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Minocycline prevents peroxidative permeabilization of cardiolipin-containing bilayer lipid membranes mediated by cytochrome c
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Minocycline prevents peroxidative permeabilization of cardiolipincontaining bilayer lipid membranes mediated by cytochrome c Alexander M. Firsov, Elena A. Kotova*, Yuri N. Antonenko Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 October 2018 Accepted 13 November 2018 Available online xxx
Peroxidase activity of cytochrome c stimulated by interaction of the protein with cardiolipin is considered to be involved in the induction of mitochondrial apoptosis associated with cytochrome c release from mitochondria. In model systems, this activity has been previously shown to cause permeabilization of cardiolipin-containing membranes. Here, we found that the antibiotic minocycline, known to have neuroprotective properties, inhibited both the binding of cyt c to cardiolipin-containing membranes and the cyt c/H2O2-induced liposome permeabilization. The results could be relevant to inhibition of cyt c release from mitochondria exerted by minocycline. © 2018 Elsevier Inc. All rights reserved.
Keywords: Apoptosis Membrane permeabilization Calcein leakage Peroxidase Cytochrome c Minocycline
1. Introduction
2. Materials and methods
In 2002 Friedlander and colleagues [1] reported pronounced suppression of mitochondrial cytochrome c (cyt c) release in vivo, in cells and in isolated mitochondria by minocycline, which was supported by later studies [2e4]. These findings obviously were of high importance due to the known broad inhibition of cell death by this clinically available antibiotic and crucial role of cyt c release in apoptosis. Despite enormous body of knowledge on beneficial effects of minocycline in various models of neurological disorders [5], accumulated during the last 15 years, as well as numerous data on minocycline interaction with mitochondria [4,6e11], the mechanism of the minocycline-induced inhibition of cyt c release from mitochondria is not well understood at present [12]. According to Kagan and colleagues [13], strong peroxidase activity acquired by cyt c upon its interaction with cardiolipin plays a significant role in promoting the release of this protein from mitochondria in the course of apoptosis initiation. Previously peroxidase activity of cyt c has been shown to cause permeabilization of artificial cardiolipincontaining lipid membranes [14e17]. Here, we report data on substantial prevention of such permeabilization by minocycline, which could be of relevance to its inhibiting effect on cyt c release from mitochondria.
2.1. Chemicals Most chemicals including equine heart cyt c, bovine heart cardiolipin, calcein, sulforhodamine B were from Sigma. 2.2. Preparation of liposomes Dye-loaded liposomes were prepared by evaporation under a stream of nitrogen of a 2% solution of a mixture of lipids in chloroform (4 mg soybean phosphatidylcholine (Sigma, Type II-S) and 1 mg bovine heart cardiolipin) followed by hydration with a buffer solution containing appropriate fluorescent marker. Two different marker solutions (0.5 ml each) were used: 1) 50 mM calcein, 50 mM KCl, 5 mM Tris, 5 mM MES, pH 7.4 and 2) 1 mM sulforhodamine B in 100 mM KCl, 10 mM Tris, 10 mM MES, pH 7.4. The mixture was vortexed, passed through several cycles of freezing and thawing, and extruded through 0.1-mm pore size Nucleopore polycarbonate membranes using an Avanti Mini-Extruder. The unbound marker was then removed by passage through a Sephadex G-50 coarse column with a buffer solution containing 100 mM KCl, 10 mM Tris, 10 mM MES, pH 7.4. 2.3. Liposome permeabilization assays
* Corresponding author. E-mail address: [email protected] (E.A. Kotova).
