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Experimental Therapeutics |
Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [N. T., W. L., D. B., Y. G., Y. W-T., T-C. C., K. W. S., J. R. B.]; Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [N. T., M. F. B.]; and Department of Surgery, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan [N. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Breast cancer is the second leading cause of cancer deaths in American women (10) . Currently there is no curative therapy for metastatic breast cancer. Although many active cytotoxic agents are used in the treatment of this disease, their use is limited by inherent or acquired tumor cell drug resistance. In addition, these cytotoxic agents are also associated with often severe, dose-limiting, systemic toxicities. Therefore, the need for development of novel therapeutic agents active against breast cancer remains an important goal.
The work presented in this paper describes the in vitro and in vivo activity of ET-743 alone and as well as in combination with several antitumor drugs against three human breast cancer cell lines, MX-1, MCF7, and MCF7/ADR that overexpresses P-gp. The combination effects of ET-743 with these agents were analyzed by the CI (11, 12, 13, 14, 15, 16) . Our goal was to determine whether combinations of these agents would produce enhanced antitumor effects, which could suggest potential combination chemotherapy therapeutic strategies for the treatment of breast cancers.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
MCF7 cells were obtained from the American Type Culture Collection (Manassas, VA). The MCF7/ADR cell line that overexpresses P-gp and the MX-1 cell line were kindly provided by T. C. Chou. MCF7 and MCF7/ADR cells were maintained as monolayer cultures in MEM containing 10% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, and 1.5 g/liter of sodium bicarbonate. MX-1 was grown in Dulbecco’s Modified Eagle High Glucose (contains 4500 mg/liter D-glucose) media plus 10% FBS and 2 mM L-glutamine. Cell lines were maintained at 37°C in 5% CO2.
SRB Cytotoxicity Assay.
Cytotoxicity to drugs was determined by the SRB cytotoxicity assay using 96-well microtiter plates as described previously (17)
. In this assay, cells are fixed with tricloroacetic acid and are stained for 30' with SRB, which measures the intracellular protein concentration and provides a sensitive measure of drug-induced cytotoxicity. Cells were plated in duplicate wells (5000 cells/well) and exposed to drugs at different concentrations. After 96 h of incubation with drugs, cells were fixed with 50% tricloroacetic acid solution for 1 h and 0.4% SRB (Sigma Chemical Co.) was added to each well. After a 30-min incubation, the plates were washed and 10 mM Tris buffer was added, and the solution was read at 570 nm on a BioWhittaker microplate reader (2001). The wells with cells containing no drugs and with media plus drugs but no cells were used as positive and negative controls, respectively.
Concurrent Exposure to ET-743 with other Antineoplastic Drugs.
Cells were seeded into 96-well plates as described previously. Cells were treated with serial dilutions of each drug individually and with both drugs simultaneously at a fixed ratio of doses that typically corresponded to 0.125, 0.25, 0.5, 1, 2, and 4 times the individual IC50s. After 96 h of exposure, growth inhibition was measured using the SRB assay. For the in vivo experiments, narrow range combination ratios were used (e.g., in Table 3
, paclitaxel/ET-743 ratios were 1:4, 1:2.67, and 1:2).
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, paclitaxel/ET-743 ratios were 1:4, 1:2.67, and 1:2).
Determination of Synergism and Antagonism and Construction of Isobolograms.
The CI was calculated by the Chou-Talalay equation, which takes into account both potency (Dm or IC50) and the shape of the dose-effect curve (the m value; Refs. 11, 12, 13
). The general equation for the classic isobologram (CI = 1) is given by:
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The (Dx)1 or (Dx)2 can be readily calculated from the median-effect equation of Chou et al. (13
, 14)
:
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a/(1 - a)] or Dm = 10-(Y-intercept)/m, and m is the slope of median-effect plot. Computer software of Chou et al. (15)
and Chou et al. (16)
allows automated calculation of m, Dm, Dx, and CI values. From (Dm)1, (Dx)2, and D1 + D2, it becomes easy to construct isobolograms automatically based on the first equation (11
, 15)
.
