| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics, Preclinical Pharmacology |
1 Department of Pharmacology and Experimental Therapeutics, 2 Division of Biostatistics, and 4 University of Maryland Greenebaum Cancer Center and Department of Oncology, University of Maryland School of Medicine, Baltimore, Maryland; and 3 Mayo Clinic College of Medicine, Rochester, Minnesota
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Experimental: Aromatase-transfected human estrogen receptor-positive breast cancer cells (MCF-7Ca) were grown as tumors in ovariectomized athymic mice. Animals received subcutaneous injection with vehicle, tamoxifen, exemestane, tamoxifen plus exemestane, and letrozole. Tumor volumes were measured weekly.
Results: All treatments were effective initially in suppressing tumor growth as first-line therapy compared with vehicle treatment. Exemestane suppressed tumor growth to a greater extent than tamoxifen. However, the combination of tamoxifen plus exemestane was more effective than either drug alone. After tumor volumes doubled on initial treatment, the mice were crossed over to receive exemestane or tamoxifen. Tumor growth slowed briefly in mice treated with tamoxifen and crossed over to exemestane, but tumor growth continued unabated in those changed from exemestane to tamoxifen. However, letrozole was effective in both groups as third-line therapy for a limited period. Letrozole as initial single agent was the best overall treatment in terms of the degree of tumor suppression and the length of effectiveness of treatment.
Conclusion: Exemestane was more effective in controlling tumor growth than tamoxifen. In addition, the combination of exemestane plus tamoxifen was clearly more effective than sequential use of these agents in the tumor model. However, the nonsteroidal aromatase inhibitor letrozole as first-line therapy was overall the most effective treatment in controlling tumor growth.
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Current treatment for postmenopausal hormone-dependent breast cancers includes two strategies to reduce the effects of estrogen on tumor growth. One strategy is inhibition of estrogen action with antiestrogens, and the other is inhibition of estrogen synthesis with aromatase inhibitors. The antiestrogen tamoxifen has been a primary treatment for breast cancer since the 1970s (2) and has been shown to delay recurrences and contra-lateral breast cancer. The agent is well tolerated and can be taken for 5 years (3) . However, tamoxifen is a partial agonist as well as an antagonist and can cause endometrial hyperplasia and occasionally endometrial cancer as well as increased risk of stroke because of its estrogenic effects (4) . In addition, the compound may not be optimally effective in terms of its antagonistic actions on the tumor. For these reasons, we have investigated a different approach using inhibition of estrogen production with aromatase inhibitors (5, 6, 7) . The two approaches have been compared in recent clinical trials and the results suggest that aromatase inhibitors are more effective than tamoxifen as first-line therapy for postmenopausal patients with hormone-responsive breast cancer (8, 9, 10) and also for patients with early breast cancer (11, 12) . Two classes of aromatase inhibitors, steroidal (formestane, exemestane), and nonsteroidal (anastrozole, letrozole) compounds have been developed. Steroidal, irreversible inhibitors (also known as suicide inhibitors) of aromatase interact with the substrate-binding site of the enzyme, binding irreversibly and causing enzyme inactivation (13) . Thus, no estrogen can be synthesized until new enzyme is produced. In contrast, nonsteroidal compounds interact with the heme-iron group of the cytochrome P450 aromatase in a reversible reaction (13) .
To investigate the effectiveness of aromatase inhibitors, our laboratory developed a xenograft tumor model using human hormone responsive (estrogen-receptor positive) breast cancer cells stably transfected with the human aromatase gene (MCF-7Ca). In this model, MCF-7Ca cells (14) are grown as tumors in the ovariectomized, immune suppressed mice (15 , 16) . The tumor serves as an autocrine source of estrogen that stimulates its growth, and it is sensitive to both the antiproliferative effects of antiestrogens and of aromatase inhibitors (17, 18, 19) . The model simulates the postmenopausal breast cancer patient as a major source of the hormone after menopause is breast tissue and where the aromatase enzyme is not under gonadotropin regulation. As the production of adrenal androgen precursors for aromatization is deficient in these mice (20) , we supplement them with androstenedione. We have shown previously that androstenedione does not affect the growth of tumors directly. Thus, growth of tumors of MCF-7 cells without aromatase transfection are not stimulated by androstenedione (15) .
