Nonsteroidal Anti-inflammatory Drugs Diclofenac and Celecoxib Attenuates Wnt/β-Catenin/Tcf Signaling Pathway in Human Glioblastoma Cells

Glioblastoma, the most common and aggressive primary brain tumors, carry a bleak prognosis and often recur even after standard treatment modalities. Emerging evidence suggests that deregulation of the Wnt/β-catenin/Tcf signaling pathway contributes to glioblastoma progression. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit tumor cell proliferation by suppressing Wnt/β-catenin/Tcf signaling in various human malignancies. In this study, we sought to inhibit Wnt/β-catenin/Tcf signaling in glioblastoma cells by the NSAIDs diclofenac and celecoxib. Both diclofenac and celecoxib significantly reduced the proliferation, colony formation and migration of human glioblastoma cells. Diclofenac and celecoxib downregulated β-catenin/Tcf reporter activity. Western and qRT-PCR analysis showed that diclofenac and celecoxib reduced the expression of β-catenin target genes Axin2, cyclin D1 and c-Myc. In addition, the cytoplasmic accumulation and nuclear translocation of β-catenin was significantly reduced following diclofenac and celecoxib treatment. Furthermore, diclofenac and celecoxib significantly increased phosphorylation of β-catenin and reduced the phosphorylation of GSK3β. These results clearly indicated that diclofenac and celecoxib are potential therapeutic agents against glioblastoma cells that act by suppressing the activation of Wnt/β-catenin/Tcf signaling.     

Activation of calpains, calpastatin and spectrin cleavage in the brain during the pathology of fatal murine cerebral malaria

Neuronal calpains appear to be activated uncontrollably by sustained elevation of cytosolic calcium levels under pathological conditions as well as neurodegenerative diseases. In the present study, we have characterized calpain activation in cytosolic extract of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Pathology of FMCM resulted in the increase in activity of calpains in both cerebral cortex and cerebellum. Western blot analysis revealed an increase in the levels of mu-calpain (calpain-1) in the cytosolic fraction of infected cerebral cortex and cerebellum although a decrease in the level of m-calpain was observed in the cytosolic fraction of infected cerebellum and cerebral cortex. Calpain activation was further confirmed by monitoring the formation of calpain-specific spectrin breakdown products (SBDP). Protease-specific SBDP revealed the formation of calpain-generated 150kDa product in the infected cerebral cortex and cerebellum. The specific signature fragment of calpain activation and spectrin breakdown after Plasmodium berghei ANKA infection provide a strong evidence of the role of calpains during the cell death in cerebral cortex and cerebellum. Given the role of calpains in neurodegeneration and cell death, our results strongly suggest that calpains are important mediators of cell injury and neurological sequelae associated with FMCM.     

Cell Death is Associated with Reduced Base Excision Repair During Chronic Alcohol Administration in Adult Rat Brain

The cell death cascades in different brain regions namely hippocampus and frontal cortex of rats fed with 10% (v/v) ethanol for 12 weeks, was examined. After Western blotting, different cell death associated proteins displayed differential activation in the two regions observed. In hippocampus, activated caspase-3 and caspase-7 resulted in subsequent cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). Cytochrome c release to cytosol and apoptosis inducing factor (AIF) translocation to nucleus was marginal. B-cell leukemia/lymphoma-2 (Bcl-2) translocation to cytosol was significant whereas Bcl-2-associated X protein (Bax) and Bcl-associated death protein (Bad) were largely located in cytosol. Further, upregulation of N-methyl d-aspartate receptor subunit 1 (NMDAR1), N-methyl d-aspartate receptor subunit 2B (NMDAR2B), N-methyl d-aspartate receptor subunit 2C (NMDAR2C) and activation of calpains were observed. In frontal cortex, caspase-3 activation, cleavage of PARP-1 and nuclear translocation of AIF were more pronounced. Moreover, cytochrome c release to cytosol, Bcl-2 translocation to cytosol was evident. However, levels of Bax, Bad, NMDA receptor subunits, and calpains were unaffected. Apoptosis was further substantiated by in situ staining for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). Results of the current study revealed that frontal cortex exhibits a higher level of ethanol-induced apoptosis relative to hippocampus. DNA polymerase beta assay and immunoblot showed significant loss in base excision repair in ethanol treated group.     

pERK, pAkt and pBad: A Possible Role in Cell Proliferation and Sustained Cellular Survival During Tumorigenesis and Tumor Progression in ENU Induced Transplacental Glioma Rat Model

Gliomas remain to be an unresolved medical problem. Better understanding of complex regulation and key molecules involved in glioma pathology are needed for designing new and effective treatment modalities. Activation of mitogen-activated protein kinase/extracellular signal regulated kinase (ERK) pathway is known to be having a critical role in cell proliferation and differentiation during the invasion and metastasis of the tumor cells. In the present study, N-ethyl N-nitrosourea induced glioma rat model was used to understand the role of ERK1/2 and Akt pathways in the progression of tumor malignancy. Twenty-four glioma rat brains of early (P90) and progressive (P180) stages were used for histological and immunoblot analysis. Results have shown increased levels of activated ERK1/2, activated Akt or protein kinase B, Bcl-2 and pBad in the glioma rats. This study may indicate increased cell proliferation and angiogenesis, mediated through activation of both ERK and Akt pathways along with increased levels of pBad. Further, pAkt and Bcl-2 levels in the progressive stage glioma rats may indicate existence of sustained tumor cell survival signals. Moreover, enhanced pBad levels in tumor may indicate that there are anti-apoptotic mechanisms, further making the malignant cells resistant to apoptosis.     

Differential PARP Cleavage: An Indication of Heterogeneous Forms of Cell Death and Involvement of Multiple Proteases in the Infarct of Focal Cerebral Ischemia in Rat

Aim Poly (ADP-ribose) polymerase (PARP) is a nuclear repair enzyme whose role is widely depicted in various physiological and pathological processes. In the present study, we wanted to check the status of PARP and the role of various cell death proteases involved in apoptotic and non-apoptotic forms of cell death during transient focal cerebral ischemia in rat model. The activation of these proteases can result in the production of PARP fragments which can be treated as specific signature fragments to the particular protease involved in the pathology and hence the type of cell death. Results In the ischemic samples, we observed activation of calpain, cathepsin-b, caspase-3, and granzyme-b which were known to act on and cleave PARP to produce specific signature fragments by Western blot and immunohistochemical analysis. Cresyl violet staining showed the presence of apoptotic and necrotic cell deaths. Further we observed interaction of AIF and gra-b with PARP in double immunofluorescence and co-immunoprecipitation experiments. Conclusion Activation of calpains, cathepsin-b, caspase-3, and granzyme-b correlated with either apoptotic or necrotic cell deaths in cresyl violet staining. The appearance of PARP signature fragments gives a clear idea on the involvement of particular protease in the pathology. Appearance of signature fragments like 89- and 50-kDa indicates the involvement of apoptotic and necrotic cell death in the pathology. Further interaction of AIF and gra-b with PARP also indicates the involvement of non-apoptotic modes of cell death during the pathology of focal cerebral ischemia.     

Increased β-Catenin/Tcf Signaling in Pilocytic Astrocytomas: A Comparative Study to Distinguish Pilocytic Astrocytomas from Low-Grade Diffuse Astrocytomas

Although pilocytic and diffuse grade II astrocytomas considered as low-grade tumors, the distinction between them is still a major clinical problem. Previously we reported the activation of Wnt/β-catenin/Tcf signaling pathway in diffuse astrocytomas, however its role in pilocytic astrocytomas is not well understood. In this study, we investigated the Wnt/β-catenin/Tcf pathway in pilocytic astrocytomas and compared with diffuse astrocytomas. We observed the differential expression of β-catenin, Tcf4, Lef1 and c-Myc in astrocytomas particularly higher levels were observed in pilocytic astrocytomas and GBM while very little expression was documented in grade II tumors. Further, immunohistochemical analysis revealed the strong positivity of β-catenin, Tcf4, Lef1 and c-Myc in pilocytic astrocytomas than that of grade II tumors and also exhibited the strong positivity in vascular endothelial cells of pilocytic astrocytomas and GBM. Hence, Wnt/β-catenin/Tcf signaling pathway is differentially expressed in astrocytomas, activation of this pathway might be helpful in separating pilocytic astrocytomas from low-grade diffuse astrocytomas.     

Differential changes in cell morphology,macromolecular composition and membrane protein profiles of neurons and astrocytes in chronic ethanol treated rats

Cellular morphology, macromolecular composition, (DNA, RNA and Protein content) marker enzyme activities for neurons [neuron specific enolase (NSE)] and astrocytes [glutamine synthetase (GS)] and plasma membrane protein profiles in the bulk isolated neurons and astrocytes from control and ethanol treated rats were studied. One month aged Wistar rats were given ethanol as sole drinking fluid for 10 weeks. Scanning electron microscopy revealed a characteristic cell surface smoothening in astrocytes due to ethanol treatment. DNA levels were unaltered, while RNA and Protein contents were decreased in astrocytes and neurons. Further,³H-leucine incorporation into proteins was decreased in neurons and astrocytes derived from ethanol treated rats indicating reduced protein synthesis in neurons and astrocytes. GS activity was affected severely suggesting impairment in astrocytic functions. Plasma membrane protein composition was analyzed by 2-D electrophoresis. The analysis indicated several protein defects in the plasma membranes of neurons and astrocytes, which might be involved in membrane disorder during ethanol challenge.¹²⁵I-Wheat Germ agglutinin binding studies showed three prominent proteins (160, 116 and 97 kDa) in astrocyte membrane fraction suggesting the possible involvement of N-terminal glycoproteins in altered astrocyte morphology during ethanol ingestion. Impairment in the astrocyte cell functions, protein changes in plasma membrane and cellular morphology studies suggest that astrocytes may be more vulnerable than neurons for ethanol effects.     

Ethanol induced alterations in plasma membrane protein phosphorylation of neurons and astrocytes

The endogenous protein phosphorylation patterns in plasma membranes of bulk isolated neurons and astroglia from control and chronic ethanol treated rats have been investigated. Chronic ethanol treatment resulted in increased phosphorylation of specific proteins with molecular weights 116, 63 and 60 kDa in both neurons and astrocytes. These proteins were further resolved by 2-DE and the analysis suggested an increased phosphorylation of 10–15 proteins, of which 116 kDa protein is phosphorylated to a higher extent by ethanol. Further, decreased phosphorylation was noticed in D-95 and D-63 proteins in neurons and D-78 and D-54 proteins in astrocytes. Alkali stability experiments for identifying the phosphoamino acid involved in phosphorylation of 116, 63 and 60 kDa proteins suggested that tyrosine and threonine are not involved and probably serine is the likely site for phosphorylation during chronic ethanol treatment. The phosphorylation of specific membrane proteins during chronic ethanol treatment might contribute to ethanol evoked cellular dysfunction.