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Clinical Cancer Research Vol. 7, 391-399, February 2001
© 2001 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Combination of Standard Cytotoxic Agents with Polyamine Analogues in the Treatment of Breast Cancer Cell Lines1

Hillary A. Hahm2, Valerie R. Dunn, Kimberly A. Butash3, Wendy L. Deveraux, Patrick M. Woster, Robert A. Casero, Jr. and Nancy E. Davidson4

The Johns Hopkins Oncology Center, Johns Hopkins University, Baltimore, Maryland 21231 [H. A. H., V. R. D., K. A. B., W. L. D., R. A. C., N. E. D.], and Wayne State University, Detroit, Michigan 48202 [P. M. W.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyamines are essential for cell growth and differentiation. Structural polyamine analogues have been shown to have antitumor activity in experimental models including breast cancer. The ability of polyamine analogues to alter activity of cytotoxic chemotherapeutic agents in breast cancer models has not been evaluated. This study evaluates the ability of two polyamine analogues, N1-ethyl-N11-[(cyclopropyl)methyl]-4,8-diazaundecane (CPENSpm) and N1-ethyl-N11-[(cycloheptyl)methyl]4,8-diazaundecane (CHENSpm) to synergize with cytotoxics in five human breast cancer cell lines. Antagonism, additivity, or synergy of the combinations was determined using the median effect/combination index model. The chemotherapeutic agents chosen, cisdiaminechloroplatinum(II), doxorubicin, 5-fluorouracil, fluorodeoxyuridine, 4-hydroperoxycyclophosphamide, paclitaxel, docetaxel, and vinorelbine, all have antitumor activity in breast cancer and represent a spectrum of mechanisms. Three treatment schedules of polyamine analogue and cytotoxic were tested in MCF-7 and MDA-MB-468 lines, demonstrating a schedule-dependence of synergistic growth inhibition. Cytotoxic agent alone for 24 h followed by polyamine analogue alone for 96 h resulted in the most synergistic combinations and the greatest synergy. This schedule was then tested in three additional breast cancer lines, and several synergistic combinations were again identified. Two cytotoxics, vinorelbine and the fluoropyrimidines, showed the most promise in combination with the polyamine analogues. They were able to synergize with one or both polyamine analogues in most of the breast cancer cell lines. CPENSpm was also able to synergize with virtually all of the cytotoxics in the estrogen receptor {alpha}-positive MCF-7 and T-47D lines. These preclinical data demonstrate a treatment schedule and combinations of polyamine analogues and cytotoxics that will be important to study mechanistically and clinically for breast cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyamines are essential for cell growth and differentiation and the finding that polyamine levels are increased in malignant versus normal tissues (1, 2, 3) has implicated the polyamine metabolic pathway as a target for antineoplastic therapy (2 , 4 , 5) . Investigators have synthesized structural analogues that can mimic the natural polyamines in their self-regulatory role, yet are unable to substitute for polyamines in terms of supporting cell growth and differentiation (5 , 6) . These analogues have been shown to have antitumor activity in multiple experimental model systems including breast cancer (7, 8, 9, 10, 11, 12, 13, 14) . In vitro growth of several breast cancer cell lines is inhibited by several spermine analogues including the n-alkylated, symmetrically substituted analogues, DESpm5 (also known as BESpm) and DENSPM (also known as BENSpm), and the unsymmetrically substituted compounds, CPENSpm and CHENSpm (9 , 14) . In addition, several of these analogues induce PCD in breast cancer cell lines (14) .

The polyamine analogue DENSPM has been evaluated in Phase I clinical trials (15 , 16) . Using a once-daily infusion schedule (for 5 days, repeated every 21 days), the drug was well tolerated, and gastrointestinal toxicity was the dose-limiting toxicity. There was no significant hematological toxicity, and this schedule is currently being evaluated in Phase II studies. Pharmacokinetic analysis using this dosing schedule demonstrated patient plasma concentrations in the micromolar range that are consistent with concentrations required in vitro for inhibition of cell growth and induction of PCD. The antitumor activity of polyamine analogues in multiple experimental model systems, as well as the preliminary clinical data available for the analogue DENSPM, attest to the therapeutic potential of this class of agents.

Although all of the roles of polyamines in cell proliferation are not known, the capacity to interact with DNA (17) and affect DNA conformation (18) are thought to play a role in their normal cellular function. Therefore, several investigators have evaluated whether depletion of polyamine pools can modulate the activity of DNA-reactive drugs in tumor model systems. These studies have generally combined chemotherapeutic agents with compounds that deplete polyamine pools via inhibition of key biosynthetic enzymes such as ornithine decarboxylase or S-adenosylmethionine decarboxylase (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) . The results of these experiments have been mixed, with some combinations demonstrating synergistic or additive activity and others demonstrating antagonism.

Only two combination studies have been done in breast cancer models. Thomas and Kiang (36) evaluated the activity of antiestrogens in combination with DFMO, an inhibitor of ornithine decarboxylase in the breast cancer cell line MCF-7 and demonstrated additive activity of these agents on cell growth inhibition. Das et al. (35) showed that concomitant or pre- or post-paclitaxel exposure of MCF-7 cells to DFMO resulted in antagonism of paclitaxel-induced cell growth inhibition and apoptosis.

Combined effects of cytotoxic chemotherapeutic agents and polyamine analogues in preclinical models of breast cancer have not been assessed. In this report, we evaluate the activity of the polyamine analogues, CPENSpm and CHENSpm, in combination with multiple cytotoxic chemotherapeutic agents currently in use in the treatment of breast cancer in breast cell lines, in vitro.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Compounds, Cell Lines, and Culture Conditions.
CPENSpm and CHENSpm were synthesized as described previously (37) . For all of the experiments, a concentrated solution (10 mM in water, stored at -20°C) was diluted in medium to desired concentration. 5-FU, c-DDP, and vinorelbine were obtained from the Johns Hopkins Oncology Center pharmacy. 5-FU and c-DDP were stored at -20°C, and vinorelbine at 4°C. Paclitaxel (a gift from Bristol-Myers/Squibb) was stored at 4°C as a 10-mM solution in DMSO. Docetaxel, a gift from Rhone Poulenc/Rorer, was stored at -20°C in absolute ethanol. 4HC was a gift from Dr. O. Michael Colvin (Duke Cancer Center, Durham, NC). 4HC was stored in powder form at -20°C and dissolved in fresh cell culture medium immediately prior to its use. FdURd and doxorubicin were obtained from the Sigma Co. and stored at -20°C in water and DMSO, respectively. All of the drugs were diluted in cell culture medium to desired final concentrations except for docetaxel, which was initially diluted in water and then in cell culture medium. The acquisition and maintenance of the breast cancer cell lines, MCF-7, T-47D, MDA-MB-468, Hs 578T, and MDA-MB-231, have been previously described (38) .

Growth Inhibition Assays.
Cells in the exponential growth phase were plated at 1–5 x 104 cells/cm2 in 24-well tissue culture plates. After attachment overnight, the medium was changed, and the cells were incubated with or without drugs for the desired exposure times. After 120 h, the cells were detached by trypsinization and counted using a Coulter counter. Percentage growth inhibition was determined by comparison of cell number per well in treated versus control cells.

Growth inhibition was also assessed using the MTT (Sigma Chemical Co.) dye assay (39) . For the MTT assay, cells were plated in 96-well dishes and treated as above. On completion of the treatment period, the media was discarded, and 100 µl of MTT (5 mg/ml in culture medium filter sterilized) was added to each well for 4 h at 37°C. The MTT solution was then removed, and the formazan crystals were dissolved in 200 µl/well of a 1:1 (v/v) solution of DMSO:ethanol for 20 min at ambient temperature. Change in absorbance was determined at A540 nm. Results were compared with wells that contained culture medium but no cells, and percentage growth inhibition was calculated by comparison of the A540 nm reading from treated versus control cells. Drug concentrations that resulted in an IC50 were determined from the plots of percentage growth inhibition versus the logarithm of the drug concentration. All of the experiments were plated in triplicate wells and were carried out at least twice. Prior to the usage of the MTT assay in experiments, the results were validated by direct comparison of results from MTT assay and conventional cell growth assays; results were consistently comparable.

Clonogenic Assay.
For colony formation assay, 200 MCF-7 cells per 60-mm tissue culture dish were allowed to attach overnight. The chemotherapeutic drug of interest was then added on day 0. After 24 h, media were removed, and the cell monolayer washed with drug- and serum-free media. The cells were then exposed for the remainder of the culture period to media containing the polyamine analogue alone. After 10 days, the cell monolayer was washed once with PBS, stained with crystal violet [0.5% crystal violet in a 3:1 (v/v) mixture of water to methanol], washed with water, and allowed to dry at ambient temperature. Visible colonies were counted.

Synergy Studies.
The median effect/CI Analysis (40) was used to determine antagonism, additivity, or synergy of combination exposures to both polyamine analogues and cytotoxic drugs. Cell cultures were treated with each agent individually at its IC50 concentration and at fixed multiples (two and three times) and fractions (0.75, 0.50, and 0.25) of the IC50 concentrations. The agents (polyamine analogue and drug) were also combined in these same dose-fixed ratios to determine CI. Antagonism was defined as any CI value above 1, additivity as CI = 1, and synergy as <1 ± SD. Experiments were done in triplicate, and each experiment yielded one CI value. Experiments that yielded a CI of less than 1 were repeated at least three times to allow for determination of SD for the CI values obtained. Experiments that yielded CI values of >1 were repeated once if the results were consistent, and the CI value shown is a representative value from one of these experiments. CI values are shown only for fractional growth inhibition levels of 0.50 or greater, because dose intensity is known to be important in breast cancer treatment (41) .

Treatment Schedules.
Three different treatment schedules were used to mimic schedules that are potentially clinically relevant. The first treatment schedule used simultaneous exposure to both polyamine analogue and cytotoxic drug for 120 h. In the second treatment schedule, the cells were exposed to 24 h of cytotoxic drug (starting on day 0). The medium was then discarded, the cell monolayer washed once with drug-free medium, and fresh medium containing the polyamine analogue was added for the remainder of the culture period (96 h). The third treatment schedule evaluated cell exposure to polyamine analogue alone for 24 h followed by removal of the medium and addition of medium containing both polyamine analogue and drug for the remainder of the culture period (96 h). Sustained exposure to the polyamine analogue was used in all of the treatment schedules because other studies have shown that lengthy exposure is necessary for optimal polyamine analogue activity (5) .

Analysis of Polyamine Content.
The polyamine content of treated and untreated cells was determined by precolumn dansylation, reversed-phase, high-performance liquid chromatographic methods of Kabra et al. (42) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The estrogen receptor positive, wild-type p53 MCF-7 cells and the estrogen receptor negative, mutant p53 MDA-MB-468 cells were chosen for these studies because they are representative of hormone-dependent and -independent breast cancer cells. Six chemotherapeutic agents (c-DDP, doxorubicin, 5-FU, vinorelbine, paclitaxel, and docetaxel) were tested in combination with the two polyamine analogues (CPENSpm and CHENSpm) in both lines using the three different treatment schedules. In addition, FdURd and 4HC were tested using the treatment schedule of cytotoxic drug followed by polyamine analogue.

Effects of CPENSpm or CHENSpm and Chemotherapeutic Drugs on MCF-7 Cells.
The schedule of drug exposure for 24 h followed by CPENSpm for 96 h in MCF-7 cells showed a synergistic effect on growth for all of the eight cytotoxic drugs at a fractional growth inhibition of 0.50 or greater (Table 1)Citation . The greatest degree of synergy was seen with the fluoropyrimidines and vinorelbine. In contrast, concurrent exposure to drug and CPENSpm for 120 h resulted in synergistic growth inhibition only with 5-FU and vinorelbine at fractional growth inhibition of 0.75. Similarly, the schedule of CPENSpm, followed by CPENSpm and drug, led to synergistic growth effects only with doxorubicin, c-DDP, paclitaxel, and docetaxel. The degree of synergy and the range of fractional growth inhibitions for which synergy was seen were also less with this sequence than those seen with the schedule of drug exposure followed by CPENSpm.


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  		Table 1 Effects of CPENSpm and chemotherapeutic drugs on MCF-7 cellsa

 
Because these studies were performed using the MTT assay, similar studies were performed using the colony formation assay rather than growth inhibition as an end point to validate the MTT assay (data not shown). Agreement between results of the clonogenic assay and the growth inhibition studies was seen, thereby validating the use of the MTT assay.

Identical studies were undertaken using CHENSpm and cytotoxics in MCF-7 cells (Table 2)Citation . Evidence for synergistic interaction was seen only with the sequence of cytotoxic agent followed by CHENSpm. Unlike CPENSpm, synergy was seen with fewer drugs including c-DDP and paclitaxel at a fractional growth inhibition of 0.90, 5-FU and FdURd at a fractional growth inhibition of >=0.75, and vinorelbine at a fractional growth inhibition of >=0.50. No synergistic combinations were seen with the treatment schedule of concurrent treatment or CHENSpm followed by the combination of cytotoxic and CHENSpm.


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  		Table 2 Effects of CHENSpm and chemotherapeutic drugs on MCF-7 cellsa

 
Endogenous Polyamine Levels and Analogue Levels in Combination Studies in the MCF-7 Cells.
A key question is whether the observed growth inhibitory effects of combinations of polyamine analogues and cytotoxics simply reflects effects on intracellular polyamines or on polyamine analogue levels. Therefore, the effect of cytotoxic drugs for 24 h followed by analogue alone for the remainder of the culture period on polyamine levels and analogue levels was assessed. Cells were harvested on day 5 for measurement of polyamines and analogues. In all of the experiments, CHENSpm alone did not substantially perturb endogenous polyamine pools, whereas CPENSpm treatment resulted in depletion of spermidine and spermine. Studies using paclitaxel, docetaxel, and 4HC in combination with CHENSpm or CPENSpm showed that these drugs had no effect on polyamine levels alone or in combination with either analogue (data not shown). Table 3Citation shows the results of similar studies with the fluoropyrimidines, doxorubicin, c-DDP, and vinorelbine. None of these drugs altered polyamine levels when used alone. In contrast, 5-FU or FdURd in combination with CHENSpm or CPENSpm resulted in elevations in both analogue levels compared with treatment with CHENSpm or CPENSpm alone. CHENSpm levels were increased 3- to 6-fold, but this increase was not associated with substantial changes in the polyamine pools. CPENSpm levels increased only 1.1- to 3.5-fold, but these changes were consistently associated with further reduction in all of the three polyamine levels compared with analogue alone. For the combination studies with doxorubicin, c-DDP, and vinorelbine, there was no consistent change in intracellular analogue levels or polyamine levels for the combination of CHENSpm plus cytotoxic when compared with changes seen with CHENSpm or drug alone. But, like fluoropyrimidines, these agents also demonstrated further depletion of polyamine pools in association with a 1.2- to 2.0-fold increase in CPENSpm intracellular concentrations when combined with CPENSpm. It should be noted that the increase in CPENSpm accumulation is offset by an almost exact reduction in charge-complement of the natural polyamines as observed previously by Bergeron et al. (43) .


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  		Table 3 Polyamine and polyamine analogue levelsa on day 5 in the MCF-7 cell line

Cells were treated with drug alone on day 0 for 24 hours; then the medium was removed and the cell monolayer washed with drug- and serum-free medium. Then medium with or without analog was added for the remainder of the culture period (120 h).

 
Effects of CPENSpm or CHENSpm and Chemotherapeutic Drugs on MDA-MB-468 Cells.
Similar studies were carried out using the estrogen receptor-negative MDA-MB-468 cell line. No evidence for synergy was seen for any treatment schedule or with any chemotherapeutic drug in combination with CPENSpm in this cell line (Table 4Citation ). Studies with CHENSpm demonstrated synergy only with treatment with either fluoropyrimidine for 24 h followed by CHENSpm for 96 h at a fractional growth inhibition of >=0.75 for 5-FU and >=0.90 for FdURd (Table 5)Citation .


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  		Table 4 Effects of CPENSpm and chemotherapeutic drugs on MDA-MB-468 cellsa

 

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  		Table 5 Effects of CHENSpm and chemotherapeutic drugs on MDA-MB-468 cellsa

 
Combination Studies Using Other Breast Cancer Cell Lines.
Combination studies using the treatment schedule of cytotoxic drug for 24 h followed by CPENSpm for 96 h led to the greatest number of synergistic combinations in both MCF-7 and MDA-MB-468 cells. Therefore, this treatment strategy was evaluated in three additional breast cancer cell lines, the estrogen receptor-negative MDA-MB-231 and Hs 578T cells and the estrogen receptor-positive T-47D cells as shown in Table 6Citation . Synergistic growth inhibition was seen with all of the drugs except doxorubicin in T-47D cells and with all of the drugs except for doxorubicin and c-DDP in Hs 578T cells. In contrast, only one combination demonstrated synergy in the MDA-MB-231 cells, CPENSpm followed by vinorelbine.


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  		Table 6 Effects of CPENSpm and chemotherapeutic drugs on MDA-MB-231, Hs-578T, and T-47D cells

Cells were treated with drug alone on day 0 for 24 h, then the media was removed and the cell monolayer washed with drug- and serum-free medium. Then medium with CPENSpm was added for the remainder of the culture period (120 h).

 
Finally, because only the two fluoropyrimidines demonstrated synergy when given before CHENSpm in MCF-7 and MDA-MB-468 cells, the sequence of 5-FU or FdURd for 24 h followed by CHENSpm for 96 h was evaluated in MDA-MB-231, Hs 578T, and T-47D cells. Synergy between 5-FU and CHENSpm was seen in all of the three lines, whereas FdURd and CHENSpm interacted in a synergistic fashion only in MDA-MB-231 and Hs 578T cells (Table 7)Citation .


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  		Table 7 Effects of CHENSpm and 5-FU or FdURd on MDA-MB-231, Hs 578T, and T-47D cellsa

Cells were treated with 5-FU or FdURd alone on day 0 for 24 h, then the medium was removed and the cell monolayer washed with drug- and serum-free medium. Then medium with CHENSpm was added for the remainder of the culture period (120 h).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Polyamine analogues have been shown to have antitumor activity as single agents in multiple experimental model systems (7, 8, 9, 10, 11, 12, 13, 14) . Their ability to modulate response to chemotherapeutic agents is worthy of study. This study addressed the activity of two polyamine analogues, CPENSpm and CHENSpm, in combination with multiple chemotherapeutic agents in breast cancer cell lines. The chemotherapeutic agents used were selected because they: (a) have antitumor activity in breast cancer; (b) are currently in use in the treatment of breast cancer; and (c) represent a broad spectrum of mechanisms of action. They include alkylating agents (4HC), topoisomerase II inhibitors (doxorubicin), antimetabolites (5-FU and FdURd), antimitotic agents (vinorelbine, paclitaxel, and docetaxel), and the DNA-reactive agent, c-DDP, which causes both intra- and interstrand DNA adducts.

Synergistic combinations were identified using one or both of the polyamine analogues in all of the cell lines evaluated. There was a schedule dependence for synergy, with the sequence of cytotoxic drug exposure for 24 h followed by polyamine analogue for 96 h resulting in the greatest number of synergistic combinations as well as the greatest magnitude of synergy for the MCF-7 and MDA-MB-468 cell lines. It is unclear why this schedule is superior even when using diverse chemotherapeutic agents in combination with either polyamine analogue in the multiple breast cancer cell lines. Most of the previous combination studies with DFMO and/or MGBG and chemotherapeutic agents have focused on treatment with the enzyme inhibitor initially to perturb polyamine pools before drug therapy based on the hypothesis that resultant changes in DNA conformation may allow for greater drug access. Despite its biological rationale, this schedule gave inconsistent results with some studies demonstrating synergism (21 , 25 , 33) for some DNA-directed agents, whereas others demonstrated antagonism (23 , 25 , 31 , 34 , 35) .

Only two studies published to date have evaluated the activity of combination studies with polyamine analogues and chemotherapeutic agents in in vitro tumor model systems. One study (24) combined the spermidine analogue BESpd with 4'-(9-acridinylamino)methanesulfon-M anisidide (m-AMSA) in a human lung cancer cell line to evaluate the induction of topoisomerase II-dependent drug-induced cleavable DNA complexes. Unfortunately, it did not address cell growth inhibition or colony-forming ability with combination therapy versus drug alone. Marverti et al. (44) studied the effect of the spermine analogue, BESpm, and c-DDP on the growth of c-DDPsensitive and -resistant ovarian carcinoma cells. In the c-DDP-sensitive cell line, concomitant exposure to c-DDP and BESpm demonstrated synergy, whereas the c-DDP-resistant cell line was found to be cross-resistant to BESpm. However, when the colony-forming ability was evaluated after concurrent treatment with both of the agents, there was a synergistic interaction as determined by median effect/CI analysis. Of note, c-DDP in our breast cancer models demonstrated synergy in combination with CPENSpm and CHENSpm in the MCF-7 cell line, and CPENSpm in the T-47D cell line.