Apoptosis Assay.
Two different assays were used to determine cell death, staining with DAPI to measure apoptosis and cell cycle analysis for sub-G1 cells, a measure of dead cells. In the DAPI assay, the cells were first washed twice with PBS and fixed with 4% formaldehyde for 1 h. The fixed cells then washed again with PBS and stained with 10 µg/ml DAPI (Sigma Chemical Co.) for 15 min. The cells containing condensed or fragmented nuclei were examined under a fluorescence microscope, and 200 cells were scored for the percent of apoptotic cells.
In the other assay for apoptotic cells, cells were harvested after treatment and fixed with ice-cold 70% ethanol. DNA content of the sub-G1 fraction, which indicates dead cells, was determined by fluorescence-activated cell sorting analysis after propidium iodine staining.
MPM-2/PI Bivariate Flow Cytometry.
The MPM-2 antibody recognizes the phosphorylated epitope found in phosphoproteins such as MAP2, HSP70, cdc25, and DNA topoisomerase II 
, most of which are phosphorylated at the onset of mitosis. The positive labeling of MPM-2 correlates with entry into mitosis, whereas the dephosphorylation of these proteins correlates with the onset of anaphase. The cells containing 4N DNA content and labeling positive for MPM-2 are likely to be in M phase (18)
. MX-1 and MCF7 cells were treated with or without drugs for the indicated times. Cells were then collected and fixed with ice-cold 70% ethanol. After washing with PBS containing 0.05% Tween 20 and 1% FBS, cells were labeled with MPM-2 antibody (final concentration of 1 µg of MPM-2 antibody/ml) for 1 h at 4°C. Cells were washed once with PBS and incubated with antimouse IgG conjugated with FITC antibody (Santa Cruz, CA) for 1 h at room temperature in the dark. After washing with PBS, cells were resuspended in 5 µg/ml PI containing 50 µg/ml RNase A. Samples were analyzed on a FACScan (Becton Dickinson, Mountain View, CA), and data were analyzed using CellQuest software. MI was defined as percentage of MPM-2-positive cells.
Animals.
Athymic nude mice bearing the nu/nu gene were used for MX-1 xenografts. Mice were obtained from Taconic Farms (outbred, Swiss background). Female mice 5–6 weeks old, weight 18–20 g, were used. For i.v. injection, the drug was administered via the tail vein. Tumor volume was assessed by measuring length x width x height using a caliper. All animal studies were conducted in accordance with the guidelines of the NIH "Guide for Care and Use of Animals" and an approved protocol reviewed by the Memorial Sloan-Kettering Cancer Center’s Institutional Animal Care and Use Committee.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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summarizes the IC50s for three cell lines after 96 h of exposure to ET-743 or other antineoplastic drugs. The three breast cancer cell lines were more sensitive to ET-743 than to the other antineoplastic agents tested. The IC50 of ET-743 was 2.5-fold higher in MCF7/DXR cells that overexpressed P-gp than in parental MCF7 cells. These IC50 concentrations were used to generate fixed ratios for combination studies.
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and 2A
, left), except for concomitant treatment of MCF7/DXR cells. The CI values calculated for the ET-743 followed by paclitaxel sequence were consistently >1 (Fig. 2A
, left), indicating that the effect of the two agents are less than additive using these two schedules in three cell lines. A different picture emerged when paclitaxel preceded ET-743 (Fig. 2A
, right). With this sequence, ET-743 enhanced the cytotoxicity of paclitaxel. The apparent IC50 of paclitaxel was diminished by ET-743 when the two agents were combined in this sequence: CI values were 0.73 ± 0.01, 0.64 ± 0.07, 0.56 ± 0.06, and 0.51 ± 0.07 at 50, 75, 90, and 95% cell kill, respectively, in MX-1 cells; 5.43 ± 1.34, 0.57 ± 0.33, 0.42 ± 0.13, and 0.53 ± 0.06 at 50, 75, 90, and 95% cell kill, respectively, in MCF7 cells; and 1.05 ± 0.32, 0.95 ± 0.16, 0.87 ± 0.02, and 0.81 ± 0.06 at 50, 75, 90, and 95% cell kill, respectively, in MCF7/DXR cells. Each CI is presented as a mean ± SD (Fig. 2A
, right and Table 2
).
