Introduction
The aromatase inhibitor letrozole has been shown to be superior to tamoxifen (TAM) in the first-line treatment of metastatic breast cancer [
1,
2]. A randomized clinical trial comparing these two drugs in the adjuvant setting is currently ongoing, and should soon provide insight into the relative efficacy of letrozole and TAM. Another adjuvant trial, the MA.17 study, evaluated patients from the National Cancer Institute of Canada Clinical Trials Group who were disease-free after initially being treated with 5 years of TAM and then being randomized to receive either 5 years of placebo or 5 years of letrozole treatment. As compared with placebo, letrozole therapy after the completion of TAM treatment significantly improved disease-free survival [
3].
Postoperative radiotherapy (RT) decreases the risk of locoregional recurrence. RT is also associated with improved survival in high-risk premenopausal and postmenopausal women with breast cancer given either adjuvant chemotherapy [
4] or TAM [
5], respectively. Whether letrozole sensitizes breast cancer cells to RT has not been determined. The present study has examined this question
in vitro, using a human breast cancer cell line stably transfected with the human aromatase gene. We present herein the capacity of letrozole to sensitize these cells to RT using clonogenic, MTT, and cell-count assays. Cell-cycle analyses showed a G
1 phase arrest and a decrease of the S phase with letrozole as compared with control cells. Moreover, compared with RT alone, combined RT and letrozole produced a significant decrease in radiation-induced G
2 phase arrest and in the number of cells in the S phase, with cell redistribution in the G
1 phase.
Materials and methods
Cell line, culture, and letrozole
MCF-7 human breast cancer cells stably transfected with the human aromatase/CYP19 gene (MCF-7CA) were kindly provided by Dr S Chen (City of Hope, Duarte, CA, USA [
6]). These cells were maintained in DMEM/NUT MIX F-12 containing 10% heat-inactivated FCS (Gibco Laboratories, Cergy Pontoise, France), 500 μg/ml geneticin (G418), 300 μg/ml glutamine, 0.25 μg/ml fungizone, 100 μg/ml streptomycin, and 100 units/ml penicillin G. The two cell lines, MCF-7 wild type and MCF-7CA, were adherent and grew as monolayers at 37°C in a humidified 5% CO
2 incubator. The cells were harvested with 0.5 g/l trypsin (Gibco Laboratories) and 0.2 g/l EDTA (Gibco Laboratories) for 3 min. Cultures were checked for the absence of mycoplasma every month.
Steroids were removed from the FCS by two treatments with dextran-coated charcoal (DCC) [
7]. Cells were cultured as monolayers in 60-mm Petri dishes with phenol-red-free medium and DCC FCS.
Letrozole used in the study was synthesized in the laboratories of Novartis Pharma AG (Basel, Switzerland). In all experiments, letrozole was added to cultures that were nonconfluent at concentrations of 7 nM–0.7 μM in 10 μl ethanol 6 days before RT.
Aromatase tritiated water assay
Aromatase activity was determined using the
3H
2O release method reported by Zhou and colleagues [
6], based on the procedure described by Thompson and Siiteri [
8]. Cells were maintained for 5 days in 10% FCS phenol-red-free medium and then seeded at 700,000 cells per well in six-well dishes with fresh DCC-treated medium. Three days later, culture plates were washed twice with PBS. One millitre of serum-free medium containing 40 nM 1β-
3H(N)-androst-4-ene-3,17-dione (NET-926; NEN PerkinElmer Life Sciences, Inc., Courtaboeuf, France) as substrate (specific activity, 25.9 Ci/mmol) with or without 100 nM letrozole was then added to each well. After 6 hours of incubation at 37°C, the reaction mixture was removed and extracted with two volumes of chloroform to terminate the reaction and to extract unused substrate and steroids. After 5 min centrifugation at 100 ×
g, the aqueous phase was then treated with an equal volume of 5% charcoal suspension to eliminate residual steroids. After 5 min centrifugation at 15,000 ×
g, radioactivity was assessed by liquid scintillation counting. The protein concentration was determined by the Bradford method [
9] after cell extraction with Reporter Lysis 5X Buffer according to the manufacturer's instructions (PROMEGA Corp., Lyon, France). Aromatase activity was then calculated from the disintegrations per minute as picomoles of oestrogen produced by milligrams of protein per hour
Radiation modalities
Cells were plated in 10 ml DCC and phenol-red-free medium (to ensure homogeneous energy deposition within each dish using 60-mm Petri dishes) and irradiated with a cobalt-60 source (γ irradiation, ELITE 100; Theratronics, Ottawa, ON, Canada) in the Radiation Department. The radiation was delivered as a single dose ranging from 0 to 4 Gy in an 11 cm × 11 cm field size at a dose rate of 0.5 Gy/min. A 3-cm polystyrene block was used under the Petri dishes during each irradiation to allow homogeneous backscattering γ-rays. The source-half-depth distance was initially calculated to obtain a constant dose rate of 0.5 Gy/min, and was adapted monthly to the cobalt-60 source radioactivity decay. Control cells were removed from the incubator and were placed for the same period of time under the cobalt-60 source but without RT. In the combined treatment modality studies, letrozole was added 6 days prior to RT.
