Extracellular calcium increases bisphosphonate-induced growth inhibition of breast cancer cells

The MCF-7 (HTB-22), MDA-MB-231 (HTB-26), T-47D (HTB-133), ZR-75-1 (CRL-1500), Hs 578T (HTB-126) and BT-549 (HTB-122) breast cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured at 37°C in a humidified 95% air and 5% carbon dioxide atmosphere. For routine maintenance, cells were propagated in 75 cm² flasks containing complete RPMI medium consisting of RPMI 1640 with phenol red, supplemented with 5% heat-inactivated foetal bovine serum and with L-glutamine, penicillin and streptomycin at standard concentrations (all from Gibco BRL, Life Technologies, Merelbeke, Belgium). Cells were harvested by trypsinization (0.1% trypsin, 0.02% EDTA) and subcultured twice weekly.

For experiments, cells were plated in complete RPMI medium. One day after seeding, the culture medium was replaced by fresh medium adjusted to the final desired calcium concentrations (from 0.6 to 2.0 mmol/l) by adding CaCl₂.2H₂O (Sigma, St Louis, MO, USA) along with contain drugs and/or other chemicals. Of note, standard RPMI medium contains 0.6 mmol/l calcium, as calculated from the manufacturer's data. Ibandronate and [¹⁴C]ibandronate (102 mCi/mmol) were supplied by Roche Diagnosics GmbH (Penzberg, Germany) and F. Hoffmann-La Roche Ltd (Basel, Switzerland), respectively. Zoledronic acid was supplied by Novartis (Basel, Switzerland). Paclitaxel, ethylene glycol tetra-acetic acid (EGTA) and MgCl₂ were from Sigma. Calcium-sensing receptor (CaR) agonist (NPS R-467) and antagonist were kindly provided by Dr J Fox (NPS Pharmaceuticals, Salt Lake City, UT, USA). Cells were treated for 1 to 72 hours, as specified in Results (see below).

Cell number was assessed indirectly by staining with crystal violet dye, as described in a previous report [21]. Briefly, cells were seeded in 96-well plates at a density of 2,500 cells/well in complete RPMI medium and cultured for 24 hours. Cells were then exposed for 72 hours or less (pulse exposures) to calcium, drugs and other compound(s) alone or in combination, as described in Results (see below). Medium was removed, cells were gently rinsed with phosphate-buffered saline (PBS), fixed with 1% glutaraldehyde/PBS for 15 minutes and stained with 0.1% crystal violet (weight/vol in double distilled H₂O) for 30 minutes. Cells were destained under running tap water for 15 minutes and subsequently lysed with 0.2% Triton X-100 (vol/vol in double distilled H₂O). The absorbance was measured at 550 nm using a Microplate Autoreader EL309 (BIO-TEK Instruments, Winooski, VT, USA). Blank wells containing medium alone were used for background subtraction and sham-treated cells were cultured in parallel as controls.

Apoptotic cell death was assessed using annexin V-phycoerythrin (PE) apoptosis detection kit I (BD Pharmingen, Erembodegem, Belgium), in accordance with the manufacturer's recommendations. Briefly, cells were seeded in six-well plates (density 50,000 cells/well), cultured for 24 hours and treated with bisphosphonates in presence or absence of additional calcium concentrations for 72 hours, as described in Results (see below). Cell monolayers were washed twice in PBS, harvested by trypsinization, centrifuged and resuspended in 100 μl 1× Binding Buffer (BD Pharmingen). After addition of 5 μl annexin V-PE and 5 μl of 7-amino-actinomycin (7-AAD, a vital dye), cell suspensions were incubated for 15 minutes at room temperature in darkness. Finally, cell samples were diluted with 400 μl 1× binding buffer and analyzed in a flow cytometer (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ, USA) within 1 hour. Data are presented as percentages of annexin V-PE positive and 7-AAD negative cells.

