A potential antitumor peptide therapeutic derived from antineoplastic urinary protein

Peptides 25 (2004) 543–549

A potential antitumor peptide therapeutic derived from antineoplastic urinary protein Kathleen M. Hehir∗ , Alexander Baguisi, Sarah E. Pennington, Janna M. Bates, Paul A. DiTullio TranXenoGen Inc., Technology Development, 800 Boston Turnpike, Shrewsbury, MA 01545, USA Received 12 November 2003; received in revised form 5 February 2004; accepted 11 February 2004 Available online 2 April 2004

Abstract New therapies in cancer treatment are focusing on multifaceted approaches to starve and kill tumors utilizing both antiangiogenic and chemotherapeutic compounds. Antineoplastic Urinary Protein (ANUP), a 32 kDa protein normally secreted in human urine, has been previously described as a molecule possessing both antiproliferative and antiangiogenic activities. Two synthetic peptides complimentary to the N-terminus of ANUP were designed to test their ability to reproduce these beneficial effects but ultimately to provide a more useful small molecule therapeutic. The results show that the peptides reduced tumor burden by up to 70% in a nude mouse model and demonstrated the ability to inhibit blood vessel formation in a chick chorioallantoic membrane assay (CAM). © 2004 Elsevier Inc. All rights reserved. Keywords: Angiogenesis; Antineoplastic urinary protein; ANUP; CAM

1. Introduction Cancer is a disease manifested by uncontrolled cell growth that presents over 100 distinct clinical pathologies [10]. The development of an effective therapy for such a broad spectrum of disease states represents a unique scientific challenge. Chemotherapeutic agents and radiation, which cause DNA mutations in actively dividing cells, were intended to selectively kill cancer cells while having limited effect on normal cells. Unfortunately, these cytotoxic agents, while effective in managing certain types of cancer, were limited in their utility due to their toxicity on normal dividing cell populations resulting in adverse side effects. Advancement in our understanding of cell biology and cancer has lead to the advent of new more selective treatments providing hope for cancer patients. Recent clinical investigations have shown the benefit of combination therapies that target not only cancer but also its ability to stimulate new blood vessel growth [8]. Angiogenesis, new blood vessel formation, is critical for neoplastic growth and metastasis but is essentially quiescent in adults [4,5]. A number of angiostatic factors such as endostatin [13], angiostatin [14], and thrombospondin [12] have been ∗

Corresponding author. Tel.: +1-508-936-4218; fax: +1-508-842-2786. E-mail address: [email protected] (K.M. Hehir).

0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.02.003

identified and act to block various steps in the pathway. However, difficulties encountered in production, lack of specificity, and low activity levels have combined to detract from the clinical utility of many of these factors. Identification of new, more specific molecules that inhibit this process will provide more treatment options that should be well tolerated and more effective when paired with existing therapies. In this paper, we describe the characterization of two 20 amino acid peptides complementary to the N-terminal sequence of antineoplastic urinary protein (ANUP). Originally identified as a 32 kDa dimer in human urine by Sloane et al. [17], ANUP inhibited endothelial cell migration and tumor cell proliferation, as well as demonstrated antiangiogenic properties by inhibition of bFGF- and VEGF-mediated blood vessel growth in chick chorioallantoic membrane (CAM) assays [11]. ANUP possesses an N-terminal pyroglutamate blockage as well as aggregation properties that indicate conformational requirements for activation of the molecule [17] but which also could complicate production and delivery as a therapeutic. In previous publications, the N-terminal region of ANUP had been shown to inhibit tumor cell proliferation using a synthetic nonapeptide, although the peptide’s activity was only a fraction of the level of the intact protein [16,18]. We designed these experiments to evaluate whether a larger peptide would be more active, as well as contain the antiangiogenic activities of the full protein, while also provid-

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ing the advantages of improved delivery options and ease of production, which would enable its use in a clinical setting.