Because both antiestrogens and aromatase inhibitors are effective in controlling tumor growth, blocking both estrogen receptors and estrogen synthesis might have an additive effect and result in better control of tumor growth. In this study, we have investigated whether the steroidal inhibitor exemestane combined with tamoxifen might be more effective than each alone. In our previous studies, we have compared the effects of combining tamoxifen with nonsteroidal aromatase inhibitors anastrozole and letrozole on tumor growth (18 , 19) . Although there was no benefit in combining treatment compared with the aromatase inhibitors alone, the effect of treatment with the combination was similar to that of tamoxifen alone. These data predicted the outcome of the clinical Anastrozole, Tamoxifen, and the Combination trial (11) , which found that anastrozole was more effective than tamoxifen and the combination of the two agents in reducing recurrences in patients with early stage breast cancer. To date, it is not known whether combining a steroidal aromatase inhibitor with the antiestrogen tamoxifen will be more effective in slowing tumor progression compared with treatment with either of these drugs alone. We were particularly interested in examining the value of combining exemestane plus tamoxifen in this model as it had been reported previously that the combination is superior to either agent alone in the rat model with carcinogen [7,12-dimethylbenz(a)anthracene]-induced mammary tumors (21) .
In the current study, we have also investigated several strategies with the goal of defining regimens that will be the basis of clinical trials to optimize treatment for postmenopausal breast cancer patients with these agents. Thus, we have investigated the effect of sequential treatment with tamoxifen and exemestane as second-line therapy and the effect of letrozole alone and in sequential therapy after tumors progressed on exemestane and tamoxifen.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
4A), tamoxifen, and hydroxypropyl cellulose were obtained from Sigma Chemical Company (St. Louis, MO). Matrigel was obtained from BD Biosciences (Bedford, MA). Letrozole was kindly provided by Dr. Dean Evans (Novartis Pharma, Basel, Switzerland). Exemestane was kindly provided by Dr. Lori Hollis (Pharmacia/Pfizer, Inc.).
Cell Culture.
MCF-7 human breast cancer cells stably transfected with the human aromatase gene (MCF-7Ca; ref. 14
) were cultured in DMEM with 5% fetal bovine serum, 1% penicillin/streptomycin solution, and 750 µg/mL G418. The culture medium was changed twice weekly.
Ovariectomized Female Athymic Nude Mice.
Female BALB/c athymic ovariectomized mice 4 to 6 weeks old were obtained from NCI (Frederick, MD). The animals were housed in a pathogen-free environment under controlled conditions of light and humidity and received food and water ad libitum. Animals were allowed to acclimatize for 48 hours after shipment and before tumor inoculation was done.
Postmenopausal Intratumoral Aromatase Model.
