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• W1970395087 abstract "Decorin is not only a regulator of matrix assembly but also a key signaling molecule that modulates the activity of tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR). Decorin evokes protracted internalization of the EGFR via a caveolar-mediated endocytosis, which leads to EGFR degradation and attenuation of its signaling pathway. In this study, we tested if systemic delivery of decorin protein core would affect the biology of an orthotopic squamous carcinoma xenograft. After tumor engraftment, the animals were given intraperitoneal injections of either vehicle or decorin protein core (2.5-10 mg kg-1) every 2 days for 18-38 days. This regimen caused a significant and dose-dependent inhibition of the tumor xenograft growth, with a concurrent decrease in mitotic index and a significant increase in apoptosis. Positron emission tomography showed that the metabolic activity of the tumor xenografts was significantly reduced by decorin treatment. Decorin protein core specifically targeted the tumor cells enriched in EGFR and caused a significant down-regulation of EGFR and attenuation of its activity. In vitro studies showed that the uptake of decorin by the A431 cells was rapid and caused a protracted down-regulation of the EGFR to levels similar to those observed in the tumor xenografts. Furthermore, decorin induced apoptosis via activation of caspase-3. This could represent an additional mechanism whereby decorin might influence cell growth and survival. Decorin is not only a regulator of matrix assembly but also a key signaling molecule that modulates the activity of tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR). Decorin evokes protracted internalization of the EGFR via a caveolar-mediated endocytosis, which leads to EGFR degradation and attenuation of its signaling pathway. In this study, we tested if systemic delivery of decorin protein core would affect the biology of an orthotopic squamous carcinoma xenograft. After tumor engraftment, the animals were given intraperitoneal injections of either vehicle or decorin protein core (2.5-10 mg kg-1) every 2 days for 18-38 days. This regimen caused a significant and dose-dependent inhibition of the tumor xenograft growth, with a concurrent decrease in mitotic index and a significant increase in apoptosis. Positron emission tomography showed that the metabolic activity of the tumor xenografts was significantly reduced by decorin treatment. Decorin protein core specifically targeted the tumor cells enriched in EGFR and caused a significant down-regulation of EGFR and attenuation of its activity. In vitro studies showed that the uptake of decorin by the A431 cells was rapid and caused a protracted down-regulation of the EGFR to levels similar to those observed in the tumor xenografts. Furthermore, decorin induced apoptosis via activation of caspase-3. This could represent an additional mechanism whereby decorin might influence cell growth and survival. The growth of human cancer cells is often dependent or facilitated by the overexpression of receptor tyrosine kinase, such as the EGFR, 2The abbreviations used are: EGFR, epidermal growth factor receptor; CT, computerized tomography; PET, positron emission tomography; 18FDG, [18F]fluorodeoxyglucose; TUNEL, transferase dUTP nick end labeling; FACS, fluorescence-activated cell sorter.2The abbreviations used are: EGFR, epidermal growth factor receptor; CT, computerized tomography; PET, positron emission tomography; 18FDG, [18F]fluorodeoxyglucose; TUNEL, transferase dUTP nick end labeling; FACS, fluorescence-activated cell sorter. that provide a growth advantage to the growing and infiltrating neoplasms (1Carpenter G. BioEssays. 2000; 22: 697-707Crossref PubMed Scopus (303) Google Scholar). To prevent the dire consequences of uncontrolled activation of EGFR, a number of negative feedback mechanisms, both extracellular and intracellular, have evolved (2Moghal N. Sternberg P.W. Curr. Opin. Cell Biol. 1999; 11: 190-196Crossref PubMed Scopus (286) Google Scholar, 3Carraway III, K.L. Sweeney C. Curr. Opin. Cell Biol. 2001; 13: 125-130Crossref PubMed Scopus (48) Google Scholar). The prominent role of the EGFR as a crucial relay station among various inputs from the environment and cellular responses has raised the significance of this signaling-transducing receptor to a new level and offers new possibilities for therapeutic intervention (4Mendelsohn J. Baselga J. Oncogene. 