Donglin Wang

Expression of far upstream element (FUSE) binding protein 1 in human glioma is correlated with c-Myc and cell proliferation

Glioma is one of the most common type of primary intracranial tumor. Although great advances have been achieved in treatment of glioma, the underlying molecular mechanisms remain largely unknown. Previous studies demonstrated that FBP1 is a transcriptional regulator of c‐Myc and acts as an important prognostic indicator in many cancers. Our study aimed to assess the expression and function of FBP1 in human glioma. Immunohistochemical and Western blot analysis were performed in human glioma and normal brain tissues. High FBP1 expression (located in cell nuclei) was observed in 70 samples and its level was correlated with the grade of malignancy. A strongly positive correlation was observed between FBP1 and c‐Myc (P = 0.005) and Ki‐67 expression (P = 0.009). In a multivariate analysis, high FBP1 and c‐Myc expressions were showed to be associated with poor prognosis in glioma. While in vitro, following serum stimulation of starved U87MG cells, the expression of FBP1 was upregulated, as well as c‐Myc and PCNA. Moreover, knockdown of FBP1 by siRNA transfection diminished the expression of c‐Myc and arrested cell growth at G1 phase. Collectively, our results shows that the expression of FBP1 is in close correlation with c‐Myc level and cell proliferation in glioma and provides a potential strategy to develop FBP1 inhibitors as novel anti‐tumor agents.

Increased Expression of Calcium/Calmodulin-Dependent Protein Kinase Type II Subunit Delta after Rat Traumatic Brain Injury

Many cellular responses to Ca2+ signals are mediated by Ca2+/calmodulin-dependent enzymes, among which is the Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII was originally described in rat brain tissue. In rat brain, four different subunits of the kinase have been identified: α, β, γ, and δ. This study aims to investigate changes of CaMKIIδ after traumatic brain injury and its possible role. Rat traumatic brain injury (TBI) model was established by controlled cortical injury system. In the present study, we mainly investigated the expression and cellular localization of CaMKIIδ after traumatic brain injury. Western blot analysis revealed that CaMKIIδ was present in normal rat brain cortex. It gradually increased, reached a peak at the third day after TBI, and then decreased. Importantly, more CaMKIIδ was colocalized with neuron. In addition, Western blot detection showed that the third day postinjury was also the apoptosis peak indicated by the elevated expression of caspase-3.Importantly, immunohistochemistry analysis revealed that injury-induced expression of CaMKIIδ was colabeled by caspase-3 (apoptosis cells marker). Moreover, pretreatment with the CaMKII inhibitor (KN62) reduced the injury-induced activation of caspase-3. Noticeably, the CaMKII inhibitor KN-62 could reduce TBI-induced cell injury assessed with lesion volume and attenuate behavioral outcome evaluated by motor test. These data suggested that CaMKIIδ may be implicated in the apoptosis of neuron and the recovery of neurological outcomes. However, the inherent mechanisms remained unknown. Further studies are needed to confirm the exact role of CaMKIIδ after brain injury.

Increased expression of transcription initiation factor IIB after rat traumatic brain injury

The protein TFIIB is a general transcription initiation factor that plays a pivotal role in the preinitiation complex (PIC) and selects the transcription initiation site. However, its distribution and function in the central nervous system (CNS) remains unclear. In the present study, we mainly investigated the expression and cellular localization of TFIIB during traumatic brain injury (TBI). Western blot analysis revealed that TFIIB was present in normal rat brain cortex. It gradually increased, reached a peak at the 5th day after TBI, and then decreased. Importantly, more TFIIB was colocalized with astrocytes and microglia, which are largely proliferated. In addition, Western blot detection showed that the 5th day post injury was also the proliferation peak indicated by the elevated expression of PCNA. Importantly, injury-induced expression of TFIIB was colabelled by proliferating cell nuclear antigen (proliferating cells marker). These data suggested that TFIIB may be implicated in the proliferation of astrocytes and microglia and the recovery of neurological outcomes. But the inherent mechanisms remained unknown. Further studies are needed to confirm the exact role of TFIIB after brain injury.

