Zhi-Qiang Li

Upregulation of SATB1 is associated with the development and progression of glioma

BackgroundSpecial AT-rich sequence-binding protein-1 (SATB1) has been reported to be expressed in several human cancers and may have malignant potential. This study was aimed at investigating the expression and potential role of SATB1 in human glioma.MethodThe relationship between SATB1 expression, clinicopathological parameters, Ki67 expression and MGMT promoter methylation status was evaluated, and the prognostic value of SATB1 expression in patients with gliomas was analyzed. SATB1-specific shRNA sequences were synthesized, and U251 cells were transfected with SATB1 RNAi plasmids. Expression of SATB1 mRNA and protein was investigated by RT-PCR and immunofluoresence staining and western blotting. The expression of c-Met, SLC22A18, caspase-3 and bcl-2 protein was determined by western blotting. U251 cell growth and adherence was detected by methyl thiazole tetrazolium assay. The apoptosis of U251 cells was examined with a flow cytometer. The adherence, invasion, and in vitro angiogenesis assays of U251 cells were done. The growth and angiogenesis of SATB1 low expressing U251 cells was measured in an in vivo xenograft model.ResultsOf 70 tumors, 44 (62.9%) were positive for SATB1 expression. SATB1 expression was significantly associated with a high histological grade and with poor survival in univariate and multivariate analyses. SATB1 expression was also positively correlated with Ki67 expression but negatively with MGMT promoter methylation in glioma tissues. SATB1 shRNA expression vectors could efficiently induce the expression of SLC22A18 protein, increase the caspase-3 protein, inhibit the expression of SATB1, c-Met and bcl-2 protein, the growth, invasion, metastasis and angiogenesis of U251 cells, and induce apoptosis in vitro. Furthermore, the tumor growth of U251 cells expressing SATB1 shRNA were inhibited in vivo, and immunohistochemical analyses of tumor sections revealed a decreased vessel density in the animals where shRNA against SATB1 were expressed.ConclusionsSATB1 may have an important role as a positive regulator of glioma development and progression, and that SATB1 might be a useful molecular marker for predicting the prognosis of glioma.

Correlation of low SLC22A18 expression with poor prognosis in patients with glioma.

We investigated the expression of the putative tumor suppressor SLC22A18 to evaluate it as a prognostic marker in glioma patients. Immunohistochemical and Western blot analyses of clinical tissue samples obtained from 120 patients with glioma were performed. Low expression of SLC22A18 was observed in 71.7% of patients. Loss of SLC22A18 expression in glioma was significantly related to pathological grade (p = 0.003). High pathological grade (World Health Organization III-IV) was correlated with negative (low or absent) expression of SLC22A18, which was correlated with a significantly shorter overall patient survival than in those with positive (high) expression (p = 0.007). Multivariate Cox regression analysis indicated that SLC22A18 expression level is an independent survival prognostic factor for patients with glioma (p = 0.011). Western blotting analysis confirmed decreased expression of SLC22A18 in glioma tissues compared with adjacent brain tissues. This study suggests that SLC22A18 functions as a tumor suppressor in glioma and represents a candidate biomarker for long-term survival in this disease.

Promoter methylation and downregulation of SLC22A18 are associated with the development and progression of human glioma

BackgroundDownregulation of the putative tumor suppressor gene SLC22A18 has been reported in a number of human cancers. The aim of this study was to investigate the relationship between SLC22A18 downregulation, promoter methylation and the development and progression of human glioma.MethodSLC22A18 expression and promoter methylation was examined in human gliomas and the adjacent normal tissues. U251 glioma cells stably overexpressing SLC22A18 were generated to investigate the effect of SLC22A18 on cell growth and adherence in vitro using the methyl thiazole tetrazolium assay. Apoptosis was quantified using flow cytometry and the growth of SLC22A18 overexpressing U251 cells was measured in an in viv o xenograft model.ResultsSLC22A18 protein expression is significantly decreased in human gliomas compared to the adjacent normal brain tissues. SLC22A18 protein expression is significantly lower in gliomas which recurred within six months after surgery than gliomas which did not recur within six months. SLC22A18 promoter methylation was detected in 50% of the gliomas, but not in the adjacent normal tissues of any patient. SLC22A18 expression was significantly decreased in gliomas with SLC22A18 promoter methylation, compared to gliomas without methylation. The SLC22A18 promoter is methylated in U251 cells and treatment with the demethylating agent 5-aza-2-deoxycytidine increased SLC22A18 expression and reduced cell proliferation. Stable overexpression of SLC22A18 inhibited growth and adherence, induced apoptosis in vitro and reduced in vivo tumor growth of U251 cells.ConclusionSLC22A18 downregulation via promoter methylation is associated with the development and progression of glioma, suggesting that SLC22A18 is an important tumor suppressor in glioma.