Cytotoxicity assays
Cultures were trypsinized and washed, and cells were plated in quintuplicate at a density of 100 cells per 60-mm Petri dish after dilution. Letrozole was added at concentrations ranging from 7 nM to 0.7 μM 24 hours after the cells were plated to allow for cell attachment. Cells were incubated at 37°C in a humidified chamber containing 5% CO2 for 12 days. The colonies were then fixed with a 1:3 (v/v) acetic acid methanol solution and were stained with 10% Giemsa (Sigma Chemical Co., St Louis, MO, USA); colonies of greater than 50 cells were scored. The plating efficiency was calculated with and without letrozole.
The cytotoxicity of RT against asynchronous, exponentially growing cells was also determined from colony formation assays. Before irradiation, the cell density was determined using appropriate dilutions (100, 100, 200, 300, and 400 cells for 0, 1, 2, 3, and 4 Gy, respectively), and cells were plated onto five replicate 60-mm Petri dishes. Cells were irradiated as already described 24 hours after plating to allow for cell attachment before the administration of RT. The letrozole-containing medium was given at a concentration of 0.7 μM 6 days before RT. Cultures were irradiated with the drug present in the medium, and were immediately returned to the incubator after irradiation. Colonies were counted after 12 days.
Experimentally derived data points are the mean of five experiments. The dose–response curves were fitted to a four-parameter model, where the response R varies with the dose D according to the equation: R = a/[1 + (D/b)
c
] + R∞, where a is the difference between the maximum and minimum response, b is the concentration of drug needed to obtain 50% of the maximal effect, c is a slope factor, and R∞ is the maximal effect. The multitarget model survival curves were fitted to the data using a least-squares regression to the linear-quadratic model: S = S
0 exp(-αD
1 - βD
1
2), where D
1 is the radiation dose, S is the surviving fraction, and S
0 is a normalizing parameter.
MTT and cell number assays
The antiproliferative effect of letrozole with or without RT on the MCF-7CA cell line was evaluated using the MTT assay as described by Mosmann [
10]. Briefly, exponentially growing cells were seeded into 96-well plates and were incubated in medium containing letrozole from 7 nM to 0.7 μM for 48 hours at 37°C. Duplicate plates containing six replicate wells/assay condition were seeded in 0.1 ml medium at densities of 5000, 15,000, and 35,000 cells per well, and were then treated with letrozole alone, with RT (2 Gy) ± letrozole (0.7 μM), and with RT (4 Gy) ± letrozole (0.7 μM), respectively. Twelve days after letrozole ± RT (2 or 4 Gy) treatment, the viability of the cells was analysed using the tetrazolium salt (Sigma) MTT colorimetric assay. Briefly, 50 μl of a 0.5% MTT solution was added to each well, followed by incubation for 4 hours at 37°C to allow MTT metabolization. The crystals formed were dissolved by adding 100 μl/well isopropylic alcohol, 1 N HCl. The absorbance at 540 nm was measured on a Microtiter
® Plate Reader MRX (Dynatech Laboratories, Chantilly, VA, USA). The results were expressed with respect to control values (cells without any treatment).
