Ellora Sen - Academia.edu (original) (raw)

Papers by Ellora Sen

Evolution-adjusted Tumor Pathophysiology:, 2013

Poised in a seemingly prepared calm stasis with trust in the intangible, cancer cell makes decisi... more Poised in a seemingly prepared calm stasis with trust in the intangible, cancer cell makes decisions from a point where philosophy seems to be its major compass. As pure biological presuppositions cannot access or fathom this invisible philosophical realm of a cancer cell; syllogism rooted in scientific ratiocination alone without philosophical analysis will elude understanding of the objectivity of a cancer cell. Appreciation of this undiscovered unseen dimension (which is but a counterpart of the comprehensible visible), will unravel the deep interconnectedness between these two and provide insight far greater than empiric epistemology.

Hypoxic preconditioning reprogrammes the brain's response to subsequent H/I (hypoxia-ischaemia) i... more Hypoxic preconditioning reprogrammes the brain's response to subsequent H/I (hypoxia-ischaemia) injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O 2). Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic protein)positive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase), EAAT-1 (excitatory amino acid transporter-1; also known as GLAST), MCT-1 (monocarboxylate transporter-1) and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP), which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.

Mental Retardation and Developmental Disabilities Research Reviews, 2006

There is an increasing awareness that the astrocytes in the immature periventricular white matter... more There is an increasing awareness that the astrocytes in the immature periventricular white matter are vulnerable to ischemia and respond to inflammation. Here we provide a synopsis of the articles that have evaluated the causes and consequences of developmental brain injuries to white matter astrocytes as well as the consequences of several genetic mutations that result in abnormal astrocyte development. Emerging data suggest that the astrocytes are not simply responding to the injury but are likely victims as well as culprits. Given the important roles that astrocytes play in maintaining ionic, neurotransmitter, and metabolic homeostasis in the brain, a more thorough understanding of the mechanisms that lead to their incapacitation, demise, or reactions as well as a better understanding of the stimuli that regulate their neuroprotective and regenerative properties will enable these cells to be manipulated to preserve the integrity of white matter and to potentially provide therapeutics to enhance neonatal regeneration and recovery from brain injury. MRDD Research Reviews 2006;12:97–104. © 2006 Wiley-Liss, Inc.

PLOS One, 2010

Background: In children born prematurely and those surviving cerebral ischemia there are white ma... more Background: In children born prematurely and those surviving cerebral ischemia there are white matter abnormalities that correlate with neurological dysfunction. Since this injury occurs in the immature brain, when the majority of subventricular zone (SVZ) cells generate white matter oligodendrocytes, we sought to study the effect this injury has on gliogenesis from the SVZ. We hypothesized that there is aberrant glial cell generation from the SVZ after neonatal hypoxia ischemia (H/I) that contributes to an increased astrogliogenesis with concomitant oligodendroglial insufficiency. Mechanistically we hypothesized that an increase in specific locally produced cytokines during recovery from injury were modifying the differentiation of glial progenitors towards astrocytes at the expense of the more developmentally-appropriate oligodendrocytes.

PLOS One, 2010

Background: In children born prematurely and those surviving cerebral ischemia there are white ma... more Background: In children born prematurely and those surviving cerebral ischemia there are white matter abnormalities that correlate with neurological dysfunction. Since this injury occurs in the immature brain, when the majority of subventricular zone (SVZ) cells generate white matter oligodendrocytes, we sought to study the effect this injury has on gliogenesis from the SVZ. We hypothesized that there is aberrant glial cell generation from the SVZ after neonatal hypoxia ischemia (H/I) that contributes to an increased astrogliogenesis with concomitant oligodendroglial insufficiency. Mechanistically we hypothesized that an increase in specific locally produced cytokines during recovery from injury were modifying the differentiation of glial progenitors towards astrocytes at the expense of the more developmentally-appropriate oligodendrocytes.

Journal of Virology, 2002

The function of the 5 region of the upstream regulatory region (URR) in regulating E6/E7 expressi... more The function of the 5 region of the upstream regulatory region (URR) in regulating E6/E7 expression in cancer-associated papillomaviruses has been largely uncharacterized. In this study we used linker-scanning mutational analysis to identify potential cis regulatory elements contained within a portion of the 5 region of the URR that are involved in regulating transcription of the E6/E7 promoter at different stages of the viral life cycle. The mutational analysis illustrated differences in the transcriptional utilization of specific regions of the URR depending on the stage of the viral life cycle. This study identified (i) viral cis elements that regulate transcription in the presence and absence of any viral gene products or viral DNA replication, (ii) the role of host tissue differentiation in viral transcriptional regulation, and (iii) cis regulatory regions that are effected by induction of the protein kinase C pathway. Our studies have provided an extensive map of functional elements in the 5 region (nuncleotides 7259 to 7510) of the human papillomavirus type 31 URR that are involved in the regulation of p99 promoter activity at different stages of the viral life cycle.

Journal of Virology, 2006

Adeno-associated virus type 2 (AAV2) seropositivity is negatively correlated with the development... more Adeno-associated virus type 2 (AAV2) seropositivity is negatively correlated with the development of human papillomavirus (HPV)-associated cervical cancer. We have begun analysis of the molecular mechanisms underlying AAV2-mediated oncosuppression through cell cycle regulation in HPV-infected keratinocytes isolated from a low-grade cervical lesion. AAV2 superinfection of HPV type 31b (HPV31b)-positive cells at early times postinfection resulted in degradation of the cyclin-dependent kinase (CDK) inhibitor p21 WAF1 protein in a proteosome-dependent manner. Downstream consequences of lowering p21 WAF1 levels included a proportional loss of cyclin E/CDK2 complexes bound to p21 WAF1 .

