- Research article
- Open Access
Additive growth inhibitory effects of ibandronate and antiestrogens in estrogen receptor-positive breast cancer cell lines
Breast Cancer Research volume 8, Article number: R2 (2005)
Bisphosphonates are inhibitors of osteoclast-mediated tumor-stimulated osteolysis, and they have become standard therapy for the management of bone metastases from breast cancer. These drugs can also directly induce growth inhibition and apoptosis of osteotropic cancer cells, including estrogen receptor-positive (ER+) breast cancer cells.
We examined the anti-proliferative properties of ibandronate on two ER+ breast cancer cell lines (MCF-7 and IBEP-2), and on one ER negative (ER-) cell line (MDA-MB-231). Experiments were performed in steroid-free medium to assess ER regulation and the effect of ibandronate in combination with estrogen or antiestrogens.
Ibandronate inhibited cancer cell growth in a dose- and time-dependent manner (approximate IC50: 10-4 M for MCF-7 and IBEP-2 cells; 3 × 10-4 M for MDA-MB-231 cells), partly through apoptosis induction. It completely abolished the mitogenic effect induced by 17β-estradiol in ER+ breast cancer cells, but affected neither ER regulation nor estrogen-induced progesterone receptor expression, as documented in MCF-7 cells. Moreover, ibandronate enhanced the growth inhibitory action of partial (4-hydroxytamoxifen) and pure (ICI 182,780, now called fluvestrant or Faslodex™) antiestrogens in estrogen-sensitive breast cancer cells. Combination analysis identified additive interactions between ibandronate and ER antagonists.
These data constitute the first in vitro evidence for additive effects between ibandronate and antiestrogens, supporting their combined use for the treatment of bone metastases from breast cancer.
Over 80% of women suffering from advanced breast cancer ultimately develop bone metastases [1, 2]. As revealed by observations published more than a decade ago , patients with estrogen receptor (ER)-positive neoplasms are more prone to develop skeletal secondaries. Metastatic breast cancer cells stimulate osteoclast-mediated bone resorption, inducing a marked osteolysis that is responsible for considerable morbidity [4, 5].
Bisphosphonates are potent inhibitors of osteoclast-mediated osteolysis  and have, therefore, emerged as a rational approach for the management of bone metastases [7, 8]. These drugs are synthetic analogs of pyrophosphate. They show high affinity for bone mineral and preferentially accumulate at sites of active bone remodeling. The most potent bisphosphonates are nitrogen-containing compounds (e.g. ibandronate, zoledronic acid) that interfere with the mevalonate pathway, leading to inhibition of the post-translational prenylation of proteins [9, 10]. From cell culture studies, it is known that they inhibit the resorptive activity and induce the apoptosis of mature osteoclasts [10, 11].
Moreover, there is now compelling in vitro evidence that bisphosphonates may also act directly on tumor cells. They inhibit proliferation and induce apoptosis in cell lines derived from various neoplasms, such as breast [12, 13] and prostate carcinomas [14, 15]. Bisphosphonates may also antagonize the growth stimulation induced by bone-derived growth factors on human breast cancer cells . Furthermore, recent animal data indicate that bisphosphonates inhibit bone metastasis growth through promotion of apoptosis in cancer cells [17, 18]. Bisphosphonates also reduce tumor cell invasiveness  and cell adhesion to bone .
In the clinical setting, bisphosphonates are often combined with conventional endocrine agents for the treatment of patients with metastatic bone disease, especially as endocrine therapy is often preferred to chemotherapy for patients with soft tissue and bone metastases . The extent to which such bisphosphonate and antiestrogen combination affects tumor cell growth has not yet been examined, however, and it is unknown which interactions are operating. The triphenylethylene antiestrogen tamoxifen is the classic hormonal treatment for the management of breast cancers expressing ERs . On the other hand, ICI 182,780  (now called fulvestrant or Faslodex™) is the only steroidal antiestrogen that has reached clinical development . Both compounds are competitive inhibitors for the binding of 17β-estradiol (E2) to ER, but their mechanisms of action are quite different . Tamoxifen, a partial ER antagonist, inhibits the activation function-2 (AF-2)-mediated transactivation, probably via the recruitment of corepressors [26, 27]. Yet this type of antagonist does not interfere with AF-1-mediated transactivation. Tamoxifen, as well as its active metabolite 4-hydroxytamoxifen, has also been shown to cause ER nuclear accumulation . By contrast, ICI 182,780, a pure ER antagonist, suppresses both AF-1 and AF-2 ER transactivation functions, and prevents nuclear transport of the receptor . In addition, such pure antagonists reduce the half-life of ER protein, leading to a decrease in receptor content (down-regulation) .
