Cathepsin B and uPAR regulate self-renewal of glioma-initiating cells through GLI-regulated Sox2 and Bmi1 expression.

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.

Urokinase-type plasminogen activator receptor (uPAR)-mediated regulation of WNT/ß-catenin signaling is enhanced in irradiated medulloblastoma cells.

Urokinase plasminogen activator receptor (uPAR) is known to promote invasion, migration, and metastasis in cancer cells. In this report, we showed that ionizing radiation (IR)-induced uPAR has a role in WNT-ß-catenin signaling and mediates induction of cancer stem cell (CSC)-like properties in medulloblastoma cell lines UW228 and D283. We observed that IR induced the expression of uPAR and CSC markers, such as Musashi-1 and CD44, and activated WNT-7a-ß-catenin signaling molecules. Overexpression of uPAR alone or with IR treatment led to increased WNT-7a-ß-catenin-TCF/LEF-mediated transactivation, thereby promoting cancer stemness. In contrast, treatment with shRNA specific for uPAR (pU) suppressed WNT-7a-ß-catenin-TCF/LEF-mediated transactivation both in vitro and in vivo. Quercetin, a potent WNT/ß-catenin inhibitor, suppressed uPAR and uPAR-mediated WNT/ß-catenin activation, and furthermore, addition of recombinant human WNT-7a protein induced uPAR, indicating the existence of a mutual regulatory relationship between uPAR and WNT/ß-catenin signaling. We showed that uPAR was physically associated with the WNT effector molecule ß-catenin on the membrane, cytoplasm, and nucleus of IR-treated cells and CSC. Most interestingly, we demonstrated for the first time that localization of uPAR in the nucleus was associated with transcription factors (TF) and their specific response elements. We observed from uPAR-ChIP, TF protein, and protein/DNA array analyses that uPAR associates with activating enhancer-binding protein 2α (AP2a) and mediates ß-catenin gene transcription. Moreover, association of uPAR with the ß-catenin·TCF/LEF complex and various other TF involved during embryonic development and cancer indicates that uPAR is a potent activator of stemness, and targeting of uPAR in combination with radiation has significant therapeutic implications.

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.

Knockdown of cathepsin B and uPAR inhibits CD151 and α3ß1 integrin-mediated cell adhesion and invasion in glioma.

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 ß1. Co-immunoprecipitation experiments revealed that downregulation of cathepsin B and uPAR decreased the interaction of CD151 with uPAR cathepsin B, and α3ß1 integrin. Studies on the downstream signaling cascade of uPAR/CD151/α3ß1 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 α3ß1 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.

Design, synthesis and preclinical evaluation of NRC-AN-019.

Imatinib mesylate is the first tyrosine kinase inhibitor developed and approved for the treatment of chronic myeloid leukemia (CML). In the past few years development of resistance towards imatinib mesylate has been reported. To overcome this problem a series of phenyl amino pyrimidine derivatives have been designed, prepared and evaluated for anti-proliferative activity against the BCR­ABL­positive leukemia cell line K562. Among these phenyl amino pyrimidine derivatives, NRC­AN­019 has been found to be a promising new lead compound for the therapy of imatinib mesylate-resistant chronic myeloid leukemia. In this communication, we describe the design, preparation and preclinical studies of NRC­AN­019.

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.

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.

EGFR and c-Met Cross Talk in Glioblastoma and Its Regulation by Human Cord Blood Stem Cells.

Receptor tyrosine kinases (RTK) and their ligands control critical biologic processes, such as cell proliferation, migration, and differentiation. Aberrant expression of these receptor kinases in tumor cells alters multiple downstream signaling cascades that ultimately drive the malignant phenotype by enhancing tumor cell proliferation, invasion, metastasis, and angiogenesis. As observed in human glioblastoma (hGBM) and other cancers, this dysregulation of RTK networks correlates with poor patient survival. Epidermal growth factor receptor (EGFR) and c-Met, two well-known receptor kinases, are coexpressed in multiple cancers including hGBM, corroborating that their downstream signaling pathways enhance a malignant phenotype. The integration of c-Met and EGFR signaling in cancer cells indicates that treatment regimens designed to target both receptor pathways simultaneously could prove effective, though resistance to tyrosine kinase inhibitors continues to be a substantial obstacle. In the present study, we analyzed the antitumor efficacy of EGFR inhibitors erlotinib and gefitinib and c-Met inhibitor PHA-665752, along with their respective small hairpin RNAs (shRNAs) alone or in combination with human umbilical cord blood stem cells (hUCBSCs), in glioma cell lines and in animal xenograft models. We also measured the effect of dual inhibition of EGFR/c-Met pathways on invasion and wound healing. Combination treatments of hUCBSC with tyrosine kinase inhibitors significantly inhibited invasion and wound healing in U251 and 5310 cell lines, thereby indicating the role of hUCBSC in inhibition of RTK-driven cell behavior. Further, the EGFR and c-Met localization in glioma cells and hGBM clinical specimens indicated that a possible cross talk exists between EGFR and c-Met signaling pathway.

SPARC overexpression inhibits cell proliferation in neuroblastoma and is partly mediated by tumor suppressor protein PTEN and AKT.

Secreted protein acidic and rich in cysteine (SPARC) is also known as BM-40 or Osteonectin, a multi-functional protein modulating cell-cell and cell-matrix interactions. In cancer, SPARC is not only linked with a highly aggressive phenotype, but it also acts as a tumor suppressor. In the present study, we sought to characterize the function of SPARC and its role in sensitizing neuroblastoma cells to radio-therapy. SPARC overexpression in neuroblastoma cells inhibited cell proliferation in vitro. Additionally, SPARC overexpression significantly suppressed the activity of AKT and this suppression was accompanied by an increase in the tumor suppressor protein PTEN both in vitro and in vivo. Restoration of neuroblastoma cell radio-sensitivity was achieved by overexpression of SPARC in neuroblastoma cells in vitro and in vivo. To confirm the role of the AKT in proliferation inhibited by SPARC overexpression, we transfected neuroblastoma cells with a plasmid vector carrying myr-AKT. Myr-AKT overexpression reversed SPARC-mediated PTEN and increased proliferation of neuroblastoma cells in vitro. PTEN overexpression in parallel with SPARC siRNA resulted in decreased AKT phosphorylation and proliferation in vitro. Taken together, these results establish SPARC as an effector of AKT-PTEN-mediated inhibition of proliferation in neuroblastoma in vitro and in vivo.

uPAR and cathepsin B inhibition enhanced radiation-induced apoptosis in gliomainitiating cells.

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.

Suppression of the uPAR-uPA system retards angiogenesis, invasion, and in vivo tumor development in pancreatic cancer cells.

Despite existing chemotherapy and surgical resection strategies, pancreatic cancer is one of the major causes of mortality in the United States with a 5-year mean survival rate of less than 5%. The activation of the urokinase-type plasminogen activator receptor-urokinase-type plasminogen activator (uPAR-uPA) system in the development of pancreatic ductal adenocarcinoma has been well established. In the present study, we used 2 pancreatic cancer cell lines, MIA PaCa-2 and PANC-1 to show the effects of uPAR and uPA downregulation. From the results, we observed that RNAi expressing plasmids efficiently downregulated mRNA and protein expression of uPAR and uPA. In vitro and in vivo angiogenic assays revealed a significant decrease in the angiogenic potential of MIA PaCa-2 and PANC-1 cells that were downregulated for both uPAR and uPA. From the angiogenesis antibody array analysis, we observed that the simultaneous downregulation of uPAR and uPA resulted in the downregulation of angiogenin and overexpression of RANTES. Further, FACS analysis showed that the simultaneous downregulation of uPAR and uPA caused the accumulation of cells in the sub-G(0/1) phase in both MIA PaCa-2 and PANC-1 cells. In addition, Western blot analysis revealed that downregulation of uPAR and uPA caused the activation of caspase 8 and CAD, which is indicative of apoptosis, and in vivo TUNEL assay confirmed these results. Finally, we observed the nuclear localization of uPA and that uPA interacts with the transcription factor Lhx-2. Taken together, the results of the present study show that the targeting of the uPAR-uPA system has therapeutic potential.