Spy1 induces de-ubiquitinating of RIP1 arrest and confers glioblastoma's resistance to tumor necrosis factor (TNF-α)-induced apoptosis through suppressing the association of CLIPR-59 and CYLD

Glioblastoma multiforme (GBM), a grade-IV glioma, is resistant to TNF-α induced apoptosis. CLIPR-59 modulates ubiquitination of RIP1, thus promoting Caspase-8 activation to induce apoptosis by TNF-α. Here we reported that CLIPR-59 was down-regulated in GBM cells and high-grade glioma tumor samples, which was associated with decreased cancer-free survival. In GBM cells, CLIPR-59 interacts with Spy1, resulting in its decreased association with CYLD, a de-ubiquitinating enzyme. Moreover, experimental reduction of Spy1 levels decreased GBM cells viability, while increased the lysine-63-dependent de-ubiquitinating activity of RIP1 via enhancing the binding ability of CLIPR-59 and CYLD in GBM, thus promoting Caspase-8 and Caspase-3 activation to induce apoptosis by TNF-α. These findings have identified a novel Spy1-CLIPR-59 interplay in GBM cells resistance to TNF-α-induced apoptosis revealing a potential target in the intervention of malignant brain tumors.

Overexpression of CCT8 and its significance for tumor cell proliferation, migration and invasion in glioma

Overexpression of chaperonin containing t-complex polypeptide 1 (TCP1), or CCT, has been reported in various classes of malignancies. However, little is known about the expression of t-complex protein subunits TCP1theta (CCT8) in gliomas. In this study, the expression of CCT8 protein was detected using blotting analysis and immunohistochemistry. CCT8 was found to be overexpressed in gliomas and to correlate with the WHO grade of gliomas. To further investigate the biological function of CCT8 in gliomas, CCT8-silenced U87 glioblastoma multiforme (GBM) and U251MG cells were constructed using a small interference RNA (siRNA) sequence. The knockdown effect of CCT8 on proliferation and invasion in these cells was analyzed using the CCK8, flow cytometry cycle, scratch, transwell invasion and fluorescence assays. Compared with the controls, the glioma cells expressing CCT8-siRNA exhibited a significantly decreased proliferation and invasion capacity, as well as a dysregulated cell cytoskeleton. This study showed that high CCT8 protein expression might be related to poor outcome of glioma, and that CCT8 regulates the proliferation and invasion of glioblastomas.

Glycinamide ribonucleotide formyl transferase is frequently overexpressed in glioma and critically regulates the proliferation of glioma cells.

AIMS Current treatments for the most common form of brain tumor, glioma, are disappointing in their effectiveness. Low expression levels of GART, an enzyme in the core nucleotide metabolism, significantly correlate with chemosensitivity, conferring a survival advantage to tumor cells. Our study aimed to explore the expression and function of GART in glioma. METHODS Immunohistochemical and Western blot analysis were performed in 70 cases of human gliomas and normal brain tissues. We mainly used cell growth assay and multicellular tumor spheroid formation assay to evaluate the proliferation and chemosensitivity of glioma cells. RESULTS High GART expression (most cancer cells cytoplasm stained) was observed in 70 specimens and was related to the grade of malignancy. We also reviewed each grade of tumors separately and investigated whether GART expression predicted patient survival within each subgroup. In brief, GART overexpression was significantly associated with overall survival (P=0.03). Interestingly, transfecting cells with GART-siRNA suppressed proliferation and enhanced temozolomide (TMZ)-induced apoptosis in glioma cells. CONCLUSION The current results showed that GART expression was associated with glioma grade and that high GART protein expression might be related to poor outcome.

