Developmental changes in Notch1 and NLE1 expression in a genetic model of absence epilepsy.

Childhood absence epilepsy (CAE) is an epilepsy syndrome with seizures occurring in the early childhood, highlighting that seizures susceptibility in CAE is dependent on brain development. The Notch 1 signalling pathway is important in brain development, yet the role of the Notch1 signalling pathway in CAE remains elusive. We here explored Notch1 and its modulator notchless homologue 1 (NLE1) expression in WAG/Rij and control rats using immunohistochemistry. Functional Notch 1 effects were assessed in WAG/Rij rats in vivo. WAG/Rij rats lack the developmental increase in cortical Notch1 and NLE 1 mRNA expression seen in controls, and Notch 1 and NLE1 mRNA and protein expression were lower in somatosensory cortices of WAG/Rij rats when compared to controls. This coincided with an overall decreased cortical GFAP expression in the early development in WAG/Rij rats. These effects were region-specific as they were not observed in thalamic tissues. Neuron-to-glia ratio as a marker of the impact of Notch signalling on differentiation was higher in layer 4 of somatosensory cortex of WAG/Rij rats. Acute application of Notch 1 agonist Jagged 1 suppressed, whereas DAPT, a Notch antagonist, facilitated spike and wave discharges (SWDs) in WAG/Rij rats. These findings point to Notch1 as an important signalling pathway in CAE which likely shapes architectural organization of the somatosensory cortex, a region critically involved in developmental epileptogenesis in CAE. More immediate effects of Notch 1 signalling are seen on in vivo SWDs in CAE, pointing to the Notch 1 pathway as a possible treatment target in CAE.

Effects of folate on notch signaling and cell proliferation in neural stem cells of neonatal rats in vitro.

The aim of the present study was to determine if folate alters Notch signaling and cell proliferation in neural stem cells (NSCs). NSCs were isolated from neonatal rats and grown in serum-free suspension culture. The cells were identified as NSCs by their expression of immunoreactive nestin. Individual cultures were assigned to one of three treatment groups: vehicle control, low-dose folate group (Folate-L, liquid media contained 4 mg/L folate), or high-dose folate group (Folate-H, liquid media contained 40 mg/L folate). Proliferating cells were identified by labeling with 5-bromo-2'-deoxyuridine (BrdU). Cell proliferation was quantitated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Gene expression of components of the Notch signaling system (Notch1, Hes1 and Mash1) was quantified by real-time polymerase chain reaction (PCR) assay. We observed that Nestin-positive NSCs grew as neurospheres in the serum-free suspension cultures. Folate increased the rate of cell proliferation compared to vehicle control (p<0.05). During cell proliferation, folate also increased Notch1 and Hes1 expression and decreased Mash1 expression compared to vehicle control (p<0.05). These results suggest that NSCs cultured from neonatal rats respond to folate with altered Notch signaling and increased cell proliferation.

Synthetic triterpenoids inhibit growth and induce apoptosis in human glioblastoma and neuroblastoma cells through inhibition of prosurvival Akt, NF-kappaB and Notch1 signaling.

Glioblastomas are high-risk primary brain tumors that are generally unresponsive or only weakly responsive to the currently available antineoplastic agents. Thus novel therapeutic strategies and agents are urgently needed to treat these incurable cancers. Oleanolic acid and ursolic acid are naturally occurring triterpenoids that have been used in traditional Asian medicine as anti-inflammatory and anti-cancer agents. Recently, synthetic oleanolic acid triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its C-28 methyl ester (CDDO-Me) and C-28 imidazole (CDDO-Im) derivatives have been shown to exhibit potent antitumor activity against diverse types of tumor cell lines, including leukemia, multiple myeloma, osteosarcoma, breast, lung, and pancreatic cancer cell lines; however, the anticancer activity of these agents for brain tumors has not been reported. In the present study, we investigated the apoptosis-inducing activity of CDDOs in glioblastoma (U87MG, U251MG) and neuroblastoma (SK-N-MC) cell lines. Cell growth/viability (MTS) and cytotoxicity (LDH release) assays demonstrated that glioblastoma cell lines are least sensitive to CDDO, but are highly sensitive to CDDO-Me and CDDO-Im at concentrations of 2.5-10 muM. CDDO-Im and CDDO-Me were equipotenent in their growth inhibitory activity. The primary mode of tumor cell destruction was apoptosis as demonstrated by significant increase in the number of hypo-diploid (sub-G0) cells and annexin V-FITC binding. Induction of apoptosis was associated with the activation of procaspases-3, -8, and -9, mitochondrial depolarization and the release of cytochrome c from mitochondria. Furthermore, CDDO-Me inhibited the levels of anti-apoptotic and prosurvival p-Akt, NF-kappaB (p65) and Notch1 signaling molecules. These studies provide rationale for clinical evaluation of these novel agents for the management of lethal brain neoplasms.