Suppression of MMP-2 attenuates TNF-α induced NF-κB activation and leads to JNK mediated cell death in glioma.

BACKGROUND: Abrogation of apoptosis for prolonged cell survival is essential in cancer progression. In our previous studies, we showed the MMP-2 downregulation induced apoptosis in cancer cell lines. Here, we attempt to investigate the exact molecular mechanism of how MMP-2 depletion leads to apoptosis in glioma xenograft cell lines. METHODOLOGY/PRINCIPAL FINDINGS: MMP-2 transcriptional suppression by MMP-2siRNA (pM) induces apoptosis associated with PARP, caspase-8 and -3 cleavage in human glioma xenograft cells 4910 and 5310. Western blotting and cytokine array showed significant decrease in the cellular and secreted levels of TNF-α with concomitant reduction in TNFR1, TRADD, TRAF2, RIP, IKKß and pIκBα expression levels resulting in inhibition of p65 phosphorylation and nuclear translocation in pM-treated cells when compared to mock and pSV controls. In addition MMP-2 suppression led to elevated Fas-L, Fas and FADD expression levels along with increased p38 and JNK phosphorylation. The JNK-activity assay showed prolonged JNK activation in pM-transfected cells. Specific inhibition of p38 with SB203580 did not show any effect whereas inhibition of JNK phosphorylation with SP600125 notably reversed pM-induced cleavage of PARP, caspase-8 and -3, demonstrating a significant role of JNK in pM-induced cell death. Supplementation of rhMMP-2 counteracted the effect of pM by remarkably elevating TNF-α, TRADD, IKKß and pIκBα expression and decreasing FADD, Fas-L, and phospho-JNK levels. The EMSA analysis indicated significant reversal of pM-inhibited NF-κB activity by rhMMP-2 treatment which rescued cells from pM-induced cell death. In vivo studies indicated that pM treatment diminished intracranial tumor growth and the immuno histochemical analysis showed decreased phospho-p65 and enhanced phospho-JNK levels that correlated with increased TUNEL-positive apoptotic cells in pM-treated tumor sections. CONCLUSION/SIGNIFICANCE: In summary, our study implies a role of MMP-2 in the regulation of TNF-α mediated constitutive NF-κB activation and Fas-mediated JNK mediated apoptosis in glioma xenograft cells in vitro and in vivo.

Antitumor activity of NRC-AN-019 in a pre-clinical breast cancer model.

Breast cancer is the second most frequently diagnosed tumor in women. Overexpression of human epidermal growth factor receptors (EGFRs) represents a biological subclass of breast cancer with distinct molecular alterations, clinical behavior and response to systemic therapy. In this study, we describe a novel compound (NRC-AN-019), which has better antitumor activity than Lapatinib. Here, we demonstrate that NRC-AN-019 is more effective in inhibiting angiogenic potential and proliferation of both MDAMB231 and HTB20/BT474 cells. FACS analysis shows that NRC-AN-019 treatment caused the accumulation of MDAMB231 and BT474 cells in the sub G0/1 phase in a dose-dependent manner and was accompanied by increased PARP cleavage, which is indicative of apoptosis. In addition, we observed inhibition of EGFR phosphorylation in both MDAMB231 and BT474 cells. From our animal studies using SCID mice implanted with BT474 cells, we observed dose-dependent inhibition of tumor growth in NRC-AN-019-treated animals compared to controls or Lapatinib-treated mice at comparable concentrations. The dose-dependent inhibition of EGFR phosphorylation was confirmed by immunohistochemical analysis of tumor sections. In vitro results demonstrate that NRC-AN-019 is superior to Lapatinib in EGFR-overexpressing cells and has strong anti-angiogenic, anti-proliferative and pro-apoptotic properties in an EGFR-overexpressing background (BT474). In vivo studies demonstrate that the antitumor activity of NRC-AN-019 is better over Lapatinib. These results suggest that NRC-AN-019 has greater therapeutic potential in the treatment of Her-2-positive breast cancer.

