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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Int J Cancer. Author manuscript; available in PMC Jan 1, 2011.
Published in final edited form as:
PMCID: PMC2784023
NIHMSID: NIHMS138042

Bisphosphonates suppress insulin-like growth factor 1-induced angiogenesis via the HIF-1α/VEGF signaling pathways in human breast cancer cells

Abstract

Adjunctive chemotherapy with bisphosphonates has been reported to delay bone metastasis and improve overall survival in breast cancer. Aside from its anti-resorptive effect, bisphosphonates exhibit antitumor activities, in vitro and in vivo, via several mechanisms including anti-angiogenesis. In this study, we investigated the potential molecular mechanisms underlying the anti-angiogenic effect of non nitrogen-containing and nitrogen-containing bisphosphonates, clodronate and pamidronate, in IGF-1 responsive human breast cancer cells. We tested whether bisphosphonates had any effects on HIF-1α/VEGF axis that plays a pivotal role in tumor angiogenesis, and our results showed that both pamidronate and clodronate significantly suppressed IGF-1 induced HIF-1α protein accumulation and VEGF expression in MCF-7 cells. Mechanistically, we found that either pamidronate or clodronate did not affect mRNA expression of HIF-1α, but apparently promoted the degradation of IGF-1 induced HIF-1α protein. Meanwhile, we found that the presence of pamidronate and clodronate led to a dose-dependent decease in the newly-synthesized HIF-1α protein induced by IGF-1 in breast cancer cells after proteasomal inhibition, thus indirectly reflecting the inhibition of protein synthesis. In addition, our results indicated that the inhibitory effects of bisphosphonates on the HIF-1α/VEGF axis are associated with the inhibition of the PI-3K/AKT/mTOR signaling pathways. Consistently, we demonstrated that pamidronate and clodronate functionally abrogated both in vitro and in vivo tumor angiogenesis induced by IGF-1 stimulated MCF-7 cells. These findings have highlighted an important mechanism of the pharmacological action of bisphosphonates in the inhibition of tumor angiogenesis in breast cancer cells.

Keywords: bisphosphonates, breast cancer, IGF-1, HIF-1α, VEGF, PI-3K/Akt, angiogenesis

Introduction

Breast cancer is the leading cancer affecting millions of women worldwide with aggressive osteolytic bone metastases in the advanced diseases 1, 2 and longstanding morbidity or skeletal complications, including bone pain, pathological fracture, hypercalcemia, spinal cord or nerve root compression syndrome.3

Bisphosphonates are synthetic analogs of inorganic pyrophosphate, containing a phosphorus-carbon-phosphorus (P-C-P) backbone and variable side chains that determine the specific potency for inhibition of bone resorption.4,5 Bisphosphonates that lack a nitrogen functional group in the R2 side chain (such as clodronate) condense with an aminoacyladenylate to form nonhydrolyzable analogues of ATP that inhibit ATP-dependent intracellular enzymes.6, 7 On the other hand, nitrogen-containing bisphosphonates (NBPs; such as pamidronate) inhibit the activity of farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway.6, 7 Due to their potent anti-resorptive activity, some bisphosphonates (clodronate, pamidronate, ibandronaye, zoledronate) have emerged in recent years as a highly effective therapeutic option for the prevention and treatment of skeletal complications secondary to bone metastases.5, 7, 8

Aside from the anti-resorptive activity, bisphosphonates also exhibit potent antitumor effects, both in vitro and in vivo,9 via several mechanisms including inhibition of tumor cell adhesion to bone,10 tumor cell invasion,11,12 proliferation, survival and apoptosis,1116 as well as enhancement of the tumoricidal effect of various anti-neoplastic agents.11, 17 Moreover, bisphosphonates have been reported to suppress tumor angiogenesis by directly suppressing endothelial cell adhesion and proliferation, decreasing capillary-like tube formation, and inhibiting the formation of blood vessels in several animal models.11, 1821 These findings, together with recent reports that bisphosphonates altered circulating angiogenic factors in metastatic breast cancer patients,22,23 suggest that bisphosphonates are also potent anti-angiogenic agents. However, the underlying mechanism by which bisphosphonates suppress tumor angiogenesis remains largely unknown.

Central to cellular response to hypoxia is the increased expression and activity of hypoxia-inducible factor (HIF)-1α, which regulates a large subset of target genes essential for cellular adaptation to low oxygen conditions.24, 25 Under normoxic condition, HIF-1α is modified at the proline residues (pro564 and pro402) by prolyl hydroxylases, interacts with the von Hippel-Lindau tumor suppressor protein (VHL), a recognition component of an E3 ubiquitin-protein ligase, and is targeted for proteosomal degradation.26, 27 However, under hypoxic condition, HIF-1α protein is stabilized through the inactivation of an oxygen-dependent HIF-1α-prolyl hydroxylase,26,27 and translocates to the nucleus where it dimerizes with HIF-1β unit. In addition to intratumoral hypoxia, HIF-1α activity is also frequently upregulated in several cancers in response to a variety of non-hypoxic signals including the inactivation of several tumor suppressors such as p53, pVHL, and PTEN, the activation of oncogenic pathways such as Src, HER/2, and Ha-Ras,2830 and the stimulation of certain hormones, cytokines, and growth factors,3135 and viral oncoproteins.36, 37 Cumulative evidence has indicated that almost all of the hallmarks of cancer, including tumorigenosis,38,39 increased resistance of tumor cells to radio- and chemotherapies,40,41,42, increased angiogenesis 25,43 and invasion/metastasis,38,44 are closely associated with the HIF-1α pathway.45, 46

