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Lovastatin-induced apoptosis is modulated by geranylgeraniol in a neuroblastoma cell line
Int. J. Devl Neuroscience 30 (2012) 451–456
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Lovastatin-induced apoptosis is modulated by geranylgeraniol in a neuroblastoma cell line Annalisa Marcuzzi a,∗ , Valentina Zanin a , Elisa Piscianz a , Paola Maura Tricarico b , Josef Vuch a , Martina Girardelli a , Lorenzo Monasta a , Anna Monica Bianco a , Sergio Crovella a,b a b
Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Trieste, Italy University of Trieste, Trieste, Italy
a r t i c l e
i n f o
Article history: Received 13 April 2012 Received in revised form 20 June 2012 Accepted 24 June 2012 Keywords: Mevalonate kinase deficiency Geranylgeraniol Neuronal apoptosis Lovastatin Isoprenoids
a b s t r a c t Mevalonic aciduria (MA), the most severe form of mevalonate kinase deficiency (MKD), is still an orphan drug disease and the pathogenetic mechanisms underlying neuronal dysfunction is still poorly understood. In our study we have investigated the apoptotic mechanism mediated by the exposure of the cultured neuroblastoma cell line, SH-SY5Y, to lovastatin in absence or in presence of the isoprenoid, geranylgeraniol, with the aim of unraveling the pathogenesis of MA. Lovastatin, blocks the mevalonate pathway inhibiting the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CR), an enzyme of the mevalonate pathway upstream the mevalonate kinase enzyme, reproducing biochemical features similar to those found in MKD. We demonstrate that apoptosis in neuronal lovastatin treated-cells is induced by the mitochondrial pathway, with caspase-9 as the initiator and caspase-3 as the effector caspase. The presence of geranylgeraniol modulates both the caspase-9 and caspase-3 activity in a dose-dependent way, confirming that this isoprenoid enters the mevalonate pathway, is metabolized and finally is able to by-pass the statin biochemical block reconstituting the mevalonate pathway. According to our findings, it should not be the time course adopted that modulates the apoptotic response but rather the isoprenoid itself. Being aware that our results have been obtained using a biochemical model of MKD, and not cells from patients with the disease, we believe our findings increase the knowledge of MA pathogenesis, and may possibly contribute to the development of novel therapeutic strategies. © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction Mevalonate kinase deficiency (MKD) is a rare autosomal recessive disease (OMIM #610377) caused by mutations affecting the second enzyme of mevalonate pathway (mevalonate kinase, MK/MVK), and the consequent shortage of intermediate compounds as well as final products of the metabolic route (Goldstein and Brown, 1990; Haas and Hoffmann, 2007). Different degrees of disease severity, according to the residual activity of MK, were observed ranging from auto-inflammatory hyper immunoglobulinemia D and periodic fever syndrome (HIDS, OMIM #260920), with a 1–8% residual MK activity, to mevalonic aciduria (MA, OMIM #610377) in which MK activity is below the level of detection (Haas and Hoffmann, 2006).
∗ Corresponding author at: Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Via dell’Istria, 65/1, 34137 Trieste, Italy. Tel.: +39 040 3785422; fax: +39 040 3785422. E-mail address: [email protected] (A. Marcuzzi). 0736-5748/$36.00 © 2012 ISDN. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijdevneu.2012.06.002
Patients with HIDS phenotype present recurrent episodes of fever and associated inflammatory symptoms, whereas patients with MA show, in addition to these episodes, developmental delay, dysmorphic features, ataxia, cerebellar atrophy, psychomotor retardation, and may die in early childhood. Despite the many efforts made in the past decades to understand the molecular events linking mevalonate pathway impairment to the inflammatory clinical phenotype, no specific treatment has yet been developed for MKD: the molecular events leading to neurologic impairment in the mevalonic aciduria are still unknown. Moreover, at present there are no pharmacological treatments aimed at improving the neuronal damage of mevalonic aciduria. Recently, it has been reported that the lack of isoprenoid intermediate geranylgeranyl-pyrophosphate (GGPP) is associated with the activation of caspase-1 and the release of interleukin-1 beta (IL1), the major cytokine responsible for the inflammatory systemic effects observed in MKD patients (Mandey et al., 2006). We previously showed, in mouse and cellular MKD models, that the inhibition of the mevalonate pathway with statin leads to augmented susceptibility to pathogens (De Leo et al., 2010). Compounds, such as lovastatin (Lova), block the mevalonate pathway by
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Fig. 1. Schematic representations of the mevalonate pathway. Compounds used in the experiments (lovastatin and isoprenoids) are indicated along the pathway.
inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CR), an enzyme of the mevalonate pathway upstream MK, reproducing MKD biochemical features (Fig. 1). Statins, in addition to decreasing the levels of isoprenoids, reduce the synthesis of the side chain of ubiquinone and induce the translocation of cytosolic Bax to mithocondria, impairing the mitochondrial metabolism. Ubiquinone is an antioxidant molecule that plays a crucial role in the respiratory chain, while Bax is required to permeabilize the mitochondrial outer membrane during apoptosis (Campia et al., 2009; Herrero-Martin and Lòpez-Rivas, 2008). Exogenous isoprenoids, such as geranylgeraniol, are able to enter the mevalonate pathway, to be metabolized by the FPPS and to rescue the shortage of intermediate isoprenoids in MKD models (De Leo et al., 2010). We already demonstrated that increased programmed cell death (PCD), and not only IL-1ß production, is caused by the mevalonate pathway inhibition: our study showed that cell death, induced by the block of the pathway, was in part sustained by the activation of caspase-3 and partially of caspase-1 (Marcuzzi et al., 2010, 2011). Recently, Mailman et al. (2011) investigated the neuronal mechanisms of cholesterol biosynthesis showing that treatment with Lova was not sufficient to block the mevalonate pathway, since cholesterol biosynthesis generates a sufficient provision of isoprenoid and cholesterol. It is commonly assumed that statins play a neuro-protective role in neurons even if the mechanism is still unclear. Indeed, the use of statins, such as Lova, has been proposed in neurodegenerative diseases, because of their ability to cross the blood-brain barrier and act directly on the brain (Sierra et al., 2011). Considering the very poor understanding of the pathogenetic mechanisms underlying neuronal dysfunction in mevalonic aciduria, we studied the role of isoprenoids such as geranylgeraniol
in the neuronal cellular model of MKD. We demonstrated mechanism of apoptosis in neuroblastoma cells (SH-SY5Y cells), using mevalonate pathway inhibitors. Even if the ideal, to understand neuronal dysfuncion in mevalonic aciduria, would be the use of cell lines from MKD patients, we excluded this approach because it would require a brain biopsy both on cases and controls. Up-to-date evidences described the levels of both FPP and GGPP in this neuronal cell line (Hooff et al., 2010). Since a non-apoptotic role of caspase-3 has been suggested, indicating this enzyme is involved in neural stem cell differentiation (D’Amelio et al., 2010), we investigated the role of caspase-3, normally considered as one of the last steps in cell death. We verified the hypothesis of an alternative role of caspase-3 possibly contributing to the expression of the neurological complications characterizing MKD patients. 2. Materials and methods 2.1. Reagents Geranylgeraniol (GGOH) and Lova (Mevinolin from Aspergillus terreus) were obtained from Sigma Chemical Co. Aldrich (St. Louis, MO) and dissolved in saline solution (Diaco SpA, Trieste, Italy).
