5-Fluorouracil Interferes with Paclitaxel Cytotoxicity Against Human Solid Tumor Cells1

Vol. 3, 1 739-1745. October 1997 Clinical Cancer Research 1739

5-Fluorouracil Interferes with Paclitaxel Cytotoxicity against Human Solid Tumor Cells1

Korey R. Johnson, Liming Wang, ( 1 ). This naturally occurring antimitotic drug has shown great Merrill C. Miller III, Mark C. Willingham, and promise in the treatment of certain human solid tumors, particularly in metastatic breast cancer and drug-refractory ovarian Weimin Fan2 cancer (2-4), although the exact mechanism of the cytotoxicity Department of Pathology and Laboratory Medicine 1K. R. J.. of paclitaxel against tumor cells is not entirely clear. Previous M. C. M.. M. C. W.. W. Fl, Experimental Oncology IL. WI, and Medicine 1W. Fl, Medical University of South Carolina, Charleston, studies demonstrated that paclitaxel is a unique antimicrotubule South Carolina 29425 agent, and its antitumor effects result mainly from interference with the normal function of microtubules and the blockage of cell cycle progression in later G,-M phases via prevention of ABSTRACT mitotic spindle formation (5, 6). However, recent studies have Paclitaxel, a naturally occurring antimitotic agent, has demonstrated that paclitaxel, at clinically relevant concentra- shown efficacy in the treatment of certain solid tumors, tions, was able to induce internucleosomal DNA fragmentation particularly metastatic breast carcinoma and drug-refrac- and the typical morphological features of apoptosis in a number tory ovarian cancers. Recent studies have demonstrated that of solid tumor cells (7-10). These results clearly indicate that paclitaxel, in addition to its effects on microtubules and cell taxol, in addition to its classical activity against microtubules cycle arrest, possesses significant cell-killing activity in solid and cell cycle arrest, also possesses cell-killing activity by tumor cells by the induction of apoptosis. However, the induction of apoptosis. mechanism by which paclitaxel leads to cell death and its Although it is well recognized that paclitaxel can cause relationship with paclitaxel-induced mitotic arrest is pres- both mitotic arrest and apoptotic cell death, it remains unclear ently unclear. In this study, we attempted to determine whether paclitaxel-induced cell death is a secondary event re- whether pre-arresting tumor cells at other phases of the cell suIting from mitotic arrest or represents a novel mechanism of cycle could affect paclitaxel-induced apoptosis. We found action for paclitaxel against tumor cells. Morphologically. a that 5-fluorouracil (5-FU), another antineoplastic agent that sustained block of mitosis seems to be required for taxol- usually arrests tumor cells at the -S phase of the cell cycle, induced apoptosis in most solid tumor cells because, by using could significantly repress the cell-killing activity of pacli- time-lapse video microscopy, most apoptotic events were ob- taxel in solid tumor cells, even when it was added simulta- served to occur in cells showing prior mitotic arrest ( 10). How- neously with paclitaxel. Further studies indicated that 5-FU ever, this observation does not absolutely prove that paclitaxel- actually inhibits the cytotoxic effects of paclitaxel on both induced apoptosis is a secondary event resulting from mitotic mitotic arrest and apoptotic cell death, suggesting that 5-FU arrest. In fact, several lines of evidence from our recent exper- might interfere with paclitaxel cytotoxicity at an early stage, iments and other laboratories have suggested that paclitaxel- probably by preventing tumor cells from entering G2-M induced apoptosis might take place via an independent pathway phase. Because recent clinical trials have used a combination ( 10-12). For example, recent experiments in our laboratory of paclitaxel and 5-FU in the treatment of metastatic breast demonstrated that paclitaxel-induced apoptotic cell death, but cancers, our results also suggest that the combination of not mitotic arrest, was significantly inhibited when the cells these two drugs might not be as valuable in clinical chemo- were pretreated with glucocorticoids (12, 13). Unless glucocor- therapy. ticoids specifically disrupt the downstream events of mitotic arrest, this selective inhibitory action of glucocorticoids on INTRODUCTION paclitaxel cytotoxicity implied that paclitaxel-induced apoptotic Paclitaxel (Taxol#{174}), a novel antineoplastic agent, was orig- cell death may occur via a signaling pathway independent of inally isolated from the bark of the pacific yew, Taxus brei’:ftlia mitotic arrest. In this study, to investigate the molecular basis of paclitaxel-induced apoptotic cell death and its possible correlation with mitotic arrest, we have investigated whether pre-arresting Received 3/28/97; revised 5/30/97; accepted 6/19/97. or synchronizing tumor cells at other phases of the cell cycle The costs of publication of this article were defrayed in part by the would affect taxol-induced apoptotic cell death. We subse- payment of page charges. This article must therefore be hereby marked quently found that S-FU,3 another antineoplastic agent that can advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. arrest tumor cells at the G1-S phase of the cell cycle ( 14), could

I This work was supported by NIH Grants CA7l85l and CA58846 (to significantly inhibit the cell-killing activity of paclitaxel in W. F.) and The Health Science Foundation of the Medical University of human breast cancer and other solid tumor cells. Further inves- South Carolina.

2 To whom requests for reprints should be addressed, at Department of Pathology and Laboratory Medicine, Medical University of South Caro- lina, 171 Ashley Avenue, Charleston, SC 29425. Phone: (803) 972- 5108: Fax: (803) 792-4157. 3 The abbreviations used are: 5-FU. 5-fluorouracil; MTT. thiazolyl blue.

tigations indicate that 5-FU can actually repress the cytotoxic Morphological Observations by Phase Contrast Mi- effects of paclitaxel on both mitotic arrest and apoptosis. These croscopy. Cells in 35-mm dishes were placed on the stage of findings suggest that 5-FU might interfere with the cytotoxicity a warmed inverted microscope and examined using phase con- of paclitaxel at an early stage. On the other hand, because recent trast microscopy as described previously (10). This microscope clinical trials have used a combination of paclitaxel and 5-FU in is equipped with a heater/recirculation device that maintains the treatment of metastatic breast cancers (15-17), our results stage temperature at 37 ± 0.5#{176}C. In addition, this system is have also raised a question as to the value of this combination in supplied with a constant through-flow of 95% air/S% CO,. Time clinical chemotherapy. lapse video recordings were prepared at a 1 :720 time lapse ratio. Individual cells in each field were chosen to examine the fre- quency of mitotic arrest, apoptotic blebbing, and micronucle- MATERIALS AND METHODS ation events. Duplicated dishes treated with S-FU, paclitaxel, or Drugs and Cell Culture. Paclitaxel was purchased from both or untreated were examined and photographed using a Calbiochem (La Jolla, CA) and dissolved in 100% DMSO to regular phase contrast microscope. make a stock solution of 1 .0 mrvi, which was then diluted in MTT Assay. BCap37 and KB cells were harvested with culture medium to obtain the desired concentration. 5-FU (Sig- trypsin and resuspended to a final concentration of 2 X i04 ma Chemical Co., St. Louis, MO) was also dissolved in DMSO. cells/ml in fresh medium with 10% FCS. Aliquots of 100 p.1 The human breast tumor BCap37 (10) and KB human epider- from cell suspension were distributed evenly into 96-well tissue culture plates with lids (Falcon, Oxnard, CA). Designated col- moid carcinoma cell lines (American Type Culture Collection, umns were treated with 10 p.M 5-FU or 100 nM paclitaxel, or Rockville, MD) were propagated in RPMI 1640 supplemented their combination. One column from each plate contained me- with 10% FCS (Hyclone, Logan, UT). As described previously dium alone, and another contained cells without drug. Cell ( 10, 12), paclitaxel and/or 5-FU were usually added when the viability was quantified by the method of Carmichael et al. (19). cells reached approximately 60 -70% confluence. After 24, 48, and 72 h, 100 p.1 of a 1 mg/mm MTT solution were Determination of Internucleosomal DNA Cleavage. added to each well, and the plate was incubated for 4 h, allowing Internucleosomal DNA fragmentation was assayed by a modi- viable cells to reduce the yellow MU into dark-blue formazan fication of methods described previously (8). After treatment crystals, which were dissolved in 100 pA of DMSO. The ab- with 100 flM paclitaxel or 10 p.M 5-FU, or their combination, sorbance in individual wells was determined at 560 nm by a cells were harvested and suspended in lysis solution containing microplate reader (Molecular Devices, CA). SO mM Tris-HC1, 10 mi EDTA, 0.5% N-lauroylsarcosine, and Western Blots. Cells treated with I p.M paclitaxel and/or 0.5 mg/ml proteinase K for 1 h at 50#{176}C.Crude DNA samples 10 p.M 5-FU were harvested by trypsinization at the times were extracted twice with equal volumes of phenol buffered indicated. Extraction of cellular proteins and Western blots of with 0. 1 M Tris-HC1 (pH 7.4), followed by an equal volume bcl-2 were performed as described previously (13). The reactive

extraction with chloroform:isoamyl alcohol (24: 1 ). The remain- bcl-2 band was identified using a chemiluminescent substrate to ing steps for DNA fragmentation analysis were performed cx- horseradish peroxidase (Amersham Corp.). actly as described (8). DNA samples were analyzed by electrophoresis in a I .5% agarose slab gel containing 0.2% ethidium RESULTS bromide and visualized under UV illumination. 5-FU Inhibits Paclitaxel-induced Apoptotic Cell Death. Flow Cytometry Analysis. Cell sample preparation and An important hallmark of apoptotic cell death is the fragmen- propidium iodide staining were performed according to the tation of genomic DNA into integer multiples of 180-bp units, method described by Nicoletti et al. (18). Cells were treated producing a characteristic ladder on agarose gel electrophoresis with 10 .LM 5-FU or 100 nM paclitaxel, or their combination, for (20). To investigate the possible influence of 5-FU on pacli- 24 and 48 h. Cells were then harvested by trypsinization and taxel-induced apoptotic cell death, we first examined whether washed twice with PBS. Cells were fixed in 1% formaldehyde paclitaxel-induced DNA fragmentation would be affected by in PBS and then dehydrated in 70% ethanol diluted in PBS. cotreatment with 5-FU. Fig. I shows the DNA fragmentation of Approximately 1 h before flow cytometry analysis, cells were BCap37 and KB cells exposed to 100 flM paclitaxel or 10 p.M, or incubated in PBS containing 100 p.g/ml RNase A and 40 i.g/ml their combination, for 48 or 72 h. As reported previously (10, propidium iodide at 37#{176}C.Cell cycle distribution was deter- 12), the characteristic DNA fragmentation ladders were ob- mined using a Coulter Epics V instrument (Coulter Corp.), with served in both BCap37 and KB cells following treatment with an argon laser set to excite at 488 nm. 100 nM paclitaxel alone for 72 h (Fig. 1, Lane 4). When those Cytospin Preparation. Cells treated with 10 p.M S-Hi or cells were treated with 10 p.M 5-FU alone, essentially no clear 100 nM paclitaxel, and their combination, were harvested by DNA fragmentation was observed (Fig. 2, Lane 6), although trypsinization at the times indicated and washed twice with morphological observation by video microscopy indicated that a Opti-MEM reduced serum medium. Cell numbers were deter- small number of cells may actually undergo apoptosis (see mined with a Coulter counter, and approximately below). However, when those cells were treated with both 5-FU 50,000-100,000 cells from each group were used for cytospin and paclitaxel simultaneously, taxol-induced DNA fragmenta- preparations. Slides were air dried and fixed in acetone prior to tion was inhibited dramatically (Fig. 2, Lanes 7 and 8), suggest- Giemsa staining and examined using bright-field microscopy ing that 5-FU could repress paclitaxel-induced apoptotic cell (8). death in both cell lines. Furthermore, this 5-FU-mediated inhi-

