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Antimicrobial Agents and Chemotherapy, August 1998, p. 2036-2040, Vol. 42, No. 8
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Paclitaxel Arrests Growth of Intracellular
Toxoplasma gondii
Randee
Estes,1,2
Nicolas
Vogel,1,3
Douglas
Mack,1,2 and
Rima
McLeod1,2,4,5,6,*
Department of Medicine, Michael Reese
Hospital, Chicago, Illinois 606161;
Departments of Medicine,4
Ophthalmology and Visual Sciences,2 and
Pathology5 and
Committee on
Immunology,6 The University of Chicago,
Chicago, Illinois 60637; and
Department of Medicine,
University of Illinois at Chicago, Chicago, Illinois
606803
Received 16 January 1998/Returned for modification 10 April
1998/Accepted 3 June 1998
 |
ABSTRACT |
Addition of paclitaxel (Taxol) at a concentration of 1 µM to
Toxoplasma gondii-infected human foreskin
fibroblasts arrested parasite multiplication. Division of the
T. gondii tachyzoite nucleus was inhibited, leading to
syncytium-like parasite structures within the fibroblasts by 24 h
after infection and treatment of the cultures. By 4 days after
infection and treatment of the cultures with paclitaxel, this
inhibition was irreversible, since the arrested intracellular form was
incapable of leaving the host cell, infecting new cells, and initiating
the growth of tachyzoites with normal morphology. Specifically,
when paclitaxel was added to infected cells for 4 days and then removed
by washing and the infected, paclitaxel-treated cells were cultured for
4 more days, there were no remaining T. gondii organisms
with normal morphology. Syncytium-like structures in the cultures that
were infected and treated with paclitaxel for 8 days were similar in
appearance to those in preparations of infected paclitaxel-treated
fibroblasts that had been cultured for 24 to 48 h. Pretreatment of
the tachyzoites for 1 h with paclitaxel followed by the
removal of the paclitaxel by repeatedly centrifuging and resuspending
the parasites in fresh medium without paclitaxel and then adding fresh
medium prior to culture of the parasites with fibroblasts did not
prevent their invasion of fibroblasts but did affect their subsequent
ability to replicate within fibroblasts. Pretreatment of the
fibroblasts with paclitaxel also diminished subsequent replication of
T. gondii in such host cells after 8 days. Thus, paclitaxel
alters the ability of T. gondii to replicate in host cells.
Inhibition of parasite microtubules by such compounds at concentrations
which do not interfere with the function of host cell microtubules may
be useful for development of novel medicines to treat T. gondii infections in the future.
| |
INTRODUCTION |
Paclitaxel (Taxol) is a diterpene
plant product derived from the western yew Taxus brevifolia
(15). It induces tubulin polymerization, resulting in the
formation of unstable and nonfunctional microtubules (10,
11), has antineoplastic properties (10), is used to treat certain human malignancies (4), and has been
found to inhibit the growth of Plasmodium falciparum
(7), which, like Toxoplasma gondii, is an
apicomplexan parasite. The report of the inhibitory effect of
paclitaxel on P. falciparum (7), its approval by
the Food and Drug Administration for the treatment of various
human malignancies, the "plant-like" properties of protozoal
microtubules (14) and calmodulin (8), and
inhibition of protozoal replication by herbicides which are inhibitors
of plant microtubules (1, 2, 14) provided the basis for the studies described here of the effect of paclitaxel on T. gondii in vitro.
| |
MATERIALS AND METHODS |
Host cells.
Human foreskin fibroblasts (HFF) (Viromed
Laboratories, Inc., Minneapolis, Minn.) were cultured in four-chamber
Lab Tek tissue culture chamber slides (Miles Laboratories, Naperville,
Ill.) or in 96-well flat-bottom tissue culture plates (Sarstedt, Inc., Newton, N.C.). They were cultured in Dulbecco's modified Eagle medium
(DMEM) (Gibco, Grand Island, N.Y.) that contained 10% heat-inactivated (60 min, 56°C) fetal calf serum (Hyclone Laboratories, Logan, Utah),
100 U of penicillin/ml, 100 µg of streptomycin/ml, 0.25 µg of
Fungizone (Gibco)/ml, and 0.292 mg of L-glutamine
(Gibco)/ml (DMEM-FCS). The fibroblasts were incubated at 37°C in 5%
CO2. After the monolayers reached confluence they were
maintained at 33°C in 5% CO2. When cultures were
maintained for only 24 h, they were incubated at 37°C, and when
cultures were maintained for 8 days, they were incubated at 33°C, in
5% CO2 in both cases.