2.2. Cell culture SH-SY5Y (human derived neuroblastoma cell line), kindly provided by Prof. S. Gustincich (Department of Neurobiology, International School for Advanced Studied S.I.S.S.A.-I.S.A.S. Trieste, Italy) were cultured in 44.5% MEM/EBSS (Euroclone, Italy), 44.5% HAM’S/F12 (Euroclone, Italy), supplemented with 10% fetal bovine serum (FBS, Euroclone, Italy), non-essential amino acid solution 1× (NEAA, Euroclone, Italy), 2 mM glutamine and penicillin streptomycin anphotericin B 1× solution (Sigma Chemical Co. Aldrich St. Louis, MO).48 h after seeding in Petri dish, cells were stimulated with different doses of Lova (0.1, 1, 5, 10 and 50 M) and GGOH (1, 5, 10, 30, 50 and 100 M).
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Fig. 2. Dose-response Lova and GGOH treatment in neuronal cells. Lova induced an incremental programmed cell death (PCD), on the contrary GGOH did not produce significant differences. PCD is expressed as a percentage of Annexin V positive cells (A+) and bars represent the means % (A+) ± SD of three independent experiments. (A) Cells were incubated with increasing concentrations of Lova (0.1–10–50 M) for 48 h. (B) Cells were incubated with increasing concentrations of GGOH (1–50 M) for 48 h. Analyses were performed with one-way ANOVA and Bonferroni correction comparing untreated cells with other experimental conditions; ***p < 0.001.
2.3. Programmed cell death assays The programmed cell death of SH-SY5Y was analyzed by flow cytometry using Annexin V (A) and Propidium Iodide (PI) staining. Cells were stained with FITCconjugated Annexin V and Propidium Iodide (Annexin V- FITC Apoptosis Detection Kit, Immunostep, Spain) following the customer indications. Briefly, 48 h after the stimulation, cells were harvested from the Petri dish with 0.5% Trypsin-EDTA solution (Sigma Chemical Co. Aldrich St. Louis, MO) and washed with PBS. 5 × 105 cells were stained with A and PI for 15 min, then washed with customer binding buffer. Fluorescence was acquired with CyAn ADP analyzer and Summit software (Beckman Coulter, Ca, USA), then analyzed with FlowJo software (version 7.6, Treestar, Inc. OR, USA). Debris was excluded from the plot based on the scatter; the apoptotic and necrotic cells were characterized on the basis of the fluorescence emitted. 2.4. Caspase-3/caspase-9 and caspase-8 assays Caspases were quantified with ApoAlert caspase-3 Colorimetric Assay Kit (Clontech Laboratories, Inc. USA, cat 630216), caspase-8 (Chemicon International, Inc. USA, APT171) and caspase-9 (Chemicon International, Inc. USA, APT 173) Colorimetric Activity Assay Kit. Briefly, 48 h after stimulation, 1.5 × 106 cells were harvested from the 6 wells plate with 0.5% Trypsin-EDTA solution (Sigma Chemical Co. Aldrich St. Louis, MO) and lysed with Cell Lysis Buffer. Each sample was centrifuged, the supernatant collected and mixed with a solution containing the specific substrate, then incubated at 37 ◦ C for 1 h in water bath. Absorbance was acquired using the LT-400 Microplate Reader (Labtech, UK) at 405 nm. 2.5. Data analysis All results are expressed as the mean ± standard deviation (SD). Statistical significance was calculated using a two-way analysis of variance (ANOVA) and Bonferroni post-test correction in the case of multiple comparisons. Analysis was performed using GraphPad Prism software (version 5.0).
3. Results 3.1. Dose-response Lova and GGOH treatment in neuronal cells We evaluated the modulation of programmed cell death (PCD) in neuronal SH-SY5Y cells by testing several doses of Lova (0.1, 1.0, 2.5, 5.0, 10.0 and 50.0 M) as we did in previously published experiments (Roensch et al., 2007). Neuronal cells treated with Lova 10.0–50.0 M showed a significant PCD increase if compared to untreated cells (Lova 10 M: 48.53 ± 2.69%, p < 0.001; Lova 50 M: 60.55 ± 14.75%, p < 0.001; untreated cells: 21.10 ± 1.64%) while no significant difference was detected at lower concentrations (Fig. 2A). We found that 10.0 M Lova was the minor dose with a statistically significant result (Fig. 2A), thus we decided to choose this dose for the following experiments, also in agreement with the literature (Arnold et al., 2010). Instead, no significant difference in PCD was found between SH-SY5Y cells treated with different GGOH concentrations (from 1 to 50 M) and untreated cells (Fig. 2B).
