Introduction
The proteasome is a multi-catalytic, multi-subunit protease complex that is responsible for the ubiquitin-dependent turnover of cellular proteins [
1‐
3]. The proteolytic component of this system, the 26S proteasome, consists of two 19S regulatory particles, involved in substrate recognition and unfolding, and a core particle, the 20S proteasome [
4]. The proteolytic activity of the proteasome measured against fluorogenic substrates illustrates three distinct cleavage preferences, termed chymotryptic-like activities, tryptic-like activities, and caspase-like activities [
5,
6]. Catalytic activity of each proteasome active site is associated with the N-terminal threonine residue, which acts as a nucleophile in hydrolysis [
3,
7,
8]. Since proteasomes play a central role in the cytoplasmic turnover of the vast majority of proteins, the manipulation of proteasomal activity is a key goal in controlling the stability of regulatory proteins [
3,
9]. Inhibition of the proteasome results in abnormal accumulation of many intracellular proteins, thereby disrupting cellular homeostasis [
10], and results in the induction of tumor cell apoptosis [
11,
12].
The most studied and best characterized proteasome inhibitor is Bortezomib (PS-341, Velcade
®; Millenium Pharmaceuticals Inc., Cambridge, MA, USA and Johnson Pharmaceutical Research and Development, LLC, Raritan, NJ, USA), a dipeptide boronic acid that works by reversibly inhibiting the effects of the proteasome and inducing apoptosis in several tumor cell lines and animal models [
13‐
15]. Bortezomib has a wide range of molecular effects, including stabilization of cell cycle regulatory proteins, inhibition of NF-κB activation, induction of apoptosis, and override of Bcl-2 resistance and angiogenesis [
14,
16]. The mechanism of action of Bortezomib has been shown to involve the inhibition of the β
5-subunit (chymotrypsin-like activity) and the β
1-subunit (caspase-like activity), with the β
5-subunit as the predominant target [
17].
Bortezomib has been approved by the US Food and Drug Administration for the treatment of chemorefractory multiple myeloma patients [
18‐
20] and for some forms of non-Hodgkin's lymphoma [
21,
22], and the inhibitor is in further clinical development in multiple tumor types, including breast cancer [
23‐
25]. Despite its clinical success, dose-limiting toxicities including grade 4 arthralgia, diarrhea, vomiting, grade 3 thrombocytopenia, anemia, febrile neutropenia, gastrointestinal toxicity, pain, fatigue, neuropathy, and electrolyte disturbances [
26‐
28] have restricted Bortezomib to a twice-weekly day 1/day 4 dosing schedule to allow complete recovery of proteasome activity between doses [
26‐
29].
These observations suggest that the search for additional proteasome inhibitors is warranted. We have recently designed and synthesized a new proteasome inhibitor, BU-32, a bis-dipeptidyl boronic acid analog of Bortezomib containing an additional dipeptide boronic acid moiety on the pyrazine ring, intended to potentially achieve stronger binding affinity and increased potency . Bivalent proteasome inhibitors, either hetero-bivalent or homo-bivalent, have been reported to increase inhibition potency by as much as two orders of magnitude compared with the monovalent analogs, although in these compounds the active moieties are typically separated by a linker of 18 to 22 carbon atoms long [
30‐
32]
In the present study, we describe the in vitro and in vivo characterization of BU-32 in breast cancer cell lines and xenograft and metastatic models. In order to test the activity of BU-32, irrespective of estrogen receptor status, we used a panel of breast cancer cell lines: MCF-7 (estrogen receptor-positive, progesterone receptor-positive), MDA-MB-231 (estrogen receptor-negative, progesterone receptor-negative, HER2-negative) and SKBR3 (HER-positive). We show that BU-32 is a potent and selective inhibitor of the chymotrypsin-like activity of the proteasome in vitro. In addition, we show that BU-32 modulates cell-cycle-dependent kinase inhibitors, upregulates p53 and proapoptotic factors Bax and Bid, downregulates NF-κB expression at the protein level, induces apoptosis, and inhibits various angiogenic factors in a panel of breast cancer cell lines. In addition, we show that BU-32 has in vivo anti-tumor activity and inhibits breast cancer-initiated bone metastasis in experimental animals.