Unprenylated Rap1A and total Rap1 were determined by Western blotting. Cells were plated at a density of 10⁴ cells/cm² in 60 cm² Petri dishes containing complete RPMI medium, cultured for 24 hours and then incubated for 24 additional hours with calcium (0.6 or 2.0 mmol/l) and ibandronate (1 to 1,000 μmol/l) or vehicle as specified in Results (see below). Cell monolayers were rinsed twice with Tris-buffered saline (50 mmol/l Tris-HCl [pH 7.5], 150 mmol/l NaCl), lysed in Tris-buffered saline containing 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 50 mmol/l NaF, 0.1 mM Na₃VO₄ and 5 mmol/l EDTA with freshly added proteolysis inhibitors, and finally centrifuged. Protein concentrations in cell lysate supernatants were determined by the BCA Protein Assay (Pierce, Rockford, IL, USA) using bovine serum albumin as standard. Equal amounts of protein were subjected to Western blotting using a goat polyclonal anti-human Rap1A antibody (SC-1482; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:1,000, which recognizes the unprenylated form of the small GTPase Rap1A [22], and a rabbit polyclonal anti-human Rap1 antibody (SC-65; Santa Cruz Biotechnology) diluted 1:500. Peroxidase-labelled anti-goat IgG antibody (1:1,500; Pierce) and peroxidase-labelled anti-rabbit IgG antibody (1:2,500; Amersham Pharmacia Biotech, Roosendaal, The Netherlands) were used as secondary reagents to detect corresponding primary antibodies. Bound peroxidase activity was revealed using the SuperSignal^® West Pico Chemiluminescent Substrate (Pierce). Immunostaining signals were digitalized with a PC-driven LAS-3000 CCD camera (Fujifilm, Tokyo, Japan), using software specifically designed for image acquisition (Image Reader, Raytest^®, Straubenhardt, Germany).

Bisphosphonate accumulation by breast cancer cells was determined by using [¹⁴C]ibandronate. Cells were seeded in 12-well plates at a density of 40,000 cells/well in complete RPMI medium and cultured for 24 hours. Cells were then incubated in fresh medium supplemented or not supplemented with calcium and containing 10 μmol/l [¹⁴C]ibandronate (102 mCi/mmol) for 4 hours, as described in Results (seee below). Cell monolayers were rinsed and lysed as described under Western blot analysis (see above). The amounts of [¹⁴C]ibandronate associated with cell lysate supernatants were determined using a liquid scintillation counter (Wallac 1409; PerkinElmer, Waltham, MA, USA). Protein concentrations in the same samples were measured by the BCA Protein Assay (Pierce), using bovine serum albumin as standard. Results were expressed as picomoles of ibandronate per milligram of protein.

Data are reported as means ± standard deviation, and statistical analysis was performed by analysis of variance. Dunnett post hoc test was used to compare treated conditions with the untreated condition (control), and Tukey post hoc test was performed for multiple comparisons between groups. The level of statistical significance was arbitrarily set at 0.01. All analyses were conducted using SPSS software (SPSS Inc., Paris, France).

Breast cancer cells were cultured in complete RPMI medium containing 5% foetal bovine serum and supplemented or not supplemented with CaCl₂, to achieve calcium concentrations from 0.6 to 2.0 mmol/l. Cell growth was assessed after 72 hours of treatment with ibandronate.

In the presence of 0.6 mmol/l calcium, 30 μmol/l ibandronate had no effect on MDA-MB-231 cell growth, whereas it slightly inhibited MCF-7 cell growth by 13.6 ± 6.6% (Figure 1). Higher extracellular calcium concentrations enhanced the inhibitory effects of ibandronate in a dose-dependent manner. Indeed, in the presence of 2.0 mmol/l calcium, 30 μmol/l ibandronate dramatically inhibited cell proliferation by 55.5 ± 7.8% and 76.1 ± 4.6% (P < 0.01) in MDA-MB-231 and MCF-7 cells, respectively. Half maximal inhibitory concentration (IC₅₀) values for growth inhibition by ibandronate were determined in presence of 0.6 and 1.6 mmol/l calcium. Dose-response curves revealed that an increase in calcium concentration decreased the IC₅₀ values of ibandronate from 150 to 60 μmol/l in MDA-MB-231 cells and from 80 to 10 μmol/l in MCF-7 cells (Figure 2 and Table 1). Similar effects were also documented in four additional breast cancer cell lines (T-47D, ZR-75-1, Hs-578T and BT-549; Table 1), indicating that these observations are not restricted to particular cell lines. Of note, a higher sensitivity of tumour cells to ibandronate appeared to be associated with the oestrogen receptor (ER) status, but appeared to be independent of the type of bone lesions that the tumour cells may develop in nude mice. Altogether, these data indicated that high calcium concentration enhanced the growth inhibitory potency of ibandronate in breast cancer cells.

Annexin V-PE labelling was used to detect apoptotic cell death. Cell apoptosis rates were low (<3% in both cell lines) in the control condition (0.6 mmol/l calcium) and did not significantly change in presence of 1.6 mmol/l calcium (Figure 3). Ibandronate (30 μmol/l) did not induce significant cell apoptosis in presence of 0.6 mmol/l calcium. By contrast, it significantly increased the percentage of annexin V-PE-positive cells in the presence of 1.6 mmol/l calcium (11.4% and 32.9% in MDA-MB-231 and MCF-7 cells, respectively; Figure 3). Similar observations were obtained with zoledronic acid, except that the latter drug provoked more extensive apoptosis, particularly in presence of 1.6 mmol/l calcium (17% and 52% for MDA-MB-231 and MCF-7 cells, respectively).

