Arsenic trioxide induces different gene expression profiles of genes related to growth and apoptosis in glioma cells dependent on the p53 status

We have previously reported that As(2)O(3) affected cell cycle progression and cyclins D1 and B1 expression in two glioma cell lines differing in p53 status (U87MG-wt; T98G-mutated). In the present study, we further demonstrated that As(2)O(3) affected proliferation, viability and apoptosis of the two cell lines in a dose- and time-dependent manner, and T98G cells were more sensitive than U87MG cells to As(2)O(3) -induced apoptosis and inhibition of proliferation and viability. We further investigated the expression profiles of genes related with apoptosis and cell cycle in the two cell lines with a human cDNA-microarray (SuperArray) spotted with 267 genes of apoptosis and cell cycle. Thirty five genes were upregulated and 15 genes downregulated at least 2-fold by As(2)O(3) in U87-MG cells; whereas, 38 genes were upregulated and 21 genes downregulated at least 2-fold in T98G cells by As(2)O(3). After As(2)O(3) treatment, p53 expression was upregulated 56.5-fold in T98G cells, but only 6.0-fold in U87MG cells. The results indicate that As(2)O(3) suppresses the growth of U87MG cells mainly by regulating expression of genes of cell cycle arrest, stress and toxicity; whereas As(2)O(3) affects T98G cells mainly by regulating expression of genes belonging to Bcl-2, tumor necrotic factor receptor and ligand families. The data may be helpful for optimizing As(2)O(3) as an anti-cancer drug in the treatment of gliomas.     

Genomic organization, chromosomal localization and regulation of expression of the neuronal nuclear matrix protein NRP/B in human brain tumors

The nuclear matrix and its role in cell physiology are largely unknown, and the discovery of any matrix constituent whose expression is tissue- and/or cell-specific offers a new avenue of exploration. Studies of the novel neuronal nuclear matrix protein, NRP/B, reveal that it is an early and highly specific marker of neuronal induction and development in vertebrates, since its expression is restricted mainly to the developing and mature nervous system. These studies also show that NRP/B is involved in neuronal differentiation. To further examine the structure-function of NRP/B, we have cloned and characterized the murine Nrp/b gene. The murine gene consists of four exons interrupted by three introns that span 7.6kb of DNA. The complete open reading frame is localized in exon 3, suggesting that NRP/B is highly conserved during evolution. Chromosomal analysis shows that NRP/B is localized to chromosome 13 in mouse and chromosome 5q12-13 in human.Since our previous studies demonstrated that NRP/B is expressed in primary hippocampal neurons but not in primary astrocytes, we have characterized NRP/B mRNA and protein expression in various brain cell lines and in human brain tumors. Abundant expression of NRP/B mRNA and protein was observed in human neuroblastoma cell lines (IMR32, SKN-MC, SKN-SH), in glioblastoma cell lines (A172, T98G, U87-MG, U118-MG, U138-MG, and U373-MG), in neuroglioma (H4) and astrocytoma cell lines (CCF-STTG1 and SW1088). Confocal analysis of NRP/B in U87-MG glioblastoma cells indicated nuclear localization of NRP/B. NRP/B expression was also observed in human primary brain tumors including glioblastoma multiformae and astrocytomas (total of five cases). These results suggest that NRP/B expression is upregulated in human brain tumors including glioblastomas and astrocytomas, while under normal conditions NRP/B expression is restricted to neurons. This study implicates a role for NRP/B in brain tumor development.     

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

ABSTRACT: BACKGROUND: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. RESULTS: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. CONCLUSIONS: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.     

The effects of cytomegalovirus on human immunodeficiency virus replication in brain-derived cells correlate with permissiveness of the cells for each virus.

Human cytomegalovirus (HCMV) is commonly found in the brains of patients with AIDS and in some cases can be detected in the same cells as can human immunodeficiency virus type 1 (HIV-1). In this study, we analyzed the patterns of replication of HIV-1 and HCMV in singly infected cells and the effects of dual infection in human brain-derived cell lines of three different origins: neuroblastoma cell lines SK-N-MC and SY5Y; astrocytoma/glioblastoma cell lines U373-MG and Hs 683; and undifferentiated glioblastoma cell lines A172 and T98G. To bypass the restriction at the adsorption/penetration step in these CD4-negative cells, we used HIV-1 (amphotropic retrovirus) pseudotypes. These HIV-1 pseudotypes infected the majority of the cells in the cultures and expressed high levels of HIV-1 gene products in all except the SY5Y cells. The cell lines differed in the ability to support HCMV infection, but coinfection with HIV-1 had no effect on HCMV replication. The A172 cells were completely nonpermissive for HCMV gene expression, while HCMV replication in the singly infected T98G and SK-N-MC cell lines was restricted at the level of some early gene products. This resulted in complete and partial inhibition, respectively, of viral DNA synthesis. Dual infection of the A172, T98G, and SK-N-MC cells had no effect on HIV-1 replication. The other three cell lines, U373-MG, Hs 683, and SY5Y, were fully permissive for HCMV replication. In the U373-MG and Hs 683 cells, HCMV markedly inhibited the synthesis of HIV-1 gene products. In contrast, a transient stimulation of HIV-1 production followed by a repression was observed in the dually infected SY5Y cells. We conclude from these results that under conditions in which both HIV-1 and HCMV can undergo fully permissive infection, HCMV can repress HIV-1 gene expression. In cells in which HCMV replication is limited but HIV-1 replicates well, there is no effect on HIV-1 gene expression. However, activation of HIV-1, at least transiently, may occur in cells in which HIV-1 gene expression is limited. These studies suggest that a threshold level of some HIV-1 gene product(s) may obscure activation or promote repression of HIV replication by HCMV.     