In the present study, we assessed the anti-proliferative properties of ibandronate, a newly developed nitrogen-containing bisphosphonate, on ER-positive breast cancer cells. These in vitro experiments were conducted in steroid-free medium (SFM) to allow for the assessment of estrogenic responses and for the measurement of ER content and activity. Besides, it is known that ER antagonists exert a growth-inhibitory effect on MCF-7 cells even in the absence of estrogenic stimulation [31–34]. We thus tested ibandronate in combination with antiestrogens in order to identify possible additive or synergistic interactions.
Materials and methods
Cell culture conditions
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  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 . 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.
Crystal violet staining
Cell number was assessed indirectly by staining with crystal violet dye as previously described . 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.
Western blot analysis
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 . 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.
Combination index calculations
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  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:
CIA+B = [(DA/A+B)/DA] + [(DB/A+B)/DB] + [α(DA/A+AB × DB/A+B)/DADB]
where CIA+B = CI for a fixed effect (F) for the combination of cytotoxic A and cytotoxic B; DA/A+B = concentration of cytotoxic A in the combination A + B giving an effect F; DB/A+B = concentration of cytotoxic B in the combination A + B giving an effect F; DA = concentration of cytotoxic A alone giving an effect F; DB = 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).
Effects of ibandronate on breast cancer cell growth in steroid-free medium
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.
Influence of ibandronate on the mitogenic effect of E2, and on estrogen receptor regulation and activity in MCF-7 cells
As previously reported , 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  (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 . 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 , 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.
Combination of ibandronate and antiestrogens
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 . 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.
Bisphosphonates, including ibandronate, are widely used for the treatment of bone diseases involving enhanced osteoclast-mediated bone resorption, such as osteoporosis, tumor-induced hypercalcemia and, most importantly, cancer-induced bone disease [7, 41]. Bisphosphonates thus constitute a major advance in the supportive care of cancer patients who develop skeletal metastases . They exert a clinically significant analgesic activity and reduce by up to 40% the frequency of cancer-induced bone complications in patients with bone metastasis from breast cancer . The beneficial effects of ibandronate, a new and potent bisphosphonate, in patients sufffering from breast cancer-induced osteolysis have recently been reported [44–46]. A substantial morbidity nevertheless persists even with the use of such new potent agents, indicating that bisphosphonates are not fully effective in blocking tumor-induced osteolysis. More studies are needed, notably to better understand the activity of bisphosphonates on tumor cells.
Recent data from in vitro work on breast cancer-derived cell lines indicate that nitrogen-containing bisphosphonates can directly inhibit tumor cell growth, primarily by inducing apoptosis [12, 13, 47–49]. However, these studies were performed in medium supplemented with whole serum, where the presence of steroids does not allow the evaluation of possible interactions of bisphosphonates with ER-mediated signaling. In the present study, we investigated the effects of ibandronate on the growth of MCF-7, IBEP-2 and MDA-MB-231 cells in medium supplemented with charcoal-stripped serum, providing a steroid-free environment suitable for the assessment of estrogenic responses. In fact, these experimental conditions may reflect more accurately the clinical situation because the vast majority of patients suffering from breast cancer are postmenopausal women with low circulating estrogen levels.
