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1.  Knockdown of Cathepsin B and uPAR Inhibits CD151 and α3β1 Integrin-mediated Cell Adhesion and Invasion in Glioma 
Molecular carcinogenesis  2012;52(10):777-790.
Glioma is a highly complex brain tumor characterized by the dysregulation of proteins and genes that leads to tumor metastasis. Cathepsin B and uPAR are overexpressed in gliomas and they are postulated to play central roles in glioma metastasis. In this study, efficient downregulation of cathepsin B and uPAR by siRNA treatments significantly reduced glioma cell adhesion to laminin as compared to vitronectin, fibronectin, or collagen I in U251 and 4910 glioma cell lines. Brain glioma tissue array analysis showed high expression of CD151 in clinical samples when compared with normal brain tissue. Cathepsin B and uPAR siRNA treatment led to the downregulation of CD151 and laminin-binding integrins α3 and β1. Co- immunoprecipitation experiments revealed that downregulation of cathepsin B and uPAR decreased the interaction of CD151 with uPAR and α3β1 integrin. Studies on the downstream signaling cascade of uPAR/CD151/α3β1 integrin have shown that phosphorylation of FAK, SRC, paxillin and expression of adaptor cytoskeletal proteins talin and vinculin were reduced with knockdown of cathepsin B, uPAR and CD151. Treatment with the bicistronic construct reduced interactions between uPAR and CD151 as well as lowering α3β1 integrin, talin, and vinculin expression levels in pre-established glioma tumors of nude mice. In conclusion, our results show that downregulation of cathepsin B and uPAR alone and in combination inhibit glioma cell adhesion by downregulating CD151 and its associated signaling molecules in vitro and in vivo. Taken together, the results of the present study show that targeting the uPAR-cathepsin B system has possible therapeutic potential.
doi:10.1002/mc.21915
PMCID: PMC3525767  PMID: 22495828
CD151; Integrin; Laminin; Adhesion
2.  Cathepsin B and uPAR regulate self-renewal of glioma-initiating cells through GLI-regulated Sox2 and Bmi1 expression 
Carcinogenesis  2012;34(3):550-559.
Cancer-initiating cells comprise a heterogeneous population of undifferentiated cells with the capacity for self-renewal and high proliferative potential. We investigated the role of uPAR and cathepsin B in the maintenance of stem cell nature in glioma-initiating cells (GICs). Simultaneous knockdown of uPAR and cathepsin B significantly reduced the expression of CD133, Nestin, Sox2 and Bmi1 at the protein level and GLI1 and GLI2 at the messenger RNA level. Also, knockdown of uPAR and cathepsin B resulted in a reduction in the number of GICs as well as sphere size. These changes are mediated by Sox2 and Bmi1, downstream of hedgehog signaling. Addition of cyclopamine reduced the expression of Sox2 and Bmi1 along with GLI1 and GLI2 expression, induced differentiation and reduced subsphere formation of GICs thereby indicating that hedgehog signaling acts upstream of Sox2 and Bmi1. Further confirmation was obtained from increased luciferase expression under the control of a GLI-bound Sox2 and Bmi1 luciferase promoter. Simultaneous knockdown of uPAR and cathepsin B also reduced the expression of Nestin Sox2 and Bmi1 in vivo. Thus, our study highlights the importance of uPAR and cathepsin B in the regulation of malignant stem cell self-renewal through hedgehog components, Bmi1 and Sox2.
doi:10.1093/carcin/bgs375
PMCID: PMC3581601  PMID: 23222817
4.  Notch signaling regulates tumor-induced angiogenesis in SPARC-overexpressed neuroblastoma 
Angiogenesis  2012;16(1):85-100.
Despite existing aggressive treatment modalities, the prognosis for advanced stage neuroblastoma remains poor with significant long-term illness in disease survivors. Advance stage disease features are associated with tumor vascularity, and as such, angiogenesis inhibitors may prove useful along with current therapies. The matricellular protein, secreted protein acidic and rich in cysteine (SPARC), is known to inhibit proliferation and migration of endothelial cells stimulated by growth factors. Here, we sought to determine the effect of SPARC on neuroblastoma tumor cell-induced angiogenesis and to decipher the molecular mechanisms involved in angiogenesis inhibition. Conditioned medium from SPARC-overexpressed neuroblastoma cells (pSPARC-CM) inhibited endothelial tube formation, cell proliferation, induced programmed cell death and suppressed expression of pro-angiogenic molecules such as VEGF, FGF, PDGF, and MMP-9 in endothelial cells. Further analyses revealed that pSPARC-CM-suppressed expression of growth factors was mediated by inhibition of the Notch signaling pathway, and cells cultured on conditioned medium from tumor cells that overexpress both Notch intracellular domain (NICD-CM) and SPARC resumed the pSPARC-CM-suppressed capillary tube formation and growth factor expression in vitro. Further, SPARC overexpression in neuroblastoma cells inhibited neo-vascularization in vivo in a mouse dorsal air sac model. Furthermore, SPARC overexpression-induced endothelial cell death was observed by co-localization studies with TUNEL assay and an endothelial marker, CD31, in xenograft tumor sections from SPARC-overexpressed mice. Our data collectively suggest that SPARC overexpression induces endothelial cell apoptosis and inhibits angiogenesis both in vitro and in vivo.
doi:10.1007/s10456-012-9301-1
PMCID: PMC3527677  PMID: 22956186
Neuroblastoma; SPARC; Angiogenesis; Apoptosis
5.  Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration 
BMC Cancer  2013;13:590.
