Influence of Caloric Restriction on Constitutive Expression of NF-κB in an Experimental Mouse Astrocytoma

Many of the current standard therapies employed for the management of primary malignant brain cancers are largely viewed as palliative, ultimately because these conventional strategies have been shown, in many instances, to decrease patient quality of life while only offering a modest increase in the length of survival. We propose that caloric restriction (CR) is an alternative metabolic therapy for brain cancer management that will not only improve survival but also reduce the morbidity associated with disease. Although we have shown that CR manages tumor growth and improves survival through multiple molecular and biochemical mechanisms, little information is known about the role that CR plays in modulating inflammation in brain tumor tissue.Phosphorylation and activation of nuclear factor κB (NF-κB) results in the transactivation of many genes including those encoding cycloxygenase-2 (COX-2) and allograft inflammatory factor-1 (AIF-1), both of which are proteins that are primarily expressed by inflammatory and malignant cancer cells. COX-2 has been shown to enhance inflammation and promote tumor cell survival in both in vitro and in vivo studies. In the current report, we demonstrate that the p65 subunit of NF-κB was expressed constitutively in the CT-2A tumor compared with contra-lateral normal brain tissue, and we also show that CR reduces (i) the phosphorylation and degree of transcriptional activation of the NF-κB-dependent genes COX-2 and AIF-1 in tumor tissue, as well as (ii) the expression of proinflammatory markers lying downstream of NF-κB in the CT-2A malignant mouse astrocytoma, [e.g. macrophage inflammatory protein-2 (MIP-2)]. On the whole, our date indicate that the NF-κB inflammatory pathway is constitutively activated in the CT-2A astrocytoma and that CR targets this pathway and inflammation.CR could be effective in reducing malignant brain tumor growth in part by inhibiting inflammation in the primary brain tumor

Time dependent neuroprotection of mycophenolate mofetil: effects on temporal dynamics in glial proliferation, apoptosis, and scar formation

BACKGROUND: Immunosuppressants such as mycophenolate mofetil (MMF) have the capacity to inhibit microglial and astrocytic activation and to reduce the extent of cell death after neuronal injury. This study was designed to determine the effective neuroprotective time frame in which MMF elicits its beneficial effects, by analyzing glial cell proliferation, migration, and apoptosis. METHODS: Using organotypic hippocampal slice cultures (OHSCs), temporal dynamics of proliferation and apoptosis after N-methyl-D-aspartate (NMDA)-mediated excitotoxicity were analyzed by quantitative morphometry of Ki-67 or cleaved caspase-3 immunoreactive glial cells. Treatment on NMDA-lesioned OHSCs with mycophenolate mofetil (MMF)100 μg/mL was started at different time points after injury or performed within specific time frames, and the numbers of propidium iodide (PI)(+) degenerating neurons and isolectin (I)B(4)(+) microglial cells were determined. Pre-treatment with guanosine 100 μmol/l was performed to counteract MMF-induced effects. The effects of MMF on reactive astrocytic scar formation were investigated in the scratch-wound model of astrocyte monolayers. RESULTS: Excitotoxic lesion induction led to significant increases in glial proliferation rates between 12 and 36 hours after injury and to increased levels of apoptotic cells between 24 and 72 hours after injury. MMF treatment significantly reduced glial proliferation rates without affecting apoptosis. Continuous MMF treatment potently reduced the extent of neuronal cell demise when started within the first 12 hours after injury. A crucial time-frame of significant neuroprotection was identified between 12 and 36 hours after injury. Pre-treatment with the neuroprotective nucleoside guanosine reversed MMF-induced antiproliferative effects on glial cells. In the scratch-wound model, gap closure was reached within 48 hours in controls, and was potently inhibited by MMF. CONCLUSIONS: Our data indicate that immunosuppression by MMF significantly attenuates the extent of neuronal cell death when administered within a crucial time frame after injury. Moreover, long-lasting immunosuppression, as required after solid-organ transplantation, does not seem to be necessary. Targeting inosine 5-monophosphate dehydrogenase, the rate-limiting enzyme of purine synthesis, is an effective strategy to modulate the temporal dynamics of proliferation and migration of microglia and astrocytes, and thus to reduce the extent of secondary neuronal damage and scar formation

Broader implications of SILAC-based proteomics for dissecting signaling dynamics in cancer

Large-scale transcriptome and epigenome analyses have been widely utilized to discover gene alterations implicated in cancer development at the genetic level. However, mapping of signaling dynamics at the protein level is likely to be more insightful and needed to complement massive genomic data. Stable isotope labeling with amino acids in cell culture (SILAC)-based proteomic analysis represents one of the most promising comparative quantitative methods that has been extensively employed in proteomic research. This technology allows for global, robust and confident identification and quantification of signal perturbations important for the progress of human diseases, particularly malignancies. The present review summarizes the latest applications of in vitro and in vivo SILAC-based proteomics in identifying global proteome/phosphoproteome and genome-wide protein-protein interactions that contribute to oncogenesis, highlighting the recent advances in dissecting signaling dynamics in cancer