Journal of Virology, 2003

Glucocorticoids have been shown to play a role in the transforming abilities of human papillomavi... more Glucocorticoids have been shown to play a role in the transforming abilities of human papillomaviruses (HPVs), and glucocorticoid response elements (GREs) have been identified in the upstream regulatory regions (URRs) of various HPV types. These findings have made glucocorticoids potential therapeutic targets for HPV infection. We have previously shown that the URR of HPV type 31 (HPV31) is insensitive to induction by the synthetic glucocorticoid dexamethasone (dex) in monolayer culture, despite the identification of three potential GREs in the 5 region of the URR. Due to the fact that the HPV life cycle is intimately linked to the differentiation of the host tissue, we chose to determine whether the URR of HPV31 was inducible by dex under differentiating conditions. Upon suspension of cells in a semisolid medium of methylcellulose, we found that the URR of HPV31 was inducible by dex. The three GREs appear to play roles as independent repressors of this inducibility. By 5 deletion analysis, the element(s) responsible for this induction was localized to nucleotides (nt) 7238 to 7557. Furthermore, we found that the region between nt 7883 and 7900 appears to act as a repressor of dex inducibility. These findings indicate that epithelial differentiation has a profound effect on the action of dex on the URR of HPV31, suggesting that glucocorticoids play an important role in the differentiation-dependent life cycle of HPV.

Cellular Signalling, 2011

The insulin-like growth factor (IGF-1) induces hypoxia inducible factor (HIF-1α) regulated genes ... more The insulin-like growth factor (IGF-1) induces hypoxia inducible factor (HIF-1α) regulated genes in glioblastoma multiforme (GBM). As HIF-1α links inflammatory and oncogenic pathways in GBM, we investigated whether IGF-1 affects HIF-1α to regulate inflammatory response in glioma cells under normoxia. IGF-1 induced Ras and Calmodulin-dependent kinase II (CaMKII) regulated HIF-1α transcriptional activity in glioma cells. Increase in HIF-1α was concurrent with decreased Toll-like receptor (TLR9) and CXCR4 expression and elevated suppressor of cytokine signaling (SOCS3) levels. Interestingly, while synthetic CpG containing oligodeoxynucleotide TLR9 agonist (CpG DNA) decreased IGF-1 mediated increase in HIF-1α activity, siRNA mediated knockdown of HIF-1α decreased TLR9 levels. This suggested that IGF-1 induced HIF-1α-TLR9 axis is regulated by both positive and negative feedback loops. Importantly, TLR9 agonist reversed the effect of IGF-1 on CXCR4 and SOCS3 expression. While knockdown of HIF-1α abrogated IGF-1 mediated increase in SOCS3 it elevated IGF-1 induced decrease in CXCR4 levels. Thus HIF-1α positively and negatively regulates SOCS3 and CXCR4 expression respectively, in glioma cells. Though TLR9 agonist had no additive effect on IGF-1 mediated increase in pro-inflammatory cytokines IL-1β, IL-6 and IL-8, treatment with TLR9 agonist alone elevated expression of these pro-inflammatory cytokines. Our studies indicate that a complex HIF-1α-TLR9 cross-talk sustains a self-regulating cycle of inflammatory response through intrinsic negative and positive feedback mechanisms.

Free Radical Biology and Medicine, 2009

The poor prognosis of glioblastoma multiforme and lack of effective therapy have necessitated the... more The poor prognosis of glioblastoma multiforme and lack of effective therapy have necessitated the identification of new treatment strategies. We have previously reported that elevation of oxidative stress induces apoptosis of glioma cells. Because the farnesyltransferase inhibitor manumycin is known to induce reactive oxygen species (ROS) generation, we evaluated the effects of manumycin on glioma cells. Manumycin induced glioma cell apoptosis by elevating ROS generation. Treatment with the ROS inhibitor N-acetylcysteine blocked manumycin-induced apoptosis, caspase-3 activity, and PARP expression, indicating the involvement of increased ROS in the proapoptotic activity of manumycin. This heightened ROS level was accompanied by a concurrent decrease in antioxidants such as superoxide dismutase (SOD-1) and thioredoxin (TRX-1). SOD-1 overexpression protects glioma cells from manumycin-induced apoptosis. In addition, small interfering RNA-mediated knockdown of SOD-1 and TRX-1 expression also increased ROS generation and sensitivity of glioma cells to manumycin-induced cell death. Interestingly, suppressing ROS generation prevented manumycin-induced Ras inhibition. This study reports for the first time that Ras inhibition by manumycin is due to heightened ROS levels. We also report for the first time that manumycin inhibits the phosphorylation of signal transducer and activator of transcription 3 and telomerase activity in a ROS-dependent manner, which plays a crucial role in glioma resistance to apoptosis. In addition manumycin (i) induced the DNA-damage repair response, (ii) affected cell-cycle-regulatory molecules, and (iii) impaired the colony-forming ability of glioma cells in a ROS-dependent manner.

We investigated the pro-inflammatory response mediated by TNFa in glioblastoma and whether treatm... more We investigated the pro-inflammatory response mediated by TNFa in glioblastoma and whether treatment with organoselenium Ebselen (2-phenyl-1,2-benzisoselenazol- 3(2H)one) can affect TNFa induced inflammatory response. Exposure to TNFa increased the expression of pro-inflammatory mediator interleukin IL-6, IL-8, monocyte chemoat- tractant protein-1 (MCP-1) and cyclooxygenase (COX-2). Treatment with Ebselen abrogated TNFa induced increase in pro-inflammatory mediators. Ebselen not only abrogated TNFa induced

