Abstract
Background: Osteodex is a novel bi-functional macromolecular polybisphosphonate developed for treatment of bone metastases in prostate and breast cancer. High efficacy of osteodex has been demonstrated both in vitro and in vivo. The present study investigates whether osteodex is also efficacious on soft tissue tumor lesions. Materials and Methods: Twelve female nude mice were injected with MDA-MB-231 cells orthotopically. Osteodex was administered i.v. at 2.5 mg/kg, once per week for five weeks. Tumor volumes were measured during the treatment period, the animals were sacrificed, and samples collected for proteomic analysis. Results: The non-treated mice developed multiple tumors greater than 4 cm with pronounced ulceration, while the treated mice had tumors smaller than 1 cm, without ulceration. While general condition of treated mice was good, non-treated animals were in poor condition. Sixteen out of 300 identified proteins were differentially expressed, with statistically significant expression changes of more than two-fold differences between treated and non-treated groups. These proteins were identified using non-gel based nano-liquid chromatography coupled with a Synapt G2 instrument. Conclusion: We conclude that osteodex showed significant treatment efficacy on soft tissue tumor implants. The study provides a global view of changes in protein expression profiles following osteodex treatment. Some functions of the identified proteins might be used to explain the specific treatment efficacy of osteodex.
Prostate and breast cancer most commonly develop metastases in the skeleton. At an advanced metastatic stage, more than 70% of patients develop bone metastases. Stephen Paget's seed and soil hypothesis, stating that circulating cancer cells can only grow where the microenvironment is permissive for growth, is still appropriate. The continuous re-modeling of bone, involving osteoclasts and osteoblasts, generates the release of multiple cytokines, chemokines and growth factors that are favorable for tumor cell growth (1-3). This, in the presence of a locally rich vasculature and high permeability surrounding of the bone marrow might explain the propensity for bone metastasis in advanced prostate and breast cancer. Even though bone is the most common site of metastasis, soft tissue metastasis often occurs at advanced stage of both prostate and breast cancer. The most common sites are lymphatic nodes, liver and lung (4-9). Lymph nodes close to the primary tumor are often the first sites of tumor spread. In prostate cancer, spinal metastasis often precedes liver and lung metastasis. Approximately half of women with metastatic breast cancer develop liver metastases. In general, detection of positive lymph nodes indicates a poor prognosis and might also be an indicator of an aggressive tumor phenotype. Both advanced prostate and breast cancer are treated with combinations of chemotherapy, radiation, hormonal, and biological therapies (10, 11).
Osteodex is a bi-functional polybisphosphonate recently developed for the treatment of bone metastases. Osteodex is comprised of a carbohydrate polymer with bisphosphonate and guanidine moieties linked to the polymeric backbone. Previous in vitro and in vivo studies have demonstrated significant bi-functional efficacy i.e. inhibition of bone resorption and antitumor efficacy (12, 13). Its mode of action depends on three primary effects: inhibition of the mevalonate pathway, induction of apoptosis, and general cytotoxicity. Clinical phase I study of osteodex was recently completed in patients with castration-resistant prostate cancer (CRPC) and a phase II study is planned for 2014. Osteodex is a macromolecule with certain electrostatic charge characteristics and, therefore, it seems reasonable that it might also be able to accumulate in breast tumor lesions dependent on the enhanced permeability and retention effect (14).
The present study investigated whether osteodex might have efficacy against soft tissue lesions. Proteomic analysis was the primary method used to investigate the effects on the proteome after osteodex treatment.
Materials and Methods
Osteodex synthesis. Osteodex was prepared as described previously (13). Briefly, Dextran 40 PhEUR (Pharmacosmos AS, Holbaek, Denmark) was oxidized with sodium meta-periodate (Merck AG, Darmstadt, Germany) and amino guanidine and alendronate (Sigma-Aldrich, Stockholm, Sweden) were subsequently conjugated. Sodium borohydride (Chemicon, Stockholm, Sweden) was used for reductive amination. PD-10 disposable Sephadex G-25 columns were used for separation and purification (G&E, Biotech AB, Onsala, Sweden). The conjugation yield was determined by elemental analysis (total nitrogen, total phosphorous; Mikrokemi AB, Uppsala, Sweden).