In the fluorescence dequenching assay, fluorescence of calcein-
https://doi.org/10.1016/j.bbrc.2018.11.078 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: A.M. Firsov et al., Minocycline prevents peroxidative permeabilization of cardiolipin-containing bilayer lipid membranes mediated by cytochrome c, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.078
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loaded liposomes was monitored at 520 nm (excitation at 490 nm) with a Panorama Fluorat 02 spectrofluorimeter (Lumex, Russia). The extent of calcein efflux was calculated as (Ft e F0)/(F100-F0), where F0 and Ft represent the initial fluorescence intensity and the fluorescence intensity at the time t, and F100 is the fluorescence intensity after complete disruption of liposomes by addition of the detergent Triton-X100 (final concentration, 0.1% w/w). Cyt c/H2O2-induced leakage of sulforhodamine B was monitored with a fluorescence correlation spectroscopy (FCS) setup, as described previously [16]. 2.4. Chemiluminescence assay of peroxidase activity Peroxidase activity was assessed by chemiluminescence response [13,14,18] arising from H2O2-induced oxidation of luminol in the presence of cyt c. Chemiluminescence was detected with a Lum-100 luminometer, as described in Ref. [19]. 2.5. Centrifugation binding assay Protein-liposome ultracentrifugation binding assays were performed as previously described [20e22], with certain modifications [23]. In particular, liposomes were prepared in the buffer containing 200 mM sucrose and subsequently dispersed in the buffer containing 200 mM glucose. Samples were mixed by pipetting and incubated 30 min prior to centrifugation. The samples were centrifuged for 60 min using a Beckman J2-21 ultracentrifuge equipped with a Beckman JA-21 rotor. The centrifuge was run at 50000g using compressed nitrogen. After completion of each run, the supernatant was immediately removed and its absorption spectrum measured. Bound (pelleted) protein was calculated as total minus free. 3. Results and discussion Based on the inhibiting effect of minocycline on peroxidase activity of cyt c found by Fiorucci and colleagues [24], we decided to study the influence of this antibiotic on permeabilization of cardiolipin-containing liposomes induced by cyt c in the presence of hydrogen peroxide. To measure permeabilization, we used two assays: 1) an increase in fluorescence intensity (removal of fluorescence self-quenching) caused by leakage into the bulk solution of the fluorescent dye calcein entrapped in liposomes at a high concentration (50 mM) [23,25] and 2) a decrease in the amplitude
Fig. 1. Effect of minocycline on calcein leakage from cardiolipin-containing liposomes induced by cytochrome c and H2O2. Concentrations: cyt c, 1 mM; H2O2, 1.5 mM. Triton X-100 (0.1%) was added at the end of the experiment to obtain 100% leakage. The solution was 200 mM sucrose, 10 mM Tris, 10 mM MES, pH 7.4. Lipid concentration, 10 mg/ml.
of the autocorrelation function of the fluorescent dye sulforhodamine B resulting from an increase in the number of fluorescent particles due to release of numerous dye molecules from liposomes [16]. Fig. 1 shows that cyt c in combination with H2O2 caused a pronounced increase in calcein fluorescence reflecting the dye leakage from liposomes, in agreement with our previous data [23,25]. The addition of minocycline at a concentration of 1 mM caused moderate suppression of the calcein fluorescence response. The inhibiting effect of minocycline increased with concentration, so that at 10 mM of the antibiotic, the inhibition of the dye leakage became strongly pronounced and at 100 mM the leakage was completely suppressed. These data were confirmed in FCS experiments with sulforhodamine B leakage from liposomes (data not shown). Fig. 2A illustrates the effect of minocycline on the intensity of luminol chemiluminescence in the presence of the cardiolipincontaining liposomes reflecting the peroxidase activity of cyt c [13]. It is seen that minocycline, at a concentration of 1 mM, substantially inhibited the luminol chemiluminescence elicited by cyt c/H2O2, while a significant inhibition of the luminol
Fig. 2. Effect of minocycline on the intensity of luminol chemiluminescence induced by cytochrome c/H2O2 (A) or Fe/ascorbate (B) in the presence of cardiolipin-containing liposomes. Cyt c, 50 nM; H2O2, 1 mM; FeSO4, 100 mM; ascorbate, 1 mM; luminol, 1mM. The buffer solution of 10 mM Tris, 10 mM MES, pH 8.5 contained 200 mM sucrose. Lipid concentration, 10 mg/ml.
