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Taxol transport in Taxus baccata cell suspension cultures
Plant Physiol. Biochem. 40 (2002) 81–88 www.elsevier.com/locate/plaphy
Taxol transport in Taxus baccata cell suspension cultures Silvia Fornalè a, Davide Degli Esposti a, Alberto Navia-Osorio b, Rosa M. Cusidò b, Javier Palazòn b, M. Teresa Piñol b, Nello Bagni a,* a
Dipartimento di Biologia Evoluzionistica Sperimentale e Centro Interdipartimentale di Biotecnologie, Università di Bologna, via Irnerio 42, 40126 Bologna, Italy b Laboratorio de Fisiologia Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avda. Diagonal 643, 08028 Barcelona, Spain Received 10 May 2001; accepted 28 July 2001
Abstract Taxol transport in Taxus baccata L. cell suspension cultures was studied using [14C]-taxol as a tracer. The time course of uptake showed a saturable absorption that reached a maximum within 20 min. The uptake depended on its exogenous concentration and its accumulation was highly stimulated in the presence of 10–15 µM exogenous taxol. The absorbed molecule was found to localise both in the cell walls (20 %) and in the cell protoplasts (80 %), suggesting an accumulation within the vacuoles. Taxol uptake was strongly inhibited by Na-orthovanadate and verapamil, while Ca2+ was found to be one of the factors required for the active absorption of the molecule, since in the absence of this cation, the uptake was reduced by about 40 % and occurred mainly through a non-energy dependent mechanism. Taxol release into the culture medium was demonstrated not to depend on cell lysis, occurred through a mechanism that reached its maximum after 10–15 min and was strongly enhanced by treatment with Na-orthovanadate and verapamil, although the effect was found to be transient. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Cell cultures; Metabolic inhibitors; Release; Taxol; Taxus baccata; Uptake
1. Introduction Taxol (paclitaxel, NSC-125973) is a highly functionalized diterpenoid produced by the genus Taxus. It is a well-established anti-tumour drug, whose properties are based on the ability to bind and stabilise microtubules, thus leading to the block of cell replication in the late G2-M phase of the cell cycle [14]. This plant secondary metabolite promotes the formation of microtubules that are resistant to disassembly, in contrast to that observed for other antimitotic agents such as colchicine and Vinca alkaloids [26]. Taxol was approved in 1992 by the US Food and Drug Administration for the treatment of ovarian and breast
Abbreviations: BAP, 6-benzylamino-purine; CC, cell cluster; DCCD, dicyclohexylcarbodiimide; 2,4 DNP, 2,4-dinitrophenol; NAA, αnaphthaleneacetic acid; NEM, N-ethylmaleimide * Corresponding author. Fax +39 051 242576. E-mail address: [email protected] (N. Bagni). © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 8 1 - 9 4 2 8 ( 0 1 ) 0 1 3 3 2 - 8
cancer, and it was shown to be active against a variety of other cancers such as lung, gastrointestinal, neck and head as well as malignant melanoma [1,16,34]. This compound was originally obtained from the bark of the pacific yew (Taxus brevifolia), but the low yield (0.01 % of dry weight) led researchers to look for alternative sources [5,38]. Total synthesis was achieved by Nicolau et al. [21] and by Holton et al. [12,13], but its high cost makes this approach not commercially feasible, so that it is currently produced by semisynthesis [22]. Much attention has been given to plant tissue culture, which is one of the approaches available to provide large amounts and a stable supply of pharmaceutically valuable compounds [15]. Basic protocols for the induction and maintenance of taxol-yielding callus and cell cultures of different Taxus species have been developed during the past decade [9,29,39] and a lot of attention is currently being dedicated to improving taxol production and release into the culture medium. Factors that regulate and limit taxol biosynthesis and the in vitro cell growth of Taxus cell cultures have been intensively investigated [2,10,17,19,27,31], but almost no information is available on how taxol is transported by Taxus cells. In fact, no
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studies have been published on the molecular mechanism underlying taxol release into the culture medium, and not even the ability of Taxus cells to take up and accumulate this metabolite at subcellular level is known. In fact, studies performed by Srinivasan et al. [32] and Hirasuna et al. [11] indicated that more than 90 % of taxol is routinely excreted into the culture medium, while Wickremesinhe and Arteca [39] supposed that taxol presence in the culture medium could be due to cell lysis rather than active secretion. Taxol is present in virtually all parts of the yew tree [4], but preferentially accumulates in bark and needles. However nothing is known about its subcellular localisation or the existence of a translocation system within the plant. Strobel et al. [33] reported the presence of taxol in the sapwood and heartwood of T. brevifolia even though only traces of its biosynthesis could be demonstrated in the xylem. They suggested that taxol might be mobilised from its place of greatest biosynthetic activity (vascular-cambial region), possibly via ray-parenchyma, and translocated to the xylem. Moreover, taxol affects microtubules in many organisms, including higher plants [18,20], so that it can be considered toxic to Taxus itself. Thus, a strategy to avoid taxol effects, such as its excretion, must be present in Taxus cells, and the elucidation of such a mechanism could be crucial to improving the productivity of Taxus plant and cell cultures. Immunocytochemical studies performed by Russin et al. [25] showed that taxol seems to accumulate almost exclusively in the cell wall, rather than in cell compartments such as vacuoles. The precise localisation of taxol in plant tissues or cell cultures raises numerous questions, since the molecule is displaced during classical fixation procedures. For this reason, no clear evidence has been achieved so far. Numerous studies showed the lack of any relationship between the endogenous content of taxol and its release in Taxus cell cultures [11,23,28,29,32]. This seems to indicate the presence of regulated processes that, at present, remain to be investigated. More information is available in the animal field, where studies on taxol transport in human cells [37] gave indications of the possible involvement of active transport mechanisms and several putative taxol-binding proteins have been identified [24]. All of the evidence currently in the literature suggests the existence of a taxol transport system. Hence, the aim of this work was to investigate how Taxus cells transport taxol and if this process presents energy-dependent features. Most of the studies on taxol published so far showed the ability of Taxus long-term cell cultures to release this compound into the culture medium, but no information is available on its uptake. Thus far no studies have been made on the molecular mechanism by which taxol is transported in and out of Taxus cells. It is our opinion that such knowledge could offer a new tool to improve taxol productivity in Taxus cell cultures. In fact the identification of proteins able to bind and transport the molecule in and out of the cell could represent a new approach to enhance taxol release.
Fig. 1. Time course of biomass accumulation and cell viability in T. baccata cell suspension cultures. Data are expressed both as fresh (■) and dry weight (·) accumulation. Cell viability (bars) was measured by the selective labelling of living/dead cells as explained in Methods and expressed as % of living cells. Each value is the mean of three replicates ± SD.
2. Results 2.1. Determination of cell growth and viability A time course was performed on T. baccata cell suspension culture over a 21-d growth period. A first rapid increase of both FW and DW was observed between days 2 and 7 (Fig. 1). This increase was then followed by a stationary phase of growth, with no significant changes in biomass. Cell viability remained very high (from 98 to 91 %) until day 13 and slightly decreased in the following days, reaching 80 % at the end of the culture period considered (day 21). 2.2. Taxol uptake in T. baccata cell suspension cultures 2.2.1. General characteristics of taxol uptake Time course assays of taxol uptake were performed on T. baccata cell suspension culture in the linear phase of growth using labelled taxol as a tracer. Taxol was found to be transported into the cells, reaching maximum absorption after 20–30 min (Fig. 2). Considering the total numbers of moles absorbed compared to those supplied, we calculated that in these conditions, cells were able to absorb up to 8 % of the exogenous supplied taxol. In order to verify the occurrence of a real taxol uptake, experiments were performed to study the localisation of the absorbed taxol. For this reason, a T. baccata cell suspension culture was first divided into two aliquots one of which was used for cell wall isolation as described in the Methods section. A time course using [14C]-taxol as a tracer was then performed on the remaining aliquot and on the cell wall suspension and the results compared (Fig. 2). A time-dependent interaction of taxol with cell wall components was observed: in the described conditions, cell walls adsorbed taxol reaching maximum adsorption after 20 min. Comparing the maximum absorption of cell cultures to the corresponding cell wall fraction, we estimated that about 20 % of the total radioactivity recovered in the cells (8 %) was due to taxol
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Fig. 2. Distribution of supplied [14C]-taxol between T. baccata cell suspension culture and its correspondent cell wall fraction. A cell suspension culture was separated into two aliquots, one of which was used for the extraction of the cell walls. A time course of taxol uptake was performed by adding 3.4 kBq labelled compound to the cell cultures (■) and their correspondent cell wall fractions (•). The experiments were performed several times with similar results: the curves are typical of one experiment. Data are expressed as pmoles absorbed by 103 cell clusters and represent the mean ± SD of three replicates.