Materials and methods
Proteasome inhibitor BU-32
BU-32 (pyrazyl-2,5-bis-CONH(CHPhe)CONH(CHisobutyl)B(OH)2) was synthesized in our laboratory using standard synthetic procedures and was characterized by 1H and 13C nuclear magnetic resonance and mass spectrometry. The detailed synthetic procedure will be reported elsewhere. BU-32 was dissolved in dimethylsulfoxide and stored at -20°C until use. BU-32 was diluted in culture media immediately before use. Both BU-32 and control media contained < 0.1% dimethylsulfoxide.
Cell culture
MCF-7 and MDA-MB-231 human breast cancer cells were maintained in DMEM supplemented with 10% FBS. SKBR3 human breast cancer cell line obtained from the American Type Culture Collection (Manassas, VA, USA) was maintained in McCoys5A, 10% FBS.
Animals
Two-week-old to 4-week old Balb/c female nude mice (obtained from Harlan Sprague Dawley, Inc., Indianapolis, IN, USA) were used in the in vivo animal experiments. All animal protocols were approved and monitored by the Institutional Animal Care and Use Committee. The animals were housed under specific pathogen-free conditions.
Cell viability
Cell viability was determined by quantification of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases. In brief, cells (4 × 104 cells per well) were incubated with 4 to 18 nM Bortezomib and BU-32 for 48 hours. Thereafter, MTT in a final concentration of 1.2 mmol/l was added and further incubated for 3 hours at 37°C. The formazan dye solubilized in 20% SDS in 50% dimethyl formamide was added and the plate was incubated at room temperature for 1 hour. The absorption was measured at 550 nm versus 690 nm in a microplate reader (Molecular Devices, Corning, New York, USA).
20S proteasome assay
Proteasome chymotrypsin-like activities, caspase-like activities, and trypsin-like activities were determined using the Proteasome-Glo™ Assay System (Promega, Madison, Wisconson, USA) according to the manufacturer's protocol. Proteasome-Glo™ buffer was mixed with luciferin detection reagent, and then the substrate was added to the mixture and incubated at room temperature for 1 hour. An equal volume of proteasome-Glo™ reagent was added to the samples and further incubated for 15 minutes. The luminogenic substrate containing the Suc-LLVY sequence is recognized by the proteasome. Following proteasome cleavage, the substrate for luciferase (aminoluciferin) was released, allowing the luciferase reaction to proceed and produce light. The luminescence was measured with a luminometer (Thermo Fisher Scientific, Waltham, MA, USA).
Western blotting
Cell lines were harvested into a lysis buffer containing a cocktail of protease inhibitors (Sigma, St Louis, MO, USA). The protein content was assessed by the Bradford assay. Equal protein concentrations were prepared for loading with Laemmli sample buffer and were run on SDS-PAGE. Separated proteins were then transferred to nitrocellulose membrane, and the protein-bound membranes were incubated for 2 hours at room temperature with Tris-buffered saline containing 0.05% Triton X-100 and 5% nonfat dry milk to block nonspecific antibody binding. The membranes were then incubated with the respective primary antibodies in Tris-buffered saline milk overnight at 4°C, and specific binding was visualized using species-specific IgG followed by enhanced chemiluminescent detection (ECL kit; Amersham Bioscience, Pittsburg, PA, USA) and exposure to enhanced chemiluminescent X-ray film. Antibodies against β-actin, p53 and NF-κB were purchased from Santa Rosa (Santa Rosa, CA, USA). Antibodies against p21 and p27 were obtained from Lab Vision (Thermo Fisher Scientific, Fermont, CA, USA). Antibodies against p44/42, phospho p44/42, Bid and Bax were purchased from Cell Signalling Technology (Danvers, MA, USA).