Involvement of ROS-alpha v beta 3 integrin-FAK/Pyk2 in the inhibitory effect of melatonin on U251 glioma cell migration and invasion under hypoxia

BackgroundMelatonin, a well-known antioxidant, has been shown to possess anti-invasive properties for glioma. However, little is known about the effect of melatonin on glioma cell migration and invasion under hypoxia, which is a crucial microenvironment for tumor progress. In addition, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration and invasion. Therefore, we investigated the possible role of these kinases and its related signaling in the regulation of human U251 glioma cells behavior by melatonin under hypoxia.MethodsThe abilities of migration and invasion of U251 glioma cells were determined by wound healing and transwell assay in vitro. The intracellular production of reactive oxygen species (ROS) was measured by using the fluorescent probe 6-carboxy-2′, 7′-dichorodihydrofluorescein diacetate (DCFH-DA). Immunofluorescence experiments and western blotting analysis were used to detect the expression level of protein. Small interfering RNAs (siRNA) was used to silence specific gene expression.ResultsThe pharmacologic concentration (1 mM) of melatonin significantly inhibited the migration and invasion of human U251 glioma cells under hypoxia. The inhibitory effect of melatonin was accompanied with the reduced phosphorylation of FAK and Pyk2, and decreased expression of alpha v beta 3 (αvβ3) integrin. Additionally, inhibition of αvβ3 integrin by siRNA reduced the phosphorylation of FAK/Pyk2 and demonstrated the similar anti-tumor effects as melatonin, suggesting the involvement of αvβ3 integrin- FAK/Pyk2 pathway in the anti-migratory and anti-invasive effect of melatonin. It was also found that melatonin treatment decreased the ROS levels in U251 glioma cells cultured under hypoxia. ROS inhibitor apocynin not only inhibited αvβ3 integrin expression and the phosphorylation levels of FAK and Pyk2, but also suppressed the migratory and invasive capacity of U251 glioma cells under hypoxia.ConclusionsThese results suggest that melatonin exerts anti-migratory and anti-invasive effects on glioma cells in response to hypoxia via ROS-αvβ3 integrin-FAK/Pyk2 signaling pathways. This provides evidence that melatonin may be a potential therapeutic molecule targeting the hypoxic microenvironment of glioma.

Overactivation of NR2B-containing NMDA receptors through entorhinal-hippocampal connection initiates accumulation of hyperphosphorylated tau in rat hippocampus after transient middle cerebral artery occlusion

Middle cerebral artery occlusion (MCAO) induces secondary damages in the hippocampus that is remote from primary ischemic regions. Tau hyperphosphorylation is an important risk for neurodegenerative diseases. Increased tau phosphorylation has been identified in ischemic cortex, but little is known regarding the changes in the hippocampus. We showed that unilateral transient MCAO induced accumulation of hyperphosphorylated tau and concurrent dephosphorylation of glycogen synthase kinase‐3β at Ser 9 in the ipsilateral hippocampus. These MCAO‐induced changes were not reproduced when glutamatergic inputs from the entorhinal cortex to the hippocampus were transected; however, the changes were mimicked by intrahippocampal N‐methyl‐d‐aspartate (NMDA) administration. Inhibition of NMDA receptor (NMDAR) subunit NR2B, but not NR2A activity in the hippocampus attenuated the accumulation of hyperphosphorylated tau and spatial cognitive impairment in MCAO rats. Together, our data suggest that overactivation of NR2B‐containing NMDARs through entorhinal–hippocampal connection plays an important role in the accumulation of hyperphosphorylated tau in the hippocampus following MCAO. Glycogen synthase kinase‐3β is an important protein kinase involved in NMDARs‐mediated tau hyperphosphorylation. This study indicates that early inhibition of NR2B‐containing NMDARs may represent a potential strategy to prevent or delay the occurrence of post‐stroke dementia.

Microarray Analysis of the Hypoxia-induced Gene Expression Profile in Malignant C6 Glioma Cells

Hypoxia is commonly featured during glioma growth and plays an important role in the processes underlying tumor progression to increasing malignancy. Here we compared the gene expression profiles of rat C6 malignant glioma cells under normoxic and hypoxic conditions by cDNA microarray analysis. Compared to normoxic culture conditions, 180 genes were up-regulated and 67 genes were down-regulated under hypoxia mimicked by CoCl2 treatment. These differentially expressed genes were involved in mutiple biological functions including development and differentiation, immune and stress response, metabolic process, and cellular physiological response. It was found that hypoxia significantly regulated genes involved in regulation of glycolysis and cell differentiation, as well as intracellular signalling pathways related to Notch and focal adhesion, which are closely associated with tumor malignant growth. These results should facilitate investigation of the role of hypoxia in the glioma development and exploration of therapeutic targets for inhibition of glioma growth.

