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This paper is aimed at a comparative investigation on two different velocity profiles for piston movement namely Sinusoidal and Trapezoidal Profiles for an IC Engine. In conventional IC Engine, velocity profile of piston motion is Sinusoidal. It has many disadvantages such as high mean velocity that leads to high inertial force, frictional losses, wear and high rate of heat leakages. Nearly 20% of the total power produced by the engine is dissipated into heat because of friction. Of this 20%, about 75% is due to friction of piston rings on the cylinder walls. This is an irreversible loss and can be seen as a consequence of high mean piston velocity associated with the existing Sinusoidal Piston Velocity Profile. In addition, varying velocity profile can cause rapid acceleration and finally jerks which lead to considerable mechanical vibration and noise. As a result the mechanical strength of engine material will be high to withstand the inertial force, friction and wear. To overcome these difficulties, an extensive attempt is made to improve the piston movement by restructuring the piston velocity profile with reduced mean velocity which is constant for most of the crank angle. A comprehensive experimental examination is conducted for the Sinusoidal velocity profile, which are utilized in arriving at an optimal CFD procedure through validation study. A proposed connecting rod configuration with internal gear and pinion arrangement is proposed to achieve different Trapezoidal Profiles. The optimum CFD procedure found from validation study is used to analyze and understand the engine with modified Trapezoidal Velocity Profiles. There is almost 20% reduction of mean piston velocity that considerably improves hydro-thermo dynamic and mechanical characteristics of the existing engine.

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. 2A, right panel, row 3, columns 2, 3 and 4 and Fig. 4D, left panel, row 5, columns 1, 2 and 3; Fig. 4A, row 1, columns 2, 3 and 4, and Fig. 4C, row 1, columns 5, 6 and 7; and Fig. 6C, row 1, column 3, 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 40: 1615-1624, 2012; DOI: 10.3892/ijo.2011.987].

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: 973‑983, 2011; DOI: 10.3892/ijo.2011.934], , .

Tumor recurrence in glioblastoma (GBM) is, in part, attributed to increased epithelial-to-mesenchymal transition (EMT) and enhanced tumor cell dissemination in adjacent brain parenchyma after ionizing radiation (IR). EMT is associated with aggressive behavior, increased stem-like characteristics and treatment resistance in malignancies; however, the underlying signaling mechanisms that regulate EMT are poorly understood. We identified grade-dependent p21-activated kinases 4 (PAK4) upregulation in gliomas and further determined its role in mesenchymal transition and radioresistance. IR treatment significantly elevated expression and nuclear localization of PAK4 in correlation with induction of reactive oxygen species (ROS) and mesenchymal transition in GBM cells. Stable PAK4 overexpression promoted mesenchymal transition by elevating EMT marker expression in these cells. Of note, transcription factor-DNA-binding arrays and chromatin immunoprecipitation experiments identified the formation of a novel nuclear PAK4/PPARγ complex which was recruited to the promoter of Nox1, a peroxisome proliferator-activated receptor gamma (PPARγ) target gene. In addition, IR further elevated PAK4/PPARγ complex co-recruitment to Nox1 promoter, and increased Nox1 expression and ROS levels associated with mesenchymal transition in these cells. Conversely, specific PAK4 downregulation decreased PPARγ-mediated Nox1 expression and suppressed EMT in IR-treated cells. In vivo orthotopic tumor experiments showed inhibition of growth and suppression of IR-induced PPARγ and Nox1 expression by PAK4 downregulation in tumors. Our results provide the first evidence of a novel role for PAK4 in IR-induced EMT and suggest potential therapeutic efficacy of targeting PAK4 to overcome radioresistance in gliomas.

