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Toxicity evaluation of some traditional African spices on breast cancer cells and isolated rat hepatic mitochondria
Food and Chemical Toxicology 50 (2012) 4199–4208
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Toxicity evaluation of some traditional African spices on breast cancer cells and isolated rat hepatic mitochondria Aphrodite T. Choumessi a,b, Rute Loureiro c, Ana M. Silva c, Ana C. Moreira c, Anatole C. Pieme b, Asonganyi Tazoacha b, Paulo J. Oliveira c,⇑, Véronique B. Penlap a a b c
Department of Biochemistry, Faculty of Science, University of Yaoundé I, B.P. 812 Yaoundé, Cameroon Department of Physiological Sciences, Faculty of Medicine and Biomedical Sciences, University of Yaoundé 1, Yaoundé, Cameroon CNC – Center for Neuroscience and Cellular Biology, University of Coimbra, P-3004-517 Coimbra, Portugal
a r t i c l e
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Article history: Received 19 May 2012 Accepted 5 August 2012 Available online 10 August 2012 Keywords: African spices Cancer cells Cytotoxicity Apoptosis Mitochondrial toxicity
a b s t r a c t Background: Fagara leprieuri (FL), Fagara xanthoxyloïdes (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) are used in many African countries as food spices or in traditional medicine to treat several maladies. In this work, we (a) investigate whether the crude spice extracts present selective cytotoxicity for breast cancer cell lines and (b) investigate whether the same extracts affect the bioenergetics and calcium susceptibility of isolated liver mitochondrial fractions. Results: All extracts were cytotoxic to the cell lines studied, with the exception of MW, which was less toxic for a normal cell line. Interestingly, some of the extracts did not depolarize mitochondria in intact breast cancer MCF-7 cells, although this effect was observed in a normal breast cancer cell line (MCF12A). All extracts increased hepatic mitochondrial state 2/4 respiration and decreased the respiratory control ratio and the transmembrane electric potential. Also, the extracts induced the mitochondrial permeability transition (MPT). Conclusions: Mitochondrial toxicity may be part of the mechanism by which the spices tested cause inhibition of proliferation and death in the cell lines tested. This study also warrants caution in the excessive use of these spices for human consumption. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Around 80% of the population in African and some Asian countries still rely in plant extracts with dietary importance to treat several health problems including cancer, hepatic and cardiovascular diseases (Balunas and Kinghorn, 2005; Mishra and Tiwari, 2011). Fagara leprieuri (FL), Fagara xanthoxyloïdes (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) are spices used in traditional African dishes, as well as in folk medicine to treat several illnesses including cancer (Kuete et al., 2011). Previous research has shown the cytotoxic activity of X. aethiopica methanolic extract on pancreatic cancer cells MiaPaCa-2, on leukemia cells CCRF-CEM cells and on cervical cancer C-33A cell lines (Adaramoye et al., 2011b; Kuete et al., 2011). The cytotoxic activity of its ethanolic Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; DW, mitochondrial transmembrane electric potential; FL, extract from Fagara leprieuri; FX, extract from Fagara xanthoxyloïdes; MPT, mitochondrial permeability transition; MW, extract from Mondia whitei; XA, extract from Xylopia aethiopica. ⇑ Corresponding author at: Center for Neuroscience and Cell Biology, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal. Tel.: +351 239855760; fax: +351 239853409. E-mail address: [email protected] (P.J. Oliveira). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.08.008
extract has also been described on HCT 116 colon cancer cells and on KG1A, U937 leukemia cells (Choumessi et al., 2012). The antimicrobial and antifungic properties of the essential oil of F. leprieuri, F. xanthoxyloïdes and X. aethiopica were also previously demonstrated (Konning et al., 2004; Tatsadjieu et al., 2003). In light of these findings, the first aim of the present work was to study whether the ethanolic extract of fruits from F. xanthoxyloïdes and F. leprieuri, dried roots of M. whitei and dried cloves of X. aethiopica present selective cytotoxicity on breast/normal cancer cell lines. Secondly, we aimed at investigating for the first time the interaction with hepatic mitochondrial bioenergetics and calcium susceptibility, as a mechanism to explain possible toxic effects of the different extracts. To this latter purpose, we have used isolated liver mitochondrial fractions, a well-known model to investigate drug-induced toxicity (Nadanaciva and Will, 2011; Pereira et al., 2009) and mechanisms involving toxicity of anti-cancer drugs (Oliveira et al., 2004; Pereira et al., 2007; Serafim et al., 2008). In fact, mitochondria have also been recognized as important in the context of carcinogenic transformation (Chatterjee et al., 2011) and efficacy of anti-cancer therapies (Barbosa et al., 2012; Wenner, 2011). Moreover, several compounds found in extracts used in traditional cuisine and/or medicine have been demonstrated to
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induce cancer cell death through a mitochondrial-mediated mechanism (Cheng et al., 2011; Park et al., 2012; Qi et al., 2012). Understanding the pharmacological and toxicological effects of traditional African spices, especially the role of mitochondrial bioenergetics modulation in their mechanisms of action, will allow developing new suitable therapies for human conditions, while avoiding undesirable mitochondrial toxicity. 2. Materials and methods 2.1. Reagents All reagents used were of analytical grade or higher, and all aqueous solutions were prepared in ultrapure (type I) water. Dulbecco’s modified Eagle’s medium (DMEM)/Ham-s Nutrient Mixture F12 and penicillin–streptomycin 100 solution were acquired from Sigma–Aldrich, St. Louis, MO. Fetal bovine serum (FBS) was acquired from GIBCO (New York, NY, USA). Rotenone (in dimethylsulfoxide), adenosine triphosphate (ATP, in water), and succinate (in water) solutions were obtained from Sigma–Aldrich, prepared as indicated, and stored as aliquots at 20 °C. Hoechst 33342 and tetramethylrhodaminemethylester (TMRM) was obtained from Molecular Probes-Invitrogen (Eugene, OR) and Caspase substrate (#235400) was obtained from Calbiochem – EMD (Darmstadt, Germany). 2.2. Animal care and ethical statement All experiments involving animals were conducted in accordance with the European convention for the protection of vertebrate animal used for experimental and other scientific purposes (EEC Directive of 1986; 86/609/EEC) and the Commission Recommendation of 18 June 2007 on guidelines for the accommodation and care of animals used for experimental and other scientific purposes (C (2007) 2525). Work was performed in accredited facilities and the senior author P. Oliveira is accredited by the Federation of Laboratory Animal Science Associations (FELASA) for animal experimentation. Male Wistar Han IGS rats at 8 weeks of age were purchased from Charles River (France). Animals were group-housed in type III-H cages (Tecniplast, Italy) with appropriated corn cob bedding and environmental enrichment. Rats were maintained in proper environmental requirements including room temperature set at 22 °C, relative humidity at 45–65%, ventilation with 15–20 changes/h, 12 h light/dark cycle, noise level <55 dB, and ad libitum access to standard rodent food (4RF21 GLP certificate, Mucedola, Italy) and acidified water (pH 2.6 with HCl, to prevent bacterial growth). All animals were acclimated 10–14 days prior to sacrifice. 2.3. Plant material and extraction Dried fruits of F. xanthoxyloïdes (FX, fr. bouche beante) and F. leprieuri (FL, sand knobwood), roots of M. whitei (MW, sweet root) and cloves of X. aethiopica (XA, negro pepper or Ethiopian pepper) were obtained commercially from spices sellers and the voucher specimen were identified at the Cameroon National herbarium. The dried materials were ground and extracted with 70% ethanol (100 mg/ml) for 2 h at 55 °C with stirring every 30 min. The suspension was centrifuged at 4000 rpm for 10 min at 25 °C. The extracts obtained were filtered and dried using vacuum. The dried extracts were then weighted and 10 mg/ml solutions were prepared and stored at 20 °C until use. In the different assays, the volume used of the ethanolic solution was always lower than 0.3% (v/v) without any effects per se. Table 1 describes some of the compounds present in the different spices. 2.4. Cell culture All cell lines used were obtained from American Type Culture Collection (ATCC) (Manassas, VA). Human MDA-MB-231 breast cancer cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum; 1% antibiotic/antimicotic and incubated at 37 °C with 5% CO2. MCF-7, another human breast cancer cell line, was cultured in DMEM with 10% fetal bovine serum; 1% antibiotic/antimicotic and 0.5% insulin at 37 °C with 5% CO2. MCF-12A, a normal human breast cell line, was cultured in DMEM – F12 with 5% fetal bovine serum, 1% antibiotic/antimitotic, 0.01 mg/ml insulin, 20 ng/ml human epidermal growth factor, 500 mg/ml hydrocortisone and 100 ng/ml cholera toxin from vibriocholerae and 0.365 mg/ml L/glutamine at 37 °C with 5% CO2. 2.5. Cell proliferation assays Cell proliferation was assessed by the Sulforhodamine B assay, as previously described (Sardao et al., 2009). For this assay different cell lines were seeded in 24-well plates at 104 cells per well. Twenty-four hours after seeding, cells were treated at different concentrations of the extracts (7.5, 15 and 30 lg/ml, selected from preliminary data using one experiment with this technique) in 70% ethanol, which was used as vehicle and added alone in control cells. After 24, 48 and 96 h
of treatment, the medium was removed and wells rinsed with 1% PBS. Cells were then fixed by adding 1% acetic acid in 100% methanol for at least 2 h at 20 °C. Later, the fixation solution was discarded and the plates dried in an oven at 37 °C. Two hundred and fifty microliters of 0.5% SRB in 1% acetic acid solution were added and incubated at 37 °C for 1 h. The wells were then washed with 1% acetic acid in water and dried. Then, 500 ll of Tris, pH 10 was added and the plates were shaken for 30 min. Finally, 200 ll of each supernatant was transferred in 96-well plates and optical density was recorded at 540 nm. 2.6. Assessment of caspase-3 activity Caspase-3 activity was investigated by using a colorimetric method (Sardao et al., 2009) in breast cancer MDA-MB-231 and normal breast MCF-12A cells, which were seeded at 3 105 cells/60 mm plates. 24 h after seeding, cells were treated with increasing extract concentrations (7.5, 15 and 30 lg/ml) and incubated for 24 and 48 h. After incubation, cell growth was stopped and cells collected using collecting medium (2 mM DTT, 100 lM phenylmethylsulfonyl fluoride (PMSF), 20 mM HEPES/NaOH pH 7.5, 250 mM Sucrose, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA) and stored at 80 °C until analysis. Prior to caspase-3 activity assay, protein quantification was made using the Bradford method with BSA 1 mg/ml as standard. For caspase-3-like activity assay, 25 lg of protein were added to different tubes containing assay buffer (25 mM HEPES pH 7.5, 0.1% CHAPS, 10% sucrose and 10 mM DTT) for a total volume of 195 ll. Five microliter of 4 mM substrate (DEVD-pNA) were added to each tube at the final concentration of 100 lM. The blank was prepared without sample and tubes were incubated at 37 °C for 2 h. Tubes were homogenized and 100 ll of each sample transferred into 96 wells plate and optical densities were read at 405 nm. 2.7. Determination of mitochondrial membrane potential on normal and cancer breast cell lines The mitochondrial membrane potential in MCF-12A and MCF-7 cells was investigated by measuring TMRM fluorescence by flow cytometry (Becton Dickinson FACScalibur). MCF-12A and MCF-7 cells were seeded at 3 105 cells/60 mm plates. Twenty-four hours after seeding, cells were treated with 30 lg/ml extract concentrations and incubated for 24 h. After incubation, cells were trypsinized and treated with TMRM (150 nM) for 30 min and then evaluated for mean cell fluorescence by flow cytometry. The uncoupler FCCP (2 lM) was used as a control and was added 15 min after TMRM treatment to promote mitochondrial depolarization. 2.8. Apoptotic DNA condensation assay DNA condensation assay was carried out using DNA labeling with Hoechst 33342. To this respect MDA-MB-231 cells were seeded at 3 105 cells/60 mm plates containing glass coverslips and left to attach for 24 h. Thereafter, cells were treated with different concentrations of extracts or with the vehicle and incubated for 24 h. After incubation, 2 lg/ml Hoechst 33342 was added for 30 min at 37 °C. The medium was then discarded, slides rinsed with 1% PBS mounted using mounting medium and submitted to microscopic analysis using a Zeiss Axioskop 2 Plus fluorescence microscope with a Zeiss AxioCam MRC (Zeiss, Göttingen, Germany). 2.9. Isolation of rat liver mitochondria Rats were sacrificed by cervical dislocation and the livers were removed, minced and rinsed in ice-cold buffer made up of 10 mM HEPES pH 7.4, 250 mM sucrose, 1 mM EGTA and 0.1% fat-free bovine serum albumin (BSA). The free-blood tissue obtained was then homogenized in a Teflon-potter homogenizer and the homogenate obtained was centrifuged at 800g for 10 min at 4 °C. The supernatant obtained was next centrifuged at 10,000g for 10 min at 4 °C and the mitochondrial pellet resuspended using a washing medium (pH 7.2) prepared like the homogenization medium without EGTA and BSA. After isolation, protein contents was determined using the Biuret method (Gornall et al., 1949) using BSA as standard. Mitochondria suspension obtained (approximately 50 mg of proteins/ml) was stored on ice and used within 5 h after 20 min of recovery period. 2.10. Mitochondrial transmembrane electric potential Mitochondria transmembrane electric potential (DW) was estimated indirectly by the mitochondrial accumulation of the lipophilic cation tetraphenylphosphonium (TPP+) detected using a TPP+-selective electrode in combination with an Ag/AgCl-saturated reference electrode. The reference electrode and the TPP+ electrode were inserted into an open vessel (with magnetic stirring) and connected to a pH meter, and signals were registered by a potentiometric recorder. Under constant stirring and at 30 °C, 1 mg mitochondrial protein was suspended in 1 ml of reaction medium (2.5 mM MgCl2, 5 mM HEPES, 5 mM KH2PO4, 65 mM KCl, and 125 mM sucrose pH 7.4 supplemented with 3 lM TPP+) and 10 mM succinate and 3 lM rotenone were added to supply electrons to the mitochondrial respiratory chain via Complex II.
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Table 1 Some of the compound found in the spice extracts used in the present work. No specific flavonoid characterization was performed. The studies were conducted with extraction methods different from the one used in the present work. Spices
Part use in the study
Compound identified
Effects
References
Fagara leprieuri
Fruits
Acridone derivative: 3-hydroxy-1,4-dimethoxy-10-methyl-9-acridone; 3-hydroxy-1,2-dime-thoxy-10-methyl-9-acridone and benzo[c]phenantridine, 10-O-demethyl-12-O-methylarnottianamide
The two first show a moderate effect on cancer cell lines (A549; DLD-1) while the third did not show any effect
Ngoumfo et al. (2010)
Fagara xanthoxyloides
Mondia whitei
Fruits
Roots
Xylopia aethiopica Fruits (Pods)
Phenols (34.59 ± 0.19 g GAE/100 g powder) and flavonoids (4.46 ± 0.40 g/100 g powder) 3,4-O-divanilloylquinic acid, 3,5-O-divanilloylquinic acid and 4,5-O-divanilloylquinic acid isolated from roots extracts Phenols (5.76 ± 0.25 g GAE/100 g powder) and flavonoids (0.44 ± 0.03 g/100 g powder) No particular compound isolated Phenols (6.40 ± 0.11 g GAE/100 g powder) and flavonoids (2.99 ± 0.01 g/100 g powder) ent-15-oxokaur-16-en-19-oic acid Oxoaporphine alkaloids
Abdou et al. (2010) No activity on cancer cells
Abdou et al. (2010)
Abdou et al. (2010) Cytotoxic and DNA damaging Possible DNA topoisomerase inhibitor
Phenols (12.74 ± 0.22 g GAE/100 g powder) and flavonoids (1.94 ± 0.03 g/100 g powder)
When investigating calcium-induced DW depolarization, the maximum mitochondria potential was recorded for 1 min and then calcium (10 ll, 10 mM stock) was added in two sequential additions, with a lag time between both additions to allow for mitochondrial repolarization. The DW was measured 30–45 s after the second calcium pulse in order to estimate the susceptibility of the mitochondrial suspension to calcium in the absence and presence of the test extracts. One micromolar cyclosporin A was used to inhibit the mitochondrial permeability transition and thus prevent calcium-induced mitochondrial depolarization (Broekemeier et al., 1989). The effect of different extracts was obtained by adding different concentrations (1.5, 2, 2.5 and 3 lg/ml, based on preliminary data) to mitochondrial suspensions. Assuming Nernst distribution of the TPP cation across the electrode membrane, absolute values of membrane potential (in millivolts) were determined according to Kamo et al. (1979).
Ouattara et al. (2004)
Choumessi et al. (2012) Harrigan et al. (1994) Abdou et al. (2010)
2.11. Measurement of mitochondrial oxygen consumption Oxygen consumption of isolated liver mitochondria was polarographically monitored with a Clark oxygen electrode connected to an appropriate recorder in a 1 ml thermostated water-jacketed and closed chamber at 30 °C with continuous stirring. The experiment was carried out by successively adding 1 ml of respiratory medium (2.5 mM MgCl2, 5 mM HEPES, 5 mM KH2PO4, 65 mM KCl, and 135 mM sucrose, pH 7.2), 5 mM succinate and 3 lM rotenone. One milligram of mitochondrial protein was added to initiate state 2 respiration, while state 3 respiration was initiated by adding 125 nmol ADP. After complete ATP phosphorylation, state 4 respiration was initiated. A second ADP (125 nmol) pulse followed by oligomycin (1 lg) was added to determine the oligomycin-induced respiratory state, in which an inhibition of passive proton flux through the ATP synthase exists. Finally, FCCP (1 lM)
Fig. 1. Effect of 70% ethanol extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) on MDA-MB-231 cell mass. Data are expressed as percentage of the control (vehicle only) for each time point. Data are means ± SD of five different experiments, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control.
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Fig. 2. Effect of 70% ethanol extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) on MCF-7 cell mass. Data are expressed as percentage of the control (vehicle only) for each time point. Data are means ± SD of five different experiments, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 control.
was added to the system to uncouple respiration. For the test samples, the extracts at different concentrations (1.5, 2, 2.5 and 3 lg/ml) were concurrently added in the chamber and the different respiration states were recorded. The respiratory control ratio (RCR) and ADP/O ratios were calculated as previously described (Chance and Williams, 1956).
2.12. Statistical analysis Data obtained are presented as figures or tables and values are mean ± SD from three to five independent experiments using distinct mitochondrial isolations. Statistical analyses were performed by using One-Way ANOVA followed by Newman– Keuls post-test. Significance was considered at p < 0.05.
