The ER-positive MCF-7 breast cancer cell line (ATCC HTB-22) was initially obtained in 1977 from the Michigan Cancer Foundation (Detroit, MI, USA). The IBEP-2 cell line was previously established in our laboratory from a pleural effusion due to metastatic breast carcinoma 35 and also expresses functional ER. MDA-MB-231 breast carcinoma cells (ATCC HTB-26) lack ER expression.

All experiments were performed in plastic flasks, dishes and multi-well plates obtained from Nunc (Naperville, IL, USA). Cells were cultured at 37°C in a humidified 95% air and 5% CO₂ atmosphere. For routine maintenance, cells were cultured in 75 cm² flasks containing RPMI medium 1640 (Gibco BRL, Life Technologies, Merelbeke, Belgium) with Phenol Red, supplemented with 10% (v/v) heat-inactivated FCS, and containing standard concentrations of L-glutamine, penicillin and streptomycin (Gibco BRL). Cells were harverested by trypsinization (0.05% (w/v) trypsin, 0.53 mM EDTA.4Na) twice a week. For experiments, cells were plated in SFM made up of RPMI medium 1640 without Phenol Red supplemented with 10% (v/v) FCS stripped of endogenous estrogens by a dextran-coated charcoal treatment as previously described 36. One day later, the seeding medium was replaced by fresh SFM containing ibandronate (gift from Hoffmann-LaRoche (Basel, Switzerland), E₂ (Sigma, St Louis, MO, USA), 4-hydroxytamoxifen (Sigma), ICI 182,780 (Tocris, Bristol, UK) or vehicle for 1 to 6 days.

Cell number was assessed indirectly by staining with crystal violet dye as previously described 37. Briefly, cancer cells were seeded in 96-well plates (density 5,000 cells/well) in SFM, and cultured for 24 h. Cells were then exposed to compounds or vehicle at various concentrations and time incubations as described in Results. Medium was removed, cells were gently washed with PBS, fixed with 1% (v/v) glutaraldehyde/PBS for 15 minutes and stained with 0.1% crystal violet (w/v in ddH₂O) for 30 minutes. Cells were destained under running tap water for 15 minutes and subsequently lysed with 0.2% Triton X-100 (v/v in ddH₂O). The absorbance was measured at 550 nm using a Microplate Autoreader EL309 (BIO-TEK Instruments, Winooski, VT, USA). Blank wells lacked cells and drugs. The IC₅₀ value refers to drug concentrations producing 50% inhibition of growth.

Cell growth was also assessed by cell count. Cells were plated in 12-well dishes at a density of 10⁴ cells/cm² in SFM. At day 1, the seeding medium was replaced by fresh SFM containing 10^-4 M ibandronate and/or 10^-8 M E₂. After three days of incubation, cells were dislodged from the vessel bottom by treatment with a trypsin-EDTA solution. After vigorous pipetting, concentrations of cells in suspension were determined in an electronic cell counter (model Z1 Coulter counter, Beckman Coulter, Fullerton, CA, USA).

Apoptotic cell death was assessed using annexin-V fluorescein isothiocyanate (FITC) and propidium iodide double staining (ApoTarget™, Annexin-V FITC Apoptosis Kit, BioSource Europe, Nivelles, Belgium), according to the manufacturer's recommendations. This method is based on apoptosis-related cell membrane modifications and relies on selective binding of annexin-V to phosphatidylserine expressed in the outer membrane leaflet during the early stages of apoptosis. Propidium iodide staining reveals cell surface membrane permeability associated with necrosis or late stage of apoptosis. Briefly, MCF-7 cells were seeded in 6-well plates (density 50,000 cells/well) in SFM, and cultured for 24 h. Cells were then exposed to compounds or vehicle for 3 to 6 days at concentrations as described in Results. Medium was renewed at day 3. Cells were washed twice in PBS, harvested by treatment with a trypsin-EDTA solution, centrifuged and resuspended in 100 μl annexin-V binding buffer. Cell suspensions received 5 μl of FITC-labeled annexin-V and 10 μl of propidium iodide buffer, were incubated for 15 minutes at room temperature in darkness, and, finally, were diluted with 400 μl annexin-V binding buffer. Cells were then analyzed by using a flow cytometrer (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA). Data are presented as dot plots showing fluorescence intensity of annexin-V FITC versus propidium iodide. Percentages of apoptotic cells are percentages of annexin-V positive and propidium iodide negative cells.