Parasites.
Tachyzoites of the RH strain of T. gondii were used to challenge fibroblasts in the presence or
absence of paclitaxel. They were obtained from T. gondii organisms continuously passaged in confluent fibroblast
monolayers in 24-well cell culture plates (Costar, Cambridge, Mass.).
The challenge ratio was one T. gondii tachyzoite to
one fibroblast. Pretreatment of the tachyzoites with paclitaxel was
performed in a 15-ml conical tube in a 37°C incubator with 5%
CO2 for 1 h.
Paclitaxel.
Paclitaxel was obtained from Sigma Chemical Co.
(St. Louis, Mo.). It was dissolved in dimethyl sulfoxide (DMSO) at a
concentration of 5 µg/ml and stored in 50-µl aliquots at 
70°C.
Just before use, the paclitaxel was diluted 1:5 in ethanol, and final
dilutions were made in DMEM-FCS. Control wells contained media with
diluent (DMSO-ethanol) equivalent to the amount present in the highest concentration of paclitaxel used in each experiment. Concentrations of
paclitaxel varied between experiments and ranged from 0.25 to 10 µg/ml. Paclitaxel was added to fibroblasts 1 h prior to challenge with T. gondii tachyzoites or 1 h after challenge. In some experiments the paclitaxel was removed by
washing after 1 h, and in some experiments the paclitaxel remained
in culture for the duration of the experiment.
Pyrimethamine and sulfadiazine.
Pyrimethamine and
sulfadiazine were obtained from Sigma Chemical Co. Prior to use,
pyrimethamine and sulfadiazine were diluted and mixed together to
attain the final concentrations, as previously described
(3).
Toxicity.
To determine the highest concentration of
paclitaxel that was not toxic to the monolayer, fibroblasts were
cultured at varying concentrations in 96-well flat-bottom microtiter
plates and allowed to adhere for 24 h. Paclitaxel was added for
24 h or 8 days. For the last 18 h of culture 1.25 µCi of
[3H]thymidine (Amersham) was added to each well. Before
being processed, the plates were viewed on an inverted-phase microscope
to assure that the monolayer was preserved as well as not confluent.
Cells were collected with a PHD cell harvester (Cambridge Technology Inc.) and processed as previously described (3).
At 3, 24, and 48 h and 8 days, when the cultures in Lab Tek tissue
culture chamber slides were processed, the monolayers were washed three
times with DMEM-FCS, stained with Giemsa stain as previously described
(6), and evaluated by light microscopy. Relative
densities were noted, as well as whether the appearance of the
fibroblasts was altered due to treatment with paclitaxel. Treated
HFF cultures were compared with untreated HFF cultures.
Assessment of outcome of infection with and without
paclitaxel.
Replication of the parasites was assessed by
measurement of [3H]uracil uptake as previously described
(3). Briefly, confluent monolayers were challenged 1:1 with
the RH strain of T. gondii for 1 h and then
treated with paclitaxel at various concentrations. [3H]uracil (2.5 µCi; Amersham) was added to each well
for the last 18 h of culture. The cells were harvested with a PHD
cell harvester and counted with a liquid scintillation
spectrophotometer.
At 3, 24, and 48 h and 8 days, when the cultures in Lab Tek tissue
culture chamber slides were processed, the monolayers were
washed three
times with DMEM-FCS, stained with Giemsa stain as
previously described
(
6), and evaluated by light microscopy.
Relative densities,
the percentage of cells that were infected,
and the appearance of the
organism and parasitophorous vacuoles
were observed and compared to
those of untreated control cultures.
