Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
431
production was obtained; whereas a re-
duction in the yield of the main taxane
in this species, taxuyunnanine C, was
observed. These results suggested that dif-
ferent branches of a metabolic pathway
might have different optimal operating
temperature conditions, and the temper-
ature shifting strategy must be tailored to
the speciFc target compound.
6.4
Elicitation and Metabolic Inhibitors
Elicitation is the stimulation of secondary
product formation by speciFc compounds
or physical conditions often mimicking
environmental stresses. It has been suc-
cessfully applied to improve Taxol
yields
in cell cultures. Jasmonate, a lipid-derived
hormone, known as a signal molecule for
herbivore and pathogen defense responses
in plants, and related compounds have
been used to induce taxane overproduc-
tion in
T. media, T. baccata and T. brevifolia
cell suspensions. Methyl jasmonate (MJ)
added at 100
µ
M in medium at the time of
cell inoculation has promoted Taxol
and
baccatin III accumulation to various ex-
tents (e.g. Taxol
from 0.120 to 0.606% dw
in
T. media
and 0.002 to 0.229% dw in
T. baccata
), with only moderate decreases
in cell yield. Optimal conFguration of MJ
stereoisomers has been investigated to
maximize taxane induction and minimize
cell growth inhibition. A crude fungal ex-
tract of
Fusarium oxysporum
caused apop-
tosis in cells of
T. chinensis
var.
mairei
,as
detected by microscopic and DNA analy-
ses; Taxol
yields tripled in treated cells.
Abiotic
elicitors
such
as
the
heavy
metals silver, cobalt, cerium, and lantha-
nium have also been used to stimulate
taxane production in cell cultures. En-
hanced Taxol
production and release in
T. chinensis
cell cultures was obtained by
a combination of ultrasound (US) treat-
ment, MJ exposure and
in situ
solvent
extraction (two-phase culture, see below).
Ultrasound applied for 2 min once or
twice during a four-week culture period
increased volumetric Taxol
yield 1.5- to
1.8-fold; MJ at 60 to 120
µ
Mresu
ltedin5
-
fold increase, and
in situ
solvent extraction,
7- to 9-fold. The most effective combined
approach was the application of US or
MJ, followed by
in situ
solvent extraction,
affording 33- to 35 mgL
1
of Taxol
,a
17-fold increase over control levels.
Suspension cultures are generally het-
erogeneous in the cell cycle phase of in-
dividual cells, which contribute to variable
and relatively low magnitude production
responses upon elicitation. Stable and op-
timized Taxol
yields have been achieved
in
T. chinensis
cultures by synchronization
induced with phosphate starvation and low
temperature treatment. Phosphate star-
vation was induced by transferring 15d
old cells to phosphate-free medium for
25 days, followed by a second transfer
to phosphate-free medium for additional
25 days. Low temperature treatment in-
volved two cycles of 72 h exposure to 4
C,
each followed by transfer to 24
C for 24 h.
Synchronized cultures, particularly by low
temperature treatment, doubled Taxol
yields in response to elicitation with MJ
relative to asynchronous cultures elicited
in the same way.
Metabolic inhibitors have been exam-
ined as a means to divert resources from
competing branch pathways towards the
main pathway leading to the compound of
interest. Since phenylalanine acts as a pre-
cursor to the phenylisoserine side chain
of Taxol
and the benzoyl moiety at C-2,
attempts were made to inhibit the phenyl-
propanoid pathway at the level of pheny-
lalanine ammonia lyase (PAL), expecting
an increase in phenylalanine availability
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