Free Radical Biology & Medicine 49 (2010) 1916–1924
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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d
Allicin disrupts the cell's electrochemical potential and induces apoptosis in yeast Martin C.H. Gruhlke a, Daniela Portz a,1, Michael Stitz a, Awais Anwar b,2, Thomas Schneider b, Claus Jacob b, Nikolaus L. Schlaich a, Alan J. Slusarenko a,⁎ a b
Department of Plant Physiology (Bio III), RWTH Aachen University, D-52056 Aachen, Germany Division of Bioorganic Chemistry, School of Pharmacy, Saarland University, D-66041 Saarbrücken, Germany
a r t i c l e
i n f o
Article history: Received 3 March 2010 Revised 9 September 2010 Accepted 20 September 2010 Available online 27 September 2010 Keywords: Redox Glutathione Garlic Allium Free radicals
a b s t r a c t The volatile substance allicin gives crushed garlic (Allium sativum) its characteristic odor and is a pro-oxidant that undergoes thiol–disulﬁde exchange reactions with –SH groups in proteins and glutathione. The antimicrobial activity of allicin is suspected to be due to the oxidative inactivation of essential thiol-containing enzymes. We investigated the hypothesis that at threshold inhibitory levels allicin can shunt yeast cells into apoptosis by altering their overall redox status. Yeast cells were treated either with chemically synthesized, pure allicin or with allicin in garlic juice. Allicin-dependent cell oxidation was demonstrated with a redox-sensitive GFP construct and the shift in cellular electrochemical potential (Ehc) from less than −215 to −181 mV was calculated using the Nernst equation after the glutathione/glutathione disulﬁde couple (2GSH/GSSG) in the cell was quantiﬁed. Caspase activation occurred after allicin treatment, and yeast expressing a human antiapoptotic Bcl-XL construct was rendered more resistant to allicin. Also, a yeast apoptosis-inducing factor deletion mutant was more resistant to allicin than wild-type cells. We conclude that allicin in garlic juice can activate apoptosis in yeast cells through its oxidizing properties and that this presents an alternative cell-killing mechanism to the previously proposed speciﬁc oxidative inactivation of essential enzymes. © 2010 Elsevier Inc. All rights reserved.
Garlic (Allium sativum L.) has been prized for centuries as a culinary herb and for its antiseptic and medicinal qualities. Indeed, the earliest written reference to garlic is in the Codex Ebers, an Egyptian papyrus from the 16th century BC, in which 22 of 800 medicinal recipes include garlic [1–3]. The chemistry of the sulfur compounds in garlic and the various reactions that occur when Allium species are wounded are complex . Nevertheless, one can differentiate the effects on the pulmonary system, thought to be largely due to the anticoagulatory effects of the allicin breakdown/follow-on product ajoene, from the biocidal action of the thiosulﬁnate allicin. The major antimicrobial agent in fresh garlic juice was identiﬁed as allicin by Cavallito [4,5], and this has been conﬁrmed subsequently several times [e.g., 6]. It may come as a surprise to many to learn that this common foodstuff has an extremely potent antibiotic potential. For many test organisms allicin's antimicrobial activity is comparable to that of conventional antibiotics such as penicillin, kanamycin, and ampicillin [4,8]. Allicin is active against human pathogens such as Helicobacter pylori and bacteria with multiple antibiotic resistances, e.g., vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus [9,10], and against many plant pathogenic
⁎ Corresponding author. E-mail address: email@example.com (A.J. Slusarenko). 1 Present address: Bayer CropScience AG, Alfred-Nobel-Strasse 50, D-40789 Monheim, Germany. 2 Present address: ECOspray Ltd, Grange Farm, Hilborough, Norfolk IP26 5BT, UK. 0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2010.09.019
bacteria and fungi [8,11–13]. Garlic can yield approximately 100 mg allicin from a 50-g composite bulb . Because allicin is a natural substance and a widespread dietary component, there is potential for its development as a therapeutic both in medicine and in agriculture. Thus, for instance, because of regulatory constraints there is a need in organic farming for fungicides derived from natural products . Notably, approximately 50% of drugs in clinical use are also of natural product origin . Allicin (diallylthiosulﬁnate), which is largely responsible for the odor of freshly crushed garlic, is produced when tissues are damaged and the substrate alliin (S-allyl-l-cysteine sulfoxide) mixes with the enzyme alliin lyase (EC 188.8.131.52; Scheme 1). Allicin has been demonstrated to oxidize and inactivate several essential enzymes in vitro, via a thiol–disulﬁde exchange mechanism, and this has been suggested to be the basis of allicin's biocidal activity in vivo [7,15–17]. With an average calculated logP value of 1.28 ± 0.57 (http://www. vcclab.org), allicin is taken up readily by cells and it has been proposed that this property contributes to its effectiveness as an antimicrobial agent . Allicin is the major sulfur-containing constituent in freshly homogenized aqueous garlic preparations but it can quickly react to form further di-, tri-, tetra-, and polysulﬁdes. For example, diallyldisulﬁde (DADS) is a major product from allicin produced in aged garlic juice preparations . Because allicin is a readily membrane-permeative pro-oxidant we reasoned that it is likely to have pronounced effects on the redox status of cells as a whole, in addition to its effect on speciﬁc thiol-containing
M.C.H. Gruhlke et al. / Free Radical Biology & Medicine 49 (2010) 1916–1924
+ 2pyruvate + 2NH3
allicin Scheme 1.
proteins and enzymes. The redox environment of the cell is linked to its biological status and several redox-regulated “nano switches” are switched on or off as a cell moves from a normal, highly reducing, to a more oxidizing state [19–21]. As these switches are activated or deactivated cellular activities go from proliferation through differentiation to apoptosis and ﬁnally, if the degree of oxidation is too great for the coordinated metabolic competence necessary for apoptosis, the cells necrose [19,20]. Because the redox status of the cell has such important consequences for the regulation of cellular activities, it must be carefully controlled. Quantitatively, the most important redox buffer in the cell is the glutathione/glutathione disulﬁde couple (2GSH/GSSG) and changes in the redox status of the cell are indicated by changes in the 2GSH/GSSG ratio [19,20]. However, to know the absolute redox potential in terms of the overall half-cell potential (Ehc) in millivolts, it is necessary to know the concentrations of oxidized and reduced glutathione within the cell and not simply their relative proportions. In a fully reduced state the standard redox potential of the GSH/GSSG couple is less than −240 mV, and if the absolute concentrations of GSH and GSSG in the cell are known, the Nernst equation can be used to calculate the actual Ehc of the cell. A convenient way to monitor cellular redox changes in real time is with cells transformed with a redox-sensitive green ﬂuorescent protein construct (roGFP) [22–25]. Apoptosis is a form of programmed cell death, which can be mediated by the activation of cysteine proteases called caspases  or via the apoptosis-inducing factor (AIF) in caspase-independent pathways . Although originally viewed as a characteristic of multicellular organisms it is now clear that apoptosis occurs in yeast, which has become a model organism for apoptosis research [28–31]. Apoptosis can, among other stimuli, be induced by oxidative stress, e.g., by exposure to H2O2 [32–34]. It was recently reported that allicin was able to induce apoptosis in murine and human tumor cell lines  and the allicin breakdown product DADS was reported to deplete GSH and kill Candida albicans cells via an oxidative mechanism [36,37]. For human epithelial carcinoma cells it was shown that allicin is able to induce apoptosis in a caspase-independent manner. In this pathway, protein kinase A plays a crucial role . In mammalian cells, proteins of the Bcl2 family exert either positive or negative controls on apoptosis. Interestingly, although Saccharomyces cerevisiae has no Bcl2 homologue , these proteins are active in yeast cells [40–44]. In animals, the AIF, a ﬂavoprotein, is a central regulatory factor of caspaseindependent apoptosis and serves as a positive intrinsic regulator of cell death that translocates from the cytosol to the nucleus upon exposure to an apoptosis-inducing agent and leads to the condensation of chromatin and DNA fragmentation [27,45]. An AIF orthologue has been described for yeast (Ynr074cp), with comparable functions in nuclear degradation and DNA fragmentation although the apoptogenic action of Ynr074cp is partially caspase dependent . In the work reported here we used the model organism S. cerevisiae (baker's yeast) to test the hypothesis that allicin in garlic juice can kill cells by acting as a redox toxin, increasing Ehc and pushing cells into apoptosis. We reasoned that were this to be the case we should be able to observe an allicin-dependent redox shift, an activation of the yeast apoptotic machinery, and protection of cells against allicin by antiapoptotic proteins. Furthermore, to our knowledge, this is the ﬁrst report of allicin-induced apoptosis in a fungus.
Materials and methods Garlic juice extraction Garlic bulbs were purchased from the supermarket and stored at 4 °C in the dark until required. Axillary buds from the composite garlic bulb were peeled and weighed and a domestic juicer (Turmix Fabr. No. 1068; Turmix AG, Jona, Switzerland) was used to extract the juice. The juice was poured into a sterile 50-ml Falcon tube and centrifuged at 5000 rpm (3000 g) for 10 min to separate the majority of the pulp from the liquid (Megafuge 1.0R; Heraeus Instruments, Osterode, Germany). Floating debris was scooped off the top of the liquid with a spatula and discarded. Filtering under pressure separated the remaining pulp from the pure extract (diaphragm vacuum pump; Vacuubrand GmbH, Wertheim, Germany). The ﬁltrate was transferred into a second sterile 50-ml Falcon tube and sealed. The average yield was approximately 1 ml of extract from 3 g fresh weight of garlic tissue and typically contained approximately 5 mg ml− 1 allicin (determined by HPLC). The garlic extract was used immediately after appropriate dilution. Dilutions were carried out with deionized water. Synthesis of allicin Allicin was obtained by oxidation of diallyldisulﬁde with H2O2 according to the method of Lawson and Wang , which was improved to increase purity and yield. Diallyldisulﬁde, sodium hydrogen carbonate, MgSO4, and H2O2 were purchased from Sigma–Aldrich (Munich, Germany). Diallyldisulﬁde was distilled at 1 mbar under vacuum before use. Deionized water (Millipore; 18.2 MΩ cm− 1) was used unless stated otherwise. Two grams (2 g=13 mmol) of freshly distilled diallyldisulﬁde (about 99% pure) was dissolved in 5 ml of cold (4 °C) glacial acetic acid, to which 3 ml of cold 30% hydrogen peroxide was added slowly. After 30 min, the temperature was allowed to increase to room temperature and stirring continued for 2 h. The reaction was stopped with addition of 25 ml of water and was extracted with 30 ml of dichloromethane. Acetic acid was removed by washing the extract several times with 5% NaHCO3 and then washing with water to pH 6–7. Solvent was evaporated in vacuo and the yellow oil obtained was redissolved in 200 ml of water. Unreacted DADS was removed by double extraction with 0.1 vol of hexane and allicin was extracted with dichloromethane again, dried over MgSO4, and concentrated in vacuo. It was puriﬁed further using silica gel chromatography petrol ether (40–65 °C):ethyl acetate (95:5). Allicin was characterized using 1 H NMR (500 MHz, CDCl3, δ) 3.72– 3.89 (m, 4 H, CH2), 5.16–5.46 (m, 4 H, CH2=), 5.71–5.92 (m, 2 H, – CH=); 13 C NMR (125 MHz, CDCl3, δ) 34.80, 56.32, 116.65, 122.65, 124.87, 129.50. The data are in agreement with reported values for allicin . Purity was further conﬁrmed using HPLC. Determination of allicin in garlic juice by HPLC The method used was based on that of Krest and Keusgen . Garlic juice was diluted 1:10 with HPLC-grade water and 1.5 ml of a 0.05 mg ml− 1 solution (in methanol) of butyl-4-hydroxybenzoate (internal standard). To protect the column this mixture was ﬁrst ﬁltered
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through a polyethersulfon membrane (0.2-μm pore size, Steriﬂip; Millipore) before 20 μl was injected into the HPLC (Kontron system with diode array detector; Kontron Instruments GmbH, Neufahrn, Germany). Using the HPLC software Geminyx (version 1.91) a mixed-gradient elution (solvent A, 30% (v/v) HPLC grade methanol adjusted to pH 2.0 with 85% (v/v) orthophosphoric acid; solvent B, 100% HPLC grade methanol) was carried out. During elution spectra were recorded between 200 and 600 nm, and for the chromatogram detection was at 254 nm. Yeast strains and cultivation Yeast strains RS453 (MATa/MATα,ade2/ade2,his2/his3,leu2/leu2,trp1/ trp1,ura3/ura3 ) and SS330 (MATa,ade2-101,ura3-52,hisΔ200,tyr1 ) were grown in shake culture in YPD (10 g yeast extract, 20 g tryptone, 20 g glucose L− 1) at 28 °C and 210 rpm. The concentration of allicin used was titrated for each yeast strain such that the rate of growth measured as increase in A600 was approximately 50% that of untreated controls. Both strains were differentially sensitive to allicin and were used in the caspase activation assay. Only RS453 was used in the GSH/GSSG glutathione reductase (GR) assay. BY4742 (Matα,his3Δ1,leu2Δ0,lys2Δ0,ura3Δ0 ) was transformed with roGFP and Bcl-XL constructs. YNR074c (Δaif) in the BY4742 background was obtained from Euroscarf (University of Frankfurt, Germany). The working concentrations of allicin for the various yeast strains were determined empirically such that growth rate of the cultures was reduced to approx 50% of untreated controls. For RS453 (GSH/GSSG determination, FLICA assay) garlic juice was added to give a ﬁnal concentration of 200 μg ml− 1 (1.3 mM) allicin, and for SS330 (FLICA assay) 50 μg ml− 1 (0.33 mM) allicin. For the BY4742 (roGFP2 and Bcl-XL expression, deletion strain Δaif) cell survival experiments a range of allicin concentrations (0.13, 0.18, and 0.25 mM, equal to 20, 30, and 40 μg ml− 1 allicin, respectively) were tested. Plasmids and constructs The roGFP2  insert was released by EcoRI and ClaI digestion from a construct in pBluescript containing human GRX1, a linker, and roGFP and subcloned into the yeast expression vector pRS413 (ADH1 promoter and URA3 selection) . Bcl-XL and Bcl-XLΔC constructs were used as described in . Both Bcl-XL and Bcl-XLΔC were controlled by the GAL1 promoter. Transformed BY4742 yeast cells were selectively cultivated on SD medium: 7 g L− 1 yeast nitrogen base with (NH4)2SO4 (Formedium Ltd., Norwich, UK), 40 g L− 1 glucose, 0.8 g L− 1 CSM (−Ura) supplement (Bio101, Vista, CA, USA); for solid medium 20 g L− 1 agar was added. Fluorescence microscopy of roGFP-expressing yeast cells After 48 h cultivation at 28 °C on SD medium, roGFP2-expressing cells were scraped from the petri plate with an inoculating loop and resuspended in 50 μl PBS (0.2 g L− 1 KCl, 0.2 g L− 1 KH2PO4, 1.5 g L− 1 Na2HPO4, 8 g L− 1 NaCl, pH 7.4) containing allicin from garlic juice in an appropriate concentration. Droplets (10 μl) were placed on a slide and examined with a ﬂuorescence microscope (DM RBE; Leica GmbH, Wetzlar, Germany), under excitation at 480 nm and ﬂuorescence detection from 505 nm. Results were documented with a digital camera (KY-F75U; JVC Germany GmbH, Friedberg, Germany).
nitro-5-thiobenzoic acid formation was monitored at 412 nm and the total glutathione present was read from a standard curve prepared using GSH (AppliChem). In any given sample the assay can be rendered to reﬂect only GSSG levels by utilizing 2-vinylpyridine to remove GSH from the GSSG/2GSH couple . The system was calibrated using standard curves prepared with GSH and GSSG purchased from AppliChem and GR from Sigma (E 3664). Under the test conditions used the detection limit for GSH was 1.25 μg ml− 1. Synthetic allicin was added to an actively growing culture of RS453 (OD600 0.4) to 100 or 200 μg ml− 1. After thorough mixing, cells were collected immediately from four separate 50-ml culture aliquots by centrifugation for 10 min at 25 °C and 4330 g. The packed cell volumes were measured and the cells washed twice by centrifugation in (143 mM) phosphate buffer (pH 7.5) containing 6.3 mM EDTA. The pellets were resuspended in 1 ml buffer and approximately 200 μl 0.2-mm-diameter glass beads was added. Cells were broken open by vortexing for 3×1 min, cooled on ice, and centrifuged at 15,800 g for 1 min. Aliquots of the supernatant were then taken to determine the GSH and GSSG contents as described above . The reaction mixture contained 12.5 μl supernatant, 5 μl GR (20 units ml− 1), 50 μl 6 mM DTNB, and 350 μl 0.3 mM NADPH, all in phosphate buffer; water was added to give a ﬁnal volume of 750 μl (ﬁnal phosphate buffer 80 mM, 3.5 mM EDTA). The cell content of GSH/ GSSG was calculated by taking the total quantity in the supernatant and calculating this as a concentration in the packed cell volume assuming that the cell walls occupied 15% and that the cells had either no vacuole or a vacuole occupying 20% of the cell volume , giving minimum and maximum GSH/GSSG concentrations, respectively. Cellular concentrations of GSH and GSSG were then substituted in the Nernst equation as described by [19,20] to calculate the Ehc of the GSH/GSSG redox couple. An analysis of variance was carried out on the results and signiﬁcance determined with the all pairwise multiple comparison procedure (Holm– Sidak method) at Pb 0.05. Cellular apoptosis assay and confocal laser microscopy Cells undergoing apoptosis were detected using an SR-FLICA Poly Caspase Detection Kit (Immunochemistry Technologies, Bloomington, MN, USA) according to the manufacturer's instructions. The SR-FLICA reagent (sulforhodamine-ﬂuorescence-labeled inhibitor of caspases) binds covalently only to activated caspases and is removed by washing from nonapoptotic cells. Apoptotic cells containing activated caspases retain the reagent on washing and ﬂuoresce red after excitation. Garlic juice, or chemically synthesized pure allicin, was added to 50 ml of an actively growing culture (OD600 0.4–0.6) of either RS453 or SS330 to give an allicin concentration of 200 or 50 μg ml− 1, respectively. Cultures were shaken further overnight and then diluted with YPD medium to OD600 0.4–0.6 and the cells from 300-μl aliquots were collected by centrifugation (5 min, 11,600 g, room temperature). Pellets were washed twice with YPD and resuspended in 300 μl of fresh medium to which 10 μl of SR-FLICA working solution was added and further incubated for 1.5 h at 28 °C. Cells were collected by centrifugation and washed twice with SRFLICA assay buffer and ﬁnally resuspended in 200 μl of assay buffer. Positive control cells were treated with 4 mM H2O2 to induce apoptosis. Cells were viewed at 630× magniﬁcation with a confocal laser scanning microscope (Leica DM R) under excitation at 488 and 568 nm and ﬂuorescence detection from 590 to 610 nm. Cells were photographed using a JVC digital camera (KY-F75U) and Discus software (version 32; Hilgers Co., Königswinter, Germany). Transformation procedure for roGFP2 and Bcl-XL
Glutathione determination Glutathione levels were determined using a standard enzymatic recycling assay based on GR (, modiﬁed by ). GSH was sequentially oxidized to GSSG by 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB; Merck) and reduced by NADPH (AppliChem) in the presence of GR. The rate of 2-
S. cerevisiae was transformed by electroporation as described in  using a Bio-Rad (Hercules, CA, USA) micropulser at “Sc” settings. Transformants were plated on solid SD-Ura medium containing 1 M sorbitol. The petri plates were incubated at 28 °C for up to 5 days until colonies were visible.
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Bcl-XL- and Bcl-XLΔC-expressing strains were plated out to determine the number of colony-forming units (cfu). To determine the percentage of surviving cells, the number of cfu from each treatment was divided by the cfu from an untreated control. Results Allicin treatment affects the redox state of roGFP2 in yeast cells To test the oxidizing properties of allicin in vivo, roGFP2 was used as a qualitative marker for the redox state of the cytoplasm. Cells were treated with allicin in garlic juice at a ﬁnal concentration of 50 μg ml− 1 and roGFP ﬂuorescence was compared to that of untreated control cells. The ﬂuorescence of the control was greater than in the allicin-treated cells, indicating a shift in cellular redox to a more oxidizing state (Fig. 1). Cells oxidized by treatment with H2O2 (positive control) similarly showed a decrease in the intensity of ﬂuorescence. Although the reduction in roGFP ﬂuorescence conﬁrmed an allicin-dependent cellular redox shift, it is not possible from this procedure (in yeast) to estimate the absolute redox potential changes because of interfering autoﬂuorescence at the extra excitation wavelengths necessary. To ﬁnd the absolute Ehc it is necessary to know the concentrations of GSH and GSSG in the cell. Glutathione determination
Fig. 1. Effect of garlic juice treatment on BY4742 yeast cells expressing cytosol-targeted roGFP2. (A) untreated control cells and (B) cells treated with allicin in garlic juice adjusted to a ﬁnal concentration of 50 μg ml− 1 (0.33 mM). Reduced ﬂuorescence indicates a more oxidized state.
Quantiﬁcation of cell survival of Bcl-XL- and Bcl-XLΔC-expressing and Δaif cells Bcl-XL, Bcl-XLΔC, and Δaif cells were grown over night in YPD medium in shake cultures as described above. The cells were harvested by centrifugation at 2500 g (Beckman Megafuge 1 OR) and washed twice with sterile distilled water. The cells were resuspended in YPGal medium and grown at 28 °C at 210 rpm for 8 h to induce the expression of Bcl-XL and Bcl-XLΔC. Garlic juice (allicin concentration 20, 30, and 40 μg ml− 1) or 1 mM H2O2 ﬁnal concentration as positive control was added and the cells were incubated for 16 h.
Using a standard glutathione reductase enzymatic recycling assay, in the two experiments reported here untreated RS453 cells were determined to have a glutathione pool of 1.8–2.3 and 1.4–1.6 mM, respectively. Maximum and minimum values in each experiment were calculated by assuming either that a vacuole was absent from the cells or that a vacuole occupied 20% of the protoplast volume (Table 1). In actively growing, untreated cells N99% was present as GSH, indicating, as expected, that the cells were in a highly reduced state with a calculated electrochemical potential of less than −215 mV. When synthetic allicin was added to cultures of RS453 cells to give a ﬁnal concentration of 100 or 200 μg ml− 1 a small, statistically nonsigniﬁcant (Pb 0.05), reduction in the absolute concentration of glutathione in the cells, to 0.28–0.38 mM, was observed (Table 1). Furthermore, after treatment with 100 μg ml− 1 allicin ~3.2–3.4%, and after 200 μg ml− 1 allicin ~9.3–9.6%, of the total glutathione pool was present as GSSG, showing that the cells had been pushed into a more oxidized state by allicin treatment. Using the Nernst equation to calculate the redox potential of the GSSG/ GSH couple it seems that after treatment with synthetic allicin, yeast cells underwent a transition from a highly reducing (Ehc b −215 mV) to a more oxidizing state (Ehc =−181 to −200 mV) (Table 1). Whether this degree of change in the electrochemical half-cell potential could be associated with the induction of apoptosis was investigated by cellular and genetic tests.
Table 1 Effects of treating RS453 yeast cells with synthetic allicin on the glutathione pool and cellular electrochemical potential Expt I
Total cellular conc. of glutathione* GSH GSSG Calculated Ehc Theoretical cellular status (after Schafer and Buettner [19,20])
1.84a ± 0.26 to 2.30a ± 0.33 mM 99.6–99.8% 0.2–0.4% b − 215c mV Proliferation
1.19b ± 0.14 to 1.49ab ± 0.17 mM 96.6–96.8% 3.2–3.4% − 196d to − 200d mV Transition from proliferation to apoptosis
1.35ab ± 0.27 to 1.59ab ± 0.29 mM 99.1–99.2% 0.8–0.9% b − 215c mV Proliferation
1.18b ± 0.12 to 1.50b ± 0.16 mM 90.4–90.7% 9.3–9.6% − 181d to − 184d mV Border of apoptosis
In experiment I cells were treated with 100 μg ml− 1 (0.65 mM) synthetic allicin and in experiment II 200 μg ml− 1 (1.3 mM) synthetic allicin was used. *Minimum and maximum values assuming that cells lack a vacuole or assuming 20% of the cell volume is vacuole, respectively. Mean of four replicates. The differences between absolute amounts of GSH in allicin-treated and untreated cells were not signiﬁcant (P b 0.05). Values with different letters are signiﬁcantly different (P b 0.05). Maximal resolution of the enzymatic test is 0.03 mM.