Fig. 3. Effect of 70% ethanol extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) on MCF-12 cell mass. Data are expressed as percentage of the control (vehicle only) for each time point. Data are means ± SD of five different experiments, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control.
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Table 2 IC50 obtained for the different extracts in the different cell lines studied. Data was determined from data present in Figs. 1–3. Results are expressed in lg/ml and are means ± SD (n = 5). FL
FX
MW
XA
MDA-MB-231
24 h 48 h 96 h
8.8 ± 5.4 8.6 ± 3.9 6.5 ± 1.1
n.d. 6.7 ± 1.4 6.0 ± 0.9
>30 >30 12.3 ± 4.1
n.d. 5.6 ± 0.2 5.2 ± 0.9
MCF-7
24 h 48 h 96 h
7.2 ± 1.4 6.1 ± 1.4 5.5 ± 0.3
n.d. 6.9 ± 1.8 5.5 ± 0.8
>30 >30 12.5 ± 3.6
6.0 ± 0.6 5.7 ± 1.0 5.2 ± 0.1
MCF-12A
24 h 48 h 96 h
7.3 ± 0.9 6.0 ± 0.4 5.7 ± 0.4
9.9 ± 2.5 7.2 ± 0.5 6.0 ± 0.3
>30 >30 >30
6.4 ± 0.9 5.2 ± 0.1 5.3 ± 0.2
N.d., not determined.
3. Results 3.1. Cytotoxicity of tested spice extracts on MDA-MB-231, MCF-7, MCF-12A cell proliferation The first aim in this work was to verify if the four test extracts presented cytotoxicity against two breast cancer cell lines, MDAMB-231 and MCF-7, as compared with the normal breast cell line MCF-12A. As shown in Fig. 1 (MDA-MB-231 cells), Fig. 2 (MCF-7 cells) and Fig. 3 (MCF-12 cells) all four extracts showed cytotoxic effects for the cell lines tested. Nevertheless, XA, FL and FX extracts appear to be more toxic than the MW extract. All four extracts showed a dose and time-dependent effect in all cell lines investigated, although MW was less toxic in the non-cancer cell line MCF-12. Table 2 represents the inhibitory concentration that resulted in 50% of inhibition of cell proliferation (IC50) at the different time-points. The data reveals that for FL, FX and XA the
IC50 is lower than 20 lg/ml for the cancer cells studied, although a toxic effect was also observed for non-tumor cells. On the other hand, the calculated IC50 value calculated confirms that MW is the least toxic of all extracts tested, with an even lower effect on non-cancer MCF-12.
3.2. Effect of XA, FL, FX and MW on apoptotic caspase-3-like activity and DNA fragmentation Since the SRB assay cannot distinguish between inhibition of cell cycle and induction of cell death, the next step was to investigate if the test extracts induce cell death as part of their mechanism of toxicity. From the three cell lines tested, we selected the breast cancer cell line MBA-MD-231 and normal breast cell line MCF-12A to measure caspase-3-like activity, one hallmark of apoptosis induction.
Fig. 4. Caspase-3-like activity of 70% ethanol extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) on MDA-MB-231 cells, after 24 and 48 h of treatment. Results are express in [pNA] released (nM/lg protein), with pNA used as standard. Data represent means ± SD of three independent different experiments, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control.
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Caspase 3-like activity was determined by using a colorimetric method and extracts were incubated with cells for 24 and 48 h (Figs. 4 and 5). The extracts used induced an increase in caspase 3-like activity in a dose and time-dependent manner in the breast cancer cell line (Fig. 4). The most potent extracts to induce an increase in caspase 3-like activity were FX and FL, which displayed a strong increase already for 24 h in MBA-MD-231 cells. Interestingly, 7.5 lg/ml XA caused an inhibition of caspase 3-like activity, which was statistically significant. This decrease was time dependent but only occurred for that concentration (Fig. 4). In order to have a control for a non-tumor cell line, we performed the same assay on normal breast cells (MCF-12A, Fig. 5). Caspase-3-like activity measured in the normal breast cell line shows a significant increase at both 24 and 48 h when treated with FL and FX extracts for the maximum concentration. FL seems to be more efficient in increasing caspase 3-like activity since it already shows a significant result for 15 lg/ml at 48 h. On the other hand, MCF-12A cells treated with XA did not show any significant alteration on caspase 3-like activity (Fig. 5). An extract from MW was not evaluated since from all four extracts tested for cytotoxicity (SRB assay), it was the one with higher IC50 (Table 2). Furthermore, detection of apoptotic DNA condensation was performed by using the fluorescent probe Hoechst 33342. Several apoptotic nuclei were detected for 24 h of treatment with FL, FX and XA. MW treatment, confirming data from caspase 3-like activity, resulted in a lower number of nuclei showing condensed chromatin when compared with the other extracts (Fig. 6). Doxorubicin was used as an apoptosis positive control to induce chromatin condensation.
end-point was FL, in which no membrane repolarization post-calcium occurred (Fig. 8C). Very mild effects were observed regarding this parameter for MW (Fig. 8C), showing lower toxicity in this regard. Cyclosporin A prevented calcium-induced depolarization (data not shown) in the various experimental groups. To better understand the effect of the extracts on isolated hepatic mitochondria, respiration assays were also carried out (Fig. 9). The results present in Fig. 9 confirm that FL is the most toxic extract, showing a significant dose-dependent effect increasing state 2 (Fig. 9A) and state 4 respiration (Fig. 9C), in a oligomycinindependent manner (Fig. 9D). Also, FL caused a very large decrease in the RCR (Fig. 9F). The effects of FL were observed even for the lowest concentration tested (1.5 lg/ml). The second most toxic extract regarding effects on mitochondrial respiration was FX, which showed an increase in state 2 and in state 4 respiration in the presence of oligomycin. None of the extracts tested had any effect on uncoupled (Fig. 9E) or state 3 respiration (Fig. 9B), with the exception of the highest concentration tested of XA, which caused a small yet statistically significant decrease of uncoupled respiration. Besides FL, all other extracts caused a reduction in the RCR, which was mostly caused by increases in
3.3. Effects of XA, FL and FX extracts on MCF-12A and MCF-7 cells mitochondrial membrane electric potential We used a breast cancer cell (MCF-7) and its direct normal equivalent (MCF-12A) treated with the three more potent extracts for 24 h to investigate alterations of the mitochondrial membrane potential. Interestingly, the results show that the three extracts tested decrease TMRM fluorescence in the normal cell line, with XA being the more potent (Fig. 7, lower panel), whereas no effects were observed for that time point (24 h) in the cancer cell line (Fig. 7, upper panel). The uncoupler FCCP caused mitochondrial depolarization in both cell lines, as expected. 3.4. Direct Effects of XA, FL, FX and MW extracts on bioenergetics and calcium tolerance of isolated hepatic mitochondrial fractions Direct mitochondrial effects of the extracts were investigated on isolated liver mitochondria from Wistar rats. Effects on mitochondrial bioenergetic parameters and calcium susceptibility were investigated in the presence of the different spices. Mitochondrial DW fluctuations were evaluated with a TPP+selective electrode and results presented as percentage of control (no extract added, vehicle only). Although all extracts presented a dose-dependent effect on the maximal DW developed, FL, FX and XA were clearly more potent than MW (Fig. 8A). The test extracts were then assayed in their effects in decreasing mitochondrial ability to accumulate calcium. This end-point was studied by investigating whether calcium-induced depolarization was increased in the presence of the extracts and if recovery occurred after the second calcium pulse. The results (Fig. 8B) show that FX, MW and XA showed similar effects in terms of mitochondrial depolarization induced by calcium. FL, was the most toxic extract, increasing mitochondrial depolarization induced by calcium. The most critical end-point regarded the repolarization DW post-calcium. The toxic effects of the extracts were confirmed, although it is noticeable that the most toxic extract in this
Fig. 5. Caspase-3-like activity of 70% ethanol extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX) and Xylopia aethiopica (XA) on MCF-12A cells, after 24 and 48 h of treatment. Results are express in [pNA] released (nM/lg protein), with pNA used as standard. Data represent means ± SD of three independent different experiments, ⁄ p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control.
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Fig. 6. DNA condensation assay, as measured by nuclear incorporation of Hoechst 33342. Images were collected under an epifluorescence microscope (400 total magnification). MDA-MB-231 cells were treated for 24 h at 7.5, 15 and 30 lg/ml of each extract. Doxorubicin (10 lM) was used as positive control to induce apoptotic chromatic condensation. The white arrows indicate apoptotic nuclei.
state 4 respiration. The stimulation of state 4 respiration with FX, MW and XA was not significant due to the large variability observed, although a tendency was clearly observed. Finally, none of the extracts caused any effect on the ADP/O ratio (Fig. 9G). 4. Discussion Medicinal plants have been used worldwide to treat various illnesses including cancer treatment with several antineoplastics (such as etoposide, paclitaxel and vinblastine) isolated from plants (Mans et al., 2000). Other phytochemicals including berberine (Diogo et al., 2011) and sanguinarine (Serafim et al., 2008) have been tested in several models as potential anti-cancer agents. F. leprieuri (FL), F. xanthoxyloïdes (FX), M. whitei (MW) and X. aethiopica (XA) are four plants used in some African countries as spices in food preparation and in folk medicine against some diseases including cancer (Kuete et al., 2011). In this study, we used two cancer cell lines (MCF-7, MDA-MB-231) and one normal breast cell line (MCF-12A) to evaluate the antiproliferative properties of extracts from FL, FX, MW and XA. We also investigated the direct toxicity of these extracts on isolated hepatic mitochondrial fractions, which are a recognized model to investigate compound toxicity (Pereira et al., 2009). The results regarding antiproliferative activity of the tested extracts on MCF-7, MDA-MB-231 and MCF-12A cells showed that all extracts inhibit cell proliferation, although MW presented less toxicity towards the non-tumor cell line (Figs. 1–3). Our findings are in line with the antiproliferative properties of XA and FL previously reported on some other cancer cell lines (Adaramoye et al., 2011a,b; Kuete et al., 2011). Calculated IC50 (Table 2) values confirmed the results and indicated that FL, FX and XA have cytotoxic activity as the doses needed to decrease cell mass by 50% is lower than 20 lg/ml. The content in phytochemicals may explain the different cytotoxicity observed (Pittella et al., 2009). In fact, the presence of phenols and flavonoids have been described in these spices (Abdou et al., 2010) which may explain antiproliferative activity on cancer cell lines (Karna et al., 2011; Moghaddam et al., 2011). Also, ent-15-oxokaur-16-en-19-oic acid has been described as the main cytotoxic compound of XA (Choumessi et al., 2012). Until now, no through characterization of the flavonoid content in the different extracts has been obtained.
Fig. 7. Mitochondrial transmembrane electric potential, assessed as TMRM relative fluorescence measured by flow cytometry in MCF-7 and MCF-12A cell lines. Both cell lines were incubated with extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX) and Xylopia aethiopica (XA) during 24 h. Graphs represent mean ± SD of three independent different experiments, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control. CLT: Control
The results obtained are indicative that the extracts tested are cytotoxic for the cell lines studied, with only MW showing what appears to be a minor selectivity towards a non-tumor cell. Cell toxicity also involved caspase 3-like signaling, suggesting
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apoptosis (Fig. 4). Interestingly, assays done in a non-tumor cell line (MCF-12A) revealed also caspase-3-like activation in a dosedependent manner (Fig. 5), with the exception of the XA extract. This is an interesting observation since this extract caused a large decrease in MCF-12A cell mass (Fig. 3). Both results appears to indicate that this particular extract causes inhibition of MCF-12A cell proliferation through inhibition of cell cycle progression or through cell death involving caspase-independent mechanisms. The involvement of apoptosis in the mechanisms of cytotoxicity of the extracts in another cell line, MDA-MB-231, was confirmed by Hoechst nuclear labeling, demonstrating chromatic condensation that resembles apoptosis (Fig. 6). Although several pathways can trigger caspase-3 activation and apoptosis (Fuchs and Steller, 2011), we decided to focus on mitochondrial involvement. In the first assay aimed an obtaining information regarding this point, we measured mitochondrial membrane electric potential using the fluorimetric probe TMRM in cancer MCF-7 and normal MCF-12A cells. The results were surprising, since the three extracts used (XA, FL and FX) caused mitochondrial depolarization in the normal (MCF-12A) but not in the tumor (MCF-7) cell line, at least for the time point measured. When comparing these results with data from the cell proliferation assays (Figs. 2 and 3), it appears clear that toxicity at the 24 h time point is slightly higher for MCF-12A cells that for their tumor counterparts (Fig. 7). This may be suggestive that the extracts may arrest cell cycle previous to directly affecting mitochondrial bioenergetics, at least for the time point and tumor cell studied. In opposition, mitochondrial depolarization may occur earlier and be a significant event. To confirm possible mitochondrial effects, we investigated extract toxicity directly on isolated hepatic mitochondrial fractions. The results were somewhat unexpected. Interestingly, most of the effects of the different extracts regarded state 2/state 4 respiration (Fig. 9), which translated to a decrease in the RCR.
The most impressive effect was for FL extract, which caused an about 3-fold increase in state 2/state 4 respiration. Minor or no effects were observed in uncoupled respiration, suggesting no inhibitory effects on the respiratory chain, and on the ADP/O value, showing that the efficiency of oxidative phosphorylation was not affected. Interestingly, there were no statistical differences regarding state 3 respiration, again confirming stronger effects in non-phosphorylative respiration. The fact that oligomycin did not inhibit the effects on state 4 respiration (and in some cases even resulted in significant differences) indicate that the extracts are having effects at the mitochondrial membrane level, thus increasing proton influx and increasing respiration (Silva and Oliveira, 2011). The results are in agreement with DW measurements (Fig. 8) in which all extracts, with different degrees of efficacy, decreased the maximal DW generated. The results suggested that the compounds increase proton influx through the inner mitochondrial membrane, contributing to decrease DW and stimulate state 2 and state 4 respiration. Also, from the data obtained, FL was the only extract that showed a higher toxicity. This was particularly visible in the higher DW depolarization induced by calcium (Fig. 8B), with DW completely lost after the second calcium pulse (Fig. 8C). The effects of FL and other extracts, although milder, together with the observed inhibitory effect of cyclosporin A, the specific inhibitor of the mitochondrial transition pore (Broekemeier et al., 1989), suggested that some of the extracts may cause cell death through induction of the MPT (Rasola and Bernardi, 2007). Nevertheless, it is not to exclude that some of the extracts may cause mitochondrial depolarization by MPT-independent mechanisms (e.g., by increasing inner membrane passive permeability to protons), which would translate also in disruption of energy production and cell death. In conclusion, this study demonstrated for the first time direct mitochondrial effects of the tested extracts and supplied some
Fig. 8. Effect of different extracts on mitochondrial membrane potential (DW). (A) Maximal DW generated with succinate in the presence of rotenone as substrate. Two calcium pulses of 100 nmol were sequentially added and calcium-induced depolarization in the second pulse (B) and repolarization post-calcium pulse (C) were evaluated. Values represent mitochondrial DW expressed in% of the control. Data are means ± SD of three independent mitochondrial preparations, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control. Values for control: Maximal DW developed: 237.0 ± 0.7 mV, calcium-induced depolarization: 181.0 ± 1.3 mV and mitochondrial membrane repolarization: 228.0 ± 1.1 mV.
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Fig. 9. Effect of extracts on mitochondrial respiration. Respiratory parameters were studied after energizing mitochondria through complex II. (A) State 2 respiration, (B) state 4 respiration, (C) state 3 respiration, (D) oligomycin (state 4 respiration under the presence of oligomycin), (E) uncoupled respiration (FCCP state), (F) ADP/O and (G) respiratory control ratio (RCR). Results are shown as% of control (vehicle only). Data are means ± SD of three distinct mitochondrial isolations, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄ p < 0.0005 versus control. Values for control: state 2: 22.8 ± 3.6 natoms O/min/mg protein, state 4: 20.7 ± 4.6 natoms O/min/mg protein, state 3: 84.7 ± 9.9 natoms O/min/ mg protein, oligomycin-state: 8.0 ± 1.5 natoms O/min/mg protein, FCCP-state: 216.1 ± 16.5 natoms O/min/mg protein, ADP/O: 1.5 ± 0.2 natoms O/min/mg protein and RCR: 5.1 ± 0.9 natoms O/min/mg protein.
clues to their anti-proliferative actions on normal and cancerous breast cells. Further investigations would intend to identify the specific chemical(s) in each spice extract responsible for the observed activities, especially for the strong induction of proton permeabilization in the inner mitochondrial membrane. Although it is very difficult to assess how the concentrations used in this study correlate with plasma levels during human consumption, since no studies are available, equivalent concentrations were used in other previous studies (Adaramoye et al., 2011a,b; Choumessi et al., 2012). The present results are important in the context of basic toxicity assessment of these extracts, warranting caution in their use in high doses as diet additives. Nevertheless, increased mitochondrial proton reflow through the inner mitochondrial membrane may have impact in diseases such as obesity (Grundlingh et al., 2011; Harper et al., 2001).
Author’s contributions A.C., R.L., A.S. and A.M. performed the experiments; A.P., A.T. and V.P. collected and processed the spices used in this work; V.P., A.P., A.T. and P.O. designed the study; A.C., R.L., A.S. and A.M. interpreted the data; P.O. and A.C. wrote the manuscript; all authors revised and approved the contents of the manuscript.
Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements Funding for this work was obtained from the Portuguese Foundation for Science and Technology (FCT), grant PTDC/QUI-QUI/ 101409/2008 (to P.J.O.), co-supported by COMPETE/FEDER and Portuguese National Funds. Aphrodite T. Choumessi was supported by a research fellowship from the ‘Coimbra Group’. The funding agencies had no role in the decision to publish this work or in data analysis and conclusions. We thank Jenna R. Erickson, M.S., Chicago Medical School, North Chicago, IL, USA for proofreading the manuscript for English language. References Abdou, B.A., Njintang, Y.N., Scher, J., Mbofung, C.M.F., 2010. Phenolic compounds and radical scavenging potential of twenty Cameroonian species. Agric. Biol. J. N. Am. 1, 213–224. Adaramoye, O.A., Adedara, I.A., Popoola, B., Farombi, E.O., 2011a. Extract of Xylopia aethiopica (Annonaceae) protects against gamma-radiation induced testicular damage in Wistar rats. J. Basic Clin. Physiol. Pharmacol. 21, 295–313.