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. When MCF7 cells were treated with ET-743 and paclitaxel concomitantly, antagonism was observed again. A moderate synergistic effect was observed when ET-743 was administrated either 6 or 24 h after paclitaxel; the greatest reduction in the IC90s was observed when ET-743 was given 24 h after paclitaxel (Fig. 3B)
.
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and 5
). Cells treated with paclitaxel alone for 24 h show approximately a 7-fold increase in the percentage of cells in G2-M phase of cell cycle compared with control (Fig. 4)
, and the MPM-2 antibody assay showed that 43 percent of the cells treated with paclitaxel alone for 24 h accumulated in M phase (Fig. 5)
. Exposure to ET-743 alone at the IC90 concentration for 72 h led to pronounced S-G2-M accumulation, and the MI of cells measured by the MPM-2 antibody treated with ET-743 alone for 72 h was very low, indicating that ET-743 treated cells accumulate in late S to G2 phase. Concurrent exposure to ET-743 and paclitaxel for 24 h reduced the extent of paclitaxel-induced M-phase accumulation (Fig. 5)
. Moderate interference with paclitaxel-mediated M-phase arrest was noted also when ET-743 was added 6 h after paclitaxel, however, 
18% of the cells in this sequence had already entered M phase at 24 h. Interestingly, an increase in the G1 phase for cells treated in this manner was observed (Fig. 4)
, as compared with cells treated with paclitaxel alone. These data suggest that the cells might have more of a chance to be affected by the sequence paclitaxel
ET-743. Cells accumulating in M phase will be first killed by paclitaxel, and then surviving cells entering G1-phase cells may be next killed by ET-743.
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, indicating that these cells were presumably killed by necrosis.
Sequential Treatment of Paclitaxel followed by ET-743 of Mice Bearing MX-1 Breast Xenografts.
To further evaluate the potential therapeutic effects of combination therapy using ET-743 and paclitaxel and to extend our observations to in vivo studies, human breast cancer xenografts in athymic mice were tested. Treatment was withheld until the 10th day after implantation to allow measurements of tumor regression. When paclitaxel was injected q3dx3 alone (20 mg/kg), the average tumor volume was reduced by 98.5% by day 21 relative to the control, however, 2 of 9 animals died of toxicity (Table 3)
. When ET-743 was injected q3dx3 alone (50 µg/kg), the average tumor volume was decreased by 82.3% by day 21, relative to the control without toxic deaths. To determine additive effects or synergism, the concentration of ET-743 was fixed at 40 µg/kg, and the sequencing effect of paclitaxel followed by ET-743 treatment was studied, and the data analyzed using nonconstant molar ratio analysis. Results are summarized in Table 3
. Synergistic cytotoxic effects were observed without an increase in lethal toxicity. When 40 µg/kg ET-743 was given 6 h after 20 mg/kg of paclitaxel, the CI value was 0.74, indicating moderate synergy. In addition, the dose reduction index values indicated that the paclitaxel and ET-743 concentration could be reduced 2.1- and 3.8-fold, respectively, to achieve the same degree of tumor regression. Moreover, the number of tumor-free mice increased from three of nine to eight of nine with sequential treatment.
Effect of Combining ET-743 with DXR.
When cells were treated with ET-743 and DXR concomitantly or in the sequence DXR followed by ET-743, a less than additive effect was observed in the three cell lines (Figs. 1
and 2B
, right). In contrast, cytotoxic synergy was seen when ET-743 preceded DXR against MCF7 cell lines, CI values were 1.06 ± 0.4, 0.66 ± 0.22, 0.41 ± 0.12, and 0.30 ± 0.08 at 50, 75, 90, and 95% cell kill, respectively; however, this sequence-dependent synergy was not observed in MX-1 and MCF7/DXR cell lines.
Effect of Combining ET-743 with 5-FU or CPT.