Background: Neuroblastoma is the most common extracranial pediatric solid tumor. Intermittent hypoxia, which is characterized by cyclic periods of hypoxia and reoxygenation, has been shown to positively modulate tumor development and thereby induce tumor growth, angiogenic processes, and metastasis. Bone is one of the target organs of metastasis in advanced neuroblastoma Neuroblastoma cells produce osteoclast-activating factors that increase bone resorption by the osteoclasts. The present study focuses on how intermittent hypoxia preconditioned SH-SY5Y neuroblastoma cells modulate osteoclastogenesis in RAW 264.7 cells compared with neuroblastoma cells grown at normoxic conditions., Methods: We inhibited HIF-1α and HIF-2α in neuroblastoma SH-SY5Y cells by siRNA/shRNA approaches. Protein expression of HIF-1α, HIF-2α and MAPKs were investigated by western blotting. Expression of osteoclastogenic factors were determined by real-time RT-PCR. The influence of intermittent hypoxia and HIF-1α siRNA on migration of neuroblastoma cells and in vitro differentiation of RAW 264.7 cells were assessed. Intratibial injection was performed with SH-SY5Y stable luciferase-expressing cells and in vivo bioluminescence imaging was used in the analysis of tumor growth in bone., Results: Upregulation of mRNAs of osteoclastogenic factors VEGF and RANKL was observed in intermittent hypoxia-exposed neuroblastoma cells. Conditioned medium from the intermittent hypoxia-exposed neuroblastoma cells was found to enhance osteoclastogenesis, up-regulate the mRNAs of osteoclast marker genes including TRAP, CaSR and cathepsin K and induce the activation of ERK, JNK, and p38 in RAW 264.7 cells. Intermittent hypoxia-exposed neuroblastoma cells showed an increased migratory pattern compared with the parental cells. A significant increase of tumor volume was found in animals that received the intermittent hypoxia-exposed cells intratibially compared with parental cells., Conclusions: Intermittent hypoxic exposure enhanced capabilities of neuroblastoma cells in induction of osteoclast differentiation in RAW 264.7 cells. Increased migration and intratibial tumor growth was observed in intermittent hypoxia-exposed neuroblastoma cells compared with parental cells.

Background: Neuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells., Methods and Findings: Cell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest/sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1α and HIF-2 α proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acid-induced differentiation of neuroblastoma cells., Conclusions: Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells.

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are overexpressed in gliomas. Deregulation of the G1 phase cell cycle machinery is a common feature of cancers. p27(Kip1) (p27) is one of the major cyclin-CDK regulators in the G1 phase. uPAR and cathepsin B downregulation was recently shown to induce p27 expression through PI3K/Akt/FOXO3a signaling. Since uPAR and cathepsin B knockdown also decreased phosphorylation of ERK, we hypothesized that ERK also has a role to play in p27 induction. As induction of p27 is due to an increase in gene transcription, we investigated the roles of c-Myc and E2F1 transcription factors which have been shown to potently affect p27 promoter activity. In the present study, shRNA against cathepsin B and uPAR as well as specific inhibitors, Wortmannin (10 μM) and U0126 (10 μM), were used to determine the roles of AKT and ERK signaling on p27 expression. Immunoblot analysis demonstrated that downregulation of both p-ERK and p-AKT downstream of EGFR and β1 integrin are involved in the p27 upregulation. Cathepsin B and uPAR downregulation induced E2F1 and decreased phosphorylaion of pocket proteins and c-Myc expression. CHIP analysis and luciferase expression studies confirmed the functional association of transcription factor E2F1 to the p27 promoter. Further, c-Myc-Max interaction inhibitor studies showed an inverse pattern of c-Myc and p27 expression. Also, cathepsin B and uPAR downregulation reduced tumor growth and increased p27 nuclear expression in vivo. In summary, cathepsin B and uPAR downregulation reduced p-ERK levels and c-Myc expression, increased expression of E2F1 and FOXO3a, decreased phosphorylation of pocket proteins and thus upregulated p27 expression in glioma cells., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Medulloblastomas are the most common malignant tumors of the central nervous system during childhood. Radiation-induced medulloblastoma tumor recurrences are aggressive and metastatic in nature. In the present study, we demonstrate that Gadd45a expression can sensitize medulloblastoma cells to radiotherapy. We have elucidated the role of Gadd45a in ionizing radiation (IR)-induced G2-M arrest and invasion and metastatic potential of the medulloblastoma cancer cell lines DAOY and D283. We demonstrate that Gadd45a is induced by IR and results in p53 phosphorylation. The role of IR-induced Gadd45a in G2-M arrest is demonstrated by fluorescence-activated cell sorting analysis in the cells treated with siRNA Gadd45a and Ov-exp Gadd45a. We show that Ov-exp Gadd45a aggravates G2-M blockage and also increases binding of Gadd45a to Cdc2 by immunocytochemistry analysis. Furthermore, we show the anti-tumorigenic role of Gadd45a to be mediated by the negative regulation of IR-induced cancer cell invasion and migration-associated proteins, such as matrix metallopeptidase (MMP)-9 and β-catenin. When compared with IR treatment alone, Ov-exp Gadd45a plus IR treatment resulted in decreased nuclear localization and increased membrane localization of β-catenin, and this was further confirmed by membrane distribution. We also show that Ov-exp Gadd45a resulted in downregulation of MMP-9 and suppression of epithelial-mesenchymal transition (EMT). Alternatively, inhibition of MMP-9 (pM) resulted in upregulation of Gadd45a and suppression of EMT. The anti-tumor effect of pM was correlated with increased expression of Gadd45a protein in nude mice intracranial tumors. Taken together, our studies demonstrate that upregulation of Gadd45a or suppression of MMP-9 (pM) with IR retards medulloblastoma tumor metastatic potential.

Cell motility is influenced by the microenvironment, signal transduction and cytoskeleton rearrangement. Cancer cells become resistant to these control mechanisms and gain the ability to move throughout the body and invade healthy tissues, which leads to metastatic disease. Integrins respond to context-dependent cues and promote cell migration and survival in cancer cells. In the present study, we analyzed the role of integrins in radiation-induced migration of meningioma cells. Migration and cell proliferation assays revealed that radiation treatment (7 Gy) significantly increased migration and decreased proliferation in two cell lines, IOMM-Lee and CH-157-MN. α3 and β1 integrins were overexpressed at both the protein and transcript levels after radiation treatment and a function-blocking α3β1 antibody inhibited the radiation-induced migration. Immunofluorescence studies illustrated the localization of α3 integrin and F-actin at the migration front of irradiated cells. Further, an increase in phosphorylation of FAK and ERK was observed, while both FAK phosphorylation inhibitor and FAK shRNA inhibited ERK phosphorylation and downregulated uPA and vinculin. In addition to the co-localization of FAK and ERK at the migration front, these FAK-inhibition results link the downstream effects of ERK to FAK. Correspondingly, U0126 quenched ERK phosphorylation and reduced the expression of molecules involved in migration. Furthermore, brain sections of the animals implanted with tumors followed by radiation treatment showed elevated levels of α3 integrin and active ERK. Taken together, our results show that radiation treatment enhances the migration of meningioma cells with the involvement of α3β1 integrin-mediated signaling via FAK and ERK.

Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Patients whose tumors recur after surgery and radiation therapy have limited therapeutic options. It has also been reported recently that radiation triggers DNA repair, cell survival and cell proliferation, and reduces apoptosis via the induction of cellular protective mechanisms. Earlier studies have reported that proteases such as uPA, uPAR and cathepsin B play important roles in tumor progression. In the present study, we attempted to determine the effectiveness of two bicistronic siRNA constructs pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation, both in in vitro and in vivo models. Transfection of a plasmid vector expressing double-stranded RNA for uPA, uPAR and cathepsin B significantly induced the sub-G0-G1 cell population by the mitochondrial intrinsic apoptotic pathway. Results showed that pUC efficiently enhanced sub-G0-G1 phases compared to pU2 and was more effective. Interestingly, we observed that in IOMM-Lee cell lines, combined treatment of radiation with pUC and pU2 is more effective in comparison to SF-3061 and MN cell lines. We showed that apoptosis caused by these bicistronic constructs involves Bcl-2, Bcl-xL, p53 inactivation, cytochrome c release from mitochondria and caspase-9 activation, followed by the activation of caspase-3. We also determined that apoptosis caused by pUC and pU2 involves a mechanism which includes inactivation of p53 by its translocation from nucleus to cytoplasm as confirmed by immunofluorescence, which shows the oncogenic potential of p53 in meningiomas. However, the simultaneous RNAi-mediated targeting of uPAR and cathepsin B (pUC), in combination with irradiation, has greater potential application for the treatment of human meningioma in comparison to pU2 by decreasing p53 expression both in vitro and in vivo.

Background: The normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, often is considered a rate-limiting step during cell cycle progression through the G(1) phase., Methodology/principal Findings: In the present study, human umbilical cord blood stem cells (hUCBSC) in co-cultures with glioblastoma cells (U251 and 5310) not only induced G(0)-G(1) phase arrest, but also reduced the number of cells at S and G(2)-M phases of cell cycle. Cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC as revealed by Western and FACS analyses. Inhibition of cyclin D1 activity by hUCBSC treatment is sufficient to abolish the expression levels of Cdk 4, Cdk 6, cyclin B1, β-Catenin levels. Our immuno precipitation experiments present evidence that, treatment of glioma cells with hUCBSC leads to the arrest of cell-cycle progression through inactivation of both cyclin D1/Cdk 4 and cyclin D1/Cdk 6 complexes. It is observed that hUCBSC, when co-cultured with glioma cells, caused an increased G(0)-G(1) phase despite the reduction of G(0)-G(1) regulatory proteins cyclin D1 and Cdk 4. We found that this reduction of G(0)-G(1) regulatory proteins, cyclin D1 and Cdk 4 may be in part compensated by the expression of cyclin E1, when co-cultured with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated, decreased expression of cyclin D1 in the presence of hUCBSC., Conclusions/significance: This paper elucidates a model to regulate glioma cell cycle progression in which hUCBSC acts to control cyclin D1 induction and in concert its partner kinases, Cdk 4 and Cdk 6 by mediating cell cycle arrest at G(0)-G(1) phase.

Hypoxia is known to induce overexpression of the urokinase plasminogen activator (uPA) and its receptor (uPAR) and thus overexpression promotes uPAR-mediated survival signaling in various cancers. Moreover, hypoxia/ overexpression of uPAR in cancer cells promote the epithelial-mesenchymal transition (EMT) and thereby invasiveness and metastasis. In this study, we show that intermittent hypoxia has a more pronounced effect than chronic hypoxia and contributes to EMT, invasion and migration in medulloblastoma cells. Intermittent hypoxia induced expression of mesenchymal markers (i.e., SNAIL, Vimentin and N-cadherin) and reduced expression of epithelial markers (i.e., Zo-1, E-cadherin) in medulloblastoma cells. Further, intermittent hypoxia also leads to enhancement in cell invasion, migration and angiogenesis in medulloblastoma cells. Intermittent hypoxia also inhibited expression of pro-anti-apoptotic proteins (Bax and Bad), and induced expression of anti-pro-apoptotic proteins (Bcl2 and Bcl-xL), and activation of ERK in medulloblastoma cells. Transcriptional inactivation of either uPA or uPAR inhibits the intermittent hypoxia-induced invasion and migration, and expression of Vimentin. uPA/ uPAR downregulation also induces E-cadherin expression and inhibits activation of ERK. Thus, transcriptional inactivation of either uPA or uPAR enhances the apoptotic response in medulloblastoma cells exposed to intermittent hypoxia. This study provides evidence of the anti-tumor efficacy of down-regulation of uPA or uPAR in medulloblastoma tumors to target hypoxia-induced cell EMT, invasion and migration, to achieve better therapeutic outcomes in the treatment of malignant medulloblastoma.