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Toxicity evaluation of some traditional African spices on breast cancer cells and isolated rat hepatic mitochondria Aphrodite T. Choumessi a,b, Rute Loureiro c, Ana M. Silva c, Ana C. Moreira c, Anatole C. Pieme b, Asonganyi Tazoacha b, Paulo J. Oliveira c,⇑, Véronique B. Penlap a a b c
Department of Biochemistry, Faculty of Science, University of Yaoundé I, B.P. 812 Yaoundé, Cameroon Department of Physiological Sciences, Faculty of Medicine and Biomedical Sciences, University of Yaoundé 1, Yaoundé, Cameroon CNC – Center for Neuroscience and Cellular Biology, University of Coimbra, P-3004-517 Coimbra, Portugal
a r t i c l e
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Article history: Received 19 May 2012 Accepted 5 August 2012 Available online 10 August 2012 Keywords: African spices Cancer cells Cytotoxicity Apoptosis Mitochondrial toxicity
a b s t r a c t Background: Fagara leprieuri (FL), Fagara xanthoxyloïdes (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) are used in many African countries as food spices or in traditional medicine to treat several maladies. In this work, we (a) investigate whether the crude spice extracts present selective cytotoxicity for breast cancer cell lines and (b) investigate whether the same extracts affect the bioenergetics and calcium susceptibility of isolated liver mitochondrial fractions. Results: All extracts were cytotoxic to the cell lines studied, with the exception of MW, which was less toxic for a normal cell line. Interestingly, some of the extracts did not depolarize mitochondria in intact breast cancer MCF-7 cells, although this effect was observed in a normal breast cancer cell line (MCF12A). All extracts increased hepatic mitochondrial state 2/4 respiration and decreased the respiratory control ratio and the transmembrane electric potential. Also, the extracts induced the mitochondrial permeability transition (MPT). Conclusions: Mitochondrial toxicity may be part of the mechanism by which the spices tested cause inhibition of proliferation and death in the cell lines tested. This study also warrants caution in the excessive use of these spices for human consumption. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Around 80% of the population in African and some Asian countries still rely in plant extracts with dietary importance to treat several health problems including cancer, hepatic and cardiovascular diseases (Balunas and Kinghorn, 2005; Mishra and Tiwari, 2011). Fagara leprieuri (FL), Fagara xanthoxyloïdes (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) are spices used in traditional African dishes, as well as in folk medicine to treat several illnesses including cancer (Kuete et al., 2011). Previous research has shown the cytotoxic activity of X. aethiopica methanolic extract on pancreatic cancer cells MiaPaCa-2, on leukemia cells CCRF-CEM cells and on cervical cancer C-33A cell lines (Adaramoye et al., 2011b; Kuete et al., 2011). The cytotoxic activity of its ethanolic Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; DW, mitochondrial transmembrane electric potential; FL, extract from Fagara leprieuri; FX, extract from Fagara xanthoxyloïdes; MPT, mitochondrial permeability transition; MW, extract from Mondia whitei; XA, extract from Xylopia aethiopica. ⇑ Corresponding author at: Center for Neuroscience and Cell Biology, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal. Tel.: +351 239855760; fax: +351 239853409. E-mail address: [email protected] (P.J. Oliveira). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.08.008
extract has also been described on HCT 116 colon cancer cells and on KG1A, U937 leukemia cells (Choumessi et al., 2012). The antimicrobial and antifungic properties of the essential oil of F. leprieuri, F. xanthoxyloïdes and X. aethiopica were also previously demonstrated (Konning et al., 2004; Tatsadjieu et al., 2003). In light of these findings, the first aim of the present work was to study whether the ethanolic extract of fruits from F. xanthoxyloïdes and F. leprieuri, dried roots of M. whitei and dried cloves of X. aethiopica present selective cytotoxicity on breast/normal cancer cell lines. Secondly, we aimed at investigating for the first time the interaction with hepatic mitochondrial bioenergetics and calcium susceptibility, as a mechanism to explain possible toxic effects of the different extracts. To this latter purpose, we have used isolated liver mitochondrial fractions, a well-known model to investigate drug-induced toxicity (Nadanaciva and Will, 2011; Pereira et al., 2009) and mechanisms involving toxicity of anti-cancer drugs (Oliveira et al., 2004; Pereira et al., 2007; Serafim et al., 2008). In fact, mitochondria have also been recognized as important in the context of carcinogenic transformation (Chatterjee et al., 2011) and efficacy of anti-cancer therapies (Barbosa et al., 2012; Wenner, 2011). Moreover, several compounds found in extracts used in traditional cuisine and/or medicine have been demonstrated to
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induce cancer cell death through a mitochondrial-mediated mechanism (Cheng et al., 2011; Park et al., 2012; Qi et al., 2012). Understanding the pharmacological and toxicological effects of traditional African spices, especially the role of mitochondrial bioenergetics modulation in their mechanisms of action, will allow developing new suitable therapies for human conditions, while avoiding undesirable mitochondrial toxicity. 2. Materials and methods 2.1. Reagents All reagents used were of analytical grade or higher, and all aqueous solutions were prepared in ultrapure (type I) water. Dulbecco’s modified Eagle’s medium (DMEM)/Ham-s Nutrient Mixture F12 and penicillin–streptomycin 100 solution were acquired from Sigma–Aldrich, St. Louis, MO. Fetal bovine serum (FBS) was acquired from GIBCO (New York, NY, USA). Rotenone (in dimethylsulfoxide), adenosine triphosphate (ATP, in water), and succinate (in water) solutions were obtained from Sigma–Aldrich, prepared as indicated, and stored as aliquots at 20 °C. Hoechst 33342 and tetramethylrhodaminemethylester (TMRM) was obtained from Molecular Probes-Invitrogen (Eugene, OR) and Caspase substrate (#235400) was obtained from Calbiochem – EMD (Darmstadt, Germany). 2.2. Animal care and ethical statement All experiments involving animals were conducted in accordance with the European convention for the protection of vertebrate animal used for experimental and other scientific purposes (EEC Directive of 1986; 86/609/EEC) and the Commission Recommendation of 18 June 2007 on guidelines for the accommodation and care of animals used for experimental and other scientific purposes (C (2007) 2525). Work was performed in accredited facilities and the senior author P. Oliveira is accredited by the Federation of Laboratory Animal Science Associations (FELASA) for animal experimentation. Male Wistar Han IGS rats at 8 weeks of age were purchased from Charles River (France). Animals were group-housed in type III-H cages (Tecniplast, Italy) with appropriated corn cob bedding and environmental enrichment. Rats were maintained in proper environmental requirements including room temperature set at 22 °C, relative humidity at 45–65%, ventilation with 15–20 changes/h, 12 h light/dark cycle, noise level <55 dB, and ad libitum access to standard rodent food (4RF21 GLP certificate, Mucedola, Italy) and acidified water (pH 2.6 with HCl, to prevent bacterial growth). All animals were acclimated 10–14 days prior to sacrifice. 2.3. Plant material and extraction Dried fruits of F. xanthoxyloïdes (FX, fr. bouche beante) and F. leprieuri (FL, sand knobwood), roots of M. whitei (MW, sweet root) and cloves of X. aethiopica (XA, negro pepper or Ethiopian pepper) were obtained commercially from spices sellers and the voucher specimen were identified at the Cameroon National herbarium. The dried materials were ground and extracted with 70% ethanol (100 mg/ml) for 2 h at 55 °C with stirring every 30 min. The suspension was centrifuged at 4000 rpm for 10 min at 25 °C. The extracts obtained were filtered and dried using vacuum. The dried extracts were then weighted and 10 mg/ml solutions were prepared and stored at 20 °C until use. In the different assays, the volume used of the ethanolic solution was always lower than 0.3% (v/v) without any effects per se. Table 1 describes some of the compounds present in the different spices. 2.4. Cell culture All cell lines used were obtained from American Type Culture Collection (ATCC) (Manassas, VA). Human MDA-MB-231 breast cancer cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum; 1% antibiotic/antimicotic and incubated at 37 °C with 5% CO2. MCF-7, another human breast cancer cell line, was cultured in DMEM with 10% fetal bovine serum; 1% antibiotic/antimicotic and 0.5% insulin at 37 °C with 5% CO2. MCF-12A, a normal human breast cell line, was cultured in DMEM – F12 with 5% fetal bovine serum, 1% antibiotic/antimitotic, 0.01 mg/ml insulin, 20 ng/ml human epidermal growth factor, 500 mg/ml hydrocortisone and 100 ng/ml cholera toxin from vibriocholerae and 0.365 mg/ml L/glutamine at 37 °C with 5% CO2. 2.5. Cell proliferation assays Cell proliferation was assessed by the Sulforhodamine B assay, as previously described (Sardao et al., 2009). For this assay different cell lines were seeded in 24-well plates at 104 cells per well. Twenty-four hours after seeding, cells were treated at different concentrations of the extracts (7.5, 15 and 30 lg/ml, selected from preliminary data using one experiment with this technique) in 70% ethanol, which was used as vehicle and added alone in control cells. After 24, 48 and 96 h