In the first part of this work, we showed that ibandronate (a nitrogen-containing bisphosphonate) induced a dose-dependent decrease in the growth of the three tested breast cancer cells cultured in SFM. These results were consistent with previous reports using steroid-containing medium (complete medium) [12, 13, 47], but contrast with our recent data showing that clodronate (a non-nitrogen-containing bisphosphonate) can stimulate the proliferation of MCF-7 cells in SFM . In fact, this mitogenic effect of clodronate appears to be mediated by the activation of ERs and is completely suppressed by antiestrogens. Moreover, focusing on MCF-7 cells, we showed that 10-3 M ibandronate exerted strong cytotoxicity partly through apoptosis induction. A lower concentration (10-4 M) of ibandronate exerted cytostatic effects associated with moderate apoptosis induction, suggesting that cell proliferation was exactly balanced by cell death. Of note, significant apoptosis was only observed after four days of incubation with 10-4 M ibandronate. Hence, at day 3, cell growth inhibition could be explained by cell cycle arrest, this step preceding apoptosis induction. Furthermore, in our experimental conditions, short-term exposures (four hours) of cells to 10-4 M ibandronate were sufficient to induce a significant inhibition of MCF-7 cell proliferation. In addition, the presence of ibandronate for only 24 hours led to an irreversible loss of cell proliferative capacity, as shown by measurement two days later, and growth inhibition was comparable to a full-time exposure. This is consistent with the observations of Jagdev et al.  showing that incubation of MCF-7 cells with zoledronic acid, even for a short period of time, results in a significant reduction in cell number and a sizeable increase in apoptosis. These results might be important insofar as serum ibandronate concentrations are maintained for only a few hours after oral bisphosphonate administration  and raise the exciting possibility of short-term effects of bisphosphonates in non-osseous sites. On the other hand, in the particular microenvironment of bone metastases, bisphosphonates accumulate at the surface of bone resorption sites. This creates a compartment where cells undergo prolonged exposure to high drug concentrations. In vivo data revealed that effective local concentrations of bisphosphonates at sites of active bone resorption are much higher than serum levels, and may reach up to 10-3 M in the resorption lacunae .
In the second part of our study, we examined the effects of ibandronate on estrogenic stimulation of MCF-7 and IBEP-2 cells. Interestingly, our experiments conducted in SFM showed that, at a concentration affecting cell proliferation (10-4 M) but not at a lower concentration (10-6 M), ibandronate suppressed the mitogenic effect induced by E2 and inhibited cell growth regardless of the presence of estrogen. As the nitrogen-containing bisphosphonate ibandronate is known to act through the inhibition of the mevalonate pathway and subsequent protein prenylation , the estrogenic stimulation of cell proliferation might require some prenylated proteins. Indeed, it has recently been reported that prenylated proteins play a role in estradiol-induced stimulation of cell proliferation through activation of the Src/Ras/Erk pathway . Nevertheless, as shown by the current observations using MCF-7 cells, ibandronate had no direct effect on ER expression and did not affect E2-induced receptor down-regulation. Similarly, ibandronate did not affect the baseline level of progesterone receptor and did not interfere with E2-induced expression of this receptor. Altogether, these data indicate that ibandronate did not alter the regulation and the activity of ERs in MCF-7 cells, while it could totally prevent estrogen-induced cell proliferation in ER-positive breast cancer cells, suggesting that it acts downstream of ER-mediated gene transactivation.
Cancer therapy mostly relies on the use of drug combinations, which generally improve the therapeutic index, that is, give better responses with less toxicity. In this setting, bisphosphonates are most often used concomitantly with endocrine therapy. The presence of functional ERs in MCF-7 cells exposed to ibandronate suggests that these cells should remain sensitive to antiestrogenic agents. In the third part of our work, we thus tested the effects of ibandronate, antiestrogens (4-hydroxytamoxifen and ICI 182,780) and combinations thereof on MCF-7, IBEP-2 and MDA-MB-231 cell growth. Of note, our data show that antiestrogens alone inhibited the proliferation of MCF-7 and IBEP-2 cells in SFM. Used as a negative control, MDA-MB-231 cell growth was not affected by antiestrogens. These results confirm previous studies showing that ER antagonists are able to reduce the growth of ER-positive breast cancer cells, even in the absence of estrogenic stimulation . It is conceivable that ER in estrogen-deprived MCF-7 cells is activated by phosphorylation due to cross-talk with other signaling pathways [54, 55]. Thus, antiestrogens would completely abolish this activation. Moreover, when ER-positive breast cancer cells were exposed to ibandronate and antiestrogens, we observed additive inhibition of cell proliferation. The mechanism by which this occurred has not been fully elucidated but inhibition of the mevalonate pathway and subsequent protein prenylation due to ibandronate treatment, and prevention of cell proliferation by antiestrogens might result in this additive effect. Indeed, nitrogen-containing bisphosphonates are known to act as analogs of isoprenoid diphosphate lipids and to inhibit farnesyl pyrophosphate synthase, an enzyme of the mevalonate pathway [56, 57]. This inhibition results in decreased isoprenoid lipid production (farnesyl pyrophosphate and geranylgeranyl pyrophosphate) and prevents protein prenylation . On the other hand, antiestrogens are reported to cause the arrest of MCF-7 cells in the G1 phase of the cell cycle, resulting in a lower proportion of cells in S phase [59, 60]. Moreover, cells treated with partial and pure antiestrogens show a significant decrease in cyclin D1 mRNA, which suggests that cyclin may be a target of antiestrogens, thus blocking entry into S phase . Because of these different mechanisms of action, it is not a surprise that we found additive growth inhibitory effects when ibandronate and antiestrogens were added concomitantly. The mechanisms involved in the additive effect of ibandronate and antiestrogens are still elusive, however, and require further investigations.