3.2. GGOH is able to reduce the apoptotic response in Lova-treated cells Neuronal cells were incubated with Lova (10 M) together with different concentrations of GGOH for 48 h. The percentage of PCD was significantly reduced with GGOH 5 M and 50 M (Lova + GGOH 5 M: 28.52 ± 1.03, p < 0.01; Lova + GGOH 50 M: 30.38 ± 4.09, p < 0.01), a smaller but still significant decrease of PCD was observed with Lova + GGOH 10 M (33.88 ± 3.75, p < 0.05), 30 M (30.96 ± 4.74, p < 0.05) and 100 M (32.95 ± 3.07, p < 0.05) when compared to Lova-treated cells (48.33 ± 2.28%). No significant change in PCD was observed between cells treated with concentrations of GGOH lower than 1 M (6.92 ± 3.98%) and Lovatreated cells (Fig. 3A). In order to verify if the experimental condition could affect the regulation of PCD, neuronal cells were incubated with Lova for 24 h and then with GGOH (1, 5, 10, 30, 50 M) for additional 24 h. In these conditions there was a progressive decrease of PCD associated with an increasing concentration of GGOH: the percentage of PCD was significantly reduced with Lova + GGOH 10 M (27.62 ± 1.70%, p < 0.001), GGOH 30 M (27.60 ± 3.20%, p < 0.001) and 50 M (21.49 ± 0.51 p < 0.001) when compared to Lova-treated cells (48.38 ± 2.56%). A smaller but still significant decrease of PCD was observed with Lova + GGOH 5 M (29.93 ± 3.60%, p < 0.01), and no change was observed using lower concentrations (Lova + GGOH 1 M: 42.33 ± 2.5%). In both experimental conditions the Lova-treated cells showed a significant increase of PCD when compared to untreated cells (21.10 ± 1.64%) (Fig. 3B). Thus, we found no significant differences between the two protocols, exception made for a more homogeneous dose-response result in the second case. The effect of GGOH in Lova-treated cells is also graphically evident in flow cytometric plots (left column) and confocal images (right column) of a SH-SY5Y sample treated with Lova 10 M (Fig. 4B) and Lova 10 M + GGOH 50 M (Fig. 4C) compared to untreated cells (Fig. 4A). Dot plots show the staining with Annexin V FITC and Propidium Iodide after gating on FSC/SSC to exclude the debris. The effect of Lova on PCD can be clearly observed in the upper and lower right quadrants of dot plot B and compared with the rescue by GGOH observed in the same quadrants of dot plot C. Images were acquired from a confocal microscope before conducting the PCD assay. 3.3. Effect of GGOH on caspase-3, -8, -9 activity Lova-treated cells showed an increased caspase-3 activity when compared to untreated cells (Lova 10 M: 65.9 103 ± 3.96 vs.
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Fig. 3. High concentrations of GGOH reduce PCD in Lova treated cells. (A) Cells were incubated with 10 M Lova and at the same time with different concentrations of GGOH (1–50 M) for 48 h. (B) Cells were incubated with 10 M Lova for 24 h and then with GGOH (1–50 M) for another 24 h. PCD: bars represent the mean % Annexin V positive cell (A+) ± SD of three independent experiments. Analyses were performed with one-way ANOVA and Bonferroni correction comparing Lova-treated cells with other experimental conditions; *p < 0.05 **p < 0.01 ***p < 0.001.
untreated, 25.03 103 ± 3.58, p < 0.001). Moreover GGOH also produced a statistically significant decrease of caspase-3 activities under all conditions (Lova + GGOH 5 M: 49.15 103 ± 0.17, p < 0.05; Lova + GGOH 10 M: 39.09 103 ± 0.64, p < 0.01; Lova + GGOH 30 M: 29.75 103 ± 0.26, p < 0.001; Lova + GGOH 50 M: 25.54 103 ± 0.83, p < 0.001) (Fig. 5A). We then evaluated whether caspase-3 was initiated by caspase-8 or -9. The activity of caspase-9 showed a similar trend to caspase-3: Lova treatment induced a higher increase of caspase-9 if compared to the untreated condition (Lova10 M: 61.57 103 ± 0.43 vs. untreated, 33.03 103 ± 1.03, p < 0.001). In addition, the caspase-9 activity showed a significant reduction in GGOH-treated cells (Lova + GGOH 5 M: 34.82 103 ± 1.07, p < 0.001; Lova + GGOH 10 M: 34.87 103 ± 1.98, p < 0.001; Lova + GGOH 30 M: 24.84 103 ± 3.40, p < 0.001; Lova + GGOH 50 M: 22.54 103 ± 1.36, p < 0.001) (Fig. 5C). We verified the role of caspase-3 in PCD, using a permeable reversible caspase inhibitor (Z-DEVD-FMK) that mimicked the effect of GGOH in Lova treated cells (data not shown). Caspase-8 was not significantly associated to any apoptotic event (Fig. 5B). 4. Discussion Mevalonic aciduria (MA), the most severe form of MKD, is still an orphan drug disease and the pathogenetic mechanism underlying neuronal dysfunction is still poorly understood. In particular, the link between the genetic defect and the neurological impairment is unclear and no treatment has been proven effective in curing the neurological symptoms in these severe cases of MKD (Bodar et al., 2005). The mechanisms that regulate cholesterol biosynthesis in neuronal cells have not been disclosed yet: it is of crucial importance to understand why cholesterol biosynthesis is impaired and deregulated in neuronal cells with consequent severe damage to the brain. With the aim of unraveling the MA pathogenesis, we investigated the apoptotic mechanism induced by exposure of cultured neuroblastoma cell line, SH-SY5Y, to lovastatin in absence or in presence of the isoprenoid geranylgeraniol. Lovastatin blocks the mevalonate pathway inhibiting 3-hydroxy-3-methylglutarylCoA reductase (HMG-CR), an enzyme of the mevalonate pathway upstream MK, reproducing biochemical features similar to those found in MKD.It is known that almost all cholesterol in the brain is synthesized locally. Cholesterol biosynthesis rates in the brain are higher during the embryonic development and then decrease after myelinization is completed (Dietschy and Turley, 2004): it is
commonly assumed that in mature brains most of the cholesterol is synthesized in the glia, whereas neurons down-regulate cholesterol biosynthesis (Pfrieger, 2003). Statins are potent inhibitors of the mevalonate pathway, through hampering HMG-CoA reductase, the limiting factor of this pathway; statins are extensively used as drugs for the treatment of hypercholesterolemia (Corsini et al., 1995). A recent study has described the consequences of a statininduced chronic reduction of neuronal cholesterol biosynthesis in the synapse formation and function, using Lova (Hooff et al., 2010). Moreover it has been shown that Lova induced differentiation and apoptosis in neuroblastoma cells, and the response was time- and dose-dependent. The ability of this drug to modulate the apoptosis in the neuroblastoma cell line has been confirmed in this study, by evaluating the effect of increasing doses of lovastatin on PCD and caspase activity. It is widely accepted that, in mammals, two main pathways have evolved for activating the caspase cascade, namely: the intrinsic (mitochondrial) pathway, regulated by caspase-9 (Green and Kroemer, 2004), and the extrinsic pathway, involving the death receptor pathway and caspase-8 (Schulze-Osthoff et al., 1998). In this study we have shown that Lova induces the increase of caspase-9 dependent apoptosis in the SH-SY5Y neuroblastoma cell line, and our results are concordant with previous findings reported in the literature: indeed existing data suggest that cells must proliferate in order to be sensitive to caspase-9 dependent statininduced apoptosis (Chapman-Shimshoni et al., 2003; Huang et al., 2006; Wong et al., 2002). We demonstrated that apoptosis in Lova treated neuronal cells is mediated by the mitochondrial pathway, where caspase-9 is the initiator and caspase-3 the effector caspase. Recently, several studies suggested that the statin-induced muscular neuronal damage mediated by oxidative stress might explain, at least partially, the pathogenesis of mevalonic aciduria and the apoptotic response (Moosmann and Behl, 2004). However, further studies are needed, and the potential role of adjuvant antioxidative treatment in mevalonic aciduria should be more deeply investigated (Celec and Behuliak, 2008). Geranylgeraniol modulates, in a dose-dependent manner, the activity of both caspase-9 and caspase-3, confirming that the isoprenoid enters the mevalonate pathway, is metabolized by enzymes downstream the FPPS, and finally is able to by-pass the statin biochemical block reconstituting the mevalonate pathway. Our results indicate that the time course used to deliver the isoprenoid does not modulate the apoptosis response. To our knowledge no theories have been proposed yet to explain this
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Fig. 4. Flow cytometry plots and image of cells SH-SY5Y in different experimental conditions. (A) Untreated cells; (B) cells incubated with 10 M Lova for 48 h and (C) cells incubated with 10 M Lova for 24 h and then with 50 M GGOH for another 24 h. The cytometry plot includes at least 28,000 cells. Numbers in the corners of quadrants represent the percent of the total. In the images, the horizontal bars indicate 0.2 mM. Lova induced an incremental PCD and high concentrations of GGOH reduced PCD.
trend. Further studies are needed, but we can hypothesize that different responses might be due to lovastatin metabolism. Recent developments, both in caspase research and in neurophysiology, indicate that caspase-3 is important not only in apoptosis but also in physiological processes not related to cell death (D’Amelio et al., 2010). The main non-apoptotic neuronal functions regulated by caspases include synaptic plasticity, dendrite pruning, as well as learning and memory processes. The knowledge of the biochemical pathway for non-apoptotic activation and modulation of caspase-3 has potential implications for the understanding of neurologic abnormalities (mental retardation,
ataxia and epilepsy) associated to mevalonic aciduria. Thus, we suggest that the mevalonate pathway can be a promising target for novel anti-inflammatory treatments. The involvement of apoptosis on the modulation of this pathway can possibly improve therapeutic approaches for intractable inflammation characterized mainly by severe neurological impairment, such as Alzheimer’s disease or Multiple Sclerosis. In conclusion, even if the findings presented in this study should be considered as preliminary, due to the adoption of a biochemical model of MKD, they should be taken as a first step in research aimed at better disclosing the mevalonic aciduria pathogenesis and,
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Fig. 5. Lovastatin induced an incremental caspase-3and 9 activity. Cells incubated with 10 M Lova for 24 h and then with GGOH (1–50 M) for another 24 h. (A) caspase-3 activity; (B) caspase-8 activity; (C) caspase-9 activity. Bars represent the mean caspase activity (103 unit) ± SD of three independent experiments. Analyses were performed with one-way ANOVA and Bonferroni correction comparing Lova-treated cells with other experimental conditions; *p < 0.05 **p < 0.01 ***p < 0.001.
eventually, at using the knowledge of the neuronal apoptotic mechanism for the development of novel therapeutic strategies for the still orphan drug MKD. Financial support This study was supported by a grant from the Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Trieste, Italy (RC 42/2011). Crovella S. is recipient of a fellowship grant from the European Project “Talents for an International House” within the 7th Research & Development Framework Programme PEOPLE – Marie Curie Actions – COFUND (co-funding of Regional, National and International Programmes). References Arnold, D.E., Gagne, C., Niknejad, N., McBurney, M.W., Dimitroulakos, J., 2010. Lovastatin induces neuronal differentiation and apoptosis of embryonal carcinoma and neuroblastoma cells: enhanced differentiation and apoptosis in combination with dbcAMP? Molecular and Cellular Biochemistry 345 (1–2), 1–11. Bodar, E.J., van der Hilst, J.C., Drenth, J.P., van der Meer, J.W., Simon, A., 2005. Effect of etanercept and anakinra on inflammatory attacks in the hyper-IgD syndrome: introducing a vaccination provocation model. Netherlands Journal of Medicine 63 (7), 260–264. Campia, I., Luissiana, C., Pescarmona, G., Ghigo, D., Bosia, A., Riganti, C., 2009. Geranylgeraniol prevents the cytotoxic effects of mevastatin in THP-1 cells, without decreasing the beneficial effects on cholesterol synthesis. British Journal of Pharmacology 158 (7), 1777–1786. Celec, P., Behuliak, M., 2008. The lack of non-steroid isoprenoids causes oxidative stress in patients with mevalonic aciduria. Medical Hypotheses 70 (5), 938–940. Chapman-Shimshoni, D., Yuklea, M., Radnay, J., Shapiro, H., Lishner, M., 2003. Simvastatin induces apoptosis of B-CLL cells by activation of mitochondrial caspase 9. Experimental Hematology 31 (9), 779–783. Corsini, A., Maggi, F.M., Catapano, A.L., 1995. Pharmacology of competitive inhibitors of HMG-CoA reductase. Pharmacological Research 31 (1), 9–27. D’Amelio, M., Cavallucci, V., Cecconi, F., 2010. Neuronal caspase-3 signaling: not only cell death. Cell Death and Differentiation 17 (7), 1104–1114. De Leo, L., Marcuzzi, A., Decorti, G., Tommasini, A., Crovella, S., Pontillo, A., 2010. Targeting farnesyl-transferase as a novel therapeutic strategy for mevalonate kinase deficiency: in vitro and in vivo approaches. Pharmacological Research 61 (6), 506–510. Dietschy, J.M., Turley, S.D., 2004. Thematic review series: brain lipids. Cholesterol metabolism in the central nervous system during early development and in the mature animal. Journal of Lipid Research 45 (8), 1375–1397.
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Lovastatin-induced apoptosis is modulated by geranylgeraniol in a neuroblastoma cell line Annalisa Marcuzzi a,∗ , Valentina Zanin a , Elisa Piscianz a , Paola Maura Tricarico b , Josef Vuch a , Martina Girardelli a , Lorenzo Monasta a , Anna Monica Bianco a , Sergio Crovella a,b a b
Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Trieste, Italy University of Trieste, Trieste, Italy
a r t i c l e
i n f o
Article history: Received 13 April 2012 Received in revised form 20 June 2012 Accepted 24 June 2012 Keywords: Mevalonate kinase deficiency Geranylgeraniol Neuronal apoptosis Lovastatin Isoprenoids
a b s t r a c t Mevalonic aciduria (MA), the most severe form of mevalonate kinase deficiency (MKD), is still an orphan drug disease and the pathogenetic mechanisms underlying neuronal dysfunction is still poorly understood. In our study we have investigated the apoptotic mechanism mediated by the exposure of the cultured neuroblastoma cell line, SH-SY5Y, to lovastatin in absence or in presence of the isoprenoid, geranylgeraniol, with the aim of unraveling the pathogenesis of MA. Lovastatin, blocks the mevalonate pathway inhibiting the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CR), an enzyme of the mevalonate pathway upstream the mevalonate kinase enzyme, reproducing biochemical features similar to those found in MKD. We demonstrate that apoptosis in neuronal lovastatin treated-cells is induced by the mitochondrial pathway, with caspase-9 as the initiator and caspase-3 as the effector caspase. The presence of geranylgeraniol modulates both the caspase-9 and caspase-3 activity in a dose-dependent way, confirming that this isoprenoid enters the mevalonate pathway, is metabolized and finally is able to by-pass the statin biochemical block reconstituting the mevalonate pathway. According to our findings, it should not be the time course adopted that modulates the apoptotic response but rather the isoprenoid itself. Being aware that our results have been obtained using a biochemical model of MKD, and not cells from patients with the disease, we believe our findings increase the knowledge of MA pathogenesis, and may possibly contribute to the development of novel therapeutic strategies. © 2012 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction Mevalonate kinase deficiency (MKD) is a rare autosomal recessive disease (OMIM #610377) caused by mutations affecting the second enzyme of mevalonate pathway (mevalonate kinase, MK/MVK), and the consequent shortage of intermediate compounds as well as final products of the metabolic route (Goldstein and Brown, 1990; Haas and Hoffmann, 2007). Different degrees of disease severity, according to the residual activity of MK, were observed ranging from auto-inflammatory hyper immunoglobulinemia D and periodic fever syndrome (HIDS, OMIM #260920), with a 1–8% residual MK activity, to mevalonic aciduria (MA, OMIM #610377) in which MK activity is below the level of detection (Haas and Hoffmann, 2006).
∗ Corresponding author at: Institute for Maternal and Child Health, IRCCS “Burlo Garofolo”, Via dell’Istria, 65/1, 34137 Trieste, Italy. Tel.: +39 040 3785422; fax: +39 040 3785422. E-mail address: [email protected] (A. Marcuzzi). 0736-5748/$36.00 © 2012 ISDN. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijdevneu.2012.06.002
Patients with HIDS phenotype present recurrent episodes of fever and associated inflammatory symptoms, whereas patients with MA show, in addition to these episodes, developmental delay, dysmorphic features, ataxia, cerebellar atrophy, psychomotor retardation, and may die in early childhood. Despite the many efforts made in the past decades to understand the molecular events linking mevalonate pathway impairment to the inflammatory clinical phenotype, no specific treatment has yet been developed for MKD: the molecular events leading to neurologic impairment in the mevalonic aciduria are still unknown. Moreover, at present there are no pharmacological treatments aimed at improving the neuronal damage of mevalonic aciduria. Recently, it has been reported that the lack of isoprenoid intermediate geranylgeranyl-pyrophosphate (GGPP) is associated with the activation of caspase-1 and the release of interleukin-1 beta (IL1), the major cytokine responsible for the inflammatory systemic effects observed in MKD patients (Mandey et al., 2006). We previously showed, in mouse and cellular MKD models, that the inhibition of the mevalonate pathway with statin leads to augmented susceptibility to pathogens (De Leo et al., 2010). Compounds, such as lovastatin (Lova), block the mevalonate pathway by
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Fig. 1. Schematic representations of the mevalonate pathway. Compounds used in the experiments (lovastatin and isoprenoids) are indicated along the pathway.