2000; 19: 6550-6565Crossref PubMed Scopus (1199) Google Scholar). We have previously shown that decorin, a secreted small leucine-rich proteoglycan (5Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (447) Google Scholar, 6Iozzo R.V. J. Biol. Chem. 1999; 274: 18843-18846Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar), is capable of suppressing the growth of tumor cells with various histogenetic backgrounds (7Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (205) Google Scholar, 8Santra M. Mann D.M. Mercer E.W. Skorski T. Calabretta B. Iozzo R.V. J. Clin. Invest. 1997; 100: 149-157Crossref PubMed Scopus (182) Google Scholar) by directly interacting with the EGFR (9Moscatello D.K. Santra M. Mann D.M. McQuillan D.J. Wong A.J. Iozzo R.V. J. Clin. Invest. 1998; 101: 406-412Crossref PubMed Scopus (241) Google Scholar, 10Iozzo R.V. Moscatello D. McQuillan D.J. Eichstetter I. J. Biol. Chem. 1999; 274: 4489-4492Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 11Santra M. Reed C.C. Iozzo R.V. J. Biol. Chem. 2002; 277: 35671-35681Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Decorin evokes a protracted down-regulation of EGFR tyrosine kinase (12Csordás G. Santra M. Reed C.C. Eichstetter I. McQuillan D.J. Gross D. Nugent M.A. Hajnóczky G. Iozzo R.V. J. Biol. Chem. 2000; 275: 32879-32887Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) and other members of the ErbB family of receptor tyrosine kinase (13Santra M. Eichstetter I. Iozzo R.V. J. Biol. Chem. 2000; 275: 35153-35161Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar) and causes an attenuation of the EGFR-mediated mobilization of intracellular calcium (12Csordás G. Santra M. Reed C.C. Eichstetter I. McQuillan D.J. Gross D. Nugent M.A. Hajnóczky G. Iozzo R.V. J. Biol. Chem. 2000; 275: 32879-32887Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Decorin induces expression of the endogenous cyclin-dependent kinase inhibitor p21WAF1 (14De Luca A. Santra M. Baldi A. Giordano A. Iozzo R.V. J. Biol. Chem. 1996; 271: 18961-18965Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 15Nash M.A. Loercher A.E. Freedman R.S. Cancer Res. 1999; 59: 6192-6196PubMed Google Scholar) and a subsequent arrest of the cells in the G1 phase of the cell cycle (7Santra M. Skorski T. Calabretta B. Lattime E.C. Iozzo R.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7016-7020Crossref PubMed Scopus (205) Google Scholar). These growth-suppressive properties of the soluble decorin and its protein core can also affect murine tumor cells (8Santra M. Mann D.M. Mercer E.W. Skorski T. Calabretta B. Iozzo R.V. J. Clin. Invest. 1997; 100: 149-157Crossref PubMed Scopus (182) Google Scholar) and normal human cells, such as endothelial cells (16Schönherr E. Levkau B. Schaefer L. Kresse H. Walsh K. J. Biol. Chem. 2001; 276: 40687-40692Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and macrophages (17Xaus J. Comalada M. Cardó M. Valledor A.F. Celada A. Blood. 2001; 98: 2124-2133Crossref PubMed Scopus (111) Google Scholar). A number of observations point toward a key role for decorin in the control of cell proliferation. First, decorin expression is markedly induced in most normal diploid cells at quiescence, whereas its expression is absent in most transformed cells (18Iozzo R.V. Murdoch A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (544) Google Scholar, 19Iozzo R.V. Danielson K.G. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 19-53Crossref PubMed Scopus (26) Google Scholar, 20Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1327) Google Scholar, 21Nash M.A. Deavers M.T. Freedman R.S. Clin. Cancer Res. 2002; 8: 1754-1760PubMed Google Scholar). Second, although decorin null animals do not develop spontaneous tumors (22Danielson K.G. Baribault H. Holmes D.F. Graham H. Kadler K.E. Iozzo R.V. J. Cell Biol. 1997; 136: 729-743Crossref PubMed Scopus (1160) Google Scholar), double mutant mice, lacking both decorin and the tumor suppressor gene p53, develop lymphomas at accelerated rates as compared with the p53 null animals, indicating that the absence of decorin is permissive for tumor development (23Iozzo R.V. Chakrani F. Perrotti D. McQuillan D.J. Skorski T. Calabretta B. Eichstetter I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3092-3097Crossref PubMed Scopus (120) Google Scholar). Third, transformation induced by the activating transcription factor-3 and the nuclear vSrc and vJun oncoproteins is associated with a marked suppression of decorin gene expression (24Kolettas E. Rosenberger R.F. Eur. J. Biochem. 1998; 254: 266-274Crossref PubMed Scopus (20) Google Scholar, 25Perez S. Vial E. van Dam H. Castellazzi M. Oncogene. 2001; 20: 1135-1141Crossref PubMed Scopus (53) Google Scholar, 26Vial E. Castellazzi M. Oncogene. 2000; 19: 1772-1782Crossref PubMed Scopus (30) Google Scholar). Fourth, decorin expression is differentially down-regulated in hepatocellular (27Miyasaka Y. Enomoto N. Nagayama K. Izumi N. Marumo F. Watanabe M. Sato C. Br. J. Cancer. 2001; 85: 228-234Crossref PubMed Scopus (84) Google Scholar), lung (28McDoniels-Silvers A.L. Nimri C.F. Stoner G.D. Lubet R.A. You M. Clin. Cancer Res. 2002; 8: 1127-1138PubMed Google Scholar), and ovarian (29Shridhar V. Lee J. Pandita A. Iturria S. Avula R. Staub J. Morrissey M. Calhoun E. Sen A. Kalli K. Keeney G. Roche P. Cliby W. Lu K. Schmandt R. Mills G.B. Bast Jr., R.C. James C.D. Couch F.J. Hartmann L.C. Lillie J. Smith D.I. Cancer Res. 2001; 61: 5895-5904PubMed Google Scholar) carcinomas, and reduced expression of decorin is associated with poor prognosis in invasive breast carcinoma (30Troup S. Njue C. Kliewer E.V. Parisien M. Roskelley C. Chakravarti S. Roughley P.J. Murphy L.C. Watson P.H. Clin. Cancer Res. 2003; 9: 207-214PubMed Google Scholar). Fifth, gene therapy of established tumor xenografts using decorin-expressing adenovirus vectors causes a growth inhibition of various tumors (31Reed C.C. Gauldie J. Iozzo R.V. Oncogene. 2002; 21: 3688-3695Crossref PubMed Scopus (124) Google Scholar, 32Biglari A. Bataille D. Naumann U. Weller M. Zirger J. Castro M.G. Lowenstein P.R. Cancer Gene Ther. 2004; 11: 721-732Crossref PubMed Scopus (65) Google Scholar, 33Tralhão J.G. Schaefer L. Micegova M. Evaristo C. Schönherr E. Kayal S. Veiga-Fernandes H. Danel C. Iozzo R.V. Kresse H. Lemarchand P. FASEB J. 2003; 17: 464-466PubMed Google Scholar) and prevents metastastic spreading of a breast carcinoma orthotopic tumor model (34Reed C.C. Waterhouse A. Kirby S. Kay P. Owens R.A. McQuillan D.J. Iozzo R.V. Oncogene. 2005; 24: 1104-1110Crossref PubMed Scopus (176) Google Scholar). In this study, we tested whether systemic delivery of decorin protein core would affect A431 cells grown as orthotopic skin tumor xenografts. Our results show for the first time that systemic delivery of decorin protein core suppresses in vivo tumorigenicity by specifically targeting EGFR-expressing tumor cells, thereby causing a significant inhibition of tumor metabolism and cell division and concurrent increase in apoptosis. These findings were corroborated by in vitro studies showing that decorin at very low concentrations (∼ 2 nm) caused apoptosis by activating caspase-3. Collectively, these data support a complex mode of action for decorin which culminates in tumor growth suppression and raise the possibility of an efficient protein therapy for cancer. Cell Cultures and Materials—A431 human squamous carcinoma cells were obtained from ATCC (Manassas, VA). Dulbecco's modified Eagle's medium, fetal bovine serum, 100× antibiotic-antimycotic solutions, and Dulbecco's phosphate-buffered saline were purchased from Mediatech (Herndon, VA). Nitrocellulose membrane was purchased from Bio-Rad. Antibodies include polyclonal rabbit antibodies against active-caspase-3 (Pharmingen), C terminus of perlecan (35Bix G. Fu J. Gonzalez E. Macro L. Barker A. Campbell S. Zutter M.M. Santoro S.A. Kim J.K. Höök M. Reed C.C. Iozzo R.V. J. Cell Biol. 2004; 166: 97-109Crossref PubMed Scopus (230) Google Scholar), EGFR (sc-03, Santa Cruz Biotechnology, Santa Cruz, CA), EGFR Tyr1068 (Cell Signaling Technology, Beverly, MA), and polyclonal goat recognizing decorin (Oncogene, San Diego, CA). Monoclonal antibodies recognizing the His tag (Calbiochem), β-actin (Sigma), Ki67 (DAKO, Carpinteria, CA), Tyr(P) (PY20, BD Bioscience), and EGFR (Ab-12; Neomarkers) were also used. Rhodamine- and fluorescein isothiocyanate-conjugated anti-mouse, anti-rabbit, and anti-goat IgG were purchased from Santa Cruz Biotechnology. horseradish peroxidase-conjugated secondary antibodies against mouse and rabbit were purchased from Amersham Biosciences and goat from Calbiochem. Super Signal West Pico chemiluminescence substrate was purchased from Pierce. Animal Experiments and A431 Orthotopic Tumor Xenografts— Animal experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee of Thomas Jefferson University. Orthotopic tumor xenografts were established as described previously (31Reed C.C. Gauldie J. Iozzo R.V. Oncogene. 2002; 21: 3688-3695Crossref PubMed Scopus (124) Google Scholar). Purification and characterization of biologically active decorin protein core was described before (36Ramamurthy P. Hocking A.M. McQuillan D.J. J. Biol. Chem. 1996; 271: 19578-19584Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Immunocompromized nu/nu mice (Charles River Laboratories) were examined every day until 2-3-mm tumors were visible. Tumor xenografts were measured (31Reed C.C. Gauldie J. Iozzo R.V. Oncogene. 2002; 21: 3688-3695Crossref PubMed Scopus (124) Google Scholar) and treated every other day by intraperitoneal injections of 60-200 μg of decorin protein core (2.5-10 mg kg-1) per animal for 18-38 days. Animals were sacrificed at the end of the experiment and the tumor, spleen, liver, lungs, kidneys, and heart were dissected. Tumors and organs were snap-frozen in liquid N2 and used for either immunohistochemical or biochemical analysis. Positron Emission Tomography (PET) and Computerized Tomography (CT)—PET studies of control and decorin-treated animals were performed using the MOSAIC PET scanner (Philips Medical Systems). The scanner used 2 × 2 x 10-mm3 gadolinium oxy-orthosilicate crystals coupled to 19-mm diameter photomultiplier tubes via a continuous slotted light guide. The detectors were arranged to produce a transaxial field of view of 128 mm and an axial field of view of 120 mm. The absolute coincidence sensitivity was 1.3% for a point source, and the transverse resolution was 2.2 mm at full-width-half-maximum. Images were reconstructed into 0.5 × 0.5 × 0.5-mm3 voxels using a three-dimensional RAMLA algorithm supplied with the camera. CT studies were performed using the MicroCATII CT scanner (Imtek Inc.). A 70 × 100-mm phosphor screen was optically coupled to a 2048 × 3072-pixel CCD camera. The x-ray source and detector were rotated around the subject to produce a transaxial field of view of 51.2 mm and an axial field of view of 76.8 mm. X-rays were generated at 80 kV peak and 500 μA. Images were reconstructed into 0.2 × 0.2 × 0.2-mm3 voxels using a Feldkamp cone beam reconstruction algorithm. To perform PET and CT imaging, mice were injected with 0.4-0.5 mCi of [18F]flourodeoxyglucose (18FDG) and allowed 2 h for tracer distribution. Just prior to imaging, mice were anesthetized with an injection of Ketamine, Xylazine, Acetopromazine (200, 10, 2 mg kg-1) via an intraperitoneal injection and placed in a 50-ml tube to facilitate multimodality stereo tactic positioning. PET data were acquired in a single position for 15 min followed by CT data acquisition for 5 min. The images were registered with an internally developed automated mutual information rigid registration algorithm. Volumes of interest were defined by drawing multislice regions of interest on the PET images using 50% of the full-width-half-maximum of the tumor to determine the tumor boundary. PET regions were also defined on contralateral soft tissues and compared with the CT images where necessary. The images were normalized on the average uptake of contralateral abdominal regions. Immunofluorescence Microscopy and Quantification—Frozen sections were dried for 1 h and fixed in ice-cold acetone for 5 min. After washing, the sections were blocked for 18 h with 5% (w/v) bovine serum albumin/phosphate-buffered saline at 4 °C and subsequently subjected to standard immunofluorescence protocols with various antibodies, co-stained with 4′,6-diamidino-2-phenylindole, and mounted with Vectashield medium (Vector Laboratories, Inc., Burlingame, CA). Images were acquired using an Olympus BX51 microscope driven by SPOT advanced version 4.0.9 imaging software (Diagnostic Instruments, Inc.). To quantify the fluorescence of EGFR in sections of tumor xenografts, control sections were analyzed at different exposure times and gain settings and converted into grayscale (37Zhu J.-X. Goldoni S. Bix G. Owens R.A. McQuillan D. Reed C.C. Iozzo R.V. J. Biol. Chem. 2005; 280: 32468-32479Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The distribution of the pixel intensity was studied using the histogram function of Adobe Photoshop® 7.0 (Adobe Systems Inc., San Jose, CA). The final adjustment was an exposure time of 50 ms, and a camera gain of 8, which records images not saturated in fluorescence intensity. Digital images for control (n = 11) and decorin-treated (n = 8) tumors were acquired. For fluorescence visualization, RGD color images were converted to 8-bit grayscale images, and three-dimensional surface plot analyses were generated with the Surface Plot function of ImageJ 1.34 (http://rsb.info.nih.gov/ij) to show the intensity of the representative fluorescent signals (38Parsons-Wingerter P. Kasman I.M. Norberg S. Magnussen A. Zanivan S. Rissone A. Baluk P. Favre C.J. Jeffry U. Murray R. McDonald D.M. Am. J. Pathol. 2005; 167: 193-211Abstract Full Text Full Text PDF PubMed Google Scholar). Determination of Apoptosis and Mitotic Index in Tumor Xenografts—To analyze apoptosis we used two approaches; the TUNEL assay (BD Bioscience), which labels internucleosomal DNA fragmentation and the detection of the protein active caspase-3 on frozen sections. Fluorescence signal of TUNEL staining and active caspase-3 was detected and quantified as described above. To determine the proliferative (mitotic) index, frozen sections of tumor xenografts were stained for the proliferation-associated marker Ki67 and by collecting the total pixel density of 41 individual (200×) fields (20 for control and 21 for decorin-treated tumors) from 8 mice. Pulse-Chase and Dose-Response Experiments—For the pulsechase experiment, confluent A431 cells were serum-starved for 18 h, pulsed with 30 μg ml-1 decorin protein core for 30 min at 37 °C, washed on ice, and then chased with serum-free medium for various time points (0.5, 1, 2, 3, 4, and 6 h) at 37 °C. For the dose-response experiment, confluent A431 cells were serumstarved for 18 h and pulsed with various concentrations (0, 1, 5, 10, and 30 μg ml-1) of decorin protein core. After 30 min, cells were washed with ice-cold phosphate-buffered saline and chased for 120 min with serum-free medium. Cells were harvested, and lysates were subjected to immunoblotting using specific antibodies described above. Cells were analyzed for EGFR, β-actin, and PY20-horseradish peroxidase total phosphorylation. Several x-ray films were analyzed to determine the linear range of the chemiluminescence signals and were subsequently quantified with Scion Image alpha 4.0.3.2 and normalized on β-actin. DNA Fragmentation Analysis by FACS and Active Caspase-3—Approximately 0.4 × 106 A431 cells were seeded in 6-cm dishes, cultured under standard conditions overnight, and then treated for 24 h with different concentrations of decorin protein core (0.1 ng to 90 μg ml-1) and 200 ng ml-1 EGF in medium containing 10% fetal bovine serum. As positive controls, cells were treated with etoposide (5 and 10 μm), a topoisomerase-II inhibitor and an established cell cycle-specific DNA-damaging agent (39Hande K.R. Eur. J. Cancer. 1998; 34: 1514-1521Abstract Full Text Full Text PDF PubMed Scopus (770) Google Scholar). Prior to DNA fragmentation analysis, the cells were trypsinized and fixed with 80% ethanol at 4 °C for 1 h, washed twice with phosphate-buffered saline, and resuspended in 50 μg ml-1 propidium iodide solution containing 0.5 μg ml-1 RNase A (40Krishan A. J. Cell Biol. 1975; 66: 188-193Crossref PubMed Scopus (1483) Google Scholar). Cells were stained for 3 h at 4°C and DNA fragmentation was analyzed by flow cytometry using an Epics XL-MCL (Beckman Coulter). To evaluate if the kinase activity of EGFR was required for apoptosis, cells were treated with 30 μg ml-1 decorin protein core and 1 μm AG1478 and processed as described above. To corroborate the results obtained for A431 cells, HeLa cells were also