RNA-Binding Motif Protein RBM47 Promotes Invasiveness of Glioblastoma Through Activation of Epithelial-to-Mesenchymal Transition Program.

Background: RNA-binding motif proteins (RBMs) have been widely implicated in the tumorigenesis of multiple human cancers but rarely investigated in glioblastoma (GBM). Methods: The expression level of RBM47 and its correlation with prognosis of GBM were examined using bioinformatics, quantitative reverse transcription PCR, and Western blot analysis. The colony formation assay and Cell Counting Kit-8 assay were used to determine the biological role of RBM47 in GBM. To measure invasiveness we used the wound healing assay and transwell assay. The regulatory relationship between RBM47 and the epithelial-to-mesenchymal transition (EMT) was examined by Western blot analysis and bioinformatic analysis. Results: Through integrative analysis of clinical proteomic and genomic tumor datasets, we found that RBM47 is significantly upregulated in GBM mesenchymal subtype, and its high expression is correlated with poor prognosis. In in vitro biological experiments, we observed a significant inhibitory effect of RBM47 knockdown on colony formation and cell growth using GBM cell lines. Conversely, overexpression of RBM47 restored and accelerated these processes. Moreover, in vitro, wound healing assays demonstrated the role of RBM46 in promoting and cell migration and invasion. Mechanistically, RBM47 enhances invasive capacity through the activation of the EMT program. In RBM47-knockdown cells, the expression levels of Vimentin and CD44 were suppressed, and the level of E-cadherin was increased. Conclusions: Taken together our results demonstrate the tumor promoting characteristics of RBM46 and suggest that it could be used both as a therapeutic target and prognostically.

Β-elemene inhibits Hsp90/Raf-1 molecular complex inducing apoptosis of glioblastoma cells.

ß-Elemene, an active component of herb medicine Curcuma wenyujin, has been shown to antagonize glioblastoma cells by inducing apoptosis. However, how ß-elemene induces apoptosis of these cells remains unclear. In this study, we report that ß-elemene disrupted the formation of the Hsp90/Raf-1 complex, a key step in maintaining the conformation stability of Raf-1, and caused deactivation of Raf-1 and inhibition of the ERK pathway, thereby leading to apoptosis of glioblastoma cells. Specifically, treatment of glioblastoma cell lines with ß-elemene attenuated phosphorylation of multiple members of the kinase families in the Ras/Raf/MEK/ERK cascade, including Raf-1 and ERK as well as downstream signaling targets such as Bcl-2. These results suggest that the Hsp90/Raf-1 complex could be a promising molecular target for new drug development for the treatment of glioblastoma.

Expression and methylation status of LAMA2 are associated with the invasiveness of nonfunctioning PitNET.

The laminin subunit alpha 2 (LAMA2) gene encodes an alpha 2 chain, which constitutes one of the subunits of laminin 2 (merosin) and laminin 4 (s-merosin). In the current study, we investigated the relationship between LAMA2 promoter methylation status and the invasiveness of clinically nonfunctioning pituitary adenomas (PitNETs). Specimens from patients with nonfunctioning PitNET were classified into three groups according to preoperative computed tomography (CT)/magnetic resonance imaging findings: a normal group (n = 6), non-invasive group (n = 11) and invasive group (n = 6). LAMA2 expression was assessed using quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting, and the methylation status of the LAMA2 promoter region was observed using sodium bisulfite sequencing. Furthermore, 5-aza-2-deoxycytidine was used to explore the relationship between decreased LAMA expression and methylation in PitNET cells. According to the RT-qPCR and western blotting results, LAMA2 expression was downregulated in invasive PitNET, while the methylation of the LAMA2 promoter was increased. Methylation of the LAMA2 promoter decreased the expression of LAMA2. Thus, changes in LAMA2 expression due to promoter methylation were inversely correlated with the invasiveness of PitNET and the protein functions as a tumor suppressor. In addition, overexpression and demethylation of LAMA2 suppressed the invasion of PitNET cells, partially by exerting effects on the PTEN-PI3K/AKT signaling pathway and matrix metalloproteinase-9 (MMP-9). Furthermore, a xenograft model was also generated, and LAMA2 overexpression significantly suppressed tumor growth in vivo. Thus, LAMA2 expression and methylation patterns might be used as biomarkers to predict the prognosis of patients with PitNET.

Silencing NUDT21 Attenuates the Mesenchymal Identity of Glioblastoma Cells via the NF-κB Pathway.

The proneural (PN) and mesenchymal (MES) subtypes of glioblastoma multiforme (GBM) are robust and generally consistent with classification schemes. GBMs in the MES subclass are predominantly primary tumors that, compared to PN tumors, exhibit a worse prognosis; thus, understanding the mechanism of MES differentiation may be of great benefit for the treatment of GBM. Nuclear factor kappa B (NF-κB) signaling is critically important in GBM, and activation of NF-κB could induce MES transdifferentiation in GBM, which warrants additional research. NUDT21 is a newly discovered tumor-associated gene according to our current research. The exact roles of NUDT21 in cancer incidence have not been elucidated. Here, we report that NUDT21 expression was upregulated in human glioma tissues and that NUDT21 promoted glioma cell proliferation, likely through the NF-κB signaling pathway. Gene set enrichment analysis, western blotting, and quantitative real-time reverse transcription polymerase chain reaction confirmed that NF-κB inhibitor zeta (NFKBIZ) was a downstream target affected by NUDT21 and that the MES identity genes in glioblastoma cells, CHI3L1 and FN1, were also differentially regulated. Our results suggest that NUDT21 is an upstream regulator of the NF-κB pathway and a potential molecular target for the MES subtype of GBM.