The antiproliferative effect of letrozole ± RT on the MCF-7CA cell line was also evaluated using a cell-count assay. MCF-7CA cells were allowed to grow for 24 hours, at which time letrozole was added at a concentration of 0.7 μM. RT (2 and 4 Gy) was delivered to those cells receiving the combination treatment 6 days after the addition of letrozole to the medium. Cells were counted every 6 days (days 6, 12, and 18) over an 18-day period after removing the cells from the plates with 0.5 g/l trypsin.
Flow cytometry
Cells were plated in 60-mm Petri dishes at a density of 2 × 106 cells/dish. Treatment consisted of letrozole (0.7 μM) alone, RT (2 and 4 Gy) alone, or letrozole (0.7 μM) plus RT (2 and 4 Gy). Cells were collected 15 days after plating, and were processed for cell-cycle analysis. Cells were harvested by trypsinization, were washed with PBS, and then 1 × 106 cells/dish of treated cells were fixed in 70% ethanol for 2 min. After removal of ethanol by centrifugation, cells were then stained with a solution containing 40 μg/ml propidium iodide (Sigma) and 0.1 mg/ml RNase A (Roche, Indianapolis, IN, USA). Stained nuclei were analysed for DNA-PI fluorescence using a Becton-Dickinson FACScan flow cytometer (Mountain View, CA, USA). Resulting DNA distributions were analysed using the CellQUEST software (Becton Dickinson) for the proportion of cells in the sub-G0 phase, the G1 phase, the S phase, and the G2–M phase of the cell cycle.
In a second series of experiments, cells were treated with letrozole (0.7 μM) alone and were then cultured for 21 days. Cells were stained at different time points up to 21 days and were analysed for DNA content on a FACScan as already described.
Statistical analysis
The nonparametric Wilcoxon signed-rank test was used to compare the surviving fraction for each dose between the two groups (RT alone and RT plus letrozole). All experiments were performed three times and the results are expressed as the mean ± standard error. All statistical tests were two-sided with an alpha level of 0.05. Data were analysed using the STATA 7.0 software (Stata Corporation, College Station, TX, USA).
Discussion
TAM is the most widely used endocrine agent for the adjuvant therapy of early breast cancer in postmenopausal women [
11]. TAM use nevertheless remains limited, most notably by its recognized pharmacological properties and side effects. For instance, TAM has been associated with an increased risk of both thromboembolic events and endometrial changes, including endometrial cancer [
12,
13]. It is possible, therefore, that other endocrine drugs may provide at least equivalent, if not superior, efficacy together with improved tolerability in patients with early disease. In this context, the third-generation aromatase inhibitors are now under investigation, and the initial results may substantially change our current clinical practice patterns [
3,
14,
15].
The efficacy of breast-conserving surgery with axillary dissection and breast radiation has been known for a long time [
16]. Overgaard and colleagues more recently demonstrated that postoperative radiotherapy decreases the risk of locoregional recurrence, and that it is associated with improved survival in high-risk patients with breast cancer given either adjuvant chemotherapy (in premenopausal women) [
4] or tamoxifen (in postmenopausal women) [
5].