of treatment, the medium was removed and wells rinsed with 1% PBS. Cells were then fixed by adding 1% acetic acid in 100% methanol for at least 2 h at 20 °C. Later, the fixation solution was discarded and the plates dried in an oven at 37 °C. Two hundred and fifty microliters of 0.5% SRB in 1% acetic acid solution were added and incubated at 37 °C for 1 h. The wells were then washed with 1% acetic acid in water and dried. Then, 500 ll of Tris, pH 10 was added and the plates were shaken for 30 min. Finally, 200 ll of each supernatant was transferred in 96-well plates and optical density was recorded at 540 nm. 2.6. Assessment of caspase-3 activity Caspase-3 activity was investigated by using a colorimetric method (Sardao et al., 2009) in breast cancer MDA-MB-231 and normal breast MCF-12A cells, which were seeded at 3 105 cells/60 mm plates. 24 h after seeding, cells were treated with increasing extract concentrations (7.5, 15 and 30 lg/ml) and incubated for 24 and 48 h. After incubation, cell growth was stopped and cells collected using collecting medium (2 mM DTT, 100 lM phenylmethylsulfonyl fluoride (PMSF), 20 mM HEPES/NaOH pH 7.5, 250 mM Sucrose, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA) and stored at 80 °C until analysis. Prior to caspase-3 activity assay, protein quantification was made using the Bradford method with BSA 1 mg/ml as standard. For caspase-3-like activity assay, 25 lg of protein were added to different tubes containing assay buffer (25 mM HEPES pH 7.5, 0.1% CHAPS, 10% sucrose and 10 mM DTT) for a total volume of 195 ll. Five microliter of 4 mM substrate (DEVD-pNA) were added to each tube at the final concentration of 100 lM. The blank was prepared without sample and tubes were incubated at 37 °C for 2 h. Tubes were homogenized and 100 ll of each sample transferred into 96 wells plate and optical densities were read at 405 nm. 2.7. Determination of mitochondrial membrane potential on normal and cancer breast cell lines The mitochondrial membrane potential in MCF-12A and MCF-7 cells was investigated by measuring TMRM fluorescence by flow cytometry (Becton Dickinson FACScalibur). MCF-12A and MCF-7 cells were seeded at 3 105 cells/60 mm plates. Twenty-four hours after seeding, cells were treated with 30 lg/ml extract concentrations and incubated for 24 h. After incubation, cells were trypsinized and treated with TMRM (150 nM) for 30 min and then evaluated for mean cell fluorescence by flow cytometry. The uncoupler FCCP (2 lM) was used as a control and was added 15 min after TMRM treatment to promote mitochondrial depolarization. 2.8. Apoptotic DNA condensation assay DNA condensation assay was carried out using DNA labeling with Hoechst 33342. To this respect MDA-MB-231 cells were seeded at 3 105 cells/60 mm plates containing glass coverslips and left to attach for 24 h. Thereafter, cells were treated with different concentrations of extracts or with the vehicle and incubated for 24 h. After incubation, 2 lg/ml Hoechst 33342 was added for 30 min at 37 °C. The medium was then discarded, slides rinsed with 1% PBS mounted using mounting medium and submitted to microscopic analysis using a Zeiss Axioskop 2 Plus fluorescence microscope with a Zeiss AxioCam MRC (Zeiss, Göttingen, Germany). 2.9. Isolation of rat liver mitochondria Rats were sacrificed by cervical dislocation and the livers were removed, minced and rinsed in ice-cold buffer made up of 10 mM HEPES pH 7.4, 250 mM sucrose, 1 mM EGTA and 0.1% fat-free bovine serum albumin (BSA). The free-blood tissue obtained was then homogenized in a Teflon-potter homogenizer and the homogenate obtained was centrifuged at 800g for 10 min at 4 °C. The supernatant obtained was next centrifuged at 10,000g for 10 min at 4 °C and the mitochondrial pellet resuspended using a washing medium (pH 7.2) prepared like the homogenization medium without EGTA and BSA. After isolation, protein contents was determined using the Biuret method (Gornall et al., 1949) using BSA as standard. Mitochondria suspension obtained (approximately 50 mg of proteins/ml) was stored on ice and used within 5 h after 20 min of recovery period. 2.10. Mitochondrial transmembrane electric potential Mitochondria transmembrane electric potential (DW) was estimated indirectly by the mitochondrial accumulation of the lipophilic cation tetraphenylphosphonium (TPP+) detected using a TPP+-selective electrode in combination with an Ag/AgCl-saturated reference electrode. The reference electrode and the TPP+ electrode were inserted into an open vessel (with magnetic stirring) and connected to a pH meter, and signals were registered by a potentiometric recorder. Under constant stirring and at 30 °C, 1 mg mitochondrial protein was suspended in 1 ml of reaction medium (2.5 mM MgCl2, 5 mM HEPES, 5 mM KH2PO4, 65 mM KCl, and 125 mM sucrose pH 7.4 supplemented with 3 lM TPP+) and 10 mM succinate and 3 lM rotenone were added to supply electrons to the mitochondrial respiratory chain via Complex II.
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Table 1 Some of the compound found in the spice extracts used in the present work. No specific flavonoid characterization was performed. The studies were conducted with extraction methods different from the one used in the present work. Spices
Part use in the study
Compound identified
Effects
References
Fagara leprieuri
Fruits
Acridone derivative: 3-hydroxy-1,4-dimethoxy-10-methyl-9-acridone; 3-hydroxy-1,2-dime-thoxy-10-methyl-9-acridone and benzo[c]phenantridine, 10-O-demethyl-12-O-methylarnottianamide
The two first show a moderate effect on cancer cell lines (A549; DLD-1) while the third did not show any effect
Ngoumfo et al. (2010)
Fagara xanthoxyloides
Mondia whitei
Fruits
Roots
Xylopia aethiopica Fruits (Pods)
Phenols (34.59 ± 0.19 g GAE/100 g powder) and flavonoids (4.46 ± 0.40 g/100 g powder) 3,4-O-divanilloylquinic acid, 3,5-O-divanilloylquinic acid and 4,5-O-divanilloylquinic acid isolated from roots extracts Phenols (5.76 ± 0.25 g GAE/100 g powder) and flavonoids (0.44 ± 0.03 g/100 g powder) No particular compound isolated Phenols (6.40 ± 0.11 g GAE/100 g powder) and flavonoids (2.99 ± 0.01 g/100 g powder) ent-15-oxokaur-16-en-19-oic acid Oxoaporphine alkaloids
Abdou et al. (2010) No activity on cancer cells
Abdou et al. (2010)
Abdou et al. (2010) Cytotoxic and DNA damaging Possible DNA topoisomerase inhibitor
Phenols (12.74 ± 0.22 g GAE/100 g powder) and flavonoids (1.94 ± 0.03 g/100 g powder)
When investigating calcium-induced DW depolarization, the maximum mitochondria potential was recorded for 1 min and then calcium (10 ll, 10 mM stock) was added in two sequential additions, with a lag time between both additions to allow for mitochondrial repolarization. The DW was measured 30–45 s after the second calcium pulse in order to estimate the susceptibility of the mitochondrial suspension to calcium in the absence and presence of the test extracts. One micromolar cyclosporin A was used to inhibit the mitochondrial permeability transition and thus prevent calcium-induced mitochondrial depolarization (Broekemeier et al., 1989). The effect of different extracts was obtained by adding different concentrations (1.5, 2, 2.5 and 3 lg/ml, based on preliminary data) to mitochondrial suspensions. Assuming Nernst distribution of the TPP cation across the electrode membrane, absolute values of membrane potential (in millivolts) were determined according to Kamo et al. (1979).
Ouattara et al. (2004)
Choumessi et al. (2012) Harrigan et al. (1994) Abdou et al. (2010)
2.11. Measurement of mitochondrial oxygen consumption Oxygen consumption of isolated liver mitochondria was polarographically monitored with a Clark oxygen electrode connected to an appropriate recorder in a 1 ml thermostated water-jacketed and closed chamber at 30 °C with continuous stirring. The experiment was carried out by successively adding 1 ml of respiratory medium (2.5 mM MgCl2, 5 mM HEPES, 5 mM KH2PO4, 65 mM KCl, and 135 mM sucrose, pH 7.2), 5 mM succinate and 3 lM rotenone. One milligram of mitochondrial protein was added to initiate state 2 respiration, while state 3 respiration was initiated by adding 125 nmol ADP. After complete ATP phosphorylation, state 4 respiration was initiated. A second ADP (125 nmol) pulse followed by oligomycin (1 lg) was added to determine the oligomycin-induced respiratory state, in which an inhibition of passive proton flux through the ATP synthase exists. Finally, FCCP (1 lM)
Fig. 1. Effect of 70% ethanol extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) on MDA-MB-231 cell mass. Data are expressed as percentage of the control (vehicle only) for each time point. Data are means ± SD of five different experiments, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control.
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Fig. 2. Effect of 70% ethanol extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) on MCF-7 cell mass. Data are expressed as percentage of the control (vehicle only) for each time point. Data are means ± SD of five different experiments, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 control.
was added to the system to uncouple respiration. For the test samples, the extracts at different concentrations (1.5, 2, 2.5 and 3 lg/ml) were concurrently added in the chamber and the different respiration states were recorded. The respiratory control ratio (RCR) and ADP/O ratios were calculated as previously described (Chance and Williams, 1956).
2.12. Statistical analysis Data obtained are presented as figures or tables and values are mean ± SD from three to five independent experiments using distinct mitochondrial isolations. Statistical analyses were performed by using One-Way ANOVA followed by Newman– Keuls post-test. Significance was considered at p < 0.05.
Fig. 3. Effect of 70% ethanol extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) on MCF-12 cell mass. Data are expressed as percentage of the control (vehicle only) for each time point. Data are means ± SD of five different experiments, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control.