Background
Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Our recent studies have clearly demonstrated the role of α9β1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration.
Methods
MMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis.
Results
Immunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p130Cas were reduced with simultaneous knockdown of both MMP-9 and uPAR.
Conclusions
Taken together, our results from the present and earlier studies clearly demonstrate that α9β1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9β1 integrin and iNOS levels.
doi:10.1186/1471-2407-13-590
PMCID: PMC3878845  PMID: 24325546
Glioma; Nitric oxide; Migration; Integrin; Knockdown
6.  Multifunctional roles of urokinase plasminogen activator (uPA) in cancer stemness and chemoresistance of pancreatic cancer 
Molecular Biology of the Cell  2013;24(17):2620-2632.
This research highlights the multifunctional role of uPA in pancreatic cancer as a regulator of stemness and chemoresistance. uPA is found to be physically associated with the transcription factors Lhx2 and HOXA5 in the nucleus and regulated expression of p53.
Pancreatic ductal adenocarcinoma (PDAC) is almost always lethal. One of the underlying reasons for this lethality is believed to be the presence of cancer stem cells (CSC), which impart chemoresistance and promote recurrence, but the mechanisms responsible are unclear. Recently the poor prognosis of PDAC has been correlated with increased expression of urokinase plasminogen activator (uPA). In the present study we examine the role of uPA in the generation of PDAC CSC. We observe a subset of cells identifiable as a side population (SP) when sorted by flow cytometry of MIA PaCa-2 and PANC-1 pancreatic cancer cells that possess the properties of CSC. A large fraction of these SP cells are CD44 and CD24 positive, are gemcitabine resistant, possess sphere-forming ability, and exhibit increased tumorigenicity, known characteristics of cancer stemness. Increased tumorigenicity and gemcitabine resistance decrease after suppression of uPA. We observe that uPA interacts directly with transcription factors LIM homeobox-2 (Lhx2), homeobox transcription factor A5 (HOXA5), and Hey to possibly promote cancer stemness. uPA regulates Lhx2 expression by suppressing expression of miR-124 and p53 expression by repressing its promoter by inactivating HOXA5. These results demonstrate that regulation of gene transcription by uPA contributes to cancer stemness and clinical lethality.
doi:10.1091/mbc.E12-04-0306
PMCID: PMC3756915  PMID: 23864708
7.  Essential Role of Cooperative NF-κB and Stat3 Recruitment to ICAM-1 Intronic Consensus Elements in the Regulation of Radiation-induced Invasion and Migration in Glioma 
Oncogene  2012;32(43):10.1038/onc.2012.546.
Although radiotherapy improves survival in patients, GBMs tend to relapse with augmented tumor migration and invasion even after irradiation (IR). Aberrant NF-κB and Stat3 activation and interaction has been suggested in several human tumors. However, possible NF-κB/Stat3 interaction and the role of Stat3 in maintenance of NF-κB nuclear retention in glioblastoma (GBM) still remain unknown. Stat3 and NF-κB (p65) physically interact with one another in the nucleus in glioma tumors. Most importantly, GST pull-down assays identified that Stat3 binds to the p65 transactivation domain (TAD) and is present in the NF-κB DNA-binding complex. Irradiation significantly elevated nuclear phospho-p65/phospho-Stat3 interaction in correlation with increased ICAM-1 and sICAM-1 levels, migration and invasion in human glioma xenograft cell lines 4910 and 5310. ChIP and promoter luciferase activity assays confirmed the critical role of adjacent NF-κB (+399) and Stat3 (+479) binding motifs in the proximal intron-1 in elevating IR-induced ICAM-1 expression. Specific inhibition of Stat3 and NF-κB with Stat3.siRNA or JSH-23 severely inhibited IR-induced p65 recruitment onto ICAM-1 intron-1 and suppressed migratory properties in both cell lines. On the other hand, Stat3C- or IR-induced Stat3 promoter recruitment was significantly decreased in p65-knockdown cells, thereby suggesting the reciprocal regulation between p65 and Stat3. We also observed a significant increase in NF-κB enrichment on ICAM-1 intron-1 and ICAM-1 transactivation in Stat3C overexpressing cells. In in vivo orthotopic experiments, suppression of tumor growth in Stat3.si+IR-treated mice was associated with the inhibition of IR-induced p-p65/p-Stat3 nuclear-colocalization and ICAM-1 levels. To our knowledge, this is the first study showing the crucial role of NF-κB/Stat3 nuclear association in IR-induced ICAM-1 regulation and implies that targeting NF-κB/Stat3 interaction may have future therapeutic significance in glioma treatment.
doi:10.1038/onc.2012.546
PMCID: PMC3664652  PMID: 23178493
Chromatin immunoprecipitation (ChIP); Glioma; GST pull-down; NF-κB; Stat3; Transactivation domain (TAD)
8.  Role of MMP-2 in the Regulation of IL-6/Stat3 Survival Signaling via Interaction With α5β1 Integrin in glioma 
Oncogene  2012;32(3):327-340.
MMP-2 plays 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 siRNA (pM) in human glioma xenograft cell lines 4910 and 5310 decreased cell proliferation compared to mock- and pSV-(scrambled vector)treatments, as determined by BrDU 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 GM-CSF, IL-6, IL-8, IL-10, TNF-α, angiogenin, VEGF and PDGF-BB in MMP-2 knockdown cells. Further, cDNA PCR array indicated potential negative regulation of JAK/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. EMSA and ChIP 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 rhMMP-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 to mock- or pSV-treatments. Immunoflorescence 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 indicates 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.
doi:10.1038/onc.2012.52
PMCID: PMC3368064  PMID: 22349830
MMP-2; α5β1; glioma; IL-6; STAT3; CyclinD1; c-Myc
9.  Transcriptional Repression of Mad-Max Complex by Human Umbilical Cord Blood Stem Cells Downregulates Extracellular Signal-Regulated Kinase in Glioblastoma 
Stem Cells and Development  2011;21(10):1779-1793.
Previously, we have shown that human umbilical cord blood stem cell (hUCBSC) treatment downregulate cyclin D1 in glioma cells. To study the cell cycle progression and investigate the upstream molecules regulating cyclin D1 expression, we analyzed the involvement of extracellular signal-regulated kinase (ERK) and its functionality after treatment with hUCBSC. We observed downregulation of pERK after hUCBSC treatment at both transcriptional and translational levels. Increased translocation of ERK from cytoplasm to the nucleus was observed in glioma cells, whereas hUCBSC cocultures with glioma cells showed suppressed nuclear translocation. This finding suggests that hUCBSC regulates ERK by suppressing its phosphorylation at phospho-Thr202/Tyr204 retarding pERK nuclear translocation. ERK promoter analysis has shown c-Myc binding sites, indicative of possible transcriptional interactions that regulate cyclin D1 and ERK expression levels. Treatment of U251 and 5310 glioma cells with U0126, a MEK/ERK inhibitor receded pERK and c-Myc levels. In another experiment, U251 and 5310 cells treated with 10074-G5, c-Myc/Max inhibitor displayed reduction in pERK and c-Myc levels suggestive of a positive feedback loop between ERK/c-Myc/Max molecules. In the present study, we show that glioma cells exhibit abundant c-Myc expression and increased c-Myc/Max activity. In contrast, the glioma cells cocultured with hUCBSC demonstrated high Mad1 expression that competitively binds to Max to repress the c-Myc/Max mediated gene transcription. Our studies thus elucidate the potential role of hUCBSC in controlling glioma cell cycle progression and invasion by limiting Max binding to c-Myc, thus regulating the expression of glioma cell cycle and invasion associated molecules such as ERK, integrins via increased levels of Mad1 expression.
doi:10.1089/scd.2011.0424
PMCID: PMC3376458  PMID: 21933022
10.  Temporal Regulation of Apoptotic and Anti-apoptotic Molecules After Middle Cerebral Artery Occlusion Followed by Reperfusion 
Molecular Neurobiology  2013;49(1):50-65.
A tremendous effort has been expended to elucidate the role of apoptotic molecules in ischemia. However, many agents that target apoptosis, despite their proven efficacy in animal models, have failed to translate that efficacy and specificity in clinical settings. Therefore, comprehensive knowledge of apoptotic mechanisms involving key apoptotic regulatory molecules and the temporal expression profiles of various apoptotic molecules after cerebral ischemia may provide insight for the development of better therapeutic strategies aimed at cerebral ischemia. The present study investigates the extent of apoptosis and the regulation of apoptotic molecules both at mRNA and protein levels at various time points after focal cerebral ischemia in a rat model of middle cerebral artery occlusion. In this study, we performed various techniques, such as TTC (2,3,5-triphenyltetrazolium chloride), H&E (hematoxylin and eosin), and TUNEL (terminal deoxy nucleotidyl transferase-mediated nick-end labeling) staining, along with polymerase chain reaction (PCR) microarray, antibody microarray, reverse transcription (RT)-PCR, immunofluorescence, and immunoblot analyses. Our research provided a large list of pro-apoptotic and anti-apoptotic molecules and their temporal expression profiles both at the mRNA and protein levels. This information could be very useful for designing future stroke therapies and aid in targeting the right molecules at critical time to obtain maximum therapeutic benefit.
doi:10.1007/s12035-013-8486-7
PMCID: PMC3918127  PMID: 23813097
Ischemia; Apoptosis; Stroke; Reperfusion; Occlusion; Infarction
11.  The homing of human cord blood stem cells to sites of inflammation 
Cell Cycle  2012;11(12):2303-2313.
Efficient homing of human umbilical cord blood mesenchymal stem cells (hUCBSC) to inflammation sites is crucial for therapeutic use. In glioblastoma multiforme, soluble factors released by the tumor facilitate the migratory capacity of mesenchymal stem cells toward glioma cells. These factors include chemokines and growth inducers. Nonetheless, the mechanistic details of these factors involved in hUCBSC homing have not been clearly delineated. The present study is aimed to deduce specific factors involved in hUCBSC homing by utilizing a glioma stem cell-induced inflammatory lesion model in the mouse brain. Our results show that hUCBSC do not form tumors in athymic nude mice brains and do not elicit immune responses in immunocompetent SKH1 mice. Further, hUCBSC spheroids migrate and invade glioma spheroids, while no effect was observed on rat fetal brain aggregates. Several cytokines, including GRO, MCP-1, IL-8, IL-3, IL-10, Osteopontin and TGF-β2, were constitutively secreted in the naive hUCBSC-conditioned medium, while significant increases of IL-8, GRO, GRO-α, MCP-1 and MCP-2 were observed in glioma stem cell-challenged hUCBSC culture filtrates. Furthermore, hUCBSC showed a stronger migration capacity toward glioma stem cells in vitro and exhibited enhanced migration to glioma stem cells in an intracranial human malignant glioma xenograft model. Our results indicate that multiple cytokines are involved in recruitment of hUCBSC toward glioma stem cells, and that hUCBSC are a potential candidate for glioma therapy.
doi:10.4161/cc.20766
PMCID: PMC3383591  PMID: 22684297
CD-8; CXCR-4; GRO-α; IL-8; SDF-1; cord blood stem cells; glioma stem cells
12.  uPAR and cathepsin B inhibition enhanced radiation-induced apoptosis in gliomainitiating cells 
Neuro-Oncology  2012;14(6):745-760.