Please cite this article as: A.M. Firsov et al., Minocycline prevents peroxidative permeabilization of cardiolipin-containing bilayer lipid membranes mediated by cytochrome c, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.078
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Fig. 3. Effect of minocycline on the binding of cytochrome c to cardiolipin-containing liposomes, as assessed by centrifugation of liposomes prepared in the sucrose medium. Concentrations: cyt c, 1 mM; lipid, 120 mg/ml. The incubation mixture contained 200 mM glucose, 10 mM Tris, 10 mM MES, pH 7.4.
chemiluminescence induced by Fe/H2O2, i.e. a ROS generating system not involving cyt c, was observed at 100 mM and higher concentrations of minocycline (Fig. 2B). According to docking simulation performed by Ref. [24], minocycline can specifically bind cyt c, which may disturb the binding of the protein to cardiolipin, thereby suppressing the induction of the peroxidase activity and membrane permeabilization. To test this hypothesis, we measured cyt c binding to the cardiolipincontaining liposomes by ultracentrifugation. The data of Fig. 3 show that minocycline at a concentration of 10 mM significantly prevented the cyt c binding to the liposomes (p < 0.05). The effect of minocycline on the binding resembles that of ATP, as described in Ref. [26]. Thus, here, we report data on the inhibiting effect of minocycline both on the cyt c-mediated peroxidative permeabilization of cardiolipin-containing liposomes and on the cyt c binding to the liposomes, which could be considered as a plausible mechanism of its anti-apoptotic activity. Noteworthy, minocycline has been reported previously to suppress ROS generation under certain conditions [4,27e29]. In view of these findings and our own observations (Fig. 2B), the antioxidant property of minocycline might be involved in its apoptosis-inhibitory effect. However, based on the present data (Figs. 1, 2A and 3), it can be concluded that the inhibitory effect of minocycline on membrane permeabilization via the suppression of cyt c binding to cardiolipin-containing membranes makes an important contribution to its inhibiting effect on apoptosis, especially at its low concentrations (10 mM and lower). Conflicts of interest The authors declare that there is no conflict of interest. Acknowledgements This work was financially supported by the Russian Science Foundation grant N 14-24-00107. References [1] S. Zhu, I.G. Stavrovskaya, M. Drozda, B.Y. Kim, V. Ona, M. Li, S. Sarang, A.S. Liu, D.M. Hartley, D.C. Wu, S. Gullans, R.J. Ferrante, S. Przedborski, B.S. Kristal, R.M. Friedlander, Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice, Nature 417 (2002) 74e78. [2] S. Matsuki, Y. Iuchi, Y. Ikeda, I. Sasagawa, Y. Tomita, J. Fujii, Suppression of cytochrome c release and apoptosis in testes with heat stress by minocycline,
3
Biochem. Biophys. Res. Commun. 312 (2003) 843e849. [3] T.M. Scarabelli, A. Stephanou, E. Pasini, G. Gitti, P. Townsend, K. Lawrence, C. Chen-Scarabelli, L. Saravolatz, D. Latchman, R. Knight, J. Gardin, Minocycline inhibits caspase activation and reactivation, increases the ratio of XIAP to smac/DIABLO, and reduces the mitochondrial leakage of cytochrome c and smac/DIABLO, J. Am. Coll. Cardiol. 43 (2004) 865e874. [4] A. Gieseler, A.T. Schultze, K. Kupsch, M.F. Haroon, G. Wolf, D. Siemen, P. Kreutzmann, Inhibitory modulation of the mitochondrial permeability transition by minocycline, Biochem. Pharmacol. 77 (2009) 888e896. [5] N. Garrido-Mesa, A. Zarzuelo, J. Gaelvez, Minocycline: far beyond an antibiotic, Br. J. Pharmacol. 169 (2013) 337e352. [6] K. Kupsch, S. Hertel, P. Kreutzmann, G. Wolf, C.W. Wallesch, D. Siemen, P. Schonfeld, Impairment of mitochondrial function by minocycline, FEBS J. 276 (2009) 1729e1738. [7] Y.N. Antonenko, T.I. Rokitskaya, A.J.L. Cooper, B.F. Krasnikov, Minocycline chelates Ca2þ, binds to membranes, and depolarizes mitochondria by formation of Ca2þ-dependent ion channels, J. Bioenerg. Biomembr. 