2. Methods 2.1. Peptide synthesis The 20mer peptides, E-L-K-C-Y-T-C-K-E-P-M-T-S-AS-C-R-T-I-T (MW = 2265.6) and Pyroglutamate-L-KC-Y-T-C-K-E-P-M-T-S-A-S-C-R-T-I-T (MW = 2247.8), were synthesized and HPLC purified by SynPep Corporation (Dublin, CA) to >70% purity. 2.2. Chicken chorioallantoic membrane (CAM) assay Fertilized White Leghorn chicken eggs were obtained from Charles River Associates/Spafas Laboratories and incubated at 37 ◦ C for 9–10 days in a humidified incubator (Model 1202, GQF Manufacturing, Savannah, GA). Small windows were created through the blunt end of the shell directly over the air sac of the embryos with the use of a small crafts Dremmel drill. The air sac was peeled-off, and two filter disks, saturated with either basic fibroblast growth factor (bFGF; Sigma® , St. Louis, MO) or the test peptide (or buffer control) were placed adjacent to each other on the CAM of the chick embryo. The shell windows were sealed with parafilm and eggs were returned to the incubator for 48–72 h. The CAM tissue directly below the filter disks was then resected, the tissues were washed three times with PBS and finally analyzed using a stereomicroscope (Nikon SMZ800). The number of branching blood vessels infiltrating under the filter disks were determined and documented. 2.3. Chick embryo neo-vascularization inhibition assay Small windows were created through the blunt end of the shell directly over the air sac of 3.5-day-old White Leghorn chick embryos with the use of a small crafts Dremmel drill. The air sac was peeled-off and filter disks with different concentrations of the test peptide or buffer control were placed over a section of the developing blood ring and overlapping connecting vessels of the chick embryo. The shell windows were sealed with parafilm and eggs were incubated at 37 ◦ C in a humidified egg incubator. After 48–72 h, the yolk membrane tissue directly below the filter disks was resected. Tissues were washed three times with PBS and analyzed for inhibition of neo-vascularization using a stereomicroscope with photo-digital attachment (Media Cybernetics, Silver Spring, MD). 2.4. Detection of cell death A dual fluorescent staining procedure (H-33342 and propidium iodide, 5 ␮g/ml; Sigma® ) was used to differentially

determine the incidence of apoptosis and necrosis of the resected tissues. Following staining, the tissues were washed three times in PBS and fixed in 4% formalin. The tissues were visualized under epifluorescence using a photo-digital imaging and software analysis system (Image-Pro Plus® , Media Cybernetics, Silver Spring, MD). The number of live, apoptotic, and necrotic cells were determined as previously detailed [2]. 2.5. Tumor cell culture and harvesting The HeLa human cervical cancer cell line (ATCC, Manassas, VA) was grown according to supplied directions in DMEM containing 10% fetal bovine serum (InvitrogenTM , Carlsbad, CA). Cells in log phase were harvested from flasks using trypsin-EDTA (0.05% trypsin/0.53 mM NaEDTA; InvitrogenTM ), washed in PBS and resuspended in DMEM for counting and viability assessment using trypan blue exclusion. Viability and re-plating efficiency were greater than 98 and 95%, respectively. Cells were resuspended at 5 × 107 cells/ml in PBS for injection into the mice. 2.6. In vivo nude mice experiment Nude nu/nu mice (4–5-week-old females, Charles River Labs, Wilmington, MA) were acclimated for 1 week before injection with 5 million HeLa cells subcutaneously (SQ) in a total volume of 100 ␮l. After 3 days of tumor development, the mice were injected SQ with each peptide prepared in phosphate buffered saline (PBS) containing 0.05% sodium dodecyl sulfate (SDS) or PBS + 0.05% SDS alone (Control). All doses were 200 ␮l in volume, with additional treatments on days 6, 10, and 13 post-transplantation. Treatments were alternated between flanks of the mice. Tumor size and mouse weights were measured three times per week. The results represent the average of the three mice in each group. At the end of the experiment, the mice were terminated and the tumors were excised for weight and histopathology. Histopathology was performed by IDEXX Laboratories (Grafton, MA). Pictures were taken using an Olympus IX70 inverted fluorescence microscope and ImagePro Plus capture and analysis software (Media Cybernetics, Silver Spring, MD). The study was conducted according to protocols approved by TranXenoGen’s IACUC committee which are in accordance with National Institutes of Health (NIH, USA) guidelines.

3. Results Two peptides, Peptide A (Pep A; ELKCYTCKEPMTSASCRTIT) and Peptide B (Pep B; pyroELKCYTCKEPMTSASCRTIT), which differ only by the pyroglutamate blockage, were chemically synthesized and purified using standard methods (SynPep Corp., Dublin, CA). The

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120 100

Pep A; 500ug

80

Pep A; 750ug

60

Pep B; 500ug

40

Control

20 0 0

(A)

2

4

6

8 10 12 14 16

Days Post Transplantation 180 160

Tumor Volume (mm3)

140 120 100 80 60 40 20 0 PepA; PepA; PepB; 500ug SQ 750ug SQ 500ug SQ

(B)

Control

250

Tumor Volume (mm3)

peptides were tested both in vivo (nude mice, embryonic neo-vascularization, and CAM assays) and in vitro (tumor cell proliferation). To assess the potential for inhibition or reduction of tumor burden by the peptides, we examined their activity in an active treatment nude mouse model of human cervical cancer. HeLa cells (5 × 106 cells in 100 ␮l; SQ) were implanted in the left mid-flank region of 5-week-old immunodeficient Nude nu/nu female mice and allowed to develop to palpable size (average diameter = 3.9 mm2 ; average volume = 24 mm3 ). Mice were randomized and divided into experimental groups, with three mice per group. The mice were treated with SQ injections (200 ␮l) of various doses of peptide in phosphate buffered saline (PBS) containing 0.05% sodium dodecyl sulphate (SDS) or with PBS containing 0.05% SDS alone. The presence of SDS was required for activation of the nonapeptide and the protein [11,18]. Both peptides were tested at a dose of 500 ␮g (2.2 × 10−7 moles per dose) while Pep A was also tested at 750 ␮g (3.3 × 10−7 moles per dose). The treatments were administered on days 3, 6, 10, and 13 post-transplantation. Tumors were measured three times per week until the mice were terminated on day 24 post-transplantation. All groups demonstrated an initial inhibition of tumor growth as compared to the control group (Fig. 1A and B). This trend continued throughout the treatment phase but was lost within a week after treatment stopped for Pep B while both Pep A treatment groups showed only limited tumor growth for the duration of the experiment (Fig. 1C). In the control group, tumor growth was initially rapid followed by a 10–14-day stationary period and then a renewed rapid tumor growth until experiment termination (Fig. 1C). Animal weights continued to rise during the treatment phase and there were no adverse events associated with the treatments. Earlier work with ANUP protein demonstrated a similar profile for both HeLa tumors [18] and Karposi’s sarcoma tumors [11] in nude mice to that demonstrated for Pep B in this experiment, including renewed growth of tumors after the treatment phase. Several studies were conducted to evaluate the antiangiogenic activities of the peptides and possibly elucidate the mode of action for the tumor reduction. Inhibition of neovascularization was investigated by administration of Pep A and Pep B on the proliferating blood vessels of 3.5-dayold chick embryos. Both peptides selectively suppressed or delayed neo-vascularization in a dose-dependent manner (Fig. 2). At a dose of 25 ␮g for Pep A and 50 ␮g for Pep B, the peptides disrupted/delayed capillary vessel development and reduced the growth of pre-existing blood vessels in the treatment area without affecting those vessels adjacent to the filter disks. At a dose of 100 ␮g, both peptides showed identical antiangiogenesis activity, comparable to the effects observed at 50 ␮g (data not shown). In contrast to the peptides, the buffer control had no observable effects on neo-vascularization. The viability of the embryos was not affected by any of the treatment regimens.

Tumor Volume (mm3)

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200 150

Pep A; 500ug Pep A; 750ug

100

Pep B; 500ug Control

50 0 -50 0

(C)

2

4

6

8 10 12 14 16 18 20 22

Days Post Transplantation

Fig. 1. Inhibition of HeLa tumor growth by ANUP peptides in nude mice. Five million HeLa cells were transplanted per mouse. After 3 days, mice were treated with Pep A (500 or 750 ␮g), Pep B (500 ␮g), or buffer only (200 ␮l volume; PBS + 0.05%SDS, Control) on days 3, 6, 10, and 13 (arrows). Tumor growth was measured three times per week and the mice were sacrificed on day 24 post-transplantation. (A) Inhibition of HeLa tumor growth during treatment phase. Results represent the average tumor volume of all mice (n = 3). Treatments with Pep A at 500 ␮g per dose (open square), Pep A at 750 ␮g per dose (open triangle), and Pep B (open circle) all demonstrate tumor inhibition during the treatment phase while control tumors (solid diamond) remain static. (B) Response of individual tumors to treatment with peptides. Tumors were measured on day 14 (1 day post last treatment). Individual mice tumor volumes demonstrate response of all animals treated with either Pep A or Pep B. (C) Tumor growth profiles post-treatment with Pep A and Pep B. Note slight increase in tumor burden for both Pep A treatment groups and escalation of tumor burden in the control and Pep B-treated groups subsequent to the fourth treatment.

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Fig. 2. Dose-dependent suppression of branching vessel formation on day 3.5 chick embryo by Pep A and Pep B. A single topical dose of either peptide induced the suppression of branching vessel formation and development; Pep A showed inhibition beginning at 25 ␮g vs. Pep B which showed inhibition by 50 ␮g. The number of embryos treated and the number showing inhibition is indicated in the table on the left.

Inhibition of bFGF-induced angiogenesis in a CAM assay is another hallmark of antiangiogenesis. Ten-day-old chick embryos were exposed to increasing doses of either Pep A or Pep B in the presence of 100 or 200 ng of bFGF for 48 h at 37 ◦ C. A dose of 200 ng of either peptide suppressed the angiogenesis activity induced by 100 ng of bFGF, with Pep

A showing comparatively better inhibition of blood vessel infiltration than the Pep B (Fig. 3). At a dose of 200 ng of bFGF, only Pep A showed some level of inhibition. The formation of branching vessels and smaller capillary vessels were inhibited by both peptides. Closer examination of the effects of the peptides on angiogenesis reveals the presence of discontinuous and disrupted blood vessels (Fig. 3C). To identify the mechanism of action of the peptides, the resected CAM tissues were dually labeled with fluorescent stains to show evidence of cell demise through either lysis or apoptosis. Minimal levels of lysed cells (stained with propidium iodide) were observed on the tissues treated with the peptides and were similar to that observed on the bFGFtreated controls (data not shown). There was however, a significant increase in DNA fragmentation on CAM tissues treated with Pep A as compared to either Pep B or bFGF control after staining with H-33342. Intensely staining apoptotic bodies were seen in tissues from the Pep A-treated CAMs and localized on the vascular cells. This is apparent by the outlining of the vessels against the non-vascular tissues (Fig. 4). Pep B showed minimal increase in apoptosis

Fig. 3. Inhibition of bFGF-mediated angiogenesis in early chick embryos by ANUP peptides. Micrographs showing induction of angiogenesis resulting from the topical administration of 100 ng of bFGF on the CAM of day 10 chick embryos. Neo-vascularization along the edges of the filter disks (A) and vascular infiltration (B) are seen after administration of bFGF alone. Inhibition of neo-vascularization and infiltration as well as degradation of pre-existing vessels is demonstrated by co-administration of 200 ng Pep A with 100 ng bFGF (C). Co-administration of 200 ng Pep B with 100 ng bFGF also results in inhibition of bFGF-induced angiogenesis (D).

