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Endocrinology Vol. 145, No. 7 3143-3152
Copyright © 2004 by The Endocrine Society

Molecular Mechanisms of the Effects of Low Concentrations of Taxol in Anaplastic Thyroid Cancer Cells

V. M. Pushkarev, D. V. Starenki, V. A. Saenko, H. Namba, J. Kurebayashi, M. D. Tronko and S. Yamashita

Nagasaki University Graduate School of Biomedical Sciences (V.M.P., D.V.S., V.A.S., H.N., S.Y.), 852-8523 Nagasaki, Japan; Institute of Endocrinology and Metabolism of Academy of Medical Sciences of Ukraine (V.M.P., M.D.T.), 04114 Kiev, Ukraine; and Kawasaki Medical School (J.K.), 701-0192, Okayama, Japan

Address all correspondence and requests for reprints to: Shunichi Yamashita, M.D., Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. E-mail: shun{at}net.nagasaki-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Understanding the detailed mechanisms of a chemotherapeutic agent action on cancer cells is essential for planning the clinical applications because drug effects are often tissue and cell type specific. This study set out to elucidate the molecular pathways of Taxol effects in human anaplastic thyroid cancer cells using as an experimental model four cell lines, ARO, KTC-2, KTC-3 (anaplastic thyroid cancer), and FRO (undifferentiated follicular cancer), and primary thyrocytes. All cell lines were sensitive to Taxol, although to different extent. In primary thyrocytes the drug displayed substantially lower cytotoxicity. In thyroid cancer cells, Taxol-induced changes characteristic to apoptosis such as poly (ADP-ribose) polymerase and procaspase cleavage and alteration of membrane asymmetry only within a narrow concentration range, from 6 to 50 nM. At higher concentration, other form(s) of cell death perhaps associated with mitochondrial collapse was observed. Low doses of Taxol enhanced Bcl2 phosphorylation and led to its degradation observed on the background of a sustained or increasing Bax level and accumulation of survivin and X-chromosome-linked inhibitor of apoptosis. c-jun-NH2 terminal kinase activation was essential for the apoptosis in anaplastic thyroid cancer cells, whereas Raf/MAPK kinase/ERK and phosphatidylinositol-3-OH kinase/Akt were likely to comprise main survival mechanisms. Our results suggest an importance of cautious interpreting of biological effects of Taxol in laboratory studies and for determining optimal doses of Taxol to achieve the desired therapeutic effect in anaplastic thyroid cancers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TAXOL HAS PROVED one of the most potent anticancer drugs introduced into medical practice during last several decades. It has been relatively successfully used for treatment of certain types of human malignancies such as ovarian, breast, lung, and head and neck cancer (1). Trials have been performed to expand the list of tumor diseases that can be treated with Taxol, including human thyroid cancers (2, 3, 4). In the latter, anaplastic thyroid cancer accounts for about 1% of all malignancies of this organ and comprises one of the most aggressive human cancer with extremely poor prognosis (5). So far there is an unsolved task of a search of new approaches to the disease treatment as well as that of thorough evaluation of the results of performed clinical attempts and data on drug effects.

Understanding the molecular mechanisms of a chemotherapeutic agent action in cancer and normal cells is indispensable for planning the evidence based clinical applications. Taxol is generally accepted to induce cell cycle arrest and apoptosis in most types of cancer cells. As a result of cell exposure to Taxol, events characteristic to apoptosis may take place: alteration of membrane asymmetry, caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage (6). Along with this, mechanisms of Taxol effect on a cell is concentration dependent, and molecular events may be distinct at different drug concentrations (7, 8).

Molecular pathways of Taxol-induced cytotoxicity in different cells are far from being understood. There are reports implicating MAPK cascades, first of all the c-jun-NH2 terminal kinase (JNK) pathway, as a major mediator of the programmed cell death evoked by the drug (9, 10). In some tumor cells, however, JNK signals have been shown not to play a pronounced role, conceding the cell-killing effects of Taxol to p38 and ERK (11, 12), and role of MAPK has even been denied (13, 14). Thus, it is likely that a central role for MAPKs as key factors in Taxol effects is tissue and/or cell line specific.

