Baicalein induces the apoptosis of U251 glioblastoma cell lines via the NF-kB-p65-mediated mechanism

Glioblastoma (GBM) is the most aggressive cerebral gliomas. Moreover, the overall prognosis of GBM is still little. Baicalein (BA) is a flavonoid derived from the Scutellaria baicalensis root, and has historically been used in anticancer therapies. However, its apoptosis role and related mechanisms in GBM has not yet been researched clearly. Thus, this study aimed to investigate the effects of BA on human GBM U251 cell line. The effects of BA on proliferation of U251 cells were measured by Cell Counting Kit-8 assay. Cellular apoptosis was detected by flow cytometry with annexin V-FITC/propidium iodide staining. The expression of apoptosis-related protein Bcl-2, Bax and cleaved-caspase3 was detected by quantitative real-time PCR and western blot. The expression of nuclear p65 protein, the active subunit of nuclear factor-kappa B (NF-κB), was determined by immunofluorescence and western blot. Our results showed that the viability of U251 cells significantly decreased in a time- and dose-dependent manner after treated with BA, and the apoptotic ratio of BA-treated groups was significantly higher than that of control groups. Furthermore, the expression of NF-kB-p65 in the nucleus was remarkably reduced, and the activity of NF-kB-p65 was remarkably inhibited after BA treatment. Combined treatment with a NF-kB-P65 inhibitor (QNZ) and BA resulted in the synergistic reduction of Bcl-2 expression and then increase of Bax and cleaved-caspase3 expression; and the viability of U251 cells was also inhibited. In conclusion, BA inhibits GBM cells viability and induces apoptosis via inhibit the activity of NF-kB-p65, suggesting that BA is a potential therapeutic agent for GBM.     

Bcl-2 siRