Bone Marrow-Derived Cathepsin K Cleaves SPARC in Bone Metastasis
2009; Elsevier BV; Volume: 175; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2009.080906
ISSN1525-2191
AutoresIzabela Podgorski, Bruce E. Linebaugh, Jennifer E. Koblinski, Deborah Rudy, Mackenzie K. Herroon, Mary B. Olive, Bonnie F. Sloane,
Tópico(s)Bone Metabolism and Diseases
ResumoBone metastasis is a hallmark of advanced prostate and breast cancers, yet the critical factors behind attraction of tumors to the skeleton have not been validated. Here, we investigated the involvement of cathepsin K in the progression of prostate tumors in the bone, which occurs both by direct degradation of bone matrix collagen I and by cleavage of other factors in the bone microenvironment. Our results demonstrated that bone marrow-derived cathepsin K is capable of processing and thereby modulating SPARC, a protein implicated in bone metastasis and inflammation. The coincident up-regulation of SPARC and cathepsin K occurred both in vivo in experimental prostate bone tumors, and in vitro in co-cultures of bone marrow stromal cells with PC3 prostate carcinoma cells. PC3-bone marrow stromal cell interaction increased secretion and processing of SPARC, as did co-cultures of bone marrow stromal cells with two other cancer cell lines. In addition, bone marrow stromal cells that were either deficient in cathepsin K or treated with cathepsin K inhibitors had significantly reduced secretion and cleavage of SPARC. Increases in secretion of pro-inflammatory cytokines (ie, interleukin-6, -8) coincident with overexpression of cathepsin K suggest possible mechanisms by which this enzyme contributes to tumor progression in the bone. This is the first study implicating bone marrow cathepsin K in regulation of biological activity of SPARC in bone metastasis. Bone metastasis is a hallmark of advanced prostate and breast cancers, yet the critical factors behind attraction of tumors to the skeleton have not been validated. Here, we investigated the involvement of cathepsin K in the progression of prostate tumors in the bone, which occurs both by direct degradation of bone matrix collagen I and by cleavage of other factors in the bone microenvironment. Our results demonstrated that bone marrow-derived cathepsin K is capable of processing and thereby modulating SPARC, a protein implicated in bone metastasis and inflammation. The coincident up-regulation of SPARC and cathepsin K occurred both in vivo in experimental prostate bone tumors, and in vitro in co-cultures of bone marrow stromal cells with PC3 prostate carcinoma cells. PC3-bone marrow stromal cell interaction increased secretion and processing of SPARC, as did co-cultures of bone marrow stromal cells with two other cancer cell lines. In addition, bone marrow stromal cells that were either deficient in cathepsin K or treated with cathepsin K inhibitors had significantly reduced secretion and cleavage of SPARC. Increases in secretion of pro-inflammatory cytokines (ie, interleukin-6, -8) coincident with overexpression of cathepsin K suggest possible mechanisms by which this enzyme contributes to tumor progression in the bone. This is the first study implicating bone marrow cathepsin K in regulation of biological activity of SPARC in bone metastasis. Prostate and breast cancers commonly metastasize to skeletal sites and locally disrupt normal bone remodeling. Despite recent progress in cancer detection and treatment, it remains unclear which skeletal-specific factors are among the critical determinants in preferential localization of metastatic cells to the bone. Recent clinical and experimental data suggest that accelerated bone remodeling may be responsible for homing of tumor cells to the bone.1Guise TA Mohammad KS Clines G Stebbins EG Wong DH Higgins LS Vessella R Corey E Padalecki S Suva L Chirgwin JM Basic mechanisms responsible for osteolytic and osteoblastic bone metastases.Clin Cancer Res. 2006; 12: 6213s-6216sCrossref PubMed Scopus (421) Google Scholar, 2Schneider A Kalikin LM Mattos AC Keller ET Allen MJ Pienta KJ McCauley LK Bone turnover mediates preferential localization of prostate cancer in the skeleton.Endocrinology. 