Glioblastomas present as diffuse tumors with invasion into normal brain tissue and frequently recur or progress after radiation as focal masses because of glioma-initiating cells. The role of the urokinase-type plasminogen activator receptor (uPAR) and cathepsin B in stem-like phenotype has been extensively studied in several solid tumors. In the present study, we demonstrated that selection of glioma-initiating cells using CD133 expression leads to a specific enrichment of CD133+ cells in both U87 and 4910 cells. In addition, CD133+ cells exhibited a considerable amount of other stem cell markers, such as Nestin and Sox-2. Radiation treatment significantly enhanced uPAR and cathepsin B levels in glioma-initiating cells. To downregulate radiation-induced uPAR and cathepsin B expression, we used a bicistronic shRNA construct that simultaneously targets both uPAR and cathepsin B (pCU). Downregulation of uPAR and cathepsin B using pCU decreased radiation-enhanced uPAR and cathepsin B levels and caused DNA damage-induced apoptosis in glioma cell lines and glioma-initiating cells. The most striking finding of this study is that knockdown of uPAR and cathepsin B inhibited ongoing transcription by suppressing BrUTP incorporation at γH2AX foci. In addition, uPAR and cathepsin B gene silencing inversely regulated survivin and H2AX expression in both glioma cells and glioma-initiating cells. Pretreatment with pCU reduced radiation-enhanced expression of uPAR, cathepsin B, and survivin and enhanced DNA damage in pre-established glioma in nude mice. Taken together, our in vitro and in vivo findings suggest that uPAR and cathepsin B inhibition might serve as an adjunct to radiation therapy to target glioma-initiating cells and, therefore, for the treatment of glioma.
doi:10.1093/neuonc/nos088
PMCID: PMC3367854  PMID: 22573309
apoptosis; glioma-initiating cells; γH2AX; radiation
13.  MMP-2 suppression abrogates irradiation-induced microtubule formation in endothelial cells by inhibiting αVβ3-mediated SDF-1/CXCR4 signaling 
International journal of oncology  2013;42(4):1279-1288.
The majority of glioblastoma multiformes (GBM) tumors recur after radiation (IR) treatment due to increased angiogenesis and IR-induced signaling events in endothelial cells (ECs) that are involved in tumor neovascularization; however, these signaling events have yet to be well characterized. In the present study, we observed that IR (8Gy) significantly elevated MMP-2 expression and gelatinolytic activity in 4910 and 5310 human GBM xenograft cells. In addition, ECs treated with tumor-conditioned media (CM) obtained from IR-treated 4910 and 5310 cells showed increased microtubule formation. In view of this finding, we investigated the possible anti-angiogenic effects of MMP-2 downregulation using siRNA (pM.si) in IR-treated cells. We also determined the effect of CM obtained from mock, pSV (scrambled vector) and pMMP-2.si on endothelial cell growth and vessel formation. pM.si-CM-treated ECs showed inhibited IR-CM-induced SDF-1, CXCR4, phospho-PI3K and phospho-AKT and αVβ3 expression levels. In vitro angiogenesis assays also showed that the pM.si+IR decreased IR-induced vessel formation in ECs. Immunofluorescence and immunoprecipitation experiments indicated the abrogation of αVβ3-SDF-1 interaction in pM.si-CM-treated ECs when compared to mock or pSV treatments. External supplementation of either rhMMP-2 or rhSDF-1 counteracted and noticeably reversed pM.si-inhibited SDF-1, CXCR4, phospho-PI3K, and phospho-AKT expression levels and angiogenesis, thereby confirming the role of MMP-2 in the regulation of αVβ3-mediated SDF-1/CXCR4 signaling. In addition to the in vitro results, the in vivo mouse dorsal air sac model also showed reduced angiogenesis after injection of pM.si alone or in combination with IR-treated xenograft cells. In contrast, injection of mock or pSV-treated cells resulted in robust formation of characteristic neovascularization. Collectively, our data demonstrate the role of MMP-2 in the regulation of SDF-1/CXCR4 signaling-mediated angiogenesis in ECs and show the anti-angiogenic efficacy of combining MMP-2 downregulation and IR when treating patients with GBM in the future.
doi:10.3892/ijo.2013.1806
PMCID: PMC3586295  PMID: 23381805
Angiogenesis; αVβ3 integrin; C-X-C chemokine receptor type-4 (CXCR-4); Immunoprecipitation; Matrix metalloprotease-2 (MMP-2); Radiation (IR); Stromal cell-derived factor-1 (SDF-1); tumor conditioned media
14.  Cathepsin B as a Cancer Target 
Cathepsin B is of significant importance to cancer therapy as it is involved in various pathologies and oncogenic processes in humans. Numerous studies have shown that cathepsin B overexpression is correlated with invasive and metastatic phenotypes in cancers. Studies have shown that abnormal regulation of cathepsin B causes cells to acquire an oncogenic character. Cathepsin B is normally associated with the lysosomes involved in autophagy and immune response, but its aberrant expression has been shown to lead to cancers. The proteolytic nature of cathepsin B has been attributed to the infiltrative nature of tumor cells, and it has been shown that cathepsin B is secreted into the extracellular matrix (ECM), thereby facilitating its destruction. Studies have demonstrated that suppression of the proteolytic activity of cathepsin B retards the infiltrative behavior of tumor cells. Further suppression of the expression of cathepsin B via antisense or RNAi also resulted in suppression of tumor growth. In addition, studies have shown that combined suppression of various molecules, such as proteases and protease receptors, including cathepsin B, can significantly suppress tumor growth. Therefore, targeting the activity or expression of cathepsin B is of significant therapeutic importance for cancer treatment.