42 (2010) 151e163. [8] M.D. Cuenca-Lopez, A. Karachitos, L. Massarotto, P.J. Oliveira, N. Aguirre, M.F. Galindo, H. Kmita, J. Jordan, Minocycline exerts uncoupling and inhibiting effects on mitochondrial respiration through adenine nucleotide translocase inhibition, Pharmacol. Res. 65 (2012) 120e128. [9] A. Karachitos, J. Jordan, H. Kmita, Cytoprotective activity of minocycline includes improvement of mitochondrial coupling: the importance of minocycline concentration and the presence of VDAC, J. Bioenerg. Biomembr. 44 (2012) 297e307. [10] P. Schonfeld, D. Siemen, P. Kreutzmann, C. Franz, L. Wojtczak, Interaction of the antibiotic minocycline with liver mitochondria e role of membrane permeabilization in the impairment of respiration, FEBS J. 280 (2013) 6589e6599. [11] J. Schwartz, E. Holmuhamedov, X. Zhang, G.L. Lovelace, C.D. Smith, J.J. Lemasters, Minocycline and doxycycline, but not other tetracyclinederived compounds, protect liver cells from chemical hypoxia and ischemia/ reperfusion injury by inhibition of the mitochondrial calcium uniporter, Toxicol. Appl. Pharmacol. 273 (2013) 172e179. [12] R.B. Shultz, Y. Zhong, Minocycline targets multiple secondary injury mechanisms in traumatic spinal cord injury, Neural Regen.Res. 12 (2017) 702e713. [13] V.E. Kagan, V.A. Tyurin, J. Jiang, Y.Y. Tyurina, V.B. Ritov, A.A. Amoscato, A.N. Osipov, N.A. Belikova, A.A. Kapralov, V. Kini, I.I. Vlasova, Q. Zhao, M. Zou, P. Di, D.A. Svistunenko, I.V. Kurnikov, G.G. Borisenko, Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors, Nat. Chem. Biol. 1 (2005) 223e232. [14] M.N. Puchkov, R.A. Vassarais, E.A. Korepanova, A.N. Osipov, Cytochrome c produces pores in cardiolipin-containing planar bilayer lipid membranes in the presence of hydrogen peroxide, Biochim. Biophys. Acta 1828 (2013) 208e212. [15] V.F. Antonov, M.N. Puchkov, E.A. Korepanova, O.Y. Nemchenko, V. Borodulin, Soft perforation of cardiolipin-containing planar lipid bilayer membrane by cytochrome c and H2O2, Eur. Biophys. J. 43 (2014) 469e476. [16] A.M. Firsov, E.A. Kotova, E.A. Korepanova, A.N. Osipov, Y.N. Antonenko, Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex, Biochim. Biophys. Acta 1848 (2015) 767e774. [17] J.P. Kitt, D.A. Bryce, S.D. Minteer, J.M. Harris, Raman spectroscopy reveals selective interactions of cytochrome c with cardiolipin that correlate with membrane permeability, J. Am. Chem. Soc. 139 (2017) 3851e3860. [18] R. Radi, L. Thomson, H. Rubbo, E. Prodanov, Cytochrome c-catalyzed oxidation of organic molecules by hydrogen peroxide, Arch. Biochem. Biophys. 288 (1991) 112e117. [19] Y.A. Vladimirov, E.V. Proskurnina, D.Y. Izmailov, A.A. Novikov, A.V. Brusnichkin, A.N. Osipov, V.E. Kagan, Mechanism of activation of cytochrome c peroxidase activity by cardiolipin, Biochemistry (Mosc.) 71 (2006) 989e997. [20] R. Radi, J.F. Turrens, B.A. Freeman, Cytochrome c-catalyzed membrane lipid peroxidation by hydrogen peroxide, Arch. Biochem. Biophys. 288 (1991) 118e125. [21] J. Hanske, J.R. Toffey, A.M. Morenz, A.J. Bonilla, K.H. Schiavoni, E.V. Pletneva, Conformational properties of cardiolipin-bound cytochrome c, Proc. Natl. Acad. Sci. U. S. A 109 (2012) 125e130. [22] J. Muenzner, J.R. Toffey, Y. Hong, E.V. Pletneva, Becoming a peroxidase: cardiolipin-induced unfolding of cytochrome c, J. Phys. Chem. B 117 (2013) 12878e12886. [23] A.M. Firsov, E.A. Kotova, V.N. Orlov, Y.N. Antonenko, V.P. Skulachev, A mitochondria-targeted antioxidant can inhibit peroxidase activity of cytochrome c by detachment of the protein from liposomes, FEBS Lett. 590 (2016) 2836e2843. [24] A. Patriarca, F. Polticelli, M.C. Piro, F. Sinibaldi, G. Mei, M. Bari, R. Santucci, L. Fiorucci, Conversion of cytochrome c into a peroxidase: inhibitory mechanisms and implication for neurodegenerative diseases, Arch. Biochem. Biophys. 522 (2012) 62e69. [25] A.M. Firsov, E.A. Kotova, Y.N. Antonenko, Calcein leakage as a robust assay for cytochrome c/H2O2 - mediated liposome permeabilization, Analyt. Biochem 552 (2018) 19e23. [26] M. Rytomaa, P. Mustonen, P.K. Kinnunen, Reversible, nonionic, and pHdependent association of cytochrome c with cardiolipinphosphatidylcholine liposomes, J. Biol. Chem. 267 (1992) 22243e22248.
Please cite this article as: A.M. Firsov et al., Minocycline prevents peroxidative permeabilization of cardiolipin-containing bilayer lipid membranes mediated by cytochrome c, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.078
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[27] Y. Miyachi, A. Yoshioka, S. Imamura, Y. Niwa, Effect of antibiotics on the generation of reactive oxygen species, J. Invest. Dermatol. 86 (1986) 449e453. [28] R.L. Kraus, R. Pasieczny, K. Lariosa-Willingham, M.S. Turner, A. Jiang, J.W. Trauger, Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radical-scavenging activity, J. Neurochem. 94
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Minocycline prevents peroxidative permeabilization of cardiolipincontaining bilayer lipid membranes mediated by cytochrome c Alexander M. Firsov, Elena A. Kotova*, Yuri N. Antonenko Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119991, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 October 2018 Accepted 13 November 2018 Available online xxx
Peroxidase activity of cytochrome c stimulated by interaction of the protein with cardiolipin is considered to be involved in the induction of mitochondrial apoptosis associated with cytochrome c release from mitochondria. In model systems, this activity has been previously shown to cause permeabilization of cardiolipin-containing membranes. Here, we found that the antibiotic minocycline, known to have neuroprotective properties, inhibited both the binding of cyt c to cardiolipin-containing membranes and the cyt c/H2O2-induced liposome permeabilization. The results could be relevant to inhibition of cyt c release from mitochondria exerted by minocycline. © 2018 Elsevier Inc. All rights reserved.
Keywords: Apoptosis Membrane permeabilization Calcein leakage Peroxidase Cytochrome c Minocycline
1. Introduction
2. Materials and methods
In 2002 Friedlander and colleagues [1] reported pronounced suppression of mitochondrial cytochrome c (cyt c) release in vivo, in cells and in isolated mitochondria by minocycline, which was supported by later studies [2e4]. These findings obviously were of high importance due to the known broad inhibition of cell death by this clinically available antibiotic and crucial role of cyt c release in apoptosis. Despite enormous body of knowledge on beneficial effects of minocycline in various models of neurological disorders [5], accumulated during the last 15 years, as well as numerous data on minocycline interaction with mitochondria [4,6e11], the mechanism of the minocycline-induced inhibition of cyt c release from mitochondria is not well understood at present [12]. According to Kagan and colleagues [13], strong peroxidase activity acquired by cyt c upon its interaction with cardiolipin plays a significant role in promoting the release of this protein from mitochondria in the course of apoptosis initiation. Previously peroxidase activity of cyt c has been shown to cause permeabilization of artificial cardiolipincontaining lipid membranes [14e17]. Here, we report data on substantial prevention of such permeabilization by minocycline, which could be of relevance to its inhibiting effect on cyt c release from mitochondria.
2.1. Chemicals Most chemicals including equine heart cyt c, bovine heart cardiolipin, calcein, sulforhodamine B were from Sigma. 2.2. Preparation of liposomes Dye-loaded liposomes were prepared by evaporation under a stream of nitrogen of a 2% solution of a mixture of lipids in chloroform (4 mg soybean phosphatidylcholine (Sigma, Type II-S) and 1 mg bovine heart cardiolipin) followed by hydration with a buffer solution containing appropriate fluorescent marker. Two different marker solutions (0.5 ml each) were used: 1) 50 mM calcein, 50 mM KCl, 5 mM Tris, 5 mM MES, pH 7.4 and 2) 1 mM sulforhodamine B in 100 mM KCl, 10 mM Tris, 10 mM MES, pH 7.4. The mixture was vortexed, passed through several cycles of freezing and thawing, and extruded through 0.1-mm pore size Nucleopore polycarbonate membranes using an Avanti Mini-Extruder. The unbound marker was then removed by passage through a Sephadex G-50 coarse column with a buffer solution containing 100 mM KCl, 10 mM Tris, 10 mM MES, pH 7.4. 2.3. Liposome permeabilization assays
* Corresponding author. E-mail address: [email protected] (E.A. Kotova).
In the fluorescence dequenching assay, fluorescence of calcein-
https://doi.org/10.1016/j.bbrc.2018.11.078 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: A.M. Firsov et al., Minocycline prevents peroxidative permeabilization of cardiolipin-containing bilayer lipid membranes mediated by cytochrome c, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.078
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loaded liposomes was monitored at 520 nm (excitation at 490 nm) with a Panorama Fluorat 02 spectrofluorimeter (Lumex, Russia). The extent of calcein efflux was calculated as (Ft e F0)/(F100-F0), where F0 and Ft represent the initial fluorescence intensity and the fluorescence intensity at the time t, and F100 is the fluorescence intensity after complete disruption of liposomes by addition of the detergent Triton-X100 (final concentration, 0.1% w/w). Cyt c/H2O2-induced leakage of sulforhodamine B was monitored with a fluorescence correlation spectroscopy (FCS) setup, as described previously [16]. 2.4. Chemiluminescence assay of peroxidase activity Peroxidase activity was assessed by chemiluminescence response [13,14,18] arising from H2O2-induced oxidation of luminol in the presence of cyt c. Chemiluminescence was detected with a Lum-100 luminometer, as described in Ref. [19]. 2.5. Centrifugation binding assay Protein-liposome ultracentrifugation binding assays were performed as previously described [20e22], with certain modifications [23]. In particular, liposomes were prepared in the buffer containing 200 mM sucrose and subsequently dispersed in the buffer containing 200 mM glucose. Samples were mixed by pipetting and incubated 30 min prior to centrifugation. The samples were centrifuged for 60 min using a Beckman J2-21 ultracentrifuge equipped with a Beckman JA-21 rotor. The centrifuge was run at 50000g using compressed nitrogen. After completion of each run, the supernatant was immediately removed and its absorption spectrum measured. Bound (pelleted) protein was calculated as total minus free. 3. Results and discussion Based on the inhibiting effect of minocycline on peroxidase activity of cyt c found by Fiorucci and colleagues [24], we decided to study the influence of this antibiotic on permeabilization of cardiolipin-containing liposomes induced by cyt c in the presence of hydrogen peroxide. To measure permeabilization, we used two assays: 1) an increase in fluorescence intensity (removal of fluorescence self-quenching) caused by leakage into the bulk solution of the fluorescent dye calcein entrapped in liposomes at a high concentration (50 mM) [23,25] and 2) a decrease in the amplitude
Fig. 1. Effect of minocycline on calcein leakage from cardiolipin-containing liposomes induced by cytochrome c and H2O2. Concentrations: cyt c, 1 mM; H2O2, 1.5 mM. Triton X-100 (0.1%) was added at the end of the experiment to obtain 100% leakage. The solution was 200 mM sucrose, 10 mM Tris, 10 mM MES, pH 7.4. Lipid concentration, 10 mg/ml.