binding to cell wall components, while the remaining 80 % was actually absorbed by the protoplast. Taxol uptake in relation to external concentration was measured over a 20-min incubation with [14C]-taxol as a tracer and increasing unlabelled taxol concentrations (up to 50 µM) in the presence of 1 mM Ca2+ (basic B5 medium). It should be noted that 50 µM represented the upper limit for our system, due to the low solubility of the molecule and to the toxic effect that relatively high concentrations of DMSO (over 0.6 %) exerted on T. baccata cells, as verified by cell viability assays (data not shown). The effect of increasing the external concentration (Fig. 3) was a rapid increase in the amount of taxol absorbed, which displayed saturation kinetic and reached a maximum corresponding to 10–15 µM external concentration. The great stimulation of taxol uptake reflected in the 30-fold increase observed comparing the absorption measured when only [14C]-taxol as a tracer was supplied (3 pmol per 103 CC) and the absorption in the presence of 10–15 µM exogenous taxol (about 85 pmol per 103 CC). 2.2.2. Effects of metabolic inhibitors on taxol uptake Taxol endogenous levels in T. baccata cell suspension cultures ranges from 0.01–0.03 % of the dry weight, which roughly corresponds to a concentration of about 15–20 µM; thus the uptake measured in our system seemed to occur against a concentration gradient, since the labelled taxol was added at a concentration of 0.6 µM. For this reason and to better address the nature of taxol uptake, the effect of different metabolic inhibitors was tested. Most of the compounds used were soluble in ethanol, which could itself be toxic. Hence it was used at low levels (1 %) where no effect due to the solvent alone was observed on taxol uptake (Table 1). Among the inhibitors tested, in the presence of 1 mM Na-orthovanadate taxol uptake was inhibited by
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Fig. 3. Effect of exogenous taxol on uptake. Taxol uptake was determined over a concentration range of 0.05 to 50 µM: 3.4 kBq [14C]-taxol and different concentration of the unlabelled compound were added to a T. baccata cell suspension culture in the presence (■) and absence (·) of 1 mM Ca2+. Data represent the mean ± SD of three different experiments and are expressed as pmoles absorbed by 103 cell clusters.
about 40 % with respect to the control. This substance, together with DCCD (dicyclohexylcarbodiimide), was tested because they inhibit the H+-ATPase of the plasmalemma, but no effect was observed with DCCD. Nitrate, supplied both as NaNO3 and KNO3 did not affect taxol uptake: this anion is known to inhibit the tonoplast ATPase at high concentrations (50–100 mM). A 25 % inhibition was observed after the treatment with verapamil, a phenylalkylamine which blocks Ca2+ channels, but no effect was observed using nifedipine (a 1-4-dihydropyridine), which exerts the same effect. No changes were observed after incubation with N-ethylmaleimide (NEM), a thiol-reacting compound. Cells treated with 1 mM 2,4-DNP produced a strong enhancement of the uptake, the amount of the absorbed molecule being about 30 % higher than the control’s. To better understand whether Ca2+ could influence taxol uptake, a series of experiments was done on T. baccata cell suspension cultures in the presence (basic B5 medium) or absence of 1 mM CaCl2 and verapamil (Table 2). Ca2+ absence reduced the uptake, in line with the inhibition caused by verapamil in the presence of 1 mM CaCl2. These results led us to conclude that the activity of Ca2+ channels was required for Ca2+-activated taxol transport. For this reason, a further set of experiments was done in order to study taxol uptake in relation to external concentration in the absence of external Ca2+. When the assay was performed in a Ca2+-free medium (Fig. 3), the uptake occurred through a non-energy dependent mechanism, indicating that Ca2+ seemed to be one of the factors involved in the active part of the absorption. 2.3. Taxol release into the culture medium A time course assay of taxol release was performed on T. baccata cells previously incubated with a known amount of the radioactive compound (Fig. 4). The release of labelled
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Table 1 Effect of metabolic inhibitors on taxol uptake. Cells were pre-incubated 30 min with the various compounds. They were then supplied with 3.7 kBq [14C]-taxol and 10 µM unlabelled taxol and the uptake was measured after 20 min. Data are the mean ± SD of three replicates. * The negative value indicates an enhancement of the uptake with respect to the control. Inhibitor
Concentration (mM)
Solvent
Percent of solvent
Percent of inhibition compared to control
Na-orthovanadate
0.1 1 0.5 1 0.1 1 100 100 0.5 0.1 0.1 –
H2O
– – 1 1 1 1 – – – – 1 1
0 42 ± 5 0 0 0 –30 ± 3* 0 0 0 25 ± 1 0 0
DCCD 2,4 DNP NaNO3 KNO3 NEM Verapamil Nifedipine None
Ethanol Ethanol H2O H2O H 2O H2O Ethanol Ethanol
taxol from the cells followed a time-dependent mechanism that reached saturation after 10 min from the beginning of the assay. In these conditions, T. baccata cells were able to release about 58–60 % of the total labelled compound. Taxol efflux was not influenced by Ca2+ presence into the culture medium, contrarily to what observed in the case of its uptake (data not shown). To better understand which kind of mechanism does regulate taxol transport in T. baccata cells, we decided to test the same inhibitors that influenced the uptake, namely Na-orthovanadate, verapamil and 2,4-dinitrophenol. Cells Table 2 Effect of Ca2+ and verapamil on taxol uptake. Cells were pre-incubated 30 min with verapamil and/or Ca2+, then 3.7 kBq [14C]-taxol and 10 µM unlabelled taxol were added. The radioactivity inside the cells was measured after 20 min. Data are the mean ± SD of three replicates and are expressed as pmoles absorbed by 103 cell clusters. Ca2+ (mM)
Verapamil (mM)
Taxol uptake (pmol·(103 CC)–1)
1 1 0 0
0 0.1 0 0.1
62.7 ± 5.8 45.2 ± 1.0 38.3 ± 0.2 33.2 ± 2.4
Fig. 4. Time course of taxol release by a 7-d-old cell culture of T. baccata. Cells were first incubated with 3.4 kBq labelled taxol and then transferred to fresh medium for the determination of the release. Data are the mean of three replicates ± SD and are expressed as % of the previously absorbed molecule that is released into the medium.
were first pre-incubated with the labelled molecule and then transferred into the efflux medium containing the metabolic inhibitor. When cells were treated with Na-orthovanadate (Fig. 5), a great stimulation of the efflux, which doubled the control value, was observed in correspondence of the first minutes of the assay . It has to be noted how this effect was transient, since starting from the fifth minute, the amount of released molecule reached control values. An analogous stimulation of taxol release was observed in the presence of verapamil (Fig. 6). This compound was found to be able to enhance taxol efflux from 90 % (3rd min) to 60 % (5th min) compared to control values. Even in this case the effect displayed by verapamil was found to be transient, although a little more persistent. In fact values comparable to the control were reached from 10 min after the beginning of the experiment. In contrast, no effect was observed when taxol release was measured in the presence of 1 mM 2,4-DNP (data not shown).
Fig. 5. Effect of Na-orthovanadate on taxol release. Cells were preincubated with 3.4 kBq labelled molecule and then transferred into the efflux medium in the presence (black bars) or not (white bars) of 1 mM Na3VO4 for the determination of the release. Data are the mean of three replicates ± SD and are expressed as % of the previously absorbed molecule that is released into the medium.
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Fig. 6. Effect of verapamil on taxol release. Cells were pre-incubated with 3.4 kBq the labelled molecule and then transferred into the efflux medium in the presence (black bars) or not (white bars) of 0.1 mM verapamil for the determination of the release. Data are the mean of three replicates ± SD and are expressed as % of the previously absorbed molecule that is released into the medium.
3. Discussion
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of exogenous Ca2+, we hypothesise that in the presence of this cation, taxol absorption can occur mainly through active processes and that this second mechanism can be considered more physiological. Furthermore, the high endogenous accumulation of this compound in Taxus cells suggests the involvement of vacuoles as storage compartments. These organelles represent the greatest part of T. baccata cells, as shown by ultrastructural studies performed on 7-d-old cell cultures and their involvement could not be excluded by previous immunocytochemical studies performed by Russin et al. [25]. However, even though the tonoplast is very close to the plasmalemma (data not shown), H+-ATPase does not seem directly involved in taxol transport. Taxol uptake in respect to its exogenous concentration was maximum in the presence of 10–15 µM exogenous taxol. A rough estimation of the Km for this transport gave a value of about 1.5 µM. This value fits with the one obtained by Walle and Walle [37] in human cells. Their studies were in favour of an energy dependent transport too, since taxol transport was found to be affected by verapamil, which is also a well known inhibitor of the P-glycoprotein.
3.1. General characteristics of taxol uptake The work reported in this paper intended to give evidence of the existence of a taxol transport mechanism in T. baccata cell suspension cultures. One of the main problem that still has to be solved in the biotechnological exploitation of Taxus cell cultures as natural producers of this compound is how to increase taxol release into the culture medium. Information on how Taxus cells can accumulate and transport this metabolite under physiological conditions (culture conditions in this case) could provide a powerful tool and a new approach to solve this problem. Despite numerous studies on taxol distribution in the whole Taxus plant, up to now it has been impossible to clearly identify whether a particular organ/tissue is responsible for taxol biosynthesis or if any plant cell produces this compound which is then preferentially accumulated in some tissues (e.g. bark and needles). In this light it was necessary to test first if cells can take up the molecule, and by which mechanism. This ability together with the existence of regulated release processes could indicate the presence of a transport system, whose understanding will permit biotechnological improvements. Data reported in this paper indicate how taxol can be absorbed by Taxus cells. Due to the lipophilic nature of the compound a diffusion mechanism could be hypothesised. At the same time, it has been proposed that taxol could mainly accumulate in the cell walls instead of internal cell compartments such as vacuole [25]. Our data indicated that this uptake reached saturation within 20 min, leading to the absorption of about 8 % of the exogenous supplied taxol and occurred against a concentration gradient. Moreover we demonstrated that only 20 % of the absorbed taxol seems to localise in the cell wall fraction. For these reasons, although a non-energy dependent mechanism occurs in the absence
3.2. Effect of Ca2+ and metabolic inhibitors on taxol uptake Few metabolic inhibitors were found to affect taxol uptake in T. baccata cells. Na-orthovanadate (an ATPase antagonist) and the Ca2+ channel blocker verapamil exerted an inhibitory effect, while the uncoupler 2,4-DNP leads to an increase of taxol absorption. Several other inhibitors were tested, but without effects. Since DCCD and nifedipine had to be supplied in a 1 % ethanol solution, it can not be excluded that the lack of effect could be due to the possible lack of permeation of the molecule within the cell and/or interactions with cell wall components. Regarding the role of Ca2+, this cation seems to be needed for taxol uptake. In fact, when the uptake was measured in a calcium-free medium, a strong inhibition of about 30 % was observed. A rough estimation of how Ca2+ can promote taxol uptake was allowed by the comparison of the absorption kinetics performed in the presence/absence of this cation (Fig. 3). Our data indicate that, up to 30 µM taxol exogenous concentration, Ca2+ seems to be one of the factors that regulate the energy-dependent part of the uptake which occurs, even if to a lower degree, by a passive mechanism. 3.3. General characteristics of taxol efflux and effect of metabolic inhibitors The studies performed on taxol efflux permitted to clarify how Taxus cells can release the compound by means of a cellular mechanism instead of a cell lysis phenomena. Even though the presence of this metabolite in the medium of long-term cultures can be partially due to cell necrosis (common in later stages of the growth cycle), the release
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measured in our assays, performed using a 7-d-old cultures with 97 % of viability (Fig. 1), was observed over a time that ranges from 30 s to 20 min and, therefore, it can not be related to cell death. T. baccata cells were able to release at least the 55–60 % of the labelled compound. This value resembles that reported in literature for T. cuspidata [6] and T. baccata [11,32] cell cultures, where percentages ranging from 60 to 90 % were measured at the end of the culture period. Taxol uptake was reduced in the presence of Naorthovanadate, while the release was strongly enhanced. This inhibitor acts on plasma membrane ATPase and its addition provokes the inhibition of proton pumping with the consequent depolarisation of membrane potentials needed for active transport of various solutes, including small molecules. For this reason Na-orthovanadate has been successfully used as an abiotic elicitor to induce alkaloid biosynthesis [36] and even the effect displayed on T. baccata cells can be related to an elicitor-induced stress response which leads to an enhanced release of secondary metabolites into the culture medium. The effect exerted by verapamil (uptake inhibition and enhancement of the release) appears to be related to its Ca2+ channel blocker activity. This hypothesis seems to be confirmed by the cross-assays on taxol uptake in the presence/absence of both verapamil and Ca2+. The same inhibition measured in the absence of this cation is in fact provoked by the treatment with verapamil. The role of this cation in taxol transport should be related to the complex network of the signal transduction mechanisms. Therefore, any transient alteration of its endogenous level could be responsible for the effects observed. The increased uptake measured in the presence of 2,4-DNP could be related to a reduced ability of cells to release the absorbed molecule. This inhibitor blocks the mitochondrial ATP synthesis and these organelles represent in our system the unique source of this molecule, being T. baccata cell suspensions cultured in the dark. Hence this result is in favour of an energydependent release mechanism. Nevertheless, when 2,4-DNP was added to the efflux medium, no effect was observed. This can be explained by the reduced permeation of the inhibitor inside the cells due to its poor water solubility. Moreover, this molecule acts through the depletion of ATP and therefore requires more time to be effective in respect to inhibitors acting directly on plasma membrane such as Na-orthovanadate. In conclusion, the data reported in this paper offer new information on how taxol is transported by Taxus cells. The possibility to interfere with such mechanisms by an inhibition of plasma membrane ATPase or by an alteration of Ca2+ cellular transport, provides new tools to achieve a better productivity in terms of amount of taxol released by cell cultures both at laboratory and industrial scale. Furthermore an intriguing hypothesis seems to underlie the picture offered by these results. The existence of an ATP-dependent release mechanism could indicate the involvement of an
ABC transporter, as already suggested by some authors. In fact, many plant secondary products such as vincristine and taxol are often substrates or inhibitors of mammal MDR (multi-drug resistance) proteins, which belong to the same family, suggesting a role in the synthesis and compartmentation of these compounds [35].
4. Methods 4.1. Establishment of callus cultures Callus tissues were obtained from young stem sections of Taxus baccata L. plants. After removing the needles, explants were immersed first in 70 % ethanol for 1 min, then for 15 min in 0.01 % HgCl2 and finally in sodium hypochlorite (1.5 % with few drops of Tween 20) for 30 min. After sterilisation, the explants were rinsed three times with sterile distilled water. Longitudinally-halved stem sections were placed with the inner, cut surface in contact with the culture medium. The explants were kept on Gamborg’s B5 medium [7] supplemented with 2× B5 vitamins, 3 % sucrose, 10 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 4 µM kinetin and 1 µM gibberellic acid (GA3) and solidified with 0.3 % Phytagel (Sigma, St Louis, MO, USA). The pH was adjusted to 5.8 prior to autoclaving. After 4–6 weeks, the different callus tissues formed were separated from each explant and cultured on B5 medium supplemented with 2× B5 vitamins, 0.5 % sucrose, 0.5 % fructose, 10 µM naphthaleneacetic acid (NAA) and 0.5 µM 6-benzylaminopurine (BAP). Both explants and calli were maintained at 23 °C in the darkness and subcultured every 2 weeks. 4.2. Establishment of cell suspension cultures Cell suspension cultures were established using the stem-derived callus as inoculum. Callus pieces (1 g FW) were inoculated into 100-mL flasks containing 10 mL liquid B5 medium prepared as described above (10 µM NAA, 1 µM BAP), capped with Magenta B-Cap (Sigma, St Louis, MO, USA) and placed in a rotary shaker (100 rpm) at 23 °C in the darkness. The liquid cultures were composed by cell clusters (CC) whose average number of cells was 10 to 15 per CC. 4.3. Electron microscopy Samples were fixed for 3 h at 4 °C in 1.8 % glutaraldehyde in 50 mM K-phosphate buffer (pH 7.3) and post-fixed overnight in 1 % osmium tetroxide in the same buffer at the same temperature. The specimens were dehydrated in a graded series of acetone and water and embedded in Spurr resin [30]. Sections (50–70 nm thick) were cut with an ultramicrotome (Ultracut Reichert), stained with lead citrate and observed with a transmission electron microscope (EM-301 Philips). Taxus cells were shown to be character
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ised by a thin primary wall and a vacuole which occupied about 80 to 90 % of the cell volume. 4.4. Uptake experiments Cell cultures obtained from friable callus were collected in the linear phase of growth (day 7: see Results section), filtered through a nylon filter (pore size 45 µm) and resuspended in fresh B5 medium in order to remove any exogenous taxol. Aliquots of 3 mL of this suspension (made homogeneous by gentle shaking) were transferred to glass ampoules, one of which was kept for the counting of cell clusters. The uptake was initiated by adding 3.7 kBq (in 2 µL DMSO) of [14C]-taxol (Paclitaxel-[2-benzoyl ring –UL-14C], specific activity 1.57 GBq·mmol–1, Sigma, St Louis, MO, USA) and then stopped by filtering the cells through a nylon filter (pore size 26 µm) placed on a Buchner funnel connected to a vacuum pump. Cells were washed four times with a 0.1-mM solution of unlabelled taxol and then transferred to a mortar and homogenised with 1 mL H2O. The homogenate was placed in 6 mL scintillation cocktail (Ultima Gold, Packard, Groningen, the Netherlands), and the radioactivity measured using a liquid scintillation analyser (LS 5800, Beckman Instruments Inc., Fullerton, CA, USA). In some experiments unlabelled taxol at different concentrations was supplied together with labelled one. The effect of various compounds on taxol uptake was studied by adding each substance at the required concentration and pre-incubating cells for 30 min prior to the assay. In the experiments performed without Ca2+, cells were filtered and washed with 100 mL of a 2-mM ethylene glycol bis-(βaminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA) solution and then resuspended in B5 medium without CaCl2. Data were expressed per cell cluster (CC). 4.5. Efflux experiments Cells were supplied with 3.4 kBq [14C]-taxol for 20 min, washed with 40 mL 0.1 mM solution of unlabelled taxol and transferred into fresh medium containing different concentrations of metabolic inhibitors in the presence or absence of Ca2+. The experiment was stopped as described for the uptake assays. Radioactivity was determined both in the cell homogenate and in the efflux medium, the sum of which was comparable to the uptake value of the control. 4.6. Fractionation A specific method for cell wall isolation was used [8]. Cells were filtered through a nylon filter (pore size 26 µm), ground in a mortar with 1 mM Na phosphate-citrate buffer, pH 5.8 and further ruptured with a Potter-Elvehjem apparatus. The homogenate was then sonicated three times in a MSE sonifier vibrating at maximal amplitude for 1 min and centrifuged at 100 × g for 15 min. After microscopical
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observation to verify the complete disruption of the cells, the pellet containing the cell wall fraction was washed with distilled water on a nylon filter (pore size 26 µm) and suspended in fresh B5 medium to be used for the uptake experiments. 4.7. Determination of cell growth and viability A time course of cell growth, measured both as fresh and dry weight, was performed in T. baccata cell suspension cultures. Samples were collected each 2–3 d throughout a culture period of 3 weeks, filtered, weighed and subsequently freeze-dried for determination of dry weight. A small aliquot of each sample was used for the cell viability assay, performed by incubating the cells for 5 min in B5 medium containing 75 µg·mL–1 propidium iodide (ICN Biomedicals, Costa Mesa, CA, USA), for the selective labelling of dead cells, and 75 µg·mL–1 fluorescein diacetate (Sigma, St Louis, MO, USA) for the selective labelling of live cells as described by Darzynkiewicz et al. [3]. Fluorescence was observed with a fluorescence microscope (DMRB, Leica Microsystems Inc., Wetzlar, Germany) using specific filters.
Acknowledgements This work was supported by the ex 60 % funds from MURST (Ministry of University and Scientific and Technological Research) of Italy. We are grateful to Mr N. Mele for editing of illustrations.
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[9] M.E. Goleniowski, Cell lines of Taxus species as source of the anticancer drug taxol, Biocell 24 (2000) 139–144. [10] M. Hezari, R. Croteau, Taxol biosynthesis: an update, Planta Med. 63 (1997) 291–295. [11] T.J. Hirasuna, L.J. Pestchanker, V. Srinivasan, M.L. Shuler, Taxol production in suspension cultures of Taxus baccata, Plant Cell Tissue Organ Cult. 44 (1996) 95–102. [12] R.A. Holton, H.B. Kim, C. Somoza, F. Liang, R.J. Biediger, P.D. Boatman, M.S. Shindo, C.C. Smith, S. Kim, H. Nadizadeh, Y. Suzuki, C. Tao, P. Vu, S. Tang, P. Zhang, K.K. Murthi, L.N. Gentile, J.H. Liu, First total synthesis of taxol. 2. Completion of the C and D rings, J. Am. Chem. Soc. 116 (1994) 1599–1600. [13] R.A. Holton, C. Somoza, H.B. Kim, F. Liang, R.J. Biediger, P.D. Boatman, M.S. Shindo, C.C. Smith, S. Kim, H. Nadizadeh, Y. Suzuki, C. Tao, P. Vu, S. Tang, P. Zhang, K.K. Murthi, L.N. Gentile, J.H. Liu, First total synthesis of taxol. 1. Functionalization of the B ring, J. Am. Chem. Soc. 116 (1994) 1597–1598. [14] S.B. Horowitz, L. Lothsteia, J.J. Manfredi, W. Mellado, J. Parness, S.N. Roy, P.B. Schiff, L. Sorbara, R. Zeheb, Taxol: mechanism of action and resistance, Ann. NY Acad. Sci. 466 (1986) 733–744. [15] M. Jaziri, A. Zhiri, Y.W. Guo, J.P. Dupont, K. Shimomura, H. Hamada, M. Vanhaelen, J. Homès, Taxus sp. cell, tissue and organ cultures as alternative source for taxoids production: a literature survey, Plant Cell Tissue Organ Cult. 46 (1996) 59–75. [16] D. Khayat, E.C. Antoine, D. Coeffic, Taxol in the management of cancers of the breast and the ovary, Cancer Invest. 18 (2000) 242–260. [17] G. Laskaris, R. van der Heijden, R. Verpoorte, Purification and partial characterisation of geranylgeranyl diphosphate synthase from Taxus baccata cell cultures. An enzyme that regulates taxane biosynthesis, Plant Sci. 153 (2000) 97–105. [18] J. Matur, N.H. Chua, Microtubule stabilization leads to growth reorientation in Arabidopsis trichomes, Plant Cell 12 (2000) 465–477. [19] N. Mirjalili, J.C. Linden, Methyl jasmonate induced production of taxol in suspension cultures of Taxus cuspidata: ethylene interaction and induction models, Biotechnol. Prog. 12 (1996) 110–118. [20] L.C. Morejohn, D.E. Fosket, Taxol-induced rose microtubule polymerization in vitro and its inhibition by colchicine, J. Cell Biol. 99 (1984) 141–147. [21] K.C. Nicolau, Z. Yang, J.J. Liu, H. Ueno, P.G. Nantermet, R.K. Guy, C.F. Clalborne, J. Renaud, E.A. Couladouros, K. Paulvannan, E.J. Sorensen, Total synthesis of taxol, Nature 367 (1994) 630–634. [22] R. Patel, Tour de paclitaxel: biocatalysis for semisynthesis, Annu. Rev. Microbiol. 98 (1998) 361–395. [23] L.J. Pestchanker, S.C. Roberts, M.L. Shuler, Kinetics of taxol production and nutrient use in suspension cultures of Taxus cuspidata in shake flasks and Wilson-type bioreactor, Enzyme Microb. Technol. 19 (1996) 256–260.
[24] D.J. Rodi, R.W. Janes, H.J. Sanganee, R.A. Holton, B.A. Wallace, L. Makowski, Screening of a library of phage-displayed peptides identifies human Bcl-2 as a taxol-binding protein, J. Mol. Biol. 285 (1999) 197–203. [25] W.A. Russin, D.D. Ellis, J.R. Gottwald, E.L. Zeldin, M. Brodhagen, R.F. Evert, Immunocytochemical localization of taxol in Taxus cuspidata, Int. J. Plant Sci. 156 (1995) 668–678. [26] P.B. Schiff, J. Fant, S.B. Horowitz, Promotion of microtubule assembly in vitro by taxol, Nature 277 (1979) 665–667. [27] A. Schoendorf, C.D. Rithner, R.M. Williams, R.B. Croteau, Molecular cloning of a cytochrome P450 taxane 10 beta-hydroxylase cDNA from Taxus and functional expression in yeast, Proc. Natl. Acad. Sci. USA 98 (2001) 1501–1506. [28] M. Seki, S. Furusaki, An immobilized cell system for taxol production, Chemtech 26 (1996) 41–45. [29] M. Seki, C. Ohzora, M. Takeda, S. Furusaki, Taxol (paclitaxel) production using free and immobilized cells of Taxus cuspidata, Biotechnol. Bioeng. 53 (1997) 214–219. [30] A.R. Spurr, A low-viscosity epoxy resin embedding medium for electron microscopy, J. Ultrasctruct. Res. 26 (1969) 36–43. [31] V. Srinivasan, V. Ciddi, V. Bringi, M.L. Shuler, Metabolic inhibitors, elicitors and precursors as tools for probing yield limitation in taxane production by Taxus chinensis cell cultures, Biotechnol. Prog. 12 (1996) 457–465. [32] V. Srinivasan, L. Pestchanker, S. Moser, T.J. Hirasuna, R.A. Taticek, M.L. Shuler, Taxol-production in bioreactors: kinetics of biomass accumulation, nutrient uptake and taxol production by cell suspension of Taxus baccata, Biotechnol. Bioeng. 47 (1995) 666–676. [33] G.A. Strobel, A. Stierle, W.M. Hess, Taxol formation in yew-Taxus, Plant Sci. 92 (1993) 1–12. [34] M. Suffness, M.E. Wall, Discovery and development of taxol, in: M. Suffness (Ed.), Taxol: Science and Applications, CRC, Boca Raton, Fl, 1995, pp. 3–25. [35] F.L. Theodoulou, Plant ABC transporters, Biochim. Biophys. Acta 1465 (2000) 79–103. [36] M. Villegas, M. Sommarin, P.E. Brodelius, Effects of sodium orthovanadate on benzophenanthridine alkaloid formation and distribution in cell suspension cultures of Eschscholtzia californica, Plant Physiol. Biochem. 38 (2000) 233–241. [37] U.K. Walle, T. Walle, Taxol transport by human intestinal epithelial Caco-2 cells, Drug Metab. Dispos. 26 (1998) 343–346. [38] N.C. Wheeler, K. Jech, S. Masters, S.W. Brobst, A.B. Alvarado, A.J. Hoover, K.M. Snader, Effects of genetic, epigenetic and environmental factors on taxol content in Taxus brevifolia and related species, J. Nat. Prod. 55 (1992) 432–440. [39] E.R.M. Wickremesinhe, R. Arteca, Taxus cell suspension cultures: optimizing growth and production of taxol, J. Plant Physiol. 44 (1994) 183–188.
Taxol transport in Taxus baccata cell suspension cultures Silvia Fornalè a, Davide Degli Esposti a, Alberto Navia-Osorio b, Rosa M. Cusidò b, Javier Palazòn b, M. Teresa Piñol b, Nello Bagni a,* a
Dipartimento di Biologia Evoluzionistica Sperimentale e Centro Interdipartimentale di Biotecnologie, Università di Bologna, via Irnerio 42, 40126 Bologna, Italy b Laboratorio de Fisiologia Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avda. Diagonal 643, 08028 Barcelona, Spain Received 10 May 2001; accepted 28 July 2001
Abstract Taxol transport in Taxus baccata L. cell suspension cultures was studied using [14C]-taxol as a tracer. The time course of uptake showed a saturable absorption that reached a maximum within 20 min. The uptake depended on its exogenous concentration and its accumulation was highly stimulated in the presence of 10–15 µM exogenous taxol. The absorbed molecule was found to localise both in the cell walls (20 %) and in the cell protoplasts (80 %), suggesting an accumulation within the vacuoles. Taxol uptake was strongly inhibited by Na-orthovanadate and verapamil, while Ca2+ was found to be one of the factors required for the active absorption of the molecule, since in the absence of this cation, the uptake was reduced by about 40 % and occurred mainly through a non-energy dependent mechanism. Taxol release into the culture medium was demonstrated not to depend on cell lysis, occurred through a mechanism that reached its maximum after 10–15 min and was strongly enhanced by treatment with Na-orthovanadate and verapamil, although the effect was found to be transient. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Cell cultures; Metabolic inhibitors; Release; Taxol; Taxus baccata; Uptake
1. Introduction Taxol (paclitaxel, NSC-125973) is a highly functionalized diterpenoid produced by the genus Taxus. It is a well-established anti-tumour drug, whose properties are based on the ability to bind and stabilise microtubules, thus leading to the block of cell replication in the late G2-M phase of the cell cycle [14]. This plant secondary metabolite promotes the formation of microtubules that are resistant to disassembly, in contrast to that observed for other antimitotic agents such as colchicine and Vinca alkaloids [26]. Taxol was approved in 1992 by the US Food and Drug Administration for the treatment of ovarian and breast
Abbreviations: BAP, 6-benzylamino-purine; CC, cell cluster; DCCD, dicyclohexylcarbodiimide; 2,4 DNP, 2,4-dinitrophenol; NAA, αnaphthaleneacetic acid; NEM, N-ethylmaleimide * Corresponding author. Fax +39 051 242576. E-mail address: [email protected] (N. Bagni). © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 8 1 - 9 4 2 8 ( 0 1 ) 0 1 3 3 2 - 8
cancer, and it was shown to be active against a variety of other cancers such as lung, gastrointestinal, neck and head as well as malignant melanoma [1,16,34]. This compound was originally obtained from the bark of the pacific yew (Taxus brevifolia), but the low yield (0.01 % of dry weight) led researchers to look for alternative sources [5,38]. Total synthesis was achieved by Nicolau et al. [21] and by Holton et al. [12,13], but its high cost makes this approach not commercially feasible, so that it is currently produced by semisynthesis [22]. Much attention has been given to plant tissue culture, which is one of the approaches available to provide large amounts and a stable supply of pharmaceutically valuable compounds [15]. Basic protocols for the induction and maintenance of taxol-yielding callus and cell cultures of different Taxus species have been developed during the past decade [9,29,39] and a lot of attention is currently being dedicated to improving taxol production and release into the culture medium. Factors that regulate and limit taxol biosynthesis and the in vitro cell growth of Taxus cell cultures have been intensively investigated [2,10,17,19,27,31], but almost no information is available on how taxol is transported by Taxus cells. In fact, no
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studies have been published on the molecular mechanism underlying taxol release into the culture medium, and not even the ability of Taxus cells to take up and accumulate this metabolite at subcellular level is known. In fact, studies performed by Srinivasan et al. [32] and Hirasuna et al. [11] indicated that more than 90 % of taxol is routinely excreted into the culture medium, while Wickremesinhe and Arteca [39] supposed that taxol presence in the culture medium could be due to cell lysis rather than active secretion. Taxol is present in virtually all parts of the yew tree [4], but preferentially accumulates in bark and needles. However nothing is known about its subcellular localisation or the existence of a translocation system within the plant. Strobel et al. [33] reported the presence of taxol in the sapwood and heartwood of T. brevifolia even though only traces of its biosynthesis could be demonstrated in the xylem. They suggested that taxol might be mobilised from its place of greatest biosynthetic activity (vascular-cambial region), possibly via ray-parenchyma, and translocated to the xylem. Moreover, taxol affects microtubules in many organisms, including higher plants [18,20], so that it can be considered toxic to Taxus itself. Thus, a strategy to avoid taxol effects, such as its excretion, must be present in Taxus cells, and the elucidation of such a mechanism could be crucial to improving the productivity of Taxus plant and cell cultures. Immunocytochemical studies performed by Russin et al. [25] showed that taxol seems to accumulate almost exclusively in the cell wall, rather than in cell compartments such as vacuoles. The precise localisation of taxol in plant tissues or cell cultures raises numerous questions, since the molecule is displaced during classical fixation procedures. For this reason, no clear evidence has been achieved so far. Numerous studies showed the lack of any relationship between the endogenous content of taxol and its release in Taxus cell cultures [11,23,28,29,32]. This seems to indicate the presence of regulated processes that, at present, remain to be investigated. More information is available in the animal field, where studies on taxol transport in human cells [37] gave indications of the possible involvement of active transport mechanisms and several putative taxol-binding proteins have been identified [24]. All of the evidence currently in the literature suggests the existence of a taxol transport system. Hence, the aim of this work was to investigate how Taxus cells transport taxol and if this process presents energy-dependent features. Most of the studies on taxol published so far showed the ability of Taxus long-term cell cultures to release this compound into the culture medium, but no information is available on its uptake. Thus far no studies have been made on the molecular mechanism by which taxol is transported in and out of Taxus cells. It is our opinion that such knowledge could offer a new tool to improve taxol productivity in Taxus cell cultures. In fact the identification of proteins able to bind and transport the molecule in and out of the cell could represent a new approach to enhance taxol release.