Moderate antagonism effects were observed for the combination of ET-743 and 5-FU when 5FU was administered concomitantly with ET-743; CI values were 1.08 ± 0.01, 1.14 ± 0.09, 1.21 ± 0.28 and 1.29 ± 0.31 at 50, 75, 90, and 95% cell kill, respectively, in MX-1 cells; and 1.06 ± 0.01, 1.31 ± 0.07, 1.64 ± 0.15, and 1.91 ± 0.22 at 50, 75, 90, and 95% cell kill, respectively, in MCF7 cells. Sequence-dependent synergistic interaction as seen for paclitaxel was not observed. Only when the cells were treated in the ET7435-FU sequence was slight synergy observed in the MCF7 cell line (Fig. 2C
, left).
To determine whether similar antagonistic effects could be observed with another S-phase-specific agent, the effects of ET-743 in combination with the topoisomerase I poison CPT was examined. The effect of combining ET-743 with CPT was somewhat different. In MX-1 cell lines, a slight synergistic effect was observed when ET-743 was administered with CPT concomitantly. For MCF7 cell lines, ET-743 with CPT also showed a slight synergistic effect when the two drugs were given concomitantly (Fig. 1)
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Effect of Combining ET-743 with CDDP.
Synergy was demonstrated for the combination of ET-743 and CDDP when CDDP was administered concomitantly (CI, 0.42 ± 0.33 and 0.74 ± 0.11 at 75 and 95% cell kill, respectively), following (CI, 0.99 ± 0.17 and 0.86 ± 0.13 at 75 and 95% cell kill, respectively) and preceding (CI, 0.74 ± 0.04 and 0.64 ± 0.17 at 75 and 95% cell kill, respectively) with ET-743 against MCF7 cell lines. CDDP was the only drug examined that exhibited sequence-independent cytotoxicity synergy with ET-743 against MCF7 cells. However, the examination of a reduction in drug IC50s obtained from the survival curves indicated that the most pronounced effect occurs when CDDP is administered before ET-743. In contrast, this sequence-independent synergy was not observed in MX-1 and MCF7/DXR cell lines, and a less than additive effect was noted for the combination of ET-743 and CDDP when CDDP was administered before ET-743.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The importance of sequence of administration is illustrated by the combination of paclitaxel and ET-743. When ET-743 is administered concomitantly or before paclitaxel, strong cytotoxic antagonism was observed. In contrast, the same agents cause greater than additive cytotoxicity when paclitaxel was administered before ET-743 in all three human breast cancer cell lines. These effects were not limited to breast cancer cell lines but were also observed in human soft tissue sarcoma cell lines (data not shown). In addition, in nude mice bearing human MX-1 breast cancer xenografts, the sequential combination of paclitaxel followed by ET-743 produced a greater tumor volume reduction than either of the chemotherapeutic agents alone, and a greater number of tumor-free mice were observed with this sequential treatment than with paclitaxel or ET-743 alone. In addition, when 40 µg/kg ET-743 were given after 15 mg/kg paclitaxel in tumor-bearing mice, none of the mice died of drug toxicity in contrast to treatment with 20 mg/kg paclitaxel-alone treatment.
