The PHSCN dendrimer as a more potent inhibitor of human breast cancer cell invasion, extravasation, and lung colony formation
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- Yao, H., Veine, D.M., Fay, K.S. et al. Breast Cancer Res Treat (2011) 125: 363. doi:10.1007/s10549-010-0826-y
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The α5β1 integrin fibronectin receptor is an attractive therapeutic target in breast cancer because it plays key roles in invasion and metastasis. While its inactive form is widely expressed, activated α5β1 occurs only on tumor cells and their associated vasculature. The PHSCN peptide has been shown to bind activated α5β1 preferentially, thereby blocking invasion in vitro, and inhibiting growth, metastasis and tumor recurrence in preclinical models. Moreover in a recent Phase I clinical trial, systemic PHSCN monotherapy was well tolerated, and metastatic disease failed to progress for 4–14 months in 38% of patients receiving it. A significantly more potent PHSCN derivative, the PHSCN–polylysine dendrimer (Ac-PHSCNGGK-MAP) has recently been developed. We report that it is 1280- to 6700-fold more potent than the PHSCN peptide at blocking α5β1 mediated SUM-149 PT and MDA-MB-231 human breast cancer cell invasion of naturally occurring basement membranes in vitro. Chou–Talalay analysis of these data suggested that invasion inhibition by the PHSCN dendrimer was highly synergistic. We also report that, consistent with its enhanced invasion-inhibitory potency, the PHSCN dendrimer is 700- to 1100-fold more effective than the PHSCN peptide at preventing SUM-149 PT and MDA-MB-231 extravasation in the lungs of athymic, nude mice. Our results also show that many extravasated SUM-149 PT and MDA-MB-231 cells go on to develop into metastatic colonies, and that pretreatment with the PHSCN dendrimer is more than 100-fold more effective at reducing lung colony formation. Since many patients newly diagnosed with breast cancer already have locally advanced or metastatic disease, the availability of a well-tolerated, nontoxic systemic therapy that can prevent metastatic progression by blocking invasion could be very beneficial.
KeywordsBreast cancerInvasionExtravasationLung metastasisIntegrin fibronectin receptorMMP-1
Fetal bovine serum
Dose reduction index
Hanks buffered salt solution
Matrix assisted laser desorption/ionization
Enzyme-linked immunoabsorbant assay
Standard error of the mean
Platelet endothelial cell adhesion molecule-1
Optimal cutting temperature
Metastatic breast cancer is currently incurable . Despite therapeutic advances, patients with metastatic disease have poor prognoses, and their median survival time is 2–3 years . Combination therapies can result in objective responses, but tumor resistance usually develops, and disease control cannot be maintained . Breast cancer patients with metastasis to visceral organs, such as lung, have shorter survival times than those with bone metastasis alone . Like patients with hormone receptor-negative tumors or hormone-refractory disease, patients with symptomatic visceral disease are treated with cytotoxic chemotherapetic regimens .
Invasion is a key feature of breast cancer metastasis because it promotes tumorigenesis by supporting endothelial cell invasion and neovascularization . Breast cancer cell invasion also enables tumor cells to migrate through the connective tissue surrounding a tumor, enter the circulatory system, and extravasate at distant sites to form metastatic colonies [7–9]. Thus, unrestricted invasion is a very important, debilitating aspect of the metastatic phenotype .
Extravasation is a key step in hematogenous cancer metastasis . It occurs after tumor cells arrest in microvasculature or sinusoids. Tumor cell arrest may occur by trapping, without stable adhesion to the microvasculature , or may involve preferential adhesion to the endothelial cells of specific sites . In either case, after arrest, invasion of the vascular basement membrane and underlying interstitial connective tissue is required for extravasation .
We have devised a peptide of five amino acids that is a potent inhibitor of α5β1 integrin-mediated invasion by breast cancer cells. The acetylated, amidated PHSCN peptide, whose primary structure is Ac-PHSCN-NH2 [6, 8, 9], emerged as an invasion inhibitor during the study of the structure activity relationship of the invasion-inducing PHSRN peptide . The PHSRN sequence is a known α5β1 ligand that interacts with a specific region of the α5 subunit of the α5β1 integrin, and is found in the cell binding domain of fibronectin [15, 16]. In addition to preventing prostate cancer cell invasion and metastasis , the PHSCN peptide is also a potent inhibitor of microvascular endothelial cell invasion and angiogenesis . Systemic PHSCN therapy prevented disease progression for prolonged periods in a variety of preclinical models, and in phase 1 clinical trial [8, 17–20].