used under the same conditions. Active caspase-3 activity was measured with the Caspase-Glo™ 3/7 kit (Promega, Madison, WI), a luminescence assay that measures caspase-3 and -7 activities. It is a mixture of a luminogenic substrate that contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity, luciferase activity, and cell lysis. Following caspase cleavage, a substrate for luciferase is released, resulting in the luciferase reaction and the production of light that is proportional to the amount of caspase activity present. Approximately 5 × 103 cells were cultured in 96-well plates for 18 h, following a 24-h dose-dependent treatment with decorin protein core (0.1, 1, 5, 10, 30, and 90 μg ml-1) or 200 ng ml-1 EGF in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Both fragmentation and caspase-3 activation experiments were also performed in the presence of the pan-caspase inhibitor VAD (Biovison, Mountain View, CA), which preferentially blocks caspases-1-3 (41Talanian R.V. Quinlan C. Trautz S. Hackett M.C. Mankovich J.A. Banach D. Ghayur T. Brady K.D. Wong W.W. J. Biol. Chem. 1997; 272: 9677-9682Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar). Prior to each analysis, the culture medium was supplemented with 50 μl of substrate solution and incubated for 1 h at 25 °C. Luminescence measurements were carried out with a microplate reader 1420 Victor3 (PerkinElmer Life Sciences). Results are given as means (n = 3-5) with three independent measurements for each group. Statistical evaluation was done with an unpaired Student's t test using the Sigma Plot 9 statistical package. p < 0.05 was considered as significant. Growth Inhibition of Tumor Xenografts by Systemic Delivery of Human Recombinant Decorin Protein Core—Decorin protein core was purified to homogeneity from the secretions of human embryonic kidney 293 cells and migrated as a monomer of 46-48 kDa on a silver-stained SDS gel (42Goldoni S. Owens R.T. McQuillan D.J. Shriver Z. Sasisekharan R. Birk D.E. Campbell S. Iozzo R.V. J. Biol. Chem. 2004; 279: 6606-6612Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Before injecting into the animals, each batch of filtered decorin protein core was tested for biological activity by determining the suppression of basal EGFR phosphorylation in quiescent A431 cells (37Zhu J.-X. Goldoni S. Bix G. Owens R.A. McQuillan D. Reed C.C. Iozzo R.V. J. Biol. Chem. 2005; 280: 32468-32479Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). We evaluated the effects of systemic delivery of recombinant decorin protein core on the growth kinetics of established orthotopic A431 squamous cell carcinoma xenografts. The results showed that at low dosages of decorin protein core (4.5 mg kg-1), there was a significant (p = 0.025) growth inhibition of the tumor xenografts in two independent experiments (Fig. 1A). We performed a total of four in vivo experiments utilizing 40 animals, of which four were excluded because there was no tumor engraftment. The total amount of decorin protein core injected in each mouse was 540 μg. The basis for the size difference at day 19 was primarily due to a growth rate advantage, since doubling time for the vehicle-treated tumors was significantly shorter than that of the decorin-treated animals (2.5 versus 3.75 days, respectively) (Fig. 1B). Similar growth kinetic was observed with another independent experiment using the same regimen with the exception of lower (2.5 mg kg-1) decorin protein core dosages (data not shown). Interestingly, when the dosage was increased to 10 mg kg-1, there was a greater growth inhibition (p < 0.001) that lasted for up to 38 days (Fig. 1C). In this case, a total amount of 3.8 mg of decorin protein core was injected in each mouse. While in the first 19 days of treatment the tumor doubling times were similar to those obtained with lower decorin dosages, the doubling time of the decorin-treated tumors was much greater (9 days versus 2.5 days) at a later time. Thus, these results indicate that systemic delivery of decorin protein core retards the growth of established orthotopic tumor xenografts in a dose-dependent manner. Next, we determined whether decorin could inhibit in vivo tumor growth by affecting tumor metabolism. To this end, several animals from separate