Peptide Synthesis and Handling
Peptides were produced by a commercial provider (American Peptide, Sunnyvale, CA) using solid-phase synthesis. Purity was guaranteed to be more than 95% as determined by HPLC and MS data provided by the manufacturer. A scrambled sequence was generated for SP6001 identified herein as SP6001SCR (PEFEPEIEVEL) as a separate control. For in vitro experiments, peptides were solubilized in 1% dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS) and stored at -20°C. Total DMSO content in all experiments in all wells was held at parity. For in vivo experiments, the peptide was solubilized in 5% DMSO and PBS, and a negative control of 5% DMSO and PBS was injected for tumor studies. Peptide was injected daily at 1 or 5 mg/kg intraperitoneally (i.p.) or subcutaneously (s.c.).
For in vitro experiments, human umbilical vein endothelial cells (HUVECs) were purchased from a commercial provider (Lonza, Walkersville, MD). Cells were passaged at constant ratios, and passages 3 to 6 were used in all experiments according to manufacturer's recommendations. We use the endothelial basal medium-2 and endothelial growth medium-2 bullet kit for cell passage and treatments, purchased from Lonza.
For breast xenograft assays, MDA-MB-231 breast cancer cells were provided by Dr Zaver Bhujwalla's group (JHMI Radiology and Oncology), providing the following details about the cell line: MDA-MB-231 breast cancer cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and used within 6 months of obtaining them from ATCC. The cell line was tested and authenticated by ATCC by two independent methods, the ATCC cytochrome C oxidase I polymerase chain reaction assay and short tandem repeat profiling using multiplex polymerase chain reaction. Cells were propagated in RPMI-1640 medium (Gibco, Carlsbad, CA), containing 10% FBS and 1% penicillin/streptomycin, and grown at 37°C and 5% CO2 under standard conditions.
Cell Adhesion Assay
A real-time cell adhesion assay was completed using a RT-CIMsystem (ACEA Biosciences, Inc, San Diego, CA) and E-plates (Roche, IN), which are 16-well plates suitable for cell culture and contain sensor electrodes. The change in impedance is measured by electrodes and expressed as a cell index, which is proportional to the degree of cell adhesion to the wells. HUVECs were gently trypsinized and plated at a density of 25,000 cells/well in the presence or absence of serpin peptides at 10 and 100 µM. Cells were allowed to settle at room temperature for 30 minutes and then loaded into the RT-CIM system. Values are scaled to percent increase above negative control (complete endothelial cell media) and taken at 1.5 hours. Statistical significance was determined at *P < .05 by Student's t test and compared with the negative control.
Western Blot Analysis
HUVECs were grown in complete endothelial cell media, plated in six-well tissue culture-treated plates at high density (360,000 cells/well), and allowed to adhere for 2 hours. At this point, the medium was removed; fresh endothelial cell media, SP6001 or SP6001SCR (10 µM) with VEGF, or VEGF alone at 20 ng/ml were added, and the cells were incubated at 37°C for 24 hours. The reactions were stopped by removing themedium containing VEGF and adding PBS at 4°C and cell lysis buffer (150 mM NaCl, 1 mM EDTA, 100 µl/ml protease inhibitors [Sigma, St. Louis, MO], 10 µl/ml phosphatase inhibitors [Sigma], and 1% Triton) was added after removing the cold PBS. The cell lysates were spun for 15 minutes at 14,000g to remove cell membranes and debris and were separated by SDS-PAGE; the transferred blots were blocked for 1 hour with 5% milk and 1% bovine serum albumin in Tris-buffered saline and Tween. The 4G10 pan-tyrosine phosphorylation antibody at 1:2000 dilution (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) was added, and the blot was incubated overnight. Secondary antibodies were added the next day at 1:2000 dilution and the phosphorylated protein bands were detected by chemiluminescence detection reagent (GE Healthcare, Little Chalfont, United Kingdom). Blots were stripped and probed with FAK primary antibody (Cell Signaling Technology, Inc, Danvers, MA).
