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Role of MMP-2 in the regulation of IL-6/Stat3 survival signaling via interaction with α5β1 integrin in glioma.

Kesanakurti D, Chetty C, Dinh DH, Gujrati M, Rao JS - Oncogene (2012)

Bottom Line: MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters.In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments.Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.

ABSTRACT
Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2'-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-α, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment.

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Effect of MMP-2 knockdown on proliferation in 4910 and 5310 glioma xenograft cell lines. A, Proliferation rate was measured by BrDU ELISA in mock-, pSV- and pM-treated 4910 and 5310 cells at 24, 48, 72 and 92 h after transfection. Percentage of BrDU incorporation which is directly proportional to proliferation percentage was calculated and data were represented as mean ± SE of three independent replicates with significance denoted by * at p<0.05, and ** at p<0.01. B, Cells were transfected for 48 hours and subjected to Ki-67 immunostaining as described in Materials and Methods and representative confocal images from different microscopic fields were shown. C, About 0.5×103 cells were seeded in 100 mm plates and were treated with mock, pSV and pM as described above. Plates were incubated for 10 days to allow colony formation, and stained with Hema-3. D, Colonies were counted under light microscope and percentage of colony formation was represented as mean±SE obtained form three independent experiments and significance was denoted by, * at p<0.01.
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Figure 1: Effect of MMP-2 knockdown on proliferation in 4910 and 5310 glioma xenograft cell lines. A, Proliferation rate was measured by BrDU ELISA in mock-, pSV- and pM-treated 4910 and 5310 cells at 24, 48, 72 and 92 h after transfection. Percentage of BrDU incorporation which is directly proportional to proliferation percentage was calculated and data were represented as mean ± SE of three independent replicates with significance denoted by * at p<0.05, and ** at p<0.01. B, Cells were transfected for 48 hours and subjected to Ki-67 immunostaining as described in Materials and Methods and representative confocal images from different microscopic fields were shown. C, About 0.5×103 cells were seeded in 100 mm plates and were treated with mock, pSV and pM as described above. Plates were incubated for 10 days to allow colony formation, and stained with Hema-3. D, Colonies were counted under light microscope and percentage of colony formation was represented as mean±SE obtained form three independent experiments and significance was denoted by, * at p<0.01.

Mentions: The significant inhibition in MMP-2 expression and activity in pM-treated 4910 and 5310 cells in comparison to mock- and pSV- treated cells at 48 h was confirmed by RT-PCR, Western blotting and gelatin zymography (Supplementary Figure S1A). BrDU proliferation assay at pre-determined time intervals indicated that the pM-transfected cells showed a time-dependent decrease in proliferation rate from 24 to 96 hours compared to mock and pSV-treated cells. A significant inhibition in proliferation up to 56.5% (72 hours) and 66.1% (96 hours) was documented in pM-treated 4910 cells. The 5310 cells displayed 56.2% and 65.4% decrease in BrDU incorporation at 72 and 96 hours of post-transfection, respectively (Figure 1A). Immunofluorescence showed decreased nuclear Ki-67 staining in pM-treated cells compared to mock- and pSV-treated controls (Figure 1B). Further, monolayer colony formation assay showed severe hindrance in the colony forming efficiency of pM-treated cells (Figure 1C). After 10 days, there was up to ≥70.8% and ≥71.2% inhibition in colony formation in pM-treated 4910 and 5310 cells respectively when compared to their control counterparts (Figure 1D).


Role of MMP-2 in the regulation of IL-6/Stat3 survival signaling via interaction with α5β1 integrin in glioma.

Kesanakurti D, Chetty C, Dinh DH, Gujrati M, Rao JS - Oncogene (2012)

Effect of MMP-2 knockdown on proliferation in 4910 and 5310 glioma xenograft cell lines. A, Proliferation rate was measured by BrDU ELISA in mock-, pSV- and pM-treated 4910 and 5310 cells at 24, 48, 72 and 92 h after transfection. Percentage of BrDU incorporation which is directly proportional to proliferation percentage was calculated and data were represented as mean ± SE of three independent replicates with significance denoted by * at p<0.05, and ** at p<0.01. B, Cells were transfected for 48 hours and subjected to Ki-67 immunostaining as described in Materials and Methods and representative confocal images from different microscopic fields were shown. C, About 0.5×103 cells were seeded in 100 mm plates and were treated with mock, pSV and pM as described above. Plates were incubated for 10 days to allow colony formation, and stained with Hema-3. D, Colonies were counted under light microscope and percentage of colony formation was represented as mean±SE obtained form three independent experiments and significance was denoted by, * at p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368064&req=5

Figure 1: Effect of MMP-2 knockdown on proliferation in 4910 and 5310 glioma xenograft cell lines. A, Proliferation rate was measured by BrDU ELISA in mock-, pSV- and pM-treated 4910 and 5310 cells at 24, 48, 72 and 92 h after transfection. Percentage of BrDU incorporation which is directly proportional to proliferation percentage was calculated and data were represented as mean ± SE of three independent replicates with significance denoted by * at p<0.05, and ** at p<0.01. B, Cells were transfected for 48 hours and subjected to Ki-67 immunostaining as described in Materials and Methods and representative confocal images from different microscopic fields were shown. C, About 0.5×103 cells were seeded in 100 mm plates and were treated with mock, pSV and pM as described above. Plates were incubated for 10 days to allow colony formation, and stained with Hema-3. D, Colonies were counted under light microscope and percentage of colony formation was represented as mean±SE obtained form three independent experiments and significance was denoted by, * at p<0.01.
Mentions: The significant inhibition in MMP-2 expression and activity in pM-treated 4910 and 5310 cells in comparison to mock- and pSV- treated cells at 48 h was confirmed by RT-PCR, Western blotting and gelatin zymography (Supplementary Figure S1A). BrDU proliferation assay at pre-determined time intervals indicated that the pM-transfected cells showed a time-dependent decrease in proliferation rate from 24 to 96 hours compared to mock and pSV-treated cells. A significant inhibition in proliferation up to 56.5% (72 hours) and 66.1% (96 hours) was documented in pM-treated 4910 cells. The 5310 cells displayed 56.2% and 65.4% decrease in BrDU incorporation at 72 and 96 hours of post-transfection, respectively (Figure 1A). Immunofluorescence showed decreased nuclear Ki-67 staining in pM-treated cells compared to mock- and pSV-treated controls (Figure 1B). Further, monolayer colony formation assay showed severe hindrance in the colony forming efficiency of pM-treated cells (Figure 1C). After 10 days, there was up to ≥70.8% and ≥71.2% inhibition in colony formation in pM-treated 4910 and 5310 cells respectively when compared to their control counterparts (Figure 1D).

Bottom Line: MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters.In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments.Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.

ABSTRACT
Matrix metalloproteinase-2 (MMP-2) has pivotal role in the degradation of extracellular matrix, and thereby enhances the invasive, proliferative and metastatic potential in cancer. Knockdown of MMP-2 using MMP-2 small interfering RNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared with mock and pSV (scrambled vector) treatments, as determined by 5-bromo-2'-deoxyuridine incorporation, Ki-67 staining and clonogenic survival assay. Cytokine array and western blotting using tumor-conditioned media displayed modulated secretory levels of various cytokines including granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor-α, angiogenin, vascular endothelial growth factor and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of Janus kinase/Stat3 pathway in pM-treated cells. Mechanistically, MMP-2 is involved in complex formation with α5 and β1 integrins and MMP-2 downregulation inhibited α5β1 integrin-mediated Stat3 phosphorylation and nuclear translocation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed inhibited Stat3 DNA-binding activity and recruitment at CyclinD1 and c-Myc promoters in pM-treated cells. In individual experiments, IL-6 or siRNA-insensitive MMP-2 overexpression by pM-FL-A141G counteracted and restored the pM-inhibited Stat3 DNA-binding activity, suggesting IL-6/Stat3 signaling suppression in pM-treated 4910 and 5310 cells. MMP-2/α5β1 binding is enhanced in human recombinant MMP-2 treatments, resulting in elevated Stat3 DNA-binding activity and recruitment on CyclinD1 and c-Myc promoters. Activation of α5β1 signaling by Fibronectin adhesion elevated pM-inhibited Stat3 phosphorylation whereas blocking α5β1 abrogated constitutive Stat3 activation. In vivo experiments with orthotropic tumor model revealed the decreased tumor size in pM treatment compared with mock or pSV treatments. Immunofluorescence studies in tumor sections corroborated our in vitro findings evidencing high expression and co-localization of MMP-2/α5β1, which is decreased upon pM treatment along with significantly reduced IL-6, phospho-Stat3, CyclinD1, c-Myc, Ki-67 and PCNA expression levels. Our data indicate the possible role of MMP-2/α5β1 interaction in the regulation of α5β1-mediated IL-6/Stat3 signaling activation and signifies the therapeutic potential of blocking MMP-2/α5β1 interaction in glioma treatment.

Show MeSH
Related in: MedlinePlus