Daniel Fassett

Upregulation of PTEN in Glioma Cells by Cord Blood Mesenchymal Stem Cells Inhibits Migration via Downregulation of the PI3K/Akt Pathway

Background PTEN (phosphatase and tensin homologue deleted on chromosome ten) is a tumor suppressor gene implicated in a wide variety of human cancers, including glioblastoma. PTEN is a major negative regulator of the PI3K/Akt signaling pathway. Most human gliomas show high levels of activated Akt, whereas less than half of these tumors carry PTEN mutations or homozygous deletions. The unique ability of mesenchymal stem cells to track down tumor cells makes them as potential therapeutic agents. Based on this capability, new therapeutic approaches have been developed using mesenchymal stem cells to cure glioblastoma. However, molecular mechanisms of interactions between glioma cells and stem cells are still unknown. Methodology/Principal Findings In order to study the mechanisms by which migration of glioma cells can be inhibited by the upregulation of the PTEN gene, we studied two glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310) alone and in co-culture with human umbilical cord blood-derived mesenchymal stem cells (hUCBSC). Co-cultures of glioma cells showed increased expression of PTEN as evaluated by immunofluorescence and immunoblotting assays. Upregulation of PTEN gene is correlated with the downregulation of many genes including Akt, JUN, MAPK14, PDK2, PI3K, PTK2, RAS and RAF1 as revealed by cDNA microarray analysis. These results have been confirmed by reverse-transcription based PCR analysis of PTEN and Akt genes. Upregulation of PTEN resulted in the inhibition of migration capability of glioma cells under in vitro conditions. Also, wound healing capability of glioma cells was significantly inhibited in co-culture with hUCBSC. Under in vivo conditions, intracranial tumor growth was inhibited by hUCBSC in nude mice. Further, hUCBSC upregulated PTEN and decreased the levels of XIAP and Akt, which are responsible for the inhibition of tumor growth in the mouse brain. Conclusions/Significance Our studies indicated that upregulation of PTEN by hUCBSC in glioma cells and in the nude mice tumors downregulated Akt and PI3K signaling pathway molecules. This resulted in the inhibition of migration as well as wound healing property of the glioma cells. Taken together, our results suggest hUCBSC as a therapeutic agent in treating malignant gliomas.

Mortality rates in geriatric patients with spinal cord injuries

OBJECT The authors undertook this study to evaluate the incidence of spinal cord injury (SCI) in geriatric patients (> or = 70 years of age) and examine the impact of patient age, extent of neurological injury, and spinal level of injury on the mortality rate associated with traumatic SCI. METHODS A prospectively maintained SCI database (3481 patients) at a single institution was retrospectively studied for the period from 1978 through 2005. Parameters analyzed included patient age, admission American Spinal Injury Association (ASIA) motor score, level of SCI, mechanism of injury, and mortality data. The data pertaining to the 412 patients 70 years of age and older were compared with those pertaining to the younger cohort using a chi-square analysis. RESULTS Since 1980, the number of SCI-related hospital admissions per year have increased fivefold in geriatric patients and the percentage of geriatric patients within the SCI population has increased from 4.2 to 15.4%. In comparison with younger patients, geriatric patients were found to be less likely to have severe neurological deficits (greater percentage of ASIA Grades C and D injuries), but the mortality rates were higher in the older age group both for the period of hospitalization (27.7% compared with 3.2%, p < 0.001) and during 1-year follow-up. The mortality rates in this older population directly correlate with the severity of neurological injury (1-year mortality rate, ASIA Grade A 66%, Grade D 23%, p < 0.001). The mortality rate in elderly patients with SCI has not changed significantly over the last two decades, and the 1-year mortality rate was greater than 40% in all periods analyzed. CONCLUSIONS Spinal cord injuries in older patients are becoming more prevalent. The mortality rate in this patient group is much greater than in younger patients and should be taken into account when aggressive interventions are considered and in counseling families regarding prognosis.