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Table 2 IC50 obtained for the different extracts in the different cell lines studied. Data was determined from data present in Figs. 1–3. Results are expressed in lg/ml and are means ± SD (n = 5). FL
FX
MW
XA
MDA-MB-231
24 h 48 h 96 h
8.8 ± 5.4 8.6 ± 3.9 6.5 ± 1.1
n.d. 6.7 ± 1.4 6.0 ± 0.9
>30 >30 12.3 ± 4.1
n.d. 5.6 ± 0.2 5.2 ± 0.9
MCF-7
24 h 48 h 96 h
7.2 ± 1.4 6.1 ± 1.4 5.5 ± 0.3
n.d. 6.9 ± 1.8 5.5 ± 0.8
>30 >30 12.5 ± 3.6
6.0 ± 0.6 5.7 ± 1.0 5.2 ± 0.1
MCF-12A
24 h 48 h 96 h
7.3 ± 0.9 6.0 ± 0.4 5.7 ± 0.4
9.9 ± 2.5 7.2 ± 0.5 6.0 ± 0.3
>30 >30 >30
6.4 ± 0.9 5.2 ± 0.1 5.3 ± 0.2
N.d., not determined.
3. Results 3.1. Cytotoxicity of tested spice extracts on MDA-MB-231, MCF-7, MCF-12A cell proliferation The first aim in this work was to verify if the four test extracts presented cytotoxicity against two breast cancer cell lines, MDAMB-231 and MCF-7, as compared with the normal breast cell line MCF-12A. As shown in Fig. 1 (MDA-MB-231 cells), Fig. 2 (MCF-7 cells) and Fig. 3 (MCF-12 cells) all four extracts showed cytotoxic effects for the cell lines tested. Nevertheless, XA, FL and FX extracts appear to be more toxic than the MW extract. All four extracts showed a dose and time-dependent effect in all cell lines investigated, although MW was less toxic in the non-cancer cell line MCF-12. Table 2 represents the inhibitory concentration that resulted in 50% of inhibition of cell proliferation (IC50) at the different time-points. The data reveals that for FL, FX and XA the
IC50 is lower than 20 lg/ml for the cancer cells studied, although a toxic effect was also observed for non-tumor cells. On the other hand, the calculated IC50 value calculated confirms that MW is the least toxic of all extracts tested, with an even lower effect on non-cancer MCF-12.
3.2. Effect of XA, FL, FX and MW on apoptotic caspase-3-like activity and DNA fragmentation Since the SRB assay cannot distinguish between inhibition of cell cycle and induction of cell death, the next step was to investigate if the test extracts induce cell death as part of their mechanism of toxicity. From the three cell lines tested, we selected the breast cancer cell line MBA-MD-231 and normal breast cell line MCF-12A to measure caspase-3-like activity, one hallmark of apoptosis induction.
Fig. 4. Caspase-3-like activity of 70% ethanol extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX), Mondia whitei (MW) and Xylopia aethiopica (XA) on MDA-MB-231 cells, after 24 and 48 h of treatment. Results are express in [pNA] released (nM/lg protein), with pNA used as standard. Data represent means ± SD of three independent different experiments, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control.
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Caspase 3-like activity was determined by using a colorimetric method and extracts were incubated with cells for 24 and 48 h (Figs. 4 and 5). The extracts used induced an increase in caspase 3-like activity in a dose and time-dependent manner in the breast cancer cell line (Fig. 4). The most potent extracts to induce an increase in caspase 3-like activity were FX and FL, which displayed a strong increase already for 24 h in MBA-MD-231 cells. Interestingly, 7.5 lg/ml XA caused an inhibition of caspase 3-like activity, which was statistically significant. This decrease was time dependent but only occurred for that concentration (Fig. 4). In order to have a control for a non-tumor cell line, we performed the same assay on normal breast cells (MCF-12A, Fig. 5). Caspase-3-like activity measured in the normal breast cell line shows a significant increase at both 24 and 48 h when treated with FL and FX extracts for the maximum concentration. FL seems to be more efficient in increasing caspase 3-like activity since it already shows a significant result for 15 lg/ml at 48 h. On the other hand, MCF-12A cells treated with XA did not show any significant alteration on caspase 3-like activity (Fig. 5). An extract from MW was not evaluated since from all four extracts tested for cytotoxicity (SRB assay), it was the one with higher IC50 (Table 2). Furthermore, detection of apoptotic DNA condensation was performed by using the fluorescent probe Hoechst 33342. Several apoptotic nuclei were detected for 24 h of treatment with FL, FX and XA. MW treatment, confirming data from caspase 3-like activity, resulted in a lower number of nuclei showing condensed chromatin when compared with the other extracts (Fig. 6). Doxorubicin was used as an apoptosis positive control to induce chromatin condensation.
end-point was FL, in which no membrane repolarization post-calcium occurred (Fig. 8C). Very mild effects were observed regarding this parameter for MW (Fig. 8C), showing lower toxicity in this regard. Cyclosporin A prevented calcium-induced depolarization (data not shown) in the various experimental groups. To better understand the effect of the extracts on isolated hepatic mitochondria, respiration assays were also carried out (Fig. 9). The results present in Fig. 9 confirm that FL is the most toxic extract, showing a significant dose-dependent effect increasing state 2 (Fig. 9A) and state 4 respiration (Fig. 9C), in a oligomycinindependent manner (Fig. 9D). Also, FL caused a very large decrease in the RCR (Fig. 9F). The effects of FL were observed even for the lowest concentration tested (1.5 lg/ml). The second most toxic extract regarding effects on mitochondrial respiration was FX, which showed an increase in state 2 and in state 4 respiration in the presence of oligomycin. None of the extracts tested had any effect on uncoupled (Fig. 9E) or state 3 respiration (Fig. 9B), with the exception of the highest concentration tested of XA, which caused a small yet statistically significant decrease of uncoupled respiration. Besides FL, all other extracts caused a reduction in the RCR, which was mostly caused by increases in
3.3. Effects of XA, FL and FX extracts on MCF-12A and MCF-7 cells mitochondrial membrane electric potential We used a breast cancer cell (MCF-7) and its direct normal equivalent (MCF-12A) treated with the three more potent extracts for 24 h to investigate alterations of the mitochondrial membrane potential. Interestingly, the results show that the three extracts tested decrease TMRM fluorescence in the normal cell line, with XA being the more potent (Fig. 7, lower panel), whereas no effects were observed for that time point (24 h) in the cancer cell line (Fig. 7, upper panel). The uncoupler FCCP caused mitochondrial depolarization in both cell lines, as expected. 3.4. Direct Effects of XA, FL, FX and MW extracts on bioenergetics and calcium tolerance of isolated hepatic mitochondrial fractions Direct mitochondrial effects of the extracts were investigated on isolated liver mitochondria from Wistar rats. Effects on mitochondrial bioenergetic parameters and calcium susceptibility were investigated in the presence of the different spices. Mitochondrial DW fluctuations were evaluated with a TPP+selective electrode and results presented as percentage of control (no extract added, vehicle only). Although all extracts presented a dose-dependent effect on the maximal DW developed, FL, FX and XA were clearly more potent than MW (Fig. 8A). The test extracts were then assayed in their effects in decreasing mitochondrial ability to accumulate calcium. This end-point was studied by investigating whether calcium-induced depolarization was increased in the presence of the extracts and if recovery occurred after the second calcium pulse. The results (Fig. 8B) show that FX, MW and XA showed similar effects in terms of mitochondrial depolarization induced by calcium. FL, was the most toxic extract, increasing mitochondrial depolarization induced by calcium. The most critical end-point regarded the repolarization DW post-calcium. The toxic effects of the extracts were confirmed, although it is noticeable that the most toxic extract in this
Fig. 5. Caspase-3-like activity of 70% ethanol extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX) and Xylopia aethiopica (XA) on MCF-12A cells, after 24 and 48 h of treatment. Results are express in [pNA] released (nM/lg protein), with pNA used as standard. Data represent means ± SD of three independent different experiments, ⁄ p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control.
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Fig. 6. DNA condensation assay, as measured by nuclear incorporation of Hoechst 33342. Images were collected under an epifluorescence microscope (400 total magnification). MDA-MB-231 cells were treated for 24 h at 7.5, 15 and 30 lg/ml of each extract. Doxorubicin (10 lM) was used as positive control to induce apoptotic chromatic condensation. The white arrows indicate apoptotic nuclei.
state 4 respiration. The stimulation of state 4 respiration with FX, MW and XA was not significant due to the large variability observed, although a tendency was clearly observed. Finally, none of the extracts caused any effect on the ADP/O ratio (Fig. 9G). 4. Discussion Medicinal plants have been used worldwide to treat various illnesses including cancer treatment with several antineoplastics (such as etoposide, paclitaxel and vinblastine) isolated from plants (Mans et al., 2000). Other phytochemicals including berberine (Diogo et al., 2011) and sanguinarine (Serafim et al., 2008) have been tested in several models as potential anti-cancer agents. F. leprieuri (FL), F. xanthoxyloïdes (FX), M. whitei (MW) and X. aethiopica (XA) are four plants used in some African countries as spices in food preparation and in folk medicine against some diseases including cancer (Kuete et al., 2011). In this study, we used two cancer cell lines (MCF-7, MDA-MB-231) and one normal breast cell line (MCF-12A) to evaluate the antiproliferative properties of extracts from FL, FX, MW and XA. We also investigated the direct toxicity of these extracts on isolated hepatic mitochondrial fractions, which are a recognized model to investigate compound toxicity (Pereira et al., 2009). The results regarding antiproliferative activity of the tested extracts on MCF-7, MDA-MB-231 and MCF-12A cells showed that all extracts inhibit cell proliferation, although MW presented less toxicity towards the non-tumor cell line (Figs. 1–3). Our findings are in line with the antiproliferative properties of XA and FL previously reported on some other cancer cell lines (Adaramoye et al., 2011a,b; Kuete et al., 2011). Calculated IC50 (Table 2) values confirmed the results and indicated that FL, FX and XA have cytotoxic activity as the doses needed to decrease cell mass by 50% is lower than 20 lg/ml. The content in phytochemicals may explain the different cytotoxicity observed (Pittella et al., 2009). In fact, the presence of phenols and flavonoids have been described in these spices (Abdou et al., 2010) which may explain antiproliferative activity on cancer cell lines (Karna et al., 2011; Moghaddam et al., 2011). Also, ent-15-oxokaur-16-en-19-oic acid has been described as the main cytotoxic compound of XA (Choumessi et al., 2012). Until now, no through characterization of the flavonoid content in the different extracts has been obtained.