RNA analysis and PCR
Total RNA was extracted using Tri Reagent (Sigma) according to the manufacturer's protocol. Gene expression was then examined by real-time quantitative RT-PCR, using the GeneAmp RNA PCR kit (Perkin-Elmer, Foster City, CA, USA) and platinum Taq polymerase (Invitrogen, Carlsbad, CA, USA) according to the manufacturers' instructions. Depending on the abundance of the specific mRNA species, 70 to 250 ng total RNA was used as starting template in the RT reaction mixture. To detect amplicon synthesis in the Smart Cycler real-time PCR thermal cycler (Cepheid, Sunnyvale, CA, USA), 0.25 × Cyber green dye (Roche, Indianapolis, IN, USA) was added to the reaction mixture. For quantification, the cycle threshold number (Ct) exhibiting the maximum curve growth rate was determined. The relative gene expression of each sample, normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was calculated by the formula:
Apoptosis assays
Annexin staining was conducted with the use of a kit (Annexin V-FITC Apoptosis detection kit; BD Pharmingen, San Jose, CA, USA). A sample of 106 cells per well was split and left to recover for 24 hours before exposure to the proteasome inhibitor. The cells were then treated with indicated concentrations of Bortezomib and BU-32 for 24 hours. After incubation, cells were washed with Dulbecco's phosphate buffered saline (DPBS) (Sigma, St. Louis, MO, USA) and resuspended in 100 μl binding buffer (supplied by the vendor). Cells were stained with annexin V-FITC and propydium iodide according to the manufacturer's protocol before analysis by flow cytometry. Results are presented as mean ± standard deviation of three independent experiments.
For bone metastasis experiments, we specifically used MDA-MB-231 cells that had been stably transfected with the gene-encoding green fluorescent protein (GFP) to detect tumor cells in live animals using noninvasive fluorescence imaging. MDA-MB-231-GFP cells (105 in 100 μl PBS) were injected intracardially. Animals were anesthetized with isofluorane and positioned ventral side up. The left cardiac ventricle was punctured through a percutaneous approach using a 27-gauge needle. Based on an average body weight, Bortezomib (0.02 mg/kg and 0.06 mg/kg body weight) and BU-32 (0.02 mg/kg and 0.06 mg/kg body weight) were given twice a week to animals by subcutaneous injection beginning on the day of tumor cell inoculation (day 0), and continuing until the end of the protocol (day 35). All doses of each drug were given by subcutaneous injection in 50 μl PBS with 0.2% dimethylsulfoxide (vehicle). Control mice received a daily treatment with vehicle only.
On day 35 after tumor cell inoculation, radiographs (MX-20 cabinet X-ray system; Faxitron X-ray Corp. (Lincolnshire, IL, USA)) of anesthetized mice were taken. For radiographs of mice, the animals were anesthetized deeply with isofluorane, laid in a prone position against the film and exposed to an X-ray at 35 KVP for 6 seconds. Films were developed using a RPX-OMAT processor (Eastman Kodak Company, Rochester, NY, USA. All radiographs from nude mice were evaluated in a blinded fashion. The number and area of osteolytic bone metastasis were calculated on radiographs using a computerized image analysis system.
Animals analyzed by radiography were also examined by noninvasive, whole-body fluorescence imaging using a fluorescence scanning system (Nikon-SMZ1500; Nikon, Tokyo, Japan) with a fluorescence stereoscope attached to a Cool SNAP CCD camera (Photometrics, Tucson, AZ, USA). Metastatic lesions in bone were identified on scanned images as fluorescent regions. The area of fluorescent spots was measured using the Bio-Rad densitometer image analysis system (Bio-Rad, Hercules, CA, USA). Anesthetized mice were killed by cervical dislocation after radiography and fluorescence imaging on day 35.
Human breast cancer cell xenograft
In tumorigenesis studies, MDA-MB-231-GFP tumor cells were implanted subcutaneously into the right flank of 4-week-old to 5-week-old Balb/c female nude mice that had been anesthetized by isofluorane inhalation. The subcutaneous tumor inoculation was performed using a 27-gauge needle attached to a 1 ml syringe containing tumor cells at a concentration of 10
7 cells/100 μl PBS. After 10 days, tumor bearing mice were randomly grouped (n = 10) and were treated by daily subcutaneous injection with therapeutic agents (Bortezomib and BU-32) in a dose of 0.02 or 0.06 mg/kg body weight or with vehicle (PBS, control) for 10 days. The tumor size was measured every other day using calipers, and the tumor volumes were calculated according to the standard formula:
The extent of tumor burden per animal was expressed in cubic millimeters. Mice were sacrificed after 28 days of treatment.