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Apoptosis assay (caspase activation) with garlic juice and pure allicin Yeast strains SS330 and RS453 were incubated with H2O2, garlic juice, or synthetic allicin and then treated with the FLICA reagent, which binds to active caspases. Washing the cells removes unbound reagent and nonapoptotic cells do not show red ﬂuorescence (Fig. 2A). In the positive control as recommended by the manufacturer, H2O2 treatment activates caspases, which was observed as a red ﬂuorescence (Fig. 2B). As shown in Figs. 2C–F, the oxidizing treatments with either pure allicin or allicin in garlic juice caused caspase activation in both yeast strains. We reasoned that if the allicin-treated cells were indeed dying apoptotically, then it should be possible to protect them against allicin with antiapoptotic proteins such as the Bcl2 family member Bcl-XL. Bcl-XL reduces allicin-induced cell death A typical dilution series plate test to measure cell survival rates is shown in Fig. 3. In this example an 8-h culture of wild-type BY4742 cells
was adjusted to 0.18 mM chemically synthesized allicin and grown on for a further 16 h before aliquots of a 100-fold dilution series were plated out. The allicin-treated culture shows a survival rate of 64% compared to the untreated control. To test whether the antiapoptotic Bcl-XL protein can inhibit allicininduced cell death, yeast cells transformed with a human Bcl-XL gene under the control of a galactose-inducible promoter were incubated for 16 h with various allicin concentrations. A yeast strain expressing a Cterminal-deleted, nonfunctional variant of Bcl-XL (ΔC) was used for comparison. In the case of the positive control (treatment with 1 mM H2O2) the survival rate of cells expressing a functional Bcl-XL gene was signiﬁcantly higher compared with the control (Fig. 4), conﬁrming that H2O2-treated cells were dying apoptotically. As expected, allicin, either pure or in garlic juice, caused a concentration-dependent decrease in cell survival in the control cells containing the Bcl-XLΔC variant. In contrast, the Bcl-XL-expressing strain was more resistant to allicin than the ΔC control. The tendency for increased survival of cells expressing a functional Bcl-XL protein can be seen for both synthetic allicin and allicin
Fig. 2. (A–D) SS330 cells and (E and F) RS453 cells were treated as described individually below and then treated with the FLICA reagent, which binds to activated caspases. (A) Control, (B) 4 mM H2O2, (C) garlic juice containing 50 μg ml− 1 (0.33 mM) allicin, (D) 50 μg ml− 1 synthetic allicin (0.33 mM), (E) 4 mM H2O2, (F) garlic juice containing 200 μg ml− 1 (1.3 mM) allicin. The FLICA reagent ﬂuoresces red after excitation at 488 and 568 nm.
M.C.H. Gruhlke et al. / Free Radical Biology & Medicine 49 (2010) 1916–1924
Fig. 3. Cell survival assay. Colonies are from plating out 10 μl of undiluted or 102-, 104-, and 106-fold dilutions of BY4742 (wild-type) cell culture treated with 0.18 mM (30 μg ml− 1) chemically synthesized allicin and growing for a further 16 h. The survival rate is calculated as the percentage of colonies in the treatment compared to the untreated control.
in garlic juice at all concentrations tested and was signiﬁcant (Pb 0.05) at 0.18 and 0.25 mM dosages (Fig. 4). This result conﬁrms that allicin is indeed inducing apoptotic cell death in yeast. As a further genetic test of this hypothesis, we investigated the role of the proapoptotic yeast AIF orthologue in allicin-induced cell death. Apoptosis inducing factor We reasoned that if allicin is killing cells apoptotically, then cells with a functional aif gene might be more sensitive to allicin than cells with an aif deletion. This would also implicate a caspase-independent apoptotic pathway in addition to the caspase-dependent mechanism already shown (Fig. 2). To test whether the proapoptotic yeast AIF orthologue enhances allicin-induced cell death, wild-type yeast or a Δaif mutant were incubated for 16 h with various allicin concentrations. As expected the survival rate of wild-type cells, containing active AIF, decreased in a concentration-dependent manner (Fig. 5). However, a mutant strain carrying an aif deletion showed a signiﬁcantly higher survival rate after oxidizing treatments with 1 mM H2O2 (positive control) or allicin, either as a pure substance or in garlic juice. This result further implicates apoptosis as the cytotoxic mechanism of allicin action in yeast. Discussion Garlic has many uses in folklore and medicine and here we explored the microbial killing mechanism associated with garlic juice
in relation to the oxidizing nature of the allicin it contains. The major antimicrobial substance in garlic extracts is allicin; however, the chemistry of sulfur compounds in garlic juice is complex and it is essential to compare the biological activity of garlic juice, analyzed for its allicin content, with the biological activity of the pure, chemically synthesized substance. At threshold concentrations, which reduced the population growth rate of the yeast culture by 50%, allicin caused a shift in the cellular redox potential, which was demonstrated by the reduction in ﬂuorescence of roGFP-containing yeast cells (Fig. 1). This result shows that allicin in diluted garlic juice, in this case at a concentration of 0.33 mM, in addition to potentially oxidizing speciﬁc –SH groups in proteins, affects the general redox environment within the cell. Unfortunately, it is not possible in yeast to estimate the absolute redox potential changes from roGFP ﬂuorescence because of interfering autoﬂuorescence at the extra excitation wavelengths needed for quantitative work . However, the shift in cellular redox potential we observed presents a potentially more general mechanism for allicin activity than has often been proposed [e.g., 7,15–17]. The redox environment of the cell controls many cellular processes and it has been proposed that cell metabolism is under the control of several redox-sensitive nano switches [19,20]. These nano switches can be considered redox-regulated signal pathways. The cellular redox potential depends upon the absolute GSH and GSSG concentrations and not just on their ratio [19,20]. Thus, to gain an estimate of the allicininduced cellular redox shift, we related the amount of extractable GSH/ GSSG to the packed cell volume of the yeast cells to calculate absolute cellular glutathione concentrations. Subtracting 15% for the estimated cell wall volume gave minimum cytosolic concentration estimates and subtracting a further 20% for a hypothetical vacuole volume gave estimated maximum concentrations. Using the Nernst equation to calculate cellular redox potentials in treated and untreated cells (Table 1) revealed that 100 or 200 μg ml− 1 allicin shifted the cellular redox state from less than −215 mV in a dose-dependent manner to greater than −200 and greater than −184 mV, respectively. The small, statistically not signiﬁcant, reduction in total [GSH] in the allicin-treated cells probably reﬂects the direct reaction of a proportion of the GSH with allicin to S-allylmercaptoglutathione , thus preventing its participation in the GR-cycling reaction . Changes in the electrochemical half-cell potential of the GSH/GSSG couple correlate with the biological status of the cell and in this range it is predicted that the cells are pushed to the border at which apoptosis is triggered [19,20]. However, as pointed out by
Fig. 4. Effect of antiapoptotic Bcl-XL protein on allicin-induced BY4742 yeast cell death. Expression of Bcl-XL and Bcl-XLΔC was controlled by a galactose-inducible promoter. (A) BclXL-expressing cells were signiﬁcantly more resistant to H2O2 than the control, a yeast strain expressing a C-terminal-deleted Bcl-XL gene (ΔC). (B, C, and D) Survival rates of BclXLΔC (ΔC)and Bcl-XL cells after treatment with 0.13, 0.18, and 0.25 mM allicin in garlic juice (G) or chemically synthesized pure allicin (A). The tendency for increased survival of cells expressing a functional Bcl-XL protein can be seen for both synthetic allicin and allicin in garlic juice at all concentrations tested. However, the increased survival rate was not signiﬁcant at the lowest concentration (0.13 mM) tested (one-way ANOVA). Within each treatment group bars with different letters indicate statistically signiﬁcant differences (P b 0.05) and the bars show the standard deviation.
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Fig. 5. Effect of deletion (Δaif) of functional proapoptotic AIF on allicin-induced BY4742 yeast cell death. (A) Δaif cells were signiﬁcantly more resistant to H2O2 than the wild-type (WT) cells with a functional aif gene. (B, C, and D) Survival rates of Δaif and WT cells after treatment with 0.13, 0.18, and 0.25 mM allicin in garlic juice (G) or chemically synthesized pure allicin (A). The signiﬁcantly increased survival of Δaif cells not expressing a functional AIF protein compared to WT cells can be seen for both synthetic allicin and allicin in garlic juice at all concentrations tested (one-way ANOVA, P b 0.05). Within each treatment group bars with different letters indicate statistically signiﬁcant differences (P b 0.05) and the bars show the standard deviation.