Several recent studies have demonstrated the critical role of HIF-1α in breast cancer,47 correlating HIF-1α levels with tumor grade, aggressiveness, metastasis, and an overall poor prognosis and clinical outcome in breast cancer after conventional adjuvant therapy.48, 4952 Increased HIF-1α activity has been shown to enhance the invasiveness and osteolytic bone metastases of breast cancer cells,44,53 and confers resistance to chemotherapeutic drugs.54, 55 Therefore, inhibition of HIF-1α activation using a variety of small molecules can regulate tumor growth and increase sensitivity to chemotherapy.56, 57, 58 Activation of IGF-1 receptor (IGF-R1)-mediating signaling in breast epithelial cells leads to an increase in cell proliferation, invasion/migration, a decrease in apoptosis, and resistance to antineoplastic agents,5962 suggesting that IGF-1/IGF-R1 plays an important role in mammary tumorigenesis. Previous studies have shown that IGF-1 can potently stimulate HIF-1α activity in a variety of types of cancer,3335, 63 including breast cancer cells.64, 65 These findings suggest that IGF-1/HIF-1α pathway might be a promising molecular target for chemoprevention and treatment of breast cancer.

In the present study, we investigated the effects of clodronate, a non-nitrogen containing bisphosphonate, and pamidronate, a nitrogen-containing bisphosphonate, on IGF-1-induced HIF-1α and VEGF gene expression in estrogen receptor (ER)-positive human breast cancer cells, MCF-7. We found that both clodronate and pamidronate specifically inhibited IGF-1-induced HIF-1α protein level by accelerating its protein degradation. In addition, bisphosphonates suppressed VEGF expression in MCF-7 cells by interfering with PI-3K/AKT/mTOR signaling pathways. Functionally, these bisphosphonates abrogated IGF-1-stimulated formation of capillary tube-like structures in vitro and tumor angiogenesis in vivo. These results suggest a potent anti-angiogenic property of bisphosphonates and highlight their underlying mechanisms of tumor anti-angiogenesis in breast cancer.

Materials and methods

Reagents

Pamidronate and clodronate were obtained from LKT laboratories (St. Paul, MN) and dissolved in 1×PBS (pH 7.2) (Invitrogen Corp., Carlsbad, CA). PD98059, wortmannin, and rapamycin were purchased from Calbiochem (San Diego, CA) and dissolved in DMSO. Recombinant human IGF-I, selective proteasome inhibitor MG-132 (Z-Leu-Leu-Leu-CHO), and protein synthesis inhibitor cycloheximide (CHX) were from Sigma. Antibodies include HIF-1α monoclonal antibody (BD Transduction Laboratories, San Jose, CA), total and phosphorylated extracellular signal-regulated kinase1/2 (ERK1/2; Thr202/Tyr204) or Akt (Ser473) antibodies (New England Biolabs, Ipswich, MA), total and phosphorylated mammalian target of rapamycin (mTOR) antibody (Cell Signaling Technology Inc., Danvers, MA), total and phosphorylated Mw. 70,000 ribosomal protein S6 kinase (p70S6K) (Thr421/Ser424), eukaryotic initiation factor 4E (eIF)-binding protein 1 (4E-BP1) (Ser65/Thr70) antibodies (Santa Cruz Biotechnology, CA), β-actin monoclonal antibody (Sigma), and horseradish peroxidase–conjugated secondary antibodies (Pierce, Rockford, IL). In vitro angiogenesis assay kits were from Millipore (Billerica, MA, USA).

Cell Lines and culture

Estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-468) human breast cancer cell lines (59) were obtained from American Type Culture Collection (ATCC) (Rockville, MD). Both cell lines were maintained in DMEM media (Invitrogen) supplemented with 10% fetal bovine serum (FBS), penicillin (100U/mL), streptomycin (100µg/mL) (Invitrogen) and incubated at 37°C in a humidified atmosphere with 5% CO2. Human umbilical vascular endothelial cells (HUVECs) were cultured in EGM™-2 SingleQuots® (Lonza, Walkersville, MD).

Treatment of cancer cells with bisphosphonates

Exponentially growing MCF-7 cells (about 80% confluence) were serum-starved for 24 h followed by pretreatment with different concentrations of pamidronate and clodronate for 1 h. To study the effects of pamidronate and clodronate on the half-life or degradation of IGF-1-induced HIF-1α protein accumulation, MCF-7 cells were pretreated with IGF-I at 40 ng/mL in serum-free media for 16 h followed by treatment with 10 µg/mL of CHX to inhibit further protein synthesis in the presence or absence of 50 µmol/L of pamidronate or clodronate for different time intervals. On the other hand, serum-starved cells were pretreated with 20 µmol/L of MG-132 for 30 min, followed by incubation with IGF-1 for 16 h in the presence or absence of pamidronate or clodronate. HIF-1α protein levels were determined by Western blot analysis.

