Background:Glioma stem-like cell (GSC) properties are responsible for gliomagenesis and recurrence. GSCs are invasive but its mechanism remains to be elucidated. Here, we attempted to identify the molecules that promote invasion in GSCs.Methods:Neurospheres and CD133+ cells were collected from glioblastoma (GBM) specimens and glioma cell lines by sphere-formation method and magnetic affinity cell sorting, respectively. Differential expression of gene candidates, its role in invasion and its signaling pathway were evaluated in glioma cell lines.Results:Neurospheres from surgical specimens attached to fibronectin and laminin, the receptors of which belong to the integrin family. Integrin α3 was overexpressed in CD133+ cells compared with CD133− cells in all the glioma cell lines (4 out of 4). Immunohistochemistry demonstrated the localisation of integrin α3 in GBM cells, including invading cells, and in the tumour cells around the vessels, which is believed to be a stem cell niche. The expression of integrin α3 was correlated with migration and invasion. The invasion activity of glioma cells was linked to the phosphorylation of extracellular signal–regulated kinase (ERK) 1/2.Conclusion:Our results suggest that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrin α3 a prime candidate for anti-invasion therapy for GBM.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that signals through a family of G protein-coupled receptors consisting of 5 members termed S1P 1-5 , and it regulates cellular proliferation, migration and survival. We investigated the expression and role of S1P receptors in glioma. Human glioma expressed S1P 1 , S1P 2 , S1P 3 , and S1P 5 by quantitative real-time PCR analysis. Expression of the S1P 1 was significantly lower in glioblastoma than in the normal brain (p < 0.01) and diffuse astrocytoma (p < 0.05). Immunoblotting showed that normal brain expressed more S1P 1 protein than did glioblastoma. Immunohistochemistry showed that S1P 1 was localized predominantly in the astrocytes in the normal brain, but no staining was observed in glioblastoma. Downregulation of S1P 1 expression correlated with poor survival of patients with glioblastoma (p < 0.05). S1P 1 small interfering RNA promoted cell proliferation in high-expressor glioma cell lines (T98G, G112). Cell proliferation was promoted by the pertussis toxin, which deactivates G i/o type of G proteins; the S1P 1 is exclusively coupled to these proteins. Forced expression of the S1P 1 in low-expressor cell lines (U87, U251) resulted in decreased cell growth and led to suppressed tumor growth in transplanted gliomas in vivo. Furthermore, we found a significant association between the S1P 1 expression and early growth response-1, a transcriptional factor that exhibits tumor suppression in glioblastoma cells (p < 0.05). These data indicate that the downregulation of S1P 1 expression enhances the malignancy of glioblastoma by increasing cell proliferation and correlates with the shorter survival of patients with glioblastoma.

The aim of this study was to investigate the association between patterns of jaw motor activity during sleep and clinical signs and symptoms of sleep bruxism. A total of 35 university students and staff members participated in this study after providing informed consent. All participants were divided into either a sleep bruxism group (n = 21) or a control group (n = 14), based on the following clinical diagnostic criteria: (1) reports of tooth-grinding sounds for at least two nights a week during the preceding 6 months by their sleep partner; (2) presence of tooth attrition with exposed dentin; (3) reports of morning masticatory muscle fatigue or tenderness; and (4) presence of masseter muscle hypertrophy. Video-polysomnography was performed in the sleep laboratory for two nights. Sleep bruxism episodes were measured using masseter electromyography, visually inspected and then categorized into phasic or tonic episodes. Phasic episodes were categorized further into episodes with or without grinding sounds as evaluated by audio signals. Sleep bruxism subjects with reported grinding sounds had a significantly higher total number of phasic episodes with grinding sounds than subjects without reported grinding sounds or controls (Kruskal-Wallis/Steel-Dwass tests; P < 0.05). Similarly, sleep bruxism subjects with tooth attrition exhibited significantly longer phasic burst durations than those without or controls (Kruskal-Wallis/Steel-Dwass tests; P < 0.05). Furthermore, sleep bruxism subjects with morning masticatory muscle fatigue or tenderness exhibited significantly longer tonic burst durations than those without or controls (Kruskal-Wallis/Steel-Dwass tests; P < 0.05). These results suggest that each clinical sign and symptom of sleep bruxism represents different aspects of jaw motor activity during sleep.

Although there are many reports on the clinical use of the MIB-1 labeling index (LI), which is a measure of proliferative activity in astrocytomas; its significance varies between studies. There are no known molecules that are directly linked to the MIB-1 LI in astrocytomas. We evaluated the clinical value of the MIB-1 LI in our human glioblastoma cases and determined the molecules that possibly influenced the MIB-1 LI. An immunohistochemical study of the MIB-1 protein was performed and MIB-1 LIs of 38 glioblastomas were determined. In the same cases, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor-alpha (PDGFRA), and sphingosine-1-phosphate receptor type 1 (S1P(1)), which are known regulators of glioma cell proliferation, were detected and quantified by quantitative real-time-PCR or western blotting. Kaplan-Meier survival curves for 38 patients with glioblastomas showed that a high MIB-1 LI correlated with poor survival (P < 0.05). Among the molecules tested, only the low expression of S1P(1) was significantly correlated with the high MIB-1 LI in glioblastomas (P < 0.05). Multivariate analysis revealed that the S1P(1) expression level was a significant prognostic factor. Our results indicate that the MIB-1 LI is an important prognostic factor in human glioblastomas. Furthermore, downregulation of S1P(1) expression increases proliferative activity, and thus enhances the malignancy of glioblastomas, resulting in a poor survival.

Cancer cells have acquired several pathways to escape from host immunity in the tumor microenvironment. Programmed death 1 (PD-1) receptor and its ligand PD-L1 are involved in the key pathway of tumor immune escape, and immune checkpoint therapy targeting PD-1 and PD-L1 has been approved for the treatment of patients with certain types of malignancies. Although PD-1 is a well-characterized receptor on T cells, the immune checkpoint receptor is also expressed on tumor-associated macrophages (TAM), a major immune component of the tumor microenvironment. In this study, we found significant diurnal oscillation in the number of PD-1–expressing TAMs collected from B16/BL6 melanoma-bearing mice. The levels of Pdcd1 mRNA, encoding PD-1, in TAMs also fluctuated in a diurnal manner. Luciferase reporter and bioluminescence imaging analyses revealed that a NF-κB response element in the upstream region of the Pdcd1 gene is responsible for its diurnal expression. A circadian regulatory component, DEC2, whose expression in TAMs exhibited diurnal oscillation, periodically suppressed NF-κB–induced transactivation of the Pdcd1 gene, resulting in diurnal expression of PD-1 in TAMs. Furthermore, the antitumor efficacy of BMS-1, a small molecule inhibitor of PD-1/PD-L1, was enhanced by administering it at the time of day when PD-1 expression increased on TAMs. These findings suggest that identification of the diurnal expression of PD-1 on TAMs is useful for selecting the most appropriate time of day to administer PD-1/PD-L1 inhibitors. Implications: Selecting the most appropriate dosing time of PD-1/PD-L1 inhibitors may aid in developing cancer immunotherapy with higher efficacy.