inhibiting 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CR), an enzyme of the mevalonate pathway upstream MK, reproducing MKD biochemical features (Fig. 1). Statins, in addition to decreasing the levels of isoprenoids, reduce the synthesis of the side chain of ubiquinone and induce the translocation of cytosolic Bax to mithocondria, impairing the mitochondrial metabolism. Ubiquinone is an antioxidant molecule that plays a crucial role in the respiratory chain, while Bax is required to permeabilize the mitochondrial outer membrane during apoptosis (Campia et al., 2009; Herrero-Martin and Lòpez-Rivas, 2008). Exogenous isoprenoids, such as geranylgeraniol, are able to enter the mevalonate pathway, to be metabolized by the FPPS and to rescue the shortage of intermediate isoprenoids in MKD models (De Leo et al., 2010). We already demonstrated that increased programmed cell death (PCD), and not only IL-1ß production, is caused by the mevalonate pathway inhibition: our study showed that cell death, induced by the block of the pathway, was in part sustained by the activation of caspase-3 and partially of caspase-1 (Marcuzzi et al., 2010, 2011). Recently, Mailman et al. (2011) investigated the neuronal mechanisms of cholesterol biosynthesis showing that treatment with Lova was not sufficient to block the mevalonate pathway, since cholesterol biosynthesis generates a sufficient provision of isoprenoid and cholesterol. It is commonly assumed that statins play a neuro-protective role in neurons even if the mechanism is still unclear. Indeed, the use of statins, such as Lova, has been proposed in neurodegenerative diseases, because of their ability to cross the blood-brain barrier and act directly on the brain (Sierra et al., 2011). Considering the very poor understanding of the pathogenetic mechanisms underlying neuronal dysfunction in mevalonic aciduria, we studied the role of isoprenoids such as geranylgeraniol
in the neuronal cellular model of MKD. We demonstrated mechanism of apoptosis in neuroblastoma cells (SH-SY5Y cells), using mevalonate pathway inhibitors. Even if the ideal, to understand neuronal dysfuncion in mevalonic aciduria, would be the use of cell lines from MKD patients, we excluded this approach because it would require a brain biopsy both on cases and controls. Up-to-date evidences described the levels of both FPP and GGPP in this neuronal cell line (Hooff et al., 2010). Since a non-apoptotic role of caspase-3 has been suggested, indicating this enzyme is involved in neural stem cell differentiation (D’Amelio et al., 2010), we investigated the role of caspase-3, normally considered as one of the last steps in cell death. We verified the hypothesis of an alternative role of caspase-3 possibly contributing to the expression of the neurological complications characterizing MKD patients. 2. Materials and methods 2.1. Reagents Geranylgeraniol (GGOH) and Lova (Mevinolin from Aspergillus terreus) were obtained from Sigma Chemical Co. Aldrich (St. Louis, MO) and dissolved in saline solution (Diaco SpA, Trieste, Italy).
2.2. Cell culture SH-SY5Y (human derived neuroblastoma cell line), kindly provided by Prof. S. Gustincich (Department of Neurobiology, International School for Advanced Studied S.I.S.S.A.-I.S.A.S. Trieste, Italy) were cultured in 44.5% MEM/EBSS (Euroclone, Italy), 44.5% HAM’S/F12 (Euroclone, Italy), supplemented with 10% fetal bovine serum (FBS, Euroclone, Italy), non-essential amino acid solution 1× (NEAA, Euroclone, Italy), 2 mM glutamine and penicillin streptomycin anphotericin B 1× solution (Sigma Chemical Co. Aldrich St. Louis, MO).48 h after seeding in Petri dish, cells were stimulated with different doses of Lova (0.1, 1, 5, 10 and 50 M) and GGOH (1, 5, 10, 30, 50 and 100 M).
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Fig. 2. Dose-response Lova and GGOH treatment in neuronal cells. Lova induced an incremental programmed cell death (PCD), on the contrary GGOH did not produce significant differences. PCD is expressed as a percentage of Annexin V positive cells (A+) and bars represent the means % (A+) ± SD of three independent experiments. (A) Cells were incubated with increasing concentrations of Lova (0.1–10–50 M) for 48 h. (B) Cells were incubated with increasing concentrations of GGOH (1–50 M) for 48 h. Analyses were performed with one-way ANOVA and Bonferroni correction comparing untreated cells with other experimental conditions; ***p < 0.001.
2.3. Programmed cell death assays The programmed cell death of SH-SY5Y was analyzed by flow cytometry using Annexin V (A) and Propidium Iodide (PI) staining. Cells were stained with FITCconjugated Annexin V and Propidium Iodide (Annexin V- FITC Apoptosis Detection Kit, Immunostep, Spain) following the customer indications. Briefly, 48 h after the stimulation, cells were harvested from the Petri dish with 0.5% Trypsin-EDTA solution (Sigma Chemical Co. Aldrich St. Louis, MO) and washed with PBS. 5 × 105 cells were stained with A and PI for 15 min, then washed with customer binding buffer. Fluorescence was acquired with CyAn ADP analyzer and Summit software (Beckman Coulter, Ca, USA), then analyzed with FlowJo software (version 7.6, Treestar, Inc. OR, USA). Debris was excluded from the plot based on the scatter; the apoptotic and necrotic cells were characterized on the basis of the fluorescence emitted. 2.4. Caspase-3/caspase-9 and caspase-8 assays Caspases were quantified with ApoAlert caspase-3 Colorimetric Assay Kit (Clontech Laboratories, Inc. USA, cat 630216), caspase-8 (Chemicon International, Inc. USA, APT171) and caspase-9 (Chemicon International, Inc. USA, APT 173) Colorimetric Activity Assay Kit. Briefly, 48 h after stimulation, 1.5 × 106 cells were harvested from the 6 wells plate with 0.5% Trypsin-EDTA solution (Sigma Chemical Co. Aldrich St. Louis, MO) and lysed with Cell Lysis Buffer. Each sample was centrifuged, the supernatant collected and mixed with a solution containing the specific substrate, then incubated at 37 ◦ C for 1 h in water bath. Absorbance was acquired using the LT-400 Microplate Reader (Labtech, UK) at 405 nm. 2.5. Data analysis All results are expressed as the mean ± standard deviation (SD). Statistical significance was calculated using a two-way analysis of variance (ANOVA) and Bonferroni post-test correction in the case of multiple comparisons. Analysis was performed using GraphPad Prism software (version 5.0).
