Hypoxia Regulates Cross-talk between Syk and Lck Leading to Breast Cancer Progression and Angiogenesis
2006; Elsevier BV; Volume: 281; Issue: 16 Linguagem: Inglês
10.1074/jbc.m512546200
ISSN1083-351X
AutoresGoutam Chakraborty, Hema Rangaswami, Shalini Jain, Gopal C. Kundu,
Tópico(s)Photodynamic Therapy Research Studies
ResumoHypoxia is a key parameter that controls tumor angiogenesis and malignant progression by regulating the expression of several oncogenic molecules. The nonreceptor protein-tyrosine kinases Syk and Lck play crucial roles in the signaling mechanism of various cellular processes. The enhanced expression of Syk in normal breast tissue but not in malignant breast carcinoma has prompted us to investigate its potential role in mammary carcinogenesis. Accordingly, we hypothesized that hypoxia/reoxygenation (H/R) may play an important role in regulating Syk activation, and Lck may be involved in this process. In this study, we have demonstrated that H/R differentially regulates Syk phosphorylation and its subsequent interaction and cross-talk with Lck in MCF-7 cells. Moreover, Syk and Lck play differential roles in regulating Sp1 activation and expressions of melanoma cell adhesion molecule (MelCAM), urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) in response to H/R. Overexpression of wild type Syk inhibited the H/R-induced uPA, MMP-9, and VEGF expression but up-regulated MelCAM expression. Our data also indicated that MelCAM acts as a tumor suppressor by negatively regulating H/R-induced uPA secretion and MMP-9 activation. The mice xenograft study showed the cross-talk between Syk and Lck regulated H/R-induced breast tumor progression and further correlated with the expressions of MelCAM, uPA, MMP-9, and VEGF. Human clinical specimen analysis supported the in vitro and in vivo findings. To our knowledge, this is first report that the cross-talk between Syk and Lck regulates H/R-induced breast cancer progression and further suggests that Syk may act as potential therapeutic target for the treatment of breast cancer. Hypoxia is a key parameter that controls tumor angiogenesis and malignant progression by regulating the expression of several oncogenic molecules. The nonreceptor protein-tyrosine kinases Syk and Lck play crucial roles in the signaling mechanism of various cellular processes. The enhanced expression of Syk in normal breast tissue but not in malignant breast carcinoma has prompted us to investigate its potential role in mammary carcinogenesis. Accordingly, we hypothesized that hypoxia/reoxygenation (H/R) may play an important role in regulating Syk activation, and Lck may be involved in this process. In this study, we have demonstrated that H/R differentially regulates Syk phosphorylation and its subsequent interaction and cross-talk with Lck in MCF-7 cells. Moreover, Syk and Lck play differential roles in regulating Sp1 activation and expressions of melanoma cell adhesion molecule (MelCAM), urokinase-type plasminogen activator (uPA), matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF) in response to H/R. Overexpression of wild type Syk inhibited the H/R-induced uPA, MMP-9, and VEGF expression but up-regulated MelCAM expression. Our data also indicated that MelCAM acts as a tumor suppressor by negatively regulating H/R-induced uPA secretion and MMP-9 activation. The mice xenograft study showed the cross-talk between Syk and Lck regulated H/R-induced breast tumor progression and further correlated with the expressions of MelCAM, uPA, MMP-9, and VEGF. Human clinical specimen analysis supported the in vitro and in vivo findings. To our knowledge, this is first report that the cross-talk between Syk and Lck regulates H/R-induced breast cancer progression and further suggests that Syk may act as potential therapeutic target for the treatment of breast cancer. Hypoxia plays a crucial role in regulating breast tumor progression through a multistep process that includes oncogene activation or inhibition of tumor suppressor genes (1Hockel M. Vaupel P. J. Natl. Cancer Inst. 2001; 93: 266-276Crossref PubMed Scopus (2173) Google Scholar). Most tumors develop regions of chronically or transiently hypoxic cells during growth (2Bussink J. Kaanders J.H. van der Kogel A.J. Radiother. Oncol. 2003; 67: 3-15Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Hypoxic tumor regions may show increased expression of many genes because of hypoxia-induced activation of transcription factors (3Dachs G.U. Tozer G.M. Eur. J. 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Hypoxia is a key parameter, which modulates the expression of a variety of genes that are involved in tumor angiogenesis, malignant progression, and distant metastasis (9Koong A.C. Denko N.C. Hudson K.M. Schindler C. Swiersz L. Koch C. Evans S. Ibrahim H. Le Q.T. Terris D.J. Giaccia A.J. Cancer Res. 2000; 60: 883-887PubMed Google Scholar). The signaling properties of reactive oxygen species are because of the reversible oxidation of redox-sensitive target proteins (10Fan C. Li Q. Ross D. Engelhardt J.F. J. Biol. Chem. 2003; 278: 2072-2080Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The generated reactive oxygen species act as intracellular second messengers in various signal transduction pathways and hence play a crucial role in regulating disease and stress-induced cellular injuries such as ischemia/reperfusion, UV irradiation, and inflammation (10Fan C. Li Q. Ross D. Engelhardt J.F. J. Biol. Chem. 2003; 278: 2072-2080Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Previous reports have indicated that areas of hypoxia/reoxygenation (H/R) 3The abbreviations used are: H/R, hypoxia/reoxygenation; Syk, splenic tyrosine kinase; Lck, leukocyte-specific kinase; HIF-1α, hypoxia-inducible factor1α; EMSA, electrophoretic mobility shift assay; MelCAM, melanoma cell adhesion molecule; uPA, urokinase-type plasminogen activator; MMP-9, matrix metalloproteinase-9; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; DAPI, 4,6-diamidino-2-phenylindole; WT, wild type; Mut, mutant; DN, dominant negative; NMRI, Naval Medical Research Institute. are a typical feature of rapidly growing and metastasizing tumors (11Dachs G.U. Patterson A.V. Firth J.D. Ratcliffe P.J. Townsend K.M. Stratford I.J. Harris A.L. Nat. Med. 1997; 3: 515-520Crossref PubMed Scopus (349) Google Scholar, 12Chaplin D.J. Hill S.A. Bell K.M. Tozer G.M. Semin. Radiat. Oncol. 1998; 8: 151-163Crossref PubMed Scopus (54) Google Scholar). It was also demonstrated that both hypoxia and consecutive hypoxia/reoxygenation exert a variety of influence in tumor cell biology that ultimately regulates tumor progression (13Kunz M. Ibrahim S.M. Mol. Cancer. 2003; 2: 1-13Crossref PubMed Scopus (162) Google Scholar). Earlier reports also showed that hypoxia and H/R regulate the activation of various mitogen-activated protein kinase signaling pathways (13Kunz M. Ibrahim S.M. Mol. Cancer. 2003; 2: 1-13Crossref PubMed Scopus (162) Google Scholar) and induce the activation of several transcriptional factors such as HIF-1α, NFκB, AP-1, and Sp1 (13Kunz M. Ibrahim S.M. Mol. Cancer. 2003; 2: 1-13Crossref PubMed Scopus (162) Google Scholar, 14Kunz M. Bloss G. Gillitzer R. Gross G. Goebeler M. Rapp U.R. Ludwig S. Biochem. 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Today. 2000; 21: 148-154Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). It has tandem amino-terminal Src homology 2 domains and a carboxyl-terminal kinase domain (19Yanagi S. Kurosaki T. Yamamura H. Cell. Signal. 1995; 7: 185-193Crossref PubMed Scopus (58) Google Scholar). The Src homology 2 domains bind phosphorylated immunoreceptor tyrosinebased activation motifs and hence play a significant role in immunoreceptor and cytokine signaling (20Cheng A.M. Negishi I. Anderson S.J. Chan A.C. Bolen J. Loh D.Y. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9797-9801Crossref PubMed Scopus (150) Google Scholar). The expression of Syk has also been reported in cell lines of epithelial origin (21Ulanova M. Puttagunta L. Marcet-Palacios M. Duszyk M. Steinhoff U. Duta F. Kim M.K. Indik Z.K. Schreiber A.D. Befus A.D. Am. J. Physiol. 2005; 288: 497-507Crossref PubMed Scopus (88) Google Scholar), but its function in these cells is not well understood. It has been documented that Syk is commonly expressed in normal human breast tissue, benign breast lesions, and low tumorigenic breast cancer cell lines (22Coopman P.J. Do M.T. Barth M. Bowden E.T. Hayes A.J. Basyuk E. Blancato J.K. Vezza P.R. McLeskey S.W. Mangeat P.H. Mueller S.C. Nature. 2000; 406: 742-747Crossref PubMed Scopus (287) Google Scholar). Previous data indicated that Syk suppresses cell motility and NFκB-mediated urokinase-type plasminogen activator (uPA) secretion by inhibiting phosphatidylinositol 3-kinase activity in breast cancer cells (23Mahabeleshwar G.H. Kundu G.C. J. Biol. Chem. 2003; 278: 6209-6221Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Lck, a member of the Src family nonreceptor protein-tyrosine kinase, is mostly expressed in T cells, breast cancer tissues, and cell lines and also in some B cells (24Koster A. Landgraf S. Leipold A. Sachse R. Gebhart E. Tulushan A.H. Ronay G. Schmidt C. Dingermann T. Anticancer Res. 1991; 11: 193-201PubMed Google Scholar). Lck binds to the cytoplasmic domain of CD4 and CD8 and plays an essential role in T cell activation and development (25Veillette A. Bookman M.A. Horak E.M. Bolen J.B. Cell. 1988; 55: 301-308Abstract Full Text PDF PubMed Scopus (1131) Google Scholar). Earlier reports have indicated that p72Syk plays a crucial role in activation of p56Lck through physical association and amino-terminal tyrosine phosphorylation at residues Tyr-518 and Tyr-519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation by ∼90% (26Couture C. Deckert M. Williams S. Russo F.O. Altman A. Mustelin T. J. Biol. Chem. 1996; 271: 24294-24299Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). However, the molecular mechanism by which H/R regulates Syk phosphorylation and its subsequent interaction with Lck leading to downstream signaling events in breast cancer cells are not well defined. MelCAM, previously known as MUC18 or MCAM, a newly recognized cell adhesion molecule with an apparent molecular mass of 113 kDa, belongs to the immunoglobulin superfamily (27Lehmann J.M. Holzmann B. Breitbart E.W. Schmiegelow P. Riethmuller G. Johnson J.P. Cancer Res. 1987; 47: 841-845PubMed Google Scholar, 28Lehmann J.M. Riethmuller G. Johnson J.P. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9891-9895Crossref PubMed Scopus (397) Google Scholar, 29Sers C. Kirsch K. Rothbacher U. Riethmuller G. Johnson J.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8514-8518Crossref PubMed Scopus (128) Google Scholar). The presence of putative binding sites for the transcription factors Sp1, AP-1, AP-2, and cAMP-response element-binding protein in the promoter region suggests that MelCAM expression can be modulated by exogenous factors (30Xie S. Luca M. Huang S. Gutman M. Reich R. Johnson J.P. Bar-Eli M. Cancer Res. 1997; 57: 2295-2303PubMed Google Scholar). At the cellular level, phorbol ester and cyclic AMP have been shown to modulate MelCAM expression (31Rummel M.M. Sers C. Johnson J.P. Cancer Res. 1996; 56: 2218-2223PubMed Google Scholar). Earlier reports have indicated that MelCAM acts differently in the progression of breast carcinomas. It is expressed in normal and benign proliferative breast epithelium, and its expression is frequently lost in in situ and infiltrating breast carcinomas (32Shih L.M. Hsu M.Y. Palazzo J.P. Herlyn M. Am. J. Pathol. 1997; 151: 745-751PubMed Google Scholar, 33Shih I.M. J. Pathol. 1999; 189: 4-11Crossref PubMed Scopus (240) Google Scholar). The MelCAM core promoter contains four binding sites for the Sp1 transcription factor, and deletion analyses have indicated that removal of all putative Sp1 sites reduced the promoter activity by 80%, suggesting that Sp1 is an important regulator of MelCAM expression (34Karlen S. Braathen L.R. J. Investig. Dermatol. 1999; 113: 711-719Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). However, the molecular mechanism by which H/R regulates Syk activation and Syk-dependent Lck-mediated Sp1 activation leading to the regulation of MelCAM expression in MCF-7 cells is not well defined. Degradation of extracellular matrix plays an important role in tumor metastasis. uPA is a member of the serine protease family that interacts with uPA receptor and facilitates the conversion of inert zymogen plasminogen into widely acting serine protease plasmin (35Collen D. Thromb. Haemostasis. 1999; 82: 259-270Crossref PubMed Scopus (354) Google Scholar, 36Blasi F. Thromb. Haemostasis. 1999; 82: 298-304Crossref PubMed Scopus (171) Google Scholar). MMP-9, also referred as gelatinase-B, is not only associated with invasion and metastasis but also has been implicated in angiogenesis, rheumatoid arthritis, retinopathy, and vascular stenosis and hence is considered to be a prioritized therapeutic target (37Shapiro S.D. Curr. Opin. Cell Biol. 1998; 10: 602-608Crossref PubMed Scopus (622) Google Scholar, 38Folkman J. Nat. Biotechnol. 1999; 17: 749Crossref PubMed Scopus (46) Google Scholar). Several reports have indicated the positive correlation between uPA/MMP-9 activation and the metastatic potential of tumors (39Rangaswami H. Bulbule A. Kundu G.C. J. Biol. Chem. 2004; 279: 38921-38935Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). However, the molecular mechanism by which H/R regulates Syk/Lck-dependent MelCAM expression and uPA secretion and the uPA-dependent pro-MMP-9 activation in breast carcinoma cells is not well understood. Moreover, the roles of these molecules in regulating H/R-induced tumorigenesis and its clinical implications are not well defined. Hypoxia-induced VEGF production provides one of the main driving forces that stimulate the angiogenesis, which accompanies tumor progression (40Ferrara N. Gerber H.P. LeCouter J. Nat. Med. 2003; 9: 669-676Crossref PubMed Scopus (7864) Google Scholar, 41Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3147) Google Scholar). To date, VEGF is considered as the key factor that guided and regulated tumor angiogenesis (42Lutsenko S.V. Kiselev S.M. Severin S.E. Biochemistry (Mosc.). 2003; 68: 286-300Crossref PubMed Scopus (31) Google Scholar). Tumor cell-derived VEGF binds to its specific receptors and regulates tumor progression through neovascularization via autocrine and paracrine pathways (43Matsumoto T. Claesson-Welsh L. Sci. STKE. 2001. 2001; (RE21)Google Scholar, 44Mercurio A.M. Bachelder R.E. Bates R.C. Chung J. Semin. Cancer Biol. 2004; 14: 115-122Crossref PubMed Scopus (65) Google Scholar). Recent evidence suggested that the VEGF promoter contains an Sp1-response element (45Bradbury D. Clarke D. Seedhouse C. Corbett L. Stocks J. Knox A. J. Biol. Chem. 2005; 280: 29993-30000Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). However, the role of Lck and Syk in H/R-induced VEGF expression in breast cancer is not defined clearly. In this study we have demonstrated the differential role of Syk and Lck in the H/R-induced uPA, MMP-9, and VEGF expression. Our findings suggested that H/R down-regulates Syk activation leading to enhanced uPA, MMP-9, and VEGF expression. Furthermore, overexpression of Syk restored H/R-induced down-regulation of Mel-CAM expression. In hypoxic cells, Lck also physically associates with Syk, and this association plays a crucial role in regulating the downstream signaling. The in vivo relevance of our study was further validated in a xenografted nude mice model, which also supports our in vitro findings. Clinical data also indicated that the higher grades of tumors showed significant HIF-1α expression compared with that of lower grades or normal breast tissue and also demonstrated an inverse correlation between Syk/MelCAM and uPA/MMP-9/VEGF expression, which further correlates with enhanced tumorigenic potential and neovascularization. Materials—The rabbit polyclonal anti-Syk, anti-Lck, anti-Sp1, anti-MelCAM, anti-uPA, anti-MMP-9, anti-actin, mouse monoclonal anti-Lck, anti-Syk, anti-phosphotyrosine antibodies, and MelCAM blocking peptide were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti-VEGF was from Oncogene. Rabbit polyclonal anti-vWF antibody was from Sigma. Rabbit polyclonal anti-HIF-1α was from Upstate Biotechnology, Inc. Lipofectamine Plus was obtained from Invitrogen. pp2, aminogenistein, and damnacanthal were from Calbiochem. The Sp1 consensus oligonucleotide was purchased from Bangalore Genei. Matrigel was purchased from BD Biosciences. The [γ-32P]ATP was purchased from the Board of Radiation and Isotope Technology (Hyderabad, India). The female nude mice (NMRI, nu/nu) were from National Institute of Virology (Pune, India). All other chemicals were of analytical grade. Cell Culture—The MCF-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mm glutamine in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Hypoxic/Reoxygenation Cultures—The MCF-7 cells grown to 50-70% confluence were made hypoxic in evacuation chambers by intermittent application of vacuum and sparging with 95% N2, 5% CO2. Cells were analyzed at this point or maintained under hypoxic conditions in the presence of 100 mm dithionate (an O2 scavenger) at 37 °C for the indicated time point. These cells were reoxygenated for the indicated periods by replacing the medium with fresh medium and incubating the cultures in humidified atmosphere of 5% CO2 and 95% air at 37 °C. Immunofluorescence and Immunohistochemistry—To detect the effect of H/R on cellular localization of Syk, cells grown in monolayer on glass slides were induced by hypoxia for 2 h and reoxygenated for 45 min. The cells were fixed and incubated with rabbit polyclonal anti-Syk antibody (1:50 dilution) followed by FITC-conjugated anti-rabbit IgG at room temperature. The role of H/R in regulating Syk-Lck colocalization was determined by immunofluorescence studies using a mixture of mouse monoclonal anti-Syk and rabbit polyclonal anti-Lck antibody followed by a mixture of TRITC- and FITC-conjugated IgG. The cells were washed and mounted with coverslips. All these samples were analyzed under confocal microscopy (Zeiss). The clinical specimens were analyzed by immunohistopathological studies. Formalin-fixed paraffin-embedded sections (4 μm) were subjected to antigen retrieval, and the Syk-Lck colocalization was determined by immunofluorescence studies using a mixture of rabbit polyclonal anti-Syk and mouse monoclonal anti-Lck antibodies followed by a mixture of FITC- and TRITC-conjugated IgGs. The levels of HIF-1α expression in these clinical samples were determined by Western blot analysis. The levels of MelCAM, uPA, MMP-9, and VEGF expressions in these samples were detected by immunofluorescence studies by using their specific antibodies. The tumor microvessel densities were detected by immunostaining with anti-vWF antibody. All these samples were analyzed under confocal microscopy (Zeiss). Plasmids and DNA Transfection—The dominant-negative form of Lck (DN Lck, K273R) in pcDNA3 was a kind gift from Dr. D. R. Branch (Canadian Blood Services, Toronto, Ontario). The wild type and kinase-negative Syk cDNA in pcDNA 3.1 were the generous gifts from Dr. Susette C. Mueller (Department of Oncology, Georgetown University Medical School, Washington, D. C.). MCF-7 cells were transfected with specific cDNA using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions as described previously (46Philip S. Bulbule A. Kundu G.C. J. Biol. Chem. 2001; 276: 44926-44935Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). These transfected cells were used for Syk-Lck colocalization and interaction studies, detection of Sp1, Syk, Lck, MelCAM, uPA, MMP-9, and VEGF by Western blot analysis, and in vivo tumorigenicity experiments. Zymography Experiments—The gelatinolytic activity was measured as described (46Philip S. Bulbule A. Kundu G.C. J. Biol. Chem. 2001; 276: 44926-44935Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 47Rangaswami H. Bulbule A. Kundu G.C. J. Biol. Chem. 2005; 280: 19381-19392Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). To examine whether H/R regulates MMP-9 activation and to investigate the effect of inhibition of MelCAM expression on H/R-induced MMP-9 activation, cells were either induced with H/R for 24 h or pretreated with MelCAM blocking peptide (50 μg) and then exposed with H/R. The conditioned media were collected, and the samples were analyzed by zymography as described (47Rangaswami H. Bulbule A. Kundu G.C. J. Biol. Chem. 2005; 280: 19381-19392Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Negative staining showed the zones of gelatinolytic activity. Immunoprecipitation—To delineate the role of H/R in the regulation of tyrosine phosphorylation of Syk, cells were exposed to hypoxia for 2 h followed by reoxygenation for 0-60 min. In separate experiments, cells were pretreated with Lck inhibitors, pp2 (4 nm), aminogenistein (2 μm), and damnacanthal (0.8 μm), and then exposed to H/R. Cell lysates were immunoprecipitated with rabbit polyclonal anti-Syk antibody and analyzed by Western blot using anti-phosphotyrosine antibody. The same blots were reprobed with anti-Syk antibody. To analyze whether Syk interacts with Lck and to determine whether H/R regulates this process, cells were exposed to H/R for 45 min. In other experiments, cells were transfected with wild type and kinase-negative Syk and then subjected to H/R. Cells were lysed in lysis buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mm iodoacetamide, 2 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin). The lysates containing equal amount of total proteins were immunoprecipitated with rabbit anti-Lck antibody and analyzed by Western blot using anti-Syk antibody. The same blots were reprobed with anti-Lck antibody. Western Blot Analysis—To analyze the roles of Syk and Lck in regulating MelCAM, VEGF expression, and MMP-9 activation, cells were transfected with wild type and kinase-negative Syk or dominant-negative Lck and then exposed to H/R for 24 h as described earlier. The cell lysates were analyzed by Western blot using anti-MelCAM or anti-VEGF antibody. The levels of Lck and Syk in nontransfected or transfected cell lysates were also detected by Western blot using anti-Lck or anti-Syk antibody. The level of active MMP-9 in conditioned media was detected by Western blot. To analyze the effect of inhibition of MelCAM on H/R-induced uPA secretion, cells were pretreated with MelCAM-specific blocking peptide (50 μg/ml) for 24 h and subjected to H/R treatment. Cell lysates were analyzed by Western blot using anti-uPA antibody. The level of HIF-1α expression in the normal and breast tumor specimens of different grades was also detected by Western blot using anti-HIF-1α antibody. The same blots were reprobed with anti-actin antibody as loading control. Nuclear Extracts and Western Blot—To check whether H/R regulates Sp1 expression, cells were subjected to H/R for 0-180 min at 37 °C. The nuclear extracts were prepared as described earlier (24Koster A. Landgraf S. Leipold A. Sachse R. Gebhart E. Tulushan A.H. Ronay G. Schmidt C. Dingermann T. Anticancer Res. 1991; 11: 193-201PubMed Google Scholar). Briefly, cells were incubated in hypotonic buffer (10 mm HEPES (pH 7.9), 1.5 mm MgCl2,10mm KCl, 0.2 mm phenylmethylsulfonyl fluoride, and 0.5 mm dithiothreitol) and allowed to swell on ice for 10 min. Cells were homogenized in a Dounce homogenizer. The nuclei were separated by spinning at 3300 × g for 5 min at 4 °C. The supernatant was used as cytoplasmic extract. The nuclear pellet was extracted in nuclear extraction buffer (20 mm HEPES (pH 7.9), 0.4 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 25% glycerol, 0.5 mm phenylmethylsulfonyl fluoride, and 0.5 mm dithiothreitol) and centrifuged at 12,000 × g for 30 min. The supernatant was used as nuclear extract. The nuclear extracts were resolved by SDS-PAGE, and the level of Sp1 was detected by Western blot using rabbit anti-Sp1 antibody. In separate experiments, to examine the effects of Syk and Lck on H/R-induced Sp1 expression, cells were transfected with wild type and kinase-negative Syk or DN Lck and then induced with H/R. The nuclear extracts were prepared and analyzed by Western blot using anti-Sp1 antibody. The levels of Lck and Syk in nuclear extracts were also detected by Western blot using anti-Lck or anti-Syk antibody. The level of expression of HIF-1α in hypoxia or H/R-induced nuclear extracts was detected by Western blot using anti-HIF-1α antibody. Actin was used as loading control. EMSA—To examine whether H/R induces the Sp1-DNA binding, cells were exposed to H/R for 0-180 min as described earlier. To check whether Syk and Lck play any role in regulating H/R-induced Sp1-DNA binding, cells were transfected with wild type and kinase-negative Syk or pretreated with Lck inhibitors, pp2 (4 nm), aminogenistein (2 μm), and damnacanthal (0.8 μm) followed by exposed to H/R for 1 h. The nuclear extracts were prepared as described above and incubated with 16 fmol of 32P-labeled double-stranded Sp1 oligonucleotide (5′-ATTCGATCGGGGCGGGGC-3′) in binding buffer (25 mm HEPES (pH 7.9), 0.5 mm EDTA, 0.5 mm dithiothreitol, 1% Nonidet P-40, 5% glycerol, 50 mm NaCl) containing 1 μg of poly(dI-dC). The DNA-protein complex was resolved on a native polyacrylamide gel and analyzed by autoradiography. In Vivo Xenograft Tumor Model—The tumorigenicity experiments were performed as described (47Rangaswami H. Bulbule A. Kundu G.C. J. Biol. Chem. 2005; 280: 19381-19392Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 48Sounni N.E. Devy L. Hajitou A. Frankenne F. Munaut C. Gilles C. Deroanne C. Thompson E.W. Foidart J.M. Noel A. FASEB J. 2002; 16: 555-564Crossref PubMed Scopus (241) Google Scholar). Briefly, female athymic nude mice, NMRI, nu/nu (BALB/c) obtained from National Institute of Virology (Pune, India) were housed under specific pathogen-free conditions and used for in vivo tumorigenicity studies. Cells were either exposed to H/R or transfected with wild type or mutant Syk and then exposed to H/R for 24 h. Cell viability was determined by the trypan blue exclusion test, and only a single cell suspensions (5 × 102/0.2 ml) of >90% viability were mixed with Matrigel and injected subcutaneously into the flanks of female athymic NMRI (nu/nu) mice (6-8 weeks old). Five mice were used in each set of experiments. Growth of tumors was monitored weekly by measuring the tumors with calipers. The mice were killed after 4 weeks of injection, and the levels of uPA, VEGF, and MelCAM in the tumors were analyzed by Western blot. The level of Sp1-DNA binding in the nuclear extracts of the tumor samples was determined by EMSA. The tumor samples were also processed for histopathological studies. Formalin-fixed paraffin-embedded sections (4 μm) were subjected to antigen retrieval and then stained with rabbit polyclonal anti-MMP-9, anti-uPA antibody, or mouse monoclonal anti-MelCAM antibody followed by FITC-conjugated anti-rabbit or anti-mouse IgG. The tumor microvessel densities were detected by immunostaining with anti-vWF antibody. The level of colocalization of Syk and Lck in these tumor specimens was determined by immunofluorescence using a mixture of mouse monoclonal anti-Syk and rabbit polyclonal anti-Lck antibody followed by a mixture of FITC- and TRITC-conjugated IgG. All these samples were analyzed under confocal microscopy (Zeiss). Human Breast Tumor Specimen Analysis—Human breast tumor specimens with different grades and normal breast tissues were collected from a local hospital with informed consent and were flash-frozen. The immunohist
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