p53- and Bax-Mediated Apoptosis in Injured Rat Spinal Cord

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. p53-mediated mitochondrial apoptosis is likely to be an important mechanism of cell death in spinal cord injury. However, the signaling cascades that are activated before DNA fragmentation have not yet been determined. DNA damage-induced, p53-activated neuronal cell death has already been identified in several neurodegenerative diseases. To determine DNA damage-induced, p53-mediated apoptosis in spinal cord injury, we performed RT-PCR microarray and analyzed 84 DNA damaging and apoptotic genes. Genes involved in DNA damage and apoptosis were upregulated whereas anti-apoptotic genes were downregulated in injured spinal cords. Western blot analysis showed the upregulation of DNA damage-inducing protein such as ATM, cell cycle checkpoint kinases, 8-hydroxy-2′-deoxyguanosine (8-OHdG), BRCA2 and H2AX in injured spinal cord tissues. Detection of phospho-H2AX in the nucleus and release of 8-OHdG in cytosol were demonstrated by immunohistochemistry. Expression of p53 was observed in the neurons, oligodendrocytes and astrocytes after spinal cord injury. Upregulation of phospho-p53, Bax and downregulation of Bcl2 were detected after spinal cord injury. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis taking place after spinal cord injury. In addition, we carried out immunohistochemical analysis to confirm Bax translocation into the mitochondria and activated p53 at Ser392. Expression of APAF1, caspase 9 and caspase 3 activities confirmed the intrinsic apoptotic pathway after SCI. Activated p53 and Bax mitochondrial translocation were detected in injured spinal neurons. Taken together, the in vitro data strengthened the in vivo observations of DNA damage-induced p53-mediated mitochondrial apoptosis in the injured spinal cord.

Cord Blood Stem Cell-Mediated Induction of Apoptosis in Glioma Downregulates X-Linked Inhibitor of Apoptosis Protein (XIAP)

Background XIAP (X-linked inhibitor of apoptosis protein) is one of the most important members of the apoptosis inhibitor family. XIAP is upregulated in various malignancies, including human glioblastoma. It promotes invasion, metastasis, growth and survival of malignant cells. We hypothesized that downregulation of XIAP by human umbilical cord blood mesenchymal stem cells (hUCBSC) in glioma cells would cause them to undergo apoptotic death. Methodology/Principal Findings We observed the effect of hUCBSC on two malignant glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310). In co-cultures of glioma cells with hUCBSC, proliferation of glioma cells was significantly inhibited. This is associated with increased cytotoxicity of glioma cells, which led to glioma cell death. Stem cells induced apoptosis in glioma cells, which was evaluated by TUNEL assay, FACS analyses and immunoblotting. The induction of apoptosis is associated with inhibition of XIAP in co-cultures of hUCBSC. Similar results were obtained by the treatment of glioma cells with shRNA to downregulate XIAP (siXIAP). Downregulation of XIAP resulted in activation of caspase-3 and caspase-9 to trigger apoptosis in glioma cells. Apoptosis is characterized by the loss of mitochondrial membrane potential and upregulation of mitochondrial apoptotic proteins Bax and Bad. Cell death of glioma cells was marked by downregulation of Akt and phospho-Akt molecules. We observed similar results under in vivo conditions in U251- and 5310-injected nude mice brains, which were treated with hUCBSC. Under in vivo conditions, Smac/DIABLO was found to be colocalized in the nucleus, showing that hUCBSC induced apoptosis is mediated by inhibition of XIAP and activation of Smac/DIABLO. Conclusions/Significance Our results indicate that downregulation of XIAP by hUCBSC treatment induces apoptosis, which led to the death of the glioma cells and xenograft cells. This study demonstrates the therapeutic potential of XIAP and hUCBSC to treat malignant gliomas.

MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice

Background Involvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential. Methodology/Principal Findings MMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of αVβ3, α6β1 and α9β1 integrins in xenograft cells. Treatment with bicistronic constructs reduced αVβ3, α6β1 and α9β1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments. Conclusions/Significance Downregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma.

The cervical end of an occipitocervical fusion: A biomechanical evaluation of 3 constructs - Laboratory investigation

OBJECT Stabilization with rigid screw/rod fixation is the treatment of choice for craniocervical disorders requiring operative stabilization. The authors compare the relative immediate stiffness for occipital plate fixation in concordance with transarticular screw fixation (TASF), C-1 lateral mass and C-2 pars screw (C1L-C2P), and C-1 lateral mass and C-2 laminar screw (C1L-C2L) constructs, with and without a cross-link. METHODS Ten intact human cadaveric spines (Oc-C4) were prepared and mounted in a 7-axis spine simulator. Each specimen was precycled and then tested in the intact state for flexion/extension, lateral bending, and axial rotation. Motion was tracked using the OptoTRAK 3D tracking system. The specimens were then destabilized and instrumented with an occipital plate and TASF. The spine was tested with and without the addition of a cross-link. The C1L-C2P and C1L-C2L constructs were similarly tested. RESULTS All constructs demonstrated a significant increase in stiffness after instrumentation. The C1L-C2P construct was equivalent to the TASF in all moments. The C1L-C2L was significantly weaker than the C1L-C2P construct in all moments and significantly weaker than the TASF in lateral bending. The addition of a cross-link made no difference in the stiffness of any construct. CONCLUSIONS All constructs provide significant immediate stability in the destabilized occipitocervical junction. Although the C1L-C2P construct performed best overall, the TASF was similar, and either one can be recommended. Decreased stiffness of the C1L-C2L construct might affect the success of clinical fusion. This construct should be reserved for cases in which anatomy precludes the use of the other two.