Hancock et al. , although by using the Nernst equation in this way, an overall, average cellular redox value can be calculated, it should be remembered that at different sites within the cell different potentials will be kept commensurate with local function. Nevertheless, the extremely rapid allicin-induced change in Ehc has not been reported before and suggests a new paradigm for the cytotoxic action of allicin. To test whether apoptosis was indeed being induced by allicin treatment, a cellular assay for activated caspases was employed and showed caspase activation in allicin-treated cells as well as in H2O2treated positive controls (Fig. 2). These results are consistent with reports that allicin can induce apoptosis in various tumor cell lines in which cells have reduced redox protection in the transformed state [35,38]. Hirsch et al. , using human mammary, endometrial, and colon cancer cell lines, reported that allicin in garlic juice was the active component that suppressed cell proliferation. The authors reported that allicin caused a transient decrease in GSH levels but that growth inhibition was accompanied by an accumulation of cells in the G0/G1 and G2/M phases of the cell cycle rather than a signiﬁcant increase in cell death. However, a later report suggested that allicin inhibited murine and human cancer cell growth by inducing apoptosis . If yeast cells were indeed being pushed into apoptosis by the oxidative action of allicin, we reasoned that it should be possible to protect them against allicin with antiapoptotic proteins. To test this hypothesis we transformed yeast with a clone for human Bcl-XL, a member of the Bcl2 family, or with an inactive Bcl-XL deletion construct. The Bcl-XL protein prevents the mitochondrial permeability transition leading to cytochrome c release and caspase activation, thus protecting cells against caspase-dependent cell death. As can be seen in Fig. 4, cells expressing an active Bcl-XL gene were more resistant to allicin treatment. This result supports the hypothesis that allicin-induced redox shifts can kill cells via a caspase-dependent apoptotic mechanism. The proapoptotic AIF was characterized in humans as promoting caspase-independent apoptosis . Yeast cells contain a single AIF orthologue of the human AIF gene . Oxidative stress can also lead to apoptotic cell death via caspase-independent AIF routes. We tested whether a yeast aif mutant was more resistant to allicin treatment than wild-type cells and found that this was indeed the case (Fig. 5). This result, in conjunction with the Bcl-XL experiments, suggests that allicin induces apoptosis in yeast via both caspase-independent and caspase-dependent routes.
We conclude that at threshold concentrations allicin in garlic juice kills yeast cells by acting as a redox toxin, which shunts cells into genetically programmed apoptosis. At higher dosages it would be expected that cells might become so oxidized that they are no longer metabolically competent to execute apoptosis and instead necrose [19,20]. In prokaryotes, which do not undergo apoptosis, it might be predicted that cell oxidation leading to necrosis is the mechanism of cell killing by allicin. It may be that allicin, which has a very broad range of antimicrobial activity, acting not only against fungi but also against oomycetes , also induces apoptotic cell death in these organisms, as it has been reported to do in several tumor cell lines . To our knowledge this is the ﬁrst documentation of allicininduced apoptosis in a fungus. The “standard” hypothesis for allicin toxicity was based on direct reaction of allicin with –SH groups in essential enzymes. A criticism of this mechanism was always that allicin would be buffered out by the GSH pool, which is there to protect such redox-sensitive molecules from oxidation . In the work reported here we reveal an extra facet to allicin's toxic action, i.e., the global disruption of cellular redox potential, precisely because it reacts with GSH. When the electrochemical cell potential rises from a highly reduced state to approx − 160 mV, this will have wide sweeping effects on all redox-sensitive proteins in the cell (see, e.g., the roGFP result shown in Fig. 1). Thus, our data support a “paradigm shift” in the mechanism of action of allicin and, in our opinion, explain much about allicin's toxic behavior. Furthermore, global redox perturbation may well be linked to the toxic action of many other thiol-selective reagents. Our model also conforms to the notion that allicin does not need to survive in the cell as allicin to be toxic. Indeed, in this model the removal of allicin by its reaction with GSH is a prerequisite for the toxic effects, which follow as a consequence. The model predicts that differential cell survival during allicin treatment would be related to the efﬁciency of redox protection/buffering in the cell. Testing this hypothesis is part of our continuing investigations. Acknowledgments The construct encoding the human GRX1, a linker, and roGFP2 in pBluescript was a kind gift from Dr. Tobias Dick (German Cancer Research Center, Heidelberg, Germany). Bcl-XL and Bcl-XLΔC were a kind gift from Dr. Stephen Manon (CNRS/Université de Bordeaux,
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France), and the SS330 yeast strain was from Dr. Ludwig Lehle (University of Regensburg, Germany). Financial support from the RWTH Aachen (A.J.S., D.P., M.G., M.S., N.S.), the University of Saarland (A.A., C.J.) is gratefully acknowledged. The research leading to these results has received funding from the [European Community's] Seventh Framework Programme [FP7/2007-2013] under grant agreement No. . Ulrike Noll is thanked for technical assistance and help with the preparation of the manuscript. References  Block, E. The chemistry of garlic and onions. Sci. Am. 252:94–99; 1985.  Block, E. The organosulfur chemistry of the genus Allium—implications for the organic chemistry of sulfur. Angew. Chem. Int. Ed Engl. 31:1135–1178; 1992.  Block, E. Garlic and Other Alliums: the Lore and the Science. Royal Chem. Soc, Cambridge, UK; 2010.  Cavallito, C. J.; Bailey, H. J. Allicin, the antibacterial principle of Allium sativum. I. Isolation, physical properties and antibacterial action. J. Am. 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Published on Sep 29, 2011
OriginalContribution ⁎ Correspondingauthor. E-mailaddress:firstname.lastname@example.org(A.J.Slusarenko). 1 Present address: Bayer Crop...