To date, the molecular mechanism underlying constitutive signal transducer and activator of transcription 3 (STAT3) activation in gliomas is largely unclear. In this study, we report that Smad6 is overexpressed in nuclei of glioma cells, which correlates with poor patient survival and regulates STAT3 activity via negatively regulating the Protein Inhibitors of Activated STAT3 (PIAS3). Mechanically, Smad6 interacts directly with PIAS3, and this interaction is mediated through the Mad homology 2 (MH2) domain of Smad6 and the Ring domain of PIAS3. Smad6 recruits Smurf1 to facilitate PIAS3 ubiquitination and degradation, which also depends on the MH2 domain and the PY motif of Smad6. Consequently, Smad6 reduces PIAS3-mediated STAT3 inhibition and promotes glioma cell growth and stem-like cell initiation. Moreover, the Smad6 MH2 transducible protein restores PIAS3 expression and subsequently reduces gliomagenesis. Collectively, we conclude that nuclear-Smad6 enhances glioma development by inducing PIAS3 degradation and subsequent STAT3 activity upregulation.

Widespread invasiveness, represented by the invasion and migration, is the most important characteristic of glioblastoma multiforme (GBM) and is the main reason for therapeutic failure and recurrence of the tumor. Hypoxia is one of the main microenvironment in determining tumor invasiveness. Therefore, intense efforts aimed at improved therapeutics are ongoing to demonstrate the molecular mechanisms governing GBM migration and invasion. This study aims to explore the role of phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway and its relationship with hypoxia inducible factor-1α (HIF-1α) in the migration and invasion of human glioblastoma U87 cells under hypoxia. In the study, we found that hypoxia could activate the PI3K/Akt/mTOR pathway associated with the enhancements of the migration and invasion of human glioblastoma U87 cells. When the PI3K/Akt/mTOR pathway and HIF-1α were inhibited by the siRNAs or inhibitors, the migration and invasion of human glioblastoma U87 cells were suppressed. Meanwhile, the expression of HIF-1α could be inhibited by the siRNA or inhibitors of PI3K/Akt/mTOR pathway. The aforementioned results demonstrate that hypoxia could induce enhancements of migration and invasion by activating PI3K/Akt/mTOR pathway by targeting HIF-1α in human glioblastoma U87 cells, which provide a theoretical basis for the treatments of GBM by targeting the PI3K/Akt/mTOR/HIF-1α pathway.

It has previously been demonstrated that curcumin possesses an antitumor activity, which is associated with its ability to induce G(2)/M cell cycle arrest and apoptosis. However the detailed underlying mechanisms remain unclear. The present study aimed to investigate the efficacy and underlying mechanism of curcumin-induced cell cycle arrest and apoptosis in U87 human glioblastoma cells. By immunofluorescence staining, subcellular fractionation and western blotting, the present study demonstrated that curcumin was able to induce G(2)/M cell cycle arrest and apoptosis by increasing the expression levels of cyclin G2, cleaved caspase-3 and Fas ligand (FasL), and decreasing the expression of cyclin-dependent kinase 1 (CDK1). In addition, increased expression of forkhead box protein O1 (FoxO1) and decreased expression of phosphorylated (p)-FoxO1 were detected in the curcumin-treated U87 cells. Curcumin was also able to induce the translocation of FoxO1 from the cytoplasm to the nucleus. Furthermore, following knockdown of FoxO1 expression in curcumin-treated U87 cells using FoxO1 small interfering RNA, the expression levels of cyclin G(2), cleaved caspase-3 and FasL were inhibited; however, the expression levels of CDK1 were not markedly altered. Notably, following knockdown of CDK1 expression under normal conditions, the total expression of FoxO1 was not affected; however, p-FoxO1 expression was decreased and FoxO1 nuclear expression was increased. Furthermore, curcumin-induced G(2)/M cell cycle arrest and apoptosis could be attenuated by FoxO1 knockdown. These results indicated that curcumin may induce G(2)/M cell cycle arrest and apoptosis in U87 cells by increasing FoxO1 expression. The present study identified a novel mechanism underlying the antitumor effects of curcumin, and may provide a theoretical basis for the application of curcumin in glioma treatment.

OBJECTIVEGlioblastoma multiforme (GBM), an aggressive primary brain tumor, is radioresistant and recurs despite aggressive surgery, chemotherapy, and radiotherapy. Curcumin as a potential radiosensitizer has received extensive attention in cancer treatment. To explore an effectiveness of this radiosensitizer for GBM treatment, we evaluated the radiosensitizing effect of curcumin and investigated its potential molecular mechanisms in the human glioma cell line U87.METHODSThe cytotoxic effects of curcumin on U87 cells were evaluated using the Cell Counting Kit-8 assay, and the radiosensitivity of U87 cells treated with curcumin was accessed by colony information assay. The effects of curcumin on cell proliferation and cell cycle regulation were determined using the 5-ethynyl-2-deoxyuridine incorporation assay and flow cytometry, respectively. Western blotting was applied to determine the effects of curcumin on protein expression of dual-specificity phosphatase-2 (DUSP-2), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) as well as phosphorylated ERK and JNK.RESULTSCurcumin significantly inhibited the proliferation of U87 cells in a dose-and time-dependent manner. Curcumin treatment at the concentrations of 5 µM and 10 M could significantly reduce the clonogenic activity and enhance the radiosensitivity of U87 cells with sensitive enhancement ratios (SERs) of 1.71 and 4.65, respectively. Curcumin resulted in G2/M cell cycle arrest in U87 cells, which were radiosensitive. Pre-treatment of U87-MG cells with 5 µM curcumin enhanced radiation-induced cell proliferation inhibition and apoptosis. Furthermore, we observed that curcumin increased DUSP-2 protein expression and decreased the phosphorylation of ERK and JNK.CONCLUSIONOur results suggest that low-dose curcumin may enhance the radiosensitivity of human glioma U87 cells in vitro by inducing G2/M cell cycle arrest through up-regulation of DUSP-2 expression and inhibition of ERK and JNK phosphorylation.

Within the last decade, extensive research has revealed that curcumin can increase the sensitivity of tumor cells to high-energy X-ray radiation in vitro and dual-specificity phosphatase (DUSPs) are implicated in cancer, obesity, diabetes, inflammation and Alzheimer’s disease. To date, the article about the radiosensitizing effect of curcumin via enhancement of the DUSP-2 pathway was rarely reported. Here, we discuss the radiosensitizing effects of curcumin on suppressing glioma vivo growth. BALB-c nude mice bearing subcutaneous U87 xenografts were treated with curcumin and/or local radiation to assess their vivo response. Tumor growth, real-time PCR, western blotting, immunohistochemical assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) were performed to explore the possible mechanism involved. Curcumin in combination with irradiation significantly enhanced the tumor-suppressive effect in vivo compared with local radiotherapy alone. Both mRNA and protein levels of DUSP-2 were significantly upregulated in the curcumin in combination with radiation treatment group. Curcumin pretreatment inhibited radiationinduced extracellular signal-regulated kinases (ERK)/c-Jun N-terminal kinases (JNK) phosphorylation and enhanced radiation-induced tumor cell apoptosis in subcutaneous xenografts. In conclusion, curcumin significantly increased the radiosensitivity of U87 human glioma cells in vivo. The radiosensitizing effect of curcumin was found to be closely related to its pro-apoptosis activity via enhancement of the DUSP-2 pathway. Keywords: Apoptosis, curcumin, dual specificity phosphatase 2, glioma, radiosensitization.