3. Results 3.1. Dose-response Lova and GGOH treatment in neuronal cells We evaluated the modulation of programmed cell death (PCD) in neuronal SH-SY5Y cells by testing several doses of Lova (0.1, 1.0, 2.5, 5.0, 10.0 and 50.0 M) as we did in previously published experiments (Roensch et al., 2007). Neuronal cells treated with Lova 10.0–50.0 M showed a significant PCD increase if compared to untreated cells (Lova 10 M: 48.53 ± 2.69%, p < 0.001; Lova 50 M: 60.55 ± 14.75%, p < 0.001; untreated cells: 21.10 ± 1.64%) while no significant difference was detected at lower concentrations (Fig. 2A). We found that 10.0 M Lova was the minor dose with a statistically significant result (Fig. 2A), thus we decided to choose this dose for the following experiments, also in agreement with the literature (Arnold et al., 2010). Instead, no significant difference in PCD was found between SH-SY5Y cells treated with different GGOH concentrations (from 1 to 50 M) and untreated cells (Fig. 2B).
3.2. GGOH is able to reduce the apoptotic response in Lova-treated cells Neuronal cells were incubated with Lova (10 M) together with different concentrations of GGOH for 48 h. The percentage of PCD was significantly reduced with GGOH 5 M and 50 M (Lova + GGOH 5 M: 28.52 ± 1.03, p < 0.01; Lova + GGOH 50 M: 30.38 ± 4.09, p < 0.01), a smaller but still significant decrease of PCD was observed with Lova + GGOH 10 M (33.88 ± 3.75, p < 0.05), 30 M (30.96 ± 4.74, p < 0.05) and 100 M (32.95 ± 3.07, p < 0.05) when compared to Lova-treated cells (48.33 ± 2.28%). No significant change in PCD was observed between cells treated with concentrations of GGOH lower than 1 M (6.92 ± 3.98%) and Lovatreated cells (Fig. 3A). In order to verify if the experimental condition could affect the regulation of PCD, neuronal cells were incubated with Lova for 24 h and then with GGOH (1, 5, 10, 30, 50 M) for additional 24 h. In these conditions there was a progressive decrease of PCD associated with an increasing concentration of GGOH: the percentage of PCD was significantly reduced with Lova + GGOH 10 M (27.62 ± 1.70%, p < 0.001), GGOH 30 M (27.60 ± 3.20%, p < 0.001) and 50 M (21.49 ± 0.51 p < 0.001) when compared to Lova-treated cells (48.38 ± 2.56%). A smaller but still significant decrease of PCD was observed with Lova + GGOH 5 M (29.93 ± 3.60%, p < 0.01), and no change was observed using lower concentrations (Lova + GGOH 1 M: 42.33 ± 2.5%). In both experimental conditions the Lova-treated cells showed a significant increase of PCD when compared to untreated cells (21.10 ± 1.64%) (Fig. 3B). Thus, we found no significant differences between the two protocols, exception made for a more homogeneous dose-response result in the second case. The effect of GGOH in Lova-treated cells is also graphically evident in flow cytometric plots (left column) and confocal images (right column) of a SH-SY5Y sample treated with Lova 10 M (Fig. 4B) and Lova 10 M + GGOH 50 M (Fig. 4C) compared to untreated cells (Fig. 4A). Dot plots show the staining with Annexin V FITC and Propidium Iodide after gating on FSC/SSC to exclude the debris. The effect of Lova on PCD can be clearly observed in the upper and lower right quadrants of dot plot B and compared with the rescue by GGOH observed in the same quadrants of dot plot C. Images were acquired from a confocal microscope before conducting the PCD assay. 3.3. Effect of GGOH on caspase-3, -8, -9 activity Lova-treated cells showed an increased caspase-3 activity when compared to untreated cells (Lova 10 M: 65.9 103 ± 3.96 vs.
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Fig. 3. High concentrations of GGOH reduce PCD in Lova treated cells. (A) Cells were incubated with 10 M Lova and at the same time with different concentrations of GGOH (1–50 M) for 48 h. (B) Cells were incubated with 10 M Lova for 24 h and then with GGOH (1–50 M) for another 24 h. PCD: bars represent the mean % Annexin V positive cell (A+) ± SD of three independent experiments. Analyses were performed with one-way ANOVA and Bonferroni correction comparing Lova-treated cells with other experimental conditions; *p < 0.05 **p < 0.01 ***p < 0.001.