Interestingly, in accordance with our results, recent data indicate that tamoxifen and the farnesyl transferase inhibitor FTI-277, acting through distinct pathways, exert an additive effect on MCF-7 cells, inhibiting cell cycle progression and cell proliferation . Of note, farnesyl transferase functions closely downstream of farnesyl pyrophosphate synthase (the target for nitrogen-containing bisphosphonates) as it catalyzes the covalent attachment of farnesyl groups to prenylated proteins.
Our results indicate that ibandronate inhibits breast cancer cell growth, both in the presence and absence of estrogenic stimulation. Moreover, the growth inhibition induced by classic antiestrogens, such as 4-hydroxytamoxifen and ICI 182,780, is larger on cancer cells expressing ERs when they are combined with ibandronate. These data suggest for the first time the existence of additive interactions between bisphosphonates and antiestrogens. Thus, our in vitro data provide a rationale for the combined use of ibandronate and antiestrogens in breast cancer patients suffering from bone metastases.
- E2 :
fetal calf serum
Body JJ: Metastatic bone disease: clinical and therapeutic aspects. Bone. 1992, 13 (Suppl 1): S57-62.
Coleman RE: Skeletal complications of malignancy. Cancer. 1997, 80 (8 Suppl): 1588-1594. 10.1002/(SICI)1097-0142(19971015)80:8+<1588::AID-CNCR9>3.0.CO;2-G.
Koenders PG, Beex LV, Langens R, Kloppenborg PW, Smals AG, Benraad TJ: Steroid hormone receptor activity of primary human breast cancer and pattern of first metastasis. The Breast Cancer Study Group. Breast Cancer Res Treat. 1991, 18: 27-32. 10.1007/BF01975440.
Mundy GR: Mechanisms of osteolytic bone destruction. Bone. 1991, 12 (Suppl 1): S1-6. 10.1016/8756-3282(91)90057-P.
Body JJ: Rationale for the use of bisphosphonates in osteoblastic and osteolytic bone lesions. Breast. 2003, 12 (Suppl 2): S37-44. 10.1016/S0960-9776(03)80162-5.
Fleisch H: Bisphosphonates: mechanisms of action. Endocr Rev. 1998, 19: 80-100. 10.1210/er.19.1.80.
Body JJ, Bartl R, Burckhardt P, Delmas PD, Diel IJ, Fleisch H, Kanis JA, Kyle RA, Mundy GR, Paterson AH, et al: Current use of bisphosphonates in oncology. International Bone and Cancer Study Group. J Clin Oncol. 1998, 16: 3890-3899.
Mundy GR, Yoneda T: Bisphosphonates as anticancer drugs. N Engl J Med. 1998, 339: 398-400. 10.1056/NEJM199808063390609.
Luckman SP, Hughes DE, Coxon FP, Graham R, Russell G, Rogers MJ: Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including Ras. J Bone Miner Res. 1998, 13: 581-589.
Russell RG, Rogers MJ: Bisphosphonates: from the laboratory to the clinic and back again. Bone. 1999, 25: 97-106. 10.1016/S8756-3282(99)00116-7.
Benford HL, McGowan NW, Helfrich MH, Nuttall ME, Rogers MJ: Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro. Bone. 2001, 28: 465-473. 10.1016/S8756-3282(01)00412-4.
Fromigue O, Lagneaux L, Body JJ: Bisphosphonates induce breast cancer cell death in vitro. J Bone Miner Res. 2000, 15: 2211-2221.
Senaratne SG, Pirianov G, Mansi JL, Arnett TR, Colston KW: Bisphosphonates induce apoptosis in human breast cancer cell lines. Br J Cancer. 2000, 82: 1459-1468.
Lee MV, Fong EM, Singer FR, Guenette RS: Bisphosphonate treatment inhibits the growth of prostate cancer cells. Cancer Res. 2001, 61: 2602-2608.