Animals. Twelve female nude mice (age 6-8 weeks/weighing approximately 18-25 g) were injected with approximately 1-1.5 million MDA-MB-231 cells (Manassas, VA, USA) subcutaneously around the mammary pad region. Tumors developed within 10-14 days post-injection in 9 out of 12 mice and allowed tumor growth to less than 1 cm before initiating treatment with osteodex. The nine tumor-bearing mice were divided into treated (n=5) and no-treatment (n=4) groups.
Thereafter the five tumor-bearing mice were treated with a single dose of osteodex, given intravenously via the tail vein at a dose of 2.5 mg/kg, once per week for five weeks. Serial measurements of tumor growth during the period of the experiment were taken until the animals are sacrificed. The measurements were difficult in the non-treated group due to extensive tumor growth with massive ulcerations.
After the treatment period, the animals were sacrificed and gross autopsy was performed. Tumor tissues were resected from both groups for proteomic analysis. All procedures were carried out in strict accordance with an approved protocol for the use of animals in research. The mice were handled and cared for according to Institutional Animal Care and Use Committee (IACUC) policies and guidelines of the office of Research Affairs (RAC# 2080-052), and Animal facility protocol at KFSHRC. The animals were kept in the animal facility at the Comparative Medicine Department and all procedures were performed according to standard guidelines.
Gross autopsy. The abdominal cavity was opened in all animals and gross autopsy was performed. Gross appearances of treated and non-treated animals were recorded including visible extra-abdominal tumors. The peritoneum was inspected, looking for evidence of infiltration and spread in the peritoneal cavity. Histologically-proven representative tissue samples were collected for proteomic analysis. Sample preparation and proteomic analysis. The samples were prepared immediately after the autopsy. Tumor cells were extracted by non-enzymatic method as previously described (15, 16). Briefly, fresh tumor samples were mechanically homogenized in 1-2 ml ice-cold RPMI-1640 medium supplemented with 5% calf serum and protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride (PMSF)/0.83 mM benzamidine). Tissue debris and connective tissues were removed using a 500 μm mesh filter and tumor cells were harvested following percoll centrifugation. The collected cell suspensions were washed twice in phosphate buffered saline containing protease inhibitor cocktails and then centrifuged at 800× g and 4°C for 3 min. Finally, all the samples were centrifuged for 5 min at 2700× g (4°C). The pellets were stored at -80°C for further analysis.
Protein in-solution digestion and Liquid chromatography-mass spectrometry analysis. The protein concentrations of the whole cell lysates were determined by the Bradford assay method. Protein concentrations of all samples were normalized and for each sample, 100 μg complex protein mixture was taken and exchanged twice with 500 μl of 0.1% RapiGest (Waters, Manchester, UK) (1 vial diluted in 1,000 μl 50 mM AmBic) with a 3-kDa ultra filtration device (Millipore, Bedford, MA, USA). Protein concentration of between 0.1 and 1 μg/μl was achieved at the end of digestion. Proteins were denatured in 0.1% RapiGest SF at 80°C for 15 min, reduced in 10 mM Dithiothreitol at 60°C for 30 min, spinned down and allowed to cool to room temperature and alkylated in 10 mM iodoacetamide for 40 min at room temperature in the dark. Samples were trypsin-digested at a 1:50 (w/w; 1 μg/μl trypsin concentration) enzyme:protein ratio and trypsin, for at least 4 h or overnight at 37°C with gentle shaking. The digestion was ended and RapiGest quenched with 4 μl of 12 M HCl at 37°C for 15 min and centrifuged at 17,900 RCF for 10 min. Samples were diluted to 5 pmol/μl or 5- to 10-fold with aqueous 0.1% formic acid prior to LC/MS analysis. All samples were spiked with yeast alcohol dehydrogenase (ADH; P00330) as internal standard to the digests to give 200 fmol per injection for absolute quantitation.
Protein identification by mass spectrometry-LC/MSE. Both qualitative and quantitative expression profiling were performed using 1-dimensional Nano Acquity liquid chromatography coupled with tandem mass spectrometry on a Synapt G2 instrument (Waters Scientific, Berkshire, UK). The instrument settings for Electrospray Ionization Mass Spectrometry analyses were optimized on the tune page as follows: Detectors set up using 2 ng/μl leucine enkephalin (556.277 Da), Mass (m/z) calibration was achieved on a separate infusion of 500 fmol [Glu] 1-fibrinopeptide B (GluFib, 785.843 Da), using the Mass Lynx IntelliStart. Other parameters included capillary voltage 3 kV,, sample cone 50 V, extraction cone 5 V, source temperature 80°C, cone gas 10 l/h, nano flow gas 0.5 bar and purge gas 800 l/h. All analyses were carried out on Trizaic Nano source (Waters) ionization in the positive ion mode nanoESI.
A total of 2 μg protein digest was loaded on column and samples were processed using the Acquity sample manager with mobile phase consisting of A1 (water + 0.1% formic acid) and B1 (acetonitrile + 0.1% formic acid) with sample flow rate of 0.500 μl/min. Data-independent acquisition (MSE)/iron mobility separation experiments were performed and data was acquired over a range of m/z 50-2000 Da with a scan time of 1 s, ramped transfer collision energy 20-50 V with a total acquisition time of 120 min. All samples were analyzed in triplicate runs and data were acquired using the Mass Lynx programs (version. 4.1, SCN833; Waters) operated in resolution and positive polarity modes. The acquired MS data were background-subtracted, smoothed and de-isotoped at medium threshold. Protein Lynx Global Server (PLGS) 2.2 (Waters) was used for all automated data processing and database searching. The generated peptide masses were searched against Uniprot protein sequence database using the PLGS 2.2 for protein identification (Waters).
Data analysis and informatics. TransOmics Informatics (Waters) was used to process and search the data. A human database containing 46,906 reviewed entries was downloaded from Uniprot. A decoy database was created by reversing this and concatenated to the original database prior to searching. The principle of the search algorithm is described by Li et al. (17). The following criteria were used for the search: one missed cleavage, maximum protein mass 1000 kDa, trypsin, carbamidomethyl C fixed and oxidation M variable modifications.
Normalized label-free quantification was achieved using exclusive TransOmics software, developed in collaboration with Nonlinear Dynamics (Newcastle, UK), and was used to plot Principal component analysis (PCA) analysis against data split into two groups. The data were filtered to show only statistically (ANOVA) significantly altered proteins (p≤0.05) with ≥3 peptides identified and a fold change of more than 2.
Additionally ‘Hi3’ absolute quantification was performed using ADH as an internal standard to give an absolute amount of each identified protein (Waters).
Results
Before sacrificing the animals for autopsy, it was noted that treated animals were in good and normal condition while non-treated animals were doing poorly due to extensive disease progression. Serial measurements of tumor growth/volume revealed a statistically significant difference between treated and non-treated animals (Figure 1A). Osteodex-treated animals had small well-demarcated lesions while non-treated animals presented with large infiltrating and ulcerating lesions (Figure 1B).
Gross autopsy. Non-treated animals developed multiple metastatic tumors in the peritoneal cavity, distant metastasis to the kidneys and livers with ulcerations and blood vessel infiltration. Treated animals had a few solitary small lesions in the kidneys but with no further infiltrations in the peritoneal cavity and no metastasis to the liver (Table I). Figure 2 shows representative histological slides by light microscopy of normal kidney glomeruli in a treated animal compared with histological appearance of neoplastic cell infiltrations of the entire kidney of non-treated animals.
To gain insight into the biological changes behind this massive difference in tumor growth, tumor samples from five treated and four non-treated mice were subjected to proteomic comparison.
Proteomic analysis and changes in protein expression. A label-free MS-based method as a tool for comparative protein expression profiling was used. A total of 19,420 unique peptide features were detected, filtered outside an area of 50-2000 m/z and background noise, and reduced to 11,045. Screening for features that differed significantly between treated and non-treated (p<0.05 and p<0.001) mice revealed 2,058 features with ∼3% false discovery rate (a statistical analysis method used for correction of multiple comparisons, adjusting observed p-values to avoid ‘over-interpretation’ of the significance of the observed results).
The majority of the proteins in the treated group were up-regulated, while the non-treated group had only a very small fraction of these up-regulated. These findings are similar to what was observed with PCA plot generated from 2-DE dataset (data not shown).
Differentially expressed proteins. Approximately 300 proteins from tumor tissues of treated and non-treated mice were identified. Sixteen of these proteins were differentially expressed with significant expression changes of at least two-fold between sample groups (Table II). A data set of the 16 proteins differentially expressed between treatment sample groups clearly discriminated the samples into two distinct groups by unsupervised hierarchical cluster analysis and PCA (Figure 3A and B).
Functional interpretation of the identified proteins. Further characterization of the identified proteins was explored using Ingenuity Pathway Analysis (Ingenuity Systems, Inc., Redwood, CA, USA). The 16 identified proteins were mapped and represented in three sub-signaling networks. Ten proteins were implicated in the network of humoral immune response, inflammatory response and cellular movement, while three of the molecules were involved in networks of molecular transport, hematological system development and function, hematological disease. Only one molecule (type VI protein-arginine deiminase, a member of peptidyl arginine deiminase family) is implicated in the network of cell cycle, cell death and survival, endocrine system disorders (Figure 4).
The summarized functional characteristics of some of the identified proteins are listed in Table III, derived from the Ingenuity Database program. The majority of these molecules are located in the cytoplasm and only a few are located in the extracellular space. While many of them act as enzymes, others act as cellular transporters. Ten proteins were up-regulated, while only four proteins were down-regulated between treated and non-treated groups. Furthermore, four proteins (Hemoglobin, beta adult minor chain (Hbb-b2), Hemoglobin, zeta (HBZ), ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 (ATP2A1), and Complement component 3 (C3) were not directly mapped in a network, but were functionally-implicated in cell death and survival in apoptosis of acinar gland cells, as well as in apoptosis of liver cell lines.
Discussion
The present study investigated the efficacy of osteodex treatment on tumor implants in mice and the effects on the tumor cell proteome was examined.
Previous experience from a wide range of chemotherapeutic drugs and other anticancer agents indicate that many result in induction of apoptosis of the malignant cells. Sequences of events occur as tumor cells undergo apoptosis, including DNA and protein degradation. Recently, Ueno et al. demonstrated that apoptosis of breast cancer tumor cells can create products detectable in serum (18). Because of laws regulating the patient's integrity, ethical issues etc. (Bio bank law Regulations), it is difficult to obtain pre- and post-therapy sequential biopsy samples. Consequently, knowledge and characteristics of apoptotic changes following treatment of solid tumors in humans are limited.
Clinical diagnosis of most malignancies can be made with accuracy, however prediction of treatment response is more difficult and limited. Therefore, there is a considerable need for the discovery and development of sensitive and specific biomarkers for disease prognosis and prediction of treatment response.
Protein biomarkers are often defined as specific proteins that can be quantitatively measured and evaluated as objective indicators of normal state, pathological state, and as an indicator of therapeutic response.
Recent advancements in proteomic analysis technologies have generated further interest in possibilities of translational research. Expression proteomics, often defined as large-scale differential protein profiling analyses, have resulted in identification of disease-related or tissue-specific proteins that could be potentially used as disease biomarkers (19, 20).
Classical expression-based proteomics strategies, including high-resolution two-dimensional protein separations (2-DE) coupled to highly sensitive protein identification by LC-MS-MS, can identify novel protein markers. Large amounts of differential expression data can be analyzed by artificial learning methods, yielding disease diagnosis or monitoring treatment response.
In the present study, we used hierarchical cluster analysis of qualitative and quantitative in-solution protein expression changes in an attempt to monitor and interpret changes as a result of osteodex therapy on breast tumors. Using protein expression changes coupled to protein identification by tandem mass spectrometry, 16 differentially expressed proteins associated with osteodex treatment were found. The expression of these proteins clearly discriminates between treated and non-treated groups (Figure 3 A and B). The majority of proteins in the treatment group were distinctly up-regulated in contrast to those in the non-treated group, indicating altered protein expression associated with the osteodex treatment. The proteins' functional characteristics were established using the ingenuity pathway analysis. Fifteen out of the 16 identified proteins were associated with different signaling networks, including humoral immune response, inflammatory response, cellular movement, molecular transport, hematological system development and function, hematological disease. One of the 16 proteins, absent in the treatment group and only expressed in the non-treated group, was an enzyme, type-6 protein-arginine deiminase (PAD), a member of peptidyl arginine deiminase family, associated with the network affecting cell cycle, cell death and survival. PAD belongs to a group of enzymes involved in post-translational protein de-aminations. They act as catalysts in the conversion of arginine residues into citrullines by the addition of calcium ions. PAD1, -2, -3 and -4 have so far been described in human genes and their expressions cut across a wide variety of tissues and are thought to play a role in the onset and progression of human neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis. PAD6 has not been well-characterized in human tumors (21). The observation of its high expression in the non-treated group and absence in treated tumor is interesting and might indicate a potential marker for monitoring osteodex treatment response.
