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.
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.
TABLE 1.
Effect of treatment with paclitaxel on DNA synthesis by HFF
| Expt no. | Time in culture | Paclitaxel dose (μM) | DNA synthesis (cpm)a
|
P value | |
|---|---|---|---|---|---|
| Without paclitaxelb | With paclitaxel | ||||
| 1 | 24 h | 7,987 ± 278 | |||
| 1 | 8,071 ± 790 | 7,158 ± 992 | 0.24c | ||
| 5 | 9,865 ± 150 | 7,459 ± 381 | 0.12c | ||
| 10 | 10,166 ± 739 | 7,232 ± 1,088 | 0.31c | ||
| 2 | 24 h | 2,689 ± 190 | |||
| 1 | 2,881 ± 589 | 3,038 ± 356 | 0.21c | ||
| 5 | 2,468 ± 183 | 3,044 ± 526 | 0.33c | ||
| 10 | 2,665 ± 566 | 3,115 ± 414 | 0.18c | ||
| 3 | 8 days | 2,379 ± 366 | |||
| 0.25 | 1,687 ± 582 | 0.16d | |||
| 0.5 | 1,969 ± 447 | 0.29d | |||
| 1 | 2,314 ± 866 | 0.91d | |||
Demonstrated by uptake of [3H]thymidine.
Where no paclitaxel dose is given, results are for HFF cultured in medium alone. Where a paclitaxel dose is given, the results are for HFF cultured in medium with the same amount of diluent as used with the paclitaxel.
P value for comparison of results with cultures of HFF treated with paclitaxel and HFF cultured with the corresponding amount of diluent in medium.
P value for comparison of results with cultures of HFF treated with paclitaxel and HFF cultured with medium.
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.
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.
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.
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.
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).
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.
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.
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