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Effect of taxol feeding on taxol and related taxane production in Taxus baccata suspension cultures
New Biotechnology Volume 25, Number 4 April 2009
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Research Paper
Effect of taxol feeding on taxol and related taxane production in Taxus baccata suspension cultures Oscar Expo´sito1, Mercedes Bonfill1, Miriam Onrubia2, Albert Jane´1, Elisabet Moyano2, Rosa M. Cusido´1, Javier Palazo´n1 and M. Teresa Pin˜ol1 1 2
Laboratorio de Fisiologı´a vegetal, Facultad de Farmacia, Universidad de Barcelona, Avda. Diagonal 643, E-08028 Barcelona, Spain Departament de Cie`ncies Experimentals i de la Salut, Universitat Pompeu Fabra, Avda Aiguader 80, E-08003 Barcelona, Spain
To achieve a better understanding of taxol metabolism and accumulation in Taxus cell cultures, a T. baccata cell line growing for 20 days in a selected growth medium was treated at the beginning of the experiment with several concentrations of taxol (25, 50, 100 and 200 mg L1). Compared with an untreated control, all these taxol concentrations stimulated cell-associated taxol content (up to 32.7 times in the presence of 200 mg L1 exogenous taxol), although higher concentrations significantly depressed cell viability. DNA laddering analysis revealed that the viability reduction was not related to apoptosis, suggesting that taxol itself was the primary responsible factor. On the basis of RT-PCR expression analysis of genes encoding taxadiene synthase (ts) and 1-deoxy-D-xylulose-5-phosphate synthase (dxs) from treated and nontreated T. baccata cell line cultures, it was observed that exogenous taxol clearly induced the mRNA levels of both taxane-related enzymes. Additionally, we found that exogenous taxol caused a considerable increase in taxadiene synthase activity, although in no case did this coincide with the highest levels of taxol observed at the end of the culture. The effect of exogenous taxol on the content of other related taxanes was also considered.
Introduction Taxol has been a highly successful anticancer drug since it was initially approved for the treatment of breast and ovarian cancers [1]. Other molecular targets for taxanes, such as multidrug resistance inhibition and apoptosis inhibitor binding, as well as treatments for nonsmall-cell lung cancer and AIDS-related Kaposi’s sarcoma, are currently being investigated [2]. Demands on the supply of taxol also continue to grow as a result of its expanding use in early intervention therapies and in combination with other chemotherapeutic agents. At present, taxol is successfully obtained in cell cultures of different Taxus species [3–7]. To enhance the taxol production of these cultures, the addition of several elicitor compounds has been assayed [8–14]. Some elicitors improve the taxol production, but the majority reduce cell viability and growth. This decrease of cell viability and the consequent Corresponding author: Bonfill, M. ([email protected])
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reduction of growth in highly productive Taxus cell cultures could be a cell response to the elicitor action. It could also be caused by the toxic effect of taxol itself on cell metabolism [14,15], because taxol affects microtubules in many organisms, including higher plants [16]. The production of defence compounds is closely related to the cell death mechanism in plants [12]. Certain fatty acids in octadecanoid pathways for signaling defense, such as those generating jasmonic acid, can also induce apoptosis [17]. Several reports have indicated that programmed cell death is closely related to taxol production in suspension cultures of Taxus cells [15,18,19]. Ma et al. [20] suggested that apoptosis is a major death mechanism in Taxus canadiensis cell cultures. By contrast, after carrying out DNA laddering analysis Kim et al. [14] have reported that viability reduction in T. cuspidata cell cultures does not appear to be related to apoptosis. In this context, it is worth noting that while apoptosis is a process in which the cell directs its own death, necrosis is
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New Biotechnology Volume 25, Number 4 April 2009
Materials and methods Establishment and growth conditions of the cell line culture The cell line of T. baccata was established from a stable callus line (the variance in taxane content and growth rate of each subculture was <10%, data not shown), as reported earlier [5]. To obtain low levels of taxol in our cell line, which would allow us to see more clearly how the addition of taxol might affect its accumulation, together with that of other related taxanes, we used a previously selected growth medium Gamborg’s B5 medium [24] with 0.5% sucrose + 0.5% fructose, 2 mg L1 of NAA and 0.1 mg L1 of BAP that promotes cell growth but not taxol production in T. baccata cell cultures [25]. 1 0.2 g wet weight of cells grown for 16 days in the growth medium (the length of time necessary for them to enter the stationary growth phase, Fig. 2) were transferred to 175-mL flasks (Sigma VOG33) containing 10 mL of the same medium. They were then cultured for 20 days without taxol (control) or with several concentrations of taxol (25, 50, 100 and 200 mg L1) from the beginning of the experiment. All flasks were capped with Magenta B-Caps (Sigma) and kept in the dark at 25 0.2 8C and ¨ hner AG, Schweiz). 100 1 rpm in a shaker-incubator (Adolf Ku Taxol dissolved in methanol [26] was sterilized by filtering through 0.22 mm sterile filters (Millipore) and added to the growth medium to give the final concentration considered. For analysis, four flasks from each treatment were harvested at days 4, 8, 12, 16 and 20.
Biomass accumulation and viability assay Fresh weight was determined by suction filtering of suspension cultures using Miracloth filters (Calbiochem, CA). The cells were then lyophilized to obtain dry weight and analyzed to determine the content of taxol and related taxanes. Cell viability was studied by the fluorecein diacetate staining technique [27]. The cells (0.2 g wet weight) were incubated in fresh growth medium (5 mL) containing fluorescein diacetate (0.1 mg mL1) for 30 min at room temperature. The cell fluorescence was observed with a fluorescence microscope at 520 nm and the percentage of fluorescent cells in relation to the total was assessed.
DNA laddering analysis To test if DNA laddering occurred in our T. baccata cell line cultures, DNA was extracted from untreated control and taxol supplemented cultures, as well as cultures cotreated with 0.375 mM salicylic acid and 0.012% H2O2. This cotreatment is reported to effectively induce apoptotic cell death in cell cultures of T. cuspidata [15]. In each case, we weighed 500 mg of cells and followed the manufacturer’s instructions of the ‘nexttecTM Genomic DNA Isolation Kit for Plants maxi’ (nexttec GmbH, Leverkusen, Germany). Gel was prepared with 1.5% agarose (Sigma, St. Louis, MO) and TBE (Tris–borate–EDTA) and analyzed with KODAK Gel Logic 100E (Vilber Lourmat, Marne-la-Vallee, France) after ethidium bromide staining (Sigma, St. Louis, MO).
Total RNA extraction and cDNA preparation Cell samples frozen with liquid nitrogen and stored at 80 8C were used to check the expression level of DXS and TXS. Total RNA was extracted using the RNAqueousTM RNA Isolation kit (Ambion) according to the manufacturer’s instructions. Concentration of each RNA sample was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). Only the RNA samples with 260/280 ratio between 1.9 and 2.1 were used for the analysis. The integrity of RNA samples was also assessed by agarose gel electrophoresis. Five nanograms of total RNA from each sample was reverse-transcribed by First-Strand cDNA Synthesis Using MMLV RT (Invitrogen), according to the manufacturer’s instructions.
