[Retracted] Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

Following the publication of the above paper, we were contacted by the University of Illinois at Chicago, to request the retraction of the above article. Following a formal institutional investigation, the investigation panel concluded that the images in question had falsifying elements. Regarding the above study, the specific allegations that were investigated were that of falsifying elements of Fig. 6A, row 2, columns 2 and 3. Following a review of this paper conducted independently by the Editor of International Journal of Oncology, the Editor concurred with the conclusions of the investigation panel, and therefore the above paper has been retracted from the publication. We also tried to contact the authors, but did not receive a reply. The Editor apologizes to the readership for the inconvenience caused.[the original article was published in International Journal of Oncology 40: 509­518, 2012; DOI: 10.3892/ijo.2011.1255].

[Retracted] Oncogenic role of p53 is suppressed by si­RNA bicistronic construct of uPA, uPAR and cathepsin­B in meningiomas both in vitro and in vivo.

Following the publication of the above paper, we were contacted by the University of Illinois at Chicago, to request the retraction of the above article. Following a formal institutional investigation, the investigation panel concluded that the images in question had falsifying elements. Regarding the above study, the specific allegations that were investigated were that of falsifying elements of Fig. 1B, bottom panel, columns 2 and 3; Fig. 4A, top panel, columns 4, 5 and 6, and middle panel, columns 1, 2 and 3; and Fig. 7D, row 1, column 1 and row 2, column 1.
Following a review of this paper conducted independently by the Editor of International Journal of Oncology, the Editor concurred with the conclusions of the investigation panel, and therefore the above paper has been retracted from the publication. We also tried to contact the authors, but did not receive a reply. The Editor apologizes to the readership for the inconvenience caused. [the original article was published in International Journal of Oncology 38: 973­983, 2011; DOI: 10.3892/ijo.2011.934]

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Intraperitoneal injection of MSC-derived exosomes prevent experimental bronchopulmonary dysplasia.

Mesenchymal stromal cell (MSC) derived exosomes mediate tissue protection and regeneration in many injuries and diseases by modulating cell protein production, protecting from apoptosis, inhibiting inflammation, and increasing angiogenesis. In the present study, daily intraperitoneal injection of MSC-derived exosomes protected alveolarization and angiogenesis in a newborn rat model of bronchopulmonary dysplasia (BPD) induced by 14 days of neonatal hyperoxia exposure (85% O2). Exosome treatment during hyperoxia prevented disruption of alveolar growth, increased small blood vessel number, and inhibited right heart hypertrophy at P14, P21, and P56. In vitro, exosomes significantly increased tube-like network formation by HUVEC, in part through a VEGF mediated mechanism. In summary, daily intraperitoneal injection of exosomes increased blood vessel number and size in the lung through pro-angiogenic mechanisms. MSC-derived exosomes therefore have both anti-inflammatory and pro-angiogenic mechanism to protect the lung from hyperoxia induced lung and heart disease associated with BPD.

miR-let-7f-1 regulates SPARC mediated cisplatin resistance in medulloblastoma cells.

Our previous studies indicate that Secreted Protein Acidic and Rich in Cysteine (SPARC) expression suppressed medulloblastoma tumor growth in vitro and in vivo. Here we sought to determine the effect of SPARC expression in medulloblastoma cells to chemotherapeutic agents. In this study, we show that SPARC expression induces cisplatin resistance in medulloblastoma cells. We also demonstrate that the autophagy was involved in SPARC expression mediated resistance to cisplatin. Suppression of autophagy by either autophagy inhibitor, 3-methyladenosine (3MA) or Atg5 siRNA enhanced cisplatin sensitivity in SPARC expressed cells. Further, SPARC expression suppressed miR-let-7f-1 expression which resulted in disrupted repression of High Mobility Group Box 1 (HMGB1), a critical regulator of autophagy. We also show that HMGB1 is a direct target of miR-let-7f-1 and forced expression of HMGB1 cDNA enhanced cisplatin sensitivity in SPARC expressed cells. In summary, our results suggest that SPARC modulates cisplatin resistance by modulating the Let-7f-1 miRNA/HMGB1 axis in medulloblastoma cells.

Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration.

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 α9ß1 signaling. Our recent studies have clearly demonstrated the role of α9ß1 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 α9ß1 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 α9ß1 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 α9ß1 integrin and iNOS levels.

MicroRNA 203 Modulates Glioma Cell Migration via Robo1/ERK/MMP-9 Signaling.

Glioblastoma (GBM) is the most common and malignant primary adult brain cancer. Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM, and this site was thought to harbor multiple tumor suppressor genes associated with GBM, a region that also encodes microRNA-203 (miR-203). In this study, we sought to identify the role of miR-203 as a tumor suppressor in the pathogenesis of GBM. We analyzed the miR-203 expression data of GBM patients in 10 normal and 495 tumor tissue samples derived from The Cancer Genome Atlas data set. Quantitative real-time PCR and in situ hybridization in 10 high-grade GBM and 10 low-grade anaplastic astrocytoma tumor samples showed decreased levels of miR-203 expression in anaplastic astrocytoma and GBM tissues and cell lines. Exogenous expression of miR-203 using a plasmid expressing miR-203 precursor (pmiR-203) suppressed glioma cell proliferation, migration, and invasion. We determined that one relevant target of miR-203 was Robo1, given that miR-203 expression decreased mRNA and protein levels as determined by RT-PCR and Western blot analysis. Moreover, cotransfection experiments using a luciferase-based transcription reporter assay have shown direct regulation of Robo1 by miR-203. We also show that Robo1 mediates miR-203 mediated antimigratory functions as up-regulation of Robo1 abrogates miR-203 mediated antimigratory effects. We also show that miR-203 expression suppressed ERK phosphorylation and MMP-9 expression in glioma cells. Furthermore, we demonstrate that miR-203 inhibits migration of the glioma cells by disrupting the Robo1/ERK/MMP-9 signaling axis. Taken together, these studies demonstrate that up-regulation of Robo1 in response to the decrease in miR-203 in glioma cells is responsible for glioma tumor cell migration and invasion.

Suppression of tumor cell invasiveness and in vivo tumor growth by microRNA-874 in non-small cell lung cancer.

MicroRNAs are a novel family of small non-coding RNAs that regulate the expression of several genes involved in normal development as well as human disorders including cancer. Here we show that miR-874 plays a tumor suppressor role in non-small cell lung cancer (NSCLC) in vitro and in vivo. In silico target prediction analysis revealed numerous genes associated with tumor progression including MMP-2 and uPA as the putative target genes of miR-874. Our preliminary in situ hybridization experiments demonstrated the diminution of miR-874 expression in lung cancer tissues compared to their normal counter parts. Overexpression of miR-874 in CD133-positive cancer stem cell (CSC) population led to a significant loss in CSC-phenotype and enhanced sphere de-differentiation into epithelial-like cells. Restoration of miR-874 expression drastically reduced cell invading ability in comparison to mock and control-miR-treated cells by suppressing the protein levels of MMP-2 and uPA. In in vivo experiments, miR-874 treatment decreased orthotopic tumor growth in nude mice compared to mock and control-miR treatments. Further, the immunoreactivity of human anti-MMP-2 and anti-uPA was significantly reduced in tumor sections from mice that received miR-874 treatment. In conclusion, our study highlights the possible tumor suppressor role of miR-874 in NSCLC-initiating cells and suggests miR-874 as a potential target in the treatment of NSCLC.

The secreted protein acidic and rich in cysteine (SPARC) induces endoplasmic reticulum stress leading to autophagy-mediated apoptosis in neuroblastoma.

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 attempted 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. This 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.

Notch signaling regulates tumor-induced angiogenesis in SPARC-overexpressed neuroblastoma.

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.

Epigenetic regulation of miRNA-211 by MMP-9 governs glioma cell apoptosis, chemosensitivity and radiosensitivity.

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.