In the present study, we wanted to evaluate
in vitro the effects of the combination of ionizing radiation and letrozole, one of the third-generation aromatase inhibitors. To our knowledge, our results demonstrate for the first time that letrozole could act as a potent radiosensitizer. A significant effect was observed in all three assays used in our experiments (clonogenic, MTT, and cell count). The effects of associating other hormonotherapies, such as TAM, with radiotherapy have been rarely described and remain poorly defined. TAM appears to exert its cytostatic activity at least partly through competitive inhibition of oestrogen binding at the oestrogen receptor, resulting in segregation of cells into the G
0/G
1 phase of the cell cycle [
17]. Because relatively less radiosensitivity was observed in the early G
1 phase [
18], a hypothetical concern was raised in several studies that the use of TAM during RT might result in the radioprotection of tumour clonogens of both hormonally responsive and unresponsive breast carcinoma cells at dose levels typically used in clinical practice [
19]. In these studies, however, cell cultures were grown in a medium containing phenol red and foetal bovine serum, two sources of exogenous estrogenic compounds [
20‐
23]. This fact complicates the interpretation of the resultant radiation survival curves [
24]. In contrast to these reports, no significant differences were observed in radiosensitivity for oestradiol-stimulated or 4-hydroxytamoxifen-inhibited cultures plated into growth-stimulating conditions immediately after irradiation or following an additional 24 hours in oestrogen-free conditions [
25]. Clearly, under defined hormonal conditions [
26], no protective effect of the active TAM metabolite 4-hydroxytamoxifen was observed [
25].
Our data suggest that letrozole inhibits cell proliferation by invoking a transition delay or by blocking in the G
1 phase of the cell cycle. This delay may require more than one complete passage of some cells through the cell cycle, since cell numbers more than doubled before the plateau growth phase was reached. In addition, two to three generation times were required for maximal accumulation of cells in the G
1 phase. We therefore added letrozole 6 days before RT. Under defined hormonal conditions, we found more than an additive effect [
27] using the combination of letrozole and RT at each dose tested. This result was reinforced by the poor effect of letrozole used alone at high concentration (7 μM) and tested in all combination treatments. Isobologram analyses are planned to confirm the existence of either an additive effect or a supra-additive effect for the association of these two treatment modalities in a future study, as proposed by Steel [
28].
A small percentage of cells was insensitive to letrozole, and remained in the proliferative fraction (9% in the S phase). The mechanism of unresponsiveness of these cells to letrozole will require further study. As shown with anti-oestrogens [
17], these data are consistent with the hypothesis that aromatase inhibitors such as letrozole induce regression of breast cancer
in vivo by simply blocking progression of cells through the cell cycle rather than by a direct cytotoxic effect. With cell replication inhibited, tumours might then regress because of ongoing cell shedding or death, or because of interaction with normal host defences.
In the RT–letrozole combination treatment, we observed a 50% decrease of MCF-7CA cells arrested in the G
2 phase as compared with RT alone, a proportional redistribution in the G
1 phase, and an interrupted synthesis phase for an 18-day period. This cell-cycle redistribution phenomenon may also explain the decrease in the surviving fraction in the combination treatment presented in the present study (Fig.
3).
In addition, associating irradiation and letrozole may modify the cytosolic oestrogen and progesterone receptor content in breast cancer cells, which might explain the observed changes in these cells' radiation sensitivity during hormonal treatment, as published with TAM [
29].
These results may have important clinical implications for the treatment of breast cancer. Since the adjuvant use of aromatase inhibitors is still an emerging treatment option [
3,
14,
15], the most effective time at which to commence the use of these drugs after surgery is not yet known. Also, if letrozole is simply placing hormone-responsive breast cancer cells into a 'resting' phase of the cell cycle (G
0–G
1 phase), then treatment of patients after surgery for primary breast cancer with adjuvant letrozole may have to be continued at least long enough for host defences or normal cell attrition to eradicate residual tumour cells. Premature discontinuation of therapy might lead to tumour regrowth. Finally, where letrozole is used concurrently with RT, we recommend starting letrozole at least 3 weeks before the start of RT in an attempt to obtain an optimal effect, consistent with the biological rationale described earlier. Furthermore, and in so far as the preclinical data with breast cancer cells can be extrapolated to the clinical situation, a radiosensitizing effect may be observed in the healthy breast. Nevertheless, Miller and colleagues showed a rising aromatase activity level in adipose tissue obtained from patients with breast cancer and within the quadrant involved with the tumour [
30‐
32].
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DA initiated the project and developed all first experiments. CL confirmed all previous results and performed all FACS analyses. SC and PP performed the aromatase activity assay. MO and SG made the statistical analyses. PM constructed survival curves of the clonogenic assay. DA, DBE, GR, and AP managed scientific experiments and contributed to the elaboration of the discussion.