ER and progesterone receptor (PgR) amounts were determined by western blotting. Cells were plated in 60 cm² petri dishes (density 10,000 cells/cm²) in SFM, cultured for 24 h and then incubated with compounds or vehicle as specified in Results. Cell monolayers were harvested and lysed using detergent cocktail, as previously described 37. Solubilized proteins were subjected to western blotting using polyclonal rabbit anti-human ERα antibody (HC-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:5,000, or monoclonal mouse anti-human PgR (A/B isoforms) antibody (NCL-PGR-AB, Novocastra Laboratories, Newcastle upon Tyne, UK) diluted 1:500. Peroxidase-labeled donkey anti-rabbit IgG antibody (1:5,000) or peroxidase-labeled sheep anti-mouse IgG antibody (1:5,000) (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) were used depending on the primary antibody. The bound peroxidase activity was revealed using the Lumi-Light Western Blotting Substrate (Roche, Mannheim, Germany). The immunoreactive band intensity was estimated using a computer-assisted gel scanning densitometer (GS-710 Callibrated Imaging Densitometer) and Quantity One software, both from Bio-Rad (Hercules, CA, USA).

Protein concentrations of total cell lysates were determined by the BCA Protein Assay (Pierce, Rockford, IL, USA) using bovine serum albumin as standard.

The cytotoxic effects obtained with the different combinations of ibandronate and 4-hydroxytamoxifen or ICI 182,780 were evaluated according to the method of Chou and Talalay 38 on Calcusyn software (Biosoft, Cambridge, UK). This method allows the identification of interactions between two drugs, regardless of the mechanism of action of the individual drugs. Cells were incubated with increasing concentrations of ibandronate (10^-6 to 10^-3 M) alone and in combination with increasing concentrations of 4-hydroxytamoxifen (10^-10 to 10^-7 M) or ICI 182,780 (10^-10 to 10^-7 M) for 72 h. Interaction between the double combinations was assessed by means of an automatically computed combination index (CI). CI was determined at 50% and 75% cell growth, and was defined as follows:

CI_A+B = [(D_A/A+B)/D_A] + [(D_B/A+B)/D_B] + [α(D_A/A+AB × D_B/A+B)/D_AD_B]

where CI_A+B = CI for a fixed effect (F) for the combination of cytotoxic A and cytotoxic B; D_A/A+B = concentration of cytotoxic A in the combination A + B giving an effect F; D_B/A+B = concentration of cytotoxic B in the combination A + B giving an effect F; D_A = concentration of cytotoxic A alone giving an effect F; D_B = concentration of cytotoxic B alone giving an effect F; α = parameter with value 0 when A and B are mutually exclusive and 1 when A and B are mutually non-exclusive.

The CI indicates synergism for values lower than 0.8, additivity for values included between 0.8 and 1.2, and antagonism for values higher than 1.2. Values of 0.8 and 1.2 suggest slight synergistic and additive cytotoxic activities, respectively.

Data are reported as means ± standard deviation (SD). Statistical analysis was performed by analysis of variance (ANOVA) which assumes a similar SD in different groups. Thus, a logarithmic transformation was applied before ANOVA when SD values were found to differ statistically. Dunnett post hoc test was used to compare treated conditions to the untreated condition (control) and Turkey post hoc test was performed for multiple comparisons between groups. The level of statistical significance was arbitrarily set at 0.01. All analyses used SPSS software (Paris, France).

MCF-7 cells were plated in RPMI 1640 medium supplemented with charcoal-stripped fetal bovine serum (SFM), and cultured for 24 h before exposure for 72 h to ibandronate at concentrations ranging from 10^-6 to 10^-3 M. In these conditions, ibandronate inhibited cell growth in a concentration-dependent manner, as assessed by photometry after crystal violet staining (approximate IC₅₀ 10^-4 M) (Fig. 1, upper panel). Electronic cell count after trypsinization gave quite similar results on hormone-deprived MCF-7 cells when the effect of 10^-4 M ibandronate was evaluated after 3 days of treatment (Table 1). IBEP-2 cells exhibited a similar dose-response curve to ibandronate as assessed by crystal violet staining (approximate IC₅₀ 10^-4 M), while MDA-MD-231 cells were slightly less sensitive to the bisphosphonate (approximate IC₅₀ 3 × 10^-4 M) (Fig. 1, middle and lower panels).

As shown by time-course experiments over 6 days, ibandronate at 10^-5 M only produced a weak inhibition of MCF-7 cell growth, detectable from day 4. By contrast, higher drug concentrations drastically affected cell growth kinetics: 10^-4 M ibandronate induced rapid cytostatic effects up to day 6, whereas 10^-3 M ibandronate clearly exerted cytotoxic effects (Fig. 2).

Moreover, high ibandronate concentrations induced apoptotic cell death, as documented by the detection of annexin-positive and propidium iodide-negative MCF-7 cells (Fig. 3a). The percentages of annexin-positive cells after 5 days of incubation with 10^-4 M and 10^-3 M ibandronate were 23.5 ± 2.4 (mean ± SD) and 50.0 ± 8.7, respectively, indicating that the inhibition of cell growth was caused, at least in part, by cell apoptosis (Fig. 3b). Of note, 10^-4 M ibandronate did not induce significant apoptosis at day 3. No significant cell death was observed using 10^-5 M bisphosphonate.

The influence of increasing treatment duration on cell growth estimated at 72 h is illustrated by pulse exposure studies in Fig. 4. A 2 h exposure to ibandronate did not significantly affect cell proliferation, whereas bisphosphonate exposure for 4 h resulted in a significant decrease in cell growth by 21%. In addition, a 45% inhibition of cell growth was already seen after a 24 h treatment and the effect was not significantly greater when the incubation with ibandronate was prolonged up to 48 or 72 h (43% and 53% growth inhibition, respectively). This suggests that the growth inhibition recorded after a 24 h exposure is already irreversible and that 24 to 72 h exposure does not augment inhibitory efficacy.

As previously reported 39, 10^-8 M E₂ stimulated the proliferation of MCF-7 cells in SFM (Fig. 5, upper panel; Table 1). In this context, 10^-6 M ibandronate, which failed to change basal cell proliferation, did not affect the growth stimulation induced by E₂. By contrast, 10^-4 M ibandronate completely abolished the mitogenic effect induced by estrogenic stimulation, and the growth inhibitory effects of ibandronate were unaffected by the presence of E₂. Closely similar findings were obtained using IBEP-2 cells, which have been recently reported as estrogen-responsive cells resembling MCF-7 cells 39 (Fig. 5, lower panel), indicating that the inhibition by ibandronate of the mitogenic stimulation induced by E₂ was not restricted to MCF-7 cells.

ER expression and activity were examined in the same culture conditions by western blot analysis. Ibandronate alone had no effect on ER content whereas E₂ induced a marked receptor down-regulation reflected by a 63% decline in ER steady-state level after 24 h (Fig. 6a). Ibandronate did not affect ER decrease induced by E₂. The expression of PgR was also investigated as it is estrogen-inducible and viewed as a classic marker of ER activation 40. Thus, the effect of ibandronate on PgR expression was assessed by western blot analysis using an antibody raised against the A/B isoforms of PgR. Exposure of MCF-7 cells for 72 h to 10^-9 M E₂ increased by more than three-fold the level of PgR B isoform (Fig. 6b). Ibandronate modified neither PgR baseline level nor the increase in PgR level induced by E₂. As previously reported 39, the A isoform of PgR was not detectable in untreated MCF-7 cells and only a small amount of this isoform was observed after E₂ stimulation.

Overall, these data indicate that ibandronate completely blocks the proliferative response induced by E₂, without affecting ER regulation and activity. From these results, it can be inferred that ibandronate does not directly act on the ER pathway but interferes with E₂-induced mitogenicity at steps downstream or independent of ER-mediated signaling. Indeed, in MCF-7 cells exposed to ibandronate, ER kept its transactivation ability.

We assessed the effects of ibandronate in combination with antiestrogens. Data shown above indeed suggest that bisphosphonate-treated MCF-7 cells keep a functional form of ER, which should be able to promote the anti-proliferative action of ER antagonists. Two well-known antiestrogens were used for this study: an active metabolite of the partial antiestrogen 4-hydroxytamoxifen and the pure antiestrogen ICI 182,780. In preliminary studies, these drugs alone or in combination with ibandronate were tested at fixed concentrations with regard to their inhibitory effects on MCF-7 growth in steroid-free conditions. Antiestrogens alone (10^-7 M) significantly decreased cell growth by 14% and 35% for 4-hydroxytamoxifen and ICI 182,780, respectively (Fig. 7, upper panels). Importantly, the growth inhibitory action induced by both antiestrogens was significantly enhanced by ibandronate. Thus, when using combined treatments, 4-hydroxytamoxifen plus ibandronate decreased cell growth by 51%, and ICI 182,780 plus ibandronate inhibited cell proliferation by 58%. Similarly, the growth of the ER-positive IBEP-2 cells was also inhibited by both antiestrogens, and growth inhibition was again significantly higher when ER antagonists were combined with ibandronate (Fig. 7, middle panel). By contrast, antiestrogens did not affect the proliferation of the ER-negative MDA-MB-231 cells, and they did not change the growth inhibitory effect of ibandronate (Fig. 7, lower panel). These data indicate that antiestrogen response in sensitive breast cancer cells may be enhanced by ibandronate.

To better characterize the interactions between ibandronate and ER antagonists in MCF-7 cells, drug combinations were evaluated over a wide range of concentrations (from 10^-6 to 10^-3 M for ibandronate and from 10^-10 to 10^-7 M for the antiestrogens). Dose-response curves are illustrated in Fig. 8. These data were submitted to isobolographic analysis to calculate the CIs at 50% and 75% inhibition of cell growth, according to the analytical procedure developed by Chou and Talalay 38. Results are summarized in Table 2. According to the CI values, the effects of ibandronate and antiestrogen were additive, suggesting that these compounds act through distinct mechanisms.