Dumon JC, Journe F, Kheddoumi N, Lagneaux L, Body JJ: Cytostatic and apoptotic effects of bisphosphonates on prostate cancer cells. Eur Urol. 2004, 45: 521-528. 10.1016/j.eururo.2003.12.012. discussion 528–529
Fromigue O, Kheddoumi N, Body JJ: Bisphosphonates antagonise bone growth factors' effects on human breast cancer cells survival. Br J Cancer. 2003, 89: 178-184. 10.1038/sj.bjc.6601009.
Hiraga T, Williams PJ, Mundy GR, Yoneda T: The bisphosphonate ibandronate promotes apoptosis in MDA-MB-231 human breast cancer cells in bone metastases. Cancer Res. 2001, 61: 4418-4424.
Neudert M, Fischer C, Krempien B, Bauss F, Seibel MJ: Site-specific human breast cancer (MDA-MB-231) metastases in nude rats: model characterisation and in vivo effects of ibandronate on tumour growth. Int J Cancer. 2003, 107: 468-477. 10.1002/ijc.11397.
Boissier S, Ferreras M, Peyruchaud O, Magnetto S, Ebetino FH, Colombel M, Delmas P, Delaisse JM, Clezardin P: Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases. Cancer Res. 2000, 60: 2949-2954.
Boissier S, Magnetto S, Frappart L, Cuzin B, Ebetino FH, Delmas PD, Clezardin P: Bisphosphonates inhibit prostate and breast carcinoma cell adhesion to unmineralized and mineralized bone extracellular matrices. Cancer Res. 1997, 57: 3890-3894.
Miyoshi Y, Taguchi T, Tamaki Y, Noguchi S: Current status of endocrine therapy for breast cancer. Breast Cancer. 2003, 10: 105-111.
Mouridsen H, Palshof T, Patterson J, Battersby L: Tamoxifen in advanced breast cancer. Cancer Treat Rev. 1978, 5: 131-141.
Wakeling AE, Dukes M, Bowler J: A potent specific pure antiestrogen with clinical potential. Cancer Res. 1991, 51: 3867-3873.
Bross PF, Baird A, Chen G, Jee JM, Lostritto RT, Morse DE, Rosario LA, Williams GM, Yang P, Rahman A, et al: Fulvestrant in postmenopausal women with advanced breast cancer. Clin Cancer Res. 2003, 9: 4309-4317.
Howell A, Osborne CK, Morris C, Wakeling AE: ICI 182,780 (Faslodex): development of a novel, "pure" antiestrogen. Cancer. 2000, 89: 817-825. 10.1002/1097-0142(20000815)89:4<817::AID-CNCR14>3.0.CO;2-6.
Webster NJ, Green S, Jin JR, Chambon P: The hormone-binding domains of the estrogen and glucocorticoid receptors contain an inducible transcription activation function. Cell. 1988, 54: 199-207. 10.1016/0092-8674(88)90552-1.
Lees JA, Fawell SE, Parker MG: Identification of two transactivation domains in the mouse oestrogen receptor. Nucleic Acids Res. 1989, 17: 5477-5488.
Laios I, Journe F, Laurent G, Nonclercq D, Toillon RA, Seo HS, Leclercq G: Mechanisms governing the accumulation of estrogen receptor alpha in MCF-7 breast cancer cells treated with hydroxytamoxifen and related antiestrogens. J Steroid Biochem Mol Biol. 2003, 87: 207-221. 10.1016/j.jsbmb.2003.09.011.
Dauvois S, White R, Parker MG: The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling. J Cell Sci. 1993, 106: 1377-1388.
Laios I, Journe F, Nonclercq D, Vidal DS, Toillon RA, Laurent G, Leclercq G: Role of the proteasome in the regulation of estrogen receptor alpha turnover and function in MCF-7 breast carcinoma cells. J Steroid Biochem Mol Biol. 2005, 94: 347-359. 10.1016/j.jsbmb.2005.02.005.
Katzenellenbogen BS, Kendra KL, Norman MJ, Berthois Y: Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breast cancer cells grown in the short-term and long-term absence of estrogens. Cancer Res. 1987, 47: 4355-4360.
Vignon F, Bouton MM, Rochefort H: Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens. Biochem Biophys Res Commun. 1987, 146: 1502-1508. 10.1016/0006-291X(87)90819-9.
Wang S, Zhang B, Faller DV: BRG1/BRM and prohibitin are required for growth suppression by estrogen antagonists. EMBO J. 2004, 23: 2293-2303. 10.1038/sj.emboj.7600231.