ß-elemene inhibits stemness, promotes differentiation and impairs chemoresistance to temozolomide in glioblastoma stem-like cells.

Accumulating evidence indicates that glioblastoma stem-like cells (GSCs) are key factors in tumour development, recurrence and chemoresistance. The impairment of stemness and the enhancement of differentiation contributes to the weakening of radiation and chemotherapy resistance of GSCs. We previously found that ß-elemene was an effective anti-glioblastoma agent and chemosensitizer. In this study, we examined the distribution of CD133(+) cells in human glioblastoma tissues by immunohistochemistry. Following treatment with ß-elemene, the formation of GSC spheres was investigated by manual counting, the proliferation of GSCs was measured with a Cell Counting Kit-8 (CCK-8) assay, and the dispersion of GSC spheres was observed with an inverted microscope. GSC spheres were treated with ß-elemene, and the expression levels of CD133, ATP-binding cassette subfamily G member 2 (ABCG2) and glial fibrillary acidic protein (GFAP) were examined by western blotting. After treatment with ß-elemene, the volumes and weights of GSC xenografts were measured, and the expression of CD133, ABCG2 and GFAP was evaluated through immunohistochemistry analysis. After treatment with ß-elemene and temozolomide (TMZ), GSC viability was examined by the CCK-8 assay, and the volumes and weights of xenografts were measured. We found that CD133(+) cells were assembled in some vascular walls and also sparsely distributed in other parts of glioblastoma tissues. ß-elemene decreased the formation of GSC spheres, dispersed GSC spheres and inhibited the proliferation of GSCs in vitro and in vivo. In the GSC spheres and xenografts treated with ß-elemene, the expression of CD133 and ABCG2 was significantly downregulated, and the expression of GFAP increased. Furthermore, the sensitivity of GSCs to TMZ was enhanced in vitro and in vivo. These results suggest that ß-elemene impaired the stemness of GSC spheres, promoted their differentiation and sensitized GSCs to TMZ. ß-elemene will hopefully become a valuable agent to enhance the effects of radiotherapy and chemotherapy.

ß-Elemene inhibits proliferation through crosstalk between glia maturation factor ß and extracellular signal­regulated kinase 1/2 and impairs drug resistance to temozolomide in glioblastoma cells.

ß-elemene, a plant-derived drug extracted from Curcuma wenyujin, has demonstrated marked antiproliferative effects on glioblastoma, while toxicity remains low. However, the underlying molecular mechanisms of the antitumor activity of ß-elemene remain to be elucidated. Previously, it was identified that the glia maturation factor ß (GMFß)/mitogen-activated protein kinase kinase (MAPK) 3/6/p38 pathway participates in the antiproliferative activity of ß-elemene on glioblastoma. In the present study, in order to illustrate the association of GMFß and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, U87 and U251 cells were treated with ß-elemene at various doses and for different durations, and the expression of phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, B-cell lymphoma 2 (Bcl-2), Bcl2-associated X and survivin was examined by western blot analysis. Following treatment with ß-elemene and the ERK1/2 inhibitor PD98059, U87 cell viability was evaluated using a Cell Counting Kit-8 (CCK-8) assay, and the expression levels of Bcl-2 and survivin were examined by western blot analysis. GMFß was then downregulated by RNA interference in ß-elemene-treated U87 cells, and the effect of this on the expression of ERK1/2 and p-ERK1/2 was determined by western blot analysis. Finally, the chemosensitisation of U87 cells to temozolomide (TMZ) through ß-elemene was examined using the CCK-8 assay. The results demonstrated that ß-elemene inhibited the proliferation of U87 glioblastoma cells through the GMFß­dependent inactivation of the ERK1/2-Bcl-2/survivin pathway. Furthermore, inhibition of ERK1/2 by PD98059 enhanced the antitumor effect of ß-elemene and impaired the expression levels of Bcl-2 and survivin. ß-elemene also increased the sensitivity of U87 glioblastoma cells to the chemotherapeutic TMZ, which was synergistically enhanced by PD98059. In conclusion, these results suggested that GMFß-dependent inactivation of the ERK1/2-Bcl-2/survivin pathway mediated the antiproliferative effect of ß-elemene on glioblastoma. Therefore, ß-elemene is a promising chemosensitizer or adjuvant therapeutic for TMZ against glioblastoma brain tumors.

Lysosomal membrane permeabilization contributes to elemene emulsion-induced apoptosis in A549 cells.

Elemene is a broad-spectrum antitumor agent. In the present study, lysosomal membrane permeabilization (LMP) was detected after short elemene emulsion--exposure (12 h) that preceded a decrease of the mitochondrial membrane potential and DNA damage (24 h) in A549 cells. At later time points (36 h) elemene emulsion caused the appearance of A549 cells with apoptotic features, including apoptotic morphology, phosphatidylserine exposure, and caspase-3 activation. A significant increase in protein expression for cathepsin D was also observed utilizing Western blot analysis after exposure to elemene emulsion for 12 h. The present study showed that elemene emulsion induced the increased levels of reactive oxygen species (ROS) and depletion of glutathione (GSH) in A549 cells. Cells treated with pepstatin A, an inhibitor for cathepsin D, showed a significant inhibition in DNA damage, mitochondrial membrane permeabilization, caspase-3 activation, and phosphatidylserine exposure. These results demonstrated that apoptosis induced by elemene emulsion in A549 cells is mediated in part through LMP and lysosomal protease cathepsin D.