I. I z_. L tially blocked (Fig. 3). In addition, from the flow cytometry xe assay, we could also see that an apparent apoptotic peak was ‘N detected in the group treated with paclitaxel alone for 48 h, but ‘. ‘- L - $_ t_ . .n.n.n , this apoptotic peak was no longer detectable if the cells were treated with a combination of paclitaxel and 5-FU. As reported previously ( 12), no apoptotic peak was observed at 24 h in the

*1)t*1Lt) V)It)M*1 groups treated with 100 nM taxol, because most of apoptotic events did not occur until after 48 h of paclitaxel treatment. The Antagonistic Effect of 5-FU on Paclitaxel Cytotox- ; ,‘?‘ :: icity Is Schedule Dependent. In clinical trials, 5-FU has been widely used in combination therapy with paclitaxel to treat metastatic breast carcinomas (15-17) and other solid tumors (22, 23). Thus, this inhibitory action of 5-FU on paclitaxel- induced mitotic arrest and apoptosis has raised a clinically relevant question as to whether the combination of 5-FU with paclitaxel might actually produce an antagonistic effect. To evaluate further the potential antagonistic effects between paclitaxel and 5-FU in human solid tumor cells in vitro, we have BCap37 Cells KB Cells used the MTI’ assays to assess the cytotoxic interaction of these

Fig. 1 Effects of 5-FU on paclitaxel-induced internucleosomal DNA two drugs administered at various schedules in both BCap37 fragmentation in BCap37 and KB cells. Tumor cells were initially and KB cells. This assay indicated that 5-FU could inhibit cell cultured in medium containing 100 nsi paclitaxel or 10 p.M 5-FU. or growth, although it had little effect on inducing apoptosis (Figs. both simultaneously for the indicated times. Fragmented DNAs were I and 3). In addition, results shown in Fig. 4 also indicated that prepared from all cell groups and exhibited on a 1.5% agarose slab gel pretreatment or simultaneous exposure of tumor cells with S-FU containing 0.1% ethidium bromide. could significantly antagonize paclitaxel cytotoxic efTects against solid tumor cells in vitro. However. when these tumor cells were pretreated with paclitaxel for 12 h, 5-FU-mediated bition of paclitaxel-induced cell death was confirmed by mor- inhibition of paclitaxel cytotoxicity was clearly attenuated, al- phological observation using phase contrast video microscopy. though an effect as great as paclitaxel alone was still not Typically, when BCap37 or KB cells were treated with 100 nM achieved. Furthermore, if tumor cells were exposed to paclitaxel paclitaxel, about 50-60% of cells underwent apoptosis by 72 h, for 24 h prior to 5-FU, the antagonistic effects of 5-FU on which were identified by the onset of rapid surface blebbing paclitaxel cytotoxicity were no longer observed (Fig. 4). We activity followed by cessation of cell movement (10, 21 ). How- have also performed the clonogenic survival assay; pretreatment ever, when those tumor cells were treated with both paclitaxel with 5-FU could clearly repress the cell-killing activity of pa- and 5-FU, only 10-20% of cells died via apoptosis. Most cells clitaxel in both BCap37 and Kb cells (data not shown). appeared to be cytostatic (no progression through the cell cycle), 5-FU Prevents Paclitaxel-induced bcl-2 Hyperphos- similar to the cells treated with 5-FU alone. In addition, about phorylation. Recent studies have demonstrated that paclitaxel 5-10% of cells also showed typical apoptotic features in the could induce bcl-2 hyperphosphorylation (1 3, 24). This modi- group treated with 10 p.M 5-FU alone for 72 h. fication was believed to cause attenuation of bcl-2 antiapoptosis 5-FU Also Prevents Paclitaxel-induced Mitotic Arrest. activity (25). To determine whether this paclitaxel-induced bcl-2 By morphological examination using phase contrast micros- hyperphosphorylation is also affected by 5-FU, we have used copy, we noticed that the cell number arrested at the G-,-M phase Western blotting to examine the expression of bcl-2 in both (rounded and detached from the dish) was significantly de- BCap37 and KB cells treated with the combination of paclitaxel creased when the BCap37 cells were cotreated with paclitaxel and 5-FU. In Fig. 5, it can be seen that paclitaxel induced a and 5-FU (Fig. 2). To determine whether 5-FU does indeed slower mobility form of bcl-2 protein, which was demonstrated affect the cytotoxic effects of paclitaxel on cell cycle arrest, to be a hyperphosphorylated form of bcl-2 (24). However, this cytospin slides were prepared by which mitotically arrested cells bcl-2 hyperphosphorylation caused by paclitaxel was clearly were easily identified by their morphological features, e.g., blocked when the cells were cotreated with 10 p.M 5-FU. condensed chromosomes. The results were summarized in Table