Semiquantitative RT-PCR analysis The primer sequences corresponding to the two genes under study were chosen using the BTI software Gene Tool Lite (version 1.0.0.1) and are given in Table 1. Oligonucleotides for the three genes were synthesized by TIB Molbiol Inc. (Berlin, Germany). Primers were designed to yield the bands of 568 bp for the dxs,
TABLE 1
Sequences of the primers used to amplify the maturase gene (used as a housekeeping gene) and the genes encoding for the enzymes taxadiene synthase (tx) and 1-deoxy-D-xylulose-5-phosphate synthase (dxs). Genes
Sequences
Amplicon size (bp)
tx
Forward primer 50 -CCACGGTTTCCTCAGGCCCTCAA-30 Reverse primer 50 -GCCGCCGAATTTGTCCAGCAGAT-30
552
dxs
Forward primer 50 -TGGCCCTGCACCCCCTGT-30 Reverse primer 50 -GCCCACATCAAAGCAGCGTTCT-30
568
maturase
Forward primer 50 -TTGATTCGTCGGATACGTCA-30 Reverse primer 50 -GTGGAACCAGAGCTTTCTGC-30
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essentially the opposite. It is the outcome of severe injurious changes in the environment of affected cells and is not an active gene-dependent form of cell death [21]. The studies of Eisenreich et al. [22] on taxol biosynthesis have conclusively shown that the taxane ring system is synthesized via the nonmevalonate plastidic 1-deoxy-D-xylulose-5-phosphate/2C-methyl-D-erytritol 4-phosphate (MEP) pathway. This is consistent with taxol’s diterpenic nature. In the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) controls the first step, which is general for all plastidic isoprenoids (monoterpenes, diterpenes and tetraterpenes), leading to the formation of the plastidic isopentenyl diphosphate from pyruvate and glyceraldehyde-3-phosphate. The first committed step toward taxanes and taxol biosynthesis involves taxadiene synthase (TXS), which promotes the cyclization of geranylgeranyl diphosphate leading to the taxane skeleton [23]. In this work, the effect of taxol feeding was investigated in cultures of a T. baccata cell line to explore how viability and apoptosis are related to taxol production. In addition, we have compared the production of taxol with the expression level of the genes encoding the enzymes DXS and TXS.
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Research Paper FIGURE 1
General structure of the taxanes investigated in this work. Ac, acetyl group; Ph, phenyl group.
552 bp for the ts and 418 bp for the maturase genes, which were amplified. PCR was performed with 2 mL of cDNA template using puReTag Ready-To-Go PCR Beads (Amersham Biosciences) and was carried out on a programmable thermocycler (MiniCycler, MJ Research). All the PCRs were performed under the following conditions: initial denaturation at 94 8C for 5 min, followed by multiple cycles at 94 8C for 30 s, 65 8C for dxs, 67 8C for ts and 58 8C for maturase for 30 s and 72 8C for 45 s and then a final extension at 72 8C for 6 min. The number of cycles was standardized for each gene separately, with 24 cycles for the 1-deoxy-D-xylulose-5-phosphate synthase gene (dxs), 30 cycles for the taxadiene synthase gene (ts) and 27 cycles for the maturase gene when the products were being amplified within the exponential phase. Within a biological replicate for a tissue sample, the same cDNA pool was used for RT-PCR analysis of each gene using gene-specific primers. Two replicates for each biological sample were used for RT-PCR analysis.
TXS isolation, protein determination and enzyme assay Cells samples (1 g) were harvested by filtration under vacuum, frozen in liquid nitrogen and stored at 80 8C until needed. TXS was extracted essentially as described by Hezari et al. [23]. The amount of protein was determined by the Bradford method [28], using bovine serum albumin as a standard (Merck). The enzyme activity was determined according to Hezari et al. [29] with some modifications in the presence of an optimum protein concentration (10–100 mg protein). 100 mL of the enzyme preparation was diluted to 500 mL with an assay buffer consisting of 30 mM Hepes (pH 8.5), 5 mM dithiothreitol, 1 mM MgCl2, 5 mM sodium ascorbate, 5 mM Na2S2O5 and 10% (v/v) glycerol. 15 mM [1-3H]geranylgeranyl diphosphate (0.68 mCi) was added to the diluted enzyme preparation and, after gently mixing, the mixture was incubated for 1 h at 32 8C. After the incubation, 1 mL of pentane was added and the reaction mixture was vortexed and centrifuged to separate the two phases obtained. This step was repeated twice. 254
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The hexane phases were passed through a silicagel column surmounted by a layer of anhydrous MgSO4 and eluted with 8 mL of pentane. The eluted was dried (in a stream of N2) and resuspended in 1 mL hexane. An aliquot of this hydrocarbon fraction (600 mL) was analyzed by liquid scintillation counting in a 10 mL of Omnifluor Ultima GoldTM (PackardR a Canberra Company) (3H efficiency, 50%). Activities were expressed in picokatals per milligrams of protein.
Taxane measurements Taxanes were extracted from lyophilized cells and the culture medium as described previously [5]. Quantification of paclitaxel and baccatin III was performed by high performance liquid chromatography (HPLC) using the method described by Richheimer et al. [30]. This method uses a commercially available pentafluorophenyl packing material that has particular selectivity for taxol and related taxanes (Fig. 1), permitting an efficient separation of taxol from 10-deacetyltaxol and cephalomanine (CEPH). Criteria for identification included retention time, UV spectra and cochromatography with standard and peak homogeneity by photo-diode array detector when spiked with authentic standard. The taxol and related taxanes studied were provided by Hauser Chemicals (USA).
Results and discussion Effects on cell growth and viability The time course of growth of the T. baccata cell line cultured in a selected growth medium, either without taxol (control) or with several concentrations of taxol (25, 50, 100 and 200 mg L1) is shown in Fig. 2. In the control conditions the cell line presented a linear growth until day 16 when the culture entered its stationary growth phase, having achieved a fresh weight of 217.0 g L1, although the highest biomass production was observed at the end of the culture (225.01 g L1), which corresponded to a growth index (final fresh weight/inoculum fresh weight) of 2.25.
FIGURE 2
Time course of growth of T. baccata cell line. Cells in their initial stationary growth phase were cultured for 20 days in a selected growth medium with or without the addition of taxol (25, 50, 100 and 200 mg L1). Data represent average values from four replicates SE.
FIGURE 3
Viability of cells, expressed as percentage of living cells related to total cells of a T. baccata cell line cultured for 20 days in a selected growth medium with or without the addition of taxol (25, 50, 100 and 200 mg L1). Data represent average values from four replicates SE.
Adding the different concentrations of taxol to the medium changed the growth pattern of the cell line. The biomass peaked at day 16 of culture after the treatment with 25 and 50 mg L1 of taxol and at day 12 with 100 and 200 mg L1. At the same time, all the tested taxol concentrations significantly (P 0.01; t-test) inhibited the growth capacity of the cell line. In the presence of 25, 50, 100 and 200 mg L1 of taxol the average inhibition on cell growth was of 38.5%, 41.4%, 45% and 71%, respectively, at the end of the culture. From these results it can be inferred that the
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taxol supplemented to the culture medium acted negatively on the biomass formation, growth decreasing with the increase of taxol concentration. As can be seen in Fig. 3, the cell viability of the cell line growing in the control conditions remained high (from 85% to 80%) until day 12 and decreased slightly in the following days, reaching 76% at the end of the culture period (day 20). When the culture medium was supplemented with taxol, cell viability decreased according to the concentration used, which was consistent with the reduced cell mass (Fig. 2). There was no significant change with the addition of 25 mg L1 of taxol, but the presence of 50, 100 and 200 mg L1 of taxol resulted in a decrease of 16%, 19% and 32%, respectively, at the end of the culture. As suggested by Kim and Gibson [12], the growth inhibition may be an indication of cellular differentiation leading to cell death in the secondary metaboliteproducing cells of the culture. To study the possibility that the decrease in viability observed in our taxol-treated cultures was a consequence of apoptotic cell death, we tested if DNA degradation had occurred in these cultures. As can be seen in Fig. 4, genomic DNA analysis revealed that the DNA laddering pattern related to apoptosis was not observed over the 20-day growth period considered in our taxol-treated cultures. As expected, the DNA laddering pattern was observed only in the cultures cotreated with 0.375 mM salicylic acid and 0.012% H2O2. Our results clearly show that the cell death observed after the addition of taxol to the cultured T. baccata cell line was not a consequence of an apoptotic response of cells but probably a necrotic cell death owing to the toxicity of taxol. Kim and Gibson [12] indicated that the reduction of growth and viability in Taxus sp. cultures elicited with methyl jasmonate was attributed to the toxic effect of increased taxol, rather than the elicitor itself. The apoptosis that was observed occurred only at a later stage of the culture period (day 35) and was not the major death mechanism. In this context, it is worth noting that although a very rapid cell death (usually apoptotic) was observed following elicitation [31,32], it is considered to be independent of the phytoalexin biosynthesis. Furthermore, several authors have also reported that taxol behaves like a phytoalexinic agent in Taxus plants [26,33].
FIGURE 4
Agarose gel electrophoresis of total DNA from a T. baccata cell line cultured without taxol (control), cotreated with 0.375 mM salicylic acid and 0.012% H2O2, or with several concentrations of taxol. Culture conditions were the same as seen in Fig. 2. Lane M, DNA markers of l/EcoRI and HindIII; lane C + , DNA from cultures cotreated with 0.375 mM salicylic acid and 0.012% H2O2; lane C, DNA from untreated control cultures; lanes 25, 50, 100 and 200, DNA from cultures treated with 25, 50, 100 and 200 mg L1 taxol, respectively. www.elsevier.com/locate/nbt
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Effects on taxane content
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The effects of taxol feeding on taxol content were compared (Fig. 5). As expected, given that the chosen growth medium did not promote taxol production [25], the total content (cell-associated + extracellular) in the control T. baccata cell line remained very low (from 1.9 to 0.6 mg L1) until day 16. The total content increased at day 20 (1.2 mg L1) when the culture was in its stationary growth phase, which is when taxol production in Taxus cell cultures mainly takes place [8,10]. Adding taxol to the growth medium at the beginning of the experiment increased the total taxol contents (cell-associated + extracellular taxol) in the studied cell cultures, although this increase depended on the culture age, and to a greater degree, on the concentration of the supplemented taxol. Meanwhile, the total taxol values at day 4 (7.2, 21.3, 31.3 and 38.6 mg L1 in response to taxol feeding of 25, 50, 100 and 200 mg L1, respectively) showed that a very high percentage of the added taxol had degraded (71%, 57%, 69% and 81%, respectively). Preliminary experiments had already shown taxol to be an unstable compound, with an average of 50% degrading by day 4 after being dissolved in the selected growth medium at 25 8C (data not shown). Taking into account a previous study [34], these results suggest that taxol feeding in our cultured T. baccata cell line contributed to taxol accumulation by activating its absorption during the first four days of culture. In this study, the presence of 10–15 mM of exogenous taxol stimulated the uptake and accumulation of taxol by T. baccata cell suspension cultures, 20% remaining in the cell wall and 80% in the cells, mainly in their vacuoles. As also shown in Fig. 5, the total taxol content achieved at day 4 in all cases decreased until day 16, confirming the nongrowth linked pattern of taxol accumulation in cell suspensions of Taxus spp. The inverse relationship between growth and taxol production in Taxus cell cultures has been reported previously by several authors [10,35,36]. At 20 days of culture with or without added taxol, when our cell line was in its stationary growth phase (Fig. 2) and taxol biosynthesis at its peak, the total content of the taxane increased significantly (P 0.01; t-test). In the control conditions, the total taxol content at day 20 (1.2 mg L1) was 1.7-fold higher
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than that obtained at day 16, and in the cultures treated with 25, 50, 100 and 200 mg L1 taxol the total taxane content achieved at the end of the culture period (5.29, 13.45, 24.41 and 39.27 mg L1, respectively) was 1.3-, 1.5-, 1.5- and 1.6-fold higher, respectively, than that observed at day 16. As cells in the stationary growth phase are characterized by limited rates of division, it has been considered that products from primary metabolism accumulate and/or become available for secondary metabolite production [37]. Because the external taxol was added at the beginning of the culture, and the levels of this taxane decreased until day 16 in all the conditions studied, our results could suggest that taxol was newly synthesized, at least in the later part of this experiment, achieving the highest contents at day 20. At the same time, the greater the concentration of external taxol added to the culture medium, the greater was the increase of taxol at day 20 in the treated cultures. At day 20, both cell-associated and extracellular taxol content were considerably higher in T. baccata cell line cultures with exogenous taxol than in the untreated control (Fig. 5). Cultures treated with 25, 50, 100 and 200 mg L1 taxol showed an extracellular taxol content that was 5.9-, 17.5-, 27.0- and 55.9-fold higher, respectively, than that observed in the control (0.38 mg L1). Moreover, at the end of the experiment, the taxol found in the medium was 2.3, 7.0, 10.3 and 21.3 mg L1, in the cultures treated with 25, 50, 100 and 200 mg L1 taxol, whereas at day 4 it was 1.4, 10.1, 9.9 and 13.4 mg L1, respectively. Because the total taxol was lower at the end of experiment than at the beginning (except when 200 mg L1 taxol was added), these results could suggest that there may have been an excretion from the producer cells to the medium when the culture was in its stationary period, or that the taxol found in the medium came from leakage caused by cell death, considering that cell viability decreased at the end of the culture (Fig. 3). Taking into account that taxol has a toxic effect on microtubules in plants [16], its excretion from Taxus cells could be seen as a necessary self-defence strategy. Regarding taxol accumulation in the cell fraction (Fig. 5), at day 20 it was 3.6-, 8.2-, 17.2- and 21.9-fold higher, respectively, in the
FIGURE 5
Effects of taxol feeding on the total taxol content (cell-associated + extracellular), expressed as mg L(1 in a cultured T. baccata cell line. Culture conditions were the same as seen in Fig. 2. Data represent average values from four replicates SE. 256
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cultures treated with 25, 50, 100 and 200 mg L1 taxol than in the untreated control (0.82 mg L1). This higher increase in cell-associated taxol suggests that taxol accumulating in the culture medium might trigger new biosynthetic processes and so enhance taxol production. This would agree with Yuan et al. [19] who observed that T. cuspidata cell suspension cultures responded to added taxol by increasing its biosynthesis, although these authors explain the increase as a consequence of apoptosis brought on by the taxane addition. This variation in apoptotic response to taxol feeding in T. cuspidata and our T. baccata cell line is probably because of the use of different Taxus species as well as culture conditions and feeding time. Other taxanes, such as baccatin III (BIII), 10-deacethylbaccatin III (DABIII), 10-deacethyltaxol (DAT) and CEPH, were also measured (Table 2). The total content (cell-associated + extracellular) was measurable (baccatin III and CEPH), measurable to the limit of detection (10-deacethylbaccatin III) or not detectable in any assay (10-deacetyltaxol). Baccatin III in the control T. baccata cell line peaked at day 4 with 0.62 mg L1, decreased from days 8 to 16 and was not detectable at day 20. The CEPH content was always much lower than that of taxol (Fig. 5), which can be reasonably explained by the greater increase of benzoylation than tigloyation in the control conditions of our cell line. It is known that although CEPH is very similar to taxol structurally (Fig. 1), it has a tigloyl group in position C-30 of the C-13 side chain instead of a benzoyl group. As can be seen in Table 2, adding taxol to the growth medium at the beginning of the experiment the total content of TABLE 2
Effects of 25, 50, 100 and 200 mg L1 taxol on 10-deacetylbaccatin III (DABIII), baccatin III (BIII), 10-deacetyltaxol (DAP) and cephalomanine (CEPH) production, expressed as mg L(1 in a cultured T. baccata cell line. Days
DABIII
BIII
DAT
CEPH
Control
4 8 12 16 20
Traces – – Traces –
0.62 0.03 0.08 0.02 0.10 0.02 0.02 0.00 –
– – – – –
0.19 0.02 0.14 0.01 0.13 0.01 0.07 0.00 0.02 0.00
25 mg L1
4 8 12 16 20
– – – Traces –
0.56 0.04 0.02 0.00 0.10 0.03 0.07 0.02 -
– – – – –
0.19 0.01 0.22 0.02 0.12 0.01 0.08 0.01 0.10 0.01
50 mg L1
4 8 12 16 20
– – – Traces –
0.33 0.04 0.03 0.00 0.05 0.01 – –
– – – – –
0.20 0.01 0.19 0.02 0.11 0.02 0.07 0.00 0.10 0.01
100 mg L1
4 8 12 16 20
– – – – –
0.24 0.02 0.42 0.05 0.08 0.01 – –
– – – – –
0.21 0.03 0.12 0.02 0.10 0.00 0.10 0.01 0.13 0.02
200 mg L1
4 8 12 16 20
– – – – –
0.10 0.04 0.09 0.02 0.06 0.03 – –
– – – – –
0.21 0.03 0.12 0.01 0.08 0.00 0.08 0.01 0.15 0.03
Culture conditions were the same as seen in Fig. 2. Data represent average values from four replicates SE.