Information about precise mechanisms of Taxol action in anaplastic thyroid cancer (ATC) cells is quite limited to date. We therefore attempted, using the annexin V staining and monitoring procaspase and PARP cleavage during the course of progressing cell death to determine whether it is possible to induce bona fide apoptosis in ATC cells and what concentration range of Taxol can cause it. Besides the above parameters, we evaluated changes in a number of pro- and antiapoptotic factors such as MAPK family members, Bax, X-chromosome-linked inhibitor of apoptosis (XIAP), survivin, and Bcl2, and characterized signaling cascades underlying cytotoxic effects of Taxol in various ATC cell lines. Because, along with proapoptotic pathways, Taxol can affect the survival signaling, e.g. ERK and phosphatidylinositol-3-OH kinase (PI3K)/Akt, and activate nuclear factor {kappa}B (NF{kappa}B) leading to the transactivation of some antiapoptotic genes (15), we also examined their relevance to the Taxol-induced cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and conditions of culturing
Human ATC cell line ARO and follicular undifferentiated carcinoma cells FRO were initially provided by J. A. Fagin (University of Cincinnati College of Medicine, Cincinnati, OH). ATC cell lines KTC-2 and KTC-3 were established at Kawasaki Medical School (Okayama, Japan).

Throughout all experiments cancer cell lines were grown in RPMI 1640 supplemented with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all reagents from Invitrogen Life Technologies, Paisley, UK) in a 5% CO2 humidified atmosphere at 37 C. Primary human thyroid cell culture was established as described previously (16) and maintained in DMEM:F12 (1:2 wt/wt) mixture supplemented with 3% FBS and 1% penicillin/streptomycin. After 2 d incubation, when the culture reached about 80% confluence, cells were washed twice with PBS (pH 7.4) at 37 C, and a fresh medium was added to each dish. Cells were incubated for an additional 24 h, exposed to the drug(s) as described below, and then collected at different time intervals.

Clonogenic assay
Clonogenic assay was performed as described elsewhere (17). Briefly, cells were seeded at 500 cells per 10-cm dish. Twenty-four hours later, fresh medium with Taxol was added. After exposure to the drug for 24 h, medium was changed and cells were grown for 2 wk. Giemsa-stained cell colonies were counted using the Colony V 1.1 software (Fujifilm, Tokyo, Japan).

Cell survival assay
Cultures were established in the 96-well flat-bottom microtiter plates (Nalge Nunc International, Tokyo, Japan) in RPMI 1640 containing 5% FBS. Cell suspensions (100 µl, 1000 cells/well) were added to each well and incubated for 24 h before treatment.

Taxol (Wako Chemicals, Osaka, Japan), MAPK/ERK kinase (MEK)1-inhibitor PD98059 (Cell Signaling Technology, Beverly, MA), JNK inhibitor SP600125, PI3K inhibitor LY294002, and a p38 MAPK inhibitor (Calbiochem, La Jolla, CA) dissolved in dimethylsulfoxide (DMSO) and the control (DMSO only) were added to each well in 10 µl of medium (final concentration of DMSO in the well did not exceed 0.1%) at varying concentrations, six wells for each concentration. After incubation, a water-soluble tetrazolium salt-based assay (WST) was performed as follows: 11 µl of the cell counting kit solution (CCK-8, Dojin, Osaka, Japan) were added to each well and incubated for 1 h at 37 C. OD was read at 450 nm in a microplate reader.

Annexin V/propidium iodide staining
Adherent cells were detached by trypsinization and washed once with warm PBS. 1 x 105 cells were double stained with fluoresceinisothiocyanate-conjugated annexin V and propidium iodide (PI) for 15 min at room temperature in a Ca2+-enriched binding buffer (apoptosis detection kit, Wako Chemicals) and then analyzed on the FACSCalibur flow cytometer (BDIS, Becton Dickinson, San Jose, CA). Annexin V and PI emissions were detected in the FL-1 and FL-2 channels, respectively. For each sample, data from 20,000 cells were acquired in list mode on logarithmic scales. Analysis was performed with the Cell Quest software (BDIS).

Assessment of mitochondrial membrane potential
Changes of the mitochondrial membrane potential were examined using flow cytometry analysis of cells stained with tetramethylrhodamine ethyl ester (TMRE, Molecular Probes, Eugene, OR) (18), a cell-permeable dye accumulating in mitochondria with unaltered membrane potential. Cells were harvested by trypsinization at the end of experimental protocol, and 1 x 105 cells were incubated with 100 ng/ml TMRE for 15 min at room temperature in HEPES-buffered saline (pH 7.4) followed by the analysis with the FACScan (20,000 cells/sample). The fluorescence intensity of TMRE was monitored at 582 nm (FL-2).