experiments were analyzed by CT and PET scan. This strategy allows for direct visualization and quantification of tissue metabolic activity via the administration of a radioactive sugar, 18FDG, which proportionally distributes to metabolically active tissue. Animals were imaged toward the end of each experimental protocol to allow for maximal tumor visibility in control and treated groups. The results showed a marked inhibition of 18FDG uptake in the decorin-treated tumor xenografts as compared with controls (Fig. 1D). PET scans were carried out using 200-450 μCi of 18FDG at different time points (16, 18, 34, and 36 days) including animals receiving both low and high dosages of decorin protein core, and in all cases there was a significant reduction in metabolic activity. Quantification of a mixture of animals (n = 10) by normalizing the maximal signal in each tumor to that in the abdomen of each animal showed a significantly reduced (p = 0.016) tumor metabolic rate (Fig. 1E). These findings were not due to differences in tumor “size” since we found a significant decrease in tumor metabolism even when comparing similar size tumors. Tumor identification and volumes were verified by concurrent CT scanning, and these values supported the results obtained by manual measurements (data not shown). Therefore, PET scan image analysis indicates that tumor growth inhibition evoked by decorin treatment is at least in part due to reduced tumor metabolic rate. Decorin Targets the Cancer Cells within the Orthotopic Tumor Xenografts—The central hypothesis of our research is that the functional receptor for decorin, i.e. the EGFR, needs to be present within a tumor cell population for decorin binding and suppression of the known oncogenic activity of the receptor. Having established that systemic delivery of decorin retards in vivo tumor growth, we investigated whether decorin would specifically target the EGFR-overproducing tumor cells. Using fluorescence microscopy and an anti-His antibody, which recognizes the N-terminal His tag on decorin, we discovered that decorin protein core specifically targets the tumor cells (Fig. 2). Specifically, decorin epitopes could be detected in the tumor cells proximal to the blood vessels (Fig. 2, B-D), with intervening areas lacking any reactivity. Decorin epitopes were patchy and primarily associated with the cell surface of the A431 squamous carcinoma cells (Fig. 2D), with a distribution similar to the EGFR. The gradient of the fluorescence signal obtained from staining with anti-His suggests that decorin diffuses through the vascular beds and specifically targets the tumor cells. In contrast, very little or no decorin was associated with normal organs, such as spleen and liver (see supplemental Fig. 1). Because decorin has been previously reported to inhibit in vitro angiogenesis by either interacting with thrombospondin-1 (43de Lange Daviesqq C. Melder R.J. Munn L.L. Mouta-Carreira C. Jain R.K. Boucher Y. Microvasc.Res. 2001; 62: 26-42Crossref PubMed Scopus (89) Google Scholar) or by reducing the endogenous tumor levels of VEGF (44Grant D.S. Yenisey C. Rose R.W. Tootell M. Santra M. Iozzo R.V. Oncogene. 2002; 21: 4765-4777Crossref PubMed Scopus (182) Google Scholar), which is also achieved by neutralizing antibodies against the EGFR (45Petit A.M.V. Rak J. Hung M.-C. Rockwell P. Goldstein N. Fendly B. Kerbel R.S. Am. J. Pathol. 1997; 151: 1523-1530PubMed Google Scholar), we quantitatively analyzed the blood vessel density in control and decorin-treated tumor xenografts utilizing an antibody directed toward the C terminus of perlecan (35Bix G. Fu J. Gonzalez E. Macro L. Barker A. Campbell S. Zutter M.M. Santoro S.A. Kim J.K. Höök M. Reed C.C. Iozzo R.V. J. Cell Biol. 2004; 166: 97-109Crossref PubMed Scopus (230) Google Scholar). The tumor blood vessels were specifically labeled by" @default.
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• W1970395087 date "2006-09-01" @default.
• W1970395087 modified "2023-10-18" @default.
• W1970395087 title "Decorin Protein Core Inhibits in Vivo Cancer Growth and Metabolism by Hindering Epidermal Growth Factor Receptor Function and Triggering Apoptosis via Caspase-3 Activation" @default.
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