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The stimulation of taxol production in Taxus brevifolia by various growth retardants
plan cience Plant Science 101 (1994) 115-124
ELSEVIER
The stimulation of taxol production in Taxus brevifolia by various growth retardants Gary
S t r o b e l a, A n d r e a
S t i e r l e a, W . M .
Hess b
aDepartment of Plant Pathology, Montana State University, Bozeman, MT 59717. USA bDepartment of Botany and Range Science, Brigham Young University, Provo, UT 84601, USA
Received 11 April 1994; revision received 14 June 1994; accepted 16 June 1994
Abstract
We have shown that chlorocholine chloride (CCC), succinic acid, 2,2-dimethylhydrazide (Alar~ ) and tetramethylammonium bromide (TMAB) stimulate [~4C]acetate incorporation into taxol in intact pieces of the inner bark of Taxus brevifolia (Pacific yew). Both CCC and Alar also stimulated taxol production in yew logs as measured by the incorporation of [1-~4C]acetate into [14C]taxol. An increase in taxol accumulation also occurred in silica gel placed under flaps of yew trees in the forest treated with these twocompounds. CCC also caused an increase, in an 8-week treatment period, of recoverable taxol from Pacific yew bark in a forest setting. CCC, alar and TMAB are all known as growth retardants, but may cause an effect on taxol biosynthesis via an inhibition of sterol biosynthesis. Keywords. Chlorocholine chloride; Alar; Growth retardants; Sterol synthesis inhibitors; Taxol; Taxus spp.
1. Introduction
Species of Taxus are c o m m o n l y found as ground shrubs or understory growth in pine, larch, cedar, or fir forests in Europe, Asia and N o r t h America [1]. Most often yews grow in very moist shaded areas along streams and mountain lakes or on lower slopes [2]. This slow growing tree is the source o f taxol, whose unique chemistry, mode of action, and effectiveness in clinical trials, make it an important new drug for treatment o f breast and ovarian cancer [1,3-7]. The content of taxol in dried yew tree bark is * Corresponding author.
relatively low, about 0.01-0.03% of the dry weight, making the cost of this drug relatively high [1]. Thus, it is important to understand the influences and factors affecting the taxol content of yews. Some seasonal variation exists in the taxane/taxol content of Pacific yew, with the greatest levels occurring in the late spring and early summer, at the time of the most rapid plant growth. There may be internal factors controlling taxol concentration in the plant tissues. Recently we have learned that in vitro taxol biosynthesis in pieces of bark is affected by such c o m p o u n d s as fungal elicitors and certain fungicides [2]. The plant growth retardant, chlorocholine chloride (CCC), had a marked stimulatory effect on in vitro
0168-9452/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(94)03909-S
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{/'l Strobel et all / P l a n t Sci lOI 11994' 115-124
taxol biosynthesis [2]. Thus, the purpose of this report is to expand this original observation with more complete studies in logs and intact plants and to examine other growth retardants for their effect on taxol biosynthesis. 2. Materials and methods
2.1. In vitro [14C]taxol assay This assay was done under the general conditions as previously described, with the addition of K phosphate buffer at 10 raM, pH 6.5 and various growth retardants at different concentrations as needed. [l-14C]Acetate (20 /~Ci) with specific activities varying from 47 to 54 mCi/mmol was administered to small pieces (2 x 4 mm) of inner bark tissues for 3 days at 23°C [2]. The reaction mixture was extracted with an equal volume of chloroform:methanol (10:1 v/v) The organic extract was dried under N2 and redissolved in 250 #1 of acetonitrile [2]. A 25-ml aliquot of this extract was then streaked on a Merck silica gel thin layer chromatographic plate (TLC) (20 cm x 20 cm × 0.25 mm thickness) and chromatograped in CHCI3:MeOH (7:1 v/v). A narrow band was scraped from the plate at the RF corresponding to taxol migration and eluted with acetonitrile. After solvent evaporation, 12 ml of Aquasol was added to the sample vial and the radioactivity determined. The CHCI3:MeOH system does not separate cephalomannine from taxol; thus, up to 20% of the radioactivity is contributed by other taxanes [2,3]. All growth retardants were obtained from Aldrich/Sigma Chemical Compan~¢ with the exception of succinic acid 2,2-dimethylhydrazide (Alar) which was provided by Dr A. Bell of Uniroyal Chemical Company Inc., Bethany, CT. Authentic taxol was a gift of the National Cancer Institute. 2.2. T a x o l - identification and quantification [14C]Taxol from the growth retardant assays was identified by its co-chromatographic behavior on a Merck TLC silica gel plate (0.25 mm) using authentic taxol as a standard [2]. Two-dimensional chromatography was also done on silica gel plates with [14C]taxol obtained from the in vitro assay test using ethyl acetate/isopropanol (95:5 v/v) in the first dimension (solvent A) and methylene
dichloride/tetrahydrofuran (6:2 v/v) (solvent B)in the second dimension. After the plate was dried it was overlaid with 20 × 25 cm Fuji G C U medical X-ray film and exposed for 1-2 weeks. Finally, after development of the film and treatment of the plate with 1% w/v vanillin in sulfuric acid with genfie heating [8], the spot on the film was compared in size, shape and location with the spot (authentic taxol) on the TLC plate [2,3]. [14C]Taxol from the bioassay tests was also identified on the basis of its co-crystallization with authentic taxol. To this end, a sample of [L4C]taxol arising from the in vitro assay was dissolved along with 1 mg of authentic taxol in 0.5 ml of absolute methanol. Then 0.5 ml of H20 was slowly added to the solution, which was stirred, and placed at 40°C for 1 h, after which taxol crystals formed. The solution was centrifuged and UV absorbance at 273 nm was measured and a radioactivity determination was made [6]. The process was repeated 4 times and each time the specific radioactivity of [14C]taxol was determined. Taxol was quantitated by its mM absorbance of 1.7 (I.0 cm light path) at 273 nm [6].