doi:10.1517/14728222.2013.740461
PMCID: PMC3587140  PMID: 23293836
Cathepsin B; cancer therapy; cancer target; ECM; Cathepsin B signaling
15.  uPAR and Cathepsin B Downregulation Induces Apoptosis by Targeting Calcineurin A to BAD via Bcl-2 in Glioma 
Journal of Neuro-Oncology  2011;107(1):69-80.
Cathepsin B and urokinase plasminogen activator receptor (uPAR) are postulated to play key roles in glioma invasion. Calcineurin is one of the key regulators of mitochondrial-dependent apoptosis, but its mechanism is poorly understood. Hence, we studied subcellular localization of calcineurin after transcriptional downregulation of uPAR and cathepsin B in glioma. In the present study, efficient downregulation of uPAR and cathepsin B increased the translocation of calcineurin A from the mitochondria to the cytosol, decreased pBAD (S136) expression and its interaction with 14-3-3ζ, and increased the interaction of BAD with Bcl-Xl. Co-depletion of uPAR and cathepsin B induced mitochondrial translocation of BAD and caspase 3 as well as PARP activation, cytochrome c and SMAC release. These effects were inhibited by FK506 (10 μM), a specific inhibitor of calcineurin. Calcineurin A was co-localized and also co-immunoprecipitated with Bcl-2. This interaction decreased with co-depletion of uPAR and cathepsin B and also with Bcl-2 inhibitor, HA 14-1 (20 μg/mL). Altered localization and interaction of calcineurin A with Bcl-2 was also observed in vivo when uPAR and cathepsin B were downregulated. In conclusion, downregulation of uPAR and cathepsin B induced apoptosis by targeting calcineurin A to BAD via Bcl-2 in glioma.
doi:10.1007/s11060-011-0727-x
PMCID: PMC3286222  PMID: 21964739
Calcineurin; BAD; Bcl2; glioma
16.  MMP-2 suppression abrogates irradiation-induced microtubule formation in endothelial cells by inhibiting αvβ3-mediated SDF-1/CXCR4 signaling 
International Journal of Oncology  2013;42(4):1279-1288.
The majority of glioblastoma multiforme (GBM) tumors recur after radiation (IR) treatment due to increased angiogenesis and IR-induced signaling events in endothelial cells (ECs) that are involved in tumor neovascularization; however, these signaling events have yet to be well characterized. In the present study, we observed that IR (8 Gy) significantly elevated MMP-2 expression and gelatinolytic activity in 4910 and 5310 human GBM xenograft cells. In addition, ECs treated with tumor-conditioned media (CM) obtained from IR-treated 4910 and 5310 cells showed increased microtubule formation. In view of this finding, we investigated the possible anti-angiogenic effects of MMP-2 downregulation using siRNA (pM.si) in IR-treated cells. We also determined the effect of CM obtained from mock, pSV (scrambled vector) and pMMP-2.si on endothelial cell growth and vessel formation. pM.si-CM-treated ECs showed inhibited IR-CM-induced SDF-1, CXCR4, phospho-PI3K and phospho-AKT and αvβ3 expression levels. In vitro angiogenesis assays also showed that the pM.si+IR decreased IR-induced vessel formation in ECs. Immunofluorescence and immunoprecipitation experiments indicated the abrogation of αvβ3-SDF-1 interaction in pM.si-CM-treated ECs when compared to mock or pSV treatments. External supplementation of either rhMMP-2 or rhSDF-1 counteracted and noticeably reversed pM.si-inhibited SDF-1, CXCR4, phospho-PI3K and phospho-AKT expression levels and angiogenesis, thereby confirming the role of MMP-2 in the regulation of αvβ3-mediated SDF-1/CXCR4 signaling. In addition to the in vitro results, the in vivo mouse dorsal air sac model also showed reduced angiogenesis after injection of pM.si alone or in combination with IR-treated xenograft cells. In contrast, injection of mock or pSV-treated cells resulted in robust formation of characteristic neovascularization. Collectively, our data demonstrate the role of MMP-2 in the regulation of SDF-1/CXCR4 signaling-mediated angiogenesis in ECs and show the anti-angiogenic efficacy of combining MMP-2 downregulation and IR when treating patients with GBM in the future.
doi:10.3892/ijo.2013.1806
PMCID: PMC3586295  PMID: 23381805
angiogenesis; αvβ3 integrin; C-X-C chemokine receptor type-4; immunoprecipitation; matrix metalloprotease-2; radiation; stromal cell-derived factor-1; tumor conditioned media
17.  Metabolic Remodeling Precedes Mitochondrial Outer Membrane Permeabilization in Human Glioma Xenograft Cells 
International journal of oncology  2011;40(2):509-518.
Glioma cancer cells adapt to changing microenvironment and shift from mitochondrial oxidative phosphorylation to aerobic glycolysis for their metabolic needs irrespective of oxygen availability. In the present study, we show that silencing MMP-9 in combination with uPAR/cathepsin B switch glioma cells glycolytic metabolism to oxidative phosphorylation (OXPHOS) and generate reactive oxygen species (ROS) to predispose glioma cells to mitochondrial outer membrane permeabilization. shRNA for MMP-9 and uPAR (pMU) as well as shRNA for MMP-9 and cathepsin B (pMC) activated complexes of mitochondria involved in OXPHOS and inhibited glycolytic hexokinase expression. The decreased interaction of hexokinase 2 with mitochondria in the treated cells indicated the inhibition of glycolysis activation. Overexpression of Akt reversed the pMU- and pMC-mediated glycolysis to OXPHOS switch. OXPHOS un-coupler oligomycin A altered the expression levels of the Bcl-2 family of proteins; treatment with pMU or pMC reversed this effect and induced mitochondrial outer membrane permeabilization. In addition, our results show changes in mitochondrial pore transition to release cytochrome c due to change in the VDAC-Bcl-XL and BAX-BAK interaction with pMU and pMC treatments. Taken together, our results suggest that pMU and pMC treatments switch glioma cells from glycolytic to OXPHOS pathway through an inhibitory effect on Akt, ROS induction, and an increase of cytosolic cytochrome c accumulation. These results demonstrate the potential of pMU and pMC as therapeutic candidates for treatment of glioma.
doi:10.3892/ijo.2011.1255
PMCID: PMC3232313  PMID: 22076676
Aerobic glycolysis; MMP-9; uPAR; cathepsin B; oxidative phosphorylation; reactive oxygen species; Bcl-2 protein family; BAX; Bcl-XL; cytochrome c; apoptosis
18.  uPA and uPAR shRNA Inhibit Angiogenesis via Enhanced Secretion of SVEGFR1 Independent of GM-CSF but Dependent on TIMP-1 in Endothelial and Glioblastoma Cells 
Molecular Oncology  2011;6(1):33-47.
The uPA/uPAR system is known to play a critical role in angiogenesis of glioblastoma. Previously, we have shown that shRNA against uPA and uPAR attenuates angiogenesis by blocking nuclear translocation of angiogenin, inhibition of angiopoietin/Tie2 signaling, and regulating several other pro-angiogenic, angiostatic and anti-angiogenic molecules. Further analysis revealed that GM-CSF, a pleiotropic cytokine, was significantly inhibited in U87MG and 4910 co-cultures with endothelial cells transfected with shRNA against uPA and uPAR. The role of the uPA/uPAR system in this process is not completely understood. Analysis of tumor conditioned medium of U87MG, 4910 and HMEC cells transfected with shRNA against uPA or uPAR alone or in combination (pU2) revealed inhibition of GM-CSF-enhanced secretion of SVEGFR1 as shown by Western blotting and ELISA. Moreover, phosphorylation of JAK2 and STAT5, the downstream effectors of GM-CSF signaling, was also inhibited in all three cell lines. Phosphorylation at Tyr 166 position of the GM-CSFRβ subunit, the signal activating subunit of the GM-CSF receptor, was inhibited in HMEC, U87MG and 4910 cells. Further analysis revealed that shRNA against uPA and/or uPAR increased secretion of TIMP-1, which is known to enhance SVEGFR1 secretion in endothelial cells. Moreover, addition of purified uPA (with and without GM-CSF) activated JAK2/STAT5 signaling in HMEC. Exogenous addition of SVEGFR1 to pU2 tumor conditioned medium enhanced inhibition of VEGF-induced endothelial capillary tube formation as assessed by an in vitro angiogenesis assay. To determine the significance of these events in vivo, nude mice with pre-established tumors treated with shRNA against uPA and/or uPAR showed decreased levels of GM-CSF and increased levels of SVEGFR1 and TIMP-1 when compared with controls. Enhanced secretion of SVEGFR1 by puPA, puPAR and pU2 in endothelial and GBM cells was mediated indirectly by MMP-7 and augmented by ectodomain shedding of VEGFr1 by tyrosine phosphorylation at the 1213 position. Taken together, these results suggest that the uPA/uPAR system could prove beneficial as an indirect target for inhibition of angiogenesis in glioblastoma.
doi:10.1016/j.molonc.2011.11.008
PMCID: PMC3268919  PMID: 22177802
uPA and uPAR; shRNA; angiogenesis; glioblastoma
19.  Radiation-Induced Hypomethylation Triggers Urokinase Plasminogen Activator Transcription in Meningioma Cells1 
Neoplasia (New York, N.Y.)  2013;15(2):192-203.
Our previous studies have shown the role of radiation-induced urokinase plasminogen activator (uPA) expression in the progression of meningioma. In the present study, we investigated whether modulation of DNA methylation profiles could regulate uPA expression. Initially, radiation treatment was found to induce hypomethylation in meningioma cells with a decrease in DNA (cytosine-5)-methyltransferase 1 (DNMT1) and methyl-CpG binding domain protein (MBD) expression. However, oxidative damage by H2O2 or pretreatment of irradiated cells with N-acetyl cysteine (NAC) did not show any influence on these proteins, thereby indicating a radiation-specific change in the methylation patterns among meningioma cells. Further, we identified that hypomethylation is coupled to an increase in uPA expression in these cells. Azacytidine treatment induced a dose-dependent surge of uPA expression, whereas pre-treatment with sodium butyrate inhibited radiation-induced uPA expression, which complemented our prior results. Methylation-specific polymerase chain reaction on bisulfite-treated genomic DNA revealed a diminished methylation of uPA promoter in irradiated cells. Transfection with small hairpin RNA (shRNA)-expressing plasmids targeting CpG islands of the uPA promoter showed a marked decline in uPA expression with subsequent decrease in invasion and proliferation of meningioma cells. Further, radiation treatment was found to recruit SP1 transcription factor, which was abrogated by shRNA treatment. Analysis on signaling events demonstrated the activation of MAP kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) in radiation-treated cells, while U0126 (MEK/ERK inhibitor) blocked hypomethylation, recruitment of SP1, and uPA expression. In agreement with our in vitro data, low DNMT1 levels and high uPA were found in intracranial tumors treated with radiation compared to untreated tumors. In conclusion, our data suggest that radiation-mediated hypomethylation triggers uPA expression in meningioma cells.
PMCID: PMC3579321  PMID: 23441133
20.  SPARC induces endoplasmic reticulum stress leading to autophagy-mediated apoptosis in neuroblastoma 
International journal of oncology  2012;42(1):188-196.
Our previous studies showed that overexpression of Secreted Protein Acidic and Rich in Cysteine (SPARC) induced autophagy-mediated apoptosis in PNET cells. In the present study, we attempt to elucidate the molecular mechanisms and signaling cascades associated with SPARC overexpression in combination with radiation therapy that eventually leads to autophagy-mediated apoptosis in neuroblastoma. SPARC expression in SK-N-AS and NB-1691 cells demonstrated the activation of caspase 3, cleavage of PARP and induction of apoptosis. The experiments to unravel the mechanisms associated with autophagy -apoptosis illustrated that SPARC overexpression triggered endoplasmic reticulum (ER) stress and thereby unfolded protein response (UPR). This was apparent with the activation of stress receptors, inositol-requiring enzyme (IRE 1α), RNA-dependent protein kinase (PKR)-like ER kinase (PERK) and BiP. The study further demonstrated the induction of transcription factor CHOP as a result of IRE-JNK activation in response to increased SPARC levels. Inhibition of ER stress and JNK activation led to inhibition of autophagy-mediated apoptosis. Further, the apparent expression of ER stress molecules among the orthotopic tumors treated by SPARC overexpression plasmids substantiated our in vitro observations. Taken together, these results illustrate the critical role of ER stress in regulating autophagy-mediated apoptosis in SPARC-overexpressed neuroblastoma cells and radiation treatment.
doi:10.3892/ijo.2012.1678
PMCID: PMC3538858  PMID: 23123816
SPARC; Neuroblastoma; Autophagy; Apoptosis; ER stress
21.  Integrin α9β1-mediated cell migration in glioblastoma via SSAT and Kir4.2 potassium channel pathway 
Cellular signalling  2011;24(1):272-281.
The α9β1 integrin accelerates cell migration through binding of the α9 cytoplasmic domain to SSAT, which catalyzes the catabolism of higher order polyamines, spermidine and spermine, to the lower order polyamine, putrescine. SSAT levels were downregulated at both the mRNA and protein levels by shRNA-mediated simultaneous knockdown of MMP-9 and uPAR/cathepsin B. In addition, we noted a prominent reduction in the expression of SSAT with MMP-9 and uPAR/cathepsin B knockdown in the tumor regions of 5310 injected nude mice brains. Further, SSAT knockdown in glioma xenograft cells significantly reduced their migration potential. Interestingly, MMP-9, uPAR and cathepsin B overexpression in these xenograft cells significantly elevated SSAT mRNA and protein levels. The migratory potential of MMP-9/uPAR/cathepsin B-overexpressed 4910 and 5310 cells was not affected by either glybenclamide (Kir 6.x inhibitor) or tertiapin-Q (Kir 1.1 and 3.x inhibitor) but instead was significantly inhibited by either barium or Kir4.2 siRNA treatments. Co-localization of α9 integrin with Kir4.2 was observed in both 4910 and 5310 xenograft cells. However, MMP-9 and uPAR/cathepsin B knockdown in these cells prominently reduced the co-localization of α9 with Kir4.2. Taken together, our results clearly demonstrate that α9β1 integrin-mediated cell migration utilizes SSAT and the Kir4.2 potassium channel pathway, and inhibition of the migratory potential of these glioma xenograft cells by simultaneous knockdown of MMP-9 and uPAR/cathepsin B could be attributed to the reduced SSAT levels and co-localization of α9 integrin with Kir4.2 inward rectifier potassium channels.
doi:10.1016/j.cellsig.2011.09.011
PMCID: PMC3205329  PMID: 21946432
Migration; Integrin; Glioma; Knockdown; Xenograft; Potassium channel
22.  Chk2-mediated G2/M Cell Cycle Arrest Maintains Radiation Resistance in Malignant Meningioma Cells 
Cancer letters  2011;313(1):64-75.
In continuation to our studies on radioresistance in meningioma, here we show that radiation treatment (7Gy) induces G2/M cell cycle arrest in meningioma cells. Phosphorylation of Chk2, Cdc25c and Cdc2 were found to be key events since interference with Chk2 activation and cyclin B1/Cdc2 interaction led to permanent arrest followed by apoptosis. Irradiated cells showed recovery and formed aggressive intracranial tumors with rapid spread and morbidity. Nevertheless, knock down of uPAR with or without radiation induced permanent arrest in G2/M phase and subsequent apoptosis in vitro and in vivo. In conclusion, our data suggest that combination treatment with radiation and uPAR knockdown or other inhibitors resulting in non-reversible G2/M arrest may be beneficial in the management of meningiomas.
doi:10.1016/j.canlet.2011.08.022
PMCID: PMC3196767  PMID: 21945852
meningioma; radiotherapy; G2/M cell cycle arrest; Chk2
23.  MMP-9 Silencing Regulates hTERT Expression via β1 Integrin-mediated FAK Signaling and Induces Senescence in Glioma Xenograft Cells 
Cellular signalling  2011;23(12):2065-2075.
In more than 90 percent of cancers including glioma, telomere elongation reverse transcriptase (hTERT) is overexpressed. In the present study, we sought to explore whether matrix metalloproteinase-9 (MMP-9) shRNA could alter hTERT-mediated proliferation in glioma cells. MMP-9 shRNA induced senescence and apoptosis in glioma cells by inhibiting hTERT expression and telomere activity. MMP-9 silencing decreased oncogenic c-Myc expression (hTERT activator), whereas the expression of the c-Myc antagonist MAD increased drastically (hTERT repressor); both c-Myc and MAD are transcription factors for hTERT. In addition, MMP-9 suppression turns the switch from c-Myc/MAX to MAD/MAX heterodimer binding to the hTERT promoter as determined by chromatin immunoprecipitation assay. We also show that silencing MAD via siRNA restored hTERT expression and inhibited senescence in glioma cells. MMP-9 transcriptional suppression decreased the expression of FAK, phosphor FAK and β1 integrin in glioma xenograft cells. Further, MMP-9 suppression decreased the interaction of β1 integrin/FAK and also MMP-9/β1 integrin as confirmed by immunoprecipitation analysis. Studies with either function blocking β1 integrin or FAK shRNA indicate that suppression of MMP-9 decreased β1 integrin-mediated induction of FAK, which led to decreased hTERT expression. Moreover, 4910 and 5310 glioma xenograft tissue sections from mice treated with MMP-9 shRNA showed reduced expression of FAK/c-Myc and elevated MAD levels. Decreased co-localization of β1 integrin and MMP-9 was associated with MMP-9-suppressed tumor sections. Further, immunoprecipitation analysis showed decreased association of proteins involved in telomere end repair in MMP-9 shRNA-treated glioma cells. Elevated levels of p73 and TRAIL and the results of the FACS analysis show induction of apoptosis in MMP-9-silenced glioma cells. Taken together, these data provide new insights into the mechanisms underlying MMP-9-mediated hTERT expression in glioma proliferation.
doi:10.1016/j.cellsig.2011.08.001
PMCID: PMC3184383  PMID: 21855630
MMP-9; shRNA; hTERT; c-Myc/MAX/MAD; FAK; β1 integrin; senescence; apoptosis; glioma
24.  MMP-9 and uPAR regulated glioma cell migration 
Cell Adhesion & Migration  2012;6(6):509-512.
Integrin-dependent and -independent MMP-9 and uPAR signaling plays a key role in glioma cell migration and invasion. In this article, we comment on all the possible pathways and molecules associated with MMP-9- and uPAR-mediated glioma cell migration with a special emphasis on integrins, a family of cell adhesion molecules. Our recent research investigations highlighted the substantial benefit of silencing both MMP-9 and uPAR together compared with their individual treatments in glioma. Simultaneous knockdown of both MMP-9 and uPAR regulated a majority of the molecules associated with glioma cell migration and significantly reduced the migration potential of glioma cells. Our results point out that the bicistronic construct, which can simultaneously silence both MMP-9 and uPAR offers a great therapeutic potential and is worth developing as a new drug for treating GBM patients.
doi:10.4161/cam.21673
PMCID: PMC3547895  PMID: 23076139
MMP-9; uPAR; glioma; integrin; migration; invasion; knockdown; shRNA
25.  Epigenetic Regulation of miRNA-211 by MMP-9 Governs Glioma Cell Apoptosis, Chemosensitivity and Radiosensitivity 
Oncotarget  2012;3(11):1439-1454.
Glioblastoma multiforme (GBM) is the most aggressive brain cancer, and to date, no curative treatment has been developed. In this study, we report that miR-211, a microRNA predicted to target MMP-9, is suppressed in grade IV GBM specimens. Furthermore, we found that miR-211 suppression in GBM involves aberrant methylation-mediated epigenetic silencing of the miR-211 promoter. Indeed, we observed a highly significant inverse correlation between miR-211 expression and MMP-9 protein levels, which is indicative of post-transcriptional control of gene expression. Additionally, shRNA specific for MMP-9 (pM) promoted miR-211 expression via demethylation of miR-211 promoter-associated CpG islands (-140 to +56). In independent experiments, we confirmed that miR-211 overexpression and pM treatments led to the activation of the intrinsic mitochondrial/Caspase-9/3-mediated apoptotic pathway in both glioma cells and cancer stem cells (CSC). We also investigated whether miR-211 is involved in the regulation of MMP-9 and thus plays a functional role in GBM. We found an acute inhibitory effect of miR-211 on glioma cell invasion and migration via suppression of MMP-9. Given the insensitivity of some GBMs to radiation and chemotherapy (temozolomide) along with the hypothesis that glioma CSC cause resistance to therapy, our study indicates that miR-211 or pM in combination with ionizing radiation (IR) and temozolomide significantly induces apoptosis and DNA fragmentation. Of note, miR-211- and pM-treated CSC demonstrated increased drug retention capacity, as observed by MDR1/P-gp mediated-Rhodamine 123 drug efflux activity assay. These results suggest that either rescuing miR-211 expression or downregulation of MMP-9 may have a new therapeutic application for GBM patients in the future.
PMCID: PMC3717804  PMID: 23183822
Matrix metalloproteinase-9 (MMP-9); microRNA-211 (miR-211); Cancer Stem Cells (CSC); Ionizing radiation (IR); Temozolomide (Tem)

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