Relationship between SATB1 expression and prognosis in astrocytoma

Special AT-rich-sequence-binding protein 1 (SATB1), a new type of gene regulator, has been reported to be expressed in various human cancers and may be associated with malignancy. The aim of this study was to investigate the expression of SATB1 in astrocytoma and to determine its prognostic value for the overall survival of patients with astrocytoma. The expression of SATB1 protein and messenger RNA (mRNA) in human astrocytoma specimens was examined using immunohistochemistry and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The relationship between SATB1 expression and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status was also investigated. Spearmans correlation coefficient was used to describe the association between SATB1 expression and the clinical parameters of astrocytoma patients. SATB1 protein and mRNA were expressed at significant levels in astrocytoma specimens. SATB1 expression was positively correlated with astrocytoma pathological grade but negatively correlated with the life span of astrocytoma patients. SATB1 expression was also significantly lower in astrocytoma specimens with MGMT promoter methylation than in those without MGMT promoter methylation. Our findings suggest that SATB1 may have an important role as a positive regulator of astrocytoma development and progression and that SATB1 might be a useful molecular marker for predicting the prognosis of patients with astrocytoma and could be a novel target for treating astrocytoma.

Induction of proline-rich tyrosine kinase 2 activation-mediated C6 glioma cell invasion after anti-vascular endothelial growth factor therapy.

BackgroundAnti-angiogenic therapy inhibits tumor growth and is considered as a potential clinical therapy for malignant glioma. However, inevitable recurrences and unexpected tumor resistance, particularly increased invasion ability of glioma cell, were observed after anti-angiogenic treatment. The underlying mechanism remains undetermined. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration; therefore, we investigated the possible role of these kinases in rat C6 glioma cell invasion induced by bevacizumab, a recombinant monoclonal antibody against vascular endothelial growth factor (VEGF).MethodsThe effects of bevacizumab on migration and invasion of C6 glioma cells were investigated in vitro and in vivo. The cells proliferation, migration, and invasion were determined by MTT assay, wound healing, and transwell assay, respectively. Invasive potential of glioma cells in vivo was assessed by counting vimentin-positive cells crossing the solid tumor rim by immunohistochemical staining. The total and phosphorylated protein levels of FAK and Pyk2 were detected by Western blotting.ResultsBevacizumab exposure increased migration and invasion of cultured C6 cells in a concentration-dependent manner. In addition, the continuous bevacizumab treatment also promoted tumor invasion in rat C6 intracranial glioma models. Bevacizumab treatment enhanced Pyk2 phosphorylation at Tyr402, but no effect on FAK phosphorylation at Tyr397 both in vitro and in vivo. Knockdown of Pyk2 by siRNA or inhibition of Pyk2 phosphorylation by Src kinase specific inhibitor PP1 partially inhibited bevacizumab-induced cell invasion in cultured C6 glioma cells. Furthermore, the combined administration of bevacizumab and PP1 significantly suppressed glioma cell invasion into surrounding brain tissues compared to bevacizumab treatment alone in experimental rats.ConclusionsThese results suggest that anti-VEGF treatment promotes glioma cell invasion via activation of Pyk2. Inhibition of Pyk2 phosphorylation might be a potential target to ameliorate the therapeutic efficiency of anti-VEGF treatment.

Predictive value of the SLC22A18 protein expression in glioblastoma patients receiving temozolomide therapy

BackgroundOur previous study showed that SLC22A18 downregulation and promoter methylation were associated with the development and progression of glioma and the elevated expression of SLC22A18 was found to increase the sensitivity of glioma U251 cells to the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In this study, we investigated the predictive value of SLC22A18 promoter methylation and protein expression in glioblastoma multiforme (GBM) patients receiving temozolomide (TMZ) therapy.Patients and methodsSLC22A18 promoter methylation and protein expression were examined by methylation-specific polymerase chain reaction (MSP) and Western blotting respectively, then we compared SLC22A18 promoter methylation and protein expression in tumor cell explants in regard to prediction of TMZ response and survival time of 86 GBM patients.ResultsSLC22A18 promoter methylation was detected in 61 of 86 (71%) samples, whereas 36 of 86 (42%) cases were scored positive for SLC22A18 protein expression. Overall SLC22A18 promoter methylation was significantly related to SLC22A18 protein expression, but a subgroup of cases did not follow this association. Multivariate Cox regression analysis indicated that SLC22A18 protein expression, but not promoter methylation, was significantly correlated with TMZ therapy. SLC22A18 protein expression predicted a significantly shorter overall survival in 51 patients receiving TMZ therapy, whereas no differences in overall survival were observed in 35 patients without TMZ therapy.ConclusionsThese results show that lack of SLC22A18 protein expression is superior to promoter methylation as a predictive tumor biomarker in GBM patients receiving temozolomide therapy.

Bisperoxovandium (pyridin‐2‐squaramide) targets both PTEN and ERK1/2 to confer neuroprotection

We and others have shown that inhibiting phosphatase and tensin homolog deleted on chromosome 10 (PTEN) or activating ERK1/2 confer neuroprotection. As bisperoxovanadium compounds are well‐established inhibitors of PTEN, we designed bisperoxovandium (pyridin‐2‐squaramide) [bpV(pis)] and determined whether and how bpV(pis) exerts a neuroprotective effect in cerebral ischaemia–reperfusion injury.