Thus, Ac-PHSCN-NH2 is a promising lead compound for targeted therapy of invasion and metastasis. To improve its potency, we attached eight Ac-PHSCNGGK peptide moieties to the N-termini of the multiantigenic peptide (MAP) polylysine dendrimer. We report that this creates a significantly more potent inhibitor of α5β1 integrin-mediated SUM-149 PT and MDA-MB-231 human breast cancer cell invasion and matrix metalloproteinase 1 (MMP-1) induction in vitro, and of extravasation and lung colony formation in vivo in athymic nude mice.
Materials and methods
Cell lines and cell culture
MDA-MB-231 metastatic  and SUM-149 PT  inflammatory human breast cancer cell lines were obtained from American Type Culture Collection (Manassas, VA), or from the laboratory of Dr. Stephen Ethier, respectively. Both cell lines were cultured as recommended, and frozen in liquid N2 in aliquots within 2 months of receipt. Single aliquots were subsequently resuscitated as needed, and cultured as recommended. No aliquot of cells was cultured for more than 4 months, and the morphologies of all cultures were routinely checked by phase contrast microscopy. Growth curves of all cultures were always recorded, and checked for consistency. For all assays in serum-free (SF) medium, SUM-149 PT and MDA-MB-231 cells were first serum-starved overnight.
Peptide and dendrimer synthesis
N-terminal acetylated, C-terminal amidated PHSRN, PHSCN, and HSPNC peptides (Ac-PHSRN-NH2, Ac-PHSCN-NH2, and Ac-HSPNC-NH2) were synthesized, their structures confirmed, and their purities assessed as described [6–9, 14]. Their purities were as follows: Ac-PHSRN-NH2 97%, Ac-PHSCN-NH2 98%, Ac-HSPNC-NH2 91%.
N-terminal acetylated PHSCN and HSPNC MAPs were synthesized by covalently attaching peptide C-termini to the N-termini of a polylysine dendrimer, 8 core MAP (Sigma-Aldrich, Saint Louis MO). MAPs were synthesized by Fmoc solid phase synthesis in a manual procedure with reaction monitoring by Ninhydrin test , to allow for complete coupling of each amino acid. Quality control of the MAPs was performed by amino acid analysis , followed by Edman sequencing and preview analysis to reveal any deletions in the sequences . Dendrimer purities were estimated to be as follows: Ac-PHSCNGGK-MAP 94%; Ac-HSPNCGGK-MAP 97% (not shown). The MAPs were also evaluated by MALDI for the expected mass of the fully populated dendrimer. The spectra showed the expected mass for the complete MAP and very little evidence of incomplete synthesis.
PHSCN and HSPNC peptides to be attached to polylysine dendrimers or ovalbumin were synthesized with two glycines and a lysine (GGK) on the C-terminal end (PHSCNGGK or HSPNCGGK) to provide a spacer and an attachment site for labeling. Ac-PHSCNGGK-NH2 and Ac-HSPNCGGK-NH2 had functional characteristics identical to Ac-PHSCN-NH2 and Ac-HSPNC-NH2, respectively (not shown). PHSCN-coupled ovalbumin (Ac-PHSCNGGK-Ova) was synthesized by coupling the PHSCNGGK C-terminus to ovalbumin with an attached EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) crosslinker (ThermoFisher Scientific, Waltham MA), according to established procedures . Ac-PHSCNGGK-Ova purity was 90%.
In vitro invasion assays
Naturally serum-free, selectively permeable basement membranes from sea urchin embryos were utilized as in vitro invasion substrates, as described [6–9, 14]. All cells were serum starved prior to addition of 10% FBS or 0.1 μg/ml Ac-PHSRN-NH2 to stimulate invasion. Peptides or dendrimers were briefly prebound to cells in half of the final invasion assay volume, prior to placement on basement membranes in the remaining volume of medium. Invasion assays were incubated for 16 h at 37°C prior to scoring at 400-fold magnification under phase contrast optics, as described [6–9, 14]. Data, mean invasion percentages, were analyzed using Graphpad Prism 5 software (San Diego, CA) as a function of log (inhibitor) versus normalized data, variable slope.
Data were analyzed by the Combination index (CI) method of Chou–Talalay , as described in . The analysis was based on the multiple drug effect equation derived from the median effect principle of the mass-action law. The median-effect equation, y = log(fa/fu), with respect to x = log(dose) defines the dose and effect relationship in the absence of reaction rate constants, where fa is the fraction of cells affected (invasion-inhibited), and fu is the fraction of cells unaffected (invaded). The x-intercept represents the IC50 value. CI and DRI values were determined assuming that the monomer represents the single dose, and the dendrimer (with 8 PHSCNGGK moieties) represents the combinatorial dose.