Preparation of cell extracts
Adherent cells were washed twice with an ice-cold PBS supplemented with sodium pyrophosphate and orthovanadate, scraped with a rubber policeman, collected in 1 ml PBS, and centrifuged for 3 min at 1000 rpm at 4 C. The pellet was then resuspended in 200 µl of the lysis buffer (Cell Signaling Technology) containing a cocktail of protease and phosphatase inhibitors. After 15 min on ice, lysates were centrifuged for 15 min at 15,000 x g and stored at –80 C until use. Protein concentration was determined with bicinchoninic acid assay reagent kit (Sigma, St. Louis, MO) according to manufacturer’s protocol.

Western blotting
Total cell lysates were boiled in the sample buffer (100 mM Tris-HCl, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, 10% dithiothreitol) and separated by SDS-PAGE 7.5–15% gradient gels (Biocraft, Tokyo, Japan). The homogeneous 8 and 15% gels were used when better separation of high- and low-molecular-weight proteins, respectively, was needed. Forty micrograms of protein were applied per each lane. Proteins were transferred onto 0.2-µm nitrocellulose membranes (Millipore Corp., Bedford, MA) by semidry blotting. Membranes were blocked with Tris-buffered saline/0.1% Tween 20 containing 5% nonfat dry milk and incubated with primary antibodies (Cell Signaling Technology or Santa Cruz Biotechnology, Santa Cruz, CA) as appropriate at 4 C overnight. After washing three times with Tris-buffered saline/0.1% Tween 20, the blots were incubated with horseradish peroxidase-conjugated species-specific secondary antibody (Cell Signaling Technology) for 1 h at room temperature and then again washed three times. Complexes were visualized using the ECL reagents (Amersham, Arlington Heights, IL).

Statistical analysis
All data were expressed as a mean ± SD. Differences between groups were examined for statistical significance using Student’s t test. P < 0.05 denoted the presence of a statistically significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cytotoxic effect of Taxol on thyroid cancer cells
The cell survival assay showed that all studied cells were sensitive to Taxol, but their viability differed among thyroid cancer cell lines. Undifferentiated follicular carcinoma cell line FRO and anaplastic cancer cells KTC-2 displayed higher sensitivity, whereas anaplastic cancer-derived ARO and especially KTC-3 had more resistant phenotype (Fig. 1AGo). The variations of serum content in the culture medium from 1 to 10% did not significantly change the survival rates of ATC cells (data not shown). Primary human thyrocytes exhibited significantly lower sensitivity to the drug as compared with tumor cells. The clonogenic assay showed that the number of colonies in any cell line tested was strongly reduced, even after exposure to 10 nM Taxol, and at the drug concentration above 50 nM, only few (if any) colonies could be recovered after a 2-wk culturing (Fig. 1BGo). Average IC50 and IC90 in the clonogenic assays were around 0.4 and 1.5 nM of Taxol, respectively, among the cell lines.



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  		FIG. 1. Effect of Taxol on cell viability and clonogenic survival. A, Viability of four ATC cell lines and primary thyrocytes (PT) exposed to varying concentrations of Taxol for 24 ({blacksquare}), 48 ({bullet}), and 72 ({blacktriangledown}) h determined using the WST assays in 96-well plates as described in Materials and Methods. B, Clonogenic assays of ARO and FRO cells treated with Taxol. For each concentration of Taxol, three 10-cm petri dishes were analyzed. Data represent mean ± SD value (n = 6 and n = 3 for the WST and clonogenic assays, respectively).

 
Alteration of the cell cycle
Study of the concentration-dependent effects of Taxol on the cell cycle demonstrated general cell accumulation in the G2/M phase and cell death. After a 72-h treatment, the majority of cells were arrested in the G2/M, especially at Taxol concentration 50 nM and higher (Fig. 2AGo). The arrest was observed in all cell lines except for KTC-3, which showed nearly even distribution of cells between G1 and G2/M phases (data not shown). Of note, the sub-G1 events presumably representative of apoptosis were mostly registered at Taxol concentrations around 10 nM. The annexin V staining revealed massive cell apoptosis at 6–25 nM of the drug accompanied with a negligible increase in necrotic subpopulation (Fig. 2BGo). First signs of apoptosis were apparent 12 h after the beginning of Taxol treatment in KTC-2 cells, and maximal effect was evident after 2 d incubation in all cell lines.



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  		FIG. 2. Effect of varying Taxol concentration on the cell cycle and induction of apoptosis in ATC cells. A, ARO cells were incubated with various concentrations of Taxol for 48 h and then fixed with ethanol and stained with PI. The histograms represent the emission detected at the FL-2 channel by flow cytometry. Nearly all cells were arrested in the G2 phase at the concentrations of 50 and 100 nM. A profound sub-G1 area at 10 nM of Taxol represents the apoptotic changes in the treated cells. B, Vital annexinV-fluoresceinisothiocyanate/PI (FL1-H and FL2-H, respectively) staining of KTC-2 cells. Starting from 6 nM Taxol, cells mostly display AnnexinV+/PI– staining suggestive of massive apoptosis. Similar results were obtained in all cell lines tested.

 
Taxol induces procaspase and PARP cleavage
To confirm the programmed cell death in treated cells, we analyzed the cleavage of caspases and PARP, a major substrate of the effector caspase-3 during apoptosis (19). Procaspase activation and PARP cleavage along with DNA laddering and membrane inversion are known to be the most characteristic apoptotic events. The maximal PARP and procaspase-3 cleavage occurred only within a narrow concentration range of Taxol: 10–25 nM in ARO and 10–50 nM in FRO and KTC-2 (Fig. 3AGo). The drug concentration above 50 nM led to the decrease of the caspase-3 activated form level paralleled by the attenuated PARP cleavage. As shown in Fig. 3BGo, in KTC-2 cells Taxol caused the appearance of the caspase-3 active forms (p17 and p19) and PARP cleavage after 12 h of treatment. In more resistant KTC-3 cells, these effects could be observed after 18 h and in ARO after 24 h exposure (data not shown). In all cell lines, maximal PARP cleavage coincided in time with the manifestations of the caspase-3 activation. Besides the caspase-3, which probably was the main effector caspase in the assayed cells, low concentration of Taxol induced activation of other proteolytic enzymes upstream to caspase-3, particularly caspase-9 (Fig. 3CGo). Activation of this enzyme suggests initiation of the mitochondrial apoptotic pathway. In addition, cleaved forms of caspase-6 and -7 were also detected (Fig. 3CGo). Interestingly, only KTC-2 cells displayed manifestations of a profound caspase-8 activation (Fig. 3CGo), a key effector of the death receptor-triggered apoptosis (20). In other cell lines we observed only trace amounts of the cleaved form of this caspase in response to Taxol (data not shown).



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  		FIG. 3. Taxol-induced apoptotic caspase activation and PARP cleavage. A, Procaspase-3 and PARP cleavage in FRO cells in the presence of increasing concentration of Taxol detected after 24 h incubation. The cleaved proteins were mostly detected within 6–50 nM of the drug. B, Accumulation of the cleaved forms of PARP and procaspase-3 in time exemplified on KTC-2 cells treated with 50 nM of Taxol. In A and B, similar results were obtained in all cell lines tested. C, Time-dependent increase of the levels of cleaved forms of procaspase-9, -6, -7, and -8 in KTC-2 cells exposed to 25 nM Taxol. Cleavage of procaspase-9, -6, and -7 was observed in all cell lines, whereas cleavage of procaspase-8 was detected only in KTC-2 (not shown for other cell lines). In A–C, numbers on the right of the blot images indicate the molecular weight of the products. The experiments were repeated at least two times.

 
Apparently Taxol induced classical apoptosis in thyroid cancer cells only when used at relatively low concentration, whereas in the drug excess, other form(s) of cell death may take place. These may be necrosis, mitotic catastrophe slow death, and others (21). Supportive of these observations, results of vital staining with cell-permeable mitochondrial dye demonstrated that large proportions of the organelles had altered potential at 100 nM of Taxol (Fig. 4Go). Perhaps further increase of the drug concentration may lead to mitochondrial collapse and/or necrosis.



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  		FIG. 4. Effect of Taxol concentration on mitochondrial potential. FRO cells were incubated with different doses of Taxol for 24 h and then collected and stained with TMRE for the flow cytometry assay as described under Materials and Methods. The histogram shows the accumulation of cells with decreased mitochondrial potential (indicated with an arrow) paralleled by the declining numbers of cells with retained {Delta}{psi}m with increasing drug concentration.