Fig. 7. Mitochondrial transmembrane electric potential, assessed as TMRM relative fluorescence measured by flow cytometry in MCF-7 and MCF-12A cell lines. Both cell lines were incubated with extracts of Fagara leprieuri (FL), Fagara xanthoxyloides (FX) and Xylopia aethiopica (XA) during 24 h. Graphs represent mean ± SD of three independent different experiments, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control. CLT: Control
The results obtained are indicative that the extracts tested are cytotoxic for the cell lines studied, with only MW showing what appears to be a minor selectivity towards a non-tumor cell. Cell toxicity also involved caspase 3-like signaling, suggesting
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apoptosis (Fig. 4). Interestingly, assays done in a non-tumor cell line (MCF-12A) revealed also caspase-3-like activation in a dosedependent manner (Fig. 5), with the exception of the XA extract. This is an interesting observation since this extract caused a large decrease in MCF-12A cell mass (Fig. 3). Both results appears to indicate that this particular extract causes inhibition of MCF-12A cell proliferation through inhibition of cell cycle progression or through cell death involving caspase-independent mechanisms. The involvement of apoptosis in the mechanisms of cytotoxicity of the extracts in another cell line, MDA-MB-231, was confirmed by Hoechst nuclear labeling, demonstrating chromatic condensation that resembles apoptosis (Fig. 6). Although several pathways can trigger caspase-3 activation and apoptosis (Fuchs and Steller, 2011), we decided to focus on mitochondrial involvement. In the first assay aimed an obtaining information regarding this point, we measured mitochondrial membrane electric potential using the fluorimetric probe TMRM in cancer MCF-7 and normal MCF-12A cells. The results were surprising, since the three extracts used (XA, FL and FX) caused mitochondrial depolarization in the normal (MCF-12A) but not in the tumor (MCF-7) cell line, at least for the time point measured. When comparing these results with data from the cell proliferation assays (Figs. 2 and 3), it appears clear that toxicity at the 24 h time point is slightly higher for MCF-12A cells that for their tumor counterparts (Fig. 7). This may be suggestive that the extracts may arrest cell cycle previous to directly affecting mitochondrial bioenergetics, at least for the time point and tumor cell studied. In opposition, mitochondrial depolarization may occur earlier and be a significant event. To confirm possible mitochondrial effects, we investigated extract toxicity directly on isolated hepatic mitochondrial fractions. The results were somewhat unexpected. Interestingly, most of the effects of the different extracts regarded state 2/state 4 respiration (Fig. 9), which translated to a decrease in the RCR.
The most impressive effect was for FL extract, which caused an about 3-fold increase in state 2/state 4 respiration. Minor or no effects were observed in uncoupled respiration, suggesting no inhibitory effects on the respiratory chain, and on the ADP/O value, showing that the efficiency of oxidative phosphorylation was not affected. Interestingly, there were no statistical differences regarding state 3 respiration, again confirming stronger effects in non-phosphorylative respiration. The fact that oligomycin did not inhibit the effects on state 4 respiration (and in some cases even resulted in significant differences) indicate that the extracts are having effects at the mitochondrial membrane level, thus increasing proton influx and increasing respiration (Silva and Oliveira, 2011). The results are in agreement with DW measurements (Fig. 8) in which all extracts, with different degrees of efficacy, decreased the maximal DW generated. The results suggested that the compounds increase proton influx through the inner mitochondrial membrane, contributing to decrease DW and stimulate state 2 and state 4 respiration. Also, from the data obtained, FL was the only extract that showed a higher toxicity. This was particularly visible in the higher DW depolarization induced by calcium (Fig. 8B), with DW completely lost after the second calcium pulse (Fig. 8C). The effects of FL and other extracts, although milder, together with the observed inhibitory effect of cyclosporin A, the specific inhibitor of the mitochondrial transition pore (Broekemeier et al., 1989), suggested that some of the extracts may cause cell death through induction of the MPT (Rasola and Bernardi, 2007). Nevertheless, it is not to exclude that some of the extracts may cause mitochondrial depolarization by MPT-independent mechanisms (e.g., by increasing inner membrane passive permeability to protons), which would translate also in disruption of energy production and cell death. In conclusion, this study demonstrated for the first time direct mitochondrial effects of the tested extracts and supplied some
Fig. 8. Effect of different extracts on mitochondrial membrane potential (DW). (A) Maximal DW generated with succinate in the presence of rotenone as substrate. Two calcium pulses of 100 nmol were sequentially added and calcium-induced depolarization in the second pulse (B) and repolarization post-calcium pulse (C) were evaluated. Values represent mitochondrial DW expressed in% of the control. Data are means ± SD of three independent mitochondrial preparations, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄p < 0.0005 versus control. Values for control: Maximal DW developed: 237.0 ± 0.7 mV, calcium-induced depolarization: 181.0 ± 1.3 mV and mitochondrial membrane repolarization: 228.0 ± 1.1 mV.
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Fig. 9. Effect of extracts on mitochondrial respiration. Respiratory parameters were studied after energizing mitochondria through complex II. (A) State 2 respiration, (B) state 4 respiration, (C) state 3 respiration, (D) oligomycin (state 4 respiration under the presence of oligomycin), (E) uncoupled respiration (FCCP state), (F) ADP/O and (G) respiratory control ratio (RCR). Results are shown as% of control (vehicle only). Data are means ± SD of three distinct mitochondrial isolations, ⁄p < 0.05, ⁄⁄p < 0.005, ⁄⁄⁄ p < 0.0005 versus control. Values for control: state 2: 22.8 ± 3.6 natoms O/min/mg protein, state 4: 20.7 ± 4.6 natoms O/min/mg protein, state 3: 84.7 ± 9.9 natoms O/min/ mg protein, oligomycin-state: 8.0 ± 1.5 natoms O/min/mg protein, FCCP-state: 216.1 ± 16.5 natoms O/min/mg protein, ADP/O: 1.5 ± 0.2 natoms O/min/mg protein and RCR: 5.1 ± 0.9 natoms O/min/mg protein.
clues to their anti-proliferative actions on normal and cancerous breast cells. Further investigations would intend to identify the specific chemical(s) in each spice extract responsible for the observed activities, especially for the strong induction of proton permeabilization in the inner mitochondrial membrane. Although it is very difficult to assess how the concentrations used in this study correlate with plasma levels during human consumption, since no studies are available, equivalent concentrations were used in other previous studies (Adaramoye et al., 2011a,b; Choumessi et al., 2012). The present results are important in the context of basic toxicity assessment of these extracts, warranting caution in their use in high doses as diet additives. Nevertheless, increased mitochondrial proton reflow through the inner mitochondrial membrane may have impact in diseases such as obesity (Grundlingh et al., 2011; Harper et al., 2001).
Author’s contributions A.C., R.L., A.S. and A.M. performed the experiments; A.P., A.T. and V.P. collected and processed the spices used in this work; V.P., A.P., A.T. and P.O. designed the study; A.C., R.L., A.S. and A.M. interpreted the data; P.O. and A.C. wrote the manuscript; all authors revised and approved the contents of the manuscript.
Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements Funding for this work was obtained from the Portuguese Foundation for Science and Technology (FCT), grant PTDC/QUI-QUI/ 101409/2008 (to P.J.O.), co-supported by COMPETE/FEDER and Portuguese National Funds. Aphrodite T. Choumessi was supported by a research fellowship from the ‘Coimbra Group’. The funding agencies had no role in the decision to publish this work or in data analysis and conclusions. We thank Jenna R. Erickson, M.S., Chicago Medical School, North Chicago, IL, USA for proofreading the manuscript for English language. References Abdou, B.A., Njintang, Y.N., Scher, J., Mbofung, C.M.F., 2010. Phenolic compounds and radical scavenging potential of twenty Cameroonian species. Agric. Biol. J. N. Am. 1, 213–224. Adaramoye, O.A., Adedara, I.A., Popoola, B., Farombi, E.O., 2011a. Extract of Xylopia aethiopica (Annonaceae) protects against gamma-radiation induced testicular damage in Wistar rats. J. Basic Clin. Physiol. Pharmacol. 21, 295–313.
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