CEPH increased significantly (P 0.01; t-test) at the end of the experiment, but not that of baccatin III in the T. baccata cell line studied. This result supports the view that baccatin III may not be a direct precursor of taxol, as suggested by Srinivasan et al. [8] after observing an improvement in taxol yield in T. chinensis cell cultures supplemented with 10 mM 1-aminobenzotriazole, in which baccatin III production had been suppressed. They concluded that baccatin III and taxol could be products of separate branches of the taxane pathway. However, at present it is assumed that taxol is formed after the conjugation of the b-phenylalanoylCoA side chain to baccatin III [38,39]. Although the degradation of taxol in cell cultures remains as a not well-known process, the decrease of taxol observed until day 16 could be due to degradation of this compound [40,41] or to other metabolic steps, such as still unknown multiple side-reactions and metabolic dead-ends, related with the taxol biosynthetic pathway. Nevertheless, further studies would be necessary to clarify this point.
Effects on expression level of DXS and TXS encoding genes and TXS activity To know if the expression level of two genes (dxs and ts) involved in taxol biosynthesis changed after the addition of taxol to the culture medium of our T. baccata cell line, the level of their transcripts was determined by RT-PCR analysis. The genes dxs and ts encode the enzymes DXS and TXS, respectively. DXS, the first enzyme of the MEP pathway, plays a fundamental role in the biosynthesis of plastidic terpenes, including the diterpenoid taxol, and TXS is involved in the cyclization of geranylgeranyl diphosphate, the first committed step of the biosynthetic pathway leading to the taxol taxane skeleton. The expression levels of the ts gene followed the same pattern over time in all studied conditions, but were clearly higher in cultures treated with taxol (Fig. 6A). The level peaked at day 4 with a rapid decrease at day 8, reaching a second, lower, peak at day 12. By day 16, ts mRNA was only detectable in the untreated control at a very low level. The concentration of taxol that most effectively induced ts mRNA expression was 50 mg L1: compared to the control, the transcript levels were 2.5-, 3.1- and 3.3-fold higher at 4, 8 and 12 days of culture, respectively. Because the induction of ts mRNA by taxol persisted for only 12 days and taxol biosynthesis mainly took place at day 20 (Fig. 5), the synthesis of TXS was presumably not a factor in the continued synthesis of the taxane in our T. baccata cell line cultures. To study this aspect in more detail, we measured the activity of this enzyme in all studied conditions. As also shown in Fig. 6A, a relatively high level of TXS activity was observed at day 4, when it was 2.8-, 6.8-, 3.2- and 1.7-fold higher, respectively, in the cultures treated with 25, 50, 100 and 200 mg L1 taxol than in the untreated control (6.64 pkat per mg of protein). Enzyme activity then decreased markedly from days 4 to 12 and thereafter was measurable only at a very low level until day 20. Thus, the enzyme activities for the synthesis of the side chain of taxol and its addition to the taxane skeleton apparently continued longer than those involved in the synthesis of at least one nonside-chain-containing taxane. Our results seem to show that the product of the biosynthetic step regulated by the enzyme TXS, the taxadiene, is not a limiting substrate in taxol formation. These results agree with those www.elsevier.com/locate/nbt
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Research Paper FIGURE 6
Effects in a cultured T. baccata cell line of taxol feeding on the taxadiene synthase (TXS) activity and the expression levels of genes (tx), encoding this enzyme (A) and (dxs) encoding the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (B). Culture conditions were the same as seen in Fig. 2. Data represent average values from four replicates SE.
obtained by Croteau et al. [38], who suggested that neither taxadiene nor other early pathway intermediates accumulate to any appreciable level in Taxus cell cultures, indicating a rapid conversion of these metabolites by downstream reactions. The expression levels of the dxs gene were also higher in cultures treated with taxol, and followed the same pattern over time in all studied conditions (Fig. 6B). Levels peaked at day 4, decreased rapidly at day 8, and reached a second, lower, peak at day 12. Compared to the untreated control, at day 4 the dxs mRNA expression in taxol-treated cultures was 1.4-, 1.5- and 1.6-fold higher after exposure to 50, 100 and 200 mg L1 taxol, respectively. Whereas ts mRNA was not detected in taxol-treated cultures at day 16 (Fig. 6), we found that all taxol concentrations, particularly 50 mg L1, induced the expression of dxs mRNA at days 16 and 20. Considering that the enzyme DXS has a role at the beginning of the MEP pathway and that the taxanes are end products, it seems that exogenous taxol could affect taxol production by increasing the source of the plastidic terpenoid precursor isopentenyl diphosphate for taxane biosynthesis. Although it has 258
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still not been demonstrated that the enzyme DXS has a crucial role in the biosynthesis of taxol in Taxus in vitro cultures, a series of studies shows that this enzyme plays a fundamental role in the biosynthesis of terpenes in various organisms, including bacteria and plants [42].
Concluding remarks The effect of taxol feeding was investigated using a T. baccata cell line growing for 20 days in a selected nontaxol promoting growth medium to study how viability and apoptosis are related with taxol production. Compared with an untreated control, all the tested taxol concentrations stimulated cell-associated taxol content, especially at day 20 when our cell line was in its stationary growth phase and taxol biosynthesis at its peak. Taxol content increased with the concentration of added taxol, although cell viability decreased correspondingly. Because DNA laddering analysis revealed that the viability reduction was not related to apoptosis, it is thus inferred that apoptotic cell death was not related with to the taxol production in our T. baccata cell line cultures. This would
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oxidative elaboration and side-chain addition to afford taxol. The possibility that exogenous taxol induces taxol-related genes and thus taxol production, is consistent with the view that the accumulation of taxol in Taxus cells is a biological response to specific external stimuli [26], which in our study could be the added taxol.
Acknowledgements We thank the Technical Science Service from Barcelona University for their support. This research has been supported by two grants from the Spanish MEC (BIO2005-05583). Oscar Expo´sito is grateful for his research grant FPU (AP-2004-5321) from the Spanish MEC.
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21 Kerr, J.F.R. et al. (1995) Anatomical methods in cell death. In Cell Death: Methods in Cell Biology, (Vol. 48) (Schwartz, L.M. and Osborne, B.A., eds) pp. 1–27, Academic Press, New York 22 Eisenreich, W. et al. (1996) Studies on the biosynthesis of taxol: taxane carbon skeleton is not of mevalonoid origin. Proc. Natl. Acad. Sci. U. S. A. 93, 6431–6436 23 Hezari, M. et al. (1997) Taxol production and taxadiene synthase activity in Taxus canadensis cell suspension cultures. Arch. Biochem. Biophys. 337, 185–190 24 Gamborg, O.L. and Miller, R.A. (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158 25 Palazo´n, J. et al. (2003) Inhibition of paclitaxel and baccatin III accumulation by mevinolin and fosmidomycin in suspension cultures of Taxus baccata. J. Biotechnol. 191, 157–163 26 Yukimune, Y. et al. (1996) Methyljasmonate-induced overproduction of paclitaxel and baccatin III in Taxus cell suspension cultures. Nat. Biotechnol. 14, 1129–1132 27 Widholm, J.M. (1972) The use of fluorecein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol. 47, 189–194 28 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principles of protein dye binding. Anal. Biochem. 72, 245–248 29 Hezari, M. et al. (1995) Purification and characterization of taxa-4(5),11(12)-diene synthase from Pacific yew (Taxus brevifolia) that catalyzes the first committed step of taxol biosynthesis. Arch. Biochem. Biophys. 322, 437–444 30 Richheimer, S.I. et al. (1992) High-performance liquid chromatographic assay of taxol. Anal. Chem. 64, 2323–2326 31 Koch, W. et al. (1998) Elicitor-induced cell death and phytoalexin synthesis in Daucus carota L.. Planta 206, 523–532 32 Lan, W.Z. et al. (2003) Cell death unlikely contributes to taxol production in fungal elicitor-induced cell suspension cultures of Taxus chinensis. Biotechnol. Lett. 25, 47–49 33 Ciddi, V. et al. (1995) Elicitation of Taxus sp. cell cultures for production of taxol. Biotechnol. Lett. 17, 1343–1346 34 Fornale`, S. et al. (2002) Taxol transport in Taxus baccata cell suspension cultures. Plant Physiol. Biochem. 40, 81–88 35 Taticek, R.A. et al. (1991) The scale-up of plant cell culture: engineering considerations. Plant Cell. Tiss. Org. Cult. 24, 139–158 36 Fett-Neto, A.G. et al. (1994) Kinetics of taxol production, growth and nutrient uptake in cell suspensions of Taxus cuspidata. Biotechnol. Bioeng. 44, 205–210 37 Hall, R.D. et al. (1988) Immobilization of higher plant cells. In Biotechnology in Agriculture and Forestry, Vol. 4, Medicinal and Aromatic Plants I (Bajaj, Y.P.S., ed.), pp. 136–156, Springer-Verlag, Berlin 38 Croteau, R. et al. (2006) Taxol biosynthesis and molecular genetics. Phytochem. Rev. 5, 75–97 39 Vongpaseuth, K. and Roberts, S.C. (2007) Advancements in the understanding of paclitaxel metabolism in tissue culture. Curr. Pharm. Biotechnol. 8, 219–236 40 Son, S.H. et al. (2000) Large-scale growth and taxane production in cell cultures of Taxus cuspidate (Japanese yew) using a novel bioreactor. Plant Cell Rep. 19, 628–633 41 Shimoda, K. et al. (2008) Biological degradation of taxol by action of cultured cells on 7-acetyltaxol-200 -yl glucoside. Chem. Lett. 37, 362–363 42 Guevara-Garcia, A. et al. (2005) Characterization of the Arabidopsis clb6 mutant illustrates the importance of posttranscriptional regulation of the methyl-Derythritol 4-phosphate pathway. Plant Cell 17, 628–643