1 . Compared to the cells treated with paclitaxel only, the number of cells arrested at the G,-M phase were significantly decreased DISCUSSION when the cells were treated with both 100 nM paclitaxel and 10 The initial purpose of this study was to investigate the p.M S-FU. Furthermore, flow cytometry analyses were per- possible relationship between paclitaxel-induced apoptotic cell formed to clarify that paclitaxel-induced G,-M phase arrest was death and mitotic arrest. If we assume that paclitaxel-induced prevented by 5-FU. The results depicted in Fig. 3 indicate that apoptotic cell death is a secondary event resulting from mitotic most cells were not able to enter the G,-M phase if BCap37 cells arrest, then cell death should be blocked once tumor cells are were pretreated or simultaneously treated with 5-FU. However, prevented from entering cell cycle arrest at the G2-M phase. A if the cells were pretreated with paclitaxel for 12 h prior to S-FU feasible approach to test this hypothesis was to synchronize or treatment, paclitaxel-induced G,-M phase arrest was only par- pre-arrest tumor cells at other phases of the cell cycle by using

Control S.FU Taxol 5#{149}FU+Taxol

24hr

48hr

72hr

Fig. 2 Effects of 5-FU on paclitaxel-induced morphological changes in BCap37 cells. Cells were treated with 100 nM paclitaxel or 10 p.M 5-FU, or both simultaneously. At the indicated time points, culturing cells were examined and photographed using a phase contrast microscopy.

Table I Effect of 5-FU and paclitaxel on mitotic arrest” similar or different modes of action are combined, the outcome

