence of each Coq polypeptide is essential for the assembly of the Coq polypeptide complex (Nguyen et al., 2014), which is required for biosynthesis of ubiquinone (He et al., 2015a). Indeed, deletion of any of the COQ genes leads to destabilization of several other Coq polypeptides (He et al., 2014). In humans, mutations in several COQ genes are associated with mitochondrial, cardiovascular, kidney and neurodegenerative diseases (Nguyen et al., 2014). These results indicate the interdependence of the Coq polypeptides. It is recognized that Coq4 seems to be in the centre of the CoQ-synthome as organizer, and the Coq3, Coq5, Coq6 and Coq7 polypeptides as more peripheral members of the complex (He et al., 2014; Kawamukai, 2015). Although the molecular function of the Coq4 protein has not been elucidated, it is known that the lack of COQ4 in yeast causes the instability of several Coq proteins, such as Coq7 and Coq3 (Kawamukai, 2015). It has been also reported that the over-expression of Coq8, which encodes aputative regulatory kinase, stabilizes the Coq polypeptide complex in several coq null mutants (Nguyen et al., 2014; He, Black et al., 2015a; He et al., 2014), and it has been implicated in the complex assembly through the phosphorylation of Coq3, Coq5, and Coq7 (Kawamukai, 2015). Coq9 is alipid-binding protein that associates with Coq7 to enable CoQ biosynthesis (Lohman et al., 2014). Recently, it has been identiVed anew protein associated with the CoQ biosynthetic complex, Coq11, which is not required for CoQ synthesis, but its deletion reduces the CoQ levels in S. cerevisiae (Allan et al., 2015). 6 UNCORRECTED PROOF T. Ortiz et al. European Journal of Pharmacology xxx (2017)xxx-xxx As we have already demonstrated, amitriptyline induces reactive oxygen species production and apoptosis in H460 cells, as well as decreases CoQ levels in adose-dependent manner (Cordero et al., 2010). In the present study, we have demonstrated adysregulation of CoQ biosynthesis by amitriptyline, at both mRNA and protein levels, in a human lung cancer cell line. According to the model of mitochondrial CoQ biosynthesis complex proposed by Tran &Clarke (Tran and Clarke, 2007), we studied the Coq proteins most closely associated to Coq2 protein, which serves as an anchor to the inner mitochondrial membrane. Most chemotherapeutic drugs, such as camptothecin, doxorubicin, and methotrexate, do not provoke any decrease of antioxidants. Instead, they frequently induce an increase of antioxidants as aprotecting mechanism against reactive oxygen species generation, leading to low cell death rates (Brea-Calvo et al., 2006). The fact that amitriptyline reduces CoQ levels can be very interesting in terms of oxidative therapy since it diminishes even more the already decreased antioxidant defenses present in cancer cells, making amitriptyline-induced free radicals amore effective weapon against tumor cells. In this study, we have observed adecrease of COQ4 mRNA expression levels as well as adecrease of the amount of Coq4 and Coq6 protein expression levels after amitriptyline treatment. Amitriptyline-induced high reactive oxygen species levels probably trigger these alterations, as we have detected asignificant correlation between increased reactive oxygen species levels and decreased amount of CoQ. Moreover, we have shown that CoQ supplementation restored reactive oxygen species levels increased by amitriptyline treatment and hence cell survival. However, the mechanism by which amitriptyline provoke these downregulations is still unknown and it should be investigated. It has been hypothesized in yeasts that Coq4 functions as the core component of the polypeptide CoQ biosynthetic complex, holding together the other COQ gene products, although aclear enzymatic function has not been assigned (Tran and Clarke, 2007). The human gene has probably the same function than in yeasts since it can effectively complement ayeast COQ4⁠null mutant (Casarin et al., 2008). On the other hand, knockdown of COQ4 causes CoQ deVciency in HeLa cells (Salviati et al., 2012). Our results showed that Coq2 expression was not affected by amitriptyline treatment, but Coq7 was overexpressed, probably as an attempt to compensate the reduction in Coq4 and Coq6. Increased reactive oxygen species may cause the transcriptional effects on COQ7 genes through the activation of NF-kB, which has been demonstrated to be activated by camptothecin in H460 cells (Brea-Calvo et al., 2009). However, the downregulation of Coq4 stimulated by amitriptyline could be enough to induce adecrease of CoQ levels, reducing the antioxidant capacity of tumor cells. In conclusion, we have demonstrated that amitriptyline induces a down-regulation of CoQ levels in alung cancer cell line. As CoQ is the main lipid soluble endogenous antioxidant, this effect can limit cell defenses against amitriptyline-induced reactive oxygen species production. Therefore, we added new evidences showing that amitriptyline could be considered agood candidate for future assays in antitumoral oxidative therapy since it induces high reactive oxygen species production and also diminishes the antioxidant machinery. Sources of funding This work has been supported by the Spanish research association FOICAM (Asociaci npara el Fomento de la Investigaci n en Cncer yMitocondriopat as), FIS PI10/00543 grant, Fondo Europeo de Desarrollo Regional (FEDER-Uni nEuropea), SAS 111242 grant (Servicio Andaluz de Salud-Junta de Andaluc a), and Proyecto de Investigaci nde Excelencia (Junta de Andaluc aCTS-5725). Con-ict of interest The authors of this study declare that they have no conWict of interest. References Allan,C.M.,Awad,A.M.,Johnson,J.S.,Shirasaki,D.I.,Wang,C.,Blaby-Haas,C.E.,Merchant,S.S.,Loo,J.A.,Clarke,C.F.,2015. IdentiVcation of Coq11, anew coenzyme Qbiosynthetic protein in the CoQ-synthome in Saccharomyces cerevisiae.J. Biol. Chem. 290 (12), 7517–7534.http://dx.doi.org/10.1074/jbc.M114.633131. Barros,M.H.,Johnson,A.,Gin,P.,Marbois,B.N.,Clarke,C.F.,TzagoloX,A.,2005. The Saccharomyces cerevisiae COQ10 gene encodes astart domain protein required for function of coenzyme Qin respiration.J. Biol. 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