2.3. Field and greenhouse experiments Native, mature Pacific yew shrubs used in these experiments were located on the west side of the Hungry Horse reservoir in Glacier County, MT, 2-3 miles south of the dam site [2,3]. Trees selected for study were growing in one select drainage, had excellent healthy top growth, and were at least 8-12 cm in diameter near the soil line (crown region). A l-ml Becton Dickinson No. 9602 sterile syringe was filled with a sterile 10-raM K phosphate solution (pH 6.9) made 63 mM with respect to CCC. A relatively high concentration of CCC was used since injection into the stem site can only be done imperfectly with loss of sample due to back pressure and dilution of the sample due to the large volume of water already present in the tree. Nevertheless, in a 10 × 20 cm area of bark located at the base of the tree we first sprayed a solution of 70% ethanol, allowed this to dry and then injected the plant growth retardant. Injection of 1 ml of solution was done in 5 rows of 10 insertion points. Approximately 0.02 ml was placed at each point. However, a sample (control) of II)
G. Strobel et al./Plant Sci. I01 (1994) 115-124 cm x 20 cm of bark was first taken from the tree which represented a 0 time control. Another control consisted of a bark sample in which a buffer (alone) solution had been injected. A 0 time control was also taken from each of the control trees. There were 4 experimental trees for the treatment and the two control groups. Final bark harvest was done 8 weeks after treatment in late spring. All bark pieces from each experimental group (0 time, 8 week treatment and control) were thoroughly air dried, combined and analysed for taxol and other taxanes. The experiment was done in the 1992 season and repeated again in 1993. At the Hungry Horse site we also did silica gel taxol trapping experiments in mid-summer [3]. Yews with an 8-12-cm diameter stem were located and 2 ml of a 10-mM K phosphate buffered solution of CCC (63 mM) was injected into an area of 10 x 20 cm of a previously disinfected (sprayed with 70% ethanol) area of the bark located about 0.2-0.05 M from the crown of the tree. After injection, the 10-20-cm area of bark was cut on 3 sides in order to make a flap. Under the flap was placed 5 g of Baker 60-200 mesh silica gel. The flap was closed and the stem was wrapped with duct tape. After 2 weeks the flap was opened and the silica gel carefully removed with a knife blade [3]. The collected silica gel was frozen and then lyophilized to a light brown powder. Ultimately, the powder was placed in a glass column and eluted first with chloroform (20 ml) which was discarded, and then with 20 mi of acetonitrile. The acetonitrile was taken to dryness and the taxol content of the residue determined. An identical experiment was done with alar as the treatment. In still another experiment, a control solution containing only 10 mM K phosphate buffer pH 6.9 was injected into the yew stems. Further, a 0 time experiment on a yew stem was also conducted. Taxol was recovered and quantitated from the silica obtained from the flaps of each of these treatments. Seedlings of Taxus media cv. Hicksii (a hybrid yew) were a generous gift of the Weyerhauser Co., Centralia, Washington, (Dr N. Wheeler). A set of 4 trees (30-50 cm) was each spray misted with 10 ml of buffer (10 mM K phosphate, pH 6.9), 4 trees each with 10 ml of 63 mM CCC in buffer and 4 trees injected with 1 ml of the same buffered CCC
117
solution at 20 locations from the top to the bottom of the main stem. The trees were placed in the MSU growth facility under natural lighting during March and April (8 weeks) with about a 12-h/day photoperiod in a temperature range of 20-23°C and then harvested. No significant differences were noted in the growth patterns of the treated trees. The stems and leaves were freeze dried and ultimately extracted and analysed for taxol and taxane content [9]. 2.4. [14C]Acetate labeling experiments in yew logs Yew logs about 0.6 m long x 8-10 cm in diameter were harvested in the Hungry Horse area of Northern Montana. As a check on the effects of optimized concentrations of CCC or alar on in vivo taxol biosynthesis, we injected 1 ml of a 0.63 mM solution of alar (in 10 mM K phosphate buffer, pH 6.9) into a 5 x 10 cm area using about 50 separate injection points with about 0.02 ml being injected at each site. The idenical treatment was done on two separate areas of each log. In addition, two 5 x 10 cm injection areas, serving as controls (buffer only) were also placed on each log. After treatment, the logs were placed in an incubator at 20°C. The ends of the logs were covered with parafilm to prevent massive moisture loss. In experiments involving CCC, 1 ml of a 63 mM buffered solution was injected. In both cases (CCC and Alar) at the end of 7, 14, and 28 days, exactly 0.4 g of inner bark of the treated and control bark areas was harvested and used in the in vitro assay for [14C]taxol production. Each in vitro test was replicated at least twice for each treatment. 2.5. Scanning electron microscopy Tissues were fixed and dehydrated using previously described methods [10]. The material was then critical point dried, gold coated with a sputter coater and observed and photographed with a JEOL 840 scanning electron microscope. 2.6. Radioactivity measurements All samples for radioactivity determinations were made in New England Nuclear's 'aquasol' (12 ml). The taxol samples were dissolved in 0.1-1.0 ml of methanol and cooled before counting. The pulse-height shift method was used to cor-
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G. Strobel et a l . / P l a n t Sct. l o t f 1994) 115-124
rect counts/min in samples to disingtegrations/min (d.p.m.).
A
I
H
I
I
CI-I 1| /+
H
CH~
H
2. 7. Mass spectroscopy II
Electrospray mass spectrometry was done with a VG trio-2 quadrapole instrument in methanol: H20:acetic acid (80:19:1, by v/v/v).
B All studies, if possible, were done at least twice. The population standard deviations of the data sets were calculated and are shown. The taxol/ taxane content of field-grown yews treated with growth retardants were determined on pooled bark samples in order to increase accuracy. This was done since not enough sample could be obtained from the same area of the tree to accomodate both 0 time and 8-week harvests without harming or killing the tree. Therefore, the experiment was repeated as identically as possible in the same field location in a second season. For the in vitro [14C]taxol assay system, an error of +9% is typically observed [2,3]. 3. Results and discussions
3.1. Effects of growth retardants on [14C]taxol production Alar, CCC (Fig. 1) and TMAB each caused a marked increase in [14C]taxol formation in the in vitro assay (Table 1). However, the greatest effects were noted in the CCC and alar treatments. Concentrations of 1-0.01 mM were more effective than 10mM in causing an increase in taxol production (Table 1). Other growth retardants, or derivatives thereof, were also tested in the in vitro assay. Most of these compounds, including choline, (2-bromoethyl)trimethyl ammonium bromide, and tetramethyl ammonim chloride, either had no effect on [14C]taxol formation (choline) or they caused a complete shift in the balance of lipid metabolism. This was manifested by the appearance of labeled compounds not normally observed in the in vitro test system (data not shown). [14C]Taxol was identified in each of these in vitro assays containing the growth retardants shown in Table 1. In order to identify [14C]taxol we used TLC cochromatography with authentic
O
II
2.9. Experimental confidence
H
f
H
I
CI
J
O
II
H
I
/CH
HO--C--C--C--C--N--N
HI HI
\CH
Fig. I. The chemical structures of (A) chlorocholine chloride (CCC) and (B) succinic acid 2,2-dirnethylhydrazide (Alar).
taxol followed by exposure to a Fuji X-ray film as shown for the Alar treatment in Fig. 2. In each case, in the growth retardant assay (CCC, Alar and TMAB), the spot on the X-ray film was coincident with the spot on the TLC plate that reacted with vanillin/sulfuric acid reagent (taxol) (Fig. 2). [14C]Taxol from these experiments was also crystallized to constant specific radioactivity as illustrated for the CCC treatment (Table 2). However, we did note that the [14C]taxol was slightly Table 1 Effects of CCC, Alar, and T M A B on [14C]taxol production in vitro Treatment
Concentration (mM)
Taxol (total d.p.m.)
Control Chlorocholine chloride (CCC)
0 I0 1 0.1 0.01 10 1 0.1 0.01 10 1 0.1 0.01
37 945 33 315 45 270 64 621 48 000 35 623 55 932 41 184 42 158 47 360 73 684 69 136 65 643
Tetramethyl ammonium bromide (TMAB)
N-Dimethylamino succinamic acid (alar)
The standard in vitro assay for taxol biosynthesis for [14C]acetate was applied to 0.4 g (fresh wt.) of bark for each of the plant growth retardants shown above.
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G. Strobel et al./Plant Sci. 101 (1994) 115-124
N
! !
~g
!
IW4
ETHYL ACETATE - I ~ I O F k l M L
95"S V/V
Fig. 2. A photograph of a TLC plate containing taxol isolated from an in vitro reaction mixture containing Alar and authentic taxol subjected to two-dimensional chromatography in solvent A followed by solvent B and then sprayed with the sulfuric acid vanillin reagent (left). The exposed and sprayed with the sulfuric acid vanillin developed X-ray film made of the TLC plate (right) shows one intense spot which is identical in size and location to the 'taxol spot' on the developed TLC plate. The solvent fronts of both A and B were to the margin of the plate.
contaminated with other compounds such as cephalomannine [2,3]. This is evidencd by a decrease in the constant specific radioactivity upon recrystallization of [14C]taxol (Table 2) and the appearance of some slight spots on the X-ray film of compounds other than taxol which is consistent with our previous observations (Fig. 2) [2,31.
3.2. Effect of Alar and CCC on [14C]taxol production in treated yew logs Since the growth retardants seemed to be influencing the production of [14C]taxol in the in vitro assay, we decided to test their effects in a more intact system. Initially, to do this we brought yew logs into the laboratory and injected them with an optimized concentration of either alar (1
Table 2 Cocrystallization of authentic taxol with [14C]taxol derived from chlorocholine chloride treated bark pieces in the labeling assay Crystallization
Sample size as percent of total taxol (%)
Taxol in sample assayed (#g)
Radioactivity (d.p.m.)
Ratio (d.p.m./mg)
First Second Third Fourth
20 10 8 10
239 128 99 120
152 78 51 60
0,63 0,60 0,51 0,50
Taxol obtained from an in vitro assay utilizing [14C]acetate was dissolved with I mg of authentic taxol and recrystallized in methanol/H20. Samples were taken after each crystallization and the specific radioactivity determined.
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G. Strobel et al./'Plant Sci. 101 (1994) 115-124
Fig. 3. The method used to inject a yew log with a l-ml hypodermic syringe filled with growth retardant.
ml of 0.63 mM) or CCC (1 ml of 63 mM) with a hypodermic syringe (Fig. 3) (see section 2 above). After 1-4 weeks the areas of bark treated with either of these compounds were then removed and subjected to the in vitro assay test utilizing [1-14C]acetate. The results show that both Alar and CCC caused a significant increase in the [14C]taxol being, produced up to at least 2 weeks after injection (Tables 3 and 4). Eventually, after 4 weeks there was little or no difference between the treatment and controls since it was obvious that the logs were stressed at Table 3 Influence of Alar on [14C]taxol formation in vitro after injection of Taxus brevifolia logs (8-10 cm diameter × 0.6 m length) Time of sampling
Treatment
Taxol (d.p.m.)
First week
Control Treatment Control Treatment Control Treatment
28 333 84 150 28 081 53 753 4122 5905
Second week Fourth week
± ± + ± ± +
528 2972 2150 3877 2150 2224
Harvested yew logs were brought into the laboratory and injected with 1 ml o f a 0.63-mm alar solution as described in the text. The logs were incubated at 20°C and weekly sampled for their in vitro taxol biosynthetic activity utilizing [14C]acetate. The results show the average of two experiments done on the same log at adjacent locations on the log.
the higher incubation temperature, had lost H20, and their metabolism of acetate had decreased (Tables 3 and 4). The [14C]taxol recovered from these assays (treated logs) was also checked for radiochemical identity and the results were basically the same as those from the standard assays (Fig. 2, Table 2). 3.3. In vivo taxol trapping experiments in forestgrown yews Even though the two previous experimental results (Tables 1 and 2) hinted at the positive effects of CCC and Alar on taxol biosynthesis, we felt that it was critical to obtain data from field Table 4 Influence of chlorocholine chloride on [14C]taxol formation in vitro after injection of Taxus brevifolia logs (8-10 cm diameter × 0.6 m length) Time of sampling
Treatment
Taxol (d.p.m.)
First week
Control Treatment Control Treatment Control Treatment
29 234 55 783 19 708 29 831 4287 11 458
Second week Fourth week
+ + ± ± ± ±
3140 1513 902 4210 287 1042
The treatment is the same as that described in the footnote to Table 3 except that 1 ml of-63 mM CCC was used in the injection treatment of the log.
G. Strobel et al. / Plant Sci. 101 (1994) 115-124
121
Table 5 The influence of Alar and chlorocholine chloride on the taxol content of silica gel under bark flaps (Pacific yew) treated with alar and CCC Treatment
Silica gel recovered (g)
Total taxol (p.g)
Taxol (~g/g)
0 Time control Control (buffer alone) Alar CCC
2.77 4.05 3.83 5.19
0 19.5 23.6 41.1
0 4.8 6.1 7.9
Yew stems in the field were injected with either alar, CCC, or control buffer and then a 'flap' made, under which 5 g of silica gel was placed. After 2 weeks, the silica gel was harvested and its taxol content determined [3].
observations. Several larger yew trees were located in the Hungry Horse Montana area and the stems (near ground level) were injected with Alar or CCC according to the injection technique shown in Fig. 3 and using methods already described. After two weeks, the silica gel was harvested, lyophilized, and analysed for taxol content [3]. In each case, when the bark had been previously injected with either Alar or CCC there was an increase in the amount of taxol detectable in the silica gel used as a trap (Table 5). Taxol obtained in this manner was chemically identified by its behavior in electrospray mass spectrometry yielding m/z of 854 (M+H) ÷ and 876 (M+Na) ÷ and by TLC in several solvent systems [2,11]. 3.4. Growth retardants in yew
Even though more taxol was obtained from
silica gel traps in the growth retardant treated yews this could have been the result of increased diffusion or release of taxol from the bark tissue into the silica gel. Therefore, it seemed reasonable to closely examine the actual taxol content of treated and untreated yew trees in the field for their taxol content. CCC was the growth retardant of choice because of its ready availability and its federal approval for crop application. Trees were injected with either CCC in buffer or buffer alone, and samples were taken at 0 time and 8 weeks later as described in section 2 above. The experiment was conducted in each of 2 growing seasons. The results show that CCC caused an apparent increase in the total taxol per gram dry weight of bark (Table 6). In one experiment, it was +58°/,, over the control and in the other it was about 100% (Table 6). Slight increases were also noted
Table 6 The effects of chlorocholine chloride (injected) on taxol production in field grown Pacific yew - - Taxus brevifidia Experiment number
Treatment
Taxol content (% dry wt.)
Taxol (#g/g dry wt.)
Difference (% change from 0 time)
Year 1 (24 J u n e - 3 Sept.)
0 9 0 9
time (buffer control) weeks (buffer control) time (treatment) weeks (treatment)
0.019 0.013 0.029 0.046
0.19 0.13 0.29 0.46
0 -31 0 +58
Year 2 (May 15)
0 8 0 8
time (buffer control) weeks (buffer control) time (treatment) weeks (treatment)
0.024 0.025 0.030 0.062
0.24 0.25 0.30 0.62
0 +4 0 +106
The stems of field grown T. brevifolia were injected with 1 ml of 63-mM CCC in the late spring/early summer and the bark harvested 8 - 9 weeks later. Bark samples were also taken at 0 time. Appropriate controls were used. Details of the experiments are given in the text.
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G. Strobel et al./Plant SoL 101 , /9941 115-/24
Table 7 The effect of chlorcholine chloride on taxol production in greenhouse grown seedlings of l)txus media cv. thcksii Treatment difference
Taxol content (% dry wt.)
Taxol (/~g/g dry wt.~
(",',,change from II time)
0 time (buffer control) 8 weeks (buffer control)
0.027 0.022
11.27 0.22
II -IS'l~.
0 time (treatment sprayed) 8 weeks (treatment sprayed)
0.022 0.022
0.22
I~
11.22
0
0 time (treatment injected) 8 weeks (treatment injected)
11.020 0.017
0.20 0.17
-15'!,,
0
Solutions of CCC were sprayed and injected into seeding yews. After 8 weeks the harvested, dried plants werc analysed for taxol and taxanes as described in the text. No statistical differences exist among treatments.
for cephalomannine and 10-deactylbaccatin (20-30%), respectively, in treated trees as compared with the controls (data not shown). 3.5. Growth retardant treatment o f seedling yews Taxus media cv. Hicksii, a hybrid yew, is being
considered as a plantation source of taxol and taxanes. Spray and injection treatments o f sets of these small trees in the greenhouse with C C C did not result in an increase in the production o f taxol or any other taxane (Table 7). Also, [1-14elacetate did not result in any labeling of taxol in the in vitro assay when tissues of this yew hybrid were tested (data not shown). Likewise, neither alar or CCC caused [14C]taxol to form in the in vitro assay (data not shown). It may be that alar and CCC are only effective in promoting an increase in taxol production in those yew species that readily utilize [1-14C]acetate [3]. Initially, we thought that the spray application of C C C would be adequate to enhance taxol production since C C C is applied topically to cereal grains to reduce internodal growth to prevent lodging. However, a closer look at yew leaves of both T. brevifolia and T. media cv, Hicksii revealed that the upper leaf surface contains no stomates and it is covered by a waxy cuticle (Fig. 4 A - D ) . However, stomates appear in rows of - 4 in T. brevifolia and rows of - 8 in 7'. media cv. Hicksii on the underside o f the leaves. The stomates have a unique set of raised subsidary cells (Fig. 4). Although it is not known if the
spraying of 7". brevijolia leaves with growth retardants is effective in promoting an increase in taxol production, direct injection of either alar or CCC did increase taxol production in T. brev~/blia (Tables 3, 4 and 6). Nevertheless, further studies on the utilization of growth retardants should take into account the structure of the Taxus leaf and its stomatal arrangement and organization especially with regard to T. brevifolia (Fig. 4). Adjuvants, adhesives or other substances used in the formulation of products applied to plants should also be considered. However, in regard to T. media c.v Hicksii, it appears that neither direct injection nor spray application of the growth retardants used resulted in a change in the level of taxol, Obviously other metabolic inhibitors that may be effective in promoting taxane production in this Taxus species need to be tested. Again, if such c o m p o u n d s are found and applied by spraying then leaf stomate structure will probably have a bearing on the uptake of the active substance. 3.6. Growth retardants as metabolic' inhibitors
It is generally believed that growth retardants exert their effect by interfering with gibberellic acid biosynthesis [12]. However, it is also known that some of these compounds, such as CCC, act as sterol synthesis inhibitors [13]. If sterol synthesis is being inhibited in yews one may expect to see a shift in metabolism with acetyl moieties being shunted into other compounds such as taxol and
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G. Strobel et al./Plant Sci. 101 (1994) 115-124
taxanes. An ideal example of this effect was observed in Dreschslera gigantea, a pathogenic fungus, and the dramatic increase in petasol (a sesquiterpenoid) production upon incubation with CCC [14]. The effect of CCC in enhancing taxol production in vitro and in vivo in Pacific yew very much resembles the effect of CCC on terpenoid biosynthesis in D. gigantea [14]. The effect, however, seems to be differential among Taxus spp., i.e. it enhances taxol accumulation in one, but has no effect in another (Tables 6 and 7). Further, the positive effect of Alar on taxol formation in Pacific yew represents an interesting twist in history. Alar had been used as a plant growth regulator in the apple industry and certain unfounded claims had been made about its potential carcinogenic properties [15]. It is ironic that it promotes the formation of taxol (Table 1), an important new anticancer drug [1]. Although several retardants were effective in our assays, it is possible that other compounds exist that are even more potent in promoting taxol accumulation [2]. It also seems that the effect of these retardants in yew species could also be more efficiently and quickly measured in cell suspension cultures, perhaps using other precursors in [tac]taxol biosynthesis [2,3,16] Acknowledgements The authors appreciate the drawing of the yew log provided by Suzan Strobel. Alar was a generous gift of the Uniroyal Chemical Company Inc. The support and help of the people of the U.S. Forest Service, Hungry Horse District, Flathead National Forest in acknowledged. The Yellow Bay Laboratory of the University of Montana has generously provided accommodations during our trips to Northern Montana. Taxol and other taxanes are a gift of the NC1. Some taxol and taxane analyses in this report were independently done by Hauser Chemical Company, Boulder, CO, compliments of Dr David Bailey. Financial support for this project has come from the Montana Science and Technology Alliance, the Montana Agricultural Experiment Station, and the National Cancer Institute, and Cytoclonal Company, Dallas, Texas.
References [1] H. Hartzell, The Yew Tree, Hulogosi Press, Eugene OR, 1991. [21 G.A. Strobel, A. Stierle and F.J.G.M. Van Kuijk, Factor influencing the in vitro production of radio labeled taxol by Pacific yew, Taxus brevifolia. Plant Sci., 84 (1992) 65-74. [3] G.A. Strobel, A. Stierle and W.M. Hess, Taxol formation in yew Taxus. Plant Sci., 92 (1993) 1-12. [41 P.B. Schiff, J. Fant and S.B. Horowitz, Promotion of microtubule assembly in vitro by taxol. Nature, 277 (1979) 665-667. [5] P.B. Schiff and S.B. Horowitz, Taxol stabilizes microtubles in mouse fibroblast cells. Proc. Natl. Acad. Sci. USA, 77 (1980) 1561-1565. [6] M.C. Wani, H.L. Taylor, M.E. Wall, P. Coggon and A.T. McPhail, Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus breviJblia. J. Am. Chem. Soc., 93 (1971) 2325-2327. [7] D.G.I. Kingston, The chemistry of taxol. Pharmacol. Ther., 52 (1991) 1-34. [8] J.H. Cardellina, HPLC separation of taxol and cephalomannine. J. Liquid Chromatogr., 14 (1991) 659-665. [9] N.C. Wheeler, K. Jech, S. Masters, S. Brobst, A.B. Alvarado and A.J. Hoover, Effects of genetic, epigenetic and environmental factors on taxol content in Taxus brevifolia and related species. J. Nat. Prod., 55 (1992) 432-440. [10] R.V. Upadhyay, G.A. Strobel and W.M. Hess, Morphogenesis and ultrastructure of the conidiomata of Ascochyta cypericola. Mycol. Res., 95 (1991) 785-791. [11] A. Stierle, G. Strobel and D. Stierle, Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Science, 260 (1993) 214-216. [12] A. Lang, Gibberellins: structure and metabolism. Annu. Rev. Plant Physiol., 21 (1970) 537-570. [131 J. Beutler, G.N. Churny, B.D. Hillon, S. Brobst, S.A. Look and K. Witherup, Proton 13c NMR assignments for taxol, 7-epitaxol and cephalomannine. J. Nat. Prod., 55 (1992) 414-423. [14] G.J. Bunkers, D. Kenfield and G.A. Strobel, Production of petasol by Drechslera gigantea in liquid culture. Mycol. Res., 95 (1991) 347-351. [15] M. Fumento, The politics of career testing. Am. Spectator., (1990) 18-23. [16] P.E. Fleming, U. Moak and H.G. Hess, Biosynthesis of taxoids. Mode of formation of the taxol side chain. J. Am. Chem. Soc., 115 (1993) 805-807.
G. Strobel et al./Plant Sci. lOl (1994) 115-124
12~
Fig. 4. Scanning electron micrographs of the surfaces of the leaves of T. hrevi/bha and l~ media c~. Hicksii. (At The upper surface of 72 brevifolia, (B) the lower surface of T. hrevi/olia, (C) a stomate with subsidiary cells of T. hrev(Iblia, and (Dt the lower leaf surface of T. media cv. Hicksii.