Fig. 1. Time course of biomass accumulation and cell viability in T. baccata cell suspension cultures. Data are expressed both as fresh (■) and dry weight (·) accumulation. Cell viability (bars) was measured by the selective labelling of living/dead cells as explained in Methods and expressed as % of living cells. Each value is the mean of three replicates ± SD.
2. Results 2.1. Determination of cell growth and viability A time course was performed on T. baccata cell suspension culture over a 21-d growth period. A first rapid increase of both FW and DW was observed between days 2 and 7 (Fig. 1). This increase was then followed by a stationary phase of growth, with no significant changes in biomass. Cell viability remained very high (from 98 to 91 %) until day 13 and slightly decreased in the following days, reaching 80 % at the end of the culture period considered (day 21). 2.2. Taxol uptake in T. baccata cell suspension cultures 2.2.1. General characteristics of taxol uptake Time course assays of taxol uptake were performed on T. baccata cell suspension culture in the linear phase of growth using labelled taxol as a tracer. Taxol was found to be transported into the cells, reaching maximum absorption after 20–30 min (Fig. 2). Considering the total numbers of moles absorbed compared to those supplied, we calculated that in these conditions, cells were able to absorb up to 8 % of the exogenous supplied taxol. In order to verify the occurrence of a real taxol uptake, experiments were performed to study the localisation of the absorbed taxol. For this reason, a T. baccata cell suspension culture was first divided into two aliquots one of which was used for cell wall isolation as described in the Methods section. A time course using [14C]-taxol as a tracer was then performed on the remaining aliquot and on the cell wall suspension and the results compared (Fig. 2). A time-dependent interaction of taxol with cell wall components was observed: in the described conditions, cell walls adsorbed taxol reaching maximum adsorption after 20 min. Comparing the maximum absorption of cell cultures to the corresponding cell wall fraction, we estimated that about 20 % of the total radioactivity recovered in the cells (8 %) was due to taxol
S. Fornalè et al. / Plant Physiol. Biochem. 40 (2002) 81–88
Fig. 2. Distribution of supplied [14C]-taxol between T. baccata cell suspension culture and its correspondent cell wall fraction. A cell suspension culture was separated into two aliquots, one of which was used for the extraction of the cell walls. A time course of taxol uptake was performed by adding 3.4 kBq labelled compound to the cell cultures (■) and their correspondent cell wall fractions (•). The experiments were performed several times with similar results: the curves are typical of one experiment. Data are expressed as pmoles absorbed by 103 cell clusters and represent the mean ± SD of three replicates.
binding to cell wall components, while the remaining 80 % was actually absorbed by the protoplast. Taxol uptake in relation to external concentration was measured over a 20-min incubation with [14C]-taxol as a tracer and increasing unlabelled taxol concentrations (up to 50 µM) in the presence of 1 mM Ca2+ (basic B5 medium). It should be noted that 50 µM represented the upper limit for our system, due to the low solubility of the molecule and to the toxic effect that relatively high concentrations of DMSO (over 0.6 %) exerted on T. baccata cells, as verified by cell viability assays (data not shown). The effect of increasing the external concentration (Fig. 3) was a rapid increase in the amount of taxol absorbed, which displayed saturation kinetic and reached a maximum corresponding to 10–15 µM external concentration. The great stimulation of taxol uptake reflected in the 30-fold increase observed comparing the absorption measured when only [14C]-taxol as a tracer was supplied (3 pmol per 103 CC) and the absorption in the presence of 10–15 µM exogenous taxol (about 85 pmol per 103 CC). 2.2.2. Effects of metabolic inhibitors on taxol uptake Taxol endogenous levels in T. baccata cell suspension cultures ranges from 0.01–0.03 % of the dry weight, which roughly corresponds to a concentration of about 15–20 µM; thus the uptake measured in our system seemed to occur against a concentration gradient, since the labelled taxol was added at a concentration of 0.6 µM. For this reason and to better address the nature of taxol uptake, the effect of different metabolic inhibitors was tested. Most of the compounds used were soluble in ethanol, which could itself be toxic. Hence it was used at low levels (1 %) where no effect due to the solvent alone was observed on taxol uptake (Table 1). Among the inhibitors tested, in the presence of 1 mM Na-orthovanadate taxol uptake was inhibited by
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Fig. 3. Effect of exogenous taxol on uptake. Taxol uptake was determined over a concentration range of 0.05 to 50 µM: 3.4 kBq [14C]-taxol and different concentration of the unlabelled compound were added to a T. baccata cell suspension culture in the presence (■) and absence (·) of 1 mM Ca2+. Data represent the mean ± SD of three different experiments and are expressed as pmoles absorbed by 103 cell clusters.