Epigenetic upregulation of urokinase plasminogen activator promotes the tropism of mesenchymal stem cells for tumor cells.

A major obstacle for the effective treatment of cancer is the invasive capacity of the tumor cells. Previous studies have shown the capability of mesenchymal stem cells (MSC) to target these disseminated tumor cells and to serve as therapeutic delivery vehicles. However, the molecular mechanisms that would enhance the migration of MSCs toward tumor areas are not well understood. In particular, very little is known about the role that epigenetic mechanisms play in cell migration and tropism of MSCs. In this study, we investigated whether histone deacetylation was involved in the repression of urokinase plasminogen activator (uPA) expression in MSCs derived from umbilical cord blood (CB) and bone marrow (BM). Induction of uPA expression by histone deacetylase inhibitors trichostatin A and sodium butyrate was observed in CB- and BM-derived MSCs examined. In vitro migration assays showed that induction of uPA expression by histone deacetylase inhibitors in CB- and BM-derived MSCs significantly enhanced tumor tropism of these cells. Furthermore, overexpression of uPA in CB-MSCs induced migration capacity toward human cancer cells in vitro. In addition, our results showed that uPA-uPAR knockdown in PC3 prostate cancer cells significantly inhibited tumor-specific migration of uPA-overexpressing MSCs. These results have significant implications for the development of MSC-mediated, tumor-selective gene therapies.

Radiation-inducible silencing of uPA and uPAR in vitro and in vivo in meningioma.

Stereospecific radiation treatment offers a distinct opportunity for temporal and spatial regulation of gene expression at tumor sites by means of inducible promoters. To this end, a plasmid, pCArG-U2, was constructed by incorporating nine CArG elements (in tandem) of EGR1 gene upstream to uPA and uPAR siRNA oligonucleotides in a pCi-neo vector. Radiation-induced siRNA expression was detected in a meningioma cell line (IOMM-Lee). Immunoblotting and RT-PCR analyses confirmed downregulation of uPA and uPAR. A similar effect was observed in transfected cells followed by H2O2 treatment. Moreover, pre-treatment of transfected cells with N-acetyl L-cysteine blocked the silencing of uPA and uPAR, which further confirmed the oxidative damage-mediated downregulation. Cell proliferation assays and Western blot analysis for apoptotic molecules confirmed cell death in a radiation-inducible fashion. Migration and matrigel invasion assays also revealed a marked decrease in migration and invasion. Immunocytochemistry showed a marked decrease in uPA and uPAR levels in transfected and irradiated cells. H&E staining revealed a decrease in the pre-established tumor volume among the animals treated with pCArG-U2 and radiation. Immunohistochemistry of the brain sections established with intracranial tumors also revealed a marked decrease in uPA and uPAR in a radiation-inducible fashion. Taken together, our data suggest pCArG-U2 as a suitable candidate for radiation-inducible gene therapy.

Induction of apoptosis in glioma cells requires cell-to-cell contact with human umbilical cord blood stem cells.

