Cent. Eur. J. Biol. • 6(1) • 2011 • 99–104 DOI: 10.2478/s11535-010-0100-7

Central European Journal of Biology

Climacostol inhibits motility and mitochondrial respiration

Research Article Yoshinori Muto1,*, Yumiko Tanabe2, Kiyoshi Kawai2, Yukio Okano3, Hideo Iio4

1Department of Functional Bioscience, Gifu University School of Medicine, 501-1193 Gifu, Japan 2Department of Nutrition, Faculty of Wellness, Chukyo Women’s University, 474-0011 Ohbu, Japan 3Department of Molecular Pathobiochemistry, Gifu University Graduate School of Medicine, 501-1194 Gifu, Japan 4Department of Material Science, Graduate School of Science, Osaka City University, 558-8585 Osaka, Japan

Received 08 July 2010; Accepted 01 October 2010

Abstract: Climacostol is a resorcinol derivative that is produced by the virens. Exposure to purified climacostol results in lethal damage to the predatory ciliate margaritifer and several other . To elucidate the mechanism of climacostol toxic action, we have investigated the effects of this compound on the swimming behavior of Tetrahymena thermophila and the respiratory system of isolated rat liver mitochondria. When added to living T. thermophila cells, climacostol markedly increased the turning frequency that was accompanied by a decrease in swimming velocity and subsequently by cell death. Observations by DIC and fluorescence microscopy showed morphological alterations in climacostol treated T. thermophila, indicating that climacostol might exert cytotoxic action on this organism. In the experiment with isolated rat liver mitochondria, climacostol was found to inhibit the NAD-linked respiration, but had no apparent effect on succinate-linked respiration. This finding indicates that climacostol specifically inhibits respiratory chain complex I in mitochondria. The combination of results suggest thatthe inhibition of mitochondrial respiration may be the cytotoxic mechanism of climacostol’s defenses against predatory .

Keywords: Climacostol • Apoptosis • Complex I • Mitochondria • Reactive oxygen species • Tetrahymena © Versita Sp. z o.o.

1. Introduction correlated to the unsaturation level of its aliphatic chain [3]. The complete molecular structure of climacostol was The ciliate, , has determined as 1,3-dihydroxy-5-[(Z)-2’-nonenyl]benzene numerous cortical granules containing a cytotoxic and it was chemically synthesized [1,4]. On the basis compound called climacostol [1]. This compound is of its chemical structure, climacostol is classified as used for chemical defense against predators such as a resorcinolic lipid, and is widely detected in plants, the raptorial ciliate and its cytotoxic fungi, algae, and bacteria [5,6], but only in one animal activity has been assessed on several species of ciliates species (a marine sponge) and in a ciliated protozoan such as nasutum, caudatum, [7]. Some observations clearly suggested participation and japonicum [2]. Recent observations of resorcinolic lipids in the modulation of the host- on various ciliated protozoa further revealed that the pathogen relationship in plants [5]. Until now, however, cytotoxicity level of climacostol appears to be inversely the physiological role of the resorcinolic lipids has not

* E-mail: [email protected] 99 Climacostol inhibits Tetrahymena motility and mitochondrial respiration

been fully clarified and little is known about the cytotoxic (25 mm × 50 mm) and a smaller coverslip (18 mm ×18 mm) mechanisms of climacostol on the target organisms. by introducing 10 μm diameter polystyrene beads into the Among the ciliates, Tetrahymena has been medium. This assembly was then mounted on an Olympus convenient for cell biology studies because several IX70 inverted microscope equipped with high-resolution species are easily grown axenically [8]. They possess DIC and epifluorescence optics. highly evolved cytoarchitecture as well as an intracellular messenger system regulating various cell functions 2.4 Digital image processing [9,10]. Its ultrastructure, cell physiology, development, Measurement of the motility pattern was initiated biochemistry, genetics and molecular biology have by transferring 50 μl aliquot of the cell suspension been extensively studied [11-15]. Moreover, the motile into a plastic petri dish (φ33mm) containing 0.4 ml behavior of Tetrahymena in response to external of 10 mM Mops/Tris (pH 7.2) inorganic saline chemical stimuli is easily assessed and has been solution with and without a test substance (the used as a measure of the biological activity of various final cell density, approximately 1×105 cells/ml). chemicals [16-18], making it a suitable model cell After rapid mixing of the medium, time-lapse images system for analysis of the cytotoxic mechanisms of were obtained with a C5985 video camera (Hamamatsu climacostol. In the present study, we have undertaken Photonics, Japan) in darkfield illumination using an a series of investigations into the mode of action of inverted microscope with a ×4 objective lens [18,20]. climacostol on Tetrahymena thermophila cells and its Video signals were digitized by a personal computer with effects on the respiratory chain of rat liver mitochondria. an LG-3 frame grabber (Scion, USA) and then converted Our results show that climacostol inhibits the motility of to TIFF format. The images were recorded every 0.1 s T. thermophila remarkably and induces morphological for 1 min after the cell suspension was added. For changes of the cell, being dependent at least in part quantitative analysis of the swimming pattern, sequential on mitochondrial function. A part of this work has been cell video images were subjected to image processing published in the Proceedings of the annual meeting of including filtering as well as binary (i.e. black and white the Japan Society of Protozoology [19]. only) transformation. Ten successive images of the same field were then added and converted to one fused image to show the swimming tracks of T. thermophila. All image 2. Experimental Procedures processing was performed using the public domain NIH Image program (developed at the U.S. National 2.1 Cell culture Institutes of Health and available on the Internet at Tetrahymena thermophila B1868(mating type II) was http://rsb.info.nih.gov/nih-image/). grown axenically at 25°C in 1% (w/v) proteose peptone containing 1% (w/v) yeast extract and 0.87% (w/v) 2.5 Mitochondrial preparation and glucose without shaking. Cells were harvested during the measurement of respiration mid-log phase of growth (5×105 cells/ml) by centrifugation, Rat liver mitochondria were prepared by the method of washed and resuspended to 1×106 cells/ml in 10 mM Schneider [21] using 0.25 M sucrose solution containing Mops/Tris (pH 7.2) containing 1 mM KCl, 1 mM NaCl and 0.5 mM EDTA and 10 mM Tris-HCl (pH 7.4). The

1 mM CaCl2 (inorganic saline solution). Cells were then mitochondrial respiration was measured using a Galvani- equilibrated for more than 30 min prior to measurement. type oxygen electrode. The reaction medium was essentially

composed of 0.15 M KCl, 5 mM MgCl2, 1 mM EDTA and 2.2 Climacostol 20 mM Tris-HCl in a final volume of 2 ml (pH 7.4). Reaction

Chemically-synthesized climacostol [1] was dissolved was initiated after saturating O2 in the reaction medium in ethanol (5 mg/ml) and stored protected from light at 30°C and was carried out at the same temperature. at −20°C until use. The solution was diluted with an Submitochondrial particles (SMP) were prepared from rat appropriate experimental medium at the time of the liver mitochondria according to the procedure of Ruzicka experiment. For each experiment, corresponding volume [22]. The oxygen uptake of SMP was measured by the of the vehicle ethanol was added and measurement was same method used for mitochondria. Reaction medium

performed as control. was essentially composed of 0.15 M KCl, 5 mM MgCl2, 5 mM inorganic phosphate, 0.5 mM EDTA, and 20 mM 2.3 Differential interference contrast (DIC) and Tris-HCl in a final volume of 1.9 ml (pH 7.4). The reaction fluorescence microscopy was performed at 30°C. The protein concentration was T. thermophila cells equilibrated in inorganic saline solution determined by the method of Lowry et al. [23], using bovine were slightly compressed between a large coverslip serum albumin as the standard protein.

100 Y. Muto et al.

3. Results of incubation in a solution of climacostol with a final concentration of 21 μM, T. thermophila cells initially 3.1 Motility changes of Tetrahymena lost their shape and became spherical (Figure 2A, thermophila induced by climacostol part b). Some cells were highly abnormal, exhibiting By regulating the frequency and direction of ciliary beat, cell lysis in which pellicle membranes were ruptured T. thermophila changes swimming patterns in response (Figure 2A, part c). Since almost all the cells became to various environmental stimuli. When observing the immobilized and spherical after exposure to 21 μM motility pattern of T. thermophila in darkfield illumination climacostol for 5 minutes, there might be no surviving with a microscope, it consists of smooth tracks (runs) cells in this condition. In Tetrahymena and related interrupted randomly by a turn which alters the direction ciliates, many of the mitochondria are located in the of motion (Figure 1A, part a). Incubating T. thermophila in cortex and are lined up parallel to the ciliary rows [13]. climacostol (21 μM) immediately increased their turning These mitochondrial organizations were visualized by frequency and slowed their speed of forward swimming loading it with mitochondria-specific dye MitoTracker (Figure 1A, part b). These immediate changes in the Green [24]. As seen in Figure 2B, part b, longitudinal swimming pattern were gradually recovered (Figure 1A, arrays of mitochondria were present in the control part c). However, T. thermophila cells became completely cells. In the cells with climacostol, however, the immobilized after exposure to climacostol for several mitochondrial pattern was completely disorganized minutes (Figure 1A, part d). Different concentrations of (Figure 2B, part d). These observations suggest that climacostol treatment for 5 min significantly decreased the motility defect induced by this compound might the motile cells in a concentration-dependent manner be mediated by its cytotoxic action altering cellular (Figure 1B). architecture.