ELSEVIER
The stimulation of taxol production in Taxus brevifolia by various growth retardants Gary
S t r o b e l a, A n d r e a
S t i e r l e a, W . M .
Hess b
aDepartment of Plant Pathology, Montana State University, Bozeman, MT 59717. USA bDepartment of Botany and Range Science, Brigham Young University, Provo, UT 84601, USA
Received 11 April 1994; revision received 14 June 1994; accepted 16 June 1994
Abstract
We have shown that chlorocholine chloride (CCC), succinic acid, 2,2-dimethylhydrazide (Alar~ ) and tetramethylammonium bromide (TMAB) stimulate [~4C]acetate incorporation into taxol in intact pieces of the inner bark of Taxus brevifolia (Pacific yew). Both CCC and Alar also stimulated taxol production in yew logs as measured by the incorporation of [1-~4C]acetate into [14C]taxol. An increase in taxol accumulation also occurred in silica gel placed under flaps of yew trees in the forest treated with these twocompounds. CCC also caused an increase, in an 8-week treatment period, of recoverable taxol from Pacific yew bark in a forest setting. CCC, alar and TMAB are all known as growth retardants, but may cause an effect on taxol biosynthesis via an inhibition of sterol biosynthesis. Keywords. Chlorocholine chloride; Alar; Growth retardants; Sterol synthesis inhibitors; Taxol; Taxus spp.
1. Introduction
Species of Taxus are c o m m o n l y found as ground shrubs or understory growth in pine, larch, cedar, or fir forests in Europe, Asia and N o r t h America [1]. Most often yews grow in very moist shaded areas along streams and mountain lakes or on lower slopes [2]. This slow growing tree is the source o f taxol, whose unique chemistry, mode of action, and effectiveness in clinical trials, make it an important new drug for treatment o f breast and ovarian cancer [1,3-7]. The content of taxol in dried yew tree bark is * Corresponding author.
relatively low, about 0.01-0.03% of the dry weight, making the cost of this drug relatively high [1]. Thus, it is important to understand the influences and factors affecting the taxol content of yews. Some seasonal variation exists in the taxane/taxol content of Pacific yew, with the greatest levels occurring in the late spring and early summer, at the time of the most rapid plant growth. There may be internal factors controlling taxol concentration in the plant tissues. Recently we have learned that in vitro taxol biosynthesis in pieces of bark is affected by such c o m p o u n d s as fungal elicitors and certain fungicides [2]. The plant growth retardant, chlorocholine chloride (CCC), had a marked stimulatory effect on in vitro
0168-9452/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(94)03909-S
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{/'l Strobel et all / P l a n t Sci lOI 11994' 115-124
taxol biosynthesis [2]. Thus, the purpose of this report is to expand this original observation with more complete studies in logs and intact plants and to examine other growth retardants for their effect on taxol biosynthesis. 2. Materials and methods
2.1. In vitro [14C]taxol assay This assay was done under the general conditions as previously described, with the addition of K phosphate buffer at 10 raM, pH 6.5 and various growth retardants at different concentrations as needed. [l-14C]Acetate (20 /~Ci) with specific activities varying from 47 to 54 mCi/mmol was administered to small pieces (2 x 4 mm) of inner bark tissues for 3 days at 23°C [2]. The reaction mixture was extracted with an equal volume of chloroform:methanol (10:1 v/v) The organic extract was dried under N2 and redissolved in 250 #1 of acetonitrile [2]. A 25-ml aliquot of this extract was then streaked on a Merck silica gel thin layer chromatographic plate (TLC) (20 cm x 20 cm × 0.25 mm thickness) and chromatograped in CHCI3:MeOH (7:1 v/v). A narrow band was scraped from the plate at the RF corresponding to taxol migration and eluted with acetonitrile. After solvent evaporation, 12 ml of Aquasol was added to the sample vial and the radioactivity determined. The CHCI3:MeOH system does not separate cephalomannine from taxol; thus, up to 20% of the radioactivity is contributed by other taxanes [2,3]. All growth retardants were obtained from Aldrich/Sigma Chemical Compan~¢ with the exception of succinic acid 2,2-dimethylhydrazide (Alar) which was provided by Dr A. Bell of Uniroyal Chemical Company Inc., Bethany, CT. Authentic taxol was a gift of the National Cancer Institute. 2.2. T a x o l - identification and quantification [14C]Taxol from the growth retardant assays was identified by its co-chromatographic behavior on a Merck TLC silica gel plate (0.25 mm) using authentic taxol as a standard [2]. Two-dimensional chromatography was also done on silica gel plates with [14C]taxol obtained from the in vitro assay test using ethyl acetate/isopropanol (95:5 v/v) in the first dimension (solvent A) and methylene
dichloride/tetrahydrofuran (6:2 v/v) (solvent B)in the second dimension. After the plate was dried it was overlaid with 20 × 25 cm Fuji G C U medical X-ray film and exposed for 1-2 weeks. Finally, after development of the film and treatment of the plate with 1% w/v vanillin in sulfuric acid with genfie heating [8], the spot on the film was compared in size, shape and location with the spot (authentic taxol) on the TLC plate [2,3]. [14C]Taxol from the bioassay tests was also identified on the basis of its co-crystallization with authentic taxol. To this end, a sample of [L4C]taxol arising from the in vitro assay was dissolved along with 1 mg of authentic taxol in 0.5 ml of absolute methanol. Then 0.5 ml of H20 was slowly added to the solution, which was stirred, and placed at 40°C for 1 h, after which taxol crystals formed. The solution was centrifuged and UV absorbance at 273 nm was measured and a radioactivity determination was made [6]. The process was repeated 4 times and each time the specific radioactivity of [14C]taxol was determined. Taxol was quantitated by its mM absorbance of 1.7 (I.0 cm light path) at 273 nm [6].