Brain Research Bulletin, 2007

The proinflammatory cytokine interleukin-1beta (IL-1beta) contributes to inflammation and neurona... more The proinflammatory cytokine interleukin-1beta (IL-1beta) contributes to inflammation and neuronal death in CNS injuries and neurodegenerative pathologies, and astrocytes have been implicated as the primary mediators of IL-1beta induced neuronal death. As astrocytes play an important role in supporting the survival and functions of neurons, we investigated the effect of plant flavonoids quercetin and luteolin, with known anti-inflammatory properties in modulating the response of human astrocytes to IL-1beta for therapeutic intervention. Flavonoids significantly decreased the release of reactive oxygen species (ROS) from astrocytes stimulated with IL-1beta. This decrease was accompanied by an increase in expression of superoxide dismutase (SOD-1) and thioredoxin (TRX1)-mediators associated with protection against oxidative stress. Flavonoids not only modulated the expression of astrocytes specific molecules such as glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and ceruloplasmin (CP) both in the presence and absence of IL-1beta but also decreased the elevated levels of proinflammatory cytokine interleukin-6 (IL-6) and chemokines interleukin-8 (IL-8), interferon-inducible protein (IP-10), monocyte-chemoattractant protein-1 (MCP-1), and RANTES from IL-1beta activated astrocytes. Significant decrease in neuronal apoptosis was observed in neurons cultured in conditioned medium obtained from astrocytes treated with a combination of IL-1beta and flavonoids as compared to that treated with IL-1beta alone. Our result suggests that by (i) enhancing the potential of activated astrocytes to detoxify free radical, (ii) reducing the expression of proinflammatory cytokines and chemokines, and (iii) modulating expression of mediators associated with enhanced physiological activity of astrocyte in response to injury, flavonoids confer (iv) protection against IL-1beta induced astrocyte mediated neuronal damage.

Neurochemistry International, 2011

Cancer stem-like cells (CSCs) possessing features of neural precursor cells (NPC) influence initi... more Cancer stem-like cells (CSCs) possessing features of neural precursor cells (NPC) influence initiation, recurrence and chemoresistance of glioblastoma multiforme (GBM). As inflammation is crucial for glioblastoma progression we investigated the effect of chronic IL-1b treatment on CSCs derived from glioblastoma cell line U87MG. Exposure to IL-1b for 10 days increased (i) accumulation of 8-OHdG -a key biomarker of oxidative DNA damage; (ii) DNA damage response (DDR) indicators cH2AX, ATM and DNA-PK; (iii) nuclear and cytoplasmic p53 and COX-2 levels and (iv) interaction between COX-2 and p53. Despite upregulating p53 expression IL-1b had no effect on cell cycle progression, apoptosis or self renewal capacity of CSCs. COX-2 inhibitor Celecoxib reduced self renewal capacity and increased apoptosis of both control and IL-1b treated CSCs. Therefore the ability of COX-2 to regulate proliferation of CSCs irrespective of exposure to IL-1b, warrants further investigation of COX-2 as a potential anti-glioma target.

Molecular Oncology, 2009

We investigated the pro-inflammatory response mediated by TNFalpha in glioblastoma and whether tr... more We investigated the pro-inflammatory response mediated by TNFalpha in glioblastoma and whether treatment with organoselenium Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]one) can affect TNFalpha induced inflammatory response. Exposure to TNFalpha increased the expression of pro-inflammatory mediator interleukin IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and cyclooxygenase (COX-2). Treatment with Ebselen abrogated TNFalpha induced increase in pro-inflammatory mediators. Ebselen not only abrogated TNFalpha induced enhanced invasiveness of glioma cells by down-regulating matrix metallo proteinase (MMP-9) and urokinase plasminogen (uPa) activity, but also inhibited glioma cell migration. Treatment with Ebselen also down-regulated the enhanced ROS production of TNFalpha treated glioma cells. In addition, Ebselen induced DNA damage repair signaling response in glioma cells both in the presence and absence of TNFalpha. These studies indicate that together with its known ability to sensitize glioma cell to TNFalpha induced apoptosis, Ebselen can overcome TNFalpha induced pro-inflammatory mediators to prevent a build up of a deleterious pro-inflammatory tumor microenvironment.

Molecular Cancer Therapeutics, 2007

Despite recent advances in understanding molecular mechanisms involved in glioblastoma progressio... more Despite recent advances in understanding molecular mechanisms involved in glioblastoma progression, the prognosis of the most malignant brain tumor continues to be dismal. Because the flavonoid kaempferol is known to suppress growth of a number of human malignancies, we investigated the effect of kaempferol on human glioblastoma cells. Kaempferol induced apoptosis in glioma cells by elevating intracellular oxidative stress. Heightened oxidative stress was characterized by an increased generation of reactive oxygen species (ROS) accompanied by a decrease in oxidant-scavenging agents such as superoxide dismutase (SOD-1) and thioredoxin (TRX-1). Knockdown of SOD-1 and TRX-1 expression by small interfering RNA (siRNA) increased ROS generation and sensitivity of glioma cells to kaempferol-induced apoptosis. Signs of apoptosis included decreased expression of Bcl-2 and altered mitochondrial membrane potential with elevated active caspase-3 and cleaved poly(ADP-ribose) polymerase expression. Plasma membrane potential and membrane fluidity were altered in kaempferol-treated cells. Kaempferol suppressed the expression of proinflammatory cytokine interleukin-6 and chemokines interleukin-8, monocyte chemoattractant protein-1, and regulated on activation, normal T-cell expressed and secreted. Kaempferol inhibited glioma cell migration in a ROS-dependent manner. Importantly, kaempferol potentiated the toxic effect of chemotherapeutic agent doxorubicin by amplifying ROS toxicity and decreasing the efflux of doxorubicin. Because the toxic effect of both kaempferol and doxorubicin was amplified when used in combination, this study raises the possibility of combinatorial therapy whose basis constitutes enhancing redox perturbation as a strategy to kill glioma cells. [Mol Cancer Ther 2007;6(9):2544 -53]

Glia, 2007

While a number of studies have documented the importance of microglia in central nervous system (... more While a number of studies have documented the importance of microglia in central nervous system (CNS) response to injury, infection and disease, little is known regarding its role in viral encephalitis. We therefore, exploited an experimental model of Japanese Encephalitis, to better understand the role played by microglia in Japanese Encephalitis Virus (JEV) infection. Lectin staining performed to assess microglial activation indicated a robust increase in reactive microglia following infection. A difference in the topographic distribution of activated, resting, and phagocytic microglia was also observed. The levels of various proinflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2), IL-6, IL-1β, TNF-α, and MCP-1 that have been implicated in microglial response to an activational state was significantly elevated following infection. These cytokines exhibited region selective expression in the brains of infected animals, with the highest expression observed in the hippocampus. Moreover, the expression of neuronal specific nuclear protein NeuN was markedly downregulated during progressive infection indicating neuronal loss. In vitro studies further confirmed that microglial activation and subsequent release of various proinflammatory mediators induces neuronal death following JEV infection. Although initiation of immune responses by microglial cells is an important protective mechanism in the CNS, unrestrained inflammatory responses may result in irreparable brain damage. Our findings suggest that the increased microglial activation following JEV infection influences the outcome of viral pathogenesis. It is likely that the increased microglial activation triggers bystander damage, as the animals eventually succumb to infection. © 2007 Wiley-Liss, Inc.

Journal of Molecular Medicine-jmm, 2011

We observed elevated levels of pro-inflammatory cytokine IL-1β in glioblastoma multiforme tumor s... more We observed elevated levels of pro-inflammatory cytokine IL-1β in glioblastoma multiforme tumor samples. Since hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in linking inflammatory and oncogenic pathways, we investigated the effect of IL-1β on HIF-1α expression in glioma cells under normoxia. IL-1β-mediated elevation of HIF-1α transcriptional activity was dependent on Ras-induced NF-κB activation, as IL-1β failed to induce NF-κB and HIF-1α activity in cells transfected with dominant negative RasN17. Increased Ras expression was accompanied by increased phosphorylation of Ras effectors AKT, ERK, JNK, and p38MAPK. While inhibition of these effectors individually failed to block the IL-1β-mediated increase in HIF-1α induction, co-inhibition of both AKT and ERK resulted in a significant decrease in IL-1β-induced HIF-1α activation. Interestingly, IL-1β elevated Wnt-1 expression in a Ras-dependent manner, and small interfering RNA (siRNA)-mediated knockdown of Wnt-1 decreased HIF-1α activity. Although Wnt-1-mediated HIF-1α was independent of the canonical Wnt/β-catenin signaling pathway, it regulated HIF-1α through NF-κB. siRNA-mediated HIF-1α knockdown attenuated elevated IL-1β mRNA levels induced upon IL-1β treatment. This was accompanied by increased interaction of HIF-1α with HIF responsive element on the IL-1β promoter upon IL-1β treatment, under normoxia. Our studies highlights for first time that (1) Ras is a key mediator of IL-1β-induced NF-κB and HIF-1α activation, under normoxia; (2) Wnt-1 regulates IL-1β-mediated HIF-1α induction via NF-κB; (3) Ras and Wnt-1 are intermediaries in the canonical IL-1β–NF-κB signaling pathway downstream of MyD88; and (4) IL-1β-induced HIF-1α drives a HIF-1α-IL-1β autocrine loop to maintain persistently elevated IL-1β level.

International Journal of Cancer, 2008

Resistance to tumor necrosis factor (TNFα)-induced apoptosis in various cancer cells has been att... more Resistance to tumor necrosis factor (TNFα)-induced apoptosis in various cancer cells has been attributed to the activation of the transcription factor NF-κB. Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]one)—a selenoorganic compound is known to prevent TNFα-mediated NF-κB activity. As glioblastoma are resistant to the cytotoxic effect of TNFα, we investigated the potential of Ebselen in sensitizing glioma cells to TNFα-induced apoptosis. Although treatment with Ebselen reduced viability of glioma cells, cotreatment with TNFα enhanced apoptosis further through alteration of TNFα-mediated signaling pathways. Sensitization of TNFα activated glioma cells to apoptosis by Ebselen involved 2 pathways: (i) abrogation of TNFα induced NF-κB activation and (ii) induction of Fas-associated death inducing signaling complex (DISC) formation. Ebselen inhibited the prosurvival pathway mediated by NF-κB by altering the association of TNF receptor associated factor 2 (TRAF2) with TNFα receptor associated death domain (TRADD) in the TNFR1-TRADD-TRAF2 complex —an interaction crucial for mediating NF-κB activity. Ebselen also induced the formation of DISC involving Fas, Fas-associated death domain (FADD) and active caspase 8 to transduce apoptotic signals in situations where NF-κB function was inhibited. Cotreatment with Ebselen and TNFα induced G2/M phase arrest in cell cycle and modulated the expression of molecules involved in cell cycle progression. These results raise the possibility of overcoming resistance to TNFα-induced apoptosis by cotreatment with organoselenium Ebselen as a strategy to kill glioma cells. © 2008 Wiley-Liss, Inc.

Neurochemistry International, 2010

Evolution-adjusted Tumor Pathophysiology:, 2013

Poised in a seemingly prepared calm stasis with trust in the intangible, cancer cell makes decisi... more Poised in a seemingly prepared calm stasis with trust in the intangible, cancer cell makes decisions from a point where philosophy seems to be its major compass. As pure biological presuppositions cannot access or fathom this invisible philosophical realm of a cancer cell; syllogism rooted in scientific ratiocination alone without philosophical analysis will elude understanding of the objectivity of a cancer cell. Appreciation of this undiscovered unseen dimension (which is but a counterpart of the comprehensible visible), will unravel the deep interconnectedness between these two and provide insight far greater than empiric epistemology.

Hypoxic preconditioning reprogrammes the brain's response to subsequent H/I (hypoxia-ischaemia) i... more Hypoxic preconditioning reprogrammes the brain's response to subsequent H/I (hypoxia-ischaemia) injury by enhancing neuroprotective mechanisms. Given that astrocytes normally support neuronal survival and function, the purpose of the present study was to test the hypothesis that a hypoxic preconditioning stimulus would activate an adaptive astrocytic response. We analysed several functional parameters 24 h after exposing rat pups to 3 h of systemic hypoxia (8% O 2). Hypoxia increased neocortical astrocyte maturation as evidenced by the loss of GFAP (glial fibrillary acidic protein)positive cells with radial morphologies and the acquisition of multipolar GFAP-positive cells. Interestingly, many of these astrocytes had nuclear S100B. Accompanying their differentiation, there was increased expression of GFAP, GS (glutamine synthetase), EAAT-1 (excitatory amino acid transporter-1; also known as GLAST), MCT-1 (monocarboxylate transporter-1) and ceruloplasmin. A subsequent H/I insult did not result in any further astrocyte activation. Some responses were cell autonomous, as levels of GS and MCT-1 increased subsequent to hypoxia in cultured forebrain astrocytes. In contrast, the expression of GFAP, GLAST and ceruloplasmin remained unaltered. Additional experiments utilized astrocytes exposed to exogenous dbcAMP (dibutyryl-cAMP), which mimicked several aspects of the preconditioning response, to determine whether activated astrocytes could protect neurons from subsequent excitotoxic injury. dbcAMP treatment increased GS and glutamate transporter expression and function, and as hypothesized, protected neurons from glutamate excitotoxicity. Taken altogether, these results indicate that a preconditioning stimulus causes the precocious differentiation of astrocytes and increases the acquisition of multiple astrocytic functions that will contribute to the neuroprotection conferred by a sublethal preconditioning stress.

Mental Retardation and Developmental Disabilities Research Reviews, 2006

There is an increasing awareness that the astrocytes in the immature periventricular white matter... more There is an increasing awareness that the astrocytes in the immature periventricular white matter are vulnerable to ischemia and respond to inflammation. Here we provide a synopsis of the articles that have evaluated the causes and consequences of developmental brain injuries to white matter astrocytes as well as the consequences of several genetic mutations that result in abnormal astrocyte development. Emerging data suggest that the astrocytes are not simply responding to the injury but are likely victims as well as culprits. Given the important roles that astrocytes play in maintaining ionic, neurotransmitter, and metabolic homeostasis in the brain, a more thorough understanding of the mechanisms that lead to their incapacitation, demise, or reactions as well as a better understanding of the stimuli that regulate their neuroprotective and regenerative properties will enable these cells to be manipulated to preserve the integrity of white matter and to potentially provide therapeutics to enhance neonatal regeneration and recovery from brain injury. MRDD Research Reviews 2006;12:97–104. © 2006 Wiley-Liss, Inc.

PLOS One, 2010

Background: In children born prematurely and those surviving cerebral ischemia there are white ma... more Background: In children born prematurely and those surviving cerebral ischemia there are white matter abnormalities that correlate with neurological dysfunction. Since this injury occurs in the immature brain, when the majority of subventricular zone (SVZ) cells generate white matter oligodendrocytes, we sought to study the effect this injury has on gliogenesis from the SVZ. We hypothesized that there is aberrant glial cell generation from the SVZ after neonatal hypoxia ischemia (H/I) that contributes to an increased astrogliogenesis with concomitant oligodendroglial insufficiency. Mechanistically we hypothesized that an increase in specific locally produced cytokines during recovery from injury were modifying the differentiation of glial progenitors towards astrocytes at the expense of the more developmentally-appropriate oligodendrocytes.

PLOS One, 2010

Background: In children born prematurely and those surviving cerebral ischemia there are white ma... more Background: In children born prematurely and those surviving cerebral ischemia there are white matter abnormalities that correlate with neurological dysfunction. Since this injury occurs in the immature brain, when the majority of subventricular zone (SVZ) cells generate white matter oligodendrocytes, we sought to study the effect this injury has on gliogenesis from the SVZ. We hypothesized that there is aberrant glial cell generation from the SVZ after neonatal hypoxia ischemia (H/I) that contributes to an increased astrogliogenesis with concomitant oligodendroglial insufficiency. Mechanistically we hypothesized that an increase in specific locally produced cytokines during recovery from injury were modifying the differentiation of glial progenitors towards astrocytes at the expense of the more developmentally-appropriate oligodendrocytes.

Journal of Virology, 2002

The function of the 5 region of the upstream regulatory region (URR) in regulating E6/E7 expressi... more The function of the 5 region of the upstream regulatory region (URR) in regulating E6/E7 expression in cancer-associated papillomaviruses has been largely uncharacterized. In this study we used linker-scanning mutational analysis to identify potential cis regulatory elements contained within a portion of the 5 region of the URR that are involved in regulating transcription of the E6/E7 promoter at different stages of the viral life cycle. The mutational analysis illustrated differences in the transcriptional utilization of specific regions of the URR depending on the stage of the viral life cycle. This study identified (i) viral cis elements that regulate transcription in the presence and absence of any viral gene products or viral DNA replication, (ii) the role of host tissue differentiation in viral transcriptional regulation, and (iii) cis regulatory regions that are effected by induction of the protein kinase C pathway. Our studies have provided an extensive map of functional elements in the 5 region (nuncleotides 7259 to 7510) of the human papillomavirus type 31 URR that are involved in the regulation of p99 promoter activity at different stages of the viral life cycle.

Journal of Virology, 2006

Adeno-associated virus type 2 (AAV2) seropositivity is negatively correlated with the development... more Adeno-associated virus type 2 (AAV2) seropositivity is negatively correlated with the development of human papillomavirus (HPV)-associated cervical cancer. We have begun analysis of the molecular mechanisms underlying AAV2-mediated oncosuppression through cell cycle regulation in HPV-infected keratinocytes isolated from a low-grade cervical lesion. AAV2 superinfection of HPV type 31b (HPV31b)-positive cells at early times postinfection resulted in degradation of the cyclin-dependent kinase (CDK) inhibitor p21 WAF1 protein in a proteosome-dependent manner. Downstream consequences of lowering p21 WAF1 levels included a proportional loss of cyclin E/CDK2 complexes bound to p21 WAF1 .

Journal of Virology, 2003

Glucocorticoids have been shown to play a role in the transforming abilities of human papillomavi... more Glucocorticoids have been shown to play a role in the transforming abilities of human papillomaviruses (HPVs), and glucocorticoid response elements (GREs) have been identified in the upstream regulatory regions (URRs) of various HPV types. These findings have made glucocorticoids potential therapeutic targets for HPV infection. We have previously shown that the URR of HPV type 31 (HPV31) is insensitive to induction by the synthetic glucocorticoid dexamethasone (dex) in monolayer culture, despite the identification of three potential GREs in the 5 region of the URR. Due to the fact that the HPV life cycle is intimately linked to the differentiation of the host tissue, we chose to determine whether the URR of HPV31 was inducible by dex under differentiating conditions. Upon suspension of cells in a semisolid medium of methylcellulose, we found that the URR of HPV31 was inducible by dex. The three GREs appear to play roles as independent repressors of this inducibility. By 5 deletion analysis, the element(s) responsible for this induction was localized to nucleotides (nt) 7238 to 7557. Furthermore, we found that the region between nt 7883 and 7900 appears to act as a repressor of dex inducibility. These findings indicate that epithelial differentiation has a profound effect on the action of dex on the URR of HPV31, suggesting that glucocorticoids play an important role in the differentiation-dependent life cycle of HPV.

Cellular Signalling, 2011

The insulin-like growth factor (IGF-1) induces hypoxia inducible factor (HIF-1α) regulated genes ... more The insulin-like growth factor (IGF-1) induces hypoxia inducible factor (HIF-1α) regulated genes in glioblastoma multiforme (GBM). As HIF-1α links inflammatory and oncogenic pathways in GBM, we investigated whether IGF-1 affects HIF-1α to regulate inflammatory response in glioma cells under normoxia. IGF-1 induced Ras and Calmodulin-dependent kinase II (CaMKII) regulated HIF-1α transcriptional activity in glioma cells. Increase in HIF-1α was concurrent with decreased Toll-like receptor (TLR9) and CXCR4 expression and elevated suppressor of cytokine signaling (SOCS3) levels. Interestingly, while synthetic CpG containing oligodeoxynucleotide TLR9 agonist (CpG DNA) decreased IGF-1 mediated increase in HIF-1α activity, siRNA mediated knockdown of HIF-1α decreased TLR9 levels. This suggested that IGF-1 induced HIF-1α-TLR9 axis is regulated by both positive and negative feedback loops. Importantly, TLR9 agonist reversed the effect of IGF-1 on CXCR4 and SOCS3 expression. While knockdown of HIF-1α abrogated IGF-1 mediated increase in SOCS3 it elevated IGF-1 induced decrease in CXCR4 levels. Thus HIF-1α positively and negatively regulates SOCS3 and CXCR4 expression respectively, in glioma cells. Though TLR9 agonist had no additive effect on IGF-1 mediated increase in pro-inflammatory cytokines IL-1β, IL-6 and IL-8, treatment with TLR9 agonist alone elevated expression of these pro-inflammatory cytokines. Our studies indicate that a complex HIF-1α-TLR9 cross-talk sustains a self-regulating cycle of inflammatory response through intrinsic negative and positive feedback mechanisms.

Free Radical Biology and Medicine, 2009

The poor prognosis of glioblastoma multiforme and lack of effective therapy have necessitated the... more The poor prognosis of glioblastoma multiforme and lack of effective therapy have necessitated the identification of new treatment strategies. We have previously reported that elevation of oxidative stress induces apoptosis of glioma cells. Because the farnesyltransferase inhibitor manumycin is known to induce reactive oxygen species (ROS) generation, we evaluated the effects of manumycin on glioma cells. Manumycin induced glioma cell apoptosis by elevating ROS generation. Treatment with the ROS inhibitor N-acetylcysteine blocked manumycin-induced apoptosis, caspase-3 activity, and PARP expression, indicating the involvement of increased ROS in the proapoptotic activity of manumycin. This heightened ROS level was accompanied by a concurrent decrease in antioxidants such as superoxide dismutase (SOD-1) and thioredoxin (TRX-1). SOD-1 overexpression protects glioma cells from manumycin-induced apoptosis. In addition, small interfering RNA-mediated knockdown of SOD-1 and TRX-1 expression also increased ROS generation and sensitivity of glioma cells to manumycin-induced cell death. Interestingly, suppressing ROS generation prevented manumycin-induced Ras inhibition. This study reports for the first time that Ras inhibition by manumycin is due to heightened ROS levels. We also report for the first time that manumycin inhibits the phosphorylation of signal transducer and activator of transcription 3 and telomerase activity in a ROS-dependent manner, which plays a crucial role in glioma resistance to apoptosis. In addition manumycin (i) induced the DNA-damage repair response, (ii) affected cell-cycle-regulatory molecules, and (iii) impaired the colony-forming ability of glioma cells in a ROS-dependent manner.

We investigated the pro-inflammatory response mediated by TNFa in glioblastoma and whether treatm... more We investigated the pro-inflammatory response mediated by TNFa in glioblastoma and whether treatment with organoselenium Ebselen (2-phenyl-1,2-benzisoselenazol- 3(2H)one) can affect TNFa induced inflammatory response. Exposure to TNFa increased the expression of pro-inflammatory mediator interleukin IL-6, IL-8, monocyte chemoat- tractant protein-1 (MCP-1) and cyclooxygenase (COX-2). Treatment with Ebselen abrogated TNFa induced increase in pro-inflammatory mediators. Ebselen not only abrogated TNFa induced

Brain Research Bulletin, 2007

The proinflammatory cytokine interleukin-1beta (IL-1beta) contributes to inflammation and neurona... more The proinflammatory cytokine interleukin-1beta (IL-1beta) contributes to inflammation and neuronal death in CNS injuries and neurodegenerative pathologies, and astrocytes have been implicated as the primary mediators of IL-1beta induced neuronal death. As astrocytes play an important role in supporting the survival and functions of neurons, we investigated the effect of plant flavonoids quercetin and luteolin, with known anti-inflammatory properties in modulating the response of human astrocytes to IL-1beta for therapeutic intervention. Flavonoids significantly decreased the release of reactive oxygen species (ROS) from astrocytes stimulated with IL-1beta. This decrease was accompanied by an increase in expression of superoxide dismutase (SOD-1) and thioredoxin (TRX1)-mediators associated with protection against oxidative stress. Flavonoids not only modulated the expression of astrocytes specific molecules such as glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and ceruloplasmin (CP) both in the presence and absence of IL-1beta but also decreased the elevated levels of proinflammatory cytokine interleukin-6 (IL-6) and chemokines interleukin-8 (IL-8), interferon-inducible protein (IP-10), monocyte-chemoattractant protein-1 (MCP-1), and RANTES from IL-1beta activated astrocytes. Significant decrease in neuronal apoptosis was observed in neurons cultured in conditioned medium obtained from astrocytes treated with a combination of IL-1beta and flavonoids as compared to that treated with IL-1beta alone. Our result suggests that by (i) enhancing the potential of activated astrocytes to detoxify free radical, (ii) reducing the expression of proinflammatory cytokines and chemokines, and (iii) modulating expression of mediators associated with enhanced physiological activity of astrocyte in response to injury, flavonoids confer (iv) protection against IL-1beta induced astrocyte mediated neuronal damage.

Neurochemistry International, 2011

Cancer stem-like cells (CSCs) possessing features of neural precursor cells (NPC) influence initi... more Cancer stem-like cells (CSCs) possessing features of neural precursor cells (NPC) influence initiation, recurrence and chemoresistance of glioblastoma multiforme (GBM). As inflammation is crucial for glioblastoma progression we investigated the effect of chronic IL-1b treatment on CSCs derived from glioblastoma cell line U87MG. Exposure to IL-1b for 10 days increased (i) accumulation of 8-OHdG -a key biomarker of oxidative DNA damage; (ii) DNA damage response (DDR) indicators cH2AX, ATM and DNA-PK; (iii) nuclear and cytoplasmic p53 and COX-2 levels and (iv) interaction between COX-2 and p53. Despite upregulating p53 expression IL-1b had no effect on cell cycle progression, apoptosis or self renewal capacity of CSCs. COX-2 inhibitor Celecoxib reduced self renewal capacity and increased apoptosis of both control and IL-1b treated CSCs. Therefore the ability of COX-2 to regulate proliferation of CSCs irrespective of exposure to IL-1b, warrants further investigation of COX-2 as a potential anti-glioma target.

Molecular Oncology, 2009

We investigated the pro-inflammatory response mediated by TNFalpha in glioblastoma and whether tr... more We investigated the pro-inflammatory response mediated by TNFalpha in glioblastoma and whether treatment with organoselenium Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]one) can affect TNFalpha induced inflammatory response. Exposure to TNFalpha increased the expression of pro-inflammatory mediator interleukin IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1) and cyclooxygenase (COX-2). Treatment with Ebselen abrogated TNFalpha induced increase in pro-inflammatory mediators. Ebselen not only abrogated TNFalpha induced enhanced invasiveness of glioma cells by down-regulating matrix metallo proteinase (MMP-9) and urokinase plasminogen (uPa) activity, but also inhibited glioma cell migration. Treatment with Ebselen also down-regulated the enhanced ROS production of TNFalpha treated glioma cells. In addition, Ebselen induced DNA damage repair signaling response in glioma cells both in the presence and absence of TNFalpha. These studies indicate that together with its known ability to sensitize glioma cell to TNFalpha induced apoptosis, Ebselen can overcome TNFalpha induced pro-inflammatory mediators to prevent a build up of a deleterious pro-inflammatory tumor microenvironment.

Molecular Cancer Therapeutics, 2007

Despite recent advances in understanding molecular mechanisms involved in glioblastoma progressio... more Despite recent advances in understanding molecular mechanisms involved in glioblastoma progression, the prognosis of the most malignant brain tumor continues to be dismal. Because the flavonoid kaempferol is known to suppress growth of a number of human malignancies, we investigated the effect of kaempferol on human glioblastoma cells. Kaempferol induced apoptosis in glioma cells by elevating intracellular oxidative stress. Heightened oxidative stress was characterized by an increased generation of reactive oxygen species (ROS) accompanied by a decrease in oxidant-scavenging agents such as superoxide dismutase (SOD-1) and thioredoxin (TRX-1). Knockdown of SOD-1 and TRX-1 expression by small interfering RNA (siRNA) increased ROS generation and sensitivity of glioma cells to kaempferol-induced apoptosis. Signs of apoptosis included decreased expression of Bcl-2 and altered mitochondrial membrane potential with elevated active caspase-3 and cleaved poly(ADP-ribose) polymerase expression. Plasma membrane potential and membrane fluidity were altered in kaempferol-treated cells. Kaempferol suppressed the expression of proinflammatory cytokine interleukin-6 and chemokines interleukin-8, monocyte chemoattractant protein-1, and regulated on activation, normal T-cell expressed and secreted. Kaempferol inhibited glioma cell migration in a ROS-dependent manner. Importantly, kaempferol potentiated the toxic effect of chemotherapeutic agent doxorubicin by amplifying ROS toxicity and decreasing the efflux of doxorubicin. Because the toxic effect of both kaempferol and doxorubicin was amplified when used in combination, this study raises the possibility of combinatorial therapy whose basis constitutes enhancing redox perturbation as a strategy to kill glioma cells. [Mol Cancer Ther 2007;6(9):2544 -53]

