Calcium-sensing receptor antagonist NPS2390 attenuates neuronal apoptosis though intrinsic pathway following traumatic brain injury in rats.

Traumatic brain injury (TBI) initiates a complex cascade of neurochemical and signaling changes that leads to neuronal apoptosis, which contributes to poor outcomes for patients with TBI. Previous study indicates that calcium-sensing receptor (CaSR) activation contributes to neuron death in focal cerebral ischemia-reperfusion mice, however, its role in neuronal apoptosis after TBI is not well-established. Using a controlled cortical impact model in rats, the present study was designed to determine the effect of CaSR inhibitor NPS2390 upon neuronal apoptosis after TBI. Rats were randomly distributed into three groups undergoing the sham surgery or TBI procedure, and NPS2390 (1.5 mg/kg) was infused subcutaneously at 30 min and 120 min after TBI. All rats were sacrificed at 24 h after TBI. Our data indicated that NPS2390 significantly reduced the brain edema and improved the neurological function after TBI. In addition, NPS2390 decreased caspase-3 levels and the number of apoptotic neurons. Furthermore, NPS2390 up-regulated anti-apoptotic protein Bcl-2 expression and down-regulated pro-apoptotic protein Bax, and reduced subsequent release of cytochrome c into the cytosol. In summary, this study indicated that inhibition of CaSR by NPS2390 attenuates neuronal apoptosis after TBI, in part, through modulating intrinsic apoptotic pathway.

Neuroprotective Mechanisms of Melatonin in Hemorrhagic Stroke.

Hemorrhagic stroke which consists of subarachnoid hemorrhage and intracerebral hemorrhage is a dominant cause of death and disability worldwide. Although great efforts have been made, the physiological mechanisms of these diseases are not fully understood and effective pharmacological interventions are still lacking. Melatonin (N-acetyl-5-methoxytryptamine), a neurohormone produced by the pineal gland, is a broad-spectrum antioxidant and potent free radical scavenger. More importantly, there is extensive evidence demonstrating that melatonin confers neuroprotective effects in experimental models of hemorrhagic stroke. Multiple molecular mechanisms such as antioxidant, anti-apoptosis, and anti-inflammation, contribute to melatonin-mediated neuroprotection against brain injury after hemorrhagic stroke. This review article aims to summarize current knowledge regarding the beneficial effects of melatonin in experimental models of hemorrhagic stroke and explores the underlying mechanisms. We propose that melatonin is a promising neuroprotective candidate that is worthy of further evaluation for its potential therapeutic applications in hemorrhagic stroke.

B7-H4(B7x)-Mediated Cross-talk between Glioma-Initiating Cells and Macrophages via the IL6/JAK/STAT3 Pathway Lead to Poor Prognosis in Glioma Patients.

PURPOSE: The objective of this study was to evaluate clinical significance and immunosuppressive mechanisms of B7-H4 (B7x/B7S1), a B7 family member, in glioma. EXPERIMENTAL DESIGN: B7-H4 levels in glioma tissue/cerebral spinal fluid (CSF) were compared between different grades of glioma patients. Survival data were analyzed with Kaplan-Meier to determine the prognostic value of B7-H4. Cytokines from CD133(+) cells to stimulate the expression of B7-H4 on human macrophages (Mφs) were investigated by FACS, neutralizing antibodies, and Transwell chemotaxis assay. shRNA, reporter vector, and chromatin immunoprecipitation were used to determine the binding of STAT3 to the B7-H4 promoter. The function of B7-H4(+) Mφs in vitro was evaluated through phagocytosis, T-cell proliferation/apoptosis, and cytokine production as well as in the xenografted model for in vivo analysis. RESULTS: We found that B7-H4 expression in tumors was associated with prognosis of human glioblastoma and correlated directly with malignant grades. Mechanistically, glioma initiating CD133(+) cells and Mφs/microglia cointeraction activated expression of B7-H4 via IL6 and IL10 in both tumor cells and microenvironment supporting cells. IL6-activated STAT3 bound to the promoter of B7-H4 gene and enhanced B7-H4 expression. Furthermore, CD133(+) cells mediated immunosuppression through B7-H4 expression on Mφs/microglia by silencing of B7-H4 expression on these cells, which led to increased microenvironment T-cell function and tumor regression in the xenograft glioma mouse model. CONCLUSIONS: We have identified B7-H4 activation on Mφs/microglia in the microenvironment of gliomas as an important immunosuppressive event blocking effective T-cell immune responses. Clin Cancer Res; 22(11); 2778-90. ©2016 AACR.

DNMT1 mediates chemosensitivity by reducing methylation of miRNA-20a promoter in glioma cells.