Tubulin β6 class V, a cytoplasmic protein, was significantly down-regulated in the samples from osteodex-treated mice. Tubulins belong to a superfamily of cytoplasmic proteins. Six members have been characterized, the α-, β-, γ-, δ-, ϵ- and the ζ-tubulin. α-Tubulin and β-tubulin are associated with the cellular dynamic/stability of microtubules and are prevalent members of the tubulin family. Many anticancer agents have β-tubulin binding activity, resulting in microtubule re-arrangements and ultimately cell-cycle blockage. The expression pattern of different tubulins after anticancer treatment has been investigated to elucidate their possible usefulness as markers for treatment outcome. In general, the global expression of the different β-tubulin isotypes showed varied and complex patterns across different tumor types (22).
A recent colorectal cancer study demonstrated a link between poor survival and the expression of Tubulin, beta 3 (TUBB3)/TUBB6, and the androgen receptor (AR), especially in females. In both genders, AR is associated with TUBB3/TUBB6 expression (23).
The semi-synthetic taxane derivative cabazitaxel works by disruption of the microtubular network necessary for cellular functions, especially during mitotic and interphase of cell division. This compound was recently approved by the Food and Drug Administration (FDA) for treatment of CRPC. With this perspective regarding the importance of effects on tubulins, the observation in this study is interesting and significant. Tubulin markers once validated might become useful in the monitoring of response to treatment of patients with CRPC.
Three proteins with the most marked difference in samples between non-treated and osteodex-treated mice were fibrinogen gamma and beta chains, Myosin light polypeptide 6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The highest mean expression of myosin light polypeptide 6 was observed among the samples from treated mice, while the other three proteins were significantly highly expressed in the samples from non-treated tumors. Of particular interest a greater than three-fold inverse expression of GAPDH in the treatment group was observed. This 38-kDa protein is an important catalytic enzyme necessary for ATP production in cells. It was recently demonstrated that GAPDH is regulated to target telomerase architecture, resulting in an arrest of telomere maintenance, induction of cancer cell senescence and increased cancer cell proliferation (24). Therefore, the present results suggest that treatment with osteodex inhibits GAPDH expression, thereby resulting in inhibition of tumor growth.
Cytoskeletal proteins are actively involved in regulation of cellular structure and the ability of the cells to maintain structural integrity is a fundamental aspect of cellular responses to injury or therapeutic agents. A recent study demonstrated that myosin light chain kinase significantly enhanced the highly proliferative ability of breast cancer cells mediated by anti-apoptosis linked to the p38 pathway (25). The enhanced cell proliferation of the samples from the non-treated group, with highly up-regulated expression of myosin light polypeptide 6 and the marked expression reduction in samples from osteodex-treated animals are another interesting observation indicating the efficacy of osteodex.
In summary, the present study indicates that osteodex, although bone-specific, exerts a considerable efficacy on soft tissue lesions also. Several of the differentially expressed protein patterns indicate a broad and significant mode of action. Some of these might have the potential to serve as markers of therapeutic response.
The present study further demonstrates the versatility of proteomic analyses and its constantly evolving technical advancement.
Acknowledgments
The Authors wish to acknowledge the support of the Research Center Administration at the King Faisal Specialist Hospital & Research Center. We are thankful for the support and logistic assistance from Logistics and Facilities Management Office. This work was supported by King Faisal Specialist Hospital and Research Centre (RAC Project # 2080 052). We thankfully acknowledge Mr. Abdallah Al-Dhfyan for technical assistance. This study was supported by The Cancer Society in Stockholm, The King Gustav V Jubilee Fund, Stockholm, The Swedish Cancer Society, and Mr Svante Wadman, Stockholm.
Footnotes
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Disclosure and Conflict of Interest
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None.
- Received January 17, 2014.
- Revision received January 30, 2014.
- Accepted January 31, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved