Showing papers by "Swapan K. Ray published in 2009"

Flavonoids activated caspases for apoptosis in human glioblastoma T98G and U87MG cells but not in human normal astrocytes.

Abstract: BACKGROUND: Human glioblastoma is a deadly brain cancer that continues to defy all current therapeutic strategies. The authors induced apoptosis in human glioblastoma T98G and U87MG cells after treatment with apigenin, (−)-epigallocatechin, (−)-epigallocatechin-3-gallate (EGCG), and genistein, which did not induce apoptosis in human normal astrocytes. METHODS: Induction of apoptosis was examined using Wright staining and ApopTag assay. Production of reactive oxygen species (ROS) and increase in intracellular free Ca2+ were measured by fluorescent probes. Analysis of mRNA and Western blotting indicated increases in expression and activities of the stress kinases and cysteine proteases for apoptosis. JC-1 showed changes in mitochondrial membrane potential (ΔΨm), and use of specific inhibitors confirmed activation of kinases and proteases in apoptosis. RESULTS: Treatment of glioblastoma cells with apigenin, (−)-epigallocatechin, EGCG, or genistein triggered ROS production that induced apoptosis with phosphorylation of p38 mitogen-activated protein kinase (MAPK) and activation of the redox-sensitive c-Jun N-terminal kinase 1 pathway. Pretreatment of cells with ascorbic acid attenuated ROS production and p38 MAPK phosphorylation. Increases in intracellular free Ca2+ and activation of caspase-4 indicated involvement of endoplasmic reticulum stress in apoptosis. Other events in apoptosis included overexpression of Bax, loss of ΔΨm, mitochondrial release of cytochrome c and Smac into the cytosol, down-regulation of baculoviral inhibitor-of-apoptosis repeat-containing proteins, and activation of calpain, caspase-9, and caspase-3. (−)-Epigallocatechin and EGCG also induced caspase-8 activity. Apigenin, (−)-epigallocatechin, EGCG, and genistein did not induce apoptosis in human normal astrocytes. CONCLUSIONS: Results strongly suggest that flavonoids are potential therapeutic agents for induction of apoptosis in human glioblastoma cells. Cancer 2010. © 2010 American Cancer Society.

Therapeutic potential of melatonin in traumatic central nervous system injury

Abstract: A vast literature extolling the benefits of melatonin has accumulated during the past four decades. Melatonin was previously considered of importance to seasonal reproduction and circadian rhythmicity. Currently, it appears to be a versatile anti-oxidative and anti-nitrosative agent, a molecule with immunomodulatory actions and profound oncostatic activity, and also to play a role as a potent neuroprotectant. Nowadays, melatonin is sold as a dietary supplement with differential availability as an over-the-counter aid in different countries. There is a widespread agreement that melatonin is nontoxic and safe considering its frequent, long-term usage by humans at both physiological and pharmacological doses with no reported side effects. Endeavors toward a designated drug status for melatonin may be enormously rewarding in clinics for treatment of several forms of neurotrauma where effective pharmacological intervention has not yet been attained. This mini review consolidates the data regarding the efficacy of melatonin as an unique neuroprotective agent in traumatic central nervous system (CNS) injuries. Well-documented actions of melatonin in combating traumatic CNS damage are compiled from various clinical and experimental studies. Research on traumatic brain injury and ischemia/reperfusion are briefly outlined here as they have been recently reviewed elsewhere, whereas the studies on different animal models of the experimental spinal cord injury have been extensively covered in this mini review for the first time.

Bcl-2 siRNA Augments Taxol Mediated Apoptotic Death in Human Glioblastoma U138MG and U251MG Cells

Abstract: The anti-neoplastic drug taxol binds to beta-tubulin to prevent tumor cell division, promoting cell death. However, high dose taxol treatment may induce cell death in normal cells too. The anti-apoptotic molecule Bcl-2 is upregulated in many cancer cells to protect them from apoptosis. In the current study, we knocked down Bcl-2 expression using cognate siRNA during low-dose taxol treatment to induce apoptosis in two human glioblastoma U138MG and U251MG cell lines. The cells were treated with either 100 nM taxol or 100 nM Bcl-2 siRNA or both for 72 h. Immunofluorescent stainings for calpain and active caspase-3 showed increases in expression and co-localization of these proteases in apoptotic cells. Fluorometric assays demonstrated increases in intracellular free [Ca(2+)], calpain, and caspase-3 indicating augmentation of apoptosis. Western blotting demonstrated dramatic increases in the levels of Bax, Bak, tBid, active caspases, DNA fragmentation factor-40 (DFF40), cleaved fragments of lamin, fodrin, and poly(ADP-ribose) polymerase (PARP) during apoptosis. The events related to apoptosis were prominent more in combination therapy than in either treatment alone. Our current study demonstrated that Bcl-2 siRNA significantly augmented taxol mediated apoptosis in different human glioblastoma cells through induction of calpain and caspase proteolytic activities. Thus, combination of taxol and Bcl-2 siRNA offers a novel therapeutic strategy for controlling the malignant growth of human glioblastoma cells.

Combination of hTERT knockdown and IFN-gamma treatment inhibited angiogenesis and tumor progression in glioblastoma.

Abstract: Purpose: The limitless invasive and proliferative capacities of tumor cells are associated with telomerase and expression of its catalytic component, human telomerase reverse transcriptase (hTERT). IFN-γ modulates several cellular activities, including signaling pathways and cell cycle, through transcriptional regulation. Experimental Design: Using a recombinant plasmid with hTERT siRNA cDNA, we downregulated hTERT during IFN-γ treatment in human glioblastoma SNB-19 and LN-18 cell lines and examined whether such a combination could inhibit angiogenesis and tumor growth in nude mice. In vitro angiogenesis assay was done using coculture of tumor cells with human microvascular endothelial cells. In vivo angiogenesis assay was done using diffusion chambers under the dorsal skin of nude mice. In vivo imaging of intracerebral tumorigenesis and longitudinal solid tumor development studies were conducted in nude mice. Results: In vitro and in vivo angiogenesis assays showed inhibition of capillary-like network formation of microvascular endothelial cells and neovascularization under dorsal skin of nude mice, respectively. We observed inhibition of intracerebral tumorigenesis and s.c. solid tumor formation in nude mice after treatment with combination of hTERT siRNA and IFN-γ. Western blotting of solid tumor samples showed significant downregulation of the molecules that regulate cell invasion, angiogenesis, and tumor progression. Conclusions: Our study showed that the combination of hTERT siRNA and IFN-γ effectively inhibited angiogenesis and tumor progression through the downregulation of molecules involved in these processes. Therefore, the combination of hTERT siRNA and IFN-γ is a promising therapeutic strategy for controlling the growth of human glioblastoma. (Clin Cancer Res 2009;15(23):7186–95)

Molecular mechanisms of the combination of retinoid and interferon-gamma for inducing differentiation and increasing apoptosis in human glioblastoma T98G and U87MG cells.

Abstract: Glioblastoma is the deadliest brain tumor that remains incurable. We examined efficacy of combination of retinoid and interferon-gamma (IFN-gamma) in human glioblastoma T98G and U87MG cells. We conjectured that retinoid could induce differentiation with down regulation of telomerase activity to increase sensitivity to IFN-gamma for apoptosis in glioblastoma cells. Indeed, treatment of cells with 1 muM all-trans retinoic acid (ATRA) or 1 muM 13-cis retinoic acid (13-CRA) for 7 days induced astrocytic differentiation with upregulation of glial fibrillary acidic protein (GFAP) and down regulation of telomerase activity. Wright staining and ApopTag assay showed, respectively, morphological and biochemical features of apoptosis in glioblastoma cells following exposure to 200 units/ml IFN-gamma for 48 h. Induction of differentiation was associated with decreases in levels of nuclear factor kappa B (NFkappaB), inducible nitric oxide synthase (iNOS), and production of nitric oxide (NO) so as to increase sensitivity to IFN-gamma for apoptosis. Notably, IFN-gamma induced signal transducer and activator of transcription-1 (STAT-1) to bind to gamma-activated sequence (GAS) of the target gene. Also, IFN-gamma activated caspase-8 and cleaved Bid to truncated Bid (tBid) for translocation to mitochondria. Fura-2 assay showed increases in intracellular free [Ca2+] and activation of calpain in apoptotic cells. Besides, increases in Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and Smac into the cytosol activated caspase-9 and caspase-3 for apoptosis. Taken together, our results showed that retinoid induced astrocytic differentiation with down regulation of telomerase activity and enhanced sensitivity to IFN-gamma for increasing apoptosis in human glioblastoma cells.

Retinoids induce differentiation and downregulate telomerase activity and N-Myc to increase sensitivity to flavonoids for apoptosis in human malignant neuroblastoma SH-SY5Y cells

Abstract: Human malignant neuroblastoma is characterized by poor differentiation and uncontrolled proliferation of immature neuroblasts. Retinoids such as all-trans-retinoic acid (ATRA), 13-cis-retinoic acid (13-CRA), and N-(4-hydroxyphenyl) retinamide (4-HPR) at low doses are capable of inducing differentiation, while flavonoids such as (-)-epigallocatechin-3-gallate (EGCG) and genistein (GST) at relatively high dose can induce apoptosis. We used combination of retinoid and flavonoid for controlling growth of malignant neuroblastoma SH-SY5Y cells. Cells were treated with a retinoid (1 microM ATRA, 1 microM 13-CRA, or 0.5 microM 4-HPR) for 7 days and then with a flavonoid (25 microM EGCG or 25 microM GST) for 24 h. Treatment of cells with a low dose of a retinoid for 7 days induced neuronal differentiation with downregulation of telomerase activity and N-Myc but overexpression of neurofilament protein (NFP) and subsequent treatment with a relatively high dose of a flavonoid for 24 h increased apoptosis in the differentiated cells. Besides, retinoids reduced the levels of inflammatory and angiogenic factors. Apoptosis was associated with increases in intracellular free [Ca2+], Bax expression, cytochrome c release from mitochondria and activities of calpain and caspases. Decreases in expression of calpastatin (endogenous calpain inhibitor) and baculovirus inhibitor-of-apoptosis repeat containing (BIRC) proteins (endogenous caspase inhibitors) favored apoptosis. Treatment of SH-SY5Y cells with EGCG activated caspase-8, indicating induction of the receptor-mediated pathway of apoptosis. Based on our observation, we conclude that combination of a retinoid and a flavonoid worked synergistically for controlling the malignant growth of human neuroblastoma cells.

Combination of taxol and Bcl-2 siRNA induces apoptosis in human glioblastoma cells and inhibits invasion, angiogenesis and tumour growth.

Abstract: Taxol is a powerful chemotherapeutic agent that binds to microtubules to prevent tumour cell division. However, a traditional high dose of taxol may also induce apoptosis in normal cells. The anti-apoptotic molecule Bcl-2 is up-regulated in tumour cells to prevent apoptosis. We designed this study to determine whether use of a low dose of taxol and anti-apoptotic Bcl-2 gene silencing would effectively induce apoptosis in human glioblastoma U251MG cells and also inhibit invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. We treated the cells with either 100 nM taxol or transfected with a plasmid vector expressing Bcl-2 siRNA or both agents together for 72 h. Knockdown of Bcl-2 potentiated efficacy of taxol for cell death. Fluorescence-activated cell sorting analysis, double immunofluorescent staining and TUNEL assay demonstrated apoptosis in about 70% of the cells after treatment with the combination of taxol and Bcl-2 siRNA. In vitro Matrigel invasion assay demonstrated dramatic decrease in glioblastoma cell invasion and in vivo angiogenesis assay showed complete inhibition of neovascularization in athymic nude mice after treatment with the combination. Further, treatment with the combination of taxol and Bcl-2 siRNA caused suppression of intracranial tumour growth and subcutaneous solid tumour development. In conclusion, our results indicate that the combination of taxol and Bcl-2 siRNA effectively induces apoptosis and inhibits glioblastoma cell invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. Therefore, the combination of a low dose of taxol and Bcl-2 siRNA is a promising therapeutic strategy for controlling the aggressive growth of human glioblastoma.

Involvement of Calpain in the Process of Jurkat T Cell Chemotaxis

Abstract: T cell involvement in the disease process of multiple sclerosis (MS) (Hickey, 1991; McFarland and Martin, 2007) and its animal model experimental autoimmune encephalomyelitis (EAE) are well established (Waksman and Adams, 1962; Swanborg, 2001). T cell infiltration into the central nervous system (CNS) is greatly increased in the plaques of MS patients (Hickey, 1991) as well as of EAE animals immediately before the onset of pathological symptoms and during clinical course of the disease (Waksman and Adams, 1962). Increased activity and expression of the Ca2+-activated protease calpain has been demonstrated in inflammatory cells (Shields et al., 1998, 1999) during the disease state in EAE animals. Moreover, inhibition of calpain has been demonstrated to decrease the T cell infiltration during acute EAE (Hassen et al., 2006). Calpain has signaling functions and is involved in a multitude of cellular processes including apoptosis (Zhivotovsky et al., 1997), T cell activation (Penna et al., 1999; Schaecher et al., 2004), cell cycle (Janossy et al., 2004), and cell migration (Huttenlocher et al., 1997; Franco and Huttenlocher, 2005). T cell exclusion from the CNS is the therapeutic strategy used by the α4β1-integrin blocking monoclonal antibody natalizumab (Ransohoff, 2007), which has been approved by the U.S. Food and Drug Administration (FDA). However, use of the FDA-approved drug natalizumab has not been without problems including several patients on this medication developing progressive multi-focal leukoencephalopathy (Koralnik, 2006) leading to the temporary withdrawal of the drug from the market. In addition, alternative targets for MS therapy are urgently warranted as a result of the low effectiveness of many of the current standard therapies available (Kleinschnitz et al., 2007). Targeted exclusion of T cells from the CNS would be of great benefit not only to MS but also to many other autoimmune diseases that have a T cell component in their pathology, including rheumatoid arthritis (Cope et al., 2007) and amyotrophic lateral sclerosis (Graves et al., 2004). Inflammatory cells entering the CNS in MS and EAE have been shown to increase calpain levels (Shields et al., 1998). If calpain was indeed involved with T cell migration then calpain inhibition would provide an alternative method for reducing pathological T cell entry into the CNS. Calpain has been shown to be involved in the migration of numerous cell types including CHO cells (Huttenlocher et al., 1997), fibroblasts (Shiraha et al., 2002), myoblasts (Leloup et al., 2006), and neutrophils (Lokuta et al., 2003). The chemokine CCL2 (previously known as monocyte chemoattractant protein-1) and its predominant receptor CCR2, a G protein–coupled receptor, have been extensively associated with inflammatory pathologies (Jee et al., 2002; Mahad et al., 2006). CCL2 has been extensively shown as a specific chemoattractant for monocytes and T cells (Carr et al., 1994; Sanders et al., 2000). Even though the initiating stimulus for migration is well established, the downstream mechanism of T cell chemotaxis as of yet remains incomplete. In this study, our aim was to gather evidence for the involvement of calpain in the process of T cell migration. This analysis used the Jurkat E6-1 T cell line to model for T cells as well as the normal human peripheral blood mononuclear cells (PBMCs) to model for primary cells. We observed an increase in activity of calpain in response to the CCL2 stimulation as well as the restriction of role of calpain in involvement in the directed migration of chemotaxis. This is in contrast to other immune cell types where calpain has been demonstrated to play a role in the negative regulation of chemokinesis (Lokuta et al., 2003). Thus, our current study provides an evidence for the involvement of calpain in T cell chemotaxis. Further examination of calpain involvement in the chemotaxis of native T cells and pathological T cells from MS patients and EAE animals is supported by the findings of this study.

Genistein induces receptor and mitochondrial pathways and increases apoptosis during BCL-2 knockdown in human malignant neuroblastoma SK-N-DZ cells.

Abstract: The potent antiapoptotic molecule Bcl-2 is markedly up-regulated in a majority of cancers, including neuroblastoma. Genistein is an isoflavone with antitumor properties. The present study sought to elucidate the molecular mechanism of genistein-induced apoptosis and also to examine the effect of genistein in increasing apoptosis during Bcl-2 knockdown in human malignant neuroblastoma SK-N-DZ cells. The cells were transfected with Bcl-2 siRNA plasmid vector, treated with 10 microM genistein, or the combination, and subjected to TUNEL staining and FACS analysis. Semiquantitative and real-time RT-PCR experiments were performed for examining expression of Fas ligand (FasL), tumor necrosis factor-alpha (TNF-alpha), Fas-associated death domain (FADD), and TNFR-1-associated death domain (TRADD). The cell lysates were analyzed by Western blotting for levels of molecules involved in both receptor- and mitochondria-mediated apoptotic pathways. Treatment with the combination of Bcl-2 siRNA and genistein resulted in more than 80% inhibition of cell proliferation. TUNEL staining and FACS analysis demonstrated apoptosis in 70% of cells after treatment with the combination of both agents. Apoptosis was associated with increases in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c, and activation of caspases through the mitochondria-mediated apoptotic pathway. Genistein triggered the receptor-mediated apoptotic pathway through upregulation of TNF-alpha, FasL, TRADD, and FADD and activation of caspase-8. Combination of Bcl-2 siRNA and genistein triggered a marked increase in cleavage of DFF45 and PARP that resulted in enhanced apoptosis. Our study demonstrates that Bcl-2 knockdown during genistein treatment effectively induced apoptosis in neuroblastoma cells. Therefore, this strategy could serve as a potential therapeutic regimen to inhibit the growth of human malignant neuroblastoma.

Inhibition of calpain attenuates encephalitogenicity of MBP-specific T cells

Abstract: Multiple sclerosis (MS) is a T-cell mediated autoimmune disease of the CNS, possessing both immune and neurodegenerative events that lead to disability. Adoptive transfer (AT) of myelin basic protein (MBP)-specific T cells into naive female SJL/J mice results in a relapsing-remitting (RR) form of experimental autoimmune encephalomyelitis (EAE). Blocking the mechanisms by which MBP-specific T cells are activated before AT may help characterize the immune arm of MS and offer novel targets for therapy. One such target is calpain, which is involved in activation of T cells, migration of immune cells into the CNS, degradation of axonal and myelin proteins, and neuronal apoptosis. Thus, the hypothesis that inhibiting calpain in MBP-specific T cells would diminish their encephalitogenicity in RR-EAE mice was tested. Incubating MBP-specific T cells with the calpain inhibitor SJA6017 before AT markedly suppressed the ability of these T cells to induce clinical symptoms of RR-EAE. These reductions correlated with decreases in demyelination, inflammation, axonal damage, and loss of oligodendrocytes and neurons. Also, calpain : calpastatin ratio, production of truncated Bid, and Bax : Bcl-2 ratio, and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated. Thus, these data suggest calpain as a promising target for treating EAE and MS.

Use of cannabinoids as a novel therapeutic modality against autoimmune hepatitis.

Abstract: Autoimmune hepatitis is a severe immune mediated chronic liver disease with a prevalence range between 50 and 200 cases per million in Western Europe and North America and mortality rates of up to 80% in untreated patients The induction of CB1 and CB2 cannabinoid receptors during liver injury and the potential involvement of endocannabinoids in the regulation of this process have sparked significant interest in further evaluating the role of cannabinoid systems during hepatic disease Cannabinoids have been shown to possess significant immunosuppressive and anti-inflammatory properties Cannabinoid abuse has been shown to exacerbate liver fibrogenesis in patients with chronic hepatitis C infection involving CB1 receptor Nonetheless, CB2 receptor activation may play a protective role during chronic liver diseases Thus, differential targeting of cannabinoid receptors may provide novel therapeutic modality against autoimmune hepatitis In this review, we summarize current knowledge on the role of endocannabinoids and exocannabinoids in the regulation of autoimmune hepatitis

Brain and spinal cord trauma

Abstract: Brain Tumor Angiogenesis.- Adrenoleukodystrophy: Molecular, Metabolic, Pathologic And Therapeutic Aspects.- Hyperammonemia.- Glutamate And Cytokine-mediated Alterations Of Phospholipids In Head Injury And Spinal Cord Trauma.- Kynurenines In The Brain: Preclinical And Clinical Studies, Therapeutic Considerations.- Neurofibromatosis Type I: From Genetic Mutation To Tumor Formations.- Amyloid And Neurodegeneration: Alzheimer's Disease And Retinal Degeneration.- Diabetic Retinopathy.- Neurotransmitters And Electrophysiology In Traumatic Brain Injury.- Free Radicals And Neuroprotection In Traumatic Brain And Spinal Cord Injury.- Neuroimmunology Of Paraproteinemic Neuropathies.- Proteolytic Mechanisms Of Cell Death In The Central Nervous System.- Nitric Oxide In Experimental Allergic Encephalomyelitis.- The BBB: Biology, Development And Brain Injury.- Phospholipase A2 In CNS Disorders: Implication On Traumatic Spinal Cord And Brain Injuries.- Axonal Damage Due To Traumatic Brain Injury.- Blood-Central Nervous System Barriers: The Gateway To Neurodegeneration, Neuroprotection And Neuroregeneration.- Engineered Antibody Fragments As Potential Therapeutics Against Misfolded Proteins In Neurodegenerative Diseases.- Pathogenesis And Treatment Of HIV-Associated Dementia: Recent Studies In A SCID Mouse Model.- Stem Cell Transplantation Therapy For Neurological Diseases.- Ubiquitin/Proteasome And Autophagy/Lysosome Pathways: Comparison And Role In Neurodegeneration.- Experimental Autoimmune Encephalomyelitis In The Pathogenesis Of Optic Neuritis: Is Calpain Involved?- Protein Carbonylation In Neurodegenerative And Demyelinating CNS Diseases.- Clinical Considerations In Translational Research With Chronic Spinal Cord Injury: Intervention Readiness And Intervention Impact.- Estrogen As A Promising Multi-Active Agent For The Treatment Of Spinal Cord Injury.- Immunotherapy Strategies For Lewy Body And Parkinson's Disease.- Clinical Outcomes After Spinal Cord Injury.- Spinal Cord Injury ~ A Clinical Perspective.- Cubing The Brain: Mapping Expression Patterns Genome-Wide.

Comprar Handbook of Neurochemistry and Molecular Neurobiology · Brain and Spinal Cord Trauma | Ray, Swapan | 9780387303437 | Springer

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