Protein extract preparation and immunoblotting

Treated and untreated cells were lysed with buffer containing 50 mmol/L Tris-HCl, pH 7.5, 5 mmol/L EDTA, 150 mmol/L NaCl, 0.5% Triton X-100, 10 mmol/L sodium fluoride, 20 mmol/L β-mercaptoethanol, 250 µmol/L sodium orthovanadate, 1 mmol/L PMSF and complete protease inhibitor cocktail (Sigma), and incubated at 4 °C for 1 h. The lysates were ultra-sonicated and centrifuged at 12,000g for 10 min. The supernatants were collected and stored at −80 °C. Protein concentrations were determined by BCA methods. 50~100 µg protein was separated on 8% ~ 10% polyacrylamide-SDS gel and electroblotted onto nitrocellulose membranes (Hybond ECL, Amersham Pharmacia, Piscataway, NJ). After blocking with TBS/5% nonfat dry milk for 2 h, the membrane was incubated overnight at 4°C with antibodies against HIF-1α, total or phosphorylated ERK1/2 (Thr202/Tyr204) or Akt (Ser473), total and phosphorylated mTOR, p70S6K, or 4E-BP1, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000) (Pierce) for 45 minutes at room temperature, and the signals were visualized by enhanced chemiluminescence detection (ECL). As a loading control, the blots were re-probed with a specific antibody against human β-actin (1:5000).

RT-PCR analysis

Total RNA was isolated from cancer cells using TRIZOL® Reagent (Invitrogen) and treated with DNase I before further use. RT-PCR analysis of HIF-1α, VEGF, and β-actin mRNA levels was performed using the One-step RT-PCR Kit (QIAGEN, Valencia, CA) with primers specific to HIF-1α (forward primer 5’-TCACCACAGGACAGTACAGGATGC-3’and reverse primer 5’-CCAGCAAAGTTAAAGCATCAGGTTCC-3’), VEGF (forward primer: 5’-AGGAGG GCAGAATCATCACG-3’; and reverse primer: 5’-CAAGGCCCACAGGGATTTTCT-3’) or specific to β-actin (forward primer: 5’-TCATGAAGTGTGACGTTGACATCCGT-3’ and reverse primer: 5’-CCTAGAAG CATTTGCGGTGCACG ATG-3’). All primers were ordered from GenoMechanix, LLC (Gainesville, FL). Reactions without the addition of RNA samples were used as negative controls.

ELISA assay

The concentration of VEGF protein in the conditioned media of untreated and treated cells was determined using human VEGF ELISA Development kit (Peprotech Inc, Rocky Hill, NJ) according to the manufacturer’s instructions. Results were normalized to cell number (2 × 105). All experiments were carried out in triplicate.

Transient transfection and luciferase reporter assays

The luciferase reporter plasmids (pGL2-Luc) harboring human VEGF promoter region (−1175/+336) was kindly provided by Dr. David K. Ann (City of Hope, California). MCF-7 cells were transiently transfected with 0.5 µg of VEGF reporter plasmids for 4 h using Lipofectamine™ 2000 Transfection Reagent according to the manufacturer’s instructions (Invitrogen). One-tenth microgram of the Renilla luciferase pRL-TK plasmid was co-transfected as an indicator for normalization of transfection efficiency. To knockdown HIF-1α expression, MCF-7 cells were transfected with a SureSilencing shRNA plasmid specific for human HIF-1α or a negative control shRNA plasmid (KH01361, SABiosciences, Frederick, MD, USA) using Lipofectamine™ 2000 Transfection Reagent. After overnight recovery, cells were starved for 24 h in serum-free media. Serum-starved cells were subsequently pretreated with different concentrations of pamidronate or clodronate for 1 h in fresh serum-free media, followed by exposure to IGF-1 (40ng/mL) for 16 h before harvesting cell lysates for luciferase assays using the Dual Luciferase Reporter Assay System (Promega, Madison, WI) or Western blot analysis.

Cell viability assay

MCF-7 cells were plated in 96-well plates (104 cells /well). Cells were serum-starved for 24 h followed by treatment with IGF-1 (40ng/mL) and different concentrations of pamidronate or clodronate for different time intervals in serum-free conditional media. Viable cells were determined using 3-(4, 5-dimethylthlthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) Assay kit (Chemicon, CA) according to the manufacturer’s instructions. Each experiment was repeated in triplicate.

In vitro capillary tube formation assay

Angiogenesis in vitro was analyzed according to the manufacturer’s instruction (Millipore). Briefly, 96-well cell culture plates were coated with ECMatrix™ followed by seeding HUVECs (5 ×103cells/well) onto the surface of the polymerized ECMatrix™ with conditioned media derived from MCF-7 cells stimulated by IGF-1 (40ng/mL) in the presence or absence of pamidronate (50 µmol/L) or clodronate (50 µmol/L). The cells were incubated at 37 °C for 6~8 h and tube formation was observed under a phase-contrast microscope and quantitated according to the manufacturer’s instructions (Millipore).

In vivo Matrigel plug angiogenesis assay

6~8 week-old male nude mice (NCR nu/nu) were purchased from the National Cancer Institute (Bethesda, MD). All experiments were undertaken in conformation with the Institutional guidelines of the Institutional Animal Care and Use Committee (IACUC) of University of Southern California. MCF-7 cells were suspended in serum-free media at 1.0 × 107 cells per mL. Aliquots of MCF-7 cell suspension (0.2 mL, 2×106 cells) were premixed with clodronate (50µmol/L) and/or IGF-1 (40 ng/mL) and then mixed with 0.4 mL BD Matrigel Matrix (BD Biosciences). The Matrigel mixtures were subcutaneously injected into both flanks of nude mice (n=4). Cells in equal volumes of solvent were used as controls, whereas the Matrigel mixed with the medium alone was used as the negative control. On day 11, mice were sacrificed and the Matrigel plugs were harvested. Part of the Matrigel plugs were weighed and used to determine hemoglobin content as described previously.66 Briefly, Matrigel plugs were digested in 0.1% Triton X-100 and processed for hemoglobin content at 405 nm absorbance using a standard curve of purified hemoglobin (Sigma).