As documented above by analyzing bisphosphonate-induced apoptosis, the modulating effect of calcium was not restricted to ibandronate, because this cation also increased the growth inhibition produced by zoledronic acid (Figure 4a). Thus, dose-response curves showed that an increase in calcium level from 0.6 to 1.6 mmol/l diminished the IC₅₀ values of zoledronic acid from 50 to 15 μmol/l in MDA-MB-231 cells and from 20 to 2 μmol/l in MCF-7 cells. Interestingly, these effects were specific to bisphosphonates because calcium had no effect on the cytotoxicity induced by the structurally unrelated antimitotic agent paclitaxel (Figure 4b). In addition, the enhancement of ibandronate activity was specifically noted with calcium, inasmuch as it was not observed with the other divalent cation magnesium in the same range of concentrations (Figure 4c). On the other hand, calcium chelation by EGTA at 0.5 mmol/l, a concentration that did not affect cell growth, significantly reduced the growth inhibitory effect induced by 30 μmol/l ibandronate in culture medium containing 1.6 mmol/l calcium (Figure 5). Similarly, calcium chelation by 100 μmol/l clodronate, a pyrophosphate-resembling bisphosphonate that had no detectable effect on cell survival at this concentration, significantly reversed the growth inhibition induced by ibandronate (data not shown).

A potential role of the CaR in mediating the effects of calcium on cell growth inhibition induced by ibandronate was checked by using CaR regulators. The CaR agonist NPS R-467 at 10^-5 mol/l did not mimic the effects of high extracellular calcium concentrations, because it did not enhance growth inhibition by ibandronate (Figure 6). Likewise, the CaR antagonist at 10^-6 mol/l did not change the growth inhibitory effect of ibandronate at 1.6 mmol/l calcium concentration. Therefore, these data excluded the possibility that CaR might be involved in the modulation of bisphosphonate effect by calcium.

In order to address the effects of calcium on cellular accumulation of ibandronate, MDA-MB-231 and MCF-7 cells were cultured in medium containing 0.6 or 2.0 mmol/l calcium and were exposed to 10 μmol/l [¹⁴C]ibandronate for 4 hours (Figure 7). In the presence of 2.0 mmol/l calcium, cells accumulated more radiolabelled ibandronate than cells cultured in presence of 0.6 mmol/l calcium (about 4.6-fold and 11.4-fold increases in MDA-MB-231 and MCF-7 cells, respectively). Interestingly, MCF-7 cells accumulated threefold more [¹⁴C]ibandronate than did MDA-MB-231 cells, which could contribute to the higher sensitivity of the former cells to bisphosphonates (Figure 1 to 3).

Recent data indicate that nitrogen-containing bisphosphonates induce a dose-dependent accumulation of unprenylated Rap1A in MDA-MB-231 cells [23]. We confirmed that calcium-induced modulation of cellular accumulation of ibandronate took place by demonstrating that high calcium concentration augmented ibandronate-induced inhibition of protein prenylation (Figure 8). Indeed, in both cell lines 10 μmol/l ibandronate was sufficient to produce a detectable inhibition of Rap1A prenylation in the presence of 2.0 mmol/l calcium, whereas 100 μmol/l ibandronate was required to achieve a similar effect in presence of 0.6 mmol/l calcium. Therefore, high calcium level increased the intracellular activity of ibandronate, probably by favouring cellular drug accumulation.

Figure 9 illustrates the growth inhibitory effect of shorter exposures (1 and 6 hours) to ibandronate plus calcium at low or high concentrations. In culture medium containing 0.6 mmol/l calcium, 1 hour of ibandronate treatment had no or only a weak effect on breast cancer cell growth. As expected, continuous treatment (72 hours) increased bisphosphonate-induced inhibition of growth. In the presence of 1.6 mmol/l calcium, exposure to ibandronate for 1 hour dramatically decreased cell proliferation. Moreover, in these experimental conditions, inhibition of cell growth was more effective than after 72 hours of ibandronate treatment with low calcium concentration. Of note, 6 hours of exposure produced intermediate effects. These data brought further evidence that calcium enhances the growth inhibitory effect of ibandronate in breast cancer cells, even in the case of short treatment durations, suggesting a significant decrease in the time needed to obtain an effective cell accumulation of the bisphosphonate.