Cyclosporine a induces growth arrest or programmed cell death of human glioma cells

Human malignant gliomas are highly resistant to current therapeutic approaches. We previously demonstrated that cyclosporine A (CsA) induces an apoptotic cell death in rat C6 glioma cells. In the present study, we found the induction of growth arrest or cell death of human malignant glioma cells exposed to CsA. In studied glioma cells, an accumulation of p21Cip1/Waf1 protein, a cell cycle inhibitor, was observed following CsA treatment, even in the absence of functional p53 tumour suppressor. CsA induced a senescence-associated growth arrest, in U87-MG glioma cells with functional p53, while in U373 and T98G glioma cells with mutated p53, CsA treatment triggered cell death associated with alterations of cell morphology, cytoplasm vacuolation, and condensation of chromatin. In T98G cells this effect was completely abolished by simultaneous treatment with an inhibitor of protein synthesis, cycloheximide (CHX). Moreover, CsA-induced cell death was accompanied by activation of executory caspases followed by PARP cleavage. CsA treatment did not elevate fasL expression and had no effect on mitochondrial membrane potential. We conclude that CsA triggers either growth arrest or non-apoptotic, programmed cell death in human malignant glioma cells. Moreover, CsA employs mechanisms different to those in the action of radio- and chemotherapeutics, and operating even in cells resistant to conventional treatments. Thus, CsA or related drugs may be an effective novel strategy to treat drug-resistant gliomas or complement apoptosis-based therapies.     

Design,Synthesis, and Bioactivity Evaluation of Novel Rosin Diterpenoid Derivatives as Potential Anti-glioma Agents

Glioma is the most common brain tumor and its treatment options are limited. Abietic acid and dehydroabietic acid are tricyclic diterpenoid oxygen compounds with strong lip solubility and anti-glioma activity. In this study, novel rosin diterpenoid derivatives were designed and synthesized using abietic acid and dehydrogenated abietic acid as lead compounds and their activities against T98G, U87MG, and U251 cells were evaluated by CCK-8 methods. The in vivo activity of compounds with stronger activity in vitro was preliminarily studied through the Zebrafish model. The results showed that the IC₅₀ values of B6 , B8 , B10 , and B12 were 11.47 to 210.6 μM, which were exhibited higher antiproliferative potency against T98G, U87MG, and U251. The scratch experiment showed that B12 inhibited the migration of T98G in a time-dependent and concentration-dependent manner. The results of in vivo activity further explained that B12 could inhibit the proliferation of the T98G. The pKa values of B6 , B8 , B10 , and B12 were 7.17 to 7.35, which were within the ideal range of glioma drugs. The ADME predictions indicated that these derivatives could pass through the blood-brain barrier. In addition, molecular docking primarily explained interaction between compounds and protein. These results suggested that B12 should be a promising candidate that merits further attention in the development of anti-glioma drugs.     

Immediate early gene IEX-1 induces astrocytic differentiation of U87-MG human glioma cells

The immediate early response gene IEX-1 is involved in the regulation of apoptosis and cell growth. In order to increase the apoptotic sensitivity to chemotherapeutic drugs and gamma-ray, we attempted to establish U87-MG human glioma cell line expressing IEX-1. Unexpectedly, however, transfection of IEX-1 into U87-MG glioma cells resulted in morphological changes to astrocytic phenotype and increase in glial differentiation marker proteins, S-100 and glial fibrillary acidic protein (GFAP). Glial cell differentiation was used to examine in rat C6 glioma cell line, since this cell line express astrocytic phenotypes by increase in intracellular cAMP concentration. Stimulation of human U87-MG glioma cells by membrane-permeable dibutyryl cAMP (dbcAMP) not only elicited their morphological changes but also induced expression of IEX-1 as well as S-100 and GFAP. H89, an inhibitor of protein kinase A (PKA), blocked dbcAMP-induced morphological changes of U87-MG cells and expression of IEX-1. In contrast, morphological changes and expression of S-100 and GFAP induced by IEX-1 were not affected by H89. Morphological changes induced by dbcAMP were totally abolished by functional disruption of IEX-1 expression by anti-sense RNA. These results indicate that IEX-1 plays an important role in astrocytic differentiation of human glioma cells and that IEX-1 functions at downstream of PKA.     