Immunohistochemstry

Part of the Matrigel plugs was fixed with 10% formalin, paraffin embedded, and serial 5-µm sections were cut. Sections were processed for antigen retrieval using 0.3% hydrogen peroxide, incubated with 0.5% Triton X-100 followed by 10% normal goat serum, and mouse anti-human HIF-1α monoclonal antibody (BD Transduction) incubation at 4 °C overnight. After several washes, slides were incubated with biotinylated universal antibody using the VECTASTAIN Kit according to the manufacturer’s instructions (Vector, Burlingame, CA). .

Statistical analysis

Data are presented as the mean ± SD for 3~4 independent experiments. One-way ANOVA, Bonferroni, and Dunnett’s T3 were employed for statistical analysis using SPSS 11.0 for windows software. P<0.05 was considered to be statistically significant.

Results

Bisphosphonates inhibited IGF-1-induced HIF-1α protein accumulation in ER-positive breast cancer cells

Several studies have reported that nitrogen (N)-bisphosphonates and non-N-bisphosphonates exhibit different efficacy in reducing the viability and proliferation or in inducing apoptosis of breast cancer cells.1216 Herein, we pretreated serum-starved MCF-7 cells with pamidronate or clodronate at the concentration range according to previous reports,12, 15 followed by incubation with IGF-1 for different time intervals and cell viability was assessed using MTT assay. Our results showed no obvious reduction in cell viability of MCF-7 cells after treatment with pamidronate and clodronate at indicated concentrations in the presence or absence of IGF-1 for 16 or 24 hours (Supplementary Figure 1a and 1b; P>0.05). However, treatment with 100µM of pamidronate or clodronate for up to 48 hours decreased the cell viability by about 20% (P<0.05) and 30% (P<0.01), respectively, with respect to the control (Supplementary Figure 1c). Meanwhile, in consistent with a previous study13, pamidronate and clodronate partially abolished the stimulatory effects of IGF-1 on the proliferation of MCF-7 cells (Supplementary Figure 1c). Based on these results, to rule out the cellular toxicity associated with bisphosphonates we selected to treat breast cancer cells with pamidronate or clodronate for <24 hours.

ER-positive breast cancer cells, MCF-7, exhibit a strong growth response to IGF-1, whereas ER-negative breast cancer cells, MDA-MB-468, are not responsive to growth-stimulation by IGF-1.59 First, we examined the effect of IGF-1 on HIF-1α protein expression in these two breast cancer cell lines. To this end, cancer cells were serum-starved for 24 hours followed by stimulation with different concentrations of IGF-1. Consistent with previous studies,64, 65 our results showed that IGF-1 dramatically enhances HIF-1α protein expression in MCF-7 cells, however, exerts no effect in MDA-MB-468 cells (Fig. 1a). Next, we explored the effects of bisphosphonates on IGF-1-induced HIF-1α protein expression in MCF-7 cells. We pretreated serum-starved MCF-7 cells with different concentrations of pamidronate or clodronate, followed by incubation with IGF-1 for 16 hours. Our results indicated that both pamidronate and clodronate inhibited IGF-1-induced HIF-1α protein accumulation (Fig. 1b and 1c). Overall, the suppression of HIF-1α protein was more severe in response to treatment with clodronate as compared to pamidronate (Fig. 1b and 1c). To further investigate whether the reduction of IGF-1-induced HIF-1α protein accumulation by pamidronate and clodronate was the result of transcriptional inhibition, we evaluated HIF-1α mRNA levels by RT-PCR. As shown in Fig. 1d, there was no increase in HIF-1α mRNA level in response to IGF-1 stimulation, and treatment of MCF-7 cells with different concentrations of pamidronate and clodronate had no obvious effects on IGF-1-induced HIF-1α mRNA expression (Fig. 1d), whereas no amplified DNA products were detected in the negative control reactions (data not shown). These results suggest that pamidronate and clodronate suppressed IGF-1-induced HIF-1α protein accumulation possibly via a post-transcriptional mechanism.

Figure 1
Pamidronate and clodronate inhibited HIF-1α protein accumulation induced by IGF-1 in breast cancer cells. (a) Serum-starved MDA-MB-468 and MCF-7 cells were stimulated with different concentrations of IGF-1 for 16h, and HIF-1α protein levels ...

Bisphosphonates inhibited IGF-1-induced VEGF expression in MCF-7 cells

VEGF is a main downstream target gene of HIF-1α and plays an important role in tumor angiogenesis.25, 28, 67 Several studies have shown that IGF-1 upregulates VEGF expression via the HIF-1α pathway in different types of cancer cells.34, 35, 63, 68 Consistently, our results also showed that IGF-1 induces VEGF expression in MCF-7 cells at both transcriptional and translational levels (Fig. 2a–2d). To determine whether IGF-1-induced up-regulation of VEGF was HIF-1α-dependent, MCF-7 cells were transiently transfected with plasmids harboring a non-specific shRNA (NS-shRNA) or SureSilencing shRNA specific for human HIF-1α (shRNAHIF-1α), followed by exposure to IGF-1 for 24 hours. As shown in Fig. 2e and 2f, transfection with shRNA-HIF-1α led to 70~80% decrease in IGF-1-induced HIF-1α protein expression and VEGF secretion (P<0.001) as determined by Western blot analysis and ELISA, respectively, but no obvious decrease was observed after transfection with NS-shRNA (P>0.05). These results suggest that IGF-1 up-regulates VEGF expression via stimulating the expression of HIF-1α protein in breast cancer cells.