High expression of adenylate cyclase-associated protein 1 accelerates the proliferation, migration and invasion of neural glioma cells

Adenylate cyclase-associated protein 1 (CAP1), a conserved member of cyclase-associated proteins was reported to be associated with the proliferation, migration or invasion of the tumors of pancreas, breast and liver, and was involved in astrocyte proliferation after acute Traumatic Brain Injury (TBI). In this study, we sought to investigate the character of CAP1 in the pathological process of human glioma by detecting human glioma specimens and cell lines. 43 of 100 specimens showed high expression of CAP1 via immunohistochemistry. With statistics analysis, we found out the expression level of CAP1 was correlated with the WHO grades of human glioma and was great positively related to Ki-67 (p<0.01). In vitro, silencing CAP1 in U251 and U87MG, the glioma cell lines with the relatively higher expression of CAP1, induced the proliferation of the cells significantly retarded, migration and invasion as well. Obviously, our results indicated that CAP1 participated in the molecular pathological process of glioma indeed, and in a certain sense, CAP1 might be a potential and promising molecular target for glioma diagnosis and therapies in the future.

CHD1L Regulates Cell Cycle, Apoptosis, and Migration in Glioma

Chromodomain helicase/ATPase DNA binding protein 1-like (CHD1L) gene is a newly identified oncogene located at Chr1q21 and it is amplified in many solid tumors. In this study, we intended to investigate the clinical significance of CHD1L expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemistry analysis showed that CHD1L was overexpressed in glioma tissues and glioma cell lines. In addition, the expression level of CHD1L was positively correlated with glioma pathological grade and Ki-67 expression. Kaplan–Meier curve indicated that high expression of CHD1L may result in poor prognosis of glioma patients. Accordingly, suppression of CHD1L in glioma cells was shown to induce cell cycle arrest and increase apoptosis. In addition, knockdown of CHD1L significantly accelerated migration and invasion ability of glioma cells. Together our findings suggest that CHD1L is involved in the progression of glioma and may be a novel target for further therapy.

DTX3L is upregulated in glioma and is associated with glioma progression

Gliomas are the most common primary brain tumors of the central nervous system (CNS). Due to the poor prognosis of glioma patients, it is urgent to develop more effective therapies. Deltex-3-like (DTX3L), also known as B-lymphoma and BAL-associated protein (BBAP), has been reported to play an important role in the progression of many tumors. This study aimed to investigate the clinical significance and biological function of DTX3L in human glioma. Clinically, the protein expression level of DTX3L is increased in glioma tissues compared with that observed in normal brain tissues. Immunohistochemical analysis demonstrated that DTX3L was highly expressed in the glioma tissues and its level was correlated with the grade of malignancy. Multivariate analysis revealed the association between high expression of DTX3L and the poor prognosis of glioma patients. In addition, knockdown of DTX3L by siRNA transfection increased glioma cell apoptosis. Moreover, suppression of DTX3L expression was shown to significantly inhibit the migration and invasion of glioma cells. These data indicate that DTX3L plays an important role in the pathogenic process of glioma, suggesting that DTX3L could be a potential prognostic biomarker for glioma.

The role of Alix in the proliferation of human glioma cells

Apoptosis-linked-gene-2-interacting protein 1 (Alix) is involved in the endosome-lysosome system in the cytoplasm. The normal function of Alix may be altered by ALG-2 toward a destructive role during active cell death. Alix also may play a role in regulation of cell proliferation. However, the role of Alix in human glioma has not been elucidated yet. This study intended to clarify the relationship between Alix and glioma pathologic grades and its role in the proliferation of glioma cells. Our findings showed that Alix protein concentrations were significantly elevated in high-grade glioma tissue compared with low-grade glioma (P < .0001). Immunohistochemical study revealed that Alix was overexpressed in 75 resected glioma tissues and may forecast poor survival. Alix expression was increased in resting serum-stimulated glioma cells. Additionally, we reduced Alix expression in U251MG cells and then found that cell viability was decreased significantly when p21 expression increased. Colony formation assay and flow cytometry analysis demonstrated that reduced Alix expression may lead to growth inhibition and cell cycle arrest. In summary, our findings suggest that Alix plays an important role in the proliferation of glioma cells and may be a novel therapeutic target.