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Effects of curcumin and curcumin derivatives on mitochondrial permeability transition pore
Free Radical Biology & Medicine, Vol. 36, No. 7, pp. 919 – 929, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter
doi:10.1016/j.freeradbiomed.2003.12.018
Original Contribution EFFECTS OF CURCUMIN AND CURCUMIN DERIVATIVES ON MITOCHONDRIAL PERMEABILITY TRANSITION PORE HEIDI LIGERET,* SOPHIE BARTHELEMY, y ROLAND ZINI,* JEAN-PAUL TILLEMENT,* SERGE LABIDALLE, y and DIDIER MORIN *,z * Laboratoire de Pharmacologie, Faculte´ de Me´decine, Cre´teil, France; y De´partement de Synthe`se Organique, Faculte´ de Pharmacie, Toulouse, France; and z CNRS, Faculte´ de Me´decine, Cre´teil, France (Received 15 September 2003; Revised 24 November 2003; Accepted 19 December 2003)
Abstract—Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a natural compound with antiproliferative properties. Recent studies suggest that these properties might be due to the ability of curcumin to induce apoptosis in tumor cells by increasing the permeability of the mitochondrial membrane. In the present study, we confirm these observations and provide a molecular mechanism for the action of curcumin in rat liver mitochondria. Curcumin induced mitochondrial swelling, the collapse of Dc, and the release of cytochrome C, events associated with the opening of the permeability transition pore (PTP). Experiments were performed with chemically substituted curcumin derivatives. Some derivatives were obtained by modification of groups on the terminal aromatic rings, and others were obtained by substitution of the diketone function with the cyclohexanone function. They demonstrated that phenol and methoxy groups were essential to promote PTP opening. Curcumin and curcumin derivatives that open the PTP were able to oxidize thiol groups. In addition, PTP opening was abolished in medium devoid of O2 and decreased in the presence of catalase, ferrozine, o-phenanthroline, mannitol, or N-ethylmaleimide. These data suggest that the mechanism by which curcumin promotes PTP opening involves the reduction of Fe3+ to Fe2+, inducing hydroxyl radical (HOS) production and oxidation of thiol groups in the membrane, leading to pore opening. D 2004 Elsevier Inc. All rights reserved. Keywords—Curcumin, Rat liver, Mitochondria, Antioxidant, Permeability transition pore, Reactive oxygen species, Free radicals
Growing evidence suggests that mitochondria are involved in the induction of the cell death program [7]. It was proposed that the formation of a pore, called the permeability transition pore (PTP), could lead to cell death by releasing apoptogenic factors from mitochondria [8]. In a recent study, we showed that curcumin was able to open the PTP in liver mitochondria [9]. Curcumin induced mitochondrial swelling, calcium release, respiration impairment, and collapse of mitochondrial membrane potential, which are events related to pore opening. Therefore, we hypothesized that PTP might be a relevant target by which curcumin induced apoptosis of tumor cells. We also demonstrated that curcumin was able to inhibit the Ca2+ induction of PTP and this effect was related to its antioxidant properties, as curcumin inhibited both superoxide anion (O2 ) production and lipid peroxidation. This was in agreement with in vitro studies [10,11] and cell culture data [2]. Thus, curcumin has a dual effect, inducing PTP despite its antioxidant properties.
INTRODUCTION
Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione) is a natural yellow pigment originally isolated from turmeric (Curcuma longa L.), a rhizome used in India for centuries as a spice and medicinal agent. It possesses a wide range of pharmacological properties. Curcumin was shown to inhibit inflammatory processes [1] and to act as an antioxidant [2,3]. It also prevents tumor proliferation in cell lines [4] and animals [5]. Recently, it was suggested that this effect of curcumin could be mediated through its ability to induce apoptosis of tumor cells [6].
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Address correspondence to: Dr. Didier Morin, De´partement de Pharmacologie, Faculte´ de Me´decine de Paris XII, 8 rue du Ge´ne´ral Sarrail, F-94010, Cre´teil, France; Fax: (33) 1 49.81.35.94; E-mail: [email protected]. 919
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This study was undertaken to clarify the mechanism of action of curcumin and to determine the structural components of the molecule that are involved in PTP opening. To this end, we prepared a series of synthetic derivatives of curcumin; some were obtained by modification of the terminal aromatic rings either by electron-donating groups or by electron-attracting groups, and others by substitution of the diketone function with the cyclohexanone function. Other molecules used were metabolites of curcumin: vanillin, ferulic acid, and ferulic aldehyde [12]. All these compounds were tested for their ability to induce pore opening and react with reactive oxygen species (ROS), and their effects were compared with those of curcumin. We intend to establish a structure – function relationship which would facilitate the planning of structural modifications for the design of molecules more effective either in inhibiting or inducing PTP opening.
50 ml dry ethyl acetate and stirred under nitrogen for 30 min. The complex formed between 2,4-pentanedione (0.05 mol) and boric anhydride (0.0035 mol) was added to the solution and the reaction mixture was stirred for 24 h. After 15 min, n-butylamine (0.25 4 mol) was added dropwise every 15 min. The following day, the mixture was hydrolyzed by the addition of 75 ml 4 N HCl and heating to 70jC for 60 min. The organic layers were separated and the aqueous fraction was extracted three times with ethyl acetate. The combined organic layers were dried over magnesium sulfate. Evaporation of the solvent left a powder which was purified by recrystallization. For compound Cu09 only, 0.8 mmol curcumin was mixed with 2– 5% of Aliquat 336 and 2 mmol of potassium tert.-butoxide at 50jC. After shaking for 1 h, 46 mmol of butyl bromide was added. The reaction was left overnight at 50jC. After filtration, the powder obtained was purified by recrystallization.
MATERIALS AND METHODS
(2) Synthesis of derivatives including a cyclohexanone chain (compounds Cy01 –Cy11). Benzaldehyde 0.061 mol and cyclohexanone 3.16 ml were dissolved in absolute ethanol. After bubbling hydrogen chloride, the precipitate formed was filtered. The powder obtained was purified by recrystallization.
General synthesis of curcumin derivatives [13] Curcumin derivatives were synthesized by Synthe´Pharma (Seysses, France) according to the methods described previously [14 – 16]. The structures of the curcumin derivatives and the names of the respective benzaldehydes used for synthesis are shown in Tables 1 and 2 All benzaldehydes and reagents were commercially available. (1) Synthesis of derivatives including a b-diketone chain (curcumin, compounds Cu02 – Cu11). Benzaldehyde 0.1 mol and tributyl borate 0.2 mol were dissolved in
(3) Purification of curcumin derivatives. The structures of all compounds were confirmed by checking melting points (using a Ko¨fler banc), elemental analysis (microanalysis), and spectral studies (1H magnetic resonance performed using a Bruker 200 MHz instrument). Thinlayer chromatography experiments and flash chromatography were performed with Merck silicagel G and
Table 1. Structure of the h-Diketone Derivatives
Compound Curcumin Cu02 Cu03 Cu04 Cu05 Cu06 Cu07 Cu08 Cu09 Cu10 Cu11
R1
R2
OCH3 H H OH
OH H OH OCH3
R3
Benzaldehyde used for synthesis
Yield (%)
4-Hydroxy-3-methoxybenzaldehyde Benzaldehyde 4-Hydroxybenzaldehyde 3-Hydroxy-4-methoxybenzaldehyde 3,4-Methylenedioxybenzaldehyde 4-Hydroxy-3,5-dimethoxybenzaldehyde 3,4,5-Trimethoxybenzaldehyde 3-Indolebenzaldehyde 4-Butyloxy-3-methoxybenzaldehyde 4-Hydroxy-3-nitrobenzaldehyde 3,5-di-tert-butyl-4-hydroxybenzaldehyde
60 40 48 35 32 34 39 37 21 30 38
OCH3 OCH3
OH OCH3
H H H H H OCH3 OCH3
OCH3 NO2 t-C4H9
OC4H9 OH OH
H H t-C4H9
OCH2O
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Table 2. Structure of the Cyclohexanone Derivatives
Compound Cy01 Cy02 Cy03 Cy04 Cy05 Cy06 Cy07 Cy08 Cy10 Cy11
R1
R2
OCH3 H H OH
OH H OH OCH3
R3
Benzaldehyde used for synthesis
Yield (%)
4-Hydroxy-3-methoxybenzaldehyde Benzaldehyde 4-Hydroxybenzaldehyde 3-Hydroxy-4-methoxybenzaldehyde 3,4-Methylenedioxybenzaldehyde 4-Hydroxy-3,5-dimethoxybenzaldehyde 3,4,5-Trimethoxybenzaldehyde 3-Indolebenzaldehyde 4-Hydroxy-3-nitrobenzaldehyde 3,5-di-tert-butyl-4-hydroxybenzaldehyde
95 94 91 74 65 75 83 76 82 72
OCH3 OCH3
OH OCH3
H H H H H OCH3 OCH3
NO2 t-C4H9
OH OH
H t-C4H9
OCH2O
silicagel Amicon 60 Am, respectively. HPLC analyses were obtained using a column Symmetry C8 5A (Waters) and the percentage purity of the synthesized derivatives was higher than 98%. There was no secondary product. Percentage yields are indicated in Tables 1 and 2. Isolation of liver mitochondria Rat liver mitochondria were isolated as described previously [17]. Male Wistar rats were decapitated. The livers were removed, weighed, and cut into small pieces in ice-cold buffer A (250 mM sucrose, 50 mM Tris, 5 mM EGTA, pH 7.2, at 4jC). The pieces were rinsed two times and homogenized in the same buffer. The homogenates were then centrifuged at 600g for 10 min at 4jC. The supernatants were centrifuged at 15,000g for 5 min at 4jC and the pellets were resuspended in the same buffer and centrifuged at 15,000g for 5 min. The mitochondrial pellet was washed in buffer B (250 mM sucrose, 50 mM Tris, pH 7.2, at 4jC) and centrifuged for 5 min at 15,000g. The final pellets were suspended in the same buffer and the protein concentration was determined using the method of Lowry et al. [18]. The final pellet contained approximately 60 mg of protein/ml. All animal procedures used in this study are in strict accordance with the European Community Council Directive of 24 November 1986 (86-609/EEC) and Decree of 20 October 1987 (87-848/EEC). Mitochondrial swelling Mitochondrial swelling was assessed by measuring the change in absorbance of the suspension at 540 nm (A540) by using a Hitachi Model UV-3000 spectrophotometer. Experiments were carried out at 25jC in 1.8 ml