about 40 % with respect to the control. This substance, together with DCCD (dicyclohexylcarbodiimide), was tested because they inhibit the H+-ATPase of the plasmalemma, but no effect was observed with DCCD. Nitrate, supplied both as NaNO3 and KNO3 did not affect taxol uptake: this anion is known to inhibit the tonoplast ATPase at high concentrations (50–100 mM). A 25 % inhibition was observed after the treatment with verapamil, a phenylalkylamine which blocks Ca2+ channels, but no effect was observed using nifedipine (a 1-4-dihydropyridine), which exerts the same effect. No changes were observed after incubation with N-ethylmaleimide (NEM), a thiol-reacting compound. Cells treated with 1 mM 2,4-DNP produced a strong enhancement of the uptake, the amount of the absorbed molecule being about 30 % higher than the control’s. To better understand whether Ca2+ could influence taxol uptake, a series of experiments was done on T. baccata cell suspension cultures in the presence (basic B5 medium) or absence of 1 mM CaCl2 and verapamil (Table 2). Ca2+ absence reduced the uptake, in line with the inhibition caused by verapamil in the presence of 1 mM CaCl2. These results led us to conclude that the activity of Ca2+ channels was required for Ca2+-activated taxol transport. For this reason, a further set of experiments was done in order to study taxol uptake in relation to external concentration in the absence of external Ca2+. When the assay was performed in a Ca2+-free medium (Fig. 3), the uptake occurred through a non-energy dependent mechanism, indicating that Ca2+ seemed to be one of the factors involved in the active part of the absorption. 2.3. Taxol release into the culture medium A time course assay of taxol release was performed on T. baccata cells previously incubated with a known amount of the radioactive compound (Fig. 4). The release of labelled
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Table 1 Effect of metabolic inhibitors on taxol uptake. Cells were pre-incubated 30 min with the various compounds. They were then supplied with 3.7 kBq [14C]-taxol and 10 µM unlabelled taxol and the uptake was measured after 20 min. Data are the mean ± SD of three replicates. * The negative value indicates an enhancement of the uptake with respect to the control. Inhibitor
Concentration (mM)
Solvent
Percent of solvent
Percent of inhibition compared to control
Na-orthovanadate
0.1 1 0.5 1 0.1 1 100 100 0.5 0.1 0.1 –
H2O
– – 1 1 1 1 – – – – 1 1
0 42 ± 5 0 0 0 –30 ± 3* 0 0 0 25 ± 1 0 0
DCCD 2,4 DNP NaNO3 KNO3 NEM Verapamil Nifedipine None
Ethanol Ethanol H2O H2O H 2O H2O Ethanol Ethanol
taxol from the cells followed a time-dependent mechanism that reached saturation after 10 min from the beginning of the assay. In these conditions, T. baccata cells were able to release about 58–60 % of the total labelled compound. Taxol efflux was not influenced by Ca2+ presence into the culture medium, contrarily to what observed in the case of its uptake (data not shown). To better understand which kind of mechanism does regulate taxol transport in T. baccata cells, we decided to test the same inhibitors that influenced the uptake, namely Na-orthovanadate, verapamil and 2,4-dinitrophenol. Cells Table 2 Effect of Ca2+ and verapamil on taxol uptake. Cells were pre-incubated 30 min with verapamil and/or Ca2+, then 3.7 kBq [14C]-taxol and 10 µM unlabelled taxol were added. The radioactivity inside the cells was measured after 20 min. Data are the mean ± SD of three replicates and are expressed as pmoles absorbed by 103 cell clusters. Ca2+ (mM)
Verapamil (mM)
Taxol uptake (pmol·(103 CC)–1)
1 1 0 0
0 0.1 0 0.1
62.7 ± 5.8 45.2 ± 1.0 38.3 ± 0.2 33.2 ± 2.4
Fig. 4. Time course of taxol release by a 7-d-old cell culture of T. baccata. Cells were first incubated with 3.4 kBq labelled taxol and then transferred to fresh medium for the determination of the release. Data are the mean of three replicates ± SD and are expressed as % of the previously absorbed molecule that is released into the medium.
were first pre-incubated with the labelled molecule and then transferred into the efflux medium containing the metabolic inhibitor. When cells were treated with Na-orthovanadate (Fig. 5), a great stimulation of the efflux, which doubled the control value, was observed in correspondence of the first minutes of the assay . It has to be noted how this effect was transient, since starting from the fifth minute, the amount of released molecule reached control values. An analogous stimulation of taxol release was observed in the presence of verapamil (Fig. 6). This compound was found to be able to enhance taxol efflux from 90 % (3rd min) to 60 % (5th min) compared to control values. Even in this case the effect displayed by verapamil was found to be transient, although a little more persistent. In fact values comparable to the control were reached from 10 min after the beginning of the experiment. In contrast, no effect was observed when taxol release was measured in the presence of 1 mM 2,4-DNP (data not shown).
Fig. 5. Effect of Na-orthovanadate on taxol release. Cells were preincubated with 3.4 kBq labelled molecule and then transferred into the efflux medium in the presence (black bars) or not (white bars) of 1 mM Na3VO4 for the determination of the release. Data are the mean of three replicates ± SD and are expressed as % of the previously absorbed molecule that is released into the medium.
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Fig. 6. Effect of verapamil on taxol release. Cells were pre-incubated with 3.4 kBq the labelled molecule and then transferred into the efflux medium in the presence (black bars) or not (white bars) of 0.1 mM verapamil for the determination of the release. Data are the mean of three replicates ± SD and are expressed as % of the previously absorbed molecule that is released into the medium.
3. Discussion
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of exogenous Ca2+, we hypothesise that in the presence of this cation, taxol absorption can occur mainly through active processes and that this second mechanism can be considered more physiological. Furthermore, the high endogenous accumulation of this compound in Taxus cells suggests the involvement of vacuoles as storage compartments. These organelles represent the greatest part of T. baccata cells, as shown by ultrastructural studies performed on 7-d-old cell cultures and their involvement could not be excluded by previous immunocytochemical studies performed by Russin et al. [25]. However, even though the tonoplast is very close to the plasmalemma (data not shown), H+-ATPase does not seem directly involved in taxol transport. Taxol uptake in respect to its exogenous concentration was maximum in the presence of 10–15 µM exogenous taxol. A rough estimation of the Km for this transport gave a value of about 1.5 µM. This value fits with the one obtained by Walle and Walle [37] in human cells. Their studies were in favour of an energy dependent transport too, since taxol transport was found to be affected by verapamil, which is also a well known inhibitor of the P-glycoprotein.
3.1. General characteristics of taxol uptake The work reported in this paper intended to give evidence of the existence of a taxol transport mechanism in T. baccata cell suspension cultures. One of the main problem that still has to be solved in the biotechnological exploitation of Taxus cell cultures as natural producers of this compound is how to increase taxol release into the culture medium. Information on how Taxus cells can accumulate and transport this metabolite under physiological conditions (culture conditions in this case) could provide a powerful tool and a new approach to solve this problem. Despite numerous studies on taxol distribution in the whole Taxus plant, up to now it has been impossible to clearly identify whether a particular organ/tissue is responsible for taxol biosynthesis or if any plant cell produces this compound which is then preferentially accumulated in some tissues (e.g. bark and needles). In this light it was necessary to test first if cells can take up the molecule, and by which mechanism. This ability together with the existence of regulated release processes could indicate the presence of a transport system, whose understanding will permit biotechnological improvements. Data reported in this paper indicate how taxol can be absorbed by Taxus cells. Due to the lipophilic nature of the compound a diffusion mechanism could be hypothesised. At the same time, it has been proposed that taxol could mainly accumulate in the cell walls instead of internal cell compartments such as vacuole [25]. Our data indicated that this uptake reached saturation within 20 min, leading to the absorption of about 8 % of the exogenous supplied taxol and occurred against a concentration gradient. Moreover we demonstrated that only 20 % of the absorbed taxol seems to localise in the cell wall fraction. For these reasons, although a non-energy dependent mechanism occurs in the absence
3.2. Effect of Ca2+ and metabolic inhibitors on taxol uptake Few metabolic inhibitors were found to affect taxol uptake in T. baccata cells. Na-orthovanadate (an ATPase antagonist) and the Ca2+ channel blocker verapamil exerted an inhibitory effect, while the uncoupler 2,4-DNP leads to an increase of taxol absorption. Several other inhibitors were tested, but without effects. Since DCCD and nifedipine had to be supplied in a 1 % ethanol solution, it can not be excluded that the lack of effect could be due to the possible lack of permeation of the molecule within the cell and/or interactions with cell wall components. Regarding the role of Ca2+, this cation seems to be needed for taxol uptake. In fact, when the uptake was measured in a calcium-free medium, a strong inhibition of about 30 % was observed. A rough estimation of how Ca2+ can promote taxol uptake was allowed by the comparison of the absorption kinetics performed in the presence/absence of this cation (Fig. 3). Our data indicate that, up to 30 µM taxol exogenous concentration, Ca2+ seems to be one of the factors that regulate the energy-dependent part of the uptake which occurs, even if to a lower degree, by a passive mechanism. 3.3. General characteristics of taxol efflux and effect of metabolic inhibitors The studies performed on taxol efflux permitted to clarify how Taxus cells can release the compound by means of a cellular mechanism instead of a cell lysis phenomena. Even though the presence of this metabolite in the medium of long-term cultures can be partially due to cell necrosis (common in later stages of the growth cycle), the release
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measured in our assays, performed using a 7-d-old cultures with 97 % of viability (Fig. 1), was observed over a time that ranges from 30 s to 20 min and, therefore, it can not be related to cell death. T. baccata cells were able to release at least the 55–60 % of the labelled compound. This value resembles that reported in literature for T. cuspidata [6] and T. baccata [11,32] cell cultures, where percentages ranging from 60 to 90 % were measured at the end of the culture period. Taxol uptake was reduced in the presence of Naorthovanadate, while the release was strongly enhanced. This inhibitor acts on plasma membrane ATPase and its addition provokes the inhibition of proton pumping with the consequent depolarisation of membrane potentials needed for active transport of various solutes, including small molecules. For this reason Na-orthovanadate has been successfully used as an abiotic elicitor to induce alkaloid biosynthesis [36] and even the effect displayed on T. baccata cells can be related to an elicitor-induced stress response which leads to an enhanced release of secondary metabolites into the culture medium. The effect exerted by verapamil (uptake inhibition and enhancement of the release) appears to be related to its Ca2+ channel blocker activity. This hypothesis seems to be confirmed by the cross-assays on taxol uptake in the presence/absence of both verapamil and Ca2+. The same inhibition measured in the absence of this cation is in fact provoked by the treatment with verapamil. The role of this cation in taxol transport should be related to the complex network of the signal transduction mechanisms. Therefore, any transient alteration of its endogenous level could be responsible for the effects observed. The increased uptake measured in the presence of 2,4-DNP could be related to a reduced ability of cells to release the absorbed molecule. This inhibitor blocks the mitochondrial ATP synthesis and these organelles represent in our system the unique source of this molecule, being T. baccata cell suspensions cultured in the dark. Hence this result is in favour of an energydependent release mechanism. Nevertheless, when 2,4-DNP was added to the efflux medium, no effect was observed. This can be explained by the reduced permeation of the inhibitor inside the cells due to its poor water solubility. Moreover, this molecule acts through the depletion of ATP and therefore requires more time to be effective in respect to inhibitors acting directly on plasma membrane such as Na-orthovanadate. In conclusion, the data reported in this paper offer new information on how taxol is transported by Taxus cells. The possibility to interfere with such mechanisms by an inhibition of plasma membrane ATPase or by an alteration of Ca2+ cellular transport, provides new tools to achieve a better productivity in terms of amount of taxol released by cell cultures both at laboratory and industrial scale. Furthermore an intriguing hypothesis seems to underlie the picture offered by these results. The existence of an ATP-dependent release mechanism could indicate the involvement of an
ABC transporter, as already suggested by some authors. In fact, many plant secondary products such as vincristine and taxol are often substrates or inhibitors of mammal MDR (multi-drug resistance) proteins, which belong to the same family, suggesting a role in the synthesis and compartmentation of these compounds [35].