MMP-1 activity assays
Effects of Ac-PHSCNGGK-MAP, Ac-HSPNCGGK-MAP, or Ac-PHSCN-NH2 on MMP-1 activities secreted by adherent cells were analyzed as described [6, 7, 9]. Adherent cells were serum-starved overnight, then pretreated with Ac-PHSCN-NH2, Ac-PHSCNGGK-MAP, or Ac-HSPNCGGK-MAP at 250 μg per 1,000,000 cells for 1 h. After pretreatment, the treatment groups were stimulated with 5 ml 10% FBS or SF medium containing 250 μg per 1,000,000 cells Ac-PHSRN-NH2 for 24 h prior to assay. Treatment groups were run in triplicate. Supernatants from treated cells were collected for analysis. Human MMP-1 ELISA Kit (RayBiotech, Inc., Norcross GA) was used to quantitate MMP-1 activity, according to manufacturer’s instructions. Data are presented as means ± SD, and results were analyzed using Student’s t-test.
Fluorescent DiI and DiO labeling of cells
Confluent SUM-149 PT or MDA-MB-231 cells were washed with Hanks-buffered salt solution (HBSS; Life Technologies, Grand Island NY), harvested with 0.25% trypsin/1% EDTA (Life Technologies), rewashed in HBSS, and fluorescently labeled in 6 ml SF medium with 25 μl of the red lipophilic carbocyanine vital dye DiI, 1,1′-dilinoleyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (Invitrogen) or 25 μl of the green lipophilic vital dye DiO, 3,3′-Dioctadecyloxacarbocyanine iodide, for 20 min in dark at 37 °C, as described [29, 30]. Cells were pelleted at 1,000 rpm for 2 min, prior to resuspension in 6 ml of medium, and recovery for 72 h. DiI has been utilized as a vital dye in numerous studies, e.g., to define neural crest invasive/migratory pathways in developing embryos . DiI labeling has been reported to persist in living, cultured neurons for a year .
Extravasation in the lungs athymic mice
To evaluate the importance of α5β1 integrin in breast cancer cell extravasation, DiI labeled SUM-149 PT or MDA-MB-231 cells were incubated for 30 min on ice with 10 μg/ml or 50 μg/ml anti α5β1 MCA1187 function blocking monoclonal antibody (MAb) (Serotec, Oxford, England), as described . A total of 10,000 pretreated cells in 0.1 ml HBSS were injected into the tail vein of each 8-week, nude athymic mouse (Jackson Laboratories, Bar Harbor ME). Each treatment group consisted of 10 mice. All mice were euthanized 24 h later, and their lungs removed and thoroughly rinsed with phosphate-buffered saline (PBS) prior to fixation with 4% paraformaldehyde in PBS overnight at 4°C. Following fixation, lungs were rinsed at room temperature in PBS, and placed in 20% sucrose overnight at 4°C. The samples were cryoprotected by submerging in optimal cutting temperature (O.C.T.): 20% sucrose mixture (VWR, Batavia IL) overnight at 4°C, prior to freezing and storing at −80°C. Frozen lungs were sectioned with a thickness of 10 μm with a Zeiss Cryostat (M550). All slides were sealed with VECTASHIELD mounting medium with DAPI (VECTOR Laboratories, Burlingame CA). Twenty sections were cut from each lung in all mice of each treatment group of 10 mice, at 200 μm intervals. Thus, a total thickness of 4 mm was analyzed from each lung. All sections were examined at 400-fold magnification with a Zeiss Scanning Laser Confocal microscope (LSM510), as previously described .
To compare Ac-PHSCNGGK-MAP and Ac-HSPNCGGK-MAP as extravasation inhibitors, DiI-labeled SUM-149 PT or MDA-MB-231 cells were prebound with appropriate concentrations of dendrimer or peptide in HBSS and incubated at 37°C for 10 min. The concentrations used for prebinding were as follows: Ac-PHSCNGGK-MAP: 10, 1, 0.1, or 0.01 ng/ml; Ac-PHSCN-NH2: 100, 10, 1, or 0.1 ng/ml; Ac-HSPNCGG-MAP: 100 ng/ml. Nude mice (Jackson Laboratories) received an equivalent intravenous dose of dendrimer, peptide (0.0005 to 5.0 μg/kg), or HBSS in 0.1 ml at the appropriate concentrations via tail vein, immediately prior to intravenous injection of DiI labeled, dendrimer- or peptide-prebound SUM-149 PT or MDA-MB-231 cells. Labeled, pretreated cells were intravenously injected in 0.1 ml HBSS, and mice euthanized 24 h later, as described above. Lungs were removed, rinsed, fixed, treated, and frozen as described above. Twenty sections were cut from each lung in all mice of each treatment group of 10 mice, at 200 μm intervals. Thus, a total thickness of 4 mm was analyzed from each lung. All sections were examined at 400-fold magnification with a Zeiss Scanning Laser Confocal microscope (LSM510), as previously described .
To determine what fractions of SUM-149 PT and MDA-MB-231 cells were intravascular or extravascular, sections made as described above were stained with rat anti-mouse PECAM-1 (platelet endothelial cell adhesion molecule-1) monoclonal antibody (Millipore, Temecula CA) , and FITC-conjugated secondary antibody (Jackson Laboratories, Bar Harbor, Maine). Sections were scored as described above, with SUM-149 PT and MDA-MB-231 cells surrounded by at least 75% of their circumference with anti PECAM staining judged to be intravascular. Cells surrounded by less anti PECAM staining were judged to be extravascular, and hence to have completed extravasation.
Lung colony formation assays
To verify the enhanced potency of Ac-PHSCNGGK-MAP as an inhibitor of lung colony formation, suspended, DiI labeled SUM-149 PT or MDA-MB-231 cells were briefly prebound with 10 ng/ml Ac-PHSCNGGK-MAP, 10 ng/ml and 100 ng/ml Ac-PHSCN-NH2, or 100 ng/ml Ac-HSPNCGGK-MAP. Eight-week-old, nude mice (Jackson Laboratories) received one systemic pretreatment with the appropriate peptide or dendrimer in 0.1 ml HBSS by tail vein injection. Immediately after pretreatment of the mice, SUM-149 PT or MDA-MB-231 cells, prebound with dendrimer or peptide, or with HBSS only, were injected into tail veins. Six weeks after injection, mice were euthanized, and lungs were removed, fixed, prepared, and stored as above.
Sections of 10 μm thickness were cut with a Zeiss cryostat as previously described : 20 sections were cut from each lung in all mice of each treatment group, at 200 μm intervals. Thus, a total thickness of 4 mm was analyzed from each lung. Sections were stained with monoclonal, FITC-conjugated anti β-actin antibody (Chemicon, Temecula CA) and scored at 400-fold magnification, based on the retention of DiI red fluorescence by the prostate cancer cells, as described for other cancer types [36–38]. SUM-149 PT and MDA-MB-231 colonies were scored only if they contained more than 50 cells, according to established criteria [37–39]. Data are presented as mean numbers of colonies per lung section ± SEM of 10 mice per group.
Clonogenic survival assays were performed as described .
Inhibition of extravasation by blocking anti α5β1 monoclonal antibody
Invasion inhibition by the PHSCN polylysine dendrimer, Ac-PHSCNGGK-MAP
We utilized naturally serum-free, selectively permeable basement membranes from sea urchin embryos [6–9, 14] as in vitro invasion substrates to compare the invasion inhibitory potencies of the PHSCN polylysine dendrimer (Ac-PHSCNGGK-MAP) and the PHSCN peptide (Ac-PHSCN-NH2). To compare their effects on serum-induced invasion, SUM-149 PT and MDA-MB-231 cells were serum-starved overnight, prior to suspension in medium containing 10% FBS, and prebinding to various concentrations of the PHSCN dendrimer or PHSCN peptide or to maximal concentrations of the specificity controls: the HSPNC dendrimer (Ac-HSPNCGGK-MAP) or HSPNC peptide (Ac-HSPNC-NH2).
Our previous research showed that the PHSRN sequence of the fibronectin cell binding domain [7–9] is specifically responsible for serum- or plasma fibronectin-induced invasion by human breast and prostate cancer cell lines. In addition, we found that the PHSRN peptide is a potent inducer of invasion by normal microvascular endothelial cells, epithelial cells, and fibroblasts [6, 14]. Thus, we also compared the invasion-inhibitory potencies of PHSCN dendrimer and PHSCN peptide on PHSRN-induced invasion by serum-free (SF) SUM-149 PT and MDA-MB-231 cells. Since the PHSCN dendrimer displays the PHSCN sequence on a significantly larger molecule (7575 vs. 598 Da), we also evaluated the effect on invasion inhibitory potency of a modified protein, resulting from coupling a single Ac-PHSCNGGK sequence to ovalbumin (45.0 kDa), to make Ac-PHSCNGGK-Ova (45.9 kDa).