3.2 Morphological alterations in climacostol 3.3 The effect of climacostol on NAD-linked treated Tetrahymena thermophila respiration oxidizing L-glutamate/malate To further determine the effects of climacostol on Climacostol was next examined for its inhibitory effect on T. thermophila, the cell and mitochondrial organization the mitochondrial respiratory system of isolated rat liver were investigated. Figure 2A shows the DIC images mitochondria, because the respiratory mechanism in liver of T. thermophila after being incubated with or without mitochondria is now well understood and it is very hard climacostol for 5 min. The morphology of a normal to isolate T. thermophila mitochondria showing tightly cell was spindle-shaped (Figure 2A, part a), and the coupled respiration. Figure 3 shows the oxygraph data was recognized in the middle of the cell of mitochondrial respiration with and without climacostol. (smooth area lacking granules). However, after 5 min Curve 4, Figure 3 shows the control shows the control

A a b B

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20

0 0510 15 20 25 Clim acostol (μM)

Figure 1. The effect of climacostol on the motility of Tetrahymena thermophila. (A) Swimming tracks of T. thermophila treated with climacostol. (Part a) Typical swimming behavior of T. thermophila before the addition of climacostol; swimming tracks of T. thermophila: (Part b) 20 s after the addition of 21 μM climacostol; (Part c) 90 s after the addition of 21 μM climacostol; (Part d) 300 s after the addition of 21 μM climacostol. Ten successive images in darkfield illumination, each acquired with a 0.1 s delay, were added to produce individual fused images. (B) Concentration-dependent inhibition of the motility of T. thermophila cells by climacostol. Swimming tracks of T. thermophila were reconstructed 5 min after the addition of climacostol. Motile cells were then counted by inspecting the swimming track images. Each point is the mean ± SD obtained from at least four separate determinations (n=30).

101 Climacostol inhibits Tetrahymena motility and mitochondrial respiration

A B

Figure 2. Morphological changes of Tetrahymena thermophila induced by climacostol. (A) DIC micrographs on the effects of climacostol. (Part a) Control cells; (Part b) cells deformed and spherical 5 min after the addition of 21 μM climacostol; (Part c) abnormal cells after 5 min of climacostol treatment. Cells were visualized by DIC microscopy as described in Experimental Procedures. Bars are 10 μm in panel a, b and 15 μm in panel c. (B) Visualization of T. thermophila mitochondria by fluorescence microscopy. (a, b) Control cells; (c, d) cells deformed by the addition of 21 μM climacostol. T. thermophila cells were exposed to 10 μM of MitoTracker Green solution for 30 min at room temperature. Cells were then treated with 21 μM climacostol. Note that DIC photomicrographs are shown in panels a and c; fluorescence photomicrographs are shown in panels b and d. Bars are 10 μm in all panels.

experiment without climacostol. A limited amount of ADP induced the distinct state 3 and 4 respirations exhibiting tightly coupled respiration. The ADP-driven respiration Climacostol was strongly inhibited by climacostol, showing a dose dependency (curve 1-curve 3, Figure 3). At high concentrations of climacostol, the respiration was Climacostol (182 μM) almost completely inhibited (curve 1, Figure 3). Climacostol 3.4 Effects of climacostol on NADH and succinate oxidases in SMP Climacostol The effects of climacostol on NADH and succinate Climacostol oxidases were tested using SMP, which were membrane (109 μM) vesicles of mitochondrial inner membranes reversed to an inside out direction, to know the inhibitory effect on the respiratory chain enzymes (Figure 4A). Curve 1, Figure 4A shows the effect of climacostol on NADH Climacostol oxidase in SMP. NADH oxidase was completely inhibited (73 μM) by climacostol at 73 μM. By contrast, the inhibitory effect of climacostol on succinate oxidase was not detected at 100 nmol O2 the tested concentrations (36 and 73 μM) at all. NADH donates electrons at complex I, whereas succinate 2 min donates electrons at complex II, bypassing complex I. Control Thus, the present results indicate that climacostol selectively inhibits the electron transport at complex I (NADH ubiquinone oxidoreductase complex) of the respiratory chain. Figure 3. The effect of climacostol on oxidative phosphorylation in The effect of climacostol on the specific complex of the isolated rat liver mitochondria. Oxygraph data indicates NAD-linked respiration oxidizing L-glutamate. Reaction mitochondrial respiratory chain was further investigated medium contained 0.15 M KCl, 5 mM MgCl2, 5 mM by using the NNN’N’-tetramethyl-p-phenylenediamine inorganic phosphate 0.5 mM EDTA, 20 mM Tris-HCl in a final volume of 2.0 ml (pH 7.4). Mitochondrial protein was (TMPD) bypass [25]. Figure 4B shows the NADH oxidase 0.85 mg/ml. The final concentrations of climacostol are activity in the presence of TMPD which generates an shown in parentheses.

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electron transport bypass from complex I to cytochrome c when respiration is inhibited by rotenone or antimycin A. A Climacostol (73 μM) Curve 1, Figure 4B shows the control experiment without climacostol. In the presence of TMPD, the NADH oxidase was not completely inhibited by rotenone (curve 3, Figure 4B) or climacostol (curve 2, Figure 4B). Moreover, in the presence of ubiquinone 2 (UQ2), climacostol inhibition could be completely relieved (curve 4, Figure 4B). All these results imply that climacostol might inhibit the same Climacostol Climacostol (37 μM) (73 μM) site as rotenone, which inhibits the electron transport at the UQ site in complex I [25]. 100 nmol O2

2 min 4. Discussion

In the present study, we have shown that climacostol affects T. thermophila motility in a dose-dependent (73 μM) manner, finally inducing necrotic cell lysis. Recently, B Buonanno et al. [26] examined the effects of climacostol on the growth and proliferation of tumoral and non- tumor mammalian cell lines, and found that it effectively inhibited the growth of tumor cell lines in a dose- dependent manner. They further demonstrated that the growth inhibition was induced by programmed cell death, a process involving mitochondrial function. These (73 μM) results point to the importance of the apoptotic pathway for the climacostol action in mammalian cell lines. In 100 nmol O2 T. thermophila, however, climacostol exerts its activity in a matter of minutes and induces necrotic damage 2 min and cell lysis. Moreover, the addition of climacostol almost immediately affects the swimming behavior Figure 4. Climacostol inhibits complex I-linked respiratory chain in SMP. (A) Oxygraph data of NADH or succinate oxidase of T. thermophila as well as one of the primary target activity. Reaction condition was the same as in Figure 3. organisms such as Dileptus as previously reported SMP (0.38 mg/ml) was used instead of mitochondria. (B) [2]. These short periods of latency suggest that the Oxygraph data of NADH and succinate oxidases in the presence of TMPD. Reaction condition was the same mechanism of climacostol action on ciliated protozoa is as in (A) but contained 50 μM TMPD in the reaction mixture. In curve 4, 25 nmol of UQ was added after not part of the apoptosis-based process. 2 injection of climacostol (73 μM). The final concentrations Observations by DIC and fluorescence microscopy of climacostol are shown in parentheses. As a control, showed morphological alterations in climacostol treated baseline NADH and succinate oxidase activities of SMP without any test compounds were measured before the T. thermophila, indicating that climacostol might exert experiments. a cytotoxic action on this organism. Furthermore, climacostol inhibited NAD-linked respiration in rat liver implicated in the process of climacostol action. In this mitochondria, but had no apparent effect on succinate- context, it is intriguing that Buonanno et al. [26] reported linked respiration in submitochondrial particles. that in HL60 cells climacostol-induced apoptosis is in This finding indicates that climacostol specifically fact preceded by intracellular reactive oxygen species inhibits respiratory chain complex I in mitochondria. generation, membrane potential dissipation and caspase Mitochondria participate in a surprising number of activation. Since the response of T. thermophila to activities, including ATP generation, intracellular Ca2+ climacostol appears not to be apoptosis-based, reactive regulation, thermogenesis and the control of apoptosis oxygen species generated in the mitochondria might be [27]. In particular, mitochondria are the main cellular directly implicated in the process leading to cell lysis generators of reactive oxygen species, and may in T. thermophila cells. Although the consequences of trigger necrotic cell death under conditions of complex climacostol action on T. thermophila and mammalian I inhibition [28]. It is thus possible that the generation cells are quite different, mitochondria may be a primary of reactive oxygen species in mitochondria might be target of this toxin in both cellular systems.

103 Climacostol inhibits Tetrahymena motility and mitochondrial respiration

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