of buffer C (250 mM sucrose, 5 mM KH2PO4, pH 7.2) with addition of 2 AM rotenone and 6 mM succinate. Mitochondria (1 mg/ml) were incubated for 1 min in this buffer and swelling was induced by addition of increasing concentrations of curcumin or its derivatives. Mitochondrial swelling was also studied under deenergized conditions. Mitochondria (1 mg/ml) were preincubated for 1 min in a Tris buffer consisting of 150 mM sucrose, 5 mM Tris, 0.5 Ag/ml of rotenone, and 0.5 Ag/ml of antimycin, pH 7.4, at 25jC in a total volume of 1.8 ml prior to the addition of 100 AM CaCl2. Swelling was initiated 4 min later by introducing 20 AM curcumin. Determination of cytochrome C release by Western blot analysis Mitochondria (1 mg/ml) were suspended in 250 Al of buffer C including 2 AM rotenone and 6 mM succinate and incubated with either curcumin, curcumin derivatives, or Ca2+ in the absence or the presence of 2 AM CsA for 20 min. The mitochondrial suspension was centrifuged at 15,000 g for 10 min at 4jC and 5 Al (0.4 mg/ml) of the resulting supernatant was added to 5 Al of a buffer containing sucrose (20%), SDS (2.4%), h-mercaptoethanol (5%), and bromphenol blue (5%). Samples were boiled at 100jC, subjected to electrophoresis on a 4– 15% gradient SDS – polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk in a Tris buffer (Tris 0.02 M, NaCl 0.14 M, pH 7.6) containing 0.1% Tween 20, and incubated overnight at 4jC with mouse monoclonal anti-rat cytochrome C antibody (5/ 1000; MAB897, R&D systems, UK). After incubation with sheep anti-mouse horseradish peroxidase (1/1000; Amersham Pharmacia Biotech, Les Ulis, France) 1 h at
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room temperature, the blots were revealed by enhanced chemiluminescence reaction (Amersham ECL+) and exposed to X-ray film (Sigma, Biomax MS-1 film). Assays of radical scavenging capacity and of lipid peroxidation The free radical scavenging capacity of compounds was determined using diphenyl-2-picryl-hydrazyl stable free radical (DPPH ) [19]. Test compounds were added to an ethanol solution of DPPH (100 AM) and the decrease in absorbance was recorded against time at 515 nm in a Hitachi U-3000 spectrophotometer. The curves allowed estimation of the effective concentration of the compound tested producing a 50% decrease in DPPH concentration at steady state (IC50). Lipid peroxidation was assessed as the generation of thiobarbituric acid-reactive substances (TBARS), i.e., lipid peroxides, according to [20]. Mitochondria (0.2 mg/ml) were suspended in NaCl (0.9%) supplemented with different concentrations of curcumin or its derivatives in a total volume of 1 ml. Identical concentrations of curcumin or curcumin derivatives were diluted in NaCl without addition of mitochondria to make color controls. All tubes were incubated for 10 min at 37jC. Following addition of 100 Al of a mixture containing FeCl2 (500 AM)/FeCl3 (1500 AM), the tubes were incubated for 30 min at 37jC. After addition of 1 ml trichloracetic acid (3%), all tubes were centrifuged at 20jC for 15 min at 3000 rpm and 1 ml of each supernatant was added to 1 ml of thiobarbituric acid (1%) and incubated for 30 min at 95jC. After recooling on ice, the generation of TBARS was determined by measuring the absorbance at 530 nm.
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O2
was added to 800 Al of a solution containing 6 M guanidine, HCl, 1 M NH3, and 1 M NH4Cl (pH 9.0 at 20jC). Twenty microliters of Ellman’s reagent (20 mM) was then added and the reaction was incubated for 15 min at room temperature. Absorbance of the medium was read at 412 nm against Ellman’s reagent blank. The amount of thiol groups was estimated using the difference in absorbance at 412 nm before and after addition of Ellman’s reagent corrected for the absorbance of Ellman’s reagent. To estimate protein thiol concentration, glutathione in the concentration range 10 to 500 AM was assayed under the same conditions. Results for thiol groups are expressed as percentages and a 100% value corresponds to the reduced thiol groups, measured in control mitochondria. This value was equal to 240 F 23 nmol/mg proteins. A value lower than 100% revealed oxidation of thiol groups, whereas a higher value reflected inhibition of the spontaneous oxidation occurring during the incubation period. Values are expressed as means F SEM fo three independent experiments done in triplicate. Measure of mitochondrial membrane potential (DW) DC was monitored by means of the fluorescent dye rhodamine 123 according to [22] at excitation and emission wavelengths of 503 and 527 nm, respectively. Mitochondria (0.5 mg/ml) were incubated in 1.8 ml buffer C supplemented with 2 AM rotenone and 0.3 AM rhodamine 123. After 30 s, 6 mM succinate was added. When DC was established, 20 AM curcumin or curcumin derivatives were added to the medium and the times of DC collapses were compared. Relative changes in membrane potential were expressed in arbitrary fluorescence units and were not converted to potential values.
production
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The generation of O2 was achieved as previously reported [20] by measuring the reduction of nitroblue tetrazolium in monoformazan that absorbed at 560 nm. Mitochondria (1 mg/ml) were incubated for 1 min in 1.2 ml buffer C supplemented with 2 AM rotenone, 1 AM cyclosporin A (CsA), and 100 AM nitroblue tetrazolium. For this particular experiment, CsA was added to inhibit mitochondrial swelling, which slightly interfered with spectroscopic detection of the reduction reaction. The production of O2 was then initiated by the addition of 6 mM succinate.
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Action on complex III of the respiratory chain Mitochondrial complex III activity was determined by a spectrophotometric method measuring the rate of cytochrome C reduction at 550 nm and 37jC triggered by decylubiquinol. The reaction mixture (1 ml buffer C) contained 2 mM ethylenediaminetetraacetic acid, 0.1 mM KCN, 2 AM rotenone, 0.1 mM decylubiquinol, and 100 Ag of mitochondria. The reaction was initiated by the addition of 50 AM of cytochrome C and the rate of reduction was measured for 2 min. The experiment was performed in the absence or the presence of curcumin or curcumin derivatives.
Protein thiol determination Protein thiol content was measured according to [21] with some modifications. Briefly, mitochondria (1 mg/ml) were incubated in buffer C in the presence of curcumin or other molecules in a total volume of 1 ml for 15 min at 25jC. After this time, 200 Al of mitochondrial solution
Measurement of reduction of Fe3+ to Fe2+ Reduction of Fe3+ to Fe2+ was measured by the ferrozine complex method [23]. The reaction mixture consisted of ferrozine (100 AM) and FeCl3 (100 AM) in a final volume of 2 ml 0.9% NaCl (pH 7). The reaction was
Curcumin and mitochondria
initiated by addition of curcumin derivatives (20 AM) and the absorbance of the Fe2+ – ferrozine complex was monitored at 560 nm.
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Table 3. Curcumin Derivatives Induced Mitochondrial Swelling Compounds
Swelling Energized conditions
RESULTS
Induction of mitochondrial swelling by curcumin and curcumin derivatives Rat liver mitochondria energized with succinate were incubated in a sucrose – phosphate buffer (buffer C) before addition of increasing concentrations of curcumin. Figure 1A shows that curcumin induced mitochondrial swelling as revealed by the large decrease in absorbance of the mitochondrial suspension at 540 nm. This effect was concentration-dependent and maximal swelling was
EC50 (AM)
Curcumin Cu02 Cu03 Cu04 Cu05 Cu06 Cu07 Cu08 Cu09 Cu10 Cu11 Cy01 Cy02 Cy03 Cy04 Cy05 Cy06 Cy07 Cy08 Cy10 Cy11 Ferulic acid Ferulic aldehyde Vanillin
Deenergized conditions
Ratea (% of Ca2+ effect)
15
48
effect effect effect effect
Induction No effect Induction Induction No effect Induction No effect No effect No effect No effect No effect Induction No effect No effect Induction No effect Induction No effect No effect No effect No effect
No effect No effect
No effect No effect
No effect
No effect
No effect No effect 32
59 No effect
48
87 No No No No No
effect effect effect effect effect
36
58 No effect No effect
15
9 No effect
91
85 No No No No
a
Rate of swelling induced by curcumin derivatives compared with the rate of swelling induced by 50 AM Ca2+ (i.e., 100%). These effects were obtained at 30, 60, 200, 60, 20, and 200 AM for curcumin, Cu04, Cu06, Cy01, Cy04, and Cy06, respectively.
Fig. 1. Effect of curcumin on mitochondrial swelling and mitochondrial potential. (A) Mitochondrial swelling: Liver mitochondria (1 mg/ml) were preincubated for 1 min in buffer C supplemented with 6 mM succinate and 2 AM rotenone, pH 7.2, at 25jC. Swelling was induced by the addition of either 1 (line a), 10 (line b), 20 (line c), 30 (line d), 40 (line e), 50 (line f), or 60 (line g) AM curcumin. Line h: swelling was induced by 50 AM Ca2+. Line i: effect of 20 AM curcumin in the presence of 1 AM CsA. (B) Mitochondrial potential: Liver mitochondria were suspended in incubation buffer C supplemented with 0.3 AM rhodamine 123. DC was measured after addition of 6 mM succinate. Line a: no other addition. Line b: 20 AM curcumin. Line c: 20 AM curcumin + 1 AM CsA. Line d: addition of 1 AM of the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced complete DC depolarization. Curcumin and CsA were added just after mitochondria. The figures show representative results from four independent experiments using four independent mitochondrial preparations.
obtained around 30 AM. It was associated with complete DC depolarization as observed after addition of the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Fig. 1B). These effects were mediated by opening of the PTP as they were completely prevented by 1 AM CsA (Figs. 1A, B), a well-established inhibitor of PTP [24,25]. The kinetic rate of swelling induced by 30 AM curcumin was equivalent to 48% of that observed in the presence of 50 AM Ca2+, a well-known inducer of PTP (Fig. 1A, Table 3). It should be noted that phosphate greatly contributed to the effect of curcumin because PTP was almost completely inhibited when swelling experiments were performed in a Tris buffer devoid of phosphate ions (data not shown). When curcumin concentrations higher than 30 AM were used, a decrease in swelling was observed; at 60 AM no further swelling occurred (Fig. 1A). We used the same protocols to evaluate the ability of 20 curcumin derivatives (Tables 1 and 2) and three curcumin metabolites, ferulic acid, ferulic aldehyde, and vanillin [12], to induce PTP. Among these compounds three, Cy01, Cy04, and Cu04, showed the same biphasic profile
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as curcumin. At low concentrations, they induced a concentration-dependent swelling which disappeared for higher concentrations. The maximal swelling-inducing effect was observed at 60, 60, and 20 AM for Cu04, Cy01, and Cy04, respectively and corresponded to 59, 58, and 9% of that observed with 50 AM Ca2+, respectively (Table 3). Two other compounds, Cu06 and Cy06, were also able to promote swelling. However, for these agents the swelling was proportional to their concentrations. At 200 AM the rate and the extent of the swelling were close to those obtained with 50 AM Ca2+. Other agents were ineffective. It is now well-established that PTP opening resulted in release of the apoptotic factor cytochrome C into the cytosol [26]. Therefore, to confirm the PTP-inducing effect of curcumin derivatives, we analyzed their effect on cytochrome C release. The molecules were assayed at the concentrations that induced maximal swelling. Figure 2 shows that curcumin, Cu04, Cu06, Cy01, Cy04, and Cy06 caused the release of cytochrome C from energized mitochondria and that this effect was completely prevented by 2 AM CsA. Identical results were observed in the presence of 50 AM Ca2+.
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curcumin derivatives on O2 and hydroxyl radical (HO ) production, thiol oxidation, and DPPH reduction (Table 4). Curcumin inhibited O2 production and lipid peroxidation and was able to oxidize membrane thiol functions and to reduce DPPH . Curcumin derivatives showed properties different from those of curcumin. Indeed, some molecules inhibited O2 production and did not abolish lipid peroxidation (CuO7, Cu10, CyO3, CyO4, Cy10) whereas CuO8 and CyO8 inhibited both effects without reducing DPPH . In the same way, CyO4 induced thiol group oxidation but was ineffective in promoting lipid peroxidation. We then compared the redox properties of curcumin derivatives with their ability to induce PTP opening. The study demonstrated that no clear relation exists between the inhibition of O2 production and the induction of swelling. Indeed, several curcumin derivatives (Cu03, Cu07, Cu10, Cy03, Cy08, Cy10) revealed potent O2 -inhibiting properties without swelling induction properties, whereas Cy06, which is a potent inductor of swelling, is a poor inhibitor of O2 production under the same experimental conditions (Table 4). It should be noted that all these molecules were tested for their ability to interact directly with complex III of the respiratory chain which is the main generator of O2 in our model. They did not show any effect (data not shown). Thus, their effect might be due to scavenging of O2 as previously observed for curcumin [10,28] and not to the inhibition of O2 generation. In the same way, there was no relation between lipid peroxidation and swelling induction as
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Effects of curcumin and curcumin derivatives on reactive oxygen species Increasing amounts of evidence suggest that oxidative stress plays a key role in PTP induction [27]. Consequently, we decided to study the effects of curcumin and
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Fig. 2. Cytochrome C release from isolated liver mitochondria induced by curcumin and related derivatives. Liver mitochondria were suspended in buffer C supplemented with 6 mM succinate and 2 AM rotenone, pH 7.2, at 25jC and incubated for 20 min with curcumin, curcumin derivatives, or Ca2+ in the presence (+) or the absence () of 2 AM CsA. The results are typical of four independent experiments. The signal intensity of cytochrome C was evaluated by densitometry (n = 4 per group).
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Curcumin and mitochondria Table 4. Comparison of the Effect of Curcumin Derivatives on Mitochondrial swelling, O2 and HO Production, Thiol Oxidation, and DPPH Reduction
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S S S Thiol HOS
Molecules Swelling O2 induction inhibition inhibition (% at IC50 (AM) 20 AM) Curcumin Cu02 Cu03 Cu04 Cu05 Cu06 Cu07 Cu08 Cu09 Cu10 Cu11 Cy01 Cy02 Cy03 Cy04 Cy05 Cy06 Cy07 Cy08 Cy10 Cy11
Yes No No Yes No Yes No No No No No Yes No No Yes No Yes No No No
77 0 100 67 0 37 36 44 0 39 0 56 0 55 18 0 18 No effect 24 28
groups (%)
0.7 74.3 F 4.3 No effect 100 F 4 3.1 60.8 F 3.7 4.1 89.6 F 3.2 No effect 100 F 3 18 77.2 F 3.7 No effect 100 F 5.2 8 100 F 3.8 No effect 115.3 F 4.8 >100 109 F 1.2 0.8 100 F 2.8 4. 1 76.5 F 3.9 No effect 100 F 4.3 >100 97.3 F 1.8 >100 94.3 F 1.5 >100 110.0 F 4.1 7.3 65.4 F 2.6 No effect 107.0 F 2.0 1.8 120.1 F 6.0 No effect 100 F 3.3 Nonsoluble
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DPPH reduction IC50 (AM)
12.5 No effect 380 110 No effect 65 No effect No effect No effect No effect 65 27 No effect No effect >400 No effect >400 No effect No effect No effect
Cy04 did not inhibit lipid peroxidation whereas Cu11, which is as potent as curcumin in inhibiting lipid peroxidation, was unable to promote swelling. Thus, the effect of curcumin was not due to its interaction with O2 and/or HO which otherwise would give rise to a toxic curcumin radical. A correlation was observed between the ability to induce swelling and the ability to promote the oxidation of membrane thiol groups as all curcumin derivatives, which induced swelling under either energized or deenergized conditions or both (Table 3), induced membrane thiol group oxidation, whereas the other derivatives did not (Table 4). The hypothesis that thiol groups may be involved in swelling induction was also reinforced by the fact that the thiol substitution compound N-ethylmaleimide completely prevented the effects of curcumin (Fig. 3). However, in the same experiment monobromobimane, which as N-ethylmaleimide forms adducts with glutathione, was inactive (Fig. 3). This seems to rule out a possible role of glutathione in the mechanism of action of curcumin. It should be noted that Cu03 caused swelling under deenergized conditions but not in respiring medium. This is probably due to the fact that Cu03 acts as an uncoupling agent. Indeed, Cu03 prevented O2 formation (Table 4), increased the rate of oxygen consumption and collapsed DC (data not shown) as observed with uncouplers [29]. Deenergized mitochondria were used to study the mechanism by which molecules that induced swelling
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oxidized thiol groups. These experimental conditions have the advantage of eliminating respiration and membrane potential in the swelling process and facilitate the study of low oxygen concentrations. To this end, mitochondria were incubated in a Tris buffer in the absence of substrate and in the presence of respiratory chain inhibitors (rotenone and antimycin) and swelling was induced by high Ca2+ concentrations. Under these experimental conditions curcumin and curcumin derivatives that induced PTP opening under energized conditions were able to induce swelling but at a lower rate and extent (Table 3). When oxygen was exhausted from the incubation medium through nitrogen purging, this phenomenon was completely inhibited, indicating the requirement of ROS in curcumin-induced swelling. In the same way, PTP opening by curcumin was inhibited by an excess of exogenous catalase (Fig. 3) suggesting that hydrogen peroxide (H2O2) or ROS derived from H2O2 are involved in the mechanism. Indeed, H2O2, which is produced following dismutation of O2 , can react with mitochondrial Fe2+, resulting in the formation of HO [30]. We therefore decided to study the effect of mannitol, a well-known scavenger of HO , and of Fe2+ chelators, ferrozine and o-phenanthroline, on curcumininduced swelling. Fig. 3 shows that preincubation of mitochondria with either mannitol, ferrozine, or o-phenanthroline inhibited mitochondrial swelling. The same results were obtained with either Cu04, Cu06, Cy01, Cy04, or Cy06. In a last step, we measured a possible effect of curcumin and curcumin derivatives on the reduction of Fe3+ to Fe2+. Figure 4 shows that all curcumin derivatives that were able to induce swelling promoted Fe3+
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Fig. 3. Inhibition of curcumin-induced mitochondrial swelling under deenergized conditions. Liver mitochondria (1 mg/ml) were incubated in a Tris sucrose buffer in the absence (line a) or the presence of monobromobimane (line b), 9000 U catalase (line c), 400 AM ophenanthroline (line d), 500 AM ferrozine (line e), 50 AM Nethylmaleimide (line f), 120 AM mannitol (line g), or nitrogen purging (line h) for 1 min before addition of 100 AM Ca2 +. Four minutes later swelling was induced by the addition of 40 AM curcumin. The figure shows representative results from four independent experiments using four independent mitochondrial preparations.
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can act as a scavenger of HO or catalyze the formation of HO depending on the experimental conditions [12]. Up to now it has not been well-established which part of the molecule is responsible for the effect of curcumin in oxygen radical reactions and conflicting results exist concerning its antioxidant mechanism. However, most of the studies underline the predominant role of both the phenolic and methoxy groups in the phenyl ring and the 1,3-diketone moiety [10,12]. The purpose of the present study was to investigate the mechanism by which curcumin promotes PTP, to define the chemical functions involved in PTP-induction, and finally to select a curcumin derivative that would display only PTP-inducing or inhibiting properties. A total of 20 compounds were synthesized and two major groups could be distinguished. The first group preserved the basic skeleton of curcumin but modified the aromatic substituents (compounds Cu02 –Cu11). The second group had a cyclohexanone ring instead of the linear diketone chain with modifications of the aromatic substituents (compounds Cy01 – Cy11). The most important structural feature seems to be the presence of a hydroxyl group in the aromatic groups. Indeed, all curcumin derivatives that are able to induce swelling (Cu04, Cu06, Cy01, Cy06, and, to a lesser extent, Cy04) possess a free hydroxyl group in the phenyl ring. In addition, when the phenolic group is blocked by a methyl (Cu07, Cy07) or a methylene dioxide (Cu05, Cy05) or substituted by a butane chain (Cu09) the activity of curcumin disappeared. This indicates that the phenolic group is essential for activity. Swelling induction is more effective when the phenolic hydroxyl group is located in the para than in the ortho position (compare curcumin and Cy01 with Cu04 and Cy04, respectively). Whether the presence of a hydroxyl group seems to be a necessary criterion, it did not appear to be sufficient because no effect was obtained with other hydroxyl compounds such as Cy03, Cu10, Cy10, Cu11, and Cy11. In fact, the effect also required the presence of an electron-donating group in the ortho position near the hydroxyl function of the aromatic ring (compounds curcumin, Cy01, Cu06, Cy06, Cu11, Cy11). These substituents must not be too bulky as the ortho-di-tert-butyl derivatives (compounds Cu11 and Cy11) were ineffective. By contrast, an electron-attracting group such as NO2 (Cu10, Cy10) inhibited the effect. There is strong evidence that the h-diketone moiety is not directly involved in curcumin induced-swelling because the nonphenolic curcumin derivatives (Cu02, Cu08, Cu09) were without effect. However, the effect of curcumin and curcumin derivatives seems to require the h-diketone moiety as it was attenuated when the hdiketone was substituted by the cyclohexanone ring
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Fig. 4. Reduction of Fe3 + to Fe2 + by curcumin derivatives measured as increase in absorbance of ferrozine complex. Curcumin derivatives (20 AM) were added to a solution of 0.9% NaCl containing 100 AM ferrozine and 100 AM FeCl3. Line a: Cy01. Line b: curcumin. Line c: Cu06. Line d: Cy06. Line e: Cu04. Line f: Cy04. Other compounds were inactive. The figure is representative of three independent experiments.
reduction. Curcumin, Cu06, Cy01, and Cy06 were the most effective. Cu04 and Cy04 also stimulated Fe3+ reduction but to a lesser extent. The other compounds were without effect. DISCUSSION
The present study shows that curcumin induces swelling, a decrease in the mitochondrial potential, and the release of cytochrome C in isolated liver mitochondria. These effects can be attributed to the occurrence of PTP as they are completely inhibited by CsA, a specific inhibitor of PTP [24]. It should be noted that curcumin-induced swelling is proportional to the concentration used up to 30 AM but that higher concentrations promoted a decrease in the extent of swelling. This biphasic profile may explain the opposite effects of curcumin which showed both anti- and proapoptotic properties in different experimental models [4– 6,31,32]. Whereas a controversy persists concerning the molecular composition of this pore, there are compelling arguments that indicate that PTP is a multiproteic complex [8]. However, despite extensive work, the nature of the mitochondrial membrane conformational change leading to PTP is still unknown. PTP is caused by mitochondrial matrix accumulation of excessive quantities of Ca2+ and can be enhanced by a variety of agents or conditions [25]. Oxidative stress seems to play a key role in PTP induction [27] with the observation that the oxidation or the crosslinking of membrane protein thiol groups is a critical event [25,33]. Because PTP opening is closely related to redox phenomena, we postulated that the oxidative properties of curcumin might be responsible for its effect. Curcumin
Curcumin and mitochondria
(compounds Cy01, Cy06, and Cy04) and was not observed with ferulic acid. In summary, this structure – activity study reveals that swelling requires the presence of a phenolic hydroxyl group associated with a radical displaying electron donation properties. It is interesting to note that these structural features are those that explain the antioxidant properties of curcumin. Indeed, it has been reported that the phenolic group is essential to the antioxidant effect [10,34] of curcumin and that the presence of a methoxy group adjacent to the phenolic group increases the free radical scavenging properties [11,35,36]. In fact, the ortho substitution with an electron donating group such as a methoxy seems to increase the antioxidant activity of phenols by facilitating the formation and enhancing the stability of the phenoxyl radical. Taken together, these data suggest that the functions that are responsible for the antioxidant properties of curcumin are also responsible for PTP opening. This is further emphasized by the fact that curcumin, Cu04, Cu06, and Cy01, which display strong antioxidant activity in the DPPH test, are able to induce swelling. This study also reveals that ferulic acid, vanillin, and ferulic aldehyde, which are obtained by curcumin decomposition [12], are totally ineffective in all tests, demonstrating that curcumin effect is not due to these metabolites. In a recent article we suggested that the effect of curcumin might be related to its interaction with O2 [9]. The hypothesis was that curcumin, during mitochondrial respiration, interacted with O2 to give birth to a radical that was toxic to mitochondria. The present study did not permit confirmation of this hypothesis primarily because all compounds that induced swelling did not interact with O2 and/or HO . In fact, PTP opening may be due to thiol group oxidation because all compounds that induced swelling were found to oxidize thiol groups and the thiol substitution agent N-ethylmaleimide completely prevented PTP opening. This suggestion is consistent with previous data indicating that oxidation of critical thiol groups of the adenine nucleotide translocase may be responsible for PTP opening [33,37]. It should be noted that N-ethylmaleimide is an inhibitor of the phosphate carrier [38] but this property cannot explain the inhibitory effect of N-ethylmaleimide on PTP as prevention of PTP was observed under deenergized conditions where phosphate was absent from the reaction medium. The remaining question was how curcumin induced thiol group oxidation. Our results showing the inhibition of PTP opening either in an anaerobic medium or in the presence of catalase strongly support the notion that ROS are involved in the process. In addition, the protective
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effect conferred by the Fe2+ chelators ferrozine and ophenanthroline suggests that the Fenton reaction, which describes the nonenzymatic reduction of H2O2 to hydroxyl anion and HO by oxidation of Fe2+, could be involved in the process. This idea is reinforced by the fact that curcumin derivatives that induce swelling are able to reduce Fe3+ into Fe2+. The effect of mannitol, which inhibits curcumin- and curcumin derivative-induced PTP opening, is also supportive of HO generation. This confirms previous results indicating that curcumin is able to catalyze the formation of HO by reducing Fe3+ into Fe2+ [12,39]. Thus, on the basis of our present data, we propose that the molecular mechanism of curcumin leading to PTP opening is related to its antioxidant properties. Under our experimental conditions, curcumin reduces Fe3+ into Fe2+, which in turn reacts with H2O2 and increases the rate of HOS production. This highly reactive radical oxidizes critical thiol groups which leads to PTP opening in the presence of Ca2+. Fig. 5 summarizes the proposed molecular process involved in the opening of PTP by curcumin. This could represent a general mechanism by which several reducing agents such as polyphenols were shown to affect mitochondrial functions [40]. This may also be one of the steps of the mechanism by which curcumin induces apoptosis of tumor cells [6,41] and thus acts as an antiproliferative agent [4]. In this regard, some of the derivatives synthesized in this study such as Cu06 and Cy06 which induce a concentrationdependent opening of the PTP display interesting properties. By contrast, the compounds Cu08 and Cy08 that show antioxidant effects without PTP induction could
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Fig. 5. Scheme summarizing the proposed molecular processes by which curcumin induces PTP opening. The respiratory chain, inserted in the inner mitochondrial membrane, generates the superoxide radical (O2 ). O2 is removed by superoxide dismutase, which promotes the generation of hydrogen peroxide (H2O2). H2O2 is then reduced to water by glutathione peroxidase or catalase. When H2O2 is accumulated in quantities too large for removal, in the presence of Fe2 +, H2O2 generates a highly reactive HO radical that oxidizes thiol ( – SH) groups, leading to pore opening. Curcumin catalyzes the formation of HO by reducing Fe3 + into Fe2 +.
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represent promising curcumin derivatives as cytoprotective agents.
Acknowledgments—The authors thank Felipe Pires for his skillful technical assistance. This work was supported by the Ministe`re de l’Education Nationale (DRED EA 427).
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F.; Zini, R.; Tillement, J. P.; Labidalle, S.; Guyot, D.; ChauvetMonges, A. M.; Crevat, A.; Le Ridant, A. Structure – property relationships of trimetazidine derivatives and model compounds as potential antioxidants. Free Radic. Biol. Med. 25:113 – 120; 1998. Zini, R.; Morin, C.; Bertelli, A.; Bertelli, A. A.; Tillement, J. P. Effects of resveratrol on the rat brain respiratory chain. Drugs Exp. Clin. Res. 25:87 – 97; 1999. Morin, D.; Zini, R.; Ligeret, H.; Neckameyer, W.; Labidalle, S.; Tillement, J. P. Dual effect of ebselen on mitochondrial permeability transition. Biochem. Pharmacol. 65:1643 – 1651; 2003. Emaus, R. K.; Grunwald, R.; Lemaster, J. J. Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim. Biophys. Acta. 850:436 – 448; 1986. Cowart, R. E.; Singleton, F. L.; Hind, J. S. A comparison of bathophenanthrolinedisulfonic acid and ferrozine as chelators of iron(II) in reduction reactions. Anal. Biochem. 211:151 – 155; 1993. Broekemeier, K. M.; Dempsey, M. E.; Pfeiffer, D. R. Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria. J. Biol. Chem. 264:7826 – 7830; 1989. Zoratti, M.; Szabo’, I. The mitochondrial permeability transition. Biochim. Biophys. Acta 1241:139 – 176; 1995. Lim, M. L.; Lum, M. G.; Hansen, T. M.; Roucou, X.; Nagley, P. On the release of cytochrome c from mitochondria during cell death signaling. J. Biomed. Sci. 9:488 – 506; 2002. Kowaltowski, A. J.; Castilho, R. F.; Vercesi, A. E. Mitochondrial permeability transition and oxidative stress. FEBS Lett. 495: 12 – 15; 2001. Reddy, A. C.; Lokesh, B. R. Studies on the inhibitory effects of curcumin and eugenol on the formation of reactive oxygen species and the oxidation of ferrous iron. Mol. Cell Biochem. 137:1 – 8; 1994. Starkov, A. A.; Dedukhova, V. I.; Skulachev, V. P. 6-Ketocholestanol abolishes the effect of the most potent uncouplers of oxidative phosphorylation in mitochondria. FEBS Lett. 355:305 – 308; 1994. Kowaltowski, A. J.; Vercesi, A. E. Mitochondrial damage induced by conditions of oxidative stress. Free Radic. Biol. Med. 26:463 – 471; 1999. Somasundaram, S.; Edmund, N. A.; Moore, D. T.; Small, G. W.; Shi, Y. Y.; Orlowski, R. Z. Dietary curcumin inhibits chemotherapy-induced apoptosis in models of human breast cancer. Cancer Res. 62:3868 – 3875; 2002. Sood, A.; Mathew, R.; Trachtman, H. Cytoprotective effect of curcumin in human proximal tubule epithelial cells exposed to shiga toxin. Biochem. Biophys. Res. Commun. 283:36 – 41; 2001. Costantini, P.; Chernyak, B. V.; Petronilli, V.; Bernardi, P. Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. J. Biol. Chem. 271:6746 – 6751; 1996. Barclay, L. R.; Vinqvist, M. R.; Mukai, K.; Goto, H.; Hashimoto, Y.; Tokunaga, A.; Uno, H. On the antioxidant mechanism of curcumin: classical methods are needed to determine antioxidant mechanism and activity. Org. Lett. 2:2841 – 2843; 2000. Cuvelier, M. E.; Richart, H.; Berset, C. Comparison of antioxidant activity of some acid-phenols: structure-activity relationship. Biosci. Biotechnol. Biochem. 56:324 – 325; 1992. Sardjiman, S. S.; Reksohadiprodjo, M. S.; Hakim, L.; Van der Goot, H.; Timmerman, H. 1,5-Diphenyl-1,4-pentadienne-3-ones and cyclic analogues as antioxidative agents: synthesis and structure – activity relationship. Eur. J. Med. Chem. 32:625 – 630; 1997. McStay, G. P.; Clarke, S. J.; Halestrap, A. P. Role of critical thiol groups on the matrix surface of the adenine nucleotide translocase in the mechanism of the mitochondrial permeability transition pore. Biochem. J. 367:541 – 548; 2002. Costantini, P.; Petronilli, V.; Colonna, R.; Bernardi, P. On the effects of paraquat on isolated mitochondria: evidence that paraquat causes opening of the cyclosporin A-sensitive permeability
Curcumin and mitochondria transition pore synergistically with nitric oxide. Toxicology 99:77 – 88; 1995. [39] Kunchandy, E.; Rao, M. N. A. Effect of curcumin on hydroxyl radical generation through Fenton reaction. Int. J. Pharm. 57:173 – 176; 1989. [40] Hodnick, W. F.; Duval, D. L.; Pardini, R. S. Inhibition of mitochondrial respiration and cyanide-stimulated generation of reactive oxygen species by selected flavonoids. Biochem. Pharmacol. 47:573 – 580; 1994. [41] Kuo, M. L.; Huang, T. S.; Lin, J. K. Curcumin, an antioxidant and anti-tumor promoter, induces apoptosis in human leukemia cells. Biochim. Biophys. Acta 1317:95 – 100; 1996.
