428
Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
Tab. 3
Growth and Taxol
accumulation in cells
and in medium of suspensions of
T. cuspidata
a
.
Time
Fresh weight
Taxol
accumulated
b
[days]
[g mL
1
]
In cells
[
µ
gg
1
EDW]
In medium
[
µ
gmL
1
]
0
0.15
4.0
0.013
6
0.20
nd
0.356
12
0.24
2.0
0.060
18
0.32
nd
bd
25
0.35
0.4
bd
32
0.39
1.2
bd
38
0.28
2.8
0.100
44
0.27
3.2
0.050
a
Values are the mean of two to three
determinations; inoculi were 1/6 w/v obtained
from a single stationary phase
donor suspension.
b
EDW, estimated dry weight; nd, not
determined; bd, below limit of detection.
Taxanes such as 10-deacetyl-baccatin III
and baccatin III can be used to pro-
duce Taxol
through semisynthesis. These
taxanes
can
also
be
used
to
produce
Taxotere
, a semisynthetic Taxol
ana-
log with higher antineoplastic properties
and higher water solubility than Taxol
;
Taxotere
was frst developed by P. Potier
and associates and Rhˆone-Poulenc Rorer
in France.
Media collected ±rom six-month-old im-
mobilized cell suspensions o±
T. cuspidata
and extracted with dichloromethane may
contain trace amounts o± Taxol
and bac-
catin III as indicated by HPLC analysis. In
di±±erentiated physiologically active cells,
Taxol
may be stored inside the cells, pos-
sibly in the vacuole or other membranous
compartment. In a detailed time course
study using the FCL1F cell suspension
line o±
T. cuspidata
, the analysis o± the
volumetric total production o± Taxol
at
stationary phase indicated that 66% o± the
total Taxol
was in the medium. In con-
trast, at a given time, Taxol
accumulation
in the medium on a per-milliliter basis
was generally a small portion o± that con-
tained per gram o± extracted dry weight
basis in the cells (Table 3). The occurrence
o± a larger extracellular component o± the
total volumetric Taxol
at stationary phase
(productive phase) may be the result o±
excretion by the cells that reach highest
amounts o± Taxol
accumulation in this
phase. Studies on the production o± various
secondary products by plant cell cultures
show that the proportion o± excreted to en-
dogenously accumulated product can be
highly variable depending on species and
cell line.
Taxol
accumulation in the medium o±
cell suspensions o±
T. cuspidata
can vary
considerably with the time o± the growth
cycle. In the kinetic study just mentioned,
the accumulation o± Taxol
in the medium
had two peaks (Table 3). The frst peak o±
Taxol
in the medium (
0
.
4
µ
gmL
1
)
occurred
in
the
early
portion
the
exponential
growth
phase,
soon
a±ter
inoculation in ±resh medium. This early
release o± Taxol
in the medium could be a
response to the trans±er into ±resh medium
(‘‘trans±er e±±ect’’) and/or to the increase
o± the internal pH o± the cells due to the
absorption o± ammonium (exchanged with
H
), which takes place in this portion o±
the growth cycle. Following the frst peak,
Taxol
was degraded, possibly by enzymes
released into the medium. A second peak
o± Taxol
in the medium (0
.
1
µ
gmL
1
)
occurred at stationary phase and may have
resulted ±rom excretion by the cells that
reach their highest level o± Taxol
at
this stage and/or ±rom a small degree o±
cell lysis, common at later stages o± the
growth cycle. Baccatin III accumulation in
the medium peaked a±ter approximately
nine days o± culture at 6
.
2
µ
gmL
1
in the
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