No abstract

The precise role of different toll-like receptor (TLR) superfamily members is just beginning to get elucidated in glioblastoma multiforme (GBM). In this study, we observed heightened TLR4 levels in GBM tumor samples as compared to adjacent normal tissue. Since the pro-inflammatory cytokine tumor necrosis factor (TNF)α induces NF-κB activation in GBM, and as several common signaling mediators are involved in TNFα and TLR4-mediated NF-κB activation, we investigated the role of TLR4 in the regulation of NF-κB activation and inflammatory responses in TNFα-treated glioma cells. TNFα elevated TLR4 expression and inhibition of TLR4 signaling by either signaling inhibitor, neutralizing antibody, or small interfering RNA (siRNA)-attenuated TNFα-induced NF-κB activation. TLR4-mediated NF-κB activation was independent of canonical myeloid differentiation factor 88 signaling but involved toll/IL-1R homology domain-containing adaptor protein-inducing interferon-β. Inhibition of TLR4 signaling abrogated TNFα-induced increase in (1) transcription factors interferon (IFN) regulatory factor 3 and STAT-1 and (2) IFNβ and inflammatory cytokines/chemokines expression. Furthermore, TNFα-induced TLR4-dependent increase in AKT activation and HIF-1α transcriptional activation suggested the existence of TLR4-AKT-HIF-1α axis. Importantly, TNFα-induced TLR4 was abrogated in cells transfected with dominant negative IκB and HIF-1α siRNA. Our studies indicate that TNFα triggered TLR4-HIF-1α and NF-κB-TLR4 feed-forward loops act in tandem to sustain inflammatory response in glioma.

The anti-neoplastic property of alkyl phospholipids has been tested for the treatment of several malignancies. In this study, we evaluated the efficacy of miltefosine (Hexadecylphosphocholine--an alkyl phospholipids analogue) on glioblastoma multiforme. In this study, we demonstrate that miltefosine-induced apoptosis is accompanied by elevated Fas, Fas-associated death domain (FADD) expression, caspase-8 activity and the increased distribution of Fas and FADD towards lipid raft microdomain to form death inducing signaling complex. Treatment with miltefosine resulted in increase in Ras, extracellular signal-regulated kinase (ERK) and p38MAPK activity. Expression of dominant-negative Ras (Ras N17) attenuated miltefosine-mediated apoptosis. Although inhibition of both ERK and p38MAPK decreased the pro-apoptotic effects of miltefosine, it was the inhibition of ERK and not p38MAPK activation that decreased Fas and FADD expression. An ERK-dependent increase in the expression of gammaH2AX-involved in response to DNA double-stranded breaks was also observed. Taken together, our findings suggest the involvement of ERK activation in miltefosine-induced glioma cell apoptosis.

Resistance to tumor necrosis factor (TNFa)-induced apoptosis in various cancer cells has been attributed to the activation of the transcription factor NF-jB. Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]one)-a selenoorganic compound is known to prevent TNFa-mediated NF-jB activity. As glioblastoma are resistant to the cytotoxic effect of TNFa, we investigated the potential of Ebselen in sensitizing glioma cells to TNFa-induced apoptosis. Although treatment with Ebselen reduced viability of glioma cells, cotreatment with TNFa enhanced apoptosis further through alteration of TNFa-mediated signaling pathways. Sensitization of TNFa activated glioma cells to apoptosis by Ebselen involved 2 pathways: (i) abrogation of TNFa induced NF-jB activation and (ii) induction of Fas-associated death inducing signaling complex (DISC) formation. Ebselen inhibited the prosurvival pathway mediated by NF-jB by altering the association of TNF receptor associated factor 2 (TRAF2) with TNFa receptor associated death domain (TRADD) in the TNFR1-TRADD-TRAF2 complex -an interaction crucial for mediating NF-jB 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-jB function was inhibited. Cotreatment with Ebselen and TNFa 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 TNFa-induced apoptosis by cotreatment with organoselenium Ebselen as a strategy to kill glioma cells.

Polyphenol epigallocatechin-3-gallate (EGCG) induced apoptosis in glioma cells by elevating oxidative stress through increased reactive oxygen species (ROS) generation. Signs of apoptosis included altered mitochondrial membrane potential and elevated expression of caspase-3 and cytochrome c. The increase in ROS was concomitant with the decrease in expression of thioredoxin (TRX-1) and ceruloplasmin (CP), mediators associated with protection against oxidative stress. EGCG downregulated the levels of pro-inflammatory cytokine interleukin (IL)-6 and chemokines IL-8, monocyte-chemoattractant protein (MCP)-1 and RANTES. EGCG also decreased the invasive potential of gliomas, possibly by affecting the urokinase plasminogen activator (uPA) and cytoskeletal architecture. Our study indicates that EGCG might serve as an effective therapeutic strategy against glioma as it not only promotes cell death through redox perturbation, but also downregulates the release of proinflammatory mediators while concomitantly decreasing the invasive potential of glioma cells.