2005; 146: 1727-1736Crossref PubMed Scopus (161) Google Scholar This is evidenced by the increased metastasis in response to experimental treatment with calciotropic hormone or to androgen ablation1Guise TA Mohammad KS Clines G Stebbins EG Wong DH Higgins LS Vessella R Corey E Padalecki S Suva L Chirgwin JM Basic mechanisms responsible for osteolytic and osteoblastic bone metastases.Clin Cancer Res. 2006; 12: 6213s-6216sCrossref PubMed Scopus (421) Google Scholar, 2Schneider A Kalikin LM Mattos AC Keller ET Allen MJ Pienta KJ McCauley LK Bone turnover mediates preferential localization of prostate cancer in the skeleton.Endocrinology. 2005; 146: 1727-1736Crossref PubMed Scopus (161) Google Scholar and reduced incidence of metastasis with antiresorptive therapies.3Gnant M Mlineritsch B Schippinger W Luschin-Ebengreuth G Postlberger S Menzel C Jakesz R Seifert M Hubalek M Bjelic-Radisic V Samonigg H Tausch C Eidtmann H Steger G Kwasny W Dubsky P Fridrik M Fitzal F Stierer M Rucklinger E Greil R Marth C Endocrine therapy plus zoledronic acid in premenopausal breast cancer.N Engl J Med. 2009; 360: 679-691Crossref PubMed Scopus (939) Google Scholar Establishment of tumors in bone requires multidirectional interactions between tumor cells, bone cells, stromal cells, and inflammatory components, as well as extracellular matrix proteins. This complex interplay between tumor cells and the bone microenvironment facilitates increased bone turnover and promotes tumor cell survival. The key enzyme responsible for osteolysis of bone is the cysteine protease cathepsin K, which is the only known mammalian protease capable of degrading both the helical and non-helical regions of collagen I, the main component of the organic bone matrix.4Garnero P Buchs N Zekri J Rizzoli R Coleman RE Delmas PD Markers of bone turnover for the management of patients with bone metastases from prostate cancer.Br J Cancer. 2000; 82: 858-864Crossref PubMed Scopus (221) Google Scholar Within the bone microenvironment, cathepsin K localizes predominantly to osteoclasts and its overexpression results in increased bone turnover.5Kiviranta R Morko J Uusitalo H Aro HT Vuorio E Rantakokko J Accelerated turnover of metaphyseal trabecular bone in mice overexpressing cathepsin K.J Bone Miner Res. 2001; 16: 1444-1452Crossref PubMed Scopus (120) Google Scholar Accordingly, a deficiency in this potent collagenase results in a bone-sclerosing disorder called pycnodysostosis in man and osteopetrosis in mice.6Gelb BD Shi GP Chapman HA Desnick RJ Pycnodysostosis, a lysosomal disease caused by cathepsin K deficiency.Science. 1996; 273: 1236-1238Crossref PubMed Scopus (857) Google Scholar, 7Saftig P Hunziker E Wehmeyer O Jones S Boyde A Rommerskirch W Moritz JD Schu P von Figura K Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice.Proc Natl Acad Sci USA. 1998; 95: 13453-13458Crossref PubMed Scopus (758) Google Scholar The presence of cathepsin K has been demonstrated in many malignancies including prostate and breast cancers, both of which have a high propensity to metastasize to bone.8Brubaker KD Vessella RL True LD Thomas R Corey E Cathepsin K mRNA and protein expression in prostate cancer progression.J Bone Miner Res. 2003; 18: 222-230Crossref PubMed Scopus (146) Google Scholar, 9Littlewood-Evans AJ Bilbe G Bowler WB Farley D Wlodarski B Kokubo T Inaoka T Sloane J Evans DB Gallagher JA The osteoclast-associated protease cathepsin K is expressed in human breast carcinoma.Cancer Res. 1997; 57: 5386-5390PubMed Google Scholar, 10Le Gall C Bellahcene A Bonnelye E Gasser JA Castronovo V Green J Zimmermann J Clezardin P A cathepsin K inhibitor reduces breast cancer induced osteolysis and skeletal tumor burden.Cancer Res. 2007; 67: 9894-9902Crossref PubMed Scopus (169) Google Scholar A role for cathepsin K in advanced cancers has been attributed mainly to its ability to degrade native collagen I and facilitate the expansion of tumors in the bone. Our recent studies and data by other groups suggest that cathepsin K also cleaves and thereby modulates the biological activity of several important proteins in the bone microenvironment.11Bossard MJ Tomaszek TA Thompson SK Amegadzie BY Hanning CR Jones C Kurdyla JT McNulty DE Drake FH Gowen M Levy MA Proteolytic activity of human osteoclast cathepsin K. 