Glia, 2007

While a number of studies have documented the importance of microglia in central nervous system (... more While a number of studies have documented the importance of microglia in central nervous system (CNS) response to injury, infection and disease, little is known regarding its role in viral encephalitis. We therefore, exploited an experimental model of Japanese Encephalitis, to better understand the role played by microglia in Japanese Encephalitis Virus (JEV) infection. Lectin staining performed to assess microglial activation indicated a robust increase in reactive microglia following infection. A difference in the topographic distribution of activated, resting, and phagocytic microglia was also observed. The levels of various proinflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2), IL-6, IL-1β, TNF-α, and MCP-1 that have been implicated in microglial response to an activational state was significantly elevated following infection. These cytokines exhibited region selective expression in the brains of infected animals, with the highest expression observed in the hippocampus. Moreover, the expression of neuronal specific nuclear protein NeuN was markedly downregulated during progressive infection indicating neuronal loss. In vitro studies further confirmed that microglial activation and subsequent release of various proinflammatory mediators induces neuronal death following JEV infection. Although initiation of immune responses by microglial cells is an important protective mechanism in the CNS, unrestrained inflammatory responses may result in irreparable brain damage. Our findings suggest that the increased microglial activation following JEV infection influences the outcome of viral pathogenesis. It is likely that the increased microglial activation triggers bystander damage, as the animals eventually succumb to infection. © 2007 Wiley-Liss, Inc.

Journal of Molecular Medicine-jmm, 2011

We observed elevated levels of pro-inflammatory cytokine IL-1β in glioblastoma multiforme tumor s... more We observed elevated levels of pro-inflammatory cytokine IL-1β in glioblastoma multiforme tumor samples. Since hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in linking inflammatory and oncogenic pathways, we investigated the effect of IL-1β on HIF-1α expression in glioma cells under normoxia. IL-1β-mediated elevation of HIF-1α transcriptional activity was dependent on Ras-induced NF-κB activation, as IL-1β failed to induce NF-κB and HIF-1α activity in cells transfected with dominant negative RasN17. Increased Ras expression was accompanied by increased phosphorylation of Ras effectors AKT, ERK, JNK, and p38MAPK. While inhibition of these effectors individually failed to block the IL-1β-mediated increase in HIF-1α induction, co-inhibition of both AKT and ERK resulted in a significant decrease in IL-1β-induced HIF-1α activation. Interestingly, IL-1β elevated Wnt-1 expression in a Ras-dependent manner, and small interfering RNA (siRNA)-mediated knockdown of Wnt-1 decreased HIF-1α activity. Although Wnt-1-mediated HIF-1α was independent of the canonical Wnt/β-catenin signaling pathway, it regulated HIF-1α through NF-κB. siRNA-mediated HIF-1α knockdown attenuated elevated IL-1β mRNA levels induced upon IL-1β treatment. This was accompanied by increased interaction of HIF-1α with HIF responsive element on the IL-1β promoter upon IL-1β treatment, under normoxia. Our studies highlights for first time that (1) Ras is a key mediator of IL-1β-induced NF-κB and HIF-1α activation, under normoxia; (2) Wnt-1 regulates IL-1β-mediated HIF-1α induction via NF-κB; (3) Ras and Wnt-1 are intermediaries in the canonical IL-1β–NF-κB signaling pathway downstream of MyD88; and (4) IL-1β-induced HIF-1α drives a HIF-1α-IL-1β autocrine loop to maintain persistently elevated IL-1β level.

International Journal of Cancer, 2008

Resistance to tumor necrosis factor (TNFα)-induced apoptosis in various cancer cells has been att... more Resistance to tumor necrosis factor (TNFα)-induced apoptosis in various cancer cells has been attributed to the activation of the transcription factor NF-κB. Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]one)—a selenoorganic compound is known to prevent TNFα-mediated NF-κB activity. As glioblastoma are resistant to the cytotoxic effect of TNFα, we investigated the potential of Ebselen in sensitizing glioma cells to TNFα-induced apoptosis. Although treatment with Ebselen reduced viability of glioma cells, cotreatment with TNFα enhanced apoptosis further through alteration of TNFα-mediated signaling pathways. Sensitization of TNFα activated glioma cells to apoptosis by Ebselen involved 2 pathways: (i) abrogation of TNFα induced NF-κB activation and (ii) induction of Fas-associated death inducing signaling complex (DISC) formation. Ebselen inhibited the prosurvival pathway mediated by NF-κB by altering the association of TNF receptor associated factor 2 (TRAF2) with TNFα receptor associated death domain (TRADD) in the TNFR1-TRADD-TRAF2 complex —an interaction crucial for mediating NF-κB activity. Ebselen also induced the formation of DISC involving Fas, Fas-associated death domain (FADD) and active caspase 8 to transduce apoptotic signals in situations where NF-κB function was inhibited. Cotreatment with Ebselen and TNFα induced G2/M phase arrest in cell cycle and modulated the expression of molecules involved in cell cycle progression. These results raise the possibility of overcoming resistance to TNFα-induced apoptosis by cotreatment with organoselenium Ebselen as a strategy to kill glioma cells. © 2008 Wiley-Liss, Inc.

Neurochemistry International, 2010