Background: Meningiomas are the most commonly occurring intracranial tumors and account for approximately 15-20% of central nervous system tumors. Surgery and radiation therapy is a common treatment for brain tumors, however, patients whose tumors recur after such treatments have limited therapeutic options. Earlier studies have reported important roles of uPA, uPAR and cathepsin B in tumor progression., Methodology/principal Findings: In the present study, we examined the therapeutic significance of RNAi-mediated simultaneous down regulation of these proteolytic networks using two bicistronic siRNA constructs, pUC (uPAR/cathepsin B) and pU2 (uPA/uPAR) either alone or in combination with radiation in two different meningioma cell lines. Transfection of meningioma cells with pUC and pU2 significantly reduced angiogenesis as compared to control treatment both in vitro and in vivo nude mice model. This effect is mediated by inhibiting angiogenic molecules (Ang-1, Ang-2 and VEGF). Expression of focal adhesion kinase (FAK) is elevated in malignant meningioma, yet the role of intrinsic FAK activity in promoting tumor progression remains undefined. We found that pUC treatment reduced FAK phosphorylation at Y925 more efficiently compared to pU2 treatment. In immunoprecipitation assay, we found pronounced reduction of FAK (Y925) interaction with Grb2 in meningioma cells transfected with pUC with and without irradiation. Transient over-expression of uPAR and cathepsin B by full length uPAR/cathepsin B (FLpU/C) in pUC transfected meningioma cells promoted vascular phenotype, rescued expression of Ang-1, Ang-2, VEGF, FAK (Y925) and Grb2 both in vitro and in vivo mice model., Conclusion/significance: These studies provide the first direct proof that bicistronic siRNA construct for uPAR and cathepsin B (pUC) reduces Y925-FAK activity and this inhibition is rescued by overexpression of both uPAR and cathepsin B which clearly demonstrates that pUC could thus be a potential therapeutic approach as an anti-angiogenic agent in meningioma.

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., (©2010 AACR.)

Angiogenesis involves the formation of new blood vessels by rerouting or remodeling existing ones and is believed to be the primary method of vessel formation in gliomas. To study the mechanisms by which angiogenesis of glioma cells can be inhibited by human umbilical cord blood stem cells (hUCBSC), we studied two glioma cell lines (SNB19, U251) and a glioma xenograft cell line (5310) alone and in co-culture with hUCBSC. Conditioned media from co-cultures of glioma cells with hUCBSC showed reduced angiogenesis as evaluated by in vitro angiogenesis assay using HMEC cells. Reduction in angiogenesis was associated with downregulation of FAK and integrin αvβ3 in the co-cultures of glioma cells. Downregulation of FAK gene is correlated with downregulation of many angiogenesis-related genes, including Ang1, VEGFA and Akt. Under in vivo conditions, neovascularization by glioma cells was inhibited by hUCBSC. Further, intracranial tumor growth was inhibited by hUCBSC in athymic nude mice. Similar to in vitro results, we observed downregulation of FAK, VEGF and Akt molecules to inhibit angiogenesis in the hUCBSC-treated nude mice brains. Taken together, our results suggest that hUCBSC have the potential to inhibit the angiogenesis of glioma cells both in vitro and in vivo.

Background: Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes., Methodology/principal Findings: In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results., Conclusions/significance: In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.

Background: In our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood., Methodology/principal Findings: In the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells., Conclusions/significance: Taken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway.

Matrix Metalloproteinase-9 (MMP-9) consists of a prodomain, catalytic domain with 3 fibronectin-like type II modules and C-terminal hemopexin-like (PEX) domain. These domains play distinct roles in terms of proteolytic activity, substrate binding and interaction with inhibitors and receptors. To assess the potential of the MMP-9-PEX domain to interfere with tumor progression, we stably transfected human glioblastoma cells with an expression vector containing a cDNA sequence of the MMP-9-PEX. The selected clones exhibited decreased MMP-9 activity and reduced invasive capacity. We assessed how secretion of MMP-9-PEX by glioblastoma cells affects angiogenic capabilities of human microvascular endothelial cells (HMECs) in vitro. MMP-9-PEX conditioned medium treatment caused a reduction in migration of HMECs and inhibited capillary-like structure formation in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor-2 protein level. The suppression of HMECs survival by conditioned medium from MMP-9-PEX stable transfectants was associated with apoptosis induction characterized by an increase in cells with a sub-G0/G1 content, fragmentation of DNA, caspase-3, -8 and -9 activation and poly (ADP-ribose) polymerase (PARP) cleavage. A significant tumor growth inhibition was observed in intracranial implants of MMP-9-PEX stable transfectants in nude mice with attenuation of CD31 and MMP-9 protein expression. These results demonstrate that MMP-9-PEX inhibits angiogenic features of endothelial cells and retards intracranial glioblastoma growth., (Copyright (c) 2008 Wiley-Liss, Inc.)