929 ABBREVIATIONS
CsA — cyclosporin A CCCP — carbonyl cyanide m-chlorophenylhydrazone DPPH — diphenyl-2-picryl-hydrazyl stable free radical DC — mitochondrial membrane potential PTP — permeability transition pore ROS — reactive oxygen species TBARS — thiobarbituric acid-reactive substances
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doi:10.1016/j.freeradbiomed.2003.12.018
Original Contribution EFFECTS OF CURCUMIN AND CURCUMIN DERIVATIVES ON MITOCHONDRIAL PERMEABILITY TRANSITION PORE HEIDI LIGERET,* SOPHIE BARTHELEMY, y ROLAND ZINI,* JEAN-PAUL TILLEMENT,* SERGE LABIDALLE, y and DIDIER MORIN *,z * Laboratoire de Pharmacologie, Faculte´ de Me´decine, Cre´teil, France; y De´partement de Synthe`se Organique, Faculte´ de Pharmacie, Toulouse, France; and z CNRS, Faculte´ de Me´decine, Cre´teil, France (Received 15 September 2003; Revised 24 November 2003; Accepted 19 December 2003)
Abstract—Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a natural compound with antiproliferative properties. Recent studies suggest that these properties might be due to the ability of curcumin to induce apoptosis in tumor cells by increasing the permeability of the mitochondrial membrane. In the present study, we confirm these observations and provide a molecular mechanism for the action of curcumin in rat liver mitochondria. Curcumin induced mitochondrial swelling, the collapse of Dc, and the release of cytochrome C, events associated with the opening of the permeability transition pore (PTP). Experiments were performed with chemically substituted curcumin derivatives. Some derivatives were obtained by modification of groups on the terminal aromatic rings, and others were obtained by substitution of the diketone function with the cyclohexanone function. They demonstrated that phenol and methoxy groups were essential to promote PTP opening. Curcumin and curcumin derivatives that open the PTP were able to oxidize thiol groups. In addition, PTP opening was abolished in medium devoid of O2 and decreased in the presence of catalase, ferrozine, o-phenanthroline, mannitol, or N-ethylmaleimide. These data suggest that the mechanism by which curcumin promotes PTP opening involves the reduction of Fe3+ to Fe2+, inducing hydroxyl radical (HOS) production and oxidation of thiol groups in the membrane, leading to pore opening. D 2004 Elsevier Inc. All rights reserved. Keywords—Curcumin, Rat liver, Mitochondria, Antioxidant, Permeability transition pore, Reactive oxygen species, Free radicals
Growing evidence suggests that mitochondria are involved in the induction of the cell death program [7]. It was proposed that the formation of a pore, called the permeability transition pore (PTP), could lead to cell death by releasing apoptogenic factors from mitochondria [8]. In a recent study, we showed that curcumin was able to open the PTP in liver mitochondria [9]. Curcumin induced mitochondrial swelling, calcium release, respiration impairment, and collapse of mitochondrial membrane potential, which are events related to pore opening. Therefore, we hypothesized that PTP might be a relevant target by which curcumin induced apoptosis of tumor cells. We also demonstrated that curcumin was able to inhibit the Ca2+ induction of PTP and this effect was related to its antioxidant properties, as curcumin inhibited both superoxide anion (O2 ) production and lipid peroxidation. This was in agreement with in vitro studies [10,11] and cell culture data [2]. Thus, curcumin has a dual effect, inducing PTP despite its antioxidant properties.
INTRODUCTION
Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione) is a natural yellow pigment originally isolated from turmeric (Curcuma longa L.), a rhizome used in India for centuries as a spice and medicinal agent. It possesses a wide range of pharmacological properties. Curcumin was shown to inhibit inflammatory processes [1] and to act as an antioxidant [2,3]. It also prevents tumor proliferation in cell lines [4] and animals [5]. Recently, it was suggested that this effect of curcumin could be mediated through its ability to induce apoptosis of tumor cells [6].
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Address correspondence to: Dr. Didier Morin, De´partement de Pharmacologie, Faculte´ de Me´decine de Paris XII, 8 rue du Ge´ne´ral Sarrail, F-94010, Cre´teil, France; Fax: (33) 1 49.81.35.94; E-mail: [email protected]. 919
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This study was undertaken to clarify the mechanism of action of curcumin and to determine the structural components of the molecule that are involved in PTP opening. To this end, we prepared a series of synthetic derivatives of curcumin; some were obtained by modification of the terminal aromatic rings either by electron-donating groups or by electron-attracting groups, and others by substitution of the diketone function with the cyclohexanone function. Other molecules used were metabolites of curcumin: vanillin, ferulic acid, and ferulic aldehyde [12]. All these compounds were tested for their ability to induce pore opening and react with reactive oxygen species (ROS), and their effects were compared with those of curcumin. We intend to establish a structure – function relationship which would facilitate the planning of structural modifications for the design of molecules more effective either in inhibiting or inducing PTP opening.
50 ml dry ethyl acetate and stirred under nitrogen for 30 min. The complex formed between 2,4-pentanedione (0.05 mol) and boric anhydride (0.0035 mol) was added to the solution and the reaction mixture was stirred for 24 h. After 15 min, n-butylamine (0.25 4 mol) was added dropwise every 15 min. The following day, the mixture was hydrolyzed by the addition of 75 ml 4 N HCl and heating to 70jC for 60 min. The organic layers were separated and the aqueous fraction was extracted three times with ethyl acetate. The combined organic layers were dried over magnesium sulfate. Evaporation of the solvent left a powder which was purified by recrystallization. For compound Cu09 only, 0.8 mmol curcumin was mixed with 2– 5% of Aliquat 336 and 2 mmol of potassium tert.-butoxide at 50jC. After shaking for 1 h, 46 mmol of butyl bromide was added. The reaction was left overnight at 50jC. After filtration, the powder obtained was purified by recrystallization.
MATERIALS AND METHODS
(2) Synthesis of derivatives including a cyclohexanone chain (compounds Cy01 –Cy11). Benzaldehyde 0.061 mol and cyclohexanone 3.16 ml were dissolved in absolute ethanol. After bubbling hydrogen chloride, the precipitate formed was filtered. The powder obtained was purified by recrystallization.