4. Methods 4.1. Establishment of callus cultures Callus tissues were obtained from young stem sections of Taxus baccata L. plants. After removing the needles, explants were immersed first in 70 % ethanol for 1 min, then for 15 min in 0.01 % HgCl2 and finally in sodium hypochlorite (1.5 % with few drops of Tween 20) for 30 min. After sterilisation, the explants were rinsed three times with sterile distilled water. Longitudinally-halved stem sections were placed with the inner, cut surface in contact with the culture medium. The explants were kept on Gamborg’s B5 medium [7] supplemented with 2× B5 vitamins, 3 % sucrose, 10 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 4 µM kinetin and 1 µM gibberellic acid (GA3) and solidified with 0.3 % Phytagel (Sigma, St Louis, MO, USA). The pH was adjusted to 5.8 prior to autoclaving. After 4–6 weeks, the different callus tissues formed were separated from each explant and cultured on B5 medium supplemented with 2× B5 vitamins, 0.5 % sucrose, 0.5 % fructose, 10 µM naphthaleneacetic acid (NAA) and 0.5 µM 6-benzylaminopurine (BAP). Both explants and calli were maintained at 23 °C in the darkness and subcultured every 2 weeks. 4.2. Establishment of cell suspension cultures Cell suspension cultures were established using the stem-derived callus as inoculum. Callus pieces (1 g FW) were inoculated into 100-mL flasks containing 10 mL liquid B5 medium prepared as described above (10 µM NAA, 1 µM BAP), capped with Magenta B-Cap (Sigma, St Louis, MO, USA) and placed in a rotary shaker (100 rpm) at 23 °C in the darkness. The liquid cultures were composed by cell clusters (CC) whose average number of cells was 10 to 15 per CC. 4.3. Electron microscopy Samples were fixed for 3 h at 4 °C in 1.8 % glutaraldehyde in 50 mM K-phosphate buffer (pH 7.3) and post-fixed overnight in 1 % osmium tetroxide in the same buffer at the same temperature. The specimens were dehydrated in a graded series of acetone and water and embedded in Spurr resin [30]. Sections (50–70 nm thick) were cut with an ultramicrotome (Ultracut Reichert), stained with lead citrate and observed with a transmission electron microscope (EM-301 Philips). Taxus cells were shown to be character
S. Fornalè et al. / Plant Physiol. Biochem. 40 (2002) 81–88
ised by a thin primary wall and a vacuole which occupied about 80 to 90 % of the cell volume. 4.4. Uptake experiments Cell cultures obtained from friable callus were collected in the linear phase of growth (day 7: see Results section), filtered through a nylon filter (pore size 45 µm) and resuspended in fresh B5 medium in order to remove any exogenous taxol. Aliquots of 3 mL of this suspension (made homogeneous by gentle shaking) were transferred to glass ampoules, one of which was kept for the counting of cell clusters. The uptake was initiated by adding 3.7 kBq (in 2 µL DMSO) of [14C]-taxol (Paclitaxel-[2-benzoyl ring –UL-14C], specific activity 1.57 GBq·mmol–1, Sigma, St Louis, MO, USA) and then stopped by filtering the cells through a nylon filter (pore size 26 µm) placed on a Buchner funnel connected to a vacuum pump. Cells were washed four times with a 0.1-mM solution of unlabelled taxol and then transferred to a mortar and homogenised with 1 mL H2O. The homogenate was placed in 6 mL scintillation cocktail (Ultima Gold, Packard, Groningen, the Netherlands), and the radioactivity measured using a liquid scintillation analyser (LS 5800, Beckman Instruments Inc., Fullerton, CA, USA). In some experiments unlabelled taxol at different concentrations was supplied together with labelled one. The effect of various compounds on taxol uptake was studied by adding each substance at the required concentration and pre-incubating cells for 30 min prior to the assay. In the experiments performed without Ca2+, cells were filtered and washed with 100 mL of a 2-mM ethylene glycol bis-(βaminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA) solution and then resuspended in B5 medium without CaCl2. Data were expressed per cell cluster (CC). 4.5. Efflux experiments Cells were supplied with 3.4 kBq [14C]-taxol for 20 min, washed with 40 mL 0.1 mM solution of unlabelled taxol and transferred into fresh medium containing different concentrations of metabolic inhibitors in the presence or absence of Ca2+. The experiment was stopped as described for the uptake assays. Radioactivity was determined both in the cell homogenate and in the efflux medium, the sum of which was comparable to the uptake value of the control. 4.6. Fractionation A specific method for cell wall isolation was used [8]. Cells were filtered through a nylon filter (pore size 26 µm), ground in a mortar with 1 mM Na phosphate-citrate buffer, pH 5.8 and further ruptured with a Potter-Elvehjem apparatus. The homogenate was then sonicated three times in a MSE sonifier vibrating at maximal amplitude for 1 min and centrifuged at 100 × g for 15 min. After microscopical
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observation to verify the complete disruption of the cells, the pellet containing the cell wall fraction was washed with distilled water on a nylon filter (pore size 26 µm) and suspended in fresh B5 medium to be used for the uptake experiments. 4.7. Determination of cell growth and viability A time course of cell growth, measured both as fresh and dry weight, was performed in T. baccata cell suspension cultures. Samples were collected each 2–3 d throughout a culture period of 3 weeks, filtered, weighed and subsequently freeze-dried for determination of dry weight. A small aliquot of each sample was used for the cell viability assay, performed by incubating the cells for 5 min in B5 medium containing 75 µg·mL–1 propidium iodide (ICN Biomedicals, Costa Mesa, CA, USA), for the selective labelling of dead cells, and 75 µg·mL–1 fluorescein diacetate (Sigma, St Louis, MO, USA) for the selective labelling of live cells as described by Darzynkiewicz et al. [3]. Fluorescence was observed with a fluorescence microscope (DMRB, Leica Microsystems Inc., Wetzlar, Germany) using specific filters.
Acknowledgements This work was supported by the ex 60 % funds from MURST (Ministry of University and Scientific and Technological Research) of Italy. We are grateful to Mr N. Mele for editing of illustrations.
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