Epidemiological studies have indicated that increased consumption of cruciferous vegetables is associated with a statistically significant reduction in the risk for cancers. The major bioactive agent in these vegetables is a class of sulfur-containing glycosides called glucosinolates. Isothiocyanates, derivatives of glucosinolates, have been shown to possess anticancer properties in a variety of tumor cell lines. In this study, we evaluated the antigrowth, cell cycle modulation and proapoptotic effects of isothiocyanate iberin in human neuroblastoma cells. Treatment of neuroblastoma cells with iberin resulted in a dose- and time-dependent inhibition of growth, increased cytotoxicity, and G1 or G2 cell cycle arrest depending upon cell type. The iberin-induced cell cycle arrest in neuroblastoma cells was associated with inhibition of expression of Cdk2, Cdk4, and Cdk6 proteins. Fluorescence microscopic analysis of DNA-staining patterns with DAPI revealed an increase in apoptotic cell death in iberin-treated cells as compared with control cells. FLICA staining showed that iberin induced apoptosis, and this apoptotic induction was found to be associated with the activation of caspase-9, caspase-3, and PARP. These findings suggest that the anticancer efficacy of iberin is mediated via induction of cell cycle arrest and apoptosis in human neuroblastoma cells and has strong potential for development as a therapeutic agent against cancer.

The hypoxic microenvironment of solid tumors is associated with malignant progression and it renders tumors more resistant to cancer therapies. Endothelial cell damage may occur following hypoxic conditions and lead to dysfunction; however, endothelial cells in tumors survive hypoxic conditions providing nutrients and oxygen to facilitate tumor growth. In this study, we investigated the effects of tumor-conditioned medium on hypoxia-induced changes in endothelial cell growth, migration and survival. Tumor conditioned medium collected from U87 human glioblastoma cells were applied to endothelial cultures in normoxia or hypoxia conditions. Hypoxia caused a reduction in clonogenic cell survival response and an increase of the sub-G1 phase of the cell cycle in endothelial cells. Cell migration was measured by spheroid and wound-induced migration assays and hypoxia compared with normoxia significantly increased the number of migrating endothelial cells. Nuclear staining with Hoechst 33258 and caspase-9 and -3 activation in endothelial cells show that hypoxia-induced apoptosis involves caspase-dependent mechanism. Exposure to hypoxia caused an increase in gene expression of VEGF and VEGFR2 and activities of MMP-2 and MMP-9. Furthermore, hypoxia induced an increase in capillary-like structure formation in endothelial cells seeded into Matrigel. Tumor conditioned medium enhanced survival and rescued endothelial cells from apoptosis induced by hypoxia. These molecular changes in endothelial cells could, in part, contribute to the angiogenic response that occurs during hypoxia-induced angiogenesis in glial tumors.

In this study, we evaluated the antiproliferative and proapoptotic effects of the isothiocyanate iberin, a bioactive agent in Brassicaceae species, in human glioblastoma cells. The human glioblastoma cell cultures were treated with different concentrations of iberin and tested for growth inhibition, cytotoxicity, induction of apoptosis, and activation of caspases. Iberin inhibited growth of tumor cells in cell proliferation assays, enhanced cytotoxicity, and induced apoptosis by activation of caspase-3 and caspase-9. Findings from this study could provide a basis for potential usefulness of the diet-derived isothiocyanate iberin as a promising therapeutic micronutrient in the prevention/intervention of brain tumors.

Neuroblastomas are the most common extra-cranial tumors of childhood and well known for their heterogeneous clinical behavior associated with certain genetic aberrations. Radiation therapy is an important modality for the treatment of high-risk neuroblastomas. In this study, we investigated whether ionizing irradiation modulate the migration and invasiveness of human neuroblastoma cells and expression of proangiogenic molecules known to be involved in tumor progression and metastasis. Irradiation of neuroblastoma cells resulted in increased migration and invasion as measured by spheroid migration and matrigel invasion assay respectively. Zymographic analysis revealed an increase in enzyme activity of MMP-9 and uPA in conditioned medium of irradiated neuroblastoma cells compared with non-irradiated cells. An increase in VEGF levels was also found in lysates of irradiated neuroblastoma cells. The up-regulation of uPA, MMP-9 and VEGF transcripts was also confirmed by RT-PCR analysis. Next, we examined the irradiated tumor cell-mediated modulation of endothelial cell behavior. Conditioned media from irradiated neuroblastoma cells enhanced capillary-like structure formation of microvascular endothelial cells. In a coculture system, irradiation of neuroblastoma cells enhanced endothelial cell invasiveness through Matrigel matrix. Endothelial cells treated with irradiated tumor cell conditioned medium were also analyzed for expression of uPA, MMP-9 and VEGF and compared to cells treated with non-irradiated tumor cell conditioned medium. These findings suggest that the irradiation effects of tumor cells could influence endothelial angiogenesis present in non-irradiated fields.

The expression of matrix metalloproteinases (MMPs), particularly MMP-9, is significantly increased during tumor progression and is thought to play a major role in mediating angiogenic process. Since microvasculature plays an important role in controlling tumor growth, we investigated the effects of MMP-9 inhibition on endothelial cell migration and tube formation, two determinants of angiogenesis. Adenoviral-mediated MMP-9 downregulation inhibited endothelial cell migration in cell wounding and spheroid migration assays. To determine the effects of MMP-9 reduction in glioblastoma/endothelial co-cultures, we used a three-dimensional co-culture assay of glioblastoma spheroids and endothelial spheroids. Untreated controls showed invasion of both cell populations into each other whereas treatment of the co-cultures with adenoviral antisense MMP-9 particles resulted in reduced invasion. Next, inhibition of MMP-9 by adenoviral vectors in endothelial cells was assessed for in vitro capillary-like structure formation either by co-culture with glioblastoma cells or exposure to glioblastoma-conditioned medium. Addition of conditioned medium from human glioblastoma cells to endothelial cells treated with antisense MMP-9 adenoviral vectors or co-cultures of glioblastoma cell lines with MMP-9-reduced endothelial cells resulted in reduced capillary-like tube formation demonstrating the key role of MMP-9 in endothelial cell network organization. Examination of in vitro capillary-like tube structure formation using Matrigel showed a significant decrease in MMP-9 downregulated endothelial cells as compared to controls. In conclusion, the inhibition of MMP-9 is required for inhibition of endothelial cell migration and tube formation and is likely to be of importance in cerebral angiogenesis for therapeutic targets.

The cysteine proteinase cathepsin B has been implicated in tumor progression by virtue of its increased mRNA and protein levels, as well as its localization at the invading front of the tumor. In this study, we examined whether blocking cathepsin B expression in human glioblastoma SNB19 cells affects angiogenesis. Stable transfectants of human glioblastoma cells with a plasmid containing antisense cathepsin B cDNA showed decreased migration rates in wound- and spheroid-migration assays. Analysis showed a reduction in VEGF protein and MMP-9 activity in the cathepsin B antisense cDNA-transfected cells. Regarding angiogenesis in vitro, we found that the conditioned medium of glioblastoma cells with downregulated cathepsin B expression reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary-like network formation. Furthermore, a marked reduction in microvasculature development was seen in an in vivo dorsal air sac assay of glioblastoma cells with downregulated cathepsin B expression. Taken together, these results provide evidence that inhibition of cathepsin B expression can suppress glioblastoma-induced neovascularization.