Knockdown of NOB1 expression by RNAi inhibits cellular proliferation and migration in human gliomas

NOB1 (NIN1/RPN12 binding protein 1 homolog), a ribosome assembly factor, is thought to be essential for the processing of the 20S pre-rRNA into the mature 18S rRNA. It is also reported to participate in proteasome biogenesis. However, the contribution of NOB1 gene dysfunction to the pathology of human diseases, such as gliomas, has not been addressed. Here, we detected expression levels of NOB1 mRNA in U251, U87, U373, and A172 cells by quantitative real-time PCR. To analyze the expression levels of NOB1 protein in glioma tissues, we performed immunohistochemistry on 56 pathologically confirmed glioma samples (7 Grade I cases, 19 Grade II cases, 16 Grade III cases, and 14 Grade IV cases). A recombinant lentivirus expressing NOB1 short hairpin RNA (shNOB1) was constructed and infected into U251 and U87-MG human glioma cells. We found that NOB1 mRNA was expressed in all four cell lines. The expression level of the NOB1 protein was significantly higher in high-grade gliomas than in low-grade gliomas. Knockdown of the NOB1 gene resulted in suppression of the proliferation and the colony-forming abilities of U251 and U87-MG cells, cell cycle arrest during the G₀/G₁ phase, and a significant enhancement of cell apoptosis. In addition, cell migration was significantly suppressed in U251 and U87-MG cells that were infected with the shNOB1-expressing lentivirus. These results suggest that NOB1 promotes glioma cell growth and migration and could be a candidate for molecular targeting during gene therapy treatments of glioma.     

p27Kip1 expression by contact inhibition as a prognostic index of human glioma

The clinical manifestations of human glioma are known to be diverse, ranging from aggressive growth and invasion to apparent dormancy; however, the molecular mechanism underlying this diversity has been largely unexplored. In the present study, we characterized four human glioma cell lines, T98G, A172, U251, and NAC6, each of which has distinct growth properties. A172 and U251 cells continue to grow after confluency, whereas the growth of T98G and NAC6 cells is contact inhibited. Northern and western blot analyses revealed that at high cell density, the expression of p27Kip1 cyclin-dependent kinase inhibitor was dramatically enhanced at both the RNA and the protein levels in T98G and NAC6 cells but not in A172 or U251. These facts together with the finding that overexpression of p27Kip1 caused G1 arrest in A172 and T98G cells suggest that the induction of p27Kip1 represents an important determinant of growth at high cell density. Immunohistochemical analyses of 42 primary gliomas revealed an inverse correlation between the level of p27 protein and the Ki-67 proliferative index. Kaplan-Meier plots demonstrated that a low level of p27 in tumors is associated with decreased overall survival. Thus, disrupted regulation of p27 expression at high cell density may play an important role in determining the clinical behavior of human gliomas as well as the prognosis for glioma patients.     

Exaggerated inflammatory responses mediated by Burkholderia cenocepacia in human macrophages derived from Cystic fibrosis patients

Glioblastoma (GBM) is a highly aggressive cancer type characterized by intense neovascularization. Several lines of evidence indicate that blood clotting enzymes play an important role in the tumor microenvironment, mainly through the activation of protease-activated receptors (PAR). In particular, PAR1 and PAR2 isoforms may activate signal transduction pathways that promote a number of pro-tumoral responses. However, little is known concerning the role of PAR1/PAR2 in GBM progression. In this study, we investigated the expression and function of PAR1 and PAR2 in the human GBM cell lines A172 and U87-MG. We also evaluated the effect of agonist peptides for PAR1 (PAR1-AP) and PAR2 (PAR2-AP) on signaling pathways and the expression of vascular endothelial growth factor (VEGF). Immunoblotting assays showed that A172 and U87-MG constitutively express PAR1 and PAR2. Treatment of GBM cells with PAR1-AP or PAR2-AP enhanced Akt (protein kinase B) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a time-dependent manner. LY29042 and PD98059, inhibitors of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, decreased PAR-mediated activation of Akt and ERK1/2, respectively. In addition, we observed that PAR2, but not PAR1, activation increased VEGF secretion in U87-MG and A172 cells. Notably, only PD98059 reduced PAR2-mediated VEGF production by GBM cells. Our results suggest that PAR2 modulates VEGF production through the MAPK/ERK1/2 pathway, and not the PI3K/Akt pathway, in human GBM cell lines. Therefore, the PAR2/MAPK signaling axis might be regarded as a relevant target for adjuvant treatment of GBM with a possible impact on tumor angiogenesis.     

5-Lipoxygenase and anandamide hydrolase (FAAH) mediate the antitumor activity of cannabidiol, a non-psychoactive cannabinoid

It has been recently reported that cannabidiol (CBD), a non-psychoactive cannabinoid, is able to kill glioma cells, both in vivo and in vitro , independently of cannabinoid receptor stimulation. However, the underlying biochemical mechanisms were not clarified. In the present study, we performed biochemical analysis of the effect of CBD both in vivo , by using glioma tumor tissues excised from nude mice, and in vitro , by using U87 glioma cells. In vivo exposure of tumor tissues to CBD significantly decreased the activity and content of 5-lipoxygenase (LOX, by ∼ 40%), and of its end product leukotriene B₄ (∼ 25%). In contrast cyclooxygenase (COX)-2 activity and content, and the amount of its end product prostaglandin E₂, were not affected by CBD. In addition, in vivo treatment with CBD markedly stimulated (∼ 175%) the activity of fatty acid amide hydrolase (FAAH), the main anandamide-degrading enzyme, while decreasing anandamide content (∼ 30%) and binding to CB₁ cannabinoid receptors (∼ 25%). In vitro pre-treatment of U87 glioma cells with MK-886, a specific 5-LOX inhibitor, significantly enhanced the antimitotic effect of CBD, whereas the pre-treatment with indomethacin (pan-COX inhibitor) or celecoxib (COX-2 inhibitor), did not alter CBD effect. The study of the endocannabinoid system revealed that CBD was able to induce a concentration-dependent increase of FAAH activity in U87 cells. Moreover, a significantly reduced growth rate was observed in FAAH-over-expressing U87 cells, compared to wild-type controls. In conclusion, the present investigation indicates that CBD exerts its antitumoral effects through modulation of the LOX pathway and of the endocannabinoid system, suggesting a possible interaction of these routes in the control of tumor growth.     

Different involvement of autophagy in human malignant glioma cell lines undergoing irradiation and temozolomide combined treatments

Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical resection, adjuvant ionizing radiation (IR) combined with Temozolomide (TMZ) drug administration is the standard therapy for GB. This currently combined radio-chemotherapy treatment resulted in glial tumor cell death induction, whose main molecular death pathways are still not completely deciphered. In this study, the autophagy process was investigated, and in vitro modulated, in two different GB cell lines, T98G and U373MG (known to differ in their radiosensitivity), after IR or combined IR/TMZ treatments. T98G cells showed a high radiosensitivity (especially at low and intermediate doses), associated with autophagy activation, assessed by Beclin-1 and Atg-5 expression increase, LC3-I to LC3-II conversion and LC3B-GFP accumulation in autophagosomes of irradiated cells; differently, U373MG cells resulted less radiosensitive. Autophagy inhibition, using siRNA against BECN1 or ATG-7 genes, totally prevented decrease in viability after both IR and IR/TMZ treatments in the radiosensitive T98G cells, confirming the autophagy involvement in the cytotoxicity of these cells after the current GB treatment, contrary to U373MG cells. However, rapamycin-mediated autophagy, that further radiosensitized T98G, was able to promote radiosensitivty also in U373MG cells, suggesting a role of autophagy process in enhancing radiosensitivity. Taken together, these results might enforce the concept that autophagy-associated cell death might constitute a possible adjuvant therapeutic strategy to enhance the conventional GB treatment.     

Glioma-specific cation conductance regulates migration and cell cycle progression

In this study, we have investigated the role of a glioma-specific cation channel assembled from subunits of the Deg/epithelial sodium channel (ENaC) superfamily, in the regulation of migration and cell cycle progression in glioma cells. Channel inhibition by psalmotoxin-1 (PcTX-1) significantly inhibited migration and proliferation of D54-MG glioma cells. Both PcTX-1 and benzamil, an amiloride analog, caused cell cycle arrest of D54-MG cells in G(0)/G(1) phases (by 30 and 40%, respectively) and reduced cell accumulation in S and G(2)/M phases after 24 h of incubation. Both PcTX-1 and benzamil up-regulated expression of cyclin-dependent kinase inhibitor proteins p21(Cip1) and p27(Kip1). Similar results were obtained in U87MG and primary glioblastoma multiforme cells maintained in primary culture and following knockdown of one of the component subunits, ASIC1. In contrast, knocking down δENaC, which is not a component of the glioma cation channel complex, had no effect on cyclin-dependent kinase inhibitor expression. Phosphorylation of ERK1/2 was also inhibited by PcTX-1, benzamil, and knockdown of ASIC1 but not δENaC in D54MG cells. Our data suggest that a specific cation conductance composed of acid-sensing ion channels and ENaC subunits regulates migration and cell cycle progression in gliomas.