MCF-7Ca cells were inoculated into the mice as described previously (15
, 18)
. Subconfluent (80%) MCF-7Ca cells were washed with Dulbecco’s PBS and scraped into Dulbecco’s PBS. Cells were collected by centrifugation and resuspended in Matrigel (10 mg/mL). Each mouse received subcutaneous injections at two sites on each flank with 0.1 mL of cell suspension (2.5 x 107 cells/mL). Because athymic mice are deficient in adrenal androgens, they were supplemented with daily injections of the aromatase substrate androstenedione (4A) 100 µg/day for the duration of the experiment. Treatment started when tumors reached a measurable size (
300 mm3) 4 weeks after inoculation. Tumors were measured weekly with calipers, and volumes were calculated with the formula 4/3
x r12 x r2 (r1 < r2), where r1 is the smaller radius. Mice were grouped for treatment (five mice per group) so that total tumor volume was similar in each group, and mice received subcutaneous injection with vehicle, nonsteroidal aromatase inhibitor letrozole (10 µg/day), antiestrogen tamoxifen (500 µg/day), and 5 different doses of steroidal aromatase inhibitor exemestane (50, 100, 250, 500, and 1,000 µg/day) prepared as suspensions in 0.3% hydroxypropyl cellulose. In the second experiment, mice were treated as above with vehicle, tamoxifen (100 µg/day), exemestane (100 and 250 µg/day), the combination of tamoxifen and exemestane at the same doses, and letrozole (10 µg/day). When tumors had doubled in volume during treatment, mice were crossed over to tamoxifen or exemestane for second-line treatment and then subsequently to letrozole. Mice treated with the combination were switched to letrozole treatment when their tumors doubled in volume. Animals were treated for the indicated times as shown in Scheme 1, after which they were sacrificed by decapitation, and tumors and uteri were excised, cleaned, and weighed.
|
The general linear models technique was used to analyze the uterine weight data. The treatment groups were compared with one another at 0.05 level of statistical significance. Either Tukey’s or Dunnett’s procedure (23 , 24) was used to make the adjustments for multiple comparisons. The results of the comparisons across treatment groups are presented as the difference in growth rate, the mean difference in tumor weight, or uterine weight with a corresponding 95% confidence interval.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. The tumor volumes in the letrozole-treated animals regressed by 15% over the 4 weeks of treatment, whereas the tamoxifen-treated tumors increased slightly by 35%. Nevertheless, although the tamoxifen-treated tumors did not regress, their growth was significantly reduced compared with the control tumors that increased by 2.8-fold during the 4 weeks of treatment. The estimated difference in the weekly growth rate was 0.15 mm3 with the 95% confidence interval from 0.08 to 0.21 mm3 (Fig. 1A)
. The inhibition of tumor growth by exemestane was shown to be dose-dependent. At doses of 50 and 100 µg/day, tumor volumes increased by 1.1 and 0.9-fold, respectively, over 4 weeks. In the animals treated with exemestane at doses of 250, 500, and 1,000 µg/day, tumor volumes remained close to pretreatment levels indicating that these doses of exemestane effectively inhibited tumor growth to a maximum extent. Exemestane at doses of 250 µg/day was superior to tamoxifen (500 µg/day; P = 0.037). When exemestane was compared with letrozole, there was no statistically significant difference between the tumor growth rates of the mice treated with higher dose (250 µg/day) of exemestane compared with those treated with letrozole (10 µg/day).
|
in the control group (344 ± 56 mg) were significantly greater than in all of the treatment groups (P = 0.05). The mean weight of letrozole-treated tumors (48 ± 7 mg) was significantly lower than that of tamoxifen-treated tumors (P < 0.001). There was a statistically significant difference in tumor volumes between letrozole and exemestane-treated tumors at doses of 250 µg/day (P = 0.03). Also, the tumor weights of the letrozole-treated group were significantly less. The difference in treatment means is 29.1 mg with the 95% confidence interval (3.5–64.7 mg). The weight of tumors treated with the lowest dosage, 50 mg of exemestane, was significantly lower than in the control group (P = 0.05) but was not significantly different from tamoxifen.
The mouse uterus is very sensitive to the effects of estrogen and can be used as a bioassay of the estrogenic effects of compounds. In the tumor model, sufficient estrogen is produced by the tumors to maintain uterine weight comparable with that of the mouse in diestrus. Treatment with aromatase inhibitors letrozole and exemestane reduced uterine weights significantly compared with those of the control and tamoxifen-treated mice (Fig. 1C)
. For instance, the estimated difference in treatment means between the control and the group treated with 50 mg of exemestane was 17.4 mg with the 95% confidence interval (6.7–27.8 mg). This result suggests that the two aromatase inhibitors cause marked reduction in estrogen levels. In contrast, tamoxifen did not cause any significant difference in uterine weight compared with control. This finding is consistent with previous reports that this antiestrogen acts as an estrogen on the uterus (25)
.
The Effect of Sequential Treatment on Tumors Progressing on Exemestane and Tamoxifen Alone and in Combination.
In the previous experiment, we identified submaximal (100 µg/day) and maximally (250 µg/day) effective doses of exemestane (Fig. 1A)
. These doses and a submaximal dose of tamoxifen (100 µg/day) were evaluated as single agents and in combination. After tumors reached a measurable size, animals were assigned to seven groups of five mice each for first-line treatment with vehicle, letrozole (10 µg/day), tamoxifen (100 µg/day), exemestane (100 µg/day), exemestane (250 µg/day), tamoxifen (100 µg/day) plus exemestane (100 µg/day), and tamoxifen (100 µg/day) plus exemestane (250 µg/day; Fig. 2
).
|
To determine the effect of these agents in sequential therapy, mice were changed to second-line treatment when tumor volume had increased by 2- to 3-fold. Control tumors doubled their initial volume after <4 weeks (Table 1)
. Tamoxifen-treated tumors had doubled after 8 weeks of treatment. Tumors treated with exemestane at the dose of 250 µg/day doubled between 9 and 10 weeks whereas those treated with the combination of tamoxifen and exemestane (250 µg/day) doubled in 16 weeks. Thus, the time of tumor progression was significantly delayed compared with either drug alone. Letrozole-treated tumors did not double until 24 weeks of treatment.
|
). Tumors became static for a brief period with exemestane and then continued to grow. After 3 weeks of treatment with exemestane, tumor volume had increased up to 3.4-fold. In a further effort to control tumor growth, mice were switched to letrozole treatment. However, tumors regressed for 3 weeks but then tumor growth resumed. When mice initially treated with exemestane (250 µg/day) were switched to treatment with tamoxifen at week 12, tumor growth continued unabated until week 16. The mice were then switched to letrozole treatment, which slowed tumor growth for 3 weeks before growth resumed. By week 21, the two groups started on exemestane or tamoxifen and receiving three sequential treatments had similar tumor volumes. Also, a second group of animals treated with exemestane (100 µg/day) for 9 weeks (data not shown) followed by second-line treatment with tamoxifen and third-line treatment with letrozole showed a similar growth pattern as tumors treated initially with the higher dose of exemestane. Combining tamoxifen plus exemestane prolonged the time of tumor doubling to 16 weeks. However, these tumors were unresponsive to second-line treatment with letrozole after 1 week. Overall, letrozole treatment alone produced the best results in delaying tumor progression (tumors did not double for 24 weeks). The weekly growth rate was less with continuous treatment with letrozole only compared with the tumor growth rate of mice treated first line with exemestane (250 µg/day) plus tamoxifen and then switched to letrozole (P = 0.046; Fig. 2
). |
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The xenograft model bearing tumors of MCF-7Ca human breast cancer cells has predicted the results of clinical trials evaluating tamoxifen and aromatase inhibitors (17
, 19)
. For example, letrozole was superior to tamoxifen in the mouse model (17
, 19) and in time to progression and time to treatment failure in breast cancer patients (8)
. Anastrozole also significantly increased time to progression compared with tamoxifen in patients (P = 0.005; refs. 8
and 9
). In addition, results of the Anastrozole, Tamoxifen, and the Combination trial (11)
confirmed our findings of combining nonsteroidal aromatase inhibitors with tamoxifen in the mouse model (17
, 19)
. Letrozole plus tamoxifen in the xenograft model was less effective than letrozole alone, and the combination was not significantly different in delaying tumor doubling than tamoxifen alone. One possible explanation for the difference in results between the two types of aromatase inhibitors is that letrozole is more potent than exemestane (Fig. 1A)
. The slight reductions reported in serum levels of nonsteroidal aromatase inhibitors (27 and 38%) when combined with tamoxifen seems unlikely to influence their activity because estrogen concentrations remain maximally suppressed (19
, 26)
. Clearance rates of exemestane and tamoxifen in combination compared with those of the drugs alone are reported not to be altered in breast cancer patients (27
, 28)
. Additional studies are therefore needed to explore whether these or other mechanisms explain the difference between the two classes of aromatase inhibitors.
When tamoxifen was used in sequence followed by exemestane, tumor growth was arrested briefly. This finding in our model of advanced breast cancer is consistent with reports that patients who progressed on tamoxifen responded to subsequent exemestane treatment (10) . Although the period of response was relatively brief in the mouse, recent studies indicate better results are achieved in early breast cancer. Thus, women who switched from tamoxifen to exemestane after 2 to 3 years had 32% fewer recurrences than those on tamoxifen (12) .
Tumors that were initially treated with exemestane and crossed over to tamoxifen grew continuously without interruption. Although this finding is not consistent with response to these drugs in patients, tumor growth was arrested for a short time (3 weeks) when mice were switched to letrozole treatment. However, when tumors were initially exposed to tamoxifen plus exemestane in combination, their response time was even shorter (1 week) to subsequent treatment with letrozole as a second-line treatment. Nevertheless, growth of tumors treated with the combination initially was reduced, and tumors did not reach the same volume as those of groups treated with three sequential therapies, for 7 additional weeks. In conclusion, exemestane was more effective in controlling tumor growth than tamoxifen. In addition, the combination of exemestane plus tamoxifen was clearly more effective than sequential use of these agents in the tumor model. However, the nonsteroidal aromatase inhibitor letrozole as first-line therapy was overall the most effective treatment in controlling tumor growth.
|
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.
Note: Presented in part at the Aromatase 2002 International Conference.
Requests for reprints: Angela M. H. Brodie, Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Health Science Facility I, Room 580G, 685 West Baltimore Street, Baltimore, MD 21201. Phone: 410-706-3137; Fax: 410-706-0032; E-mail: abrodie{at}umaryland.edu
Received 3/23/04; revised 7/29/04; accepted 8/ 5/04.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  V. C. Jordan and B. W. O'Malley Selective Estrogen-Receptor Modulators and Antihormonal Resistance in Breast Cancer J. Clin. Oncol., December 20, 2007; 25(36): 5815 - 5824. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. F. Macedo, Z. Guo, S. L. Tilghman, G. J. Sabnis, Y. Qiu, and A. Brodie Role of androgens on mcf-7 breast cancer cell growth and on the inhibitory effect of letrozole. Cancer Res., August 1, 2006; 66(15): 7775 - 7782. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. R. Hutson, R. R. Love, T. C. Havighurst, E. Rogers, and J. F. Cleary Effect of Exemestane on Tamoxifen Pharmacokinetics in Postmenopausal Women Treated for Breast Cancer Clin. Cancer Res., December 15, 2005; 11(24): 8722 - 8727. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Jelovac, G. Sabnis, B. J. Long, L. Macedo, O. G. Goloubeva, and A. M.H. Brodie Activation of Mitogen-Activated Protein Kinase in Xenografts and Cells during Prolonged Treatment with Aromatase Inhibitor Letrozole Cancer Res., June 15, 2005; 65(12): 5380 - 5389. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
, mice treated with tamoxifen (100 µg/day) as first-line therapy were switched after 9 weeks to treatment with exemestane (250 µg/day) for 3 weeks to week 12, and then to letrozole (10 µg/day) as a third-line treatment until week 21; 
, mice treated with exemestane (250 µg/day) as first-line therapy were switched after 12 weeks to tamoxifen (100 µg/day) as a second-line treatment for 4 weeks until week 16, and then to third-line treatment with letrozole (10 µg/day) until week 21; 
, mice treated with the combination of tamoxifen (100 µg/day) plus exemestane (250 µg/day) as first-line treatment were switched after 21 weeks to second-line therapy with letrozole (10 µg/day) and continued to week 28; 
, mice were treated with letrozole (10 µg/day) for 28 weeks.