In vivotoxicity study
In vivo toxicity was performed in Balb/c mice (Harlan Industries, Houston, TX, USA). Mice 2 to 4 weeks old were caged for 1 week before the experiment to acclimatize them to the environment. The mice were fed with standard mice chaw (Harlan Industries) and water ad libitum. For the experiment, mice were given a dose range of BU-32 and Bortezomib (0.1 to 0.25 mg/kg twice weekly for 2 weeks) intraperitoneal injection. Each group consisted of 10 mice, and control or untreated mice were given only vehicle (0.3% hydroxyl propyl cellulose). The animals were monitored regularly for external signs of toxicity or lethality.
Statistical analysis
Unless otherwise stated, all experiments were conducted at least three times and results are expressed as the mean ± standard deviation. Significant differences were calculated by Student's t test using SPSS statistical software (IBM Company, Chicago, IL), and significance was achieved when P < 0.05.
Discussion
In the present article we show that the novel diboronated analog of Bortezomib BU-32 (NSC D750499-S) is a potent and selective inhibitor of the chymotrypsin-like activity of the 20S proteasome and has potent
in vitro anti-tumor activity against a panel of breast cancer cells as well as
in vivo efficacy in mouse xenograft and metastasis models. In the present study, we first compared the cytotoxicity of BU-32 with Bortezomib, which has been validated clinically for the treatment of multiple myeloma and non-Hodgkin's lymphoma, and works by reversibly inhibiting the effects of the proteasome and by inducing apoptosis in several tumor cell lines and animal models [
13‐
15]. The results of our MTT experiments demonstrate that BU-32 induces strong growth arrest at clinically relevant concentrations
in vitro ( < 10 nM) with MDA-MB-231, MCF-7, and SKBR-3 cell lines, and that MDA-MB-231-GFP tumor xenografts treated with intravenous BU-32 displays substantial inhibition of proliferation
in vivo in a dose-dependent manner. Interestingly, the cytotoxicity of BU-32 against the panel of three breast cancer cell lines is comparable with that of Bortezomib in spite of the more bulky size of BU-32 and the possibility of steric hindrance at the binding pocket of the proteasome. While a definitive evidence for the nature of the interactions of BU-32 with the binding pockets of the proteasome awaits further studies, it is clear from our
in vitro and
in vivo data that there is reasonably good interaction with the active site given the observed potency of BU-32 in these cell lines.
We tested the active site selectivity of BU-32 in our panel of breast cancer cell lines, and found that it blocks all three proteasomal activities in the MDA-MB-231, MCF-7, and SKBR3 cell lines - with the chymotrypsin-like and caspase-like activities being the most predominant, and to a lesser degree the trypsin-like activity. Our observation is consistent with the active site selectivity profile reported for Bortezomib [
3,
17,
33‐
38].
Proteasome-induced cytotoxicity could potentially result from multiple events, including the stabilization and deregulated function of cyclins, cell-cycle-dependent kinase inhibitors, tumor suppressor proteins, IκB, proapoptotic proteins, and a large number of other proteins associated with cell cycle progression[
14]. Indeed, proteasome inhibition has been shown to stabilize the cyclin-dependent kinase inhibitors p21 and p27, the tumor suppressor p53, and the proapoptotic proteins Bid and Bax [
15,
39‐
45]. The increased levels of activated p21, p27, p53, Bid, and Bax result in inhibition of cell cycle progression and/or promotion of apoptosis in response to Bortezomib [
45]. As with the proteasome inhibitor Bortezomib, BU-32 exposure leads to accumulation of the cell-cycle-dependent kinase inhibitors p21 and p27, the p53 tumor suppressor gene and the proapoptotic proteins Bid and Bax, and downregulation of NFκB at the protein level in MCF-7, MDA-MB-231, and SKBR3 cell lines. There is no significant difference in the levels of expression of the mitogen-activated protein kinases p42, p44, phospho-p42 and phospho-p44 compared with the controls. Although the p21 and p27 levels increased in all three cell lines for both BU-32 and Bortezomib, our results show that the effect is cell line dependent.
These observations are consistent with an earlier report that the effect of proteasome inhibition on different markers varies by drug, cell line, and time point of analysis [
46]. BU-32 exposure caused greater accumulation of Bax in all three cell lines. It is known that p53 plays important roles in cell cycle regulation and apoptosis [
47‐
49]. Exposure to BU-32 resulted in accumulation of the p53 tumor suppressor gene in all three breast cancer cell lines we studied. The effect was more pronounced for BU-32 in the cell lines MDA-MB-231 and SKBR3 than in MCF-7 cells compared with Bortezomib. Proteasome inhibitor-induced apoptosis has been described as p53 dependent [
12]. Other reports have shown that sensitivity to proteasome inhibition was partially dependent on the p53 status of the breast [
50] and lung cancers
in vitro [
47], but Bortezomib-induced apoptosis was p53 independent in prostate cancer cells [
14], multiple myeloma [
15], and colon cancer cells [
51]. As with Bortezomib [
45], the degree of variability in the sensitivity to BU-32 with respect to p53 status appears to be cell line dependent.
We next examined whether exposure to BU-32 downregulated the transcription factor NF-κB. We found that BU-32 downregulated the expression of NF-κB in our panel of breast cancer cell lines and that the effect was comparable with Bortezomib. The initial rationale for the use of Bortezomib in multiple myeloma was inhibition of NF-κB activity by blocking proteasomal degradation of inhibitor of κBα [
52]. Interestingly, recent reports indicate that there is a paradigm shift in myeloma with respect to ascribing the mechanism of Bortezomib's anti-tumor activity to NF-κB inhibition [
53], and suggest that Bortezomib-induced cytotoxicity cannot be fully attributed to inhibition of canonical NF-κB activity [
54].
We also investigated whether exposure to BU-32 affects the expression of genes involved in the angiogenic cascade, such as VEGF, VEGF-receptor 1 (FLT1), the angiopoietin-Tie system (Ang1, Ang2, Tie1, and Tie2), epidermal growth factor receptor, and KDR in MCF7, MDA-MB-231, and SKBR3 cell lines, and compared the effect with that of Bortezomib. Their expression in endothelial cells increases with the angiogenic switch and persists throughout angiogenesis - except during vessel stabilization, when inhibitors prevail [
55]. It has been shown that VEGF triggers blood vessel formation [
56]. Ang1 and Ang2 are mandatory for vessel sprouting and remodeling [
57]. Ang1 promotes sprouting in the presence of VEGF, induces branching networks with the typical organization of mature vessels, and stabilizes perivascular endothelial cell interactions [
56]. Ang2 exerts a vessel destabilizing effect, which allows VEGF-mediated vascular reorganization [
56]. The angiopoietin-Tie2 system appears to govern maturation and stabilization of blood vessels. It is well understood from previous studies that VEGF mainly binds to and activates two different receptor tyrosine kinases: fms-like tyrosine kinase 1 (Flt-1) and KDR [
58]. It is also reported in human vascular endothelial cells that KDR mediates the majority of VEGF transcription-regulating activities [
59] and recent studies have demonstrated that KDR mediates the mitogenic, chemotactic, tubulogenic, and survival activities of VEGF [
60]. In the present article we showed that exposure to BU-32 induces a significant downregulation of these angiogenic markers in all three cell lines and that the effect was generally more pronounced for BU-32 than Bortezomib. These data suggest that BU-32 exerts anti-angiogenic activity through the downregulation of genes required for growth of endothelial cells, and that angiogenesis is a possible mechanism of its cytotoxicity.
We also investigated the anti-breast cancer activity of BU-32 in vivo in a human breast cancer mouse model. In a xenograft model of breast cancer, BU-32 treatment resulted in significant inhibition of tumor growth. We also showed that BU-32 significantly reduces breast-cancer-initiated bone metastasis in vivo. These effects were not associated with any significant toxicity when compared with Bortezomib. We also showed from our toxicity study that BU-32 has a broader safe dosing range compared with Bortezomib. From this toxicity study, BU-32 showed a promising tolerance profile and therefore can offer a wide range of therapeutic index. Even though BU-32 appears to be an appealing therapeutic candidate, further pharmacokinetic studies as well as pharmacodynamic studies need to be conducted to validate the activity and safety profiles.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JKA conceived, designed and coordinated the study, provided study material and helped to write the manuscript. HBN performed the in vitro and in vivo studies. SSR assisted in writing the manuscript. BS synthesized BU-32. RRT provided experimental support and proofread the manuscript. All authors read and approved the final manuscript.