Percentage of cells at G2-M phase” can be synergistic, additive, or antagonistic. Synergism implies that two drugs may produce greater therapeutic efficacy than an BCap37 KB expected additive effect, whereas antagonism implies that the Drug exposure 24 h 48 h 24 h 48 h actual therapeutic activity produced by two drugs may be Control 5±2 6±2 4±3 6±3 smaller than their additive effect. The promising clinical activity 5-FU 2±1 1±1 3±1 2±1 of paclitaxel has promoted considerable interest in combining Paclitaxel 38 ± 7 49 ± 10 44 ± 8 53 ± 1 1 this drug with other antitumor agents. In clinical trials, 5-FU is 5-FU paclitaxel 5 3 7 4 6 4 5 2 + ± ± ± ± one of these agents widely used with paclitaxel in the treatment “ Based on three separate experiments and presented as mean ± of metastatic breast carcinomas (15-1 7) and other solid tumors, SE. including tumors of the head and neck (22) and gastrointestinal 1. Cytospin slides were stained with Geimsa. Three hundred cells were counted from each slide, and only those cells with typical mor- tract (23). Thus, the finding of the antagonistic effect of S-FU on phological features of condensed chromosones were counted as mitot- paclitaxel cytotoxicity has also raised a clinically relevant ques- ically arrested cells. tion as to whether the combination of 5-FU with paclitaxel is really beneficial in clinical chemotherapy. By using MTT assays, we have further evaluated the in vitro cytotoxic interaction other drugs or agents and then examine whether paclitaxel still and potential antagonistic effects of these two drugs adminis- causes cell death. Because of the ability of 5-FU to arrest cells tered at different schedules in both BCap37 and KB cells. Data in G,-S phase (14), 5-FU was selected for this study. As shown generated by this assay indicated that, unless paclitaxel was in our results, paclitaxel-induced apoptosis was indeed inhibited added 24 h prior to 5-FU, the cytotoxic effect of this combina- by pretreatment of tumor cells with 5-FU. Furthermore, we tion of 5-FU and paclitaxel is not as effective as paclitaxel alone found that this antagonistic effect was still observed, although (Fig. 4). These results suggest that the combination of paclitaxel 5-FU was added simultaneously with paclitaxel (Fig. 1). In and 5-FU actually produces subadditive effects against those addition, through cytospin and flow cytometric analyses, it was solid tumor cells in vitro. In a recent report, the possible antag- demonstrated that 5-FU could predominantly arrest the tumor onistic effect between S-Ri and padlitaxel has also been ad- cells at G,-S phase and nearly completely prevent the cells from dressed by Kano et al. (26). In their study, the subadditive entering G,-M phase, unless paclitaxel was added prior to 5-Ri cytotoxic effect was also observed when MCF-7 breast cancer (Table 1 and Fig. 3). Therefore, the mechanism by which S-Ri cells were pretreated with 5-FU or 5-FU was administered represses paclitaxel-induced apoptosis was mainly through the simultaneously with paclitaxel. Thus, pretreatment with S-FU or prevention of tumor cells from entering G2-M phase. Based on coadministration of 5-FU and paclitaxel essentially produces no these findings, it may be presumed that paclitaxel-induced ap- synergistic effect against tumor cells in vitro. However, it re- optotic cell death is cell cycle dependent. mains uncertain whether an additive effect is generated when Combination therapy with multiple drugs is a common tumor cells are pretreated with paclitaxel prior to 5-FU. Kano et practice in the treatment of cancer. When anticancer agents with al. (26) reported that an additive effect occurred with this

24hr 4Shr

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It 5-FU C

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Fig. 3 Flow cytometric analysis of cell cycle distribution. BCap37 cells, treated with 100 flM paclitaxel or 10 p.M 5-FU, Taxol both drugs simultaneously, 6-h preincubation with 5-FU followed by paclitaxel, or 6-h preincubation with paclitaxel followed by 5-FU, for the indicated times, were harvested and 0 1024 stained for DNA with propidium iodide and analyzed by flow cytometry as de- GI scribed in “Materials and Methods.”

The distribution of cells in G0-G1, 5, ‘ and G,-M phases of the cell cycle and I apoptotic cells (Ap) are indicated above C 5-FU+Taxol C each corresponding peak. .

0IlIdri . 1024

C C Pre-5.FU+Taxol C .

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? G2.M Pre-Taxol+5-FU I\A . . 0 1024 0 1024

BCap37 Cells

Control

5-FU

. Taxol

0 5-FU+TX

C- U Pre 5-FU6+Taxol 0 Pre TX6+5-Fu

0 Pre TX24-5-FU Fig. 4 Cytotoxic effects of 5-FU and paclitaxel against BCap37 24 48 72 and KB cells. Approximately 2 X l0 BCap37 or KB cells were Time (Hrs) cultured in 96-well microculture plates. After 24 h, cells were cx- posed to 100 nM paclitaxel or 10 KB Cells I.I.M 5-FU, or both drugs in different sequences. After the indicated time, M’fl’ assays were per- Control i:#{149} formed as described in “Materials and Methods.” Bars. SD. 5-FU

