Mitochondrial-Targeted Protective Properties of Isolated Diterpenoids

Article pubs.acs.org/jnp

Mitochondrial-Targeted Protective Properties of Isolated Diterpenoids from Sideritis spp. in Response to the Deleterious Changes Induced by H2O2 Elena González-Burgos,† Ana Isabel Duarte,‡ Maria Emilia Carretero,† Paula Isabel Moreira,*,‡,§ and Maria Pilar Gómez-Serranillos*,† †

Department of Pharmacology, Faculty of Pharmacy, Complutense University, Madrid, Spain Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal § Laboratory of Physiology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal ‡

ABSTRACT: Mitochondrial impairment and oxidative stress are considered widely to be central events in many forms of neurodegenerative disease. The current study has evaluated for the first time the potential protective role of three diterpenoids [andalusol (1), conchitriol (2), and lagascatriol (3)] in response to the deleterious H2O2-induced changes on mitochondrial function. U373-MG human astrocytoma cells and PC12 rat adrenal pheochromocytoma cells were used as models for evaluating the cytoprotective potential of these compounds. In the absence of diterpenoids 1−3, H2O2 compromised mitochondrial function, decreasing mitochondrial membrane potential and ATP levels, increasing caspase-3 activity, and disrupting cytosolic and mitochondrial calcium homeostasis. However, treatment with the diterpenoids, prior to H2O2, prevented these mitochondrial perturbations. In particular, 1 and 3 were the most effective compounds in protecting mitochondrial function against H2O2-induced oxidative stress in U373-MG, whereas all three diterpenoids studied were significantly active against PC12 cells. Since consistent evidence has demonstrated the contribution of H2O2 on both progression and pathological development of several human diseases associated with mitochondrial function and oxidative stress responses, compounds 1−3 are worthy of further investigation.

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diterpenoids derived from Sideritis spp., have been demonstrated recently to behave as bioactive indirect antioxidants.5 However, to the best of our knowledge no prior study has analyzed the effects of these compounds on H2O2-induced mitochondrial dysfunction. Accordingly, the antioxidant activity demonstrated for these diterpenoids and the role of mitochondria in the pathogenesis of neurodegenerative diseases encouraged the authors to investigate the effect of these natural products on oxidative stress-related mitochondrial dysfunction. For this purpose, two common cell lines were used for neuroprotective strategies (U373-MG and PC12 cells). In this investigation assays for mitochondrial membrane potential (ΔΨm), cytosolic and mitochondrial calcium levels, ATP levels, and caspase-3 activity were performed.

here is strong evidence supporting the role of mitochondria as key targets for the prevention and treatment of many common age-related diseases including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease.1 The mitochondria undertake some of the most critical functions for cells including cellular bioenergetics maintenance, storage of calcium ions for buffering against their toxic effects, control of the cell cycle, monitorization of cell differentiation, growth, and development, among others.2 Reactive oxygen species (ROS) such as H2O2 have been documented to interact with mitochondrial structure and function, inducing dysfunction of this organelle and leading to subsequent cell death mainly by apoptosis. The noxious action of ROS involves the oxidation of proteins, lipids, and DNA, resulting in function impairment, as evidenced by a decrease in ATP production and calcium homeostasis perturbations.3 Antioxidants are among the substances investigated in mitochondria for therapeutic approaches and strategies against ROS-related mitochondria dysfunction. Antioxidants, through direct and indirect mechanisms, inhibit the damaged processes caused by abnormal ROS generation and accumulation.4 Andalusol (1), conchitriol (2), and lagascatriol (3), major © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The causes of neurodegeneration occurring in several diseases such as Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease, among others, are multicausal;6 however, among the different contributing factors, many in vitro, in vivo, Received: February 5, 2013

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dx.doi.org/10.1021/np400118d | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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techniques for studying the mitochondria, the potential roles of 1−3 as mitochondrial antioxidant compounds have been investigated for the first time. Hydrogen peroxide is the major ROS in the human body. There is strong and consistent evidence in different cell types that high concentrations of H2O2 can cause mitochondrial dysfunction, as indicated by decreased mitochondrial membrane potential and mitochondrial translocation, causing the release of cytochrome c, overproduction of ROS, and disturbance of calcium homeostasis.9,10 The toxicity of hydrogen peroxide is related to the capacity of this ROS to interact with ion metals such as copper and iron, producing other highly reactive ROS including hydroxyl radical.11 Moreover, high levels of hydrogen peroxide have been detected in post-mortem brain of patients suffering from Alzheimer’s disease and Parkinson’s disease.12,13 Hydrogen peroxide is therefore a therapeutic target in the prevention and treatment of mitochondria-generated ROS in the pathogenesis and progression of neurodegenerative disease associated with oxidative stress. Effect of Diterpenoids 1−3 on Mitochondrial Membrane Potential. Mitochondria are one of the intracellular ROS targets, and changes in the mitochondrial membrane potential are one of the biochemical parameters directly related to oxidative stress in mitochondria.14 Therefore, it was first investigated if 1−3 inhibit H2O2-induced depolarization of mitochondrial membrane potential. To evaluate the potential protective effect of 1−3 on H2O2-induced mitochondrial damage, U373-MG and PC12 cells were treated with these compounds 24 h before exposure to H2O2. Figure 1 shows the result of the changes in mitochondrial membrane potential in both the PC12 (Figure 1A) and U373-MG (Figure 1B) cell

and human post-mortem studies have identified oxidative stress as a common pathogenic mechanism underlying these neurodegenerative disorders.7 Oxidative stress defines a cytotoxic condition resulting from an excessive intracellular production of ROS such as superoxide anion and hydrogen peroxide that cannot be counteracted by the human antioxidant defense systems.3 Mitochondria are the major source of ROS generation in mammalian cells. Superoxide anion radical is generated in complexes I and III and hydrogen peroxide in complex IV of the mitochondrial electron-transport chain. As a consequence of insult by several extracellular environmental factors, mitochondria can produce excessive ROS, which may lead to mitochondrial dysfunction.8 Antioxidants, which can access the mitochondrial compartment, may exert a protective effect against harmful ROS produced in excess in this organelle, consequently preventing oxidative damage.4 It has been demonstrated that 1−3 act as antioxidant agents, exerting a neuroprotective role against H2O2-induced oxidative stress in neurons and astrocyte cell lines.5 Since protection of the mitochondria represents a potential therapeutic target for prevention of neurodegenerative diseases associated with oxidative stress, the search for new biologically active compounds from plants that act as mitochondria-targeted antioxidants is a challenge in this research area. Using different

Figure 1. Effect of 1−3 on mitochondrial membrane potential of (A) PC12 cells and (B) U373-MG cells. These cells were incubated with 1−3 (5 and 10 μM; 24 h) and with H2O2 (0.1 mM for PC12 cells and 1 mM for U373-MG cells; 30 min). The fluorescent cationic dye tetramethylrhodamine methyl ester (TMRM) was an indicator of mitochondrial membrane potential changes. Data are expressed as means ± SD (% of control) (n = 9; *p < 0.05 and ***p < 0.001 vs control; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs H2O2). B



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lines using the fluorescence dye tetramethylrhodamine methyl ester (TMRM). This indicator is a lipophilic cation accumulated selectively by the mitochondria, which are metabolically active with negative membrane potential. As seen in Figure 1 (panels A and B), treatment with 1−3 per se did not cause changes in mitochondrial membrane potential compared to control cells. On the other hand, after 30 min of treatment with H2O2 (0.1 mM for PC12 cells and 1 mM for U373-MG cells), a significant reduction in the mitochondrial membrane potential resulted, as shown by intracellular fluorescence decreases of 84% for PC12 cells and of 33% for U373-MG cells. Treatment with 1−3 (5 and 10 μM, 24 h), prior to H2O2 incubation, prevented the decrease of H2O2induced mitochondrial membrane potential. In particular, 1−3 in PC12 cells and 1 and 3 in U373-MG cells at both concentrations used attenuated the changes in mitochondrial membrane potential significantly. Effect of Diterpenoids 1−3 on Calcium Levels. Mitochondrial function and integrity can be affected as a consequence of a disruption of mitochondrial calcium homeostasis. Calcium overload can lead to an impairment of mitochondrial function and to an overproduction of ROS in mitochondria through different mechanisms including peroxidation of cardiolipin, dissociation of cytochrome c from the inner mitochondrial membrane, and the opening of mitochondrial permeability transition pores, among others.15 Thus, the effects of diterpenoids 1−3 were studied on calcium levels. Tables 1 and 2 show the cytosolic and mitochondrial calcium

Table 2. Effect of Compounds 1−3 on Mitochondrial Calcium Levels compound control H2O2 1 (5 μM) 1 (10 μM) 1 (5 μM + H2O2) 1 (10 μM + H2O2) 2 (5 μM) 2 (10 μM) 2 (5 μM + H2O2) 2 (10 μM + H2O2) 3 (5 μM) 3 (10 μM) 3 (5 μM + H2O2) 3 (10 μM + H2O2)

compound control H2O2 1 (5 μM) 1 (10 μM) 1 (5 μM + H2O2) 1 (10 μM + H2O2) 2 (5 μM) 2 (10 μM) 2 (5 μM + H2O2) 2 (10 μM + H2O2) 3 (5 μM) 3 (10 μM) 3 (5 μM + H2O2) 3 (10 μM + H2O2)

cytosolic calcium (nM) 51.3 395.9 56.5 56.8 185.8 174.8 53.5 60.6 188.8 192.6 63.5 51.4 202.5 187.7

± ± ± ± ± ± ± ± ± ± ± ± ± ±

11.3 15.9c 8.9 10.9 13.6e 14.6e 8.2 9.8 12.5e 12.5e 9.3 7.6 8.9e 13.6e

cytosolic calcium (nM) 91.1 482.7 92.2 98.0 189.6 166.2 93.2 98.0 479.6 430.9 102.1 98.1 222.5 208.5

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.0 1.7 1.05 1.09 1.3

± ± ± ± ±

0.02 0.01d 0.03 0.04 0.03e

mitochondrial calcium (relative to control) (nM)b 1.0 1.5 0.97 0.99 1.15

± ± ± ± ±

0.03 0.02c 0.04 0.03 0.05e

1.2 ± 0.06e

1.09 ± 0.03e

1.08 ± 0.04 1.05 ± 0.06 1.4 ± 0.04e

0.99 ± 0.03 0.97 ± 0.02 1.48 ± 0.03

1.3 ± 0.04e

1.45 ± 0.05

0.9 ± 0.03 0.98 ± 0.04 1.4 ± 0.05e

1.02 ± 0.02 1.05 ± 0.01 1.22 ± 0.03e

1.5 ± 0.02e

1.17 ± 0.03e

a

PC12 cells. bU373-MG cells. These cells were incubated with 1−3 (5 and 10 μM; 24 h) and with H2O2 (0.1 mM for PC12 cells and 1 mM for U373-MG cells; 30 min). Mitochondrial calcium level changes were indicated by the fluorescent cationic dye Rhod-2. Data are expressed as means ± SD (n = 9). cp < 0.01 vs control. dp < 0.001 vs control. ep < 0.05 vs H2O2.

Table 1. Effect of Compounds 1−3 on Cytosolic Calcium Levels a

mitochondrial calcium (relative to control) (nM)a

MG cells with andalusol (1) and lagascatriol (2) prevented the increase of induced toxicity of H2O2 (Table 1). Significant differences were also found between mitochondrial calcium levels of control cells and H2O2 treatment (an accumulation of calcium in mitochondria of 0.7-fold in PC12 cells and 0.6-fold in U373-MG cells compared to control cells). When PC12 cells were treated for 24 h with 1−3 and U373MG cells with 1 and 3, prior to H2O2 treatment, a decrease in mitochondrial calcium levels was observed. This effect was particularly marked for 1 (5 and 10 μM) and 2 (10 μM) in PC12 cells and 1 and 3 (5 and 10 μM), causing a reduction of mitochondria calcium levels similar to control (Table 2). Effect of Diterpenoids 1−3 on ATP Levels. Mitochondria produce most of the ATP in the body via oxidative phosphorylation. The impairment of mitochondrial function implies the reduction of oxidative phosphorylation and thus the reduction of ATP formation.16 Accordingly, the effects of compounds 1−3 were investigated on ATP production capacity in U373-MG and PC12 cells under H2O2-induced oxidative stress conditions. The effects on ATP levels measured by HPLC are shown in Figure 2. The treatment of PC12 and U373-MG cells with H2O2 caused a significant reduction in ATP levels by 41% and 31%, respectively, when compared to control cells. None of the diterpenoids studied show any effect on cellular ATP content. However, as shown in Figure 2, 1−3 and 1 and 3 in PC12 and U373-MG cells, respectively, protected these cells from H2O2 toxicity, by enhancing ATP generation. The maximum effect in both cell types was observed for 1 at 10 μM. Effect of Diterpenoids 1−3 on Caspase-3 Activity. Apoptosis is the process of programmed cell death characterized by an energy-dependent cascade of biochemical changes and a distinct series of morphological features including cell

b

12.5 14.5d 16.5 26.9 18.9e 20.5e 21.5 30.5 19.8 19.6 23.6 32.5 20.5e 23.0e

a

PC12 cells. bU373-MG cells. These cells were incubated with 1−3 (5 and 10 μM; 24 h) and with H2O2 (0.1 mM for PC12 cells and 1 mM for U373-MG cells; 30 min). Cytosolic calcium level changes were indicated by the fluorescent cationic dye Indo-1/AM. Data are expressed as means of concentration (nM) ± SD (n = 9). cp < 0.01 vs control. dp < 0.001 vs control. ep < 0.01 vs H2O2.

levels, respectively, in PC12 and U373-MG cells. None of the assayed compounds, at the concentrations and incubation times used, produced changes in either cytosolic or mitochondrial calcium levels. Cytosolic calcium levels were found to be significantly increased after H2O2 treatment compared to control cells (395.9 nM vs 51.3 nM in PC12 cells and 482.7 nM vs 91.1 nM in U373-MG cells). However, pretreatment prior to H2O2 of PC12 cells with the three diterpenoids studied and of U373C



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Figure 2. Effect of 1−3 on ATP levels of (A) PC12 cells and (B) U373-MG cells. These cells were incubated with 1−3 (5 and 10 μM; 24 h) and with H2O2 (0.1 mM for PC12 cells and 1 mM for U373-MG cells; 30 min). Changes in ATP levels were measured by HPLC. Data are expressed as means ± SD (% of control) (n = 9; *p < 0.05 vs control; #p < 0.05 vs H2O2).

Figure 3. Effect of 1−3 on caspase-3 activity of (A) PC12 cells and (B) U373-MG cells. These cells were incubated with 1−3 (5 and 10 μM; 24 h) and with H2O2 (0.1 mM for PC12 cells and 1 mM for U373-MG cells; 30 min). Caspase-3 activity changes were indicated by Ac-DEVD-AMC as a fluorogenic indicator. Data are expressed as means ± SD (% of control) (n = 9; *p < 0.05 vs control; #p < 0.05 vs H2O2).

shrinkage, membrane blebbing, and pyknosis.17 Apoptosis can be the major physiological form of cell death or a type of pathological cell death that may occur under stress conditions or by the action of different physical and chemical agents. High levels of reactive oxygen species such as hydrogen peroxide may mediate apoptotic cell death. These ROS can facilitate caspase activation and finally apoptosis. The protease caspase-3 acts as

an effector enzyme essential in the apoptotic signaling pathway.18,19 As a hallmark of apoptosis, caspase-3 activation was analyzed using the synthetic tetrapeptide fluorogenic substrate Ac-DEVD-AMC. Caspase-3 cleaves the substrate Ac-DEVD-AMC between Ac-DEVD and AMC, consequently quantified from the fluorescence of the released product AMC by fluorometry. D



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°C using a Spectramax Gemini EM fluorocytometer (Molecular Devices). Once the measurement was finished, fluorescence intensity was recorded initially under identical wavelength conditions for 8 min after addition of ionomycin (3 μM) and then 4 min after MnCl2 (3 mM) addition. The following formula was employed to calculate cytosolic calcium levels: [Ca2+]i = Kd × (F − Fmin)/(Fmax − F) where Kd = dissociation constant of Indo-1 (250 nM), F = relative fluorescence signal of samples, Fmin = AF + 1/12(Fmax − AF), Fmax = maximum fluorescence signal after ionomycin, and AF = autofluorescence signal obtained after MnCl2 addition. Measurement of Mitochondrial Calcium Levels. The fluorescent cationic dye Rhod-2/AM was used for the measurement of mitochondrial calcium levels.27 On treatment, cells were first incubated in Krebs normal calcium medium containing Rhod-2/AM for 40 min at 37 °C in the dark. After this incubation period, cells were maintained in Krebs normal calcium medium for 30 min at 37 °C in the dark to hydrolyze the ester completely. The fluorescence intensity was measured at a wavelength of 552 nm excitation and 581 nm emission for 4 min at 37 °C using a Spectramax Gemini EM fluorocytometer (Molecular Devices). Once the measurements were finished, the calcium ionophore A23187 (5 μM) was added and fluorescence was recorded for 15 min under wavelength conditions identical with those described above. Mitochondrial calcium levels relative to control were determined by the ratio between the maximum fluorescence signal after the calcium ionophore A23187 addition and the basal fluorescence. Measurement of ATP Levels. ATP levels were measured in total cellular extracts using an HPLC method.28 After the treatment described above, the cells were first washed twice with PBS, then scraped from the 12-well plates using 0.6 M HClO4 and 25 mM EDTA-Na+, and subsequently centrifuged at 14.000g for 2 min at 4 °C. Next, supernatants were neutralized with 3 M KOH in 1.5 M Tris (pH ≈ 7) and centrifuged at 14.000g for 2 min at 4 °C. The analysis of ATP content was performed on an HPLC 1200 Series instrument (Agilent Technologies, Inc.). The chromatographic separation was carried out on a Mediterranean Sea C18 analytical column (5 μm particle size, 15 × 0.46 cm internal diameter) (Teknokroma, Barcelona, Spain) for 6 min. The mobile phase, at a flow rate of 1 mL/min, was composed of 100 mM KH2PO4 (pH 6.5)/1% methanol in the isocratic mode. The UV detector was set at 254 nm. Concentrations of ATP were determined from a standard curve prepared from ATP in water (concentration range 0.25 to 5 μM) and were normalized by protein content. Results are expressed as percent of control. Determination of Caspase-3 Activity. Caspase-3 activity was determined in total cellular extracts (20 μg) using the colorimetric specific substrate Ac-DEVD-AMC (20 μM) (incubation for 1 h at 37 °C). Fluorescence intensity was measured at λexc 360 nm and λem 460 nm using a FLx800 fluorescence microplate reader. Results are expressed as percent of control (100%).29 Statistical Analysis. All data were determined as means ± SD. Statistical analysis was performed using one-way analysis of variance and the Bonferroni test, with p values less than 0.05 considered statistically significant.

Figure 3 shows the effect of treatment with compounds 1−3 on caspase-3 activity. The exposure of PC12 (Figure 3A) and U373-MG cells (Figure 3B) to H2O2 for 30 min at 0.1 and 1 mM, respectively, produced a significant increase in caspase-3 activity by 2.1- (PC12 cells) and 2.6-fold (U373-MG cells) compared to control cells. On the other hand, treatment with only 1−3 did not activate caspase-3, which demonstrates that under the experimental conditions used these compounds did not induce this enzyme involved in the apoptosis process. However, when cells are pretreated for 24 h with 1−3, prior to H2O2 exposure, the resultant induced caspase-3 activity increase was inhibited. This protective effect was significant statistically for 1 and 3 in U373-MG cells and for 1−3 in PC12 cells. Taken together, the present results confirm the potential protective role of diterpenoids 1−3 in the mitochondria, supporting previous work that has demonstrated an efficient protection of several labdane-type and kaurane-type diterpenoids in cardiomyocytes and macrophages, respectively.20,21

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EXPERIMENTAL SECTION

General Experimental Procedures. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), horse serum (HS), gentamicin, pyruvate, tetramethylrhodamine methyl ester (TMRM), Indo-1/AM, Rhod-2/AM, and Pluoronic F127 were obtained from Gibco-Invitrogen (Grand Island, NY, USA), and Ac-DEVD-AMC substrate was from Alexis Biochemicals. Carbonyl cyanide ptriflouromethoxyphenylhydrazone (FCCP), oligomycin, MnCl2, calcium ionophore A23187, ionomycin, and ATP were purchased from Sigma (St. Louis, MO, USA). Compound 1 was isolated from the aerial parts of Sideritis arborescens Salzm. ex Benth. (Lamiaceae), and compounds 2 and 3 were isolated from Sideritis angustifolia Lag. (Lamiaceae), all with a purity of over 98%, as previously described.22−24 Voucher specimens of S. arborescens (MAF 130879) and S. angustifolia (MAF 100516) were deposited at the Department of Plant Biology of the Faculty of Pharmacy of the University Complutense of Madrid. Cell Culture. U373-MG (human glioblastoma astrocytoma cell line) was grown in DMEM containing 10% FBS and 0.5% gentamicin (50 mg/mL). PC12 (rat adrenal pheochromocytoma cell line) was grown in DMEM containing 5% FBS, 10% HS, 0.2% pyruvate, and 0.5% gentamicin (50 mg/mL). U373-MG and PC12 cells were incubated in a CO2 incubator (5%) at 37 °C. Cell Treatment. Pretreatment with 1−3 (5 and 10 μM) was performed 24 h before H2O2 addition (0.1 mM for PC12, 1 mM for U373-MG; 30 min). Measurement of Mitochondrial Membrane Potential (ΔΨm). The fluorescent cationic dye tetramethylrhodamine methyl ester (TMRM) was used for monitoring mitochondrial membrane potential (ΔΨm).25 After being treated, cells were incubated in Krebs medium containing TMRM (150 nM), and fluorescence intensity was measured at a wavelength of 549 nm excitation and 573 nm emission for 45 min at 37 °C using a Spectramax Gemini EM fluorocytometer (Molecular Devices). Once the measurement was finished, FCCP (6 μM) and oligomycin (0.25 μg/mL) were added, resulting in maximum depolarization of membrane potential (ΔΨm). Fluorescence intensity was measured under identical wavelength conditions for 15 min. Results expressed as percent of control were calculated by subtracting the maximum fluorescence value (after FCCP and oligomycin addition) and the basal fluorescence value. Measurement of Cytosolic Calcium Levels. The fluorescent cationic dye Indo-1/AM was used for the measurement of cytosolic calcium levels.26 On treatment, cells were first incubated in Krebs medium containing Indo-1/AM (3 μM) for 45 min at 37 °C in the dark, and, subsequently, they were maintained in dye-free Krebs medium for 15 min at 37 °C in the dark to hydrolyze acetoxymethyl ester completely. The fluorescence intensity was measured at a wavelength of 350 nm excitation and 410 nm emission for 4 min at 37

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AUTHOR INFORMATION

Corresponding Author

*(P. I. Moreira) Tel: +351239480012. Fax: +351239480034. Email: [email protected]. (M. P. Gómez-Serranillos) Tel: +34913941767. Fax: +34913941624. E-mail: [email protected]. es. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a doctoral grant from the Spanish Ministry of Education and Science (FPU), awarded to E.G.B. E



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Mitochondrial-Targeted Protective Properties of Isolated Diterpenoids

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