Cell cycle analysis revealed some differences between the cytotoxic action of ET-743 and paclitaxel. When the cells were treated with paclitaxel, the IC90 concentration, cells accumulated in the G2-M phase and produced a marked M-phase accumulation after 24 h of exposure. In contrast, ET-743 treated cells showed a late S to G2-phase accumulation after 72 h of treatment. Similar observations on cell cycle progression of ET-743 treated cells have been made by D’Incalci et al. (19)
. Analysis of cell cycle progression provides a clue to the underlying mechanism of the antagonistic effect seen in MCF7 cell lines (Fig. 4)
. This study revealed that the percentage of M-phase cells associated with paclitaxel-induced cell death decreased when ET743 was administered concomitantly. The ET-743 induced abrogation of G2-M phase checkpoint with tricostatin treatment was also reported by Scotto et al. (8)
. Both MX-1 and MCF7 cell lines treated with ET-743 alone at a concentration ranging from 2 to 10 nM, respectively, accumulated slowly in the G2 phase of the cell cycle (data for MX-1 not shown). In addition, there was an increase in the sub-G1 population, which represents an increase in dead cells. ET-743 may exert a second mechanism of toxicity at a phase of the cell cycle other than G2 arrest. Interestingly, an increase in the percentage of cells in the G1 phase was seen for MCF7 cells when paclitaxel was given before ET-743 (Fig. 4)
. This increase in G1 phase cells may be associated with the synergism observed for this sequence treatment. Recently, Erba et. al. (20)
reported that the cells in G1 phase were more sensitive to ET-743 than in other phases of the cell cycle (20)
. Takebayashi et al. (21)
demonstrated that HCT-116 cells treated with a high concentration of ET-743 accumulated in G1 phase. These data support our hypothesis that an increase of cells re-entering the G1 phase from M phase by sequential treatment with paclitaxel followed by ET-743 may increase cell kill. In fact, a decrease of the number in multinuclear cells, which may relate to early paclitaxel resistance (22)
, was observed in this sequential treatment compared with paclitaxel alone treatment (data not shown).
The sequence-dependent synergy observed may be explained by more than one mechanism, including G2-M accumulation, a non-G2-M-dependent cytotoxic effect, and exposure time and concentration. A possible reason for the observed synergistic effect of ET-743 and paclitaxel was proposed by Synold et al. (23) . They reported that ET-743 inhibited the orphan nuclear receptor SXR induced by paclitaxel. SXR regulates paclitaxel metabolism and efflux by CYP34, CYP2C8, and P-gp. However, the exact mechanism of this sequence-dependent synergistic cytotoxic effect of ET-743 and paclitaxel are unclear.
Another drug used to treat breast cancer, 5-FU, appears not to be a good partner of ET-743. Moderate antagonism was observed for the combination of ET-743 and 5-FU when ET-743 was administered concomitantly. This antagonistic cytotoxicity was not improved by altering the sequence schedule. In addition, when MCF7 cells were treated with another antifolate, trimetrexate, and ET743 concomitantly, antagonistic cytotoxicity was also observed, and this antagonism was not changed by altering the schedule (data not shown).
In MCF7 cell lines, synergistic cytotoxicity was observed when ET-743 was administered before DXR (Fig. 1)
. ET-743 has been reported to inhibit transcription of stress induction of the mdr-1 gene (8
, 9)
. If ET-743 prevents mdr-1 up-regulation, administration of ET-743 and paclitaxel or DXR may exhibit synergism. Although similar synergistic results were not seen in MX-1 and MCF7/DXR cell lines, it is possible that DXR in combination with ET-743 may be an effective combination. ET-743 administration followed by DXR also resulted in synergistic cytotoxicity against human soft tissue sarcoma cell lines (data not shown).
In summary, our studies suggest that ET-743 exhibits marked sequence-dependent synergistic effects when administered after paclitaxel against human breast cancer cell lines in vitro and in vivo and provides a rationale for future clinic studies of this combination.
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FOOTNOTES |
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1 Supported by United States Public Health Service Grant PO1-CA-47179 and a fund from The Jikei University Alumni Association. 
2 To whom requests for reprints should be addressed, at 195 Little Albany Street, Room 3033, New Brunswick, NJ 08903. Phone: (732) 235-8510; Fax: (732) 235-8181; E-mail: bertinoj{at}umdnj.edu
3 The abbreviations used are: ET-743, ecteinascidin-743; 5-FU, 5-fluorouracil; FBS, fetal bovine serum; CI, combination index; SRB, sulforhodamine; DAPI, 4',6-diamidino-2-phenylindole; DXR, doxorubicin; P-gp, P-glycoprotein; MI, mitotic index; PI, propidium iodide; CPT, camptothecin; CDDP, cisplatin.
Received 9/26/01. Accepted 10/ 3/02.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), 75 (
), 90 (
), and 95 (
)% fraction killed and are means of at least three independent experiments; bars, SD.