Figure 2
Pamidronate and clodronate inhibited VEGF expression and transcriptional activation induced by IGF-1 in MCF-7 cells. (a~c) Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of pamidronate or clodronate followed by exposure ...

To determine whether pamidronate and clodronate inhibit IGF-1-induced VEGF expression in MCF-7 cells, we examined VEGF mRNA expression and protein production in the conditioned media by RT-PCR and ELISA, respectively. Our results indicated that pamidronate and clodronate suppressed IGF-1-induced VEGF protein production (Fig. 2a) and mRNA expression (Fig. 2b and 2c) in a concentration-dependent manner. Consistent with above findings on HIF-1α expression, clodronate treatment showed a stronger inhibitory effect on VEGF expression as compared to pamidronate (Fig. 2c; P<0.001). To further confirm the effect of bisphosphonates on VEGF transcriptional activation, MCF-7 cells were transiently transfected with a luciferase reporter plasmid (pGL2-Luc) harboring the human VEGF promoter followed by treatment with various concentrations of pamidronate or clodronate, and IGF-1 for 16 hours. Our results showed that IGF-1 remarkably enhanced VEGF promoter activities (Fig. 2d; P<0.01), which are abrogated by both pamidronate and clodronate in a concentration-dependent manner (Fig. 2d). There appeared no obvious changes in cell morphology and toxicity in MCF-7 cells after treatment with bisphosphonates at the indicated concentrations (data not shown).

Bisphosphonates inhibited IGF-1-induced activation of PI-3K/Akt/mTOR signaling pathways

Previous studies have shown that the PI-3K/Akt and ERK1/2 signaling pathways are involved in IGF-1-induced HIF-1α protein accumulation and VEGF expression.63, 68 Here, we also found IGF-1 stimulated Akt activation in MCF-7 cells in a time-dependent manner (Fig. 3a), but had no obvious effects on phosphorylated-ERK1/2 levels (Fig. 3a). To examine the effects of pamidronate and clodronate on IGF-1-induced activation of Akt, serum-starved MCF-7 cells were pretreated for 1 hour with different concentrations of pamidronate or clodronate followed by incubation with IGF-1. Our results showed that treatment with bisphosphonates led to a concentration dependent-decrease in IGF-I-induced activation of Akt (Fig. 3b). Treatment with clodronate remarkably suppressed IGF-1-induced phosphorylated-Akt levels to higher extent than treatment with pamidronate (Fig. 3b and supplementary Fig. 2a; P<0.01). To further confirm that PI-3K/Akt is involved in IGF-1-induced HIF-1α protein accumulation and VEGF expression, we next pretreated serum-starved MCF-7 cells with various concentrations of wortmannin (a specific inhibitor of PI-3K/Akt) followed by exposure to IGF-1 for 16 hours. Our results showed that IGF-1-induced HIF-1α protein accumulation and VEGF protein secretion were dramatically attenuated by wortmannin (Fig. 3c and 3d). However, pretreatment with PD98059, a specific inhibitor of ERK1/2, showed no obvious effects on IGF-1-induced HIF-1α protein accumulation and VEGF protein secretion (data not shown).

Figure 3
Pamidronate and clodronate inhibited PI-3K/Akt/mTOR signaling pathways activated by IGF-1 in MCF-7 cells. (a) Serum-starved MCF-7 cells were treated with IGF-1 (40 ng/mL) for different time intervals, and phosphorylated ERK1/2, AKT, mTOR, p70S6K and 4E-BP1 ...

Previous studies have shown that IGF-1-mediated activation of PI-3K/Akt is essential to regulate HIF-1α protein synthesis via activation of several protein translational regulators, including mTOR, p70S6K, and 4E-BP1.63, 68 Thus, we next investigated the effects of bisphosphonates on the protein translational machinery. Our results showed that IGF-1 stimulated activation of mTOR, p70S6K, and 4E-BP1 in MCF-7 cells in a time-dependent manner (Fig. 3a), and treatment with pamidronate or clodronate strikingly inhibited IGF-1-induced activation of mTOR, p70S6K, and 4E-BP-1 in a concentration-dependent manner (Fig. 3b). Consistent with above findings, clodronate demonstrated a stronger inhibitory effect on IGF-1-induced activation of mTOR and 4E-BP-1 than pamidronate (Supplementary Fig. 2b and 2d). To further confirm these results, we pretreated serum-starved MCF-7 cells with rapamycin, a specific inhibitor of mTOR, followed by exposure to IGF-1 for 16 hours. As expected pretreatment with rapamycin significantly suppressed IGF-1 induced HIF-1α protein accumulation (Fig. 3c) and VEGF secretion (Fig. 3e; P<0.01). Collectively, these results suggest that pamidronate and clodronate inhibited IGF-1-induced HIF-1α accumulation and its downstream target gene, VEGF, possibly by interfering with PI-3K/Akt/mTOR signaling pathways that play a pivotal role in the protein translational machinery.29, 31, 39, 46

Bisphosphonates inhibited IGF-1-induced HIF-1α protein accumulation in MCF-7 cells by promoting HIF-1α protein degradation