. Taxol .0 0 E 5-FU+Tx

C- 0 . Pre

Pre TX6+S-Fu

E1 PreTX24+S-FU

Time (Hrs)

sequential combination. but such a synergistic effect was not confirmed in our experiments. As we can see from Figs. 3 and 0 0 4, S-FU was still able to interfere with the activity of paclitaxel

in both mitotic arrest and apoptotic cell death, although S-FU was added 6 or 12 h later. However, when tumor cells were - 0 = pretreated with paclitaxel for 24 h, the cytotoxic effect of 1 + 0 =I1 paclitaxel was indeed no longer affected by 5-FU, but the , expected additive effect was still not observed. Basically, the combined cytotoxic effect of these two drugs in this schedule was not greater than when treated with paclitaxel alone (Fig. 4). Therefore, more studies, particularly in vito experiments with animal models, are necessary to address this clinically important issue. In addition, our recent studies have demonstrated that paclitaxel could induce bcl-2 hyperphosphorylation in both BCap37 and KB cells ( I 3). Due to this posttranslational modi- BCap37 Cells fication, bcl-2 has been postulated to lose its ability to block KB Cells apoptosis (25). More recently, the phosphorylation of bcl-2 has Fig. 5 Western analysis of bcl-2 protein content. Cellular proteins been also suggested to reflect the damage of microtubules, were extracted from BCap37 and KB cells treated with 1 p.M paclitaxel because this modification is commonly induced by antimicro- or 10 p.M 5-FU. or their combination. Equal amounts (100 g/lane) of cellular protein were fractionated on I 2% SDS-polyacrylamide gels and tubule agents and always occurs in the G,-M phase of the cell transferred to a nitrocellulose membrane. The membrane was then cycle (27). Moreover, Schandl et al. (28) have reported recently immunoblotted with anti-bcl-2 antibody as described in “Materials and that this phosphorylation event occurs normally as part of mi- Methods.”

tosis, suggesting that it may have no relevance to the antiapop- apoptosis and altered gene expression in human breast cancer cells. Cell. totic effects of bcl-2. Therefore, it was necessary to examine the Pharmacol., 2: 249-257, 1995. possible influence of S-Ri on paclitaxel-induced bcl-2 phos- II . Donaldson, K. L., Goolsby, G., Kiener, P. A., and WahI, A. F. Activation of p34cdc2 coincident with paclitaxel-induced apoptosis. Cell phorylation. Our results show that the hyperphosphorylation of Growth Differ., 5: 1041-1050, 1994. bcl-2 was, indeed, repressed when those tumor cells were 12. Fan, W., Cheng, L., Norris, J. S., and Willingham, M. C. Glucocor- treated simultaneously with S-Ri (Fig. 5). This finding may ticoids selectively inhibit taxol-induced apoptosis in a human breast provide another piece of evidence that S-Ri antagonizes the cancer cell line. Cell. Pharmacol., 3: 435-440, 1996. cytotoxicity of paclitaxel by preventing tumor cells from enter- 13. Fan, W., Schandl, C. A., Cheng, L., Norris, J. S., and Willingham, ing the G2-M phase of cell cycle. M. C. Glucocorticoids modulate paclitaxel cytotoxicity in human solid In summary, this study has investigated the possible influ- tumor cells. Cell. Pharmacol., 3: 343-348, 1996. ence of S-Ri on the cytotoxic effect of paclitaxel against human 14. Maybaum, J., Ullman, B., Mandel, H. G., Day, J. L., and Sadee, W. solid tumor cells in vitro. Our results demonstrated that S-Ri Regulation of RNA- and DNA-directed actions of 5-fluoropyrimidines could repress paclitaxel cytotoxicity in both apoptotic cell death in mouse T-lymphoma (S-49) cells. Cancer Res., 40: 4209-4215, 1980. and mitotic arrest. Moreover, S-Ri was found to arrest tumor I 5. Klaassen, U., Harstrick, A., Wilke, H., and Seeber, S. Preclinical and clinical study results of the combination of paclitaxel and 5-fluorouracil/ cells at the G1-S phase and inhibit paclitaxel-induced bcl-2 folinic acid in the treatment of metastatic breast cancer. Semin. 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Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 1997 American Association for Cancer Research. 5-Fluorouracil interferes with paclitaxel cytotoxicity against human solid tumor cells.

K R Johnson, L Wang, M C Miller, 3rd, et al.

Clin Cancer Res 1997;3:1739-1745.

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