Comparison of IC50, CI, and DRI values for PHSCNGGK-MAP and PHSCN as inhibitors of FBS- or PHSRN-induced invasion by SUM-149 PT or MDA-MB-231 cells
100 ng/ml Ac-PHSRN-NH2
The combination index (CI) and the DRI values, shown in Table 1, allow determination of whether the 8 PHSCN moieties on the dendrimer result in an additive or a synergistic increase in invasion inhibitory potency. Synergism is demonstrated by CI <1 and DRI >1, and additive effects are indicated by CI ≥1 and DRI ≥1 . Thus, in combination, the CI and DRI values listed in Table 1 for both cell lines show the increased efficacy of the PHSCN dendrimer, and indicate that its 8 PHSCN moieties interact synergistically to block invasion for in vitro assays.
Inhibition of serum- or PHSRN-induced MMP-1 secretion by Ac-PHSCNGGK-MAP
Inhibition of SUM-149 PT or MDA-MB-231 cell extravasation in nude mice by prebinding Ac-PHSCNGGK-MAP or Ac-PHSCN-NH2
Comparison of IC50, CI, and DRI values for PHSCNGGK-MAP and PHSCN as inhibitors of extravasation by SUM-149 PT and MDA-MB-231 cells
The results of this experiment indicate that Ac-PHSCNGGK-MAP pretreatment caused 73.3% (SUM-149 PT) or 82.0% (MDA-MB-231) of DiI-labeled breast cancer cells to remain in the lung vasculature; while 26.7% or 18.0%, respectively, appeared to have extravasated. In contrast, only 12.3% (SUM-149 PT) or 9.3% (MDA-MB-231) of untreated breast cancer cells appeared to remain inside the vasculature; whereas, 87.7% or 90.7%, respectively, appeared to have extravasated. Thus, a single, brief Ac-PHSCNGGK-MAP pretreatment at 10 ng/ml was able to reduce breast cancer cell extravasation in the lungs of nude mice by 3- to 5-fold.
Inhibition of SUM-149 PT or MDA-MB-231 lung colony formation in nude mice by prebinding Ac-PHSCNGGK-MAP or Ac-PHSCN-NH2
After pretreatment with either 10 ng/ml (17 nM) or 100 ng/ml (170 nM) of Ac-PHSCN-NH2, lung colony formation by MDA-MB-231 cells (Fig. 6b) was reduced by approximately 50%. In contrast, a dose of 10 ng/ml (1.32 nM) of Ac-PHSCNGGK-MAP achieved 80% inhibition, similar to that observed for SUM-149 PT cells. The overall reduction of lung colonization by Ac-PHSCNGGK-MAP pretreatment in both cell lines suggests at least 2 orders of magnitude in increased potency for SUM-149-PT cells, and perhaps even more in MDA-MB-231 cells (Fig. 6a, b). This allows estimation of a DRI of approximately 100–1,000-fold.
Since only growing micrometastases are functionally relevant, the total number of cells in each micrometastasis was determined for each treatment group shown in Fig. 6. Each micrometastasis included in the totals contained from 50 to 200 cells (data not shown). No micrometastases were scored unless they contained at least 50 cells, similar to criteria previously employed by others [36–38].
Chemotherapy is utilized for the treatment of breast cancer patients with symptomatic visceral metastases, and in patients without symptomatic visceral metastases whose tumors are estrogen receptor and progesterone receptor negative, thus ruling out hormonal therapy . Despite therapeutic advances, a majority of patients with node-positive breast cancer eventually develop recurrence, most frequently to the lungs, liver, or bone. Overall, patients with metastatic breast cancer have a median survival of 24 months, but those with visceral metastasis survive for significantly shorter times than those with metastasis only to bone . Thus, while there is no cure for metastatic breast cancer, occasional prolonged periods of remission have been reported. For example, among patients who had a complete remission after anthracycline-based therapy, 17% (or 3% of all treated patients) remained disease-free for 5 years . Thus, palliation of cancer-related symptoms and possible prolongation of life are primary goals of chemotherapy.