General synthesis of curcumin derivatives [13] Curcumin derivatives were synthesized by Synthe´Pharma (Seysses, France) according to the methods described previously [14 – 16]. The structures of the curcumin derivatives and the names of the respective benzaldehydes used for synthesis are shown in Tables 1 and 2 All benzaldehydes and reagents were commercially available. (1) Synthesis of derivatives including a b-diketone chain (curcumin, compounds Cu02 – Cu11). Benzaldehyde 0.1 mol and tributyl borate 0.2 mol were dissolved in
(3) Purification of curcumin derivatives. The structures of all compounds were confirmed by checking melting points (using a Ko¨fler banc), elemental analysis (microanalysis), and spectral studies (1H magnetic resonance performed using a Bruker 200 MHz instrument). Thinlayer chromatography experiments and flash chromatography were performed with Merck silicagel G and
Table 1. Structure of the h-Diketone Derivatives
Compound Curcumin Cu02 Cu03 Cu04 Cu05 Cu06 Cu07 Cu08 Cu09 Cu10 Cu11
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R2
OCH3 H H OH
OH H OH OCH3
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Benzaldehyde used for synthesis
Yield (%)
4-Hydroxy-3-methoxybenzaldehyde Benzaldehyde 4-Hydroxybenzaldehyde 3-Hydroxy-4-methoxybenzaldehyde 3,4-Methylenedioxybenzaldehyde 4-Hydroxy-3,5-dimethoxybenzaldehyde 3,4,5-Trimethoxybenzaldehyde 3-Indolebenzaldehyde 4-Butyloxy-3-methoxybenzaldehyde 4-Hydroxy-3-nitrobenzaldehyde 3,5-di-tert-butyl-4-hydroxybenzaldehyde
60 40 48 35 32 34 39 37 21 30 38
OCH3 OCH3
OH OCH3
H H H H H OCH3 OCH3
OCH3 NO2 t-C4H9
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OCH2O
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Table 2. Structure of the Cyclohexanone Derivatives
Compound Cy01 Cy02 Cy03 Cy04 Cy05 Cy06 Cy07 Cy08 Cy10 Cy11
R1
R2
OCH3 H H OH
OH H OH OCH3
R3
Benzaldehyde used for synthesis
Yield (%)
4-Hydroxy-3-methoxybenzaldehyde Benzaldehyde 4-Hydroxybenzaldehyde 3-Hydroxy-4-methoxybenzaldehyde 3,4-Methylenedioxybenzaldehyde 4-Hydroxy-3,5-dimethoxybenzaldehyde 3,4,5-Trimethoxybenzaldehyde 3-Indolebenzaldehyde 4-Hydroxy-3-nitrobenzaldehyde 3,5-di-tert-butyl-4-hydroxybenzaldehyde
95 94 91 74 65 75 83 76 82 72
OCH3 OCH3
OH OCH3
H H H H H OCH3 OCH3
NO2 t-C4H9
OH OH
H t-C4H9
OCH2O
silicagel Amicon 60 Am, respectively. HPLC analyses were obtained using a column Symmetry C8 5A (Waters) and the percentage purity of the synthesized derivatives was higher than 98%. There was no secondary product. Percentage yields are indicated in Tables 1 and 2. Isolation of liver mitochondria Rat liver mitochondria were isolated as described previously [17]. Male Wistar rats were decapitated. The livers were removed, weighed, and cut into small pieces in ice-cold buffer A (250 mM sucrose, 50 mM Tris, 5 mM EGTA, pH 7.2, at 4jC). The pieces were rinsed two times and homogenized in the same buffer. The homogenates were then centrifuged at 600g for 10 min at 4jC. The supernatants were centrifuged at 15,000g for 5 min at 4jC and the pellets were resuspended in the same buffer and centrifuged at 15,000g for 5 min. The mitochondrial pellet was washed in buffer B (250 mM sucrose, 50 mM Tris, pH 7.2, at 4jC) and centrifuged for 5 min at 15,000g. The final pellets were suspended in the same buffer and the protein concentration was determined using the method of Lowry et al. [18]. The final pellet contained approximately 60 mg of protein/ml. All animal procedures used in this study are in strict accordance with the European Community Council Directive of 24 November 1986 (86-609/EEC) and Decree of 20 October 1987 (87-848/EEC). Mitochondrial swelling Mitochondrial swelling was assessed by measuring the change in absorbance of the suspension at 540 nm (A540) by using a Hitachi Model UV-3000 spectrophotometer. Experiments were carried out at 25jC in 1.8 ml
of buffer C (250 mM sucrose, 5 mM KH2PO4, pH 7.2) with addition of 2 AM rotenone and 6 mM succinate. Mitochondria (1 mg/ml) were incubated for 1 min in this buffer and swelling was induced by addition of increasing concentrations of curcumin or its derivatives. Mitochondrial swelling was also studied under deenergized conditions. Mitochondria (1 mg/ml) were preincubated for 1 min in a Tris buffer consisting of 150 mM sucrose, 5 mM Tris, 0.5 Ag/ml of rotenone, and 0.5 Ag/ml of antimycin, pH 7.4, at 25jC in a total volume of 1.8 ml prior to the addition of 100 AM CaCl2. Swelling was initiated 4 min later by introducing 20 AM curcumin. Determination of cytochrome C release by Western blot analysis Mitochondria (1 mg/ml) were suspended in 250 Al of buffer C including 2 AM rotenone and 6 mM succinate and incubated with either curcumin, curcumin derivatives, or Ca2+ in the absence or the presence of 2 AM CsA for 20 min. The mitochondrial suspension was centrifuged at 15,000 g for 10 min at 4jC and 5 Al (0.4 mg/ml) of the resulting supernatant was added to 5 Al of a buffer containing sucrose (20%), SDS (2.4%), h-mercaptoethanol (5%), and bromphenol blue (5%). Samples were boiled at 100jC, subjected to electrophoresis on a 4– 15% gradient SDS – polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk in a Tris buffer (Tris 0.02 M, NaCl 0.14 M, pH 7.6) containing 0.1% Tween 20, and incubated overnight at 4jC with mouse monoclonal anti-rat cytochrome C antibody (5/ 1000; MAB897, R&D systems, UK). After incubation with sheep anti-mouse horseradish peroxidase (1/1000; Amersham Pharmacia Biotech, Les Ulis, France) 1 h at
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room temperature, the blots were revealed by enhanced chemiluminescence reaction (Amersham ECL+) and exposed to X-ray film (Sigma, Biomax MS-1 film). Assays of radical scavenging capacity and of lipid peroxidation The free radical scavenging capacity of compounds was determined using diphenyl-2-picryl-hydrazyl stable free radical (DPPH ) [19]. Test compounds were added to an ethanol solution of DPPH (100 AM) and the decrease in absorbance was recorded against time at 515 nm in a Hitachi U-3000 spectrophotometer. The curves allowed estimation of the effective concentration of the compound tested producing a 50% decrease in DPPH concentration at steady state (IC50). Lipid peroxidation was assessed as the generation of thiobarbituric acid-reactive substances (TBARS), i.e., lipid peroxides, according to [20]. Mitochondria (0.2 mg/ml) were suspended in NaCl (0.9%) supplemented with different concentrations of curcumin or its derivatives in a total volume of 1 ml. Identical concentrations of curcumin or curcumin derivatives were diluted in NaCl without addition of mitochondria to make color controls. All tubes were incubated for 10 min at 37jC. Following addition of 100 Al of a mixture containing FeCl2 (500 AM)/FeCl3 (1500 AM), the tubes were incubated for 30 min at 37jC. After addition of 1 ml trichloracetic acid (3%), all tubes were centrifuged at 20jC for 15 min at 3000 rpm and 1 ml of each supernatant was added to 1 ml of thiobarbituric acid (1%) and incubated for 30 min at 95jC. After recooling on ice, the generation of TBARS was determined by measuring the absorbance at 530 nm.
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was added to 800 Al of a solution containing 6 M guanidine, HCl, 1 M NH3, and 1 M NH4Cl (pH 9.0 at 20jC). Twenty microliters of Ellman’s reagent (20 mM) was then added and the reaction was incubated for 15 min at room temperature. Absorbance of the medium was read at 412 nm against Ellman’s reagent blank. The amount of thiol groups was estimated using the difference in absorbance at 412 nm before and after addition of Ellman’s reagent corrected for the absorbance of Ellman’s reagent. To estimate protein thiol concentration, glutathione in the concentration range 10 to 500 AM was assayed under the same conditions. Results for thiol groups are expressed as percentages and a 100% value corresponds to the reduced thiol groups, measured in control mitochondria. This value was equal to 240 F 23 nmol/mg proteins. A value lower than 100% revealed oxidation of thiol groups, whereas a higher value reflected inhibition of the spontaneous oxidation occurring during the incubation period. Values are expressed as means F SEM fo three independent experiments done in triplicate. Measure of mitochondrial membrane potential (DW) DC was monitored by means of the fluorescent dye rhodamine 123 according to [22] at excitation and emission wavelengths of 503 and 527 nm, respectively. Mitochondria (0.5 mg/ml) were incubated in 1.8 ml buffer C supplemented with 2 AM rotenone and 0.3 AM rhodamine 123. After 30 s, 6 mM succinate was added. When DC was established, 20 AM curcumin or curcumin derivatives were added to the medium and the times of DC collapses were compared. Relative changes in membrane potential were expressed in arbitrary fluorescence units and were not converted to potential values.
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The generation of O2 was achieved as previously reported [20] by measuring the reduction of nitroblue tetrazolium in monoformazan that absorbed at 560 nm. Mitochondria (1 mg/ml) were incubated for 1 min in 1.2 ml buffer C supplemented with 2 AM rotenone, 1 AM cyclosporin A (CsA), and 100 AM nitroblue tetrazolium. For this particular experiment, CsA was added to inhibit mitochondrial swelling, which slightly interfered with spectroscopic detection of the reduction reaction. The production of O2 was then initiated by the addition of 6 mM succinate.
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Action on complex III of the respiratory chain Mitochondrial complex III activity was determined by a spectrophotometric method measuring the rate of cytochrome C reduction at 550 nm and 37jC triggered by decylubiquinol. The reaction mixture (1 ml buffer C) contained 2 mM ethylenediaminetetraacetic acid, 0.1 mM KCN, 2 AM rotenone, 0.1 mM decylubiquinol, and 100 Ag of mitochondria. The reaction was initiated by the addition of 50 AM of cytochrome C and the rate of reduction was measured for 2 min. The experiment was performed in the absence or the presence of curcumin or curcumin derivatives.
Protein thiol determination Protein thiol content was measured according to [21] with some modifications. Briefly, mitochondria (1 mg/ml) were incubated in buffer C in the presence of curcumin or other molecules in a total volume of 1 ml for 15 min at 25jC. After this time, 200 Al of mitochondrial solution
Measurement of reduction of Fe3+ to Fe2+ Reduction of Fe3+ to Fe2+ was measured by the ferrozine complex method [23]. The reaction mixture consisted of ferrozine (100 AM) and FeCl3 (100 AM) in a final volume of 2 ml 0.9% NaCl (pH 7). The reaction was
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initiated by addition of curcumin derivatives (20 AM) and the absorbance of the Fe2+ – ferrozine complex was monitored at 560 nm.
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Table 3. Curcumin Derivatives Induced Mitochondrial Swelling Compounds
Swelling Energized conditions
RESULTS
Induction of mitochondrial swelling by curcumin and curcumin derivatives Rat liver mitochondria energized with succinate were incubated in a sucrose – phosphate buffer (buffer C) before addition of increasing concentrations of curcumin. Figure 1A shows that curcumin induced mitochondrial swelling as revealed by the large decrease in absorbance of the mitochondrial suspension at 540 nm. This effect was concentration-dependent and maximal swelling was
EC50 (AM)
Curcumin Cu02 Cu03 Cu04 Cu05 Cu06 Cu07 Cu08 Cu09 Cu10 Cu11 Cy01 Cy02 Cy03 Cy04 Cy05 Cy06 Cy07 Cy08 Cy10 Cy11 Ferulic acid Ferulic aldehyde Vanillin
Deenergized conditions
Ratea (% of Ca2+ effect)
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effect effect effect effect
Induction No effect Induction Induction No effect Induction No effect No effect No effect No effect No effect Induction No effect No effect Induction No effect Induction No effect No effect No effect No effect
No effect No effect
No effect No effect
No effect
No effect
No effect No effect 32
59 No effect
48
87 No No No No No
effect effect effect effect effect
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58 No effect No effect
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9 No effect
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85 No No No No
a
Rate of swelling induced by curcumin derivatives compared with the rate of swelling induced by 50 AM Ca2+ (i.e., 100%). These effects were obtained at 30, 60, 200, 60, 20, and 200 AM for curcumin, Cu04, Cu06, Cy01, Cy04, and Cy06, respectively.
Fig. 1. Effect of curcumin on mitochondrial swelling and mitochondrial potential. (A) Mitochondrial swelling: Liver mitochondria (1 mg/ml) were preincubated for 1 min in buffer C supplemented with 6 mM succinate and 2 AM rotenone, pH 7.2, at 25jC. Swelling was induced by the addition of either 1 (line a), 10 (line b), 20 (line c), 30 (line d), 40 (line e), 50 (line f), or 60 (line g) AM curcumin. Line h: swelling was induced by 50 AM Ca2+. Line i: effect of 20 AM curcumin in the presence of 1 AM CsA. (B) Mitochondrial potential: Liver mitochondria were suspended in incubation buffer C supplemented with 0.3 AM rhodamine 123. DC was measured after addition of 6 mM succinate. Line a: no other addition. Line b: 20 AM curcumin. Line c: 20 AM curcumin + 1 AM CsA. Line d: addition of 1 AM of the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) induced complete DC depolarization. Curcumin and CsA were added just after mitochondria. The figures show representative results from four independent experiments using four independent mitochondrial preparations.
obtained around 30 AM. It was associated with complete DC depolarization as observed after addition of the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Fig. 1B). These effects were mediated by opening of the PTP as they were completely prevented by 1 AM CsA (Figs. 1A, B), a well-established inhibitor of PTP [24,25]. The kinetic rate of swelling induced by 30 AM curcumin was equivalent to 48% of that observed in the presence of 50 AM Ca2+, a well-known inducer of PTP (Fig. 1A, Table 3). It should be noted that phosphate greatly contributed to the effect of curcumin because PTP was almost completely inhibited when swelling experiments were performed in a Tris buffer devoid of phosphate ions (data not shown). When curcumin concentrations higher than 30 AM were used, a decrease in swelling was observed; at 60 AM no further swelling occurred (Fig. 1A). We used the same protocols to evaluate the ability of 20 curcumin derivatives (Tables 1 and 2) and three curcumin metabolites, ferulic acid, ferulic aldehyde, and vanillin [12], to induce PTP. Among these compounds three, Cy01, Cy04, and Cu04, showed the same biphasic profile
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as curcumin. At low concentrations, they induced a concentration-dependent swelling which disappeared for higher concentrations. The maximal swelling-inducing effect was observed at 60, 60, and 20 AM for Cu04, Cy01, and Cy04, respectively and corresponded to 59, 58, and 9% of that observed with 50 AM Ca2+, respectively (Table 3). Two other compounds, Cu06 and Cy06, were also able to promote swelling. However, for these agents the swelling was proportional to their concentrations. At 200 AM the rate and the extent of the swelling were close to those obtained with 50 AM Ca2+. Other agents were ineffective. It is now well-established that PTP opening resulted in release of the apoptotic factor cytochrome C into the cytosol [26]. Therefore, to confirm the PTP-inducing effect of curcumin derivatives, we analyzed their effect on cytochrome C release. The molecules were assayed at the concentrations that induced maximal swelling. Figure 2 shows that curcumin, Cu04, Cu06, Cy01, Cy04, and Cy06 caused the release of cytochrome C from energized mitochondria and that this effect was completely prevented by 2 AM CsA. Identical results were observed in the presence of 50 AM Ca2+.
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curcumin derivatives on O2 and hydroxyl radical (HO ) production, thiol oxidation, and DPPH reduction (Table 4). Curcumin inhibited O2 production and lipid peroxidation and was able to oxidize membrane thiol functions and to reduce DPPH . Curcumin derivatives showed properties different from those of curcumin. Indeed, some molecules inhibited O2 production and did not abolish lipid peroxidation (CuO7, Cu10, CyO3, CyO4, Cy10) whereas CuO8 and CyO8 inhibited both effects without reducing DPPH . In the same way, CyO4 induced thiol group oxidation but was ineffective in promoting lipid peroxidation. We then compared the redox properties of curcumin derivatives with their ability to induce PTP opening. The study demonstrated that no clear relation exists between the inhibition of O2 production and the induction of swelling. Indeed, several curcumin derivatives (Cu03, Cu07, Cu10, Cy03, Cy08, Cy10) revealed potent O2 -inhibiting properties without swelling induction properties, whereas Cy06, which is a potent inductor of swelling, is a poor inhibitor of O2 production under the same experimental conditions (Table 4). It should be noted that all these molecules were tested for their ability to interact directly with complex III of the respiratory chain which is the main generator of O2 in our model. They did not show any effect (data not shown). Thus, their effect might be due to scavenging of O2 as previously observed for curcumin [10,28] and not to the inhibition of O2 generation. In the same way, there was no relation between lipid peroxidation and swelling induction as
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Effects of curcumin and curcumin derivatives on reactive oxygen species Increasing amounts of evidence suggest that oxidative stress plays a key role in PTP induction [27]. Consequently, we decided to study the effects of curcumin and
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Fig. 2. Cytochrome C release from isolated liver mitochondria induced by curcumin and related derivatives. Liver mitochondria were suspended in buffer C supplemented with 6 mM succinate and 2 AM rotenone, pH 7.2, at 25jC and incubated for 20 min with curcumin, curcumin derivatives, or Ca2+ in the presence (+) or the absence () of 2 AM CsA. The results are typical of four independent experiments. The signal intensity of cytochrome C was evaluated by densitometry (n = 4 per group).
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Curcumin and mitochondria Table 4. Comparison of the Effect of Curcumin Derivatives on Mitochondrial swelling, O2 and HO Production, Thiol Oxidation, and DPPH Reduction
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Molecules Swelling O2 induction inhibition inhibition (% at IC50 (AM) 20 AM) Curcumin Cu02 Cu03 Cu04 Cu05 Cu06 Cu07 Cu08 Cu09 Cu10 Cu11 Cy01 Cy02 Cy03 Cy04 Cy05 Cy06 Cy07 Cy08 Cy10 Cy11
Yes No No Yes No Yes No No No No No Yes No No Yes No Yes No No No
77 0 100 67 0 37 36 44 0 39 0 56 0 55 18 0 18 No effect 24 28
groups (%)
0.7 74.3 F 4.3 No effect 100 F 4 3.1 60.8 F 3.7 4.1 89.6 F 3.2 No effect 100 F 3 18 77.2 F 3.7 No effect 100 F 5.2 8 100 F 3.8 No effect 115.3 F 4.8 >100 109 F 1.2 0.8 100 F 2.8 4. 1 76.5 F 3.9 No effect 100 F 4.3 >100 97.3 F 1.8 >100 94.3 F 1.5 >100 110.0 F 4.1 7.3 65.4 F 2.6 No effect 107.0 F 2.0 1.8 120.1 F 6.0 No effect 100 F 3.3 Nonsoluble
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DPPH reduction IC50 (AM)
12.5 No effect 380 110 No effect 65 No effect No effect No effect No effect 65 27 No effect No effect >400 No effect >400 No effect No effect No effect
Cy04 did not inhibit lipid peroxidation whereas Cu11, which is as potent as curcumin in inhibiting lipid peroxidation, was unable to promote swelling. Thus, the effect of curcumin was not due to its interaction with O2 and/or HO which otherwise would give rise to a toxic curcumin radical. A correlation was observed between the ability to induce swelling and the ability to promote the oxidation of membrane thiol groups as all curcumin derivatives, which induced swelling under either energized or deenergized conditions or both (Table 3), induced membrane thiol group oxidation, whereas the other derivatives did not (Table 4). The hypothesis that thiol groups may be involved in swelling induction was also reinforced by the fact that the thiol substitution compound N-ethylmaleimide completely prevented the effects of curcumin (Fig. 3). However, in the same experiment monobromobimane, which as N-ethylmaleimide forms adducts with glutathione, was inactive (Fig. 3). This seems to rule out a possible role of glutathione in the mechanism of action of curcumin. It should be noted that Cu03 caused swelling under deenergized conditions but not in respiring medium. This is probably due to the fact that Cu03 acts as an uncoupling agent. Indeed, Cu03 prevented O2 formation (Table 4), increased the rate of oxygen consumption and collapsed DC (data not shown) as observed with uncouplers [29]. Deenergized mitochondria were used to study the mechanism by which molecules that induced swelling
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oxidized thiol groups. These experimental conditions have the advantage of eliminating respiration and membrane potential in the swelling process and facilitate the study of low oxygen concentrations. To this end, mitochondria were incubated in a Tris buffer in the absence of substrate and in the presence of respiratory chain inhibitors (rotenone and antimycin) and swelling was induced by high Ca2+ concentrations. Under these experimental conditions curcumin and curcumin derivatives that induced PTP opening under energized conditions were able to induce swelling but at a lower rate and extent (Table 3). When oxygen was exhausted from the incubation medium through nitrogen purging, this phenomenon was completely inhibited, indicating the requirement of ROS in curcumin-induced swelling. In the same way, PTP opening by curcumin was inhibited by an excess of exogenous catalase (Fig. 3) suggesting that hydrogen peroxide (H2O2) or ROS derived from H2O2 are involved in the mechanism. Indeed, H2O2, which is produced following dismutation of O2 , can react with mitochondrial Fe2+, resulting in the formation of HO [30]. We therefore decided to study the effect of mannitol, a well-known scavenger of HO , and of Fe2+ chelators, ferrozine and o-phenanthroline, on curcumininduced swelling. Fig. 3 shows that preincubation of mitochondria with either mannitol, ferrozine, or o-phenanthroline inhibited mitochondrial swelling. The same results were obtained with either Cu04, Cu06, Cy01, Cy04, or Cy06. In a last step, we measured a possible effect of curcumin and curcumin derivatives on the reduction of Fe3+ to Fe2+. Figure 4 shows that all curcumin derivatives that were able to induce swelling promoted Fe3+
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Fig. 3. Inhibition of curcumin-induced mitochondrial swelling under deenergized conditions. Liver mitochondria (1 mg/ml) were incubated in a Tris sucrose buffer in the absence (line a) or the presence of monobromobimane (line b), 9000 U catalase (line c), 400 AM ophenanthroline (line d), 500 AM ferrozine (line e), 50 AM Nethylmaleimide (line f), 120 AM mannitol (line g), or nitrogen purging (line h) for 1 min before addition of 100 AM Ca2 +. Four minutes later swelling was induced by the addition of 40 AM curcumin. The figure shows representative results from four independent experiments using four independent mitochondrial preparations.
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can act as a scavenger of HO or catalyze the formation of HO depending on the experimental conditions [12]. Up to now it has not been well-established which part of the molecule is responsible for the effect of curcumin in oxygen radical reactions and conflicting results exist concerning its antioxidant mechanism. However, most of the studies underline the predominant role of both the phenolic and methoxy groups in the phenyl ring and the 1,3-diketone moiety [10,12]. The purpose of the present study was to investigate the mechanism by which curcumin promotes PTP, to define the chemical functions involved in PTP-induction, and finally to select a curcumin derivative that would display only PTP-inducing or inhibiting properties. A total of 20 compounds were synthesized and two major groups could be distinguished. The first group preserved the basic skeleton of curcumin but modified the aromatic substituents (compounds Cu02 –Cu11). The second group had a cyclohexanone ring instead of the linear diketone chain with modifications of the aromatic substituents (compounds Cy01 – Cy11). The most important structural feature seems to be the presence of a hydroxyl group in the aromatic groups. Indeed, all curcumin derivatives that are able to induce swelling (Cu04, Cu06, Cy01, Cy06, and, to a lesser extent, Cy04) possess a free hydroxyl group in the phenyl ring. In addition, when the phenolic group is blocked by a methyl (Cu07, Cy07) or a methylene dioxide (Cu05, Cy05) or substituted by a butane chain (Cu09) the activity of curcumin disappeared. This indicates that the phenolic group is essential for activity. Swelling induction is more effective when the phenolic hydroxyl group is located in the para than in the ortho position (compare curcumin and Cy01 with Cu04 and Cy04, respectively). Whether the presence of a hydroxyl group seems to be a necessary criterion, it did not appear to be sufficient because no effect was obtained with other hydroxyl compounds such as Cy03, Cu10, Cy10, Cu11, and Cy11. In fact, the effect also required the presence of an electron-donating group in the ortho position near the hydroxyl function of the aromatic ring (compounds curcumin, Cy01, Cu06, Cy06, Cu11, Cy11). These substituents must not be too bulky as the ortho-di-tert-butyl derivatives (compounds Cu11 and Cy11) were ineffective. By contrast, an electron-attracting group such as NO2 (Cu10, Cy10) inhibited the effect. There is strong evidence that the h-diketone moiety is not directly involved in curcumin induced-swelling because the nonphenolic curcumin derivatives (Cu02, Cu08, Cu09) were without effect. However, the effect of curcumin and curcumin derivatives seems to require the h-diketone moiety as it was attenuated when the hdiketone was substituted by the cyclohexanone ring
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Fig. 4. Reduction of Fe3 + to Fe2 + by curcumin derivatives measured as increase in absorbance of ferrozine complex. Curcumin derivatives (20 AM) were added to a solution of 0.9% NaCl containing 100 AM ferrozine and 100 AM FeCl3. Line a: Cy01. Line b: curcumin. Line c: Cu06. Line d: Cy06. Line e: Cu04. Line f: Cy04. Other compounds were inactive. The figure is representative of three independent experiments.
reduction. Curcumin, Cu06, Cy01, and Cy06 were the most effective. Cu04 and Cy04 also stimulated Fe3+ reduction but to a lesser extent. The other compounds were without effect. DISCUSSION
The present study shows that curcumin induces swelling, a decrease in the mitochondrial potential, and the release of cytochrome C in isolated liver mitochondria. These effects can be attributed to the occurrence of PTP as they are completely inhibited by CsA, a specific inhibitor of PTP [24]. It should be noted that curcumin-induced swelling is proportional to the concentration used up to 30 AM but that higher concentrations promoted a decrease in the extent of swelling. This biphasic profile may explain the opposite effects of curcumin which showed both anti- and proapoptotic properties in different experimental models [4– 6,31,32]. Whereas a controversy persists concerning the molecular composition of this pore, there are compelling arguments that indicate that PTP is a multiproteic complex [8]. However, despite extensive work, the nature of the mitochondrial membrane conformational change leading to PTP is still unknown. PTP is caused by mitochondrial matrix accumulation of excessive quantities of Ca2+ and can be enhanced by a variety of agents or conditions [25]. Oxidative stress seems to play a key role in PTP induction [27] with the observation that the oxidation or the crosslinking of membrane protein thiol groups is a critical event [25,33]. Because PTP opening is closely related to redox phenomena, we postulated that the oxidative properties of curcumin might be responsible for its effect. Curcumin
Curcumin and mitochondria
(compounds Cy01, Cy06, and Cy04) and was not observed with ferulic acid. In summary, this structure – activity study reveals that swelling requires the presence of a phenolic hydroxyl group associated with a radical displaying electron donation properties. It is interesting to note that these structural features are those that explain the antioxidant properties of curcumin. Indeed, it has been reported that the phenolic group is essential to the antioxidant effect [10,34] of curcumin and that the presence of a methoxy group adjacent to the phenolic group increases the free radical scavenging properties [11,35,36]. In fact, the ortho substitution with an electron donating group such as a methoxy seems to increase the antioxidant activity of phenols by facilitating the formation and enhancing the stability of the phenoxyl radical. Taken together, these data suggest that the functions that are responsible for the antioxidant properties of curcumin are also responsible for PTP opening. This is further emphasized by the fact that curcumin, Cu04, Cu06, and Cy01, which display strong antioxidant activity in the DPPH test, are able to induce swelling. This study also reveals that ferulic acid, vanillin, and ferulic aldehyde, which are obtained by curcumin decomposition [12], are totally ineffective in all tests, demonstrating that curcumin effect is not due to these metabolites. In a recent article we suggested that the effect of curcumin might be related to its interaction with O2 [9]. The hypothesis was that curcumin, during mitochondrial respiration, interacted with O2 to give birth to a radical that was toxic to mitochondria. The present study did not permit confirmation of this hypothesis primarily because all compounds that induced swelling did not interact with O2 and/or HO . In fact, PTP opening may be due to thiol group oxidation because all compounds that induced swelling were found to oxidize thiol groups and the thiol substitution agent N-ethylmaleimide completely prevented PTP opening. This suggestion is consistent with previous data indicating that oxidation of critical thiol groups of the adenine nucleotide translocase may be responsible for PTP opening [33,37]. It should be noted that N-ethylmaleimide is an inhibitor of the phosphate carrier [38] but this property cannot explain the inhibitory effect of N-ethylmaleimide on PTP as prevention of PTP was observed under deenergized conditions where phosphate was absent from the reaction medium. The remaining question was how curcumin induced thiol group oxidation. Our results showing the inhibition of PTP opening either in an anaerobic medium or in the presence of catalase strongly support the notion that ROS are involved in the process. In addition, the protective
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effect conferred by the Fe2+ chelators ferrozine and ophenanthroline suggests that the Fenton reaction, which describes the nonenzymatic reduction of H2O2 to hydroxyl anion and HO by oxidation of Fe2+, could be involved in the process. This idea is reinforced by the fact that curcumin derivatives that induce swelling are able to reduce Fe3+ into Fe2+. The effect of mannitol, which inhibits curcumin- and curcumin derivative-induced PTP opening, is also supportive of HO generation. This confirms previous results indicating that curcumin is able to catalyze the formation of HO by reducing Fe3+ into Fe2+ [12,39]. Thus, on the basis of our present data, we propose that the molecular mechanism of curcumin leading to PTP opening is related to its antioxidant properties. Under our experimental conditions, curcumin reduces Fe3+ into Fe2+, which in turn reacts with H2O2 and increases the rate of HOS production. This highly reactive radical oxidizes critical thiol groups which leads to PTP opening in the presence of Ca2+. Fig. 5 summarizes the proposed molecular process involved in the opening of PTP by curcumin. This could represent a general mechanism by which several reducing agents such as polyphenols were shown to affect mitochondrial functions [40]. This may also be one of the steps of the mechanism by which curcumin induces apoptosis of tumor cells [6,41] and thus acts as an antiproliferative agent [4]. In this regard, some of the derivatives synthesized in this study such as Cu06 and Cy06 which induce a concentrationdependent opening of the PTP display interesting properties. By contrast, the compounds Cu08 and Cy08 that show antioxidant effects without PTP induction could
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Fig. 5. Scheme summarizing the proposed molecular processes by which curcumin induces PTP opening. The respiratory chain, inserted in the inner mitochondrial membrane, generates the superoxide radical (O2 ). O2 is removed by superoxide dismutase, which promotes the generation of hydrogen peroxide (H2O2). H2O2 is then reduced to water by glutathione peroxidase or catalase. When H2O2 is accumulated in quantities too large for removal, in the presence of Fe2 +, H2O2 generates a highly reactive HO radical that oxidizes thiol ( – SH) groups, leading to pore opening. Curcumin catalyzes the formation of HO by reducing Fe3 + into Fe2 +.
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represent promising curcumin derivatives as cytoprotective agents.
Acknowledgments—The authors thank Felipe Pires for his skillful technical assistance. This work was supported by the Ministe`re de l’Education Nationale (DRED EA 427).
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929 ABBREVIATIONS
CsA — cyclosporin A CCCP — carbonyl cyanide m-chlorophenylhydrazone DPPH — diphenyl-2-picryl-hydrazyl stable free radical DC — mitochondrial membrane potential PTP — permeability transition pore ROS — reactive oxygen species TBARS — thiobarbituric acid-reactive substances
S