 
Changes in Bax, XIAP, and survivin levels and Bcl2 phosphorylation
Most chemotherapeutic agents provoke an imbalance between the pro- and antiapoptotic factors acting at different stages of apoptosis. Depending on its capacity to withstand this imbalance, a cell either survives or dies. One of the central roles in determining cell fate belongs to the pro- and antiapoptotic proteins of Bcl2 family.

In all cell lines exposed to Taxol, we observed Bcl2 phosphorylation (Fig. 5AGo). In FRO and KTC-2 cells, which possess higher sensitivity to Taxol, Bcl2 phosphorylation was detected as early as 3 h after exposure to the drug. It is important to note that Bcl2 protein level significantly decreased after 12–24 h of exposure to Taxol in all cell lines. These data suggest that in parallel to Bcl2 phosphorylation, there was a general decrease of the Bcl2 protein level, which possibly may be a consequence of this modification.



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  		FIG. 5. Effects of Taxol on phosphorylation and protein levels of pro- and antiapoptotic factors. A, Changes of Bcl2 phosphorylation and protein level in KTC-2 cells at different time points after treatment with 25 nM Taxol. Note the increase of the phosphorylated form of Bcl2 and decrease of its content. Similar effects were observed in all cell lines tested. B, Taxol-induced accumulation of Bax, survivin, and XIAP in ATC cell lines. Cells were incubated with 25 nM Taxol. Results are representative of least two experiments.

 
Increase of Bax level was found in the three cell lines (data for KTC-3 not shown), whereas in FRO it was stably high (Fig. 5BGo). In all cell lines, Taxol treatment induced the accumulation of the inhibitor of apoptosis family proteins, survivin, and XIAP (Fig. 5BGo) that can bind and inhibit caspase-3 and -7 (22).

Role of MAPK and PI3K in Taxol effects
Taxol induced the phosphorylation of JNK1/2 in all cell lines studied. This modification was most pronounced at Taxol concentration 6–10 nM to 50 nM (Fig. 6AGo) and correlated well with the PARP cleavage (see Fig. 3AGo). The JNK1/2 phosphorylation was noticed after 6 h of exposure to Taxol in ARO or even after 3 h in more susceptible KTC-2 cells (Fig. 6BGo). Phosphorylation of JNK indicates its activation because it was accompanied by the phosphorylation of its downstream targets, transcriptional factors activating transcription factor (ATF)-2 and cJun (Fig. 6Go, A and B). In ARO and KTC-2 cell lines, Taxol enhanced phosphorylation of the MKK4, an upstream kinase to JNK, not observed in FRO cells (Fig. 6CGo). Furthermore, apoptosis signal-regulating kinase (ASK)1, an upstream kinase to MKK4, was also phosphorylated in response to the drug in corresponding cells (Fig. 6CGo).



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  		FIG. 6. Effect of Taxol on the members of MAPK pathways. A, Dose-dependent JNK and its downstream target, ATF-2, phosphorylation on the example of FRO cells after 24 h incubation. B, Prolonged phosphorylation of the JNK and its downstream target, cJun. Cell lysates were collected at different time points of incubation with 25 nM Taxol. Accumulation of phospho-cJun in KTC-2 is shown on the time scale to demonstrate parallelism with PARP and procaspase cleavage in Fig. 3Go, B and C, respectively. In A and B, similar observations were made in all cell lines tested. C, The phosphorylation of MKK4 and ASK1 increased in time in the presence of 25 nM Taxol in ARO and KTC-2 cells but not in FRO. D, ERK1/2 and cRaf-1 phosphorylation in different cell lines.

 
Effects of Taxol on ERK phosphorylation were markedly diverse in various cell lines (Fig. 6DGo). In KTC-2 cells, Taxol induced a sustained phosphorylation of both ERK1 and ERK2. In parallel, the phosphorylation of c-Raf-1, an upstream to ERK kinase, was also increased. Site of c-Raf phosphorylation, Ser 338, corresponded to the RAS pathway. In KTC-3 cells, ERK phosphorylation, especially that of ERK2, as well as was the phosphorylation of c-Raf-1was slightly decreased. In ARO cells we detected dephosphorylation of ERK at 36 h of cell exposure to Taxol when maximal caspase activation and PARP cleavage occurred. Examination of the ERK phosphorylation as a function of Taxol concentration showed that ERK2 phosphorylation in FRO cells decreased at higher drug doses, and ERK1 was activated only at Taxol concentration causing maximal apoptosis (10–50 nM) and then declined (data not shown).