CPENSpm and CHENSpm are both spermine analogues with antitumor activity and the ability to induce PCD in multiple experimental model systems, yet they apparently have different mechanisms of action. CPENSpm has been shown to superinduce the catabolic enzyme SSAT in a number of model systems (7 , 10 , 45 , 46) . This superinduction is associated with production of hydrogen peroxide, and increased oxidative stress is believed to be an important mediator in the induction of PCD by this agent in select tumor types (47) . CHENSpm, however, does not superinduce this enzyme in any model system studied; yet it has significant antitumor activity and also induces PCD. In a human lung cancer model, this agent leads to a G2-M cell cycle arrest (47) and alters tubulin polymerization (48) . These agents demonstrated different spectrums of activities in combination with chemotherapeutic drugs in the breast cancer models.

In the breast cancer cell lines studied, two classes of agents, the fluoropyrimidines and vinorelbine demonstrated the most activity in combination with CPENSpm or CHENSpm. 5-FU demonstrated synergy in combination with CPENSpm in three of the five breast cancer cell lines evaluated when drug treatment preceded polyamine analogue exposure. 5-FU and/or FdURd demonstrated synergy in combination with CHENSpm in all of the five cell lines. This class of drugs has not been previously evaluated in combination studies with polyamine analogues, although there have been several studies in epithelial tumor model systems using DFMO in combination with 5-FU with variable results (19 , 32 , 33) .

In contrast, our results in the breast cancer cell lines with two different spermine analogues demonstrate synergistic combinations with one or both analogues in several of the breast cancer cell lines evaluated. These findings warrant further evaluation in in vivo models of breast cancer and examination of the possible mechanisms responsible for synergy. 5-FU is an antimetabolite with several mechanisms of action, including inhibition of thymidylate synthase and incorporation into DNA and/or RNA (49) . Evaluation of cell cycle modulation, total intracellular 5-FU content, specific incorporation into DNA and RNA, and thymidylate synthase activity in the presence of the polyamine analogue are currently being explored. Because different schedules of treatment or the same schedule in a different cell line can result in different responses (additivity, antagonism, or synergy), evaluation of these changes using different treatment schedules and breast cancer cell lines with variable responses should help identify mechanisms that play a role in a synergistic response.

One of the possible mechanisms, the effect of drug on polyamine analogue cellular accumulation and polyamine pool depletion was extensively evaluated in the MCF-7 cell line. This cell line was chosen because it demonstrated synergy with all of the cytotoxic drugs evaluated in combination with CPENSpm as well as with several drugs in combination with CHENSpm using the treatment schedule of drug followed by analogue. Five of eight drugs tested led to increased CPENSpm levels in the cells treated with the combination compared with cells treated with analogue alone, and this increase was associated with further depletion of the polyamine levels. This change may well play an important role in the synergistic response seen with these drugs in combination. Whether the increased level of CPENSpm is attributable to decreased efflux or to alterations in metabolism will need to be evaluated. In addition, it will be interesting to see whether this phenomenon occurs as well in in vivo model systems as well, and how these changes might correlate with effects on tumor growth.

Similar studies of intracellular polyamines and CHENSpm levels in MCF-7 cells showed that substantial increases in CHENSpm levels occurred only in combination with the fluoropyrimidines. What role this may play in the synergy that is seen with those agents and CHENSpm remains to be defined. Synergistic responses to drug combinations may be attributable to more than one mechanism. Thus, the finding of changes in polyamine analogue levels in combination studies will need to be evaluated in several other breast cancer cell lines to see whether this relationship between synergy and increased intracellular analogue levels holds true. Not all of the synergistic drug combinations in the MCF-7 cell line were associated with changes in polyamine pools or analogue levels, which suggests that alternate mechanisms are involved in these synergistic responses with certain chemotherapeutic agents.