We have previously demonstrated the multipotent nature of human umbilical cord blood stem cells (hUCB). In this study, we have attempted to show the use of hUCB in glioma therapy. We used hUCB enriched in CD44 and CD133 cells for our studies and observed that glioma cells co-cultured with hUCB undergo apoptosis. To prove the role of cell-to-cell contact in the induction of apoptotic events, we used a modified 0.22 microm Boyden's chamber where the upper surface was used to culture glioma cells (SNB19 or U87) or xenografts (4910 or 5310) and the lower surface to culture hUCB. TUNEL assay was carried out to determine the degree of apoptotic induction and we observed that glioma or xenograft cells co-cultured with hUCB had a higher number of TUNEL-positive characteristics (63+/-6%) compared to the controls. Further, we co-cultured glioma cells labeled with lipophilic green fluorescent dye and hUCB labeled with lipophilic red fluorescent dye. FACS analysis of cells collected from the upper and lower surfaces revealed that glioma cells had taken up red fluorescent dye from the stem cells (70+/-3%) when compared to glioma cells co-cultured with fibroblast cells (15+/-4%). The apoptotic events in the glioma and xenograft cells co-cultured with hUCB were also confirmed by Western blot analysis for the cleavage of PARP and activation of caspase 8. In addition, elevated levels of CHK-2 levels and downregulation of MAP2K1 were observed in glioma cells co-cultured with hUCB indicating the DNA damage and decrease in cell survival. Nude mice, intracranially implanted with luciferase-expressing U87 cells followed by implantation of hUCB or human fibroblast cells showed retardation of intracranial tumors in hUCB-implanted mice. Taken together, these results demonstrate that hUCB have therapeutic potential with possible clinical implications.

Human umbilical cord blood stem cells show PDGF-D-dependent glioma cell tropism in vitro and in vivo.

Despite advances in clinical therapies and technologies, the prognosis for patients with malignant glioma is poor. Neural stem cells (NSCs) have a chemotactic tropism toward glioma cells. The use of NSCs as carriers of therapeutic agents for gliomas is currently being explored. Here, we demonstrate that cells isolated from the umbilical cord blood show mesenchymal characteristics and can differentiate to adipocytes, osteocytes, and neural cells and show tropism toward cancer cells. We also show that these stem cells derived from the human umbilical cord blood (hUCB) induce apoptosis-like cell death in the glioma cell line SNB19 via Fas-mediated caspase-8 activation. From our glioma tropism studies, we have observed that hUCB cells show tropism toward glioma cells in vitro, in vivo, and ex vivo. We determined that this migration is partially dependent on the expression levels of platelet-derived growth factor (PDGF)-D from glioma cells and have observed that local concentration gradient of PDGF-D is sufficient to cause migration of hUCB cells toward the gradient as seen from our brain slice cultures. In our animal experiment studies, we observed that intracranially implanted SNB19 green fluorescent protein cells induced tropism of the hUCB cells toward themselves. In addition, the ability of these hUCBs to inhibit established intracranial tumors was also observed. We also determined that the migration of stem cells toward glioma cells was partially dependent on PDGF secreted by glioma cells and that the presence of PDGF-receptor (PDGFR) on hUCB is required for migration. Our results demonstrate that hUCB are capable of inducing apoptosis in human glioma cells and also show that glioma tropism and hUCB tropism toward glioma cells are partially dependent on the PDGF/PGGFR system.

Inhibition of histone deacetylase activity promotes invasion of human cancer cells through activation of urokinase plasminogen activator.

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in differential regulation of urokinase plasminogen activator (uPA) gene expression are not fully understood. In this study, we investigated whether histone deacetylation was involved in repression of uPA expression in human cancer cells. Induction of uPA expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA), sodium butyrate, and scriptaid was observed in all three different types of human cancer cells examined. Chromatin immunoprecipitation assays showed that the induction of uPA expression by TSA was accompanied by a remarkable increase of acetylation of histones H3 and H4, which are associated with the uPA promoter region in human cancer cells. These results were further substantiated by the findings of a restriction enzyme accessibility assay and TSA-stimulated uPA promoter activity through the inhibition of HDAC activity. In vitro Matrigel invasion assays showed that induction of uPA expression by HDAC inhibitors in human cancer cells resulted in a significant increase of cancer cell invasion. Furthermore, HDAC1 knockdown by small interference RNA stimulated uPA expression and cancer cell invasion. In conclusion, this study demonstrates the important role of histone modifications in regulating uPA gene expression and raises a possibility that the use of HDAC inhibitors in patients as cancer therapy may paradoxically establish metastasis through up-regulation or reactivation of uPA.