2.3. Field and greenhouse experiments Native, mature Pacific yew shrubs used in these experiments were located on the west side of the Hungry Horse reservoir in Glacier County, MT, 2-3 miles south of the dam site [2,3]. Trees selected for study were growing in one select drainage, had excellent healthy top growth, and were at least 8-12 cm in diameter near the soil line (crown region). A l-ml Becton Dickinson No. 9602 sterile syringe was filled with a sterile 10-raM K phosphate solution (pH 6.9) made 63 mM with respect to CCC. A relatively high concentration of CCC was used since injection into the stem site can only be done imperfectly with loss of sample due to back pressure and dilution of the sample due to the large volume of water already present in the tree. Nevertheless, in a 10 × 20 cm area of bark located at the base of the tree we first sprayed a solution of 70% ethanol, allowed this to dry and then injected the plant growth retardant. Injection of 1 ml of solution was done in 5 rows of 10 insertion points. Approximately 0.02 ml was placed at each point. However, a sample (control) of II)
G. Strobel et al./Plant Sci. I01 (1994) 115-124 cm x 20 cm of bark was first taken from the tree which represented a 0 time control. Another control consisted of a bark sample in which a buffer (alone) solution had been injected. A 0 time control was also taken from each of the control trees. There were 4 experimental trees for the treatment and the two control groups. Final bark harvest was done 8 weeks after treatment in late spring. All bark pieces from each experimental group (0 time, 8 week treatment and control) were thoroughly air dried, combined and analysed for taxol and other taxanes. The experiment was done in the 1992 season and repeated again in 1993. At the Hungry Horse site we also did silica gel taxol trapping experiments in mid-summer [3]. Yews with an 8-12-cm diameter stem were located and 2 ml of a 10-mM K phosphate buffered solution of CCC (63 mM) was injected into an area of 10 x 20 cm of a previously disinfected (sprayed with 70% ethanol) area of the bark located about 0.2-0.05 M from the crown of the tree. After injection, the 10-20-cm area of bark was cut on 3 sides in order to make a flap. Under the flap was placed 5 g of Baker 60-200 mesh silica gel. The flap was closed and the stem was wrapped with duct tape. After 2 weeks the flap was opened and the silica gel carefully removed with a knife blade [3]. The collected silica gel was frozen and then lyophilized to a light brown powder. Ultimately, the powder was placed in a glass column and eluted first with chloroform (20 ml) which was discarded, and then with 20 mi of acetonitrile. The acetonitrile was taken to dryness and the taxol content of the residue determined. An identical experiment was done with alar as the treatment. In still another experiment, a control solution containing only 10 mM K phosphate buffer pH 6.9 was injected into the yew stems. Further, a 0 time experiment on a yew stem was also conducted. Taxol was recovered and quantitated from the silica obtained from the flaps of each of these treatments. Seedlings of Taxus media cv. Hicksii (a hybrid yew) were a generous gift of the Weyerhauser Co., Centralia, Washington, (Dr N. Wheeler). A set of 4 trees (30-50 cm) was each spray misted with 10 ml of buffer (10 mM K phosphate, pH 6.9), 4 trees each with 10 ml of 63 mM CCC in buffer and 4 trees injected with 1 ml of the same buffered CCC
117
solution at 20 locations from the top to the bottom of the main stem. The trees were placed in the MSU growth facility under natural lighting during March and April (8 weeks) with about a 12-h/day photoperiod in a temperature range of 20-23°C and then harvested. No significant differences were noted in the growth patterns of the treated trees. The stems and leaves were freeze dried and ultimately extracted and analysed for taxol and taxane content [9]. 2.4. [14C]Acetate labeling experiments in yew logs Yew logs about 0.6 m long x 8-10 cm in diameter were harvested in the Hungry Horse area of Northern Montana. As a check on the effects of optimized concentrations of CCC or alar on in vivo taxol biosynthesis, we injected 1 ml of a 0.63 mM solution of alar (in 10 mM K phosphate buffer, pH 6.9) into a 5 x 10 cm area using about 50 separate injection points with about 0.02 ml being injected at each site. The idenical treatment was done on two separate areas of each log. In addition, two 5 x 10 cm injection areas, serving as controls (buffer only) were also placed on each log. After treatment, the logs were placed in an incubator at 20°C. The ends of the logs were covered with parafilm to prevent massive moisture loss. In experiments involving CCC, 1 ml of a 63 mM buffered solution was injected. In both cases (CCC and Alar) at the end of 7, 14, and 28 days, exactly 0.4 g of inner bark of the treated and control bark areas was harvested and used in the in vitro assay for [14C]taxol production. Each in vitro test was replicated at least twice for each treatment. 2.5. Scanning electron microscopy Tissues were fixed and dehydrated using previously described methods [10]. The material was then critical point dried, gold coated with a sputter coater and observed and photographed with a JEOL 840 scanning electron microscope. 2.6. Radioactivity measurements All samples for radioactivity determinations were made in New England Nuclear's 'aquasol' (12 ml). The taxol samples were dissolved in 0.1-1.0 ml of methanol and cooled before counting. The pulse-height shift method was used to cor-
118
G. Strobel et a l . / P l a n t Sct. l o t f 1994) 115-124
rect counts/min in samples to disingtegrations/min (d.p.m.).
A
I
H
I
I
CI-I 1| /+
H
CH~
H
2. 7. Mass spectroscopy II
Electrospray mass spectrometry was done with a VG trio-2 quadrapole instrument in methanol: H20:acetic acid (80:19:1, by v/v/v).
B All studies, if possible, were done at least twice. The population standard deviations of the data sets were calculated and are shown. The taxol/ taxane content of field-grown yews treated with growth retardants were determined on pooled bark samples in order to increase accuracy. This was done since not enough sample could be obtained from the same area of the tree to accomodate both 0 time and 8-week harvests without harming or killing the tree. Therefore, the experiment was repeated as identically as possible in the same field location in a second season. For the in vitro [14C]taxol assay system, an error of +9% is typically observed [2,3]. 3. Results and discussions
3.1. Effects of growth retardants on [14C]taxol production Alar, CCC (Fig. 1) and TMAB each caused a marked increase in [14C]taxol formation in the in vitro assay (Table 1). However, the greatest effects were noted in the CCC and alar treatments. Concentrations of 1-0.01 mM were more effective than 10mM in causing an increase in taxol production (Table 1). Other growth retardants, or derivatives thereof, were also tested in the in vitro assay. Most of these compounds, including choline, (2-bromoethyl)trimethyl ammonium bromide, and tetramethyl ammonim chloride, either had no effect on [14C]taxol formation (choline) or they caused a complete shift in the balance of lipid metabolism. This was manifested by the appearance of labeled compounds not normally observed in the in vitro test system (data not shown). [14C]Taxol was identified in each of these in vitro assays containing the growth retardants shown in Table 1. In order to identify [14C]taxol we used TLC cochromatography with authentic
O
II
2.9. Experimental confidence
H
f
H
I
CI
J
O
II
H
I
/CH
HO--C--C--C--C--N--N
HI HI
\CH
Fig. I. The chemical structures of (A) chlorocholine chloride (CCC) and (B) succinic acid 2,2-dirnethylhydrazide (Alar).
taxol followed by exposure to a Fuji X-ray film as shown for the Alar treatment in Fig. 2. In each case, in the growth retardant assay (CCC, Alar and TMAB), the spot on the X-ray film was coincident with the spot on the TLC plate that reacted with vanillin/sulfuric acid reagent (taxol) (Fig. 2). [14C]Taxol from these experiments was also crystallized to constant specific radioactivity as illustrated for the CCC treatment (Table 2). However, we did note that the [14C]taxol was slightly Table 1 Effects of CCC, Alar, and T M A B on [14C]taxol production in vitro Treatment
Concentration (mM)
Taxol (total d.p.m.)
Control Chlorocholine chloride (CCC)
0 I0 1 0.1 0.01 10 1 0.1 0.01 10 1 0.1 0.01
37 945 33 315 45 270 64 621 48 000 35 623 55 932 41 184 42 158 47 360 73 684 69 136 65 643
Tetramethyl ammonium bromide (TMAB)
N-Dimethylamino succinamic acid (alar)
The standard in vitro assay for taxol biosynthesis for [14C]acetate was applied to 0.4 g (fresh wt.) of bark for each of the plant growth retardants shown above.
119
G. Strobel et al./Plant Sci. 101 (1994) 115-124
N
! !
~g
!
IW4
ETHYL ACETATE - I ~ I O F k l M L
95"S V/V
Fig. 2. A photograph of a TLC plate containing taxol isolated from an in vitro reaction mixture containing Alar and authentic taxol subjected to two-dimensional chromatography in solvent A followed by solvent B and then sprayed with the sulfuric acid vanillin reagent (left). The exposed and sprayed with the sulfuric acid vanillin developed X-ray film made of the TLC plate (right) shows one intense spot which is identical in size and location to the 'taxol spot' on the developed TLC plate. The solvent fronts of both A and B were to the margin of the plate.
contaminated with other compounds such as cephalomannine [2,3]. This is evidencd by a decrease in the constant specific radioactivity upon recrystallization of [14C]taxol (Table 2) and the appearance of some slight spots on the X-ray film of compounds other than taxol which is consistent with our previous observations (Fig. 2) [2,31.