To further explore the molecular mechanisms whereby pamidronate and clodronate inhibit IGF-1 induced HIF-1α protein accumulation, we observed their effects on the degradation kinetic of HIF-1α protein in response to IGF-1 stimulation in MCF-7 cells. To this end, serum-starved MCF-7 cells were exposed to IGF-1 for 16 hours followed by treatment with CHX to block ongoing protein synthesis in the presence or absence of bisphosphonates for different time periods. Our results showed that pamidronate and clodronate promoted the degradation of IGF-1 induced HIF-1α protein as compared with that treated with CHX alone (Fig. 4 b & 4c versus Fig 4a). The estimated half-lives of IGF-1-induced HIF-1α protein are 25.45 min and 37.48 min in MCF-7 cells treated with pamidronate and clodronate, respectively, as compared to 83.01 min in the absence of bisphosphonates, whereas pamidronate appeared to have a stronger effect in promoting the degradation rate than clodronate (Fig. 4d). These findings indicate that pamidronate and clodronate reduce IGF-1-induced HIF-1α protein accumulation, at least in part, by accelerating its degradation or reducing its stability in MCF-7 cells.

Figure 4
Pamidronate and clodronate promoted the degradation of IGF-1 induced HIF-1α protein in MCF-7 cells. (a~c) Serum-starved MCF-7 cells were exposed to IGF-1 (40 ng/mL) for 16 h followed by treatment with CHX (10 µg/mL) in the presence or ...

Previous studies have demonstrated that HIF-1α protein is rapidly degraded under normoxic conditions mainly via the ubiquitin-proteasome system26, 27 and the accumulation rate of HIF-1α protein due to proteasomal inhibition indirectly reflects the synthesis rate of the protein.26, 5658 To determine whether bisphosphonates could also affect the synthesis of HIF-1α protein, experiments with the specific proteasome inhibitor MG132 were performed as previously described5658. To this end, MCF-7 cells were pretreated with 20 µmol/L of MG-132 for 30 min followed by incubation with IGF-1 for 6 hours in the presence or absence of pamidronate or clodronate. Our results showed that in the absence of IGF-1, treatment with MG132 alone led to an increase in the ubiquitinated fraction of HIF-1α protein, but only a slight increase in the 120 kDa HIF-1α protein level (Fig. 4e, lane 2). As postulated, the increased levels of the newly synthesized 120 kDa HIF-1α protein induced by IGF-1 significantly decreased in response to pamidronate and clodronate treatment in a concentration-dependent manner, whereas clodronate appeared to have a stronger effect in reducing the synthesis rate than pamidronate (Fig. 4e and 4f). Meanwhile, our results also showed that the presence of various concentrations of bisphosphonates led to a significant increase in the formation of the polyubiquitinated fraction of HIF-1α protein (Fig. 4e), a critical step for the degradation through the proteasome pathway.26, 27, 35 Taken together, these results suggest that pamidronate and clodronate inhibited IGF-1-induced HIF-1α protein accumulation possibly by increasing the rate of its degradation and/or decreasing the rate of its synthesis in MCF-7 cells.

Bisphosphonates inhibited tumor angiogenesis stimulated by IGF-1 both in vitro and in vivo

IGF-1 has been reported to promote tumor angiogenesis in several cancer cells.34, 35 Consistent with previous findings, our results showed that conditioned media derived from IGF-1-treated MCF-7 cells were capable of stimulating capillary tube-like structures by HUVEC on Matrigels (Fig. 5a, panel IV; Fig. 5b). We next asked whether the IGF-1 induced angiogenic effect would be suppressed by treatment with bisphopshonates. Our data showed that pretreatment with 50µmol/L of pamidronate or clodronate abrogated tubule formation in both IGF-1 treated or untreated conditioned media exposed endothelial cells (Fig. 5a, panels V and VI vs IV; Fig. 5b).

Figure 5
Effects of pamidronate and clodronate on the in vitro formation of capillary tube-like structures induced by MCF-7 cells in response to IGF-1. HUVECs (5 × 103 cells/well) were seeded onto the surface of 96-well culture plates pre-coated with polymerized ...

To further determine the effect of bisphosphonates on MCF-7 cells-induced tumor angiogenesis in vivo, we performed Matrigel plug angiogenesis assay in nude mice, a commonly used in vivo model to test the antiangiogenic effects of compounds or drugs.66 Our results showed that Matrigel plugs mixed with the untreated conditioned medium did not induce angiogenesis (Fig. 6a-I), showing barely detectable or low hemoglobin levels (Fig. 6b). However, stimulation with IGF-1 greatly enhanced MCF-7 cell tumor angiogenesis (Fig. 6a-V vs Fig. 6a-II), shown here as a 20-fold higher hemoglobin levels in the angiogenic plugs as compared to control (P < 0.01; Fig. 6b). In addition, our results indicated that treatment of MCF-7 cells with clodronate significantly abrogated IGF-1-stimulated tumor angiogenesis (Fig. 6a-VI vs Fig. 6a-V) and dramatically decreased the hemoglobin levels (P < 0.01; Fig. 6b), but had no obvious effect on unstimulated tumor induced blood vessel formation (Fig. 6a-V and Fig. 6b). We also found that IGF-1 significantly increased HIF-1α protein expression in MCF-7 xenografts (Fig. 6c-III vs Fig. 6c-I), which was drastically attenuated in the presence of clodronate (Fig. 6c-IV vs Fig. 6c-III). Similar findings were observed in MCF-7 cells treated with pamidronate (data not shown). Taken together, our results indicated that clodronate and pamidronate exert their anti-angiogenic effects possibly via disrupting HIF-1α/VEGF pathway that plays an important role in tumor angiogenesis.25, 35, 43, 66

Figure 6
Clodronate inhibited IGF-1 stimulated in vivo tumor angiogenesis induced by MCF-7 cells. MCF-7 cells (0.2ml, 2×106 cells) were mixed with 0.4 ml of BD Matrigel Matrix in the presence or absence of clodronate (50 µmol/L) and/or IGF-1 (40 ...

Discussion

Numerous studies have suggested that IGF-1/IGF-1R-mediated activation of downstream signaling pathways play an important role in mammary tumorigenesis and progression of breast cancer.5962 However, due to the heterogeneity of breast tumor cells, either ER-positive or ER-negative, response to IGF-1 stimulated growth varies with cell types59. It appears that the altered response to IGF-1 stimulation might be due to the different expression levels of IGF-1-associated signaling molecules. For instance, previous studies have demonstrated that ER-positive IGF-1-responsive breast cancer cells, like MCF-7, express relatively higher levels and activation of IGF-R1 than ER-negative IGF-1-unresponsive breast cancer cells, like MDA-MB-468.59 Meanwhile, as compared to MDA-MB-468 cells, ER-positive MCF-7 cells displayed higher levels of endogenous insulin receptor substrates (IRS),59 which play a central role in IGF-1/IGF-R1 activated downstream signaling pathways.69 These findings provide a possible explanation that IGF-1 significantly enhances HIF-1α protein accumulation in ER-positive MCF-7 cells, but not in ER-negative MDA-MB-468 cells. However, further studies are required to confirm the underlying mechanisms.

Current therapeutic protocol has incorporated bisphosphonates in the treatment and prevention of skeletal complications secondary to bone metastases in breast cancer.58 In addition to their antiresorptive effects, bisphosphonates have also been shown to have direct and indirect antitumor activities through a variety of mechanisms917, including their potential anti-angiogenic activities.11, 1821 However, it is of note that there are discrepancies and even controversial reports between structurally different non nitrogen-containing bisphosphonates and nitrogen-containing bisphosphonates in terms of their efficacy in reducing cell viability/anti-proliferation and/or apoptosis induction in cancer cells.1216 For instance, several studies have shown that as compared with nitrogen-containing bisphosphonates such as pamidronate and alendronate, clodronate, a non nitrogen-containing bisphosphonate, displayed the least,15, 16 and under certain conditions, even no obvious,12 anti-proliferative and apoptotic inductive activity in breast cancer cells. On the contrary, in the present study we found that clodronate reduced the cell viability and abrogated IGF-1-stimulated proliferation of MCF-7 cells more than pamidronate (Supplementary Figure 1). This is in consistent with a previous report by Fromigue et al that clodronate exhibited stronger antagonizing effects than pamidronate on the stimulatory effects of several growth factors, including IGF-1, on the proliferation of breast cancer cells.13 Such discrepancies between non nitrogen-containing and nitrogen-containing bisphosphonates in terms of their efficacy in reducing the cell viability/anti-proliferation of cancer cells may possibly due to the different mechanisms of pharmacological actions7 and different cell contexts and experimental conditions.1216 Therefore, the detailed mechanisms warrant further investigation.

In addition to the anti-tumoral activities of both non nitrogen-containing and nitrogen-containing bisphosphonates, accumulating evidence has shown that these compounds also possess potent anti-angiogenic effects both in vitro and in vivo.1823 In agreement with these findings, our present study also showed that both clodronate and pamidronate dramatically suppressed both in vitro and in vivo tumor angiogenesis induced by MCF-7 cells in response to IGF-1 (Fig. 5; Fig. 6). However, up to date the underlying molecular mechanisms remain largely unknown. Because VEGF, as a potent mediator of new blood vessel formation or angiogenesis that plays a pivotal role in the growth and metastasis of cancer24, 25, 72, can be induced either by hypoxia or non-hypoxic stimuli via transactivation of the major hypoxia-responsive transcription activator, HIF-1α,28, 31, 63, 68 then we further explored whether bisphosphonates could disrupt this pro-angiogenic pathway in breast cancer cells. As postulated, we demonstrated for the first time that both clodronate and pamidronate indeed significantly inhibited HIF-1α protein accumulation (Fig. 1; Fig. 6c) and VEGF expression in ER-positive human breast cancer MCF-7 cells in response to IGF-1 stimulation (Fig. 2). Our findings suggest that disruption of HIF-1α/VEGF axis might contribute, at least in part, to the anti-angiogenic activity of bisphosphonates.

Previous studies have demonstrated that a variety of small molecules and compounds inhibit hypoxia- or growth factor-induced HIF-1α protein accumulation through stimulating the degradation and/or decreasing the synthesis rate of the protein.35, 5658 We then investigated the potential molecular mechanisms whereby pamidonate and clodronate inhibit IGF-1-upregulated HIF-1α protein accumulation in breast cancer cells. Firstly, we demonstrated that treatment of MCF-7 cells with clodronate or pamidronate had no obvious effects on the expression of HIF-1α mRNA (Fig. 1d), suggesting that both drugs inhibit HIF-1α protein accumulation at the translational and/or post-translational levels. Secondly, we tested whether bisphosphonates could affect the degradation of HIF-1α protein, and we found that both clodronate and pamidronate could significantly speed the degradation rate of IGF-1 induced HIF-1α protein, whereas pamidronate appeared to possess stronger effects than clodronate in this regard (Fig. 4a~d). In addition, because rapid degradation of HIF-1α protein occurs under normoxic conditions mainly via the ubiquitin-proteasome pathway,26, 27 the accumulation rate of HIF-1α protein due to proteasomal inhibition indirectly reflects the synthesis rate of the protein.26, 5658 We subsequently observed the effects of bisphosphonates on IGF-1 induced HIF-1α protein accumulation in breast cells after treatment with MG132, a specific proteasomal inhibitor. Our data showed that the presence of clodronate or pamidronate led to a dose-dependent decrease in the newly-synthesized 120 kDa HIF-1α protein levels induced by IGF-1 in cells after proteasomal inhibition. , whereas clodronate appeared to be more potent than pamidronate (Fig. 4e and 4f). Even though pamidronate and clodronate exhibited a different degree of efficacy in stimulating the degradation and decreasing the synthesis of HIF-1α protein, the overall net inhibition of IGF-1 induced HIF-1α protein accumulation caused by clodronate was more pronounced than that by pamidronate (Fig. 1). Such differences in the efficacy of clodronate and pamidronate in reducing IGF-1 induced HIF-1α protein accumulation may reflect their different molecule structures and mechanisms of pharmacological actions.7, 1226 However, further investigation was required to address the mechanism in detail.

Several lines of evidence have shown that an increased HIF-1α protein accumulation induced by non-hypoxic stimuli such as oncogenic activation, cytokines and growth factors including IGF-1 is mainly due to an increased HIF-1α protein synthesis,2835 whereby PI-3K/AKT/mTOR signaling pathways play a pivotal role.28, 29, 31, 32, 43, 46 Then we also explored whether bisphosphonates had any effect on these signaling pathways. We found that bisphosphonates significantly inhibited IGF-1-stimulated phosphorylation of AKT, mTOR, p70S6K1 and 4E-BP1 (Fig. 3b), all of which compromise essential components of the protein translational machinery cascades.70, 71 Correspondingly, treatment of MCF-7 cells with wortmannin, a specific inhibitor of PI-3K, or rapamycin, a specific inhibitor of mTOR, drastically inhibited IGF-1-induced HIF-1α protein accumulation and VEGF expression (Fig. 3c~3e). Collectively, these findings suggest that the mechanisms by which clodronate and pamidronate inhibit IGF-1-induced HIF-1α protein accumulation may involve, at least in part, the disruption of PI-3K/AKT/mTOR signaling pathways and translational machineries. Recently, Han et al reported that PI-3K/Akt pathway does not play a major role in protecting IGF-1-induced HIF-1α from degradation mediated by SCH66336, a farnesyltransferase inhibitor.35 Therefore, further studies are required to validate whether PI-3K/Akt/mTOR pathways are directly involved in bisphosphonate-mediated increased degradation and reduced synthesis of IGF-1 induced HIF-1α protein in breast cancer cells.

In summary, in the present study we have demonstrated, to our knowledge for the first time, that clodronate and pamidronate inhibited in vitro and in vivo angiogenesis mediated by IGF-1 stimulated breast cancer cells possibly through the inhibition of IGF-1-induced HIF-1α protein accumulation and VEGF expression. Meanwhile, our studies imply that the molecular mechanisms underlying the inhibitory effects of clodronate and pamidronate on IGF-1 induced HIF-1α protein accumulation might involve simultaneous inhibition of HIF-1α protein synthesis and enhancement of its degradation. Therefore, our findings have highlighted an important mechanism of the pharmacological action of bisphosphonates in the inhibition of tumor angiogenesis in breast cancer cells.

Supplementary Material

Supp Mat1

Supp Mat2

Acknowledgements

This work was supported in part by National Institute of Health Research Grant, 1S11 AR47359 and R03 CA128099 (to A. Le), the Oral and Maxillofacial Surgery Foundation Research Support Grant, OMSF002894 (to Q. Zhang), and the National Natural Science Foundation of China, 30672741 (to K. Zhou) and 30872944 (to X. Tang).

Abbreviations

HIF-1
hypoxia inducible factor-1
IGF-1
insulin-like growth factor-1
VEGF
vascular endothelial growth factor
PI-3K
phosphoinositide 3-kinase
mTOR
mammalian target of rapamycin
CHX
cycloheximide
p70S6K
phosphorylated Mw. 70,000 ribosomal protein S6 kinase
4E-BP1
eukaryotic initiation factor 4E (eIF)-binding protein 1 (4E-BP1)
HUVEC
human umbilical vascular endothelial cells

Footnotes

Novelty and Impact Statement: Pamidronate and clodronate were found to inhibit in vitro and in vivo tumor angiogenesis induced by MCF-7 cells in response to IGF-I. Both drugs were identified to suppress IGF-I-induced HIF-1α/VEGF expression in human breast cancer cells, a pathway that plays an important role in tumor angiogenesis. The inhibition of HIF-1α protein expression by bisphosphonates is apparently linked to the accelerated degradation of HIF-1α and the inhibition of the PI-3K/AKT/mTOR signaling cascade. These findings have highlighted an important signaling pathway of bisphosphonate-induced anti-angiogenic effect with potential pharmacological targets in regulating tumor angiogenesis in breast cancer.

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