Statistics.
The significance of differences was determined
by Student's t test. P values of 
0.05
were considered significant. Data which are shown are representative of
three replicate experiments.
| |
RESULTS |
Effect of treatment with paclitaxel on DNA synthesis by HFF.
Paclitaxel did not alter the growth of fibroblasts at concentrations
between 0.25 and 10 µM, as demonstrated by uptake of [3H]thymidine (Table 1)
(P > 0.05) by nonconfluent human fibroblast monolayers. Inspection of such monolayers by light microscopy with an
inverted-phase microscope showed no change in the relative densities of
fibroblast cultures due to culture with paclitaxel.
Effect of paclitaxel on intracellular T. gondii.
[3H]uracil uptake by tachyzoites of the RH strain of
T. gondii within HFF was diminished significantly
(P < 0.05) by treatment with paclitaxel. Specifically,
when 1 µM paclitaxel was added to HFF cultures 1 h after
challenge and the cultures were processed after 24 h,
[3H]uracil uptake was reduced from 10,306 ± 303 to
2,691 ± 664 cpm (Fig. 1a)
(P < 0.05). When 1 µM paclitaxel was added 1 h
after challenge and the cultures were maintained with 1 µM paclitaxel for 4 days, washed, and incubated for 4 more days in paclitaxel-free medium, [3H]uracil uptake was reduced from 37,691 ± 7,967 to 1,557 ± 986 cpm (Fig. 1b) (P < 0.05).
Control cultures were confluent fibroblasts incubated with medium plus
diluent (DMSO-ethanol) and T. gondii for either 24 h or 8 days. Challenged control cultures and challenged paclitaxel-treated cultures were also compared by light microscopy. Control monolayers were also washed free of diluent to precisely replicate the conditions of culture with paclitaxel. Control
cultures that had not been treated with paclitaxel had 29% infected
HFF cells (Fig. 2A), and cultures in
which paclitaxel had been present for 24 h (Fig. 2B) had 31%
infected HFF cells. Tachyzoites in these paclitaxel-treated
cultures were unable to replicate normally within the
parasitophorous vacuole, with resultant intracellular syncytium-like structures (Fig. 2B and
3). Cultures in which paclitaxel was
removed by washing after 1 h had 32% infected HFF cells, and the
parasites were replicating at 24 h (not shown). When these same
cultures were analyzed at 48 h, the challenged control
cultures showed multiply infected HFF cells (Fig. 2C) and
many free tachyzoites compared with paclitaxel-treated
cultures that had been washed after 1 h of exposure to paclitaxel.
These latter cultures had syncytium-like intracellular
structures and showed arrest of parasite multiplication (Fig.
2D). The control cultures that were maintained for 8 days (Fig. 2E) had
many free and intracellular tachyzoites with normal
appearance and showed destruction of the monolayer due to
parasite replication, whereas the paclitaxel-treated culture (Fig. 2F) had complete inhibition of T. gondii
replication and showed the formation of syncytium-like
intracellular structures and preservation of the
monolayer.
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FIG. 1.
Effect of paclitaxel on intracellular parasites as shown
by [3H]uracil uptake by tachyzoites of the RH strain
of T. gondii in HFF. The data are means ± standard deviations for six replicate wells. The open bars represent
control wells, and the solid bars represent paclitaxel-treated wells.
(a) Paclitaxel remained in cultures for 1 day. (b) Paclitaxel remained
in cultures for 4 days and then was removed by washing, and cultures
were analyzed after 8 days. (c) HFF were pretreated with paclitaxel for
1 h prior to challenge, and cultures were analyzed after 8 days of
culture. (d) Tachyzoites were pretreated with paclitaxel for 1 h,
washed, and added to HFF, and cultures were analyzed after 8 days of
culture. (e) Pyrimethamine (0.1 µg/ml) and sulfadiazine (25 µg/ml)
remained for the 8 days of culture.
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FIG. 2.