of the autocorrelation function of the fluorescent dye sulforhodamine B resulting from an increase in the number of fluorescent particles due to release of numerous dye molecules from liposomes [16]. Fig. 1 shows that cyt c in combination with H2O2 caused a pronounced increase in calcein fluorescence reflecting the dye leakage from liposomes, in agreement with our previous data [23,25]. The addition of minocycline at a concentration of 1 mM caused moderate suppression of the calcein fluorescence response. The inhibiting effect of minocycline increased with concentration, so that at 10 mM of the antibiotic, the inhibition of the dye leakage became strongly pronounced and at 100 mM the leakage was completely suppressed. These data were confirmed in FCS experiments with sulforhodamine B leakage from liposomes (data not shown). Fig. 2A illustrates the effect of minocycline on the intensity of luminol chemiluminescence in the presence of the cardiolipincontaining liposomes reflecting the peroxidase activity of cyt c [13]. It is seen that minocycline, at a concentration of 1 mM, substantially inhibited the luminol chemiluminescence elicited by cyt c/H2O2, while a significant inhibition of the luminol
Fig. 2. Effect of minocycline on the intensity of luminol chemiluminescence induced by cytochrome c/H2O2 (A) or Fe/ascorbate (B) in the presence of cardiolipin-containing liposomes. Cyt c, 50 nM; H2O2, 1 mM; FeSO4, 100 mM; ascorbate, 1 mM; luminol, 1mM. The buffer solution of 10 mM Tris, 10 mM MES, pH 8.5 contained 200 mM sucrose. Lipid concentration, 10 mg/ml.
Please cite this article as: A.M. Firsov et al., Minocycline prevents peroxidative permeabilization of cardiolipin-containing bilayer lipid membranes mediated by cytochrome c, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.078
A.M. Firsov et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Fig. 3. Effect of minocycline on the binding of cytochrome c to cardiolipin-containing liposomes, as assessed by centrifugation of liposomes prepared in the sucrose medium. Concentrations: cyt c, 1 mM; lipid, 120 mg/ml. The incubation mixture contained 200 mM glucose, 10 mM Tris, 10 mM MES, pH 7.4.
chemiluminescence induced by Fe/H2O2, i.e. a ROS generating system not involving cyt c, was observed at 100 mM and higher concentrations of minocycline (Fig. 2B). According to docking simulation performed by Ref. [24], minocycline can specifically bind cyt c, which may disturb the binding of the protein to cardiolipin, thereby suppressing the induction of the peroxidase activity and membrane permeabilization. To test this hypothesis, we measured cyt c binding to the cardiolipincontaining liposomes by ultracentrifugation. The data of Fig. 3 show that minocycline at a concentration of 10 mM significantly prevented the cyt c binding to the liposomes (p < 0.05). The effect of minocycline on the binding resembles that of ATP, as described in Ref. [26]. Thus, here, we report data on the inhibiting effect of minocycline both on the cyt c-mediated peroxidative permeabilization of cardiolipin-containing liposomes and on the cyt c binding to the liposomes, which could be considered as a plausible mechanism of its anti-apoptotic activity. Noteworthy, minocycline has been reported previously to suppress ROS generation under certain conditions [4,27e29]. In view of these findings and our own observations (Fig. 2B), the antioxidant property of minocycline might be involved in its apoptosis-inhibitory effect. However, based on the present data (Figs. 1, 2A and 3), it can be concluded that the inhibitory effect of minocycline on membrane permeabilization via the suppression of cyt c binding to cardiolipin-containing membranes makes an important contribution to its inhibiting effect on apoptosis, especially at its low concentrations (10 mM and lower). Conflicts of interest The authors declare that there is no conflict of interest. Acknowledgements This work was financially supported by the Russian Science Foundation grant N 14-24-00107. References [1] S. Zhu, I.G. Stavrovskaya, M. Drozda, B.Y. Kim, V. Ona, M. Li, S. Sarang, A.S. Liu, D.M. Hartley, D.C. Wu, S. Gullans, R.J. Ferrante, S. Przedborski, B.S. Kristal, R.M. Friedlander, Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice, Nature 417 (2002) 74e78. [2] S. Matsuki, Y. Iuchi, Y. Ikeda, I. Sasagawa, Y. Tomita, J. Fujii, Suppression of cytochrome c release and apoptosis in testes with heat stress by minocycline,
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Please cite this article as: A.M. Firsov et al., Minocycline prevents peroxidative permeabilization of cardiolipin-containing bilayer lipid membranes mediated by cytochrome c, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.078
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Please cite this article as: A.M. Firsov et al., Minocycline prevents peroxidative permeabilization of cardiolipin-containing bilayer lipid membranes mediated by cytochrome c, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.078