3.2. Effect of Alar and CCC on [14C]taxol production in treated yew logs Since the growth retardants seemed to be influencing the production of [14C]taxol in the in vitro assay, we decided to test their effects in a more intact system. Initially, to do this we brought yew logs into the laboratory and injected them with an optimized concentration of either alar (1
Table 2 Cocrystallization of authentic taxol with [14C]taxol derived from chlorocholine chloride treated bark pieces in the labeling assay Crystallization
Sample size as percent of total taxol (%)
Taxol in sample assayed (#g)
Radioactivity (d.p.m.)
Ratio (d.p.m./mg)
First Second Third Fourth
20 10 8 10
239 128 99 120
152 78 51 60
0,63 0,60 0,51 0,50
Taxol obtained from an in vitro assay utilizing [14C]acetate was dissolved with I mg of authentic taxol and recrystallized in methanol/H20. Samples were taken after each crystallization and the specific radioactivity determined.
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G. Strobel et al./'Plant Sci. 101 (1994) 115-124
Fig. 3. The method used to inject a yew log with a l-ml hypodermic syringe filled with growth retardant.
ml of 0.63 mM) or CCC (1 ml of 63 mM) with a hypodermic syringe (Fig. 3) (see section 2 above). After 1-4 weeks the areas of bark treated with either of these compounds were then removed and subjected to the in vitro assay test utilizing [1-14C]acetate. The results show that both Alar and CCC caused a significant increase in the [14C]taxol being, produced up to at least 2 weeks after injection (Tables 3 and 4). Eventually, after 4 weeks there was little or no difference between the treatment and controls since it was obvious that the logs were stressed at Table 3 Influence of Alar on [14C]taxol formation in vitro after injection of Taxus brevifolia logs (8-10 cm diameter × 0.6 m length) Time of sampling
Treatment
Taxol (d.p.m.)
First week
Control Treatment Control Treatment Control Treatment
28 333 84 150 28 081 53 753 4122 5905
Second week Fourth week
± ± + ± ± +
528 2972 2150 3877 2150 2224
Harvested yew logs were brought into the laboratory and injected with 1 ml o f a 0.63-mm alar solution as described in the text. The logs were incubated at 20°C and weekly sampled for their in vitro taxol biosynthetic activity utilizing [14C]acetate. The results show the average of two experiments done on the same log at adjacent locations on the log.
the higher incubation temperature, had lost H20, and their metabolism of acetate had decreased (Tables 3 and 4). The [14C]taxol recovered from these assays (treated logs) was also checked for radiochemical identity and the results were basically the same as those from the standard assays (Fig. 2, Table 2). 3.3. In vivo taxol trapping experiments in forestgrown yews Even though the two previous experimental results (Tables 1 and 2) hinted at the positive effects of CCC and Alar on taxol biosynthesis, we felt that it was critical to obtain data from field Table 4 Influence of chlorocholine chloride on [14C]taxol formation in vitro after injection of Taxus brevifolia logs (8-10 cm diameter × 0.6 m length) Time of sampling
Treatment
Taxol (d.p.m.)
First week
Control Treatment Control Treatment Control Treatment
29 234 55 783 19 708 29 831 4287 11 458
Second week Fourth week
+ + ± ± ± ±
3140 1513 902 4210 287 1042
The treatment is the same as that described in the footnote to Table 3 except that 1 ml of-63 mM CCC was used in the injection treatment of the log.
G. Strobel et al. / Plant Sci. 101 (1994) 115-124
121
Table 5 The influence of Alar and chlorocholine chloride on the taxol content of silica gel under bark flaps (Pacific yew) treated with alar and CCC Treatment
Silica gel recovered (g)
Total taxol (p.g)
Taxol (~g/g)
0 Time control Control (buffer alone) Alar CCC
2.77 4.05 3.83 5.19
0 19.5 23.6 41.1
0 4.8 6.1 7.9
Yew stems in the field were injected with either alar, CCC, or control buffer and then a 'flap' made, under which 5 g of silica gel was placed. After 2 weeks, the silica gel was harvested and its taxol content determined [3].
observations. Several larger yew trees were located in the Hungry Horse Montana area and the stems (near ground level) were injected with Alar or CCC according to the injection technique shown in Fig. 3 and using methods already described. After two weeks, the silica gel was harvested, lyophilized, and analysed for taxol content [3]. In each case, when the bark had been previously injected with either Alar or CCC there was an increase in the amount of taxol detectable in the silica gel used as a trap (Table 5). Taxol obtained in this manner was chemically identified by its behavior in electrospray mass spectrometry yielding m/z of 854 (M+H) ÷ and 876 (M+Na) ÷ and by TLC in several solvent systems [2,11]. 3.4. Growth retardants in yew
Even though more taxol was obtained from
silica gel traps in the growth retardant treated yews this could have been the result of increased diffusion or release of taxol from the bark tissue into the silica gel. Therefore, it seemed reasonable to closely examine the actual taxol content of treated and untreated yew trees in the field for their taxol content. CCC was the growth retardant of choice because of its ready availability and its federal approval for crop application. Trees were injected with either CCC in buffer or buffer alone, and samples were taken at 0 time and 8 weeks later as described in section 2 above. The experiment was conducted in each of 2 growing seasons. The results show that CCC caused an apparent increase in the total taxol per gram dry weight of bark (Table 6). In one experiment, it was +58°/,, over the control and in the other it was about 100% (Table 6). Slight increases were also noted
Table 6 The effects of chlorocholine chloride (injected) on taxol production in field grown Pacific yew - - Taxus brevifidia Experiment number
Treatment
Taxol content (% dry wt.)
Taxol (#g/g dry wt.)
Difference (% change from 0 time)
Year 1 (24 J u n e - 3 Sept.)
0 9 0 9
time (buffer control) weeks (buffer control) time (treatment) weeks (treatment)
0.019 0.013 0.029 0.046
0.19 0.13 0.29 0.46
0 -31 0 +58
Year 2 (May 15)
0 8 0 8
time (buffer control) weeks (buffer control) time (treatment) weeks (treatment)
0.024 0.025 0.030 0.062
0.24 0.25 0.30 0.62
0 +4 0 +106
The stems of field grown T. brevifolia were injected with 1 ml of 63-mM CCC in the late spring/early summer and the bark harvested 8 - 9 weeks later. Bark samples were also taken at 0 time. Appropriate controls were used. Details of the experiments are given in the text.
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G. Strobel et al./Plant SoL 101 , /9941 115-/24
Table 7 The effect of chlorcholine chloride on taxol production in greenhouse grown seedlings of l)txus media cv. thcksii Treatment difference
Taxol content (% dry wt.)
Taxol (/~g/g dry wt.~
(",',,change from II time)
0 time (buffer control) 8 weeks (buffer control)
0.027 0.022
11.27 0.22
II -IS'l~.