Comparison of infected HFF monolayers treated with
paclitaxel for various times (magnification, ×420). The arrows
indicate parasites (A, C, and E) and syncytia (B, D, and F). (A)
Control T. gondii-infected HFF after 24 h in
culture. (B) T. gondii-infected HFF treated with 1 µM
paclitaxel for 24 h after infection. (C) Control T. gondii-infected HFF after 48 h in culture. (D) T. gondii-infected HFF treated with 1 µM paclitaxel for 48 h
after infection. (E) Control T. gondii-infected HFF
after 8 days in culture. (F) T. gondii-infected HFF
treated with 1 µM paclitaxel for 4 days and then washed thoroughly to
remove the paclitaxel and cultured for 4 more days.
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FIG. 3.
Light micrographs under the same conditions as for Fig.
2 but at higher magnification (×840) demonstrating syncytium-like
structures caused by paclitaxel treatment of T. gondii-infected cultures. (A) Control untreated culture with
replicating T. gondii indicated by the arrow. (B)
Cultures treated with paclitaxel. The arrow marks the syncytium-like
remnant of a T. gondii organism. N, nucleus.
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|
Effect of preincubation of T. gondii with
paclitaxel.
To determine the effect of pretreatment of
extracellular tachyzoites with paclitaxel, tachyzoites of
the RH strain of T. gondii were incubated in a 15-ml
conical tube in DMEM-FCS with 1 µM paclitaxel or with medium alone
(sham) at 37°C with 5% CO2. After 1 h they were
washed three times with DMEM-FCS and then added to the confluent fibroblasts. Such pretreatment of tachyzoites with paclitaxel 3 h after infection did not affect their morphology or their
ability to invade fibroblasts (Fig. 4A)
when analyzed at 3 h. After 24 h they had divided and formed
parasitophorous vacuoles (Fig. 4B), but by 8 days their uptake of
[3H]uracil was significantly reduced, from 22,452 ± 9,922 to 5,938 ± 3,339 cpm (Fig. 1d) (P < 0.05).
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FIG. 4.
Comparison of results of pretreatment of HFF and of
tachyzoites with paclitaxel. (A) HFF infected with pretreated
extracellular tachyzoites after 3 h in culture. (B) HFF
infected with pretreated extracellular tachyzoites after
24 h in culture. (C) HFF pretreated with 1 µM paclitaxel
prior to challenge with T. gondii after 24 h in
culture. The arrows indicate parasites. Magnification, ×420.
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|
Effect of preincubation of HFF with paclitaxel.
Confluent fibroblast monolayers were incubated with 1 µM paclitaxel
for 1 h and washed three times with DMEM-FCS prior to challenge
with T. gondii. The percentages of infected cells in untreated and treated cultures 24 h after infection were 29 and 33%, respectively. There was no effect on the morphology of
intracellular T. gondii tachyzoites at 24 h
(Fig. 4C), but there was significant reduction of
[3H]uracil uptake (from 39,039 ± 3,268 to
7,009 ± 1,348 cpm [Fig. 1c] [P < 0.05]) at 8 days. Fibroblasts alone do not take up [3H]uracil, and
such fibroblasts were included in every experiment (data not shown).
| |
DISCUSSION |
Microtubules provide support and structure to the
cytoskeletons of T. gondii tachyzoites and HFF host
cells (12). They are involved in cell division, form
essential components of certain cell organelles, such as centrioles and
spindle fibers, and affect glucose uptake. Paclitaxel has been shown to
induce tubulin polymerization, resulting in the formation of abnormally
stable and nonfunctional microtubules (12). Paclitaxel was
found to inhibit the growth of the apicomplexan parasite P. falciparum (4, 5), and compounds that inhibit plant
microtubules were found to inhibit Leishmania and
T. gondii without adversely affecting the mammalian
host cells (1, 14). These studies suggested that paclitaxel
might also have an effect on T. gondii microtubules at
concentrations which would not adversely effect the mammalian host
cells of this obligate intracellular parasite. Pharmacological studies
of humans show maximum concentrations in plasma following a 6-h
infusion of paclitaxel at a dose of 210 to 250 mg per m2 to
be 3.1 to 4.1 µM (9).
When 1 µM of paclitaxel was added to T. gondii-infected fibroblasts for either 24 or 48 h or for the
first 4 days of an 8-day culture period, the ability of the
intracellular parasites to replicate was markedly inhibited. The effect
appeared to be irreversible, as the removal of paclitaxel after 4 days
of culture did not alter the effect on the inhibited T. gondii tachyzoites 4 days later.
Pretreatment of T. gondii tachyzoites with
paclitaxel for 1 h also inhibited their subsequent ability to
replicate, but to a lesser degree than when paclitaxel was
present throughout the experiment. This pretreatment did not
block the invasion of fibroblasts by the parasites or the ability of
the parasites to form parasitophorous vacuoles. Although paclitaxel did
not affect T. gondii's ability to invade the
host cell, it did impair the parasite's ability to replicate during
the following 8 days in culture.
To better understand when paclitaxel affected T. gondii
tachyzoites, Giemsa-stained slides were compared at 3, 24, and
48 h and 8 days after infection. At the early time points,
when paclitaxel was removed, the organisms were able to invade and
replicate, while if paclitaxel remained in culture, the organisms were
able to invade but replication was impaired. When these cultures were maintained for 8 days, the ability of the parasites to replicate was
severely impaired and the parasites formed the syncytium-like structures shown in Fig. 2F.
The most likely explanation for the inhibitory effect of paclitaxel on
T. gondii is that its effect on the microtubules of the
parasite renders the parasite incapable of forming the structures it
needs for replication and/or glucose uptake. Pretreatment of the
fibroblasts with paclitaxel does not preclude invasion of host cells by
T. gondii or multiplication during the initial 24 to
48 h after infection. However, between 48 h and 8 days after infection of host cells, such parasites can no longer multiply, which
results in the formation of syncytium-like structures by the parasites.
This suggests that either there is cell-associated paclitaxel remaining
on the host cell surface which interacts with the parasites; paclitaxel
is taken up by the host cell and slowly released to affect the
microtubules of the intracellular parasites so that they cannot divide;
or microtubules of the host cells are affected so that the parasites
cannot continue to make or maintain their parasitophorous vacuoles or
access essential nutrients from the host cells.
Pretreatment of T. gondii with paclitaxel has the same
effect; when pretreated tachyzoites are placed on untreated
fibroblasts, they are able to invade and multiply for 24 to 48 h,
but by 8 days their growth is arrested. The microtubule functions of
both the host cells and the parasites are affected by paclitaxel,
because when paclitaxel is washed away and the tachyzoites are
given the chance to multiply in previously paclitaxel-treated HFF, they cannot do so after 24 to 48 h (Fig. 4B). Similarly, when
host cells are pretreated with paclitaxel, tachyzoites infect HFF
and begin to replicate in 24 to 48 h but by 8 days they no longer replicate (Fig. 1d). Explanations for these data include the following. (i) The host cells retain paclitaxel, despite washing, and it is slowly
released and is detrimental to the parasitophorous vacuole. (ii)
Another possible explanation is that paclitaxel might coat the surface
of the host cell and damage the parasite as it enters in such a
way that over time the parasite cannot continue normal replication.
We have observed that paclitaxel, which has been approved to
treat certain human malignancies, alters the ability of T. gondii to replicate in host cells. This presents the
possibility that paclitaxel (or related inhibitors of parasite
microtubules) might be useful for the development of novel treatments
for T. gondii infections.
| |
ACKNOWLEDGMENTS |
This work was supported by National Institutes of Health
grants AI 16945, AI 27530, and F32 AI08749. Rima McLeod is the Jules and Doris Stein Research to Prevent Blindness (RPB) Professor, and
Douglas Mack is the recipient of an RPB Career Development Award.
Career Development Award.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Ophthalmology, Visual Science Center, University of Chicago, 939 East 57th St. (MC 2114), Chicago, IL, 60637. Phone: (773) 834-4152. Fax:
(773) 834-3577. E-mail: rmcleod{at}midway.uchicago.edu.
| |
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Antimicrobial Agents and Chemotherapy, August 1998, p. 2036-2040, Vol. 42, No. 8
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