untreated, 25.03 103 ± 3.58, p < 0.001). Moreover GGOH also produced a statistically significant decrease of caspase-3 activities under all conditions (Lova + GGOH 5 M: 49.15 103 ± 0.17, p < 0.05; Lova + GGOH 10 M: 39.09 103 ± 0.64, p < 0.01; Lova + GGOH 30 M: 29.75 103 ± 0.26, p < 0.001; Lova + GGOH 50 M: 25.54 103 ± 0.83, p < 0.001) (Fig. 5A). We then evaluated whether caspase-3 was initiated by caspase-8 or -9. The activity of caspase-9 showed a similar trend to caspase-3: Lova treatment induced a higher increase of caspase-9 if compared to the untreated condition (Lova10 M: 61.57 103 ± 0.43 vs. untreated, 33.03 103 ± 1.03, p < 0.001). In addition, the caspase-9 activity showed a significant reduction in GGOH-treated cells (Lova + GGOH 5 M: 34.82 103 ± 1.07, p < 0.001; Lova + GGOH 10 M: 34.87 103 ± 1.98, p < 0.001; Lova + GGOH 30 M: 24.84 103 ± 3.40, p < 0.001; Lova + GGOH 50 M: 22.54 103 ± 1.36, p < 0.001) (Fig. 5C). We verified the role of caspase-3 in PCD, using a permeable reversible caspase inhibitor (Z-DEVD-FMK) that mimicked the effect of GGOH in Lova treated cells (data not shown). Caspase-8 was not significantly associated to any apoptotic event (Fig. 5B). 4. Discussion Mevalonic aciduria (MA), the most severe form of MKD, is still an orphan drug disease and the pathogenetic mechanism underlying neuronal dysfunction is still poorly understood. In particular, the link between the genetic defect and the neurological impairment is unclear and no treatment has been proven effective in curing the neurological symptoms in these severe cases of MKD (Bodar et al., 2005). The mechanisms that regulate cholesterol biosynthesis in neuronal cells have not been disclosed yet: it is of crucial importance to understand why cholesterol biosynthesis is impaired and deregulated in neuronal cells with consequent severe damage to the brain. With the aim of unraveling the MA pathogenesis, we investigated the apoptotic mechanism induced by exposure of cultured neuroblastoma cell line, SH-SY5Y, to lovastatin in absence or in presence of the isoprenoid geranylgeraniol. Lovastatin blocks the mevalonate pathway inhibiting 3-hydroxy-3-methylglutarylCoA reductase (HMG-CR), an enzyme of the mevalonate pathway upstream MK, reproducing biochemical features similar to those found in MKD.It is known that almost all cholesterol in the brain is synthesized locally. Cholesterol biosynthesis rates in the brain are higher during the embryonic development and then decrease after myelinization is completed (Dietschy and Turley, 2004): it is
commonly assumed that in mature brains most of the cholesterol is synthesized in the glia, whereas neurons down-regulate cholesterol biosynthesis (Pfrieger, 2003). Statins are potent inhibitors of the mevalonate pathway, through hampering HMG-CoA reductase, the limiting factor of this pathway; statins are extensively used as drugs for the treatment of hypercholesterolemia (Corsini et al., 1995). A recent study has described the consequences of a statininduced chronic reduction of neuronal cholesterol biosynthesis in the synapse formation and function, using Lova (Hooff et al., 2010). Moreover it has been shown that Lova induced differentiation and apoptosis in neuroblastoma cells, and the response was time- and dose-dependent. The ability of this drug to modulate the apoptosis in the neuroblastoma cell line has been confirmed in this study, by evaluating the effect of increasing doses of lovastatin on PCD and caspase activity. It is widely accepted that, in mammals, two main pathways have evolved for activating the caspase cascade, namely: the intrinsic (mitochondrial) pathway, regulated by caspase-9 (Green and Kroemer, 2004), and the extrinsic pathway, involving the death receptor pathway and caspase-8 (Schulze-Osthoff et al., 1998). In this study we have shown that Lova induces the increase of caspase-9 dependent apoptosis in the SH-SY5Y neuroblastoma cell line, and our results are concordant with previous findings reported in the literature: indeed existing data suggest that cells must proliferate in order to be sensitive to caspase-9 dependent statininduced apoptosis (Chapman-Shimshoni et al., 2003; Huang et al., 2006; Wong et al., 2002). We demonstrated that apoptosis in Lova treated neuronal cells is mediated by the mitochondrial pathway, where caspase-9 is the initiator and caspase-3 the effector caspase. Recently, several studies suggested that the statin-induced muscular neuronal damage mediated by oxidative stress might explain, at least partially, the pathogenesis of mevalonic aciduria and the apoptotic response (Moosmann and Behl, 2004). However, further studies are needed, and the potential role of adjuvant antioxidative treatment in mevalonic aciduria should be more deeply investigated (Celec and Behuliak, 2008). Geranylgeraniol modulates, in a dose-dependent manner, the activity of both caspase-9 and caspase-3, confirming that the isoprenoid enters the mevalonate pathway, is metabolized by enzymes downstream the FPPS, and finally is able to by-pass the statin biochemical block reconstituting the mevalonate pathway. Our results indicate that the time course used to deliver the isoprenoid does not modulate the apoptosis response. To our knowledge no theories have been proposed yet to explain this
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Fig. 4. Flow cytometry plots and image of cells SH-SY5Y in different experimental conditions. (A) Untreated cells; (B) cells incubated with 10 M Lova for 48 h and (C) cells incubated with 10 M Lova for 24 h and then with 50 M GGOH for another 24 h. The cytometry plot includes at least 28,000 cells. Numbers in the corners of quadrants represent the percent of the total. In the images, the horizontal bars indicate 0.2 mM. Lova induced an incremental PCD and high concentrations of GGOH reduced PCD.
trend. Further studies are needed, but we can hypothesize that different responses might be due to lovastatin metabolism. Recent developments, both in caspase research and in neurophysiology, indicate that caspase-3 is important not only in apoptosis but also in physiological processes not related to cell death (D’Amelio et al., 2010). The main non-apoptotic neuronal functions regulated by caspases include synaptic plasticity, dendrite pruning, as well as learning and memory processes. The knowledge of the biochemical pathway for non-apoptotic activation and modulation of caspase-3 has potential implications for the understanding of neurologic abnormalities (mental retardation,
ataxia and epilepsy) associated to mevalonic aciduria. Thus, we suggest that the mevalonate pathway can be a promising target for novel anti-inflammatory treatments. The involvement of apoptosis on the modulation of this pathway can possibly improve therapeutic approaches for intractable inflammation characterized mainly by severe neurological impairment, such as Alzheimer’s disease or Multiple Sclerosis. In conclusion, even if the findings presented in this study should be considered as preliminary, due to the adoption of a biochemical model of MKD, they should be taken as a first step in research aimed at better disclosing the mevalonic aciduria pathogenesis and,
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Fig. 5. Lovastatin induced an incremental caspase-3and 9 activity. Cells incubated with 10 M Lova for 24 h and then with GGOH (1–50 M) for another 24 h. (A) caspase-3 activity; (B) caspase-8 activity; (C) caspase-9 activity. Bars represent the mean caspase activity (103 unit) ± SD of three independent experiments. Analyses were performed with one-way ANOVA and Bonferroni correction comparing Lova-treated cells with other experimental conditions; *p < 0.05 **p < 0.01 ***p < 0.001.
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