Blockade of SOX4 mediated DNA repair by SPARC enhances radioresponse in medulloblastoma.

SPARC is a matricellular glycoprotein and a putative radioresistance-reversal-gene. We therefore explored the possibility of SPARC expression on medulloblastoma radiosensitivity in vitro and in vivo. The combined treatment of the SPARC and irradiation resulted in increased cell death when compared to cells treated with irradiation alone in vitro and in vivo. SPARC expression prior to irradiation suppressed checkpoints-1,-2 and p53 phosphorylation and DNA repair gene XRCC1. We also demonstrate that SPARC expression suppressed irradiation induced SOX-4 mediated DNA repair. These results provide evidence of the anti-tumor effect of combining SPARC with irradiation as a new therapeutic strategy for the treatment of medulloblastoma.

Integrin α9ß1-mediated cell migration in glioblastoma via SSAT and Kir4.2 potassium channel pathway.

The α9ß1 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 α9ß1 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.

Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

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 the glycolytic metabolism of glioma cells 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 OXPHOS to glycolysis switch. The 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 changes 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 the glycolytic to the 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 the treatment of glioma.

RETRACTED: MMP-9 induces CD44 cleavage and CD44 mediated cell migration in glioblastoma xenograft cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor in Chief. On behalf the University of Illinois at Chicago, the Associate Vice Chancellor and Research Integrity Officer requested retraction of the article because of the elements listed here that were deemed false: Figure 5B, bottom row, Columns 1, 2, 4 and 5, top panel; Figure 5B, Row 1, Columns 1 and 2, bottom panel; Figure 5B, Row 2, Columns 3 and 4 bottom panel. Based on these circumstances the Editor in Chief has therefore decided to retract the paper. The corresponding author has been non-responsive to approaches from the Publisher.

SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells.

Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reduction in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.

Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice.

BACKGROUND: Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. CONCLUSION/SIGNIFICANCE: Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point.

MMP-9 silencing regulates hTERT expression via ß1 integrin-mediated FAK signaling and induces senescence in glioma xenograft cells.

In more than 90% 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, phospho FAK and ß1 integrin in glioma xenograft cells. Further, MMP-9 suppression decreased the interaction of ß1 integrin/FAK and also MMP-9/ß1 integrin as confirmed by immunoprecipitation analysis. Studies with either function blocking ß1 integrin or FAK shRNA indicate that suppression of MMP-9 decreased ß1 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 ß1 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.

MMP-2 siRNA inhibits radiation-enhanced invasiveness in glioma cells.

BACKGROUND: Our previous work and that of others strongly suggests a relationship between the infiltrative phenotype of gliomas and the expression of MMP-2. Radiation therapy, which represents one of the mainstays of glioma treatment, is known to increase cell invasion by inducing MMP-2. Thus, inhibition of MMP-2 provides a potential means for improving the efficacy of radiotherapy for malignant glioma. METHODOLOGY/PRINCIPAL FINDINGS: We have tested the ability of a plasmid vector-mediated MMP-2 siRNA (p-MMP-2) to modulate ionizing radiation-induced invasive phenotype in the human glioma cell lines U251 and U87. Cells that were transfected with p-MMP-2 with and without radiation showed a marked reduction of MMP-2 compared to controls and pSV-transfected cells. A significant reduction of proliferation, migration, invasion and angiogenesis of cells transfected with p-MMP-2 and in combination with radiation was observed compared to controls. Western blot analysis revealed that radiation-enhanced levels of VEGF, VEGFR-2, pVEGFR-2, p-FAK, and p-p38 were inhibited with p-MMP-2-transfected cells. TUNEL staining showed that radiation did not induce apoptosis in U87 and U251 cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to controls. Intracranial tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation compared to controls. CONCLUSION/SIGNIFICANCE: MMP-2 inhibition, mediated by p-MMP-2 and in combination with radiation, significantly reduced tumor cell migration, invasion, angiogenesis and tumor growth by modulating several important downstream signaling molecules and directing cells towards apoptosis. Taken together, our results demonstrate the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in glioma patients.