0 time (treatment sprayed) 8 weeks (treatment sprayed)
0.022 0.022
0.22
I~
11.22
0
0 time (treatment injected) 8 weeks (treatment injected)
11.020 0.017
0.20 0.17
-15'!,,
0
Solutions of CCC were sprayed and injected into seeding yews. After 8 weeks the harvested, dried plants werc analysed for taxol and taxanes as described in the text. No statistical differences exist among treatments.
for cephalomannine and 10-deactylbaccatin (20-30%), respectively, in treated trees as compared with the controls (data not shown). 3.5. Growth retardant treatment o f seedling yews Taxus media cv. Hicksii, a hybrid yew, is being
considered as a plantation source of taxol and taxanes. Spray and injection treatments o f sets of these small trees in the greenhouse with C C C did not result in an increase in the production o f taxol or any other taxane (Table 7). Also, [1-14elacetate did not result in any labeling of taxol in the in vitro assay when tissues of this yew hybrid were tested (data not shown). Likewise, neither alar or CCC caused [14C]taxol to form in the in vitro assay (data not shown). It may be that alar and CCC are only effective in promoting an increase in taxol production in those yew species that readily utilize [1-14C]acetate [3]. Initially, we thought that the spray application of C C C would be adequate to enhance taxol production since C C C is applied topically to cereal grains to reduce internodal growth to prevent lodging. However, a closer look at yew leaves of both T. brevifolia and T. media cv, Hicksii revealed that the upper leaf surface contains no stomates and it is covered by a waxy cuticle (Fig. 4 A - D ) . However, stomates appear in rows of - 4 in T. brevifolia and rows of - 8 in 7'. media cv. Hicksii on the underside o f the leaves. The stomates have a unique set of raised subsidary cells (Fig. 4). Although it is not known if the
spraying of 7". brevijolia leaves with growth retardants is effective in promoting an increase in taxol production, direct injection of either alar or CCC did increase taxol production in T. brev~/blia (Tables 3, 4 and 6). Nevertheless, further studies on the utilization of growth retardants should take into account the structure of the Taxus leaf and its stomatal arrangement and organization especially with regard to T. brevifolia (Fig. 4). Adjuvants, adhesives or other substances used in the formulation of products applied to plants should also be considered. However, in regard to T. media c.v Hicksii, it appears that neither direct injection nor spray application of the growth retardants used resulted in a change in the level of taxol, Obviously other metabolic inhibitors that may be effective in promoting taxane production in this Taxus species need to be tested. Again, if such c o m p o u n d s are found and applied by spraying then leaf stomate structure will probably have a bearing on the uptake of the active substance. 3.6. Growth retardants as metabolic' inhibitors
It is generally believed that growth retardants exert their effect by interfering with gibberellic acid biosynthesis [12]. However, it is also known that some of these compounds, such as CCC, act as sterol synthesis inhibitors [13]. If sterol synthesis is being inhibited in yews one may expect to see a shift in metabolism with acetyl moieties being shunted into other compounds such as taxol and
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taxanes. An ideal example of this effect was observed in Dreschslera gigantea, a pathogenic fungus, and the dramatic increase in petasol (a sesquiterpenoid) production upon incubation with CCC [14]. The effect of CCC in enhancing taxol production in vitro and in vivo in Pacific yew very much resembles the effect of CCC on terpenoid biosynthesis in D. gigantea [14]. The effect, however, seems to be differential among Taxus spp., i.e. it enhances taxol accumulation in one, but has no effect in another (Tables 6 and 7). Further, the positive effect of Alar on taxol formation in Pacific yew represents an interesting twist in history. Alar had been used as a plant growth regulator in the apple industry and certain unfounded claims had been made about its potential carcinogenic properties [15]. It is ironic that it promotes the formation of taxol (Table 1), an important new anticancer drug [1]. Although several retardants were effective in our assays, it is possible that other compounds exist that are even more potent in promoting taxol accumulation [2]. It also seems that the effect of these retardants in yew species could also be more efficiently and quickly measured in cell suspension cultures, perhaps using other precursors in [tac]taxol biosynthesis [2,3,16] Acknowledgements The authors appreciate the drawing of the yew log provided by Suzan Strobel. Alar was a generous gift of the Uniroyal Chemical Company Inc. The support and help of the people of the U.S. Forest Service, Hungry Horse District, Flathead National Forest in acknowledged. The Yellow Bay Laboratory of the University of Montana has generously provided accommodations during our trips to Northern Montana. Taxol and other taxanes are a gift of the NC1. Some taxol and taxane analyses in this report were independently done by Hauser Chemical Company, Boulder, CO, compliments of Dr David Bailey. Financial support for this project has come from the Montana Science and Technology Alliance, the Montana Agricultural Experiment Station, and the National Cancer Institute, and Cytoclonal Company, Dallas, Texas.
References [1] H. Hartzell, The Yew Tree, Hulogosi Press, Eugene OR, 1991. [21 G.A. Strobel, A. Stierle and F.J.G.M. Van Kuijk, Factor influencing the in vitro production of radio labeled taxol by Pacific yew, Taxus brevifolia. Plant Sci., 84 (1992) 65-74. [3] G.A. Strobel, A. Stierle and W.M. Hess, Taxol formation in yew Taxus. Plant Sci., 92 (1993) 1-12. [41 P.B. Schiff, J. Fant and S.B. Horowitz, Promotion of microtubule assembly in vitro by taxol. Nature, 277 (1979) 665-667. [5] P.B. Schiff and S.B. Horowitz, Taxol stabilizes microtubles in mouse fibroblast cells. Proc. Natl. Acad. Sci. USA, 77 (1980) 1561-1565. [6] M.C. Wani, H.L. Taylor, M.E. Wall, P. Coggon and A.T. McPhail, Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus breviJblia. J. Am. Chem. Soc., 93 (1971) 2325-2327. [7] D.G.I. Kingston, The chemistry of taxol. Pharmacol. Ther., 52 (1991) 1-34. [8] J.H. Cardellina, HPLC separation of taxol and cephalomannine. J. Liquid Chromatogr., 14 (1991) 659-665. [9] N.C. Wheeler, K. Jech, S. Masters, S. Brobst, A.B. Alvarado and A.J. Hoover, Effects of genetic, epigenetic and environmental factors on taxol content in Taxus brevifolia and related species. J. Nat. Prod., 55 (1992) 432-440. [10] R.V. Upadhyay, G.A. Strobel and W.M. Hess, Morphogenesis and ultrastructure of the conidiomata of Ascochyta cypericola. Mycol. Res., 95 (1991) 785-791. [11] A. Stierle, G. Strobel and D. Stierle, Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Science, 260 (1993) 214-216. [12] A. Lang, Gibberellins: structure and metabolism. Annu. Rev. Plant Physiol., 21 (1970) 537-570. [131 J. Beutler, G.N. Churny, B.D. Hillon, S. Brobst, S.A. Look and K. Witherup, Proton 13c NMR assignments for taxol, 7-epitaxol and cephalomannine. J. Nat. Prod., 55 (1992) 414-423. [14] G.J. Bunkers, D. Kenfield and G.A. Strobel, Production of petasol by Drechslera gigantea in liquid culture. Mycol. Res., 95 (1991) 347-351. [15] M. Fumento, The politics of career testing. Am. Spectator., (1990) 18-23. [16] P.E. Fleming, U. Moak and H.G. Hess, Biosynthesis of taxoids. Mode of formation of the taxol side chain. J. Am. Chem. Soc., 115 (1993) 805-807.
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12~
Fig. 4. Scanning electron micrographs of the surfaces of the leaves of T. hrevi/bha and l~ media c~. Hicksii. (At The upper surface of 72 brevifolia, (B) the lower surface of T. hrevi/olia, (C) a stomate with subsidiary cells of T. hrev(Iblia, and (Dt the lower leaf surface of T. media cv. Hicksii.