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Fig. 5. Histopathology of tumors isolated from Pep A-treated mice (750 ␮g). Larger areas of cell death in tumors isolated from Pep A-treated mice are evident by the increased number of dark nuclei. Magnification, 75×; scale bars, 10 ␮m.

Fig. 4. Resected CAM tissues topically treated with bFGF in the presence of Pep A and fluorescently labeled to analyze for apoptosis. The condensed apoptotic nuclei will intensely fluoresce (and will appear white) under epifluorescence visualization compared to the staining patter of the viable cells with H-33342 (blue nuclei). (A) 100 ng bFGF plus 200 ng Pep A (100×). Note the intensely staining apoptotic nuclei (white) of vascular cells resulting in the outlining of the vessels against the lighter stained non-vascular cell nuclei which at higher magnification (200×) are clearly apoptotic (B). The Pep B-treated CAM did not demonstrate the peri-vascular apoptosis and the white nuclei were randomly distributed throughout the CAM tissue (C). As expected, bFGF-treated control CAM showed minimal and randomly distributed apoptotic nuclei (not shown). Scale bars, 10 ␮m.

over the control, with a random staining pattern observed. However, there was an observable condensation of the cell nuclei in the Pep B-treated CAMs as compared to controls (not shown). Peri-vascular apoptotic cells have previously been demonstrated in mouse carcinomas using the antiangiogenic compounds Fc-angiostatin and Fc-endostatin where the combination of these compounds resulted in significant reduction of tumor volume [3]. To correlate antiangiogenic activity with the tumor reduction in the nude mouse model, tumors were isolated upon sacrifice and prepared for histopathology to examine them for evidence of cell death and antiangiogenesis. Tumor sections were reviewed for size (small, medium, or large), areas of apoptosis (size and number), vessel infiltration, and neo-vascularization/peripheral vascularization. The totals for each group were averaged and are summarized in Table 1. Both peptides demonstrate reduction in peripheral vascularization as well as reduction in tumor infiltration as compared with controls. Pep A-treated mice tumor sections were markedly reduced for both infiltration and vascularization. There was no real difference in the number of areas of cell death between the groups, however, the size of the areas of cell death were larger from the Pep A high dose tumor sections (Fig. 5). This observation requires further investigation to determine the significance, but may help explain why the tumors did not develop further after the treatment stopped.

4. Discussion ANUP may be one more representative of a growing class of antiangiogenic and antitumor substances that are derived from the proteolysis or other modification of blood proteins. Other proteins in this group include heparin-binding

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Table 1 Histopathology of tumors isolated from all mice post-sacrifice and analyzed for section size (small, medium, or large), areas of cell death (size and number), tumor infiltration, and neo-vascularization/peripheral vascularization

Section size Cell death Infiltration Peripheral vascularization

Pep A (500 ␮g SQ)

Pep A (750 ␮g SQ)

Pep B (500 ␮g SQ)

Control

M, M, M 2.7 0.3 18.0

S, M, M 12.0 0.7 14.3

M, L, L 15.3 2.7 21.0

M, L, L 12.7 6.0 49.0

The numbers represent the group average of the sections analyzed. Note the reduced infiltration and vascularization evident in the treatment groups as compared with the control group.