Although methyltransferase has been recognized as a major element that governs the epigenetic regulation of the genome during temozolomide (TMZ) chemotherapy in glioblastoma multiforme (GBM) patients, its regulatory effect on glioblastoma chemoresistance has not been well defined. This study investigated whether DNA methyltransferase (DNMT) expression was associated with TMZ sensitivity in glioma cells and elucidated the underlying mechanism. DNMT expression was analyzed by western blotting. miR-20a promoter methylation was evaluated by methylation-specific PCR. Cell viability and apoptosis were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and TdT-mediated dUTP-biotin nick end labeling assays, respectively. The results showed that compared with parental U251 cells, DNMT1 expression was downregulated, miR-20a promoter methylation was attenuated and miR-20a levels were elevated in TMZ-resistant U251 cells. Methyltransferase inhibition by 5-aza-2'-deoxycytidine treatment reduced TMZ sensitivity in U251 cells. In U251/TM cells, DNMT1 expression was negatively correlated with miR-20a expression and positively correlated with TMZ sensitivity and leucine-rich repeats and immunoglobulin-like domains 1 expression; these effects were reversed by changes in miR-20a expression. DNMT1 overexpression induced an increase in U251/TM cell apoptosis that was inhibited by the miR-20a mimic, whereas DNMT1 silencing attenuated U251/TM cell apoptosis in a manner that was abrogated by miR-20a inhibitor treatment. Tumor growth of the U251/TM xenograft was inhibited by pcDNA-DNMT1 pretreatment and boosted by DNMT1-small hairpin RNA pretreatment. In summary, DNMT1 mediated chemosensitivity by reducing methylation of the microRNA-20a promoter in glioma cells.

miR-20a mediates temozolomide-resistance in glioblastoma cells via negatively regulating LRIG1 expression.

AIMS: Resistance to temozolomide (TMZ) is a major obstacle in the treatment of glioblastoma multiforme (GBM). MiRNAs is considered as an important modulator of drug resistance in many cancers. Here, we aimed to elucidate the relationship between miR-20a, its predicted target genes leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) and TMZ resistance in GBM. MAIN METHODS: Real-time PCR or western blot was used to measure the levels of miR-20a and LRIG1. The cell viability was obtained to investigate the sensitivity of U251 cells and TMZ-resistant U251 (U251/TMZ) cells to TMZ. MiR-20a inhibitor or miR-20a mimic was used to down-regulate or up-regulate miR-20a expression. The interaction between miR-20a and its predicted target gene LRIG1 was confirmed by 3'-UTR dual-luciferase reporter assay. pcDNA-LRIG1 was used to overexpress LRIG1 [corrected]. A xenograft tumor model was used to investigate the in vivo antitumor activity. KEY FINDINGS: MiR-20a was highly expressed and LRIG1 lowly expressed in U251/TMZ cells. Knockdown of miR-20a by treatment with miR-20a inhibitor restored sensitivity of U251/TM cells to TMZ in vivo and in vitro, whereas overexpression of miR-20a by treatment with miR-20a mimic resulted in increased TMZ resistance. The levels of LRIG1 were inversely related to miR-20a levels. And the luciferase reporter assays showed that miR-20a directly targeted the 3'UTR of LRIG1. In addition, functional knock-down of LRIG1 by gene specific siRNA reversed the effect of miR-20a inhibitor. SIGNIFICANCE: MiR-20a mediated TMZ-resistance in glioblastoma cells through negatively regulating LRIG1 expression, which suggesting that miR-20a and LRIG1 would be potential therapeutic targets for glioma therapy.

B7-H4 expression is elevated in human U251 glioma stem-like cells and is inducible in monocytes cultured with U251 stem-like cell conditioned medium.

Previous studies indicated that B7-H4, the youngest B7 family, negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors. Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy. However, the link between B7-H4 and tumor stem cells is unclear. In this study, we investigated B7-H4 expression in the medium of human glioma U251 cell cultures. Immunofluorescence results showed that U251 cells cultured in serum-free medium (supplemented with 2% B27, 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor) maintained stem-like cell characteristics, including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2. In contrast, U251 cells cultured in serum-containing medium highly expressed differentiation marker glial fibrillary acidic protein. Flow cytometry analysis showed serum-free medium-cultured U251 cells expressed higher intracellular B7-H4 than serum-containing medium-cultured U251 cells (24%-35% vs. 8%-11%, P < 0.001). Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of B7-H4 after stimulation with conditioned medium from U251 cells cultured in serum-containing medium. Moreover, conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4 expression compared with serum-containing conditioned medium (P < 0.01). Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells, and conditioned medium from these cells more effectively induced monocytes to express B7-H4 than conditioned medium from U251 cells cultured in the presence of serum. Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4.