siRNA-mediated downregulation of MMP-9 and uPAR in combination with radiation induces G2/M cell-cycle arrest in Medulloblastoma
Our previous work and that of other investigators strongly suggest a relationship between the upregulation of metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR) in tumor angiogenesis and metastasis. In this study, we evaluated the role of MMP-9 and uPAR in medullobl...
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| Veröffentlicht in: | Molecular cancer research 2011-01, Vol.9 (1), p.51-66 |
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| creator | Nagaraju, Ganji Purna Chandra Nalla, Arun Kumar Gupta, Reshu Mohanam, Sanjeeva Gujrati, Meena Dinh, Dzung H Rao, Jasti S |
| description | Our previous work and that of other investigators strongly suggest a relationship between the upregulation of metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR) in tumor angiogenesis and metastasis. In this study, we evaluated the role of MMP-9 and uPAR in medulloblastoma cancer cell resistance to ionizing irradiation (IR) and tested the antitumor efficacy of siRNA (short interfering RNA) against MMP-9 [plasmid siRNA vector for MMP-9 (pM)] and uPAR [plasmid vector for uPAR (pU)] either alone or in combination [plasmid siRNA vector for both uPAR and MMP-9 (pUM)]. Cell proliferation (BrdU assay), apoptosis (in situ TUNEL for DNA fragmentation), and cell-cycle (FACS) analyses were carried out to determine the effect of siRNA either alone or in combination with IR on G2/M cell-cycle arrest in medulloblastoma cells. IR upregulated MMP-9 and uPAR expression in medulloblastoma cells; pM, pU, and pUM in combination with IR effectively reduced both MMP-9 and uPAR expression, thereby leading to increased radiosensitivity of medulloblastoma cells. siRNA treatments (pM, pU, and pUM) also promoted IR-induced apoptosis and enhanced IR-induced G2/M arrest during cell-cycle progression. While IR induces G2/M cell-cycle arrest through inhibition of the pCdc2- and cyclin B-regulated signaling pathways involving p53, p21/WAF1, and Chk2 gene expression, siRNA (pM, pU, and pUM) alone or in combination with IR induced G2/M arrest mediated through inhibition of the pCdc2- and cyclin B1-regulated signaling pathways involving Chk1 and Cdc25A gene expression. Taken together, our data suggest that downregulation of MMP-9 and uPAR induces Chk1-mediated G2/M cell-cycle arrest, whereas the disruption caused by IR alone is dependent on p53- and Chk2-mediated G2/M cell-cycle arrest. |
| doi_str_mv | 10.1158/1541-7786.MCR-10-0399 |
| format | Article |
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In this study, we evaluated the role of MMP-9 and uPAR in medulloblastoma cancer cell resistance to ionizing irradiation (IR) and tested the antitumor efficacy of siRNA (short interfering RNA) against MMP-9 [plasmid siRNA vector for MMP-9 (pM)] and uPAR [plasmid vector for uPAR (pU)] either alone or in combination [plasmid siRNA vector for both uPAR and MMP-9 (pUM)]. Cell proliferation (BrdU assay), apoptosis (in situ TUNEL for DNA fragmentation), and cell-cycle (FACS) analyses were carried out to determine the effect of siRNA either alone or in combination with IR on G2/M cell-cycle arrest in medulloblastoma cells. IR upregulated MMP-9 and uPAR expression in medulloblastoma cells; pM, pU, and pUM in combination with IR effectively reduced both MMP-9 and uPAR expression, thereby leading to increased radiosensitivity of medulloblastoma cells. siRNA treatments (pM, pU, and pUM) also promoted IR-induced apoptosis and enhanced IR-induced G2/M arrest during cell-cycle progression. While IR induces G2/M cell-cycle arrest through inhibition of the pCdc2- and cyclin B-regulated signaling pathways involving p53, p21/WAF1, and Chk2 gene expression, siRNA (pM, pU, and pUM) alone or in combination with IR induced G2/M arrest mediated through inhibition of the pCdc2- and cyclin B1-regulated signaling pathways involving Chk1 and Cdc25A gene expression. Taken together, our data suggest that downregulation of MMP-9 and uPAR induces Chk1-mediated G2/M cell-cycle arrest, whereas the disruption caused by IR alone is dependent on p53- and Chk2-mediated G2/M cell-cycle arrest.</description><identifier>ISSN: 1541-7786</identifier><identifier>EISSN: 1557-3125</identifier><identifier>DOI: 10.1158/1541-7786.MCR-10-0399</identifier><identifier>PMID: 21148633</identifier><language>eng</language><publisher>United States</publisher><subject>Angiogenesis ; Antitumor activity ; Apoptosis ; Apoptosis - genetics ; Apoptosis - radiation effects ; Blotting, Western ; Cancer ; CDC2 Protein Kinase - metabolism ; Cdc25A phosphatase ; Cell Cycle - radiation effects ; Cell Cycle Proteins - metabolism ; Cell Division - radiation effects ; Cell Line, Tumor ; Cell Proliferation ; CHK1 protein ; Chk2 gene ; Cyclin-Dependent Kinase 2 - metabolism ; Cyclin-dependent kinase inhibitor p21 ; Data processing ; DNA fragmentation ; Down-Regulation ; Flow Cytometry ; G2 Phase - radiation effects ; Gelatinase B ; Gene Expression Regulation, Neoplastic - radiation effects ; Humans ; I.R. radiation ; Matrix Metalloproteinase 9 - genetics ; Matrix Metalloproteinase 9 - metabolism ; Medulloblastoma ; Medulloblastoma - genetics ; Medulloblastoma - metabolism ; Medulloblastoma - pathology ; Metastases ; Mitogen-Activated Protein Kinases - metabolism ; p53 protein ; Phosphatidylinositol 3-Kinases - metabolism ; Phosphorylation - radiation effects ; Plasmids ; Radiosensitivity ; ras Proteins - metabolism ; Receptors, Urokinase Plasminogen Activator - genetics ; Receptors, Urokinase Plasminogen Activator - metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; RNA Interference ; RNA, Small Interfering - genetics ; Signal transduction ; siRNA ; Tumor Suppressor Protein p53 - metabolism ; Tumors ; u-Plasminogen activator</subject><ispartof>Molecular cancer research, 2011-01, Vol.9 (1), p.51-66</ispartof><rights>2010 AACR.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c372t-3486c22cdbb3dd0ce2aba820003e217cadcf744ef9163de1ed476de517332b563</citedby><cites>FETCH-LOGICAL-c372t-3486c22cdbb3dd0ce2aba820003e217cadcf744ef9163de1ed476de517332b563</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,776,780,881,3343,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21148633$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nagaraju, Ganji Purna Chandra</creatorcontrib><creatorcontrib>Nalla, Arun Kumar</creatorcontrib><creatorcontrib>Gupta, Reshu</creatorcontrib><creatorcontrib>Mohanam, Sanjeeva</creatorcontrib><creatorcontrib>Gujrati, Meena</creatorcontrib><creatorcontrib>Dinh, Dzung H</creatorcontrib><creatorcontrib>Rao, Jasti S</creatorcontrib><title>siRNA-mediated downregulation of MMP-9 and uPAR in combination with radiation induces G2/M cell-cycle arrest in Medulloblastoma</title><title>Molecular cancer research</title><addtitle>Mol Cancer Res</addtitle><description>Our previous work and that of other investigators strongly suggest a relationship between the upregulation of metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR) in tumor angiogenesis and metastasis. In this study, we evaluated the role of MMP-9 and uPAR in medulloblastoma cancer cell resistance to ionizing irradiation (IR) and tested the antitumor efficacy of siRNA (short interfering RNA) against MMP-9 [plasmid siRNA vector for MMP-9 (pM)] and uPAR [plasmid vector for uPAR (pU)] either alone or in combination [plasmid siRNA vector for both uPAR and MMP-9 (pUM)]. Cell proliferation (BrdU assay), apoptosis (in situ TUNEL for DNA fragmentation), and cell-cycle (FACS) analyses were carried out to determine the effect of siRNA either alone or in combination with IR on G2/M cell-cycle arrest in medulloblastoma cells. IR upregulated MMP-9 and uPAR expression in medulloblastoma cells; pM, pU, and pUM in combination with IR effectively reduced both MMP-9 and uPAR expression, thereby leading to increased radiosensitivity of medulloblastoma cells. siRNA treatments (pM, pU, and pUM) also promoted IR-induced apoptosis and enhanced IR-induced G2/M arrest during cell-cycle progression. While IR induces G2/M cell-cycle arrest through inhibition of the pCdc2- and cyclin B-regulated signaling pathways involving p53, p21/WAF1, and Chk2 gene expression, siRNA (pM, pU, and pUM) alone or in combination with IR induced G2/M arrest mediated through inhibition of the pCdc2- and cyclin B1-regulated signaling pathways involving Chk1 and Cdc25A gene expression. Taken together, our data suggest that downregulation of MMP-9 and uPAR induces Chk1-mediated G2/M cell-cycle arrest, whereas the disruption caused by IR alone is dependent on p53- and Chk2-mediated G2/M cell-cycle arrest.</description><subject>Angiogenesis</subject><subject>Antitumor activity</subject><subject>Apoptosis</subject><subject>Apoptosis - genetics</subject><subject>Apoptosis - radiation effects</subject><subject>Blotting, Western</subject><subject>Cancer</subject><subject>CDC2 Protein Kinase - metabolism</subject><subject>Cdc25A phosphatase</subject><subject>Cell Cycle - radiation effects</subject><subject>Cell Cycle Proteins - metabolism</subject><subject>Cell Division - radiation effects</subject><subject>Cell Line, Tumor</subject><subject>Cell Proliferation</subject><subject>CHK1 protein</subject><subject>Chk2 gene</subject><subject>Cyclin-Dependent Kinase 2 - metabolism</subject><subject>Cyclin-dependent kinase inhibitor p21</subject><subject>Data processing</subject><subject>DNA fragmentation</subject><subject>Down-Regulation</subject><subject>Flow Cytometry</subject><subject>G2 Phase - radiation effects</subject><subject>Gelatinase B</subject><subject>Gene Expression Regulation, Neoplastic - radiation effects</subject><subject>Humans</subject><subject>I.R. radiation</subject><subject>Matrix Metalloproteinase 9 - genetics</subject><subject>Matrix Metalloproteinase 9 - metabolism</subject><subject>Medulloblastoma</subject><subject>Medulloblastoma - genetics</subject><subject>Medulloblastoma - metabolism</subject><subject>Medulloblastoma - pathology</subject><subject>Metastases</subject><subject>Mitogen-Activated Protein Kinases - metabolism</subject><subject>p53 protein</subject><subject>Phosphatidylinositol 3-Kinases - metabolism</subject><subject>Phosphorylation - radiation effects</subject><subject>Plasmids</subject><subject>Radiosensitivity</subject><subject>ras Proteins - metabolism</subject><subject>Receptors, Urokinase Plasminogen Activator - genetics</subject><subject>Receptors, Urokinase Plasminogen Activator - metabolism</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>RNA Interference</subject><subject>RNA, Small Interfering - genetics</subject><subject>Signal transduction</subject><subject>siRNA</subject><subject>Tumor Suppressor Protein p53 - metabolism</subject><subject>Tumors</subject><subject>u-Plasminogen activator</subject><issn>1541-7786</issn><issn>1557-3125</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUFvFSEUhYnR2Fr9CRp2rmi5MAwzG5OXF60mHdu81DVhgGkxzFBhxqYr_7pD3rOxq66Ayzkn9-RD6D3QUwDRnIGogEjZ1KfddkeAEsrb9gU6BiEk4cDEy3I_aI7Qm5x_UsooyPo1OmIAVVNzfoz-ZL_7viGjs17PzmIb76fkbpagZx8nHAfcdVekxXqyeLna7LCfsIlj76e94N7Ptzjp4i5PP9nFuIzP2VmHjQuBmAcTHNYpuTwXc-fsEkLsg85zHPVb9GrQIbt3h_ME_fjy-Xr7lVxcnn_bbi6I4ZLNhK_rGsaM7XtuLTWO6V43jFLKHQNptDWDrCo3tFBz68DZStbWCZCcs17U_AR92ufeLf1a1rhpTjqou-RHnR5U1F49_Zn8rbqJvxWnjLW1WAM-HgJS_LWsZdToc2moJxeXrFoqoWplJZ9VNpVYUUHLVqXYK02KOSc3PO4DVBXKqhBUhaBaKZdpobz6Pvxf5tH1Dyv_C0LYpVw</recordid><startdate>20110101</startdate><enddate>20110101</enddate><creator>Nagaraju, Ganji Purna Chandra</creator><creator>Nalla, Arun Kumar</creator><creator>Gupta, Reshu</creator><creator>Mohanam, Sanjeeva</creator><creator>Gujrati, Meena</creator><creator>Dinh, Dzung H</creator><creator>Rao, Jasti S</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7TM</scope><scope>7U7</scope><scope>C1K</scope><scope>5PM</scope></search><sort><creationdate>20110101</creationdate><title>siRNA-mediated downregulation of MMP-9 and uPAR in combination with radiation induces G2/M cell-cycle arrest in Medulloblastoma</title><author>Nagaraju, Ganji Purna Chandra ; Nalla, Arun Kumar ; Gupta, Reshu ; Mohanam, Sanjeeva ; Gujrati, Meena ; Dinh, Dzung H ; Rao, Jasti S</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c372t-3486c22cdbb3dd0ce2aba820003e217cadcf744ef9163de1ed476de517332b563</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Angiogenesis</topic><topic>Antitumor activity</topic><topic>Apoptosis</topic><topic>Apoptosis - genetics</topic><topic>Apoptosis - radiation effects</topic><topic>Blotting, Western</topic><topic>Cancer</topic><topic>CDC2 Protein Kinase - metabolism</topic><topic>Cdc25A phosphatase</topic><topic>Cell Cycle - radiation effects</topic><topic>Cell Cycle Proteins - metabolism</topic><topic>Cell Division - radiation effects</topic><topic>Cell Line, Tumor</topic><topic>Cell Proliferation</topic><topic>CHK1 protein</topic><topic>Chk2 gene</topic><topic>Cyclin-Dependent Kinase 2 - metabolism</topic><topic>Cyclin-dependent kinase inhibitor p21</topic><topic>Data processing</topic><topic>DNA fragmentation</topic><topic>Down-Regulation</topic><topic>Flow Cytometry</topic><topic>G2 Phase - radiation effects</topic><topic>Gelatinase B</topic><topic>Gene Expression Regulation, Neoplastic - radiation effects</topic><topic>Humans</topic><topic>I.R. radiation</topic><topic>Matrix Metalloproteinase 9 - genetics</topic><topic>Matrix Metalloproteinase 9 - metabolism</topic><topic>Medulloblastoma</topic><topic>Medulloblastoma - genetics</topic><topic>Medulloblastoma - metabolism</topic><topic>Medulloblastoma - pathology</topic><topic>Metastases</topic><topic>Mitogen-Activated Protein Kinases - metabolism</topic><topic>p53 protein</topic><topic>Phosphatidylinositol 3-Kinases - metabolism</topic><topic>Phosphorylation - radiation effects</topic><topic>Plasmids</topic><topic>Radiosensitivity</topic><topic>ras Proteins - metabolism</topic><topic>Receptors, Urokinase Plasminogen Activator - genetics</topic><topic>Receptors, Urokinase Plasminogen Activator - metabolism</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>RNA Interference</topic><topic>RNA, Small Interfering - genetics</topic><topic>Signal transduction</topic><topic>siRNA</topic><topic>Tumor Suppressor Protein p53 - metabolism</topic><topic>Tumors</topic><topic>u-Plasminogen activator</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nagaraju, Ganji Purna Chandra</creatorcontrib><creatorcontrib>Nalla, Arun Kumar</creatorcontrib><creatorcontrib>Gupta, Reshu</creatorcontrib><creatorcontrib>Mohanam, Sanjeeva</creatorcontrib><creatorcontrib>Gujrati, Meena</creatorcontrib><creatorcontrib>Dinh, Dzung H</creatorcontrib><creatorcontrib>Rao, Jasti S</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Nucleic Acids Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Molecular cancer research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nagaraju, Ganji Purna Chandra</au><au>Nalla, Arun Kumar</au><au>Gupta, Reshu</au><au>Mohanam, Sanjeeva</au><au>Gujrati, Meena</au><au>Dinh, Dzung H</au><au>Rao, Jasti S</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>siRNA-mediated downregulation of MMP-9 and uPAR in combination with radiation induces G2/M cell-cycle arrest in Medulloblastoma</atitle><jtitle>Molecular cancer research</jtitle><addtitle>Mol Cancer Res</addtitle><date>2011-01-01</date><risdate>2011</risdate><volume>9</volume><issue>1</issue><spage>51</spage><epage>66</epage><pages>51-66</pages><issn>1541-7786</issn><eissn>1557-3125</eissn><abstract>Our previous work and that of other investigators strongly suggest a relationship between the upregulation of metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR) in tumor angiogenesis and metastasis. In this study, we evaluated the role of MMP-9 and uPAR in medulloblastoma cancer cell resistance to ionizing irradiation (IR) and tested the antitumor efficacy of siRNA (short interfering RNA) against MMP-9 [plasmid siRNA vector for MMP-9 (pM)] and uPAR [plasmid vector for uPAR (pU)] either alone or in combination [plasmid siRNA vector for both uPAR and MMP-9 (pUM)]. Cell proliferation (BrdU assay), apoptosis (in situ TUNEL for DNA fragmentation), and cell-cycle (FACS) analyses were carried out to determine the effect of siRNA either alone or in combination with IR on G2/M cell-cycle arrest in medulloblastoma cells. IR upregulated MMP-9 and uPAR expression in medulloblastoma cells; pM, pU, and pUM in combination with IR effectively reduced both MMP-9 and uPAR expression, thereby leading to increased radiosensitivity of medulloblastoma cells. siRNA treatments (pM, pU, and pUM) also promoted IR-induced apoptosis and enhanced IR-induced G2/M arrest during cell-cycle progression. While IR induces G2/M cell-cycle arrest through inhibition of the pCdc2- and cyclin B-regulated signaling pathways involving p53, p21/WAF1, and Chk2 gene expression, siRNA (pM, pU, and pUM) alone or in combination with IR induced G2/M arrest mediated through inhibition of the pCdc2- and cyclin B1-regulated signaling pathways involving Chk1 and Cdc25A gene expression. Taken together, our data suggest that downregulation of MMP-9 and uPAR induces Chk1-mediated G2/M cell-cycle arrest, whereas the disruption caused by IR alone is dependent on p53- and Chk2-mediated G2/M cell-cycle arrest.</abstract><cop>United States</cop><pmid>21148633</pmid><doi>10.1158/1541-7786.MCR-10-0399</doi><tpages>16</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Angiogenesis Antitumor activity Apoptosis Apoptosis - genetics Apoptosis - radiation effects Blotting, Western Cancer CDC2 Protein Kinase - metabolism Cdc25A phosphatase Cell Cycle - radiation effects Cell Cycle Proteins - metabolism Cell Division - radiation effects Cell Line, Tumor Cell Proliferation CHK1 protein Chk2 gene Cyclin-Dependent Kinase 2 - metabolism Cyclin-dependent kinase inhibitor p21 Data processing DNA fragmentation Down-Regulation Flow Cytometry G2 Phase - radiation effects Gelatinase B Gene Expression Regulation, Neoplastic - radiation effects Humans I.R. radiation Matrix Metalloproteinase 9 - genetics Matrix Metalloproteinase 9 - metabolism Medulloblastoma Medulloblastoma - genetics Medulloblastoma - metabolism Medulloblastoma - pathology Metastases Mitogen-Activated Protein Kinases - metabolism p53 protein Phosphatidylinositol 3-Kinases - metabolism Phosphorylation - radiation effects Plasmids Radiosensitivity ras Proteins - metabolism Receptors, Urokinase Plasminogen Activator - genetics Receptors, Urokinase Plasminogen Activator - metabolism Reverse Transcriptase Polymerase Chain Reaction RNA Interference RNA, Small Interfering - genetics Signal transduction siRNA Tumor Suppressor Protein p53 - metabolism Tumors u-Plasminogen activator |
| title | siRNA-mediated downregulation of MMP-9 and uPAR in combination with radiation induces G2/M cell-cycle arrest in Medulloblastoma |
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