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In the vast majority of human cancers, SPARC is a marker of aggressiveness and poor prognosis; however, SPARC is also anti-tumorigenic, suggesting multiple roles for this protein in tumor growth and progression.17Podhajcer OL Benedetti L Girotti MR Prada F Salvatierra E Llera AS The role of the matricellular protein SPARC in the dynamic interaction between the tumor and the host.Cancer Metastasis Rev. 2008; 27: 523-537Crossref PubMed Scopus (143) Google Scholar, 18Clark CJ Sage EH A prototypic matricellular protein in the tumor microenvironment–where there's SPARC, there's fire.J Cell Biochem. 2008; 104: 721-732Crossref PubMed Scopus (107) Google Scholar, 19Framson PE Sage EH SPARC and tumor growth: where the seed meets the soil?.J Cell Biochem. 2004; 92: 679-690Crossref PubMed Scopus (223) Google Scholar, 20Koblinski JE Kaplan-Singer BR VanOsdol SJ Wu M Engbring JA Wang S Goldsmith CM Piper JT Vostal JG Harms JF Welch DR Kleinman HK Endogenous osteonectin/SPARC/BM-40 expression inhibits MDA-MB-231 breast cancer cell metastasis.Cancer Res. 2005; 65: 7370-7377Crossref PubMed Scopus (98) Google Scholar Increased levels of SPARC in prostate cancer have been correlated with an invasive phenotype and suggested to facilitate homing of tumor cells to the bone.21De S Chen J Narizhneva NV Heston W Brainard J Sage EH Byzova TV Molecular pathway for cancer metastasis to bone.J Biol Chem. 2003; 278: 39044-39050Crossref PubMed Scopus (129) Google Scholar, 22Jacob K Webber M Benayahu D Kleinman HK Osteonectin promotes prostate cancer cell migration and invasion: a possible mechanism for metastasis to bone.Cancer Res. 1999; 59: 4453-4457PubMed Google Scholar Several proteases, including cathepsin K, have been shown to cleave SPARC in vitro,11Bossard MJ Tomaszek TA Thompson SK Amegadzie BY Hanning CR Jones C Kurdyla JT McNulty DE Drake FH Gowen M Levy MA Proteolytic activity of human osteoclast cathepsin K. Expression, purification, activation, and substrate identification.J Biol Chem. 1996; 271: 12517-12524Crossref PubMed Scopus (445) Google Scholar, 23Motamed K SPARC (osteonectin/BM-40).Int J Biochem Cell Biol. 1999; 31: 1363-1366Crossref PubMed Scopus (94) Google Scholar, 24Sasaki T Gohring W Mann K Maurer P Hohenester E Knauper V Murphy G Timpl R Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increases its affinity for collagens.J Biol Chem. 1997; 272: 9237-9243Crossref PubMed Scopus (142) Google Scholar a process that gives rise to smaller peptides with higher affinity for collagens and presumably basement membrane.24Sasaki T Gohring W Mann K Maurer P Hohenester E Knauper V Murphy G Timpl R Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increases its affinity for collagens.J Biol Chem. 1997; 272: 9237-9243Crossref PubMed Scopus (142) Google Scholar Proteolytic activation of SPARC not only enhances its binding affinity, which might be essential for matrix storage, but also leads to the release of biologically active cleavage products.25Tai IT Tang MJ SPARC in cancer biology: its role in cancer progression and potential for therapy.Drug Resist Updat. 2008; 11: 231-246Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar These bio-peptides regulate several growth factors including vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factor-2, and ultimately contribute to the enhancement of tumor-associated angiogenesis.26Lane TF Iruela-Arispe ML Johnson RS Sage EH SPARC is a source of copper-binding peptides that stimulate angiogenesis.J Cell Biol. 1994; 125: 929-943Crossref PubMed Scopus (224) Google Scholar Expression of SPARC is often coincident with induction of matrix-degrading enzymes, in particular, matrix metalloproteinases (MMPs) 1, 2, 3, and 9, and MT1-MMP, a process leading to facilitation of extracellular matrix remodeling and induction of inflammatory responses.17Podhajcer OL Benedetti L Girotti MR Prada F Salvatierra E Llera AS The role of the matricellular protein SPARC in the dynamic interaction between the tumor and the host.Cancer Metastasis Rev. 2008; 27: 523-537Crossref PubMed Scopus (143) Google Scholar, 27Shankavaram UT DeWitt DL Funk SE Sage EH Wahl LM Regulation of human monocyte matrix metalloproteinases by SPARC.J Cell Physiol. 1997; 173: 327-334Crossref PubMed Scopus (100) Google Scholar, 28McClung HM Thomas SL Osenkowski P Toth M Menon P Raz A Fridman R Rempel SA SPARC upregulates MT1-MMP expression. MMP-2 activation, and the secretion and cleavage of galectin-3 in U87MG glioma cells.Neurosci Lett. 2007; 419: 172-177Crossref PubMed Scopus (70) Google Scholar Here, we demonstrated coincident up-regulation of SPARC and the bone-resorbing enzyme cathepsin K both in vivo in experimental prostate bone tumors and in vitro in co-cultures of bone marrow stromal cells (BMSC) with prostate and breast carcinoma cells. The involvement of cathepsin K in cleavage and processing of SPARC was validated by the use of a selective cathepsin K inhibitor and cathepsin K-deficient bone marrow stromal cells. Overexpression of cathepsin K coincident with changes in pro-inflammatory cytokines was demonstrated by human cytokine antibody arrays and further confirmed by cathepsin K inhibition – a result suggesting possible mechanisms by which this enzyme contributes to tumor progression in the bone. Dulbecco’s modified Eagle’s medium, 2-[N-morpholino] ethane-sulfonic acid, piperazine-NVNV-bis[2-ethanesulfonic acid], Hanks’ salt solution, sodium bicarbonate, antibiotics, dimethyl sulfoxide, paraformaldehyde, the broad spectrum cysteine protease inhibitor E-64, a human monoclonal β-actin antibody, a human monoclonal cytokeratin antibody (clone C-11+PCK-26+CY-90+KS-1A3+M20+A53-B/A2) and other chemicals, unless otherwise stated, were obtained from Sigma (St. Louis, MO). Fetal bovine serum, trypsin-EDTA and collagenase were obtained from Invitrogen (Carlsbad, CA). Vitrogen-100 collagen type I was from Cohesion Laboratories (Palo Alto, CA). The fluorogenic substrate Z-Gly-Pro-Arg-NHMec and its cleavage product NH2Mec were purchased from Bachem (King of Prussia, PA). CA074 was purchased from Peptides International (Louisville, KY). Cathepsin K inhibitor L-87372429Li CS Deschenes D Desmarais S Falgueyret JP Gauthier JY Kimmel DB Leger S Masse F McGrath ME McKay DJ Percival MD Riendeau D Rodan SB Therien M Truong VL Wesolowski G Zamboni R Black WC Identification of a potent and selective non-basic cathepsin K inhibitor.Bioorg Med Chem Lett. 2006; 16: 1985-1989Crossref PubMed Scopus (84) Google Scholar was obtained from (Merck-Frosst, Canada). Mouse anti-human cathepsin K antibodies were purchased from Novocastra (Newcastle, UK). Mouse anti-human SPARC antibodies were purchased from Hematological Technologies (Essex Junction, VT). Mouse anti-human tartrate resistant acid phosphatase (TRAcP) antibodies were from Zymed Laboratories, and mouse anti-vascular endothelial growth factor, rabbit anti-α-smooth muscle actin, and rabbit anti-vimentin antibodies were from Abcam (Cambridge, MA); rabbit anti-CD68 antibodies were from R&D Systems (Minneapolis, MN) and mouse anti-CD31 antibodies were from Dako (Carpinteria, CA). Mouse anti-human interleukin (IL)-6, goat anti-human IL-8 antibodies, and goat anti-human receptor activator for nuclear factor κ-B ligand (RANKL) neutralizing antibodies (AF626) were from R&D Systems (Minneapolis, MN). Alexa Fluor 488- and Alexa Fluor 564-conjugated donkey anti-mouse and anti-rabbit IgG were from Molecular Probes (Eugene, OR) and normal donkey serum was obtained from Jackson ImmunoResearch (West Grove, PA). Horseradish peroxidase-labeled goat anti-rabbit IgG and Micro BCA protein kits were purchased from Pierce (Rockford, IL). RayBio Human Cytokine Antibody Arrays V were purchased from RayBiotech (Norcross, GA). Western blotting detection kits were from Amersham Pharmacia Biotechnologies (Piscataway, NJ). The Vectastain Elite ABC immunohistochemistry kit and NovaRED kit for peroxidase were purchased from Vector Laboratories, (Burlingame, CA). Peptides for polyclonal antibody production were designed according to SPARC cleavage fragments reported by Sasaki et al,24Sasaki T Gohring W Mann K Maurer P Hohenester E Knauper V Murphy G Timpl R Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increases its affinity for collagens.J Biol Chem. 1997; 272: 9237-9243Crossref PubMed Scopus (142) Google Scholar using the antigenic search website http://mobyle.pasteur.fr/cgi-bin/portal.py?form=antigenic. last accessed July 31, 2002). The peptides were synthesized at the Federal Drug Administration (Bethesda, MD). Rabbit anti-human antibodies were made at Covance (Denver, PA). PC3, an androgen-independent osteolytic line derived from a bone metastasis of a high-grade adenocarcinoma,30Kaighn ME Narayan KS Ohnuki Y Lechner JF Jones LW Establishment and characterization of a human prostatic carcinoma cell line (PC-3).Invest Urol. 1979; 17: 16-23PubMed Google Scholar and DU145, an androgen-independent osteolytic line derived from a brain metastasis31Stone KR Mickey DD Wunderli H Mickey GH Paulson DF Isolation of a human prostate carcinoma cell line (DU 145).Int J Cancer. 1978; 21: 274-281Crossref PubMed Scopus (1010) Google Scholar were purchased from American Type Culture Collection (Manassas, VA). The human prostate cancer C4-2B cell line is a derivative of the LNCaP cell line32Thalmann GN Sikes RA Wu TT Degeorges A Chang SM Ozen M Pathak S Chung LW LNCaP progression model of human prostate cancer: androgen-independence and osseous metastasis.Prostate. 2000; 44: 91-103Crossref PubMed Scopus (292) Google Scholar and was kindly provided by Dr. Leland W. K. Chung, Emory University, Atlanta, Georgia. The MDA-231BO is a bone-seeking clone derived from MDA MB-231 breast carcinoma cells33Yoneda T Williams PJ Hiraga T Niewolna M Nishimura R A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in vitro.J Bone Miner Res. 2001; 16: 1486-1495Crossref PubMed Scopus (365) Google Scholar and was kindly provided by Dr. Toshiyuki Yoneda (University of Texas Health Science Center, San Antonio, TX). All cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and maintained in a 37°C humidified incubator ventilated with 5% CO2. For derivation of human bone marrow stromal cells (hBMSC) and for in vivo tumor implantation, human male fetal femurs (16 to 19 weeks gestation) were purchased from Advanced Bioscience Resources (Alameda, CA) as previously described.34Podgorski I Linebaugh BE Sameni M Jedeszko C Bhagat S Cher ML Sloane BF Bone microenvironment modulates expression and activity of cathepsin B in prostate cancer.Neoplasia. 2005; 7: 207-223Abstract Full Text PDF PubMed Scopus (59) Google Scholar Primary hBMSCs were isolated from human fetal bones by flushing the marrow first with 0.25 ml of 0.05% trypsin-EDTA, and then twice with 0.5 ml of Dulbecco’s modified Eagle’s medium. The cell suspension was overlaid on a 10% to 30% serum gradient and centrifuged for 5 minutes at 700 × g to remove the majority of hematopoietic cells. The resulting cell pellet was resuspended in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin, and seeded in 35 mm cell culture dishes. After 24 hours, nonadherent cells were removed by replacing the medium. Cells were cultured to confluency by replacing the medium every 2 to 3 days and then expanded and used for co-culture experiments with PC3 cells. Primary mouse bone marrow cells (mBMSC) were isolated from femurs and tibiae of 6- to 8-week-old cathepsin K-null (FVB/n, N5 CTSK) and wild-type (FVB/n, N5 CTSK) mice that were kindly provided by Dr. Lisa Coussens (University of California at San Francisco, San Francisco, CA). Five-week-old male homozygous ICRSC-M severe combined immunodeficient (SCID) mice were purchased from Taconic Farms (Germantown, NY) and were allowed to acclimate in their housing for 1 week. Mice were maintained under aseptic conditions according to NIH guidelines as found in the “Guidelines for the Care and Use of Experimental Animals” (http://grants.nih.gov/grants/olaw/Guidebook.pdf, last accessed February 28, 2005). All experimental protocols were approved by the Animal Investigation Committee at Wayne State University. Implantation with human bone fragments and tumor cell injections were performed under isoflurane inhalational anesthesia according to previously published procedures.35Nemeth JA Harb JF Barroso Jr, U He Z Grignon DJ Cher ML Severe combined immunodeficient-hu model of human prostate cancer metastasis to human bone.Cancer Res. 1999; 59: 1987-1993PubMed Google Scholar Briefly, human fetal bones were cut into 1.5 cm-long cylinders and implanted into the flanks of SCID mice (2 pieces/animal). Following a 4-week engraftment period, PC3 cells (105 cells/20 μl, right flank) or PBS (control, 20 μl, left flank) were injected directly into the marrow of implanted bone fragments using a 27-gauge needle. Bone tumors and their corresponding controls were removed 2, 4, and 6 weeks after injection. Before tissue removal, mice were euthanized by CO2 inhalation, followed by cervical dislocation. Harvested bone tumors and their corresponding controls were divided into two groups. Half of the samples were immediately fixed and embedded for sectioning (see Immunohistochemistry of Human Bone Tumors section below). The remaining samples were homogenized using an electric tissue grinder in 500 μl of 250 mmol/L sucrose, 25 mmol/L 2-[N-morpholino] ethane-sulfonic acid, 1 mmol/L EDTA, pH 6.5, and 0.1% Triton X-100. The resulting extracts were centrifuged at 800 g for 10 minutes, and supernatants were collected and frozen at −80°C. Expression of cathepsin K, SPARC, and cleavage products of SPARC was assessed by immunoblotting according to our previously established and published procedures.34Podgorski I Linebaugh BE Sameni M Jedeszko C Bhagat S Cher ML Sloane BF Bone microenvironment modulates expression and activity of cathepsin B in prostate cancer.Neoplasia. 2005; 7: 207-223Abstract Full Text PDF PubMed Scopus (59) Google Scholar Activity of cathepsin K in tissue extracts was assayed against the fluorogenic substrate Z-Gly-Pro-Arg-NHMec (final concentration, 100 μmol/L). In addition to being a substrate for cathepsin K, Z-Gly-Pro-Arg-NHMec is also effectively cleaved by cathepsin B (B. E. Linebaugh and B. F. Sloane, unpublished observations), therefore we performed the reaction in the presence of the highly selective cathepsin B inhibitor CA074 (5 μmol/L).34Podgorski I Linebaugh BE Sameni M Jedeszko C Bhagat S Cher ML Sloane BF Bone microenvironment modulates expression and activity of cathepsin B in prostate cancer.Neoplasia. 2005; 7: 207-223Abstract Full Text PDF PubMed Scopus (59) Google Scholar The progress of the reaction was monitored every minute for a period of 30 minutes on a Fluoroskan II microplate reader. Results of activity assays are expressed as relative fluorescence units formed per minute per cell unit. Cell units were calculated as the protein/DNA ratio (mg protein/μg DNA).34Podgorski I Linebaugh BE Sameni M Jedeszko C Bhagat S Cher ML Sloane BF Bone microenvironment modulates expression and activity of cathepsin B in prostate cancer.Neoplasia. 2005; 7: 207-223Abstract Full Text PDF PubMed Scopus (59) Google Scholar Statistical significance was determined by a two-tailed t-test with assumed equal variance and P ≤ 0.05 was considered statistically significant. Tumors were fixed overnight in 4% paraformaldehyde, decalcified in 10% EDTA for 2 weeks and embedded in paraffin. Serial sections (4 μm) were cut, deparaffinized and rehydrated. Adjacent sections of each tumor were analyzed by H&E staining for histological changes and by immunofluorescence for expression and localization of proteins of interest [ie, mouse cathepsin K, 1:200; SPARC, 1:400; 28 kDa and 10 kDa SPARC fragments (028 and 010), 1:200; CD68, 1:200; cytokeratin, 1:300; vimentin, 1:500; α-smooth muscle actin, 1:500; TRAcP, 1:100]. Controls were run in the absence of primary antibody. For immunofluorescent staining, secondary antibodies were Alexa Fluor 488 (green)- and Alexa Fluor 564 (red)-conjugated donkey anti-rabbit IgG and donkey anti-mouse IgG. For immunohistochemical analysis of cathepsin K expression in PC3 bone tumors, biotinylated secondary antibodies conjugated with peroxidase were used along with a NovaRED kit as a substrate for the peroxidase-mediated reaction. Recombinant human platelet SPARC (500 ng) was incubated with 50 ng of recombinant human cathepsin K in the absence and presence of 10 μmol/L cysteine protease inhibitor E-64. The reaction was carried in a 50 mmol/L 2-[N-morpholino] ethane-sulfonic acid buffer, containing 2 mmol/L EDTA and 4 mmol/L dithiothreitol at pH 6.0 and pH 7.4. Samples were electrophoresed on 10% to 20% acrylamide gels and immunoblotted using antibodies to full length SPARC and antibodies to cleavage fragments of SPARC. Collagen I gel solutions were prepared according to manufacturers instructions. The individual cultures of BMSCs (1 × 106 cells/dish) and tumor cells (1 × 106 cells/dish) were mixed with 4 ml of collagen and seeded on 100-mm2 tissue culture dishes. For co-culture experiments BMSCs (9 × 105 cells/dish) and tumor cells (1 × 105 cells/dish) were mixed together and embedded in collagen I as described for individual cultures. In initial experiments, ratios of 2:1, 5:1 and 10:1 (BMSC: PC3) were tested. Based on the most significant changes in expression and activity of cathepsin K, a final ratio of 10:1 (BMSC: PC3) was chosen for all subsequent experiments. To compare the effects of three-dimensional and two-dimensional environments on the interaction of bone marrow stromal cells with prostate cancer cells, all single and mixed BMSC and PC3 cultures were also grown on uncoated dishes. All cells were cultured for 60 hours, and then serum-starved for an additional 12 hours. Cells were harvested from collagen I gels using 0.1% collagenase in PBS at 37°C. Cell pellets were washed in PBS; resuspended in 250 mmol/L sucrose, 25 mmol/L 2-[N-morpholino] ethane-sulfonic acid, 1 mmol/L EDTA, pH 6.5, and 0.1% Triton X-100 buffer, lysed by sonication; and frozen at −80°C until used. DNA concentration was determined by the method of Downs and Wilfinger,36Downs TR Wilfinger WW Fluorometric quantification of DNA in cells and tissue.Anal Biochem. 1983; 131: 538-547Crossref PubMed Scopus (442) Google Scholar and protein was determined using a Micro BCA protein assay kit. Conditioned media (unless otherwise specified) were passed through Millipore 100K concentrators (Burlington, MA) at 150 × g to remove large collagen fragments and were concentrated using Millipore 10K concentrators. Samples were analyzed by immunoblotting and activity assays as described for tissue samples (see Immunoblots and Activity Assays in Tissue Extracts section above). To assay the activity of procathepsin K in conditioned media, pepsin activation was performed as previously described37Hou WS Bromme D Zhao Y Mehler E Dushey C Weinstein H Miranda CS Fraga C Greig F Carey J Rimoin DL Desnick RJ Gelb BD Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis.J Clin Invest. 1999; 103: 731-738Crossref PubMed Scopus (140) Google Scholar and cathepsin K activity measured as described above. The RayBio Human Cytokine Antibody Array V was used for the simultaneous detection of 79 cytokines. Experiments were performed according to manufacturer’s instructions, using media conditioned by either individual BMSC or PC3 cells or BMSC-PC3 co-cultures in the absence or presence of cathepsin K inhibitor. Briefly, membranes were incubated in a blocking buffer for 30 minutes, followed by 2-hour incubation with conditioned media. Media samples (1 ml) were appropriately diluted based on the ratio of protein/DNA
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