The combination of vinorelbine, an antimitotic agent that binds tubulin resulting in microtubule depolymerization (50) , and CPENSpm demonstrates synergy in all of the breast cancer cell lines except MDA-MB-468. But when used with CHENSpm in the MCF-7 and MDA-MB-468 cell lines, vinorelbine was antagonistic in the MDA-MB-468 cell line and synergistic in the MCF-7 cell line. Polyamines may play a role in the natural dynamics of microtubules (51 , 52) , and some polyamine analogues have been shown to induce a G2-M cell cycle arrest and alter tubulin dynamics (47 , 48) . The positive interaction between polyamine analogues and vinorelbine may be mediated by further modulation of this pathway. Of note, the taxanes, paclitaxel and docetaxel, which are known to stabilize tubulin polymerization, have been shown to synergize with the polyamine analogues as well, albeit under more limited experimental conditions.

In addition, it is noteworthy that the combination of CPENSpm with virtually all of the cytotoxics had synergistic effects in the MCF-7 and T-47D cells. Because these drugs represent a spectrum of different mechanisms of action, their extensive ability to synergize with CPENSpm is intriguing. This result implies that the analogue may be modifying a common pathway by which all of the these drugs work to produce an antitumor response. It is known that most chemotherapeutic agents induce PCD (53 , 54) . Polyamine analogues are also known to induce PCD in multiple tumor types including breast cancer (13 , 14 , 55 , 56) . Studies using the CPENSpm analogue in the H157 human lung cancer cell line have shown that production of oxidative stress via hydrogen peroxide production in the cell because of SSAT induction by this analogue is a component of cell death (47) . Whether this pathway is involved in the synergistic response seen with combination therapy in these cell lines remains to be evaluated.

Finally, both the MCF-7 and T-47D cell lines are estrogen receptor positive and dependent on estradiol for cell growth. It will be important to determine whether other estrogen receptorpositive breast cancer cell lines also demonstrate synergy with CPENSpm and a broad spectrum of cancer chemotherapeutic agents. If this is the case, the role of estrogen receptor-dependent proliferation pathways in breast cancer cells and their association with the activity of polyamine analogues may be important to investigate.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from The Department of Defense Breast Cancer Program DAMD 17-99-1-9231 (to N. E. D.), Department of Defense Breast Cancer Program DAMD17-97-1-7338 (to H. A. H.), The Breast Cancer Research Foundation (to N. E. D.), The National Cancer Institute CA51085 (to R. A. C.), The National Cancer Institute CA85509 (P. M. W.), and The Pearl M. Stetler Research Fund for Women Physicians (H. A. H.). Back

2 Present address: Northwest Georgia Oncology Centers, P.C.55 Whitcher Street, Suite 300, Marietta, GA 30060. Back

3 Present address: Life Technologies, 9800 Medical Center Drive, Rockville, MD 20850. Back

4 To whom requests for reprints should be addressed, at Johns Hopkins Medical Institutions, Cancer Research Building, 1650 Orleans Street, Room 409, Baltimore, MD 21231. Phone: (410) 955-8489; E-mail: davidna{at}jhmi.edu Back

5 The abbreviations used are: DESpm, N1,N12-diethylspermine (also known as BESpm, N1,N12-bis(ethyl)spermine); DENSPM, N1-N11-diethylnorspermine (also known as BENSpm, N1,N11-bis(ethyl)norspermine); CPENSpm, N1-ethyl-N11-[(cyclopropyl)methyl]-4,8-diazaundecane and CHENSpm, N1-ethyl-N11-[(cycloheptyl)methyl]4,8-diazaundecane; BESpd,N1,N8 bis(ethyl)spermidine, c-DDP, cis-diaminechloroplatinum(II); 5-FU, 5-fluorouracil; FdURd, fluorodeoxyuridine; 4HC, 4-hydroperoxycyclophosphamide; DFMO, difluoromethylornithine; MTT, 3-(4,5-dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide; CI, combination index; PCD, programmed cell death; SSAT, spermidine/spermine N1-acetyl-transferase; MGBG, methylglyoxal-bis(guanylhydrazone). Back

Received 7/20/00; revised 11/20/00; accepted 11/20/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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