We followed a similar protocol for analysis of pJNK-1. Briefly, SP6001 or SP6001SCR were applied at 1, 10, and 30 µM for 4 hours, and cells were lysed with cell lysis buffer, separated by SDS-PAGE, and probed with pJNK-1 antibody (Cell Signaling Technology, Inc). Blots were then stripped and probed for JNK-1 (Cell Signaling Technology, Inc). Experiments were repeated at least once. The ratio of phospho-protein to total protein was quantified with ImageJ software (National Institutes of Health, Bethesda, MD).
Cell Migration Assay
We used the Oris Pro migration assay (Platypus Technologies, Madison, WI), a modified wound healing assay to assess SP6001 effects on cell migration. Plates are 96-well tissue culture plates that contain stoppers that block the center of the wells. HUVECs were plated at 20,000 cells/well in the presence or absence of peptide SP6001 at 25, 50, or 100 µM and SP6001SCR at 10, 50, or 100 µM in complete endothelial cell media and were allowed to adhere. Two hours later, the stoppers were removed, and the cells were allowed to migrate into the center of the well for 20 hours, at which point cells were stained with calcein AM (Invitrogen, Carlsbad, CA), and fluorescence was measured with a fluorescent Victor V plate reader (Perkin Elmer,Waltham, MA). In some wells, stoppers were removed only after 20 hours to determine background migration, which was negligible.
A Caspase-Glo 3/7 Assay apoptosis detection assay was used to determine whether the peptides were causing endothelial cells to undergo apoptosis (Promega, Madison, WI). Briefly, cells were plated at 5000 cells/well in opaque 96-well plates to minimize well-to-well crosstalk. The next morning, the cells were serum starved for 24 hours by replacing complete medium with serum-free media. A combination of basic fibroblast growth factor (bFGF)/VEGF (30/10 ng/ml) with or without peptide (0.1, 1 or 10 µM) was added at this point. Forty-eight hours later, the Caspase-Glo chemiluminescent substrate (100 µl/well) was added, and luminescence was measured with a Victor V plate reader (Perkin Elmer). A serum-free medium control was used as an apoptosis inducer. Each concentration of peptide was tested in triplicate, and the experiments were repeated three times. The degree of apoptosis in wells containing peptide is compared to only bFGF/VEGF-treated wells. P values for the Student's t test were calculated and considered to be significant if they are P < .01. Experimental values shown are a composite graph of n = 3 independent experiments.
MDA-MB-231 Breast Xenograft Assay
Orthotopic breast xenografts were established in severe combined immunodeficient (SCID) mice using previously established techniques [22
]. Briefly, 2 x 106
cells were injected into the mammary fat pad of animals, and tumors were allowed to grow to a palpable starting size (~100 mm3
), which usually took 2 to 3 weeks. The mice were randomized and grouped into sets of eight for control or 1- or 5-mg/kg peptide treatments. There was no statistical difference in starting values among sets as determined by P
values from Student's t
test. Tumor sizes were determined every fourth day using calipers and applying the formula V = ab2
/2, where a
is the long axis and b
is the short axis of the tumor. The peptides were administered daily i.p. or s.c. into the flank of the animal for a total of 25 days. Statistical significance was determined on termination of the experiment by Student's t
test, and P
values shown compare PBS-treated controls and peptide-treated groups.
Immunohistochemical Staining and Quantification
After the sacrifice of animals on day 25 of the experiment, the tumors were excised and stored in a zinc-based fixative (BD Biosciences, San Jose, CA) for 2 weeks and sent to the JHMI Immunohistochemistry Core Facility where they were centrally cross-sectioned and embedded in paraffin wax. Samples were then sent to Covance, Inc (Princeton, NJ), for immunohistochemical staining for the CD34 antigen. After receipt of images, they were quantified for CD34 staining by FRiDA (free software available from Johns Hopkins University, Baltimore, MD) and measured pixel intensity at 20x magnification/frame. Statistical significance was assessed at **P < .01 by Student's t test. Images shown are magnified at 4x to visually show greater surface area.