Third, MAPK, which might be implicated in Taxol effects, p38, largely known as a proapoptotic factor (11), is likely to play a secondary but not minor role in thyroid ATC cell death observed in our experiments. In all cell lines, its phosphorylation was found to be declining in time in cultures supplemented with apoptosis-inducing doses of Taxol (data not shown).

To further assess the relevance of the observed MAPK pathway activation to the cytotoxic effects of Taxol, we investigated whether the inhibition of MAPK pathways modulates cell survival after treatment. Inhibition of JNK with SP600125 significantly increased cell survival (Fig. 7AGo). The finding was further supported by the observation of the reduced procaspase-3 and PARP cleavage in the Taxol/SP600125-treated cells (Fig. 7BGo). Using PD98059, an inhibitor of MEK1, which is an upstream kinase to ERK, we observed decreased cell viability after Taxol treatment (Fig. 7AGo). The cooperation between Taxol and MEK inhibitor was especially clearly seen in the caspase-3 activation and PARP cleavage (Fig. 7BGo). In the presence of PD98059, levels of the cleaved forms of these molecules markedly increased. These data suggest that activation of the cRaf-1/ERK1/2 pathway provides a survival drive to thyroid cancer cells treated with Taxol. p38 MAPK inhibitor caused a significant increase of cell viability in ARO and KTC-2 cell lines treated with the drug (Fig. 7AGo).



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  		FIG. 7. Effect of MAPK and PI3K inhibitors on Taxol cytotoxicity. A, Cells were preincubated for 1 h with one of the following inhibitors: 5 µM of JNK inhibitor SP600125, 10 µM of p38 MAPK inhibitor, 10 µM of MEK1 inhibitor PD98059, or 10 µM of PI3K inhibitor LY294002. Then Taxol was added at specified concentrations. Cell viability was determined after 48 h incubation using the WST assay, as described in Materials and Methods. Data are mean ± SD, n = 6. B, Potentiation or inhibition of Taxol-induced apoptosis was estimated by caspase-3 activation and PARP cleavage in cells incubated 24 h with Taxol (15 nM) in the presence of MAPK inhibitors (above-mentioned concentrations) using Western blotting. Procaspase-3 cleavage is shown for ARO cells and PARP is for KTC-2.

 
Akt pathway has been mostly associated with cell proliferation and survival (23). Preincubation of cell cultures with LY294002, a specific inhibitor of PI3K, decreased cell viability in the presence of Taxol (Fig. 7AGo) and increased procaspase-3 and especially PARP cleavage (Fig. 7BGo) in all lines tested. These findings are consistent with the role of PI3K/Akt signaling as apoptosis-inhibiting mechanism.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Different concentrations of Taxol are known to evoke distinct effects on both cytoskeleton and cellular biochemical processes (7, 8, 24, 25, 26). Low concentrations of Taxol can alter microtubule dynamics and/or induce G2/M cell cycle arrest, whereas high concentrations of the drug (200 nM to 30 µM) cause massive microtubule damage (8). In general, main apoptotic mechanisms initiated at high Taxol concentration include the characteristic changes in the gene expression profile and activation of JNK, Raf-1, cyclin-dependent kinases, and caspases. At low drug concentration, apoptosis may also occur, but its mechanisms are largely unknown (8). Our data demonstrate that typical apoptosis in Taxol-treated human thyroid cancer cells can mostly be observed within a narrow range of low drug concentration, from 6–10 nM to 25–50 nM, depending on the cell line. At higher Taxol doses, we detected clear decrease of the PARP and procaspase-3 cleavage and the loss of mitochondria potential, but cell death remained profound. It is worth noting that there was no significant difference in the ARO cell viability between cultures treated with 25 nM and clinically relevant concentration, 5 µM of Taxol, after 24 h (data not shown).

Reported data suggest that Taxol can directly affect mitochondria perhaps through the interaction with tubulin associated with the mitochondrial membrane (27), and at high concentration the drug has been shown to bind the organelles and decrease mitochondrial potential (28). Another work (29) has also demonstrated that high Taxol doses caused rapid release of Ca2+ from mitochondria and loss of mitochondrial potential. In human neuroblastoma cells, 1 µM Taxol induced the release of cytochrome c, increased respiration rate, and production of reactive oxygen species in mitochondria (30). It is therefore possible that Taxol at high concentration may cause a variant of cell death other than apoptosis. Such form of cell death would be mostly associated with mitochondrial collapse.

Main caspases involved in the Taxol-induced apoptosis are caspase-8, -9, and -3 sometimes accompanied by caspase-6 and -7 and, less frequently, caspase-2 (31, 32). In the thyroid cancer cells, we found that Taxol induced the appearance of activated forms of caspase-3 and -9 in all cell lines. We also observed the presence of a cleaved form of the caspase-6, downstream to caspase-3, and that of caspase-7, which is downstream to caspase-9. KTC-2 was the only cell line that displayed cleavage of procaspase-8, which is known to mediate the death receptor pathway of apoptosis (20). Perhaps elevated sensitivity of KTC-2 cells to Taxol, the highest among ATC cell lines tested, may in part be attributed to the functioning of this additional apoptotic mechanism.

Bcl2 family proteins controlling the mitochondrial membrane permeability play an important role in the processes of drug-induced apoptosis. Overexpression of Bcl2 has been reported to increase cell resistance to Taxol (33, 34). Exposure to Taxol can stimulate Bcl2 phosphorylation in various cell lines (35, 36, 37), but current understanding of biological relevance of this modification is somewhat controversial. Several reports have demonstrated that Bcl2 phosphorylation is essential for the Taxol-induced apoptosis (35, 36, 37). One of the possible explanations of the phenomenon is that phosphorylated Bcl2 possesses attenuated ability to interact with proapoptotic mitochondrial proteins such as Bax (38) or Bad, which stimulate the release of cytochrome c and other proapoptotic factors from the organelles, leading to the formation of caspase-activating apoptotic complexes (39). On the other hand, Bcl2 phosphorylation is known to be a common event in mitosis (40). Furthermore, phosphorylation of Bcl2 has been shown to protect it from proteasomal degradation (41), and, in addition, in A2780 human ovarian cancer, cell down-regulation of Bcl2 enhanced the resistance to Taxol (28). In the Taxol-treated thyroid cancer cells, we found that Bcl2 phosphorylation correlated with PARP and procaspase cleavage. We also observed gradual decrease of Bcl2 levels in all studied cell lines. This fact taken together with corresponding changes in Bax level seen in ARO and KTC-2 cells may imply Bcl2 phosphorylation as an initiating event in mitochondria-dependent apoptosis of this type of cells exposed to Taxol.

We also observed a rise in the levels of antiapoptotic survivin and XIAP after Taxol treatment. Survivin plays an as-yet-nondetailed role during mitosis (42), and its accumulation in ATC cells may reflect the drug interference with mitotic machinery rather than exertion of survivin’s antiapoptotic function. XIAP has been recently shown to directly inhibit two members of the cell death protease family, effector caspases-3 and -7, and also prevent the activation of caspase-9 (43). The regulation of human XIAP gene expression is known to be mediated by the NF{kappa}B (44). NF{kappa}B has been shown to activate a survival pathway and cause the Taxol resistance in some models (45, 46). Further experiments are necessary to clarify exact role of NF{kappa}B in Taxol-treated ATC systems.

Activation of MAPKs, especially JNK, has proved a crucial event in the Taxol effects in most of cancer cells (9, 10, 47). These data are not clear-cut though, and in particular cell systems, JNK has been shown not to play a noticeable role in the Taxol-induced apoptosis, whereas p38 and ERK have been recognized as main factors (11, 12). Furthermore, in some cancer cells, MAPK has been shown not to be involved in Taxol effects (13, 14), suggesting a strong dependence on the cell type. In thyroid cancer cells, we observed activation of the JNK cascade documented as ASK1/MKK4/JNK/cJun-ATF-2. The activation of JNK and JNK-dependent transcription factors coincided in time with the caspase and PARP cleavage. Additionally, the JNK-inhibitor SP600125 increased cell survival and inhibited the apoptotic effect of Taxol. Taking into account these data together with reports that JNK can directly phosphorylate Bcl2 (48), one can speculate that in our experiments the JNK-mediated Taxolinduced apoptosis in thyroid cancer cells might be associated with Bcl2 phosphorylation.

The phosphorylation of ERK differed in various thyroid cancer cell lines treated with Taxol. We observed the activation of ERK1/2 and phosphorylation of its nuclear substrate Elk1 in KTC-2 cells, but there was a decrease of phosphorylation of ERK1/2 in KTC-3 and ARO cells and that of ERK2 in FRO cells. In our opinion, these facts demonstrate that inhibition or activation of ERK is not a universal effect of Taxol in thyroid cancer cells. There was no clear correlation between ras and BRAF status and diversity of ERK activation in studied cell lines because all cells have wt ras and exept KTC-3 (wt BRAF), and other cell lines have mutant BRAF (49). Because the inhibitor of MEK1, an upstream kinase to ERK, enhanced the Taxol effects, it is plausible to suppose that this ERK accounts mainly for the survival pathway in thyroid anaplastic cells, being consistent with most available reports (50, 51).

The PI3K/Akt pathway has been associated with cancer cell resistance to chemotherapeutic drugs including Taxol (23). Possible mechanisms of such resistance may be due to the inhibition of the JNK pathway via Akt interference with JIP1, a scaffolding JNK-interacting protein (52) as well as with phosphorylation and retaining in cytoplasm of the proapoptotic forkhead homolog in rhabdomyosarcoma, direct phosphorylation of Bad (53), or activation of the NF{kappa}Bdependent survival mechanisms (46, 54). Our experiments confirm this notion, and furthermore, data obtained with cell cultures incubated with the PI3K inhibitor showed that in ATC cells PI3K/Akt cascade along with MEK/ERK comprises a cell survival signal. Thus, one can assume that one of the possible mechanisms of chemoresistance in ATC cells is at least in part associated with MEK/ERK and PI3K activation, and proteins of the inhibitor of apoptosis family may represent main effectors of the cell survival.

Data obtained in our experiments show that ATC cells are sensitive to Taxol and can be effectively killed in cultures, but mechanisms of cytotoxicity are distinct at different drug concentration. As summarized in Fig. 8Go, low drug doses can activate a variety of signaling cascades, both proapoptotic and prosurvival. Within the concentration range of 6–50 nM, imbalance between these cascades evokes apoptotic changes in the cells: detectable are cleaved forms of procaspases and PARP and phosphatidylserine exposure on the outer membrane. At higher doses of Taxol, cell death forms other than apoptosis eventually become predominant as seen from the loss of mitochondrial potential and attenuation of biochemical manifestations of the programmed cell death.



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  		FIG. 8. Proposed scheme of main signaling pathways mediating effects of low doses of Taxol in thyroid cancer cells. Central role in the Taxol-induced ATC cell death seems to belong under such conditions to the JNK pathway. As a result of its activation, perhaps together with direct Taxol interaction with mitochondria, corresponding changes in Bcl2 and Bax levels occur, leading to the appearance of active forms of several caspases. In particular cases Taxol can induce activation of caspase-8, which may act in concert with other executioners activated through the mitochondria-dependent factors. The p38 MAPK cascade is likely to cooperate with JNK pathway in cell killing. Along with signaling leading to the cell death, Taxol also activates the survival mechanisms. In ATC cells, these were represented mostly by the ERK, PI3K/Akt, and perhaps NF{kappa}B pathways. MT, Microtubules; DR, death receptor; {Delta}{psi}m, mitochondrial potential. Dashed arrows represent associations between factors when direct interaction mechanism is not established or is putative.

 
The pharmacokinetics study of close to paclitaxel taxane-docetaxel showed that in an hour plasma concentration of the drug dropped from an initial 5000 nM to 100 nM and in 6 h to 10 nM (55). Thus, the average actual effective concentration of taxanes in plasma for periods from 3 to 24 h is around 10 nM. Our results may be useful for the optimization of experimental models of treatment of human anaplastic thyroid cancer and interpreting biological effects of the drug.


   Footnotes
 
This work was supported in part by Research Grants 15256004, 14380256, 15390295, 15590981 and the 21st Century Center of Excellence Program of International Consortium for Medical Care of Hibakusha and Radiation Life Science from Japanese Ministry of Education, Science, Culture, and Sports.

Abbreviations: ASK, Apoptosis signal-regulating kinase; ATC, anaplastic thyroid cancer; ATF, activating transcription factor; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; JNK, c-jun-NH2 terminal kinase; MKK4, MAPK kinase; MEK, MAPK/ERK kinase; NF{kappa}B, nuclear factor {kappa}B; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; PI3K, phosphatidylinositol-3-OH kinase; TMRE, tetramethylrhodamine ethyl ester; WST, water-soluble tetrazolium salt-based assay; XIAP, X-chromosome-linked inhibitor of apoptosis.

Received February 2, 2004.

Accepted for publication March 19, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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