However, because they target DNA structure or the microtubules involved in spindle formation , chemotherapeutic agents also adversely affect rapidly proliferating cells of the gut, the skin, and the bone marrow. Furthermore, breast cancer cells can become resistant to anthracyclines and taxanes [43, 44]. Because breast cancer invasion is a very important, debilitating aspect of the metastatic phenotype , there is a need for an effective, targeted, systemic anti-invasive agent for breast cancer.
When unchecked by normal regulatory mechanisms, the regulatory pathways initiated by α5β1 integrin may give rise to the progressive growth and invasion observed in metastatic breast cancer. For example, the interaction of α5β1 with the PHSRN sequence of the fibronectin cell binding domain is sufficient to induce interstitial collagenase expression and basement membrane invasion by microvascular endothelial cells  and metastatic breast cancer cells , as well as prostate cancer cells [8, 9] and normal epithelial cells and fibroblasts . Because fibronectin is found in all body fluids, proper regulation of α5β1-mediated invasion is very important. This is accomplished by another integrin fibronectin receptor, α4β1. When fibronectin is intact, α4β1 integrin interacts with the LDV sequence of the fibronectin connecting segment, LHGPEILDVPST, to repress α5β1-mediated interstitial collagenase expression . Fragmentation of fibronectin by urokinase plasminogen activator, which also functions in clot dissolution (reviewed in ), gives rise to α5β1 mediated invasion during wound healing [47, 48]. Thus, an important attribute of α5β1-induced invasion in normal α5β1+, α4β1+ cells is its regulation by α4β1 integrin. Although still expressing abundant surface α5β1 many metastatic breast cancer cell lines have low levels of surface α4β1 relative to mammary epithelial cells . Loss of surface α4β1, which can result from ErbB-2 oncogene overexpression in transformed mammary epithelial cells [49–51], has been shown to cause constitutive invasiveness in human metastatic breast and prostate cancer cell lines [7, 9] in the presence of the abundant pFn of blood, lymph, and interstitial fluid [52, 53].
Here, we show that attaching 8 PHSCN peptide moieties to a polylysine dendrimer results in a 60- to 305-fold increase in invasion-inhibitory potency for metastatic human breast cancer cell lines in vitro, and a significant increase in ability to inhibit extravasation and lung colony formation in vivo in athymic, nude mice. Because only highly elevated concentrations of PHSCN dendrimer affected clonogenic survival of SUM-149PT and MDA-MB-231 cells in vitro, inhibition of extravasation and colony formation by low PHSCN dendrimer concentrations was very unlikely to be due to reductions in survival.
The rationale for employing PHSCN-dendrimers as improved therapeutic agents is that their polyvalency will enhance the affinity of their interactions with their target protein, the α5β1 integrin. There are many examples of the ability of multimeric peptides to enhance binding affinity. For example, a peptide complementary to a specific region of endothelin exhibited a binding affinity that was 100-fold greater when it was synthesized as a MAP peptide . Peptide dendrimers are also used as inhibitors of increased potency. For example, peptide dendrimers have been used to inhibit the entry of malaria parasites into hepatocytes . In another example, a peptide dendrimer based on an 8 amino acid sequence from gp120, the HIV-1 surface envelope glycoprotein, has been shown to inhibit HIV-1 infection of both CD4+ and CD4− cells [56, 57]. In an application closely related to ours, the increased binding affinity of dendrimers, compared to monomeric peptides, has been used to design anti-metastatic drugs. For example, a dendrimer containing 16 copies of the YIGSR laminin peptide has been found to inhibit tumor metastasis to lung tissue, and slow tumor growth, when administered as a systemic agent in mice bearing B16F10 melanoma .
Thus, systemic therapy with a potent anti-invasive agent, such as the PHSCN–dendrimer, that effectively targets the activated α5β1 integrins of tumor cells, without affecting the unactivated α5β1 receptors of healthy tissues, could potentially allow patients with metastatic breast cancer to live for long periods of time with their disease, without many toxic side effects. Because of the PHSCN peptide’s efficacy and lack of toxicity, when used as a systemic monotherapy in Phase 1 clinical trial , more potent PHSCN peptide derivatives, such as the PHSCN-dendrimer, may also be useful additions to radiotherapy or more conventional chemotherapeutic regimens.
The authors wish to thank Dr. Philip Andrews for his suggestion of utilizing MAP peptides. All peptides were synthesized by the University of Michigan Protein Structure Facility (Dr. Henriette A. Remmer). The masses of the MAP peptides were verified by Angela Walker, Ph.D of the Michigan Proteome Consortium (Dr. Philip C. Andrews). This research was supported by a Department of the Army IDEA award, W81XWH-06-1-0371, to DLL.