fragments of fibronectin [6,7], modified antithrombin III [15], angiostatin [13], and endostatin [14]. The mechanism of the antiangiogenic activity for ANUP as well as for the ANUP peptides is unknown. The other proteins listed above are known to bind to adhesion proteins and since previous work with ANUP demonstrated inhibition of endothelial cell migration, it has been postulated that ANUP effects endothelial cell migration by binding to a cell surface receptor in this lineage [11]. The antiproliferative potential of both peptides was assessed in standard neutral red and MTT assays on several human tumor cells and neither peptide demonstrated significant activity at up to 8.8 × 10−4 M (data not shown). It is interesting that in cell culture, the ANUP protein (IC50 at 7.5 × 10−8 M on HeLa cells), but not the peptides, demonstrated antiproliferative effects on a wide variety of human tumor cells. This may indicate that the antiangiogenic region resides in the N-terminal 20 amino acids since angiogenesis is not a requirement for the proliferation of tumor cells in culture, while the antiproliferative activity for the protein resides elsewhere in the molecule or is dependent upon a conformation not possible with these peptides. A peptide without pyroglutamate (i.e. Pep A) demonstrates better suppression of tumors in nude mice and clear evidence of antiangiogenesis by a reduction in both tumor infiltration and vascularization. Pep A also requires a lower dose for neo-vascularization, is more antiangiogenic in a CAM assay and exhibits more localized apoptosis in the same assay as compared to a peptide with pyroglutamate (i.e. Pep B). The ability of both forms of the peptide to inhibit neovascularization of chick embryos as well as to inhibit angiogenesis in bFGF-mediated CAM assays lends credence to the theory that the N-terminus contains the antiangiogenic activity for the molecule. Cloning of the full-length ANUP cDNA and recombinant expression will allow detailed characterization and structure/function analysis as well as the design of additional peptide variants with anti-tumor activity. Further investigations will explore the benefit of combination therapies using one or more ANUP peptides in conjunction with other chemotherapeutic agents or protein therapeutics. The clinical data showing increased survival in colorectal cancer patients treated with Avastin by Genentech [9] have prompted the design of additional clinical trials in other cancers with this antiangiogenic molecule in

combination with other therapeutic modalities, such as Herceptin, Rituxan, and Tarceva. To obtain the best results, it may be necessary to combine ANUP Pep A with other anticancer molecules or even modify the peptide to link it with other chemotherapeutic agents [1] and, thus, deliver them more effectively. ANUP Pep A demonstrates a number of the indicators of antiangiogenesis both in vitro, and, more importantly, in vivo at doses that are reasonable for clinical application. Future studies using this peptide as well as other peptide variants will help to elucidate the mechanism of action and aid in the design of an effective delivery strategy. This peptide may be one more tool in the future arsenal of antiangiogenic drugs that holds promise for the treatment of many cancers.

Acknowledgments The authors would like to thank Dr. Nathan Sloane for his reading of the manuscript. They also thank Dr. Sean Sanders for his comments and critical review of the manuscript. References [1] Arap W, Pasqualini R, Ruoslahti E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 1998;279:377–80. [2] Baguisi A, Lonergan P, Overstrom EW, Boland MP. Vitrification of bovine embryos: incidence of apoptosis and necrosis. In: Paper presented at the Annual Conference of the International Embryo Transfer Society, Quebec City, Quebec, Canada, 10–12 January 1999; Theriogenology 1999;51:161–448. [3] Bergers G, Javaherian K, Lo KM, Folkman J, Hanahan D. Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. Science 1999;284:808–12. [4] Fidler IJ, Ellis LM. The implications of angiogenesis for the biology and therapy of cancer metastasis. Cell 1994;79:185–8. [5] Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27–31. [6] Homandberg GA, Williams JE, Grant D, Schumacher B, Eisenstein R. Heparin-binding fragments of fibronectin are potent inhibitors of endothelial cell growth. Am J Pathol 1985;120:327–32. [7] Homandberg GA, Kramer-Bjerke J, Grant D, Christianson G, Eisenstein R. Heparin-binding fragments of fibronectin are potent inhibitors of endothelial cell growth: structure–function correlations. Biochim Biophys Acta 1986;874:61–71. [8] http://www.cancer.gov/clinicaltrials/developments/anti-angio-table.

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