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agree with Kim et al. [14], who suggest that the high production of taxol may causally affect growth and viability through, as yet, undefined mechanisms. A plot of TXS activity in taxol-treated cultures clearly indicated that a pronounced decrease of activity occurred at least one week before cell-associated and extracellular taxol peaked at day 20, when the cultures were in its stationary growth phase. Thus, from day 16 to day 20, it seems that taxol formation did not depend on continued taxadiene production, suggesting that there must be other flux-limiting steps more downstream in the process, which are probably also stimulated by taxol feeding. It has been demonstrated [29] that taxadiene undergoes extensive
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Effect of taxol feeding on taxol and related taxane production in Taxus baccata suspension cultures Oscar Expo´sito1, Mercedes Bonfill1, Miriam Onrubia2, Albert Jane´1, Elisabet Moyano2, Rosa M. Cusido´1, Javier Palazo´n1 and M. Teresa Pin˜ol1 1 2
Laboratorio de Fisiologı´a vegetal, Facultad de Farmacia, Universidad de Barcelona, Avda. Diagonal 643, E-08028 Barcelona, Spain Departament de Cie`ncies Experimentals i de la Salut, Universitat Pompeu Fabra, Avda Aiguader 80, E-08003 Barcelona, Spain
To achieve a better understanding of taxol metabolism and accumulation in Taxus cell cultures, a T. baccata cell line growing for 20 days in a selected growth medium was treated at the beginning of the experiment with several concentrations of taxol (25, 50, 100 and 200 mg L1). Compared with an untreated control, all these taxol concentrations stimulated cell-associated taxol content (up to 32.7 times in the presence of 200 mg L1 exogenous taxol), although higher concentrations significantly depressed cell viability. DNA laddering analysis revealed that the viability reduction was not related to apoptosis, suggesting that taxol itself was the primary responsible factor. On the basis of RT-PCR expression analysis of genes encoding taxadiene synthase (ts) and 1-deoxy-D-xylulose-5-phosphate synthase (dxs) from treated and nontreated T. baccata cell line cultures, it was observed that exogenous taxol clearly induced the mRNA levels of both taxane-related enzymes. Additionally, we found that exogenous taxol caused a considerable increase in taxadiene synthase activity, although in no case did this coincide with the highest levels of taxol observed at the end of the culture. The effect of exogenous taxol on the content of other related taxanes was also considered.
Introduction Taxol has been a highly successful anticancer drug since it was initially approved for the treatment of breast and ovarian cancers [1]. Other molecular targets for taxanes, such as multidrug resistance inhibition and apoptosis inhibitor binding, as well as treatments for nonsmall-cell lung cancer and AIDS-related Kaposi’s sarcoma, are currently being investigated [2]. Demands on the supply of taxol also continue to grow as a result of its expanding use in early intervention therapies and in combination with other chemotherapeutic agents. At present, taxol is successfully obtained in cell cultures of different Taxus species [3–7]. To enhance the taxol production of these cultures, the addition of several elicitor compounds has been assayed [8–14]. Some elicitors improve the taxol production, but the majority reduce cell viability and growth. This decrease of cell viability and the consequent Corresponding author: Bonfill, M. ([email protected])
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reduction of growth in highly productive Taxus cell cultures could be a cell response to the elicitor action. It could also be caused by the toxic effect of taxol itself on cell metabolism [14,15], because taxol affects microtubules in many organisms, including higher plants [16]. The production of defence compounds is closely related to the cell death mechanism in plants [12]. Certain fatty acids in octadecanoid pathways for signaling defense, such as those generating jasmonic acid, can also induce apoptosis [17]. Several reports have indicated that programmed cell death is closely related to taxol production in suspension cultures of Taxus cells [15,18,19]. Ma et al. [20] suggested that apoptosis is a major death mechanism in Taxus canadiensis cell cultures. By contrast, after carrying out DNA laddering analysis Kim et al. [14] have reported that viability reduction in T. cuspidata cell cultures does not appear to be related to apoptosis. In this context, it is worth noting that while apoptosis is a process in which the cell directs its own death, necrosis is
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New Biotechnology Volume 25, Number 4 April 2009
Materials and methods Establishment and growth conditions of the cell line culture The cell line of T. baccata was established from a stable callus line (the variance in taxane content and growth rate of each subculture was <10%, data not shown), as reported earlier [5]. To obtain low levels of taxol in our cell line, which would allow us to see more clearly how the addition of taxol might affect its accumulation, together with that of other related taxanes, we used a previously selected growth medium Gamborg’s B5 medium [24] with 0.5% sucrose + 0.5% fructose, 2 mg L1 of NAA and 0.1 mg L1 of BAP that promotes cell growth but not taxol production in T. baccata cell cultures [25]. 1 0.2 g wet weight of cells grown for 16 days in the growth medium (the length of time necessary for them to enter the stationary growth phase, Fig. 2) were transferred to 175-mL flasks (Sigma VOG33) containing 10 mL of the same medium. They were then cultured for 20 days without taxol (control) or with several concentrations of taxol (25, 50, 100 and 200 mg L1) from the beginning of the experiment. All flasks were capped with Magenta B-Caps (Sigma) and kept in the dark at 25 0.2 8C and ¨ hner AG, Schweiz). 100 1 rpm in a shaker-incubator (Adolf Ku Taxol dissolved in methanol [26] was sterilized by filtering through 0.22 mm sterile filters (Millipore) and added to the growth medium to give the final concentration considered. For analysis, four flasks from each treatment were harvested at days 4, 8, 12, 16 and 20.
Biomass accumulation and viability assay Fresh weight was determined by suction filtering of suspension cultures using Miracloth filters (Calbiochem, CA). The cells were then lyophilized to obtain dry weight and analyzed to determine the content of taxol and related taxanes. Cell viability was studied by the fluorecein diacetate staining technique [27]. The cells (0.2 g wet weight) were incubated in fresh growth medium (5 mL) containing fluorescein diacetate (0.1 mg mL1) for 30 min at room temperature. The cell fluorescence was observed with a fluorescence microscope at 520 nm and the percentage of fluorescent cells in relation to the total was assessed.
DNA laddering analysis To test if DNA laddering occurred in our T. baccata cell line cultures, DNA was extracted from untreated control and taxol supplemented cultures, as well as cultures cotreated with 0.375 mM salicylic acid and 0.012% H2O2. This cotreatment is reported to effectively induce apoptotic cell death in cell cultures of T. cuspidata [15]. In each case, we weighed 500 mg of cells and followed the manufacturer’s instructions of the ‘nexttecTM Genomic DNA Isolation Kit for Plants maxi’ (nexttec GmbH, Leverkusen, Germany). Gel was prepared with 1.5% agarose (Sigma, St. Louis, MO) and TBE (Tris–borate–EDTA) and analyzed with KODAK Gel Logic 100E (Vilber Lourmat, Marne-la-Vallee, France) after ethidium bromide staining (Sigma, St. Louis, MO).
Total RNA extraction and cDNA preparation Cell samples frozen with liquid nitrogen and stored at 80 8C were used to check the expression level of DXS and TXS. Total RNA was extracted using the RNAqueousTM RNA Isolation kit (Ambion) according to the manufacturer’s instructions. Concentration of each RNA sample was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). Only the RNA samples with 260/280 ratio between 1.9 and 2.1 were used for the analysis. The integrity of RNA samples was also assessed by agarose gel electrophoresis. Five nanograms of total RNA from each sample was reverse-transcribed by First-Strand cDNA Synthesis Using MMLV RT (Invitrogen), according to the manufacturer’s instructions.
Semiquantitative RT-PCR analysis The primer sequences corresponding to the two genes under study were chosen using the BTI software Gene Tool Lite (version 1.0.0.1) and are given in Table 1. Oligonucleotides for the three genes were synthesized by TIB Molbiol Inc. (Berlin, Germany). Primers were designed to yield the bands of 568 bp for the dxs,
TABLE 1
Sequences of the primers used to amplify the maturase gene (used as a housekeeping gene) and the genes encoding for the enzymes taxadiene synthase (tx) and 1-deoxy-D-xylulose-5-phosphate synthase (dxs). Genes
Sequences
Amplicon size (bp)
tx
Forward primer 50 -CCACGGTTTCCTCAGGCCCTCAA-30 Reverse primer 50 -GCCGCCGAATTTGTCCAGCAGAT-30
552
dxs
Forward primer 50 -TGGCCCTGCACCCCCTGT-30 Reverse primer 50 -GCCCACATCAAAGCAGCGTTCT-30
568
maturase
Forward primer 50 -TTGATTCGTCGGATACGTCA-30 Reverse primer 50 -GTGGAACCAGAGCTTTCTGC-30
418
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essentially the opposite. It is the outcome of severe injurious changes in the environment of affected cells and is not an active gene-dependent form of cell death [21]. The studies of Eisenreich et al. [22] on taxol biosynthesis have conclusively shown that the taxane ring system is synthesized via the nonmevalonate plastidic 1-deoxy-D-xylulose-5-phosphate/2C-methyl-D-erytritol 4-phosphate (MEP) pathway. This is consistent with taxol’s diterpenic nature. In the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) controls the first step, which is general for all plastidic isoprenoids (monoterpenes, diterpenes and tetraterpenes), leading to the formation of the plastidic isopentenyl diphosphate from pyruvate and glyceraldehyde-3-phosphate. The first committed step toward taxanes and taxol biosynthesis involves taxadiene synthase (TXS), which promotes the cyclization of geranylgeranyl diphosphate leading to the taxane skeleton [23]. In this work, the effect of taxol feeding was investigated in cultures of a T. baccata cell line to explore how viability and apoptosis are related to taxol production. In addition, we have compared the production of taxol with the expression level of the genes encoding the enzymes DXS and TXS.
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Research Paper FIGURE 1
General structure of the taxanes investigated in this work. Ac, acetyl group; Ph, phenyl group.
552 bp for the ts and 418 bp for the maturase genes, which were amplified. PCR was performed with 2 mL of cDNA template using puReTag Ready-To-Go PCR Beads (Amersham Biosciences) and was carried out on a programmable thermocycler (MiniCycler, MJ Research). All the PCRs were performed under the following conditions: initial denaturation at 94 8C for 5 min, followed by multiple cycles at 94 8C for 30 s, 65 8C for dxs, 67 8C for ts and 58 8C for maturase for 30 s and 72 8C for 45 s and then a final extension at 72 8C for 6 min. The number of cycles was standardized for each gene separately, with 24 cycles for the 1-deoxy-D-xylulose-5-phosphate synthase gene (dxs), 30 cycles for the taxadiene synthase gene (ts) and 27 cycles for the maturase gene when the products were being amplified within the exponential phase. Within a biological replicate for a tissue sample, the same cDNA pool was used for RT-PCR analysis of each gene using gene-specific primers. Two replicates for each biological sample were used for RT-PCR analysis.
TXS isolation, protein determination and enzyme assay Cells samples (1 g) were harvested by filtration under vacuum, frozen in liquid nitrogen and stored at 80 8C until needed. TXS was extracted essentially as described by Hezari et al. [23]. The amount of protein was determined by the Bradford method [28], using bovine serum albumin as a standard (Merck). The enzyme activity was determined according to Hezari et al. [29] with some modifications in the presence of an optimum protein concentration (10–100 mg protein). 100 mL of the enzyme preparation was diluted to 500 mL with an assay buffer consisting of 30 mM Hepes (pH 8.5), 5 mM dithiothreitol, 1 mM MgCl2, 5 mM sodium ascorbate, 5 mM Na2S2O5 and 10% (v/v) glycerol. 15 mM [1-3H]geranylgeranyl diphosphate (0.68 mCi) was added to the diluted enzyme preparation and, after gently mixing, the mixture was incubated for 1 h at 32 8C. After the incubation, 1 mL of pentane was added and the reaction mixture was vortexed and centrifuged to separate the two phases obtained. This step was repeated twice. 254
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The hexane phases were passed through a silicagel column surmounted by a layer of anhydrous MgSO4 and eluted with 8 mL of pentane. The eluted was dried (in a stream of N2) and resuspended in 1 mL hexane. An aliquot of this hydrocarbon fraction (600 mL) was analyzed by liquid scintillation counting in a 10 mL of Omnifluor Ultima GoldTM (PackardR a Canberra Company) (3H efficiency, 50%). Activities were expressed in picokatals per milligrams of protein.
Taxane measurements Taxanes were extracted from lyophilized cells and the culture medium as described previously [5]. Quantification of paclitaxel and baccatin III was performed by high performance liquid chromatography (HPLC) using the method described by Richheimer et al. [30]. This method uses a commercially available pentafluorophenyl packing material that has particular selectivity for taxol and related taxanes (Fig. 1), permitting an efficient separation of taxol from 10-deacetyltaxol and cephalomanine (CEPH). Criteria for identification included retention time, UV spectra and cochromatography with standard and peak homogeneity by photo-diode array detector when spiked with authentic standard. The taxol and related taxanes studied were provided by Hauser Chemicals (USA).
Results and discussion Effects on cell growth and viability The time course of growth of the T. baccata cell line cultured in a selected growth medium, either without taxol (control) or with several concentrations of taxol (25, 50, 100 and 200 mg L1) is shown in Fig. 2. In the control conditions the cell line presented a linear growth until day 16 when the culture entered its stationary growth phase, having achieved a fresh weight of 217.0 g L1, although the highest biomass production was observed at the end of the culture (225.01 g L1), which corresponded to a growth index (final fresh weight/inoculum fresh weight) of 2.25.
FIGURE 2
Time course of growth of T. baccata cell line. Cells in their initial stationary growth phase were cultured for 20 days in a selected growth medium with or without the addition of taxol (25, 50, 100 and 200 mg L1). Data represent average values from four replicates SE.
FIGURE 3
Viability of cells, expressed as percentage of living cells related to total cells of a T. baccata cell line cultured for 20 days in a selected growth medium with or without the addition of taxol (25, 50, 100 and 200 mg L1). Data represent average values from four replicates SE.
Adding the different concentrations of taxol to the medium changed the growth pattern of the cell line. The biomass peaked at day 16 of culture after the treatment with 25 and 50 mg L1 of taxol and at day 12 with 100 and 200 mg L1. At the same time, all the tested taxol concentrations significantly (P 0.01; t-test) inhibited the growth capacity of the cell line. In the presence of 25, 50, 100 and 200 mg L1 of taxol the average inhibition on cell growth was of 38.5%, 41.4%, 45% and 71%, respectively, at the end of the culture. From these results it can be inferred that the
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taxol supplemented to the culture medium acted negatively on the biomass formation, growth decreasing with the increase of taxol concentration. As can be seen in Fig. 3, the cell viability of the cell line growing in the control conditions remained high (from 85% to 80%) until day 12 and decreased slightly in the following days, reaching 76% at the end of the culture period (day 20). When the culture medium was supplemented with taxol, cell viability decreased according to the concentration used, which was consistent with the reduced cell mass (Fig. 2). There was no significant change with the addition of 25 mg L1 of taxol, but the presence of 50, 100 and 200 mg L1 of taxol resulted in a decrease of 16%, 19% and 32%, respectively, at the end of the culture. As suggested by Kim and Gibson [12], the growth inhibition may be an indication of cellular differentiation leading to cell death in the secondary metaboliteproducing cells of the culture. To study the possibility that the decrease in viability observed in our taxol-treated cultures was a consequence of apoptotic cell death, we tested if DNA degradation had occurred in these cultures. As can be seen in Fig. 4, genomic DNA analysis revealed that the DNA laddering pattern related to apoptosis was not observed over the 20-day growth period considered in our taxol-treated cultures. As expected, the DNA laddering pattern was observed only in the cultures cotreated with 0.375 mM salicylic acid and 0.012% H2O2. Our results clearly show that the cell death observed after the addition of taxol to the cultured T. baccata cell line was not a consequence of an apoptotic response of cells but probably a necrotic cell death owing to the toxicity of taxol. Kim and Gibson [12] indicated that the reduction of growth and viability in Taxus sp. cultures elicited with methyl jasmonate was attributed to the toxic effect of increased taxol, rather than the elicitor itself. The apoptosis that was observed occurred only at a later stage of the culture period (day 35) and was not the major death mechanism. In this context, it is worth noting that although a very rapid cell death (usually apoptotic) was observed following elicitation [31,32], it is considered to be independent of the phytoalexin biosynthesis. Furthermore, several authors have also reported that taxol behaves like a phytoalexinic agent in Taxus plants [26,33].
FIGURE 4
Agarose gel electrophoresis of total DNA from a T. baccata cell line cultured without taxol (control), cotreated with 0.375 mM salicylic acid and 0.012% H2O2, or with several concentrations of taxol. Culture conditions were the same as seen in Fig. 2. Lane M, DNA markers of l/EcoRI and HindIII; lane C + , DNA from cultures cotreated with 0.375 mM salicylic acid and 0.012% H2O2; lane C, DNA from untreated control cultures; lanes 25, 50, 100 and 200, DNA from cultures treated with 25, 50, 100 and 200 mg L1 taxol, respectively. www.elsevier.com/locate/nbt
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Effects on taxane content
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The effects of taxol feeding on taxol content were compared (Fig. 5). As expected, given that the chosen growth medium did not promote taxol production [25], the total content (cell-associated + extracellular) in the control T. baccata cell line remained very low (from 1.9 to 0.6 mg L1) until day 16. The total content increased at day 20 (1.2 mg L1) when the culture was in its stationary growth phase, which is when taxol production in Taxus cell cultures mainly takes place [8,10]. Adding taxol to the growth medium at the beginning of the experiment increased the total taxol contents (cell-associated + extracellular taxol) in the studied cell cultures, although this increase depended on the culture age, and to a greater degree, on the concentration of the supplemented taxol. Meanwhile, the total taxol values at day 4 (7.2, 21.3, 31.3 and 38.6 mg L1 in response to taxol feeding of 25, 50, 100 and 200 mg L1, respectively) showed that a very high percentage of the added taxol had degraded (71%, 57%, 69% and 81%, respectively). Preliminary experiments had already shown taxol to be an unstable compound, with an average of 50% degrading by day 4 after being dissolved in the selected growth medium at 25 8C (data not shown). Taking into account a previous study [34], these results suggest that taxol feeding in our cultured T. baccata cell line contributed to taxol accumulation by activating its absorption during the first four days of culture. In this study, the presence of 10–15 mM of exogenous taxol stimulated the uptake and accumulation of taxol by T. baccata cell suspension cultures, 20% remaining in the cell wall and 80% in the cells, mainly in their vacuoles. As also shown in Fig. 5, the total taxol content achieved at day 4 in all cases decreased until day 16, confirming the nongrowth linked pattern of taxol accumulation in cell suspensions of Taxus spp. The inverse relationship between growth and taxol production in Taxus cell cultures has been reported previously by several authors [10,35,36]. At 20 days of culture with or without added taxol, when our cell line was in its stationary growth phase (Fig. 2) and taxol biosynthesis at its peak, the total content of the taxane increased significantly (P 0.01; t-test). In the control conditions, the total taxol content at day 20 (1.2 mg L1) was 1.7-fold higher
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than that obtained at day 16, and in the cultures treated with 25, 50, 100 and 200 mg L1 taxol the total taxane content achieved at the end of the culture period (5.29, 13.45, 24.41 and 39.27 mg L1, respectively) was 1.3-, 1.5-, 1.5- and 1.6-fold higher, respectively, than that observed at day 16. As cells in the stationary growth phase are characterized by limited rates of division, it has been considered that products from primary metabolism accumulate and/or become available for secondary metabolite production [37]. Because the external taxol was added at the beginning of the culture, and the levels of this taxane decreased until day 16 in all the conditions studied, our results could suggest that taxol was newly synthesized, at least in the later part of this experiment, achieving the highest contents at day 20. At the same time, the greater the concentration of external taxol added to the culture medium, the greater was the increase of taxol at day 20 in the treated cultures. At day 20, both cell-associated and extracellular taxol content were considerably higher in T. baccata cell line cultures with exogenous taxol than in the untreated control (Fig. 5). Cultures treated with 25, 50, 100 and 200 mg L1 taxol showed an extracellular taxol content that was 5.9-, 17.5-, 27.0- and 55.9-fold higher, respectively, than that observed in the control (0.38 mg L1). Moreover, at the end of the experiment, the taxol found in the medium was 2.3, 7.0, 10.3 and 21.3 mg L1, in the cultures treated with 25, 50, 100 and 200 mg L1 taxol, whereas at day 4 it was 1.4, 10.1, 9.9 and 13.4 mg L1, respectively. Because the total taxol was lower at the end of experiment than at the beginning (except when 200 mg L1 taxol was added), these results could suggest that there may have been an excretion from the producer cells to the medium when the culture was in its stationary period, or that the taxol found in the medium came from leakage caused by cell death, considering that cell viability decreased at the end of the culture (Fig. 3). Taking into account that taxol has a toxic effect on microtubules in plants [16], its excretion from Taxus cells could be seen as a necessary self-defence strategy. Regarding taxol accumulation in the cell fraction (Fig. 5), at day 20 it was 3.6-, 8.2-, 17.2- and 21.9-fold higher, respectively, in the
FIGURE 5
Effects of taxol feeding on the total taxol content (cell-associated + extracellular), expressed as mg L(1 in a cultured T. baccata cell line. Culture conditions were the same as seen in Fig. 2. Data represent average values from four replicates SE. 256
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cultures treated with 25, 50, 100 and 200 mg L1 taxol than in the untreated control (0.82 mg L1). This higher increase in cell-associated taxol suggests that taxol accumulating in the culture medium might trigger new biosynthetic processes and so enhance taxol production. This would agree with Yuan et al. [19] who observed that T. cuspidata cell suspension cultures responded to added taxol by increasing its biosynthesis, although these authors explain the increase as a consequence of apoptosis brought on by the taxane addition. This variation in apoptotic response to taxol feeding in T. cuspidata and our T. baccata cell line is probably because of the use of different Taxus species as well as culture conditions and feeding time. Other taxanes, such as baccatin III (BIII), 10-deacethylbaccatin III (DABIII), 10-deacethyltaxol (DAT) and CEPH, were also measured (Table 2). The total content (cell-associated + extracellular) was measurable (baccatin III and CEPH), measurable to the limit of detection (10-deacethylbaccatin III) or not detectable in any assay (10-deacetyltaxol). Baccatin III in the control T. baccata cell line peaked at day 4 with 0.62 mg L1, decreased from days 8 to 16 and was not detectable at day 20. The CEPH content was always much lower than that of taxol (Fig. 5), which can be reasonably explained by the greater increase of benzoylation than tigloyation in the control conditions of our cell line. It is known that although CEPH is very similar to taxol structurally (Fig. 1), it has a tigloyl group in position C-30 of the C-13 side chain instead of a benzoyl group. As can be seen in Table 2, adding taxol to the growth medium at the beginning of the experiment the total content of TABLE 2
Effects of 25, 50, 100 and 200 mg L1 taxol on 10-deacetylbaccatin III (DABIII), baccatin III (BIII), 10-deacetyltaxol (DAP) and cephalomanine (CEPH) production, expressed as mg L(1 in a cultured T. baccata cell line. Days
DABIII
BIII
DAT
CEPH
Control
4 8 12 16 20
Traces – – Traces –
0.62 0.03 0.08 0.02 0.10 0.02 0.02 0.00 –
– – – – –
0.19 0.02 0.14 0.01 0.13 0.01 0.07 0.00 0.02 0.00
25 mg L1
4 8 12 16 20
– – – Traces –
0.56 0.04 0.02 0.00 0.10 0.03 0.07 0.02 -
– – – – –
0.19 0.01 0.22 0.02 0.12 0.01 0.08 0.01 0.10 0.01
50 mg L1
4 8 12 16 20
– – – Traces –
0.33 0.04 0.03 0.00 0.05 0.01 – –
– – – – –
0.20 0.01 0.19 0.02 0.11 0.02 0.07 0.00 0.10 0.01
100 mg L1
4 8 12 16 20
– – – – –
0.24 0.02 0.42 0.05 0.08 0.01 – –
– – – – –
0.21 0.03 0.12 0.02 0.10 0.00 0.10 0.01 0.13 0.02
200 mg L1
4 8 12 16 20
– – – – –
0.10 0.04 0.09 0.02 0.06 0.03 – –
– – – – –
0.21 0.03 0.12 0.01 0.08 0.00 0.08 0.01 0.15 0.03
Culture conditions were the same as seen in Fig. 2. Data represent average values from four replicates SE.
CEPH increased significantly (P 0.01; t-test) at the end of the experiment, but not that of baccatin III in the T. baccata cell line studied. This result supports the view that baccatin III may not be a direct precursor of taxol, as suggested by Srinivasan et al. [8] after observing an improvement in taxol yield in T. chinensis cell cultures supplemented with 10 mM 1-aminobenzotriazole, in which baccatin III production had been suppressed. They concluded that baccatin III and taxol could be products of separate branches of the taxane pathway. However, at present it is assumed that taxol is formed after the conjugation of the b-phenylalanoylCoA side chain to baccatin III [38,39]. Although the degradation of taxol in cell cultures remains as a not well-known process, the decrease of taxol observed until day 16 could be due to degradation of this compound [40,41] or to other metabolic steps, such as still unknown multiple side-reactions and metabolic dead-ends, related with the taxol biosynthetic pathway. Nevertheless, further studies would be necessary to clarify this point.
Effects on expression level of DXS and TXS encoding genes and TXS activity To know if the expression level of two genes (dxs and ts) involved in taxol biosynthesis changed after the addition of taxol to the culture medium of our T. baccata cell line, the level of their transcripts was determined by RT-PCR analysis. The genes dxs and ts encode the enzymes DXS and TXS, respectively. DXS, the first enzyme of the MEP pathway, plays a fundamental role in the biosynthesis of plastidic terpenes, including the diterpenoid taxol, and TXS is involved in the cyclization of geranylgeranyl diphosphate, the first committed step of the biosynthetic pathway leading to the taxol taxane skeleton. The expression levels of the ts gene followed the same pattern over time in all studied conditions, but were clearly higher in cultures treated with taxol (Fig. 6A). The level peaked at day 4 with a rapid decrease at day 8, reaching a second, lower, peak at day 12. By day 16, ts mRNA was only detectable in the untreated control at a very low level. The concentration of taxol that most effectively induced ts mRNA expression was 50 mg L1: compared to the control, the transcript levels were 2.5-, 3.1- and 3.3-fold higher at 4, 8 and 12 days of culture, respectively. Because the induction of ts mRNA by taxol persisted for only 12 days and taxol biosynthesis mainly took place at day 20 (Fig. 5), the synthesis of TXS was presumably not a factor in the continued synthesis of the taxane in our T. baccata cell line cultures. To study this aspect in more detail, we measured the activity of this enzyme in all studied conditions. As also shown in Fig. 6A, a relatively high level of TXS activity was observed at day 4, when it was 2.8-, 6.8-, 3.2- and 1.7-fold higher, respectively, in the cultures treated with 25, 50, 100 and 200 mg L1 taxol than in the untreated control (6.64 pkat per mg of protein). Enzyme activity then decreased markedly from days 4 to 12 and thereafter was measurable only at a very low level until day 20. Thus, the enzyme activities for the synthesis of the side chain of taxol and its addition to the taxane skeleton apparently continued longer than those involved in the synthesis of at least one nonside-chain-containing taxane. Our results seem to show that the product of the biosynthetic step regulated by the enzyme TXS, the taxadiene, is not a limiting substrate in taxol formation. These results agree with those www.elsevier.com/locate/nbt
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New Biotechnology Volume 25, Number 4 April 2009
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New Biotechnology Volume 25, Number 4 April 2009
Research Paper FIGURE 6
Effects in a cultured T. baccata cell line of taxol feeding on the taxadiene synthase (TXS) activity and the expression levels of genes (tx), encoding this enzyme (A) and (dxs) encoding the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (B). Culture conditions were the same as seen in Fig. 2. Data represent average values from four replicates SE.
obtained by Croteau et al. [38], who suggested that neither taxadiene nor other early pathway intermediates accumulate to any appreciable level in Taxus cell cultures, indicating a rapid conversion of these metabolites by downstream reactions. The expression levels of the dxs gene were also higher in cultures treated with taxol, and followed the same pattern over time in all studied conditions (Fig. 6B). Levels peaked at day 4, decreased rapidly at day 8, and reached a second, lower, peak at day 12. Compared to the untreated control, at day 4 the dxs mRNA expression in taxol-treated cultures was 1.4-, 1.5- and 1.6-fold higher after exposure to 50, 100 and 200 mg L1 taxol, respectively. Whereas ts mRNA was not detected in taxol-treated cultures at day 16 (Fig. 6), we found that all taxol concentrations, particularly 50 mg L1, induced the expression of dxs mRNA at days 16 and 20. Considering that the enzyme DXS has a role at the beginning of the MEP pathway and that the taxanes are end products, it seems that exogenous taxol could affect taxol production by increasing the source of the plastidic terpenoid precursor isopentenyl diphosphate for taxane biosynthesis. Although it has 258
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still not been demonstrated that the enzyme DXS has a crucial role in the biosynthesis of taxol in Taxus in vitro cultures, a series of studies shows that this enzyme plays a fundamental role in the biosynthesis of terpenes in various organisms, including bacteria and plants [42].
Concluding remarks The effect of taxol feeding was investigated using a T. baccata cell line growing for 20 days in a selected nontaxol promoting growth medium to study how viability and apoptosis are related with taxol production. Compared with an untreated control, all the tested taxol concentrations stimulated cell-associated taxol content, especially at day 20 when our cell line was in its stationary growth phase and taxol biosynthesis at its peak. Taxol content increased with the concentration of added taxol, although cell viability decreased correspondingly. Because DNA laddering analysis revealed that the viability reduction was not related to apoptosis, it is thus inferred that apoptotic cell death was not related with to the taxol production in our T. baccata cell line cultures. This would
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oxidative elaboration and side-chain addition to afford taxol. The possibility that exogenous taxol induces taxol-related genes and thus taxol production, is consistent with the view that the accumulation of taxol in Taxus cells is a biological response to specific external stimuli [26], which in our study could be the added taxol.
Acknowledgements We thank the Technical Science Service from Barcelona University for their support. This research has been supported by two grants from the Spanish MEC (BIO2005-05583). Oscar Expo´sito is grateful for his research grant FPU (AP-2004-5321) from the Spanish MEC.
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agree with Kim et al. [14], who suggest that the high production of taxol may causally affect growth and viability through, as yet, undefined mechanisms. A plot of TXS activity in taxol-treated cultures clearly indicated that a pronounced decrease of activity occurred at least one week before cell-associated and extracellular taxol peaked at day 20, when the cultures were in its stationary growth phase. Thus, from day 16 to day 20, it seems that taxol formation did not depend on continued taxadiene production, suggesting that there must be other flux-limiting steps more downstream in the process, which are probably also stimulated by taxol feeding. It has been demonstrated [29] that taxadiene undergoes extensive
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