Buck MB, Pfizenmaier K, Knabbe C: Antiestrogens induce growth inhibition by sequential activation of p38 mitogen-activated protein kinase and transforming growth factor-beta pathways in human breast cancer cells. Mol Endocrinol. 2004, 18: 1643-1657. 10.1210/me.2003-0278.
Siwek B, Larsimont D, Lacroix M, Body JJ: Establishment and characterization of three new breast-cancer cell lines. Int J Cancer. 1998, 76: 677-683. 10.1002/(SICI)1097-0215(19980529)76:5<677::AID-IJC11>3.0.CO;2-1.
Devleeschouwer N, Legros N, Olea-Serrano N, Paridaens R, Leclercq G: Estrogen conjugates and serum factors mediating the estrogenic trophic effect on MCF-7 cell growth. Cancer Res. 1987, 47: 5883-5887.
Journe F, Chaboteaux C, Dumon JC, Leclercq G, Laurent G, Body JJ: Steroid-free medium discloses estrogenic effects of the bisphosphonate clodronate on breast cancer cells. Br J Cancer
Chou TC, Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984, 22: 27-55. 10.1016/0065-2571(84)90007-4.
Journe F, Body JJ, Leclercq G, Nonclercq D, Laurent G: Estrogen responsiveness of IBEP-2, a new human cell line derived from breast carcinoma. Breast Cancer Res Treat. 2004, 86: 39-53. 10.1023/B:BREA.0000032922.87541.31.
Fazzari A, Catalano MG, Comba A, Becchis M, Raineri M, Frairia R, Fortunati N: The control of progesterone receptor expression in MCF-7 breast cancer cells: effects of estradiol and sex hormone-binding globulin (SHBG). Mol Cell Endocrinol. 2001, 172: 31-36. 10.1016/S0303-7207(00)00397-X.
Lipton A: Bisphosphonates and breast carcinoma: present and future. Cancer. 2000, 88 (12 Suppl): 3033-3037. 10.1002/1097-0142(20000615)88:12+<3033::AID-CNCR20>3.0.CO;2-C.
Body JJ: Reducing skeletal complications and bone pain with intravenous ibandronate for metastatic bone disease. EJC Supplements. 2004, 2: 5-8.
Body JJ: Bisphosphonates in the treatment of metastatic breast cancer. J Mammary Gland Biol Neoplasia. 2001, 6: 477-485. 10.1023/A:1014795216669.
Body JJ, Diel IJ, Lichinitser MR, Kreuser ED, Dornoff W, Gorbunova VA, Budde M, Bergstrom B: Intravenous ibandronate reduces the incidence of skeletal complications in patients with breast cancer and bone metastases. Ann Oncol. 2003, 14: 1399-1405. 10.1093/annonc/mdg367.
Body JJ, Diel IJ, Lichinitzer M, Lazarev A, Pecherstorfer M, Bell R, Tripathy D, Bergstrom B: Oral ibandronate reduces the risk of skeletal complications in breast cancer patients with metastatic bone disease: results from two randomised, placebo-controlled phase III studies. Br J Cancer. 2004, 90: 1133-1137. 10.1038/sj.bjc.6601663.
Mancini I, Dumon JC, Body JJ: Efficacy and safety of ibandronate in the treatment of opoid-resistant bone pain associated with metastatic bone disease: a pilot study. J Clin Oncol. 2004, 22: 3587-3592. 10.1200/JCO.2004.07.054.
Jagdev SP, Coleman RE, Shipman CM, Rostami HA, Croucher PI: The bisphosphonate, zoledronic acid, induces apoptosis of breast cancer cells: evidence for synergy with paclitaxel. Br J Cancer. 2001, 84: 1126-1134. 10.1054/bjoc.2001.1727.
Senaratne SG, Mansi JL, Colston KW: The bisphosphonate zoledronic acid impairs Ras membrane [correction of impairs membrane] localisation and induces cytochrome c release in breast cancer cells. Br J Cancer. 2002, 86: 1479-1486. 10.1038/sj.bjc.6600297.
Senaratne SG, Colston KW: Direct effects of bisphosphonates on breast cancer cells. Breast Cancer Res. 2002, 4: 18-23. 10.1186/bcr412.
Ravn P, Neugebauer G, Christiansen C: Association between pharmacokinetics of oral ibandronate and clinical response in bone mass and bone turnover in women with postmenopausal osteoporosis. Bone. 2002, 30: 320-324. 10.1016/S8756-3282(01)00665-2.
Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD, Golub E, Rodan GA: Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest. 1991, 88: 2095-2105.
Doisneau-Sixou SF, Cestac P, Chouini S, Carroll JS, Hamilton AD, Sebti SM, Poirot M, Balaguer P, Faye JC, Sutherland RL, et al: Contrasting effects of prenyltransferase inhibitors on estrogen-dependent cell cycle progression and estrogen receptor-mediated transcriptional activity in MCF-7 cells. Endocrinology. 2003, 144: 989-998. 10.1210/en.2002-220726.
Jensen J, Kitlen JW, Briand P, Labrie F, Lykkesfeldt AE: Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium. J Steroid Biochem Mol Biol. 2003, 84: 469-478. 10.1016/S0960-0760(03)00068-2.
Bunone G, Briand PA, Miksicek RJ, Picard D: Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J. 1996, 15: 2174-2183.
Weigel NL, Zhang Y: Ligand-independent activation of steroid hormone receptors. J Mol Med. 1998, 76: 469-479. 10.1007/s001090050241.
van Beek E, Pieterman E, Cohen L, Lowik C, Papapoulos S: Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates. Biochem Biophys Res Commun. 1999, 264: 108-111. 10.1006/bbrc.1999.1499.
Bergstrom JD, Bostedor RG, Masarachia PJ, Reszka AA, Rodan G: Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch Biochem Biophys. 2000, 373: 231-241. 10.1006/abbi.1999.1502.
Rogers MJ: New insights into the molecular mechanisms of action of bisphosphonates. Curr Pharm Des. 2003, 9: 2643-2658. 10.2174/1381612033453640.
Osborne CK, Boldt DH, Clark GM, Trent JM: Effects of tamoxifen on human breast cancer cell cycle kinetics: accumulation of cells in early G1 phase. Cancer Res. 1983, 43: 3583-3585.
Osborne CK, Boldt DH, Estrada P: Human breast cancer cell cycle synchronization by estrogens and antiestrogens in culture. Cancer Res. 1984, 44: 1433-1439.
Watts CK, Sweeney KJ, Warlters A, Musgrove EA, Sutherland RL: Antiestrogen regulation of cell cycle progression and cyclin D1 gene expression in MCF-7 human breast cancer cells. Breast Cancer Res Treat. 1994, 31: 95-105. 10.1007/BF00689680.
Doisneau-Sixou SF, Cestac P, Faye JC, Favre G, Sutherland RL: Additive effects of tamoxifen and the farnesyl transferase inhibitor FTI-277 on inhibition of MCF-7 breast cancer cell-cycle progression. Int J Cancer. 2003, 106: 789-798. 10.1002/ijc.11263.
Journe F, Chaboteaux C, Laurent G, Dumon JC, Body JJ: Ibandronate inhibits the proliferation of estrogen receptor-positive breast cancer cells: evidence for additivity with antiestrogens. Bone. 2004, 34: S59-abstract 23
This study received financial support from F Hoffmann-La Roche AG (Basel, Switzerland), from the 'Fondation Medic', from the Belgian Fund for Medical Scientific Research (grants 3.4563.02 and 3.4512.03), and from 'Les Amis de l'Institut Bordet'. Guy Laurent is Senior Research Associate of the National Fund for Scientific Research. This study was presented in part at The IVth International Conference on Cancer-Induced Bone Diseases, 7 to 9 December 2003, San Antonio, TX (abstract number 67), and at the workshop 'What is New in Bisphosphonates?', 24 to 26 March 2004, Davos, Switzerland .
The authors declare that they have no competing interests.
FJ designed the experiments, performed the analysis and interpretation of the data and drafted the manuscript. CC carried out cell culture experiments, cell growth determination and western blot analysis. NM participated in combination index calculation. HD carried out apoptosis determination. GL carried out cell growth experiments and critically revised the manuscript. JJB participated in the design of the experiments, discussed the results and revised the manuscript. All authors read and approved the final manuscript
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Journe, F., Chaboteaux, C., Magne, N. et al. Additive growth inhibitory effects of ibandronate and antiestrogens in estrogen receptor-positive breast cancer cell lines. Breast Cancer Res 8, R2 (2005). https://doi.org/10.1186/bcr1363
- Estrogen Receptor
- Bone Metastasis
- Zoledronic Acid