Co-depletion of cathepsin B and uPAR induces G0/G1 arrest in glioma via FOXO3a mediated p27Kip1 upregulation

Background Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined. Methodology/Principal Findings Cathespin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27Kip1 and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by αVβ3/PI3K/AKT/FOXO pathway as observed by the decreased αVβ3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27Kip1 and FOXO3a when treated with Ly294002 (10 µM) and increased luciferase expression with the siRNA and Ly294002 treatments when the FOXO binding promoter region of p27Kip1 was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly. Conclusion/Significance Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27Kip1 accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.

Urokinase Plasminogen Activator Receptor and/or Matrix Metalloproteinase-9 Inhibition Induces Apoptosis Signaling through Lipid Rafts in Glioblastoma Xenograft Cells

Small interfering RNA (siRNA)-mediated transcriptional knockdown of urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9), alone or in combination, inhibits uPAR and/or MMP-9 expression and induces apoptosis in the human glioblastoma xenograft cell lines 4910 and 5310. siRNA against uPAR (pU-Si), MMP-9 (pM-Si), or both (pUM-Si) induced apoptosis and was associated with the cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase. Furthermore, protein levels of the Fas receptor (APO-1/CD-95) were increased following transcriptional inactivation of uPAR and/or MMP-9. In addition, Fas siRNA against the Fas death receptor blocked apoptosis induced by pU-Si, pM-Si, or pUM-Si, thereby indicating the role for Fas signaling in pU-Si–, pM-Si–, or pUM-Si–mediated apoptotic cell death of human glioma xenograft cells. Thus, transcriptional inactivation of uPAR and/or MMP-9 enhanced localization of Fas death receptor, Fas-associated death domain-containing protein, and procaspase-8 into lipid rafts. Additionally, disruption of lipid rafts with methyl β cyclodextrin prevented Fas clustering and pU-Si–, pM-Si–, or pUM-Si–induced apoptosis, which is indicative of coclustering of Fas death receptor into lipid rafts in the glioblastoma xenograft cell lines 4910 and 5310. These data indicate the crucial role of the clusters of apoptotic signaling molecule-enriched rafts in programmed cell death, acting as concentrators of death receptors and downstream signaling molecules, and as the linchpin from which a potent death signal is launched in uPAR- and/or MMP-9–downregulated cells. Mol Cancer Ther; 9(9); 2605–17. ©2010 AACR.

Recurrence-free chronic subdural hematomas: a retrospective analysis of the instillation of tissue plasminogen activator in addition to twist drill or burr hole drainage in the treatment of chronic subdural hematomas.

OBJECTIVE To evaluate whether increasing the volume drained from chronic subdural hematomas (SDHs) via either twist drill drainage (TDD) or burr hole drainage (BHD) followed by instillation of tissue plasminogen activator (tPA) is more efficacious than simple drainage alone. METHODS Patients admitted over the course of 42 months (2007-2010) to a single institution for treatment of chronic SDH were retrospectively evaluated. RESULTS There were 139 patients treated for chronic SDH; 54 patients were treated with BHD alone, 3 were treated with tPA after BHD, 85 were treated with TDD alone, and 12 were treated with tPA after TDD. Follow-up examinations were performed 1 month after treatment in 13 of 15 patients treated with tPA and 93 of 124 patients treated without tPA. Patients treated with tPA had a significantly lower rate of recurrence than patients treated without tPA (P=0.041). Patients treated with BHD had a recurrence rate of 11.8%, whereas patients treated with BHD and tPA had 0% recurrence. Patients treated with TDD had a recurrence rate of 30%, whereas patients treated with TDD and tPA had 0% recurrence. Without tPA, BHD was found to be a significantly better treatment than TDD (P=0.016). Mean drainage for TDD with tPA was 427.33 mL. There were no complications related to the administration of tPA. CONCLUSIONS This study adds another therapeutic option for patients with chronic SDH requiring treatment. In this retrospective study, the addition of tPA increased the volume of hematoma drained and significantly reduced the incidence of recurrence requiring further intervention regardless of cranial access route. No complications occurred related directly or indirectly to the administration of tPA. Further study of this technique is warranted.

Low-profile pelvic fixation with sacral alar-iliac screws

BackgroundIn the last years, a new technique for pelvic fixation using ‘sacral alar-iliac’ screws has been proposed as an alternative to the classic ‘iliac wing’ screws and the old Galveston technique.MethodsThe authors provide a ‘step-by-step’ review of the technical details regarding this technique, as well as some important anatomical and radiological landmarks for its successful performance.Conclusions‘Sacral alar-iliac’ screws provide several advantages in relation to pelvic fixation with iliac bolts such as: better alignment with the rods, use of a ‘low-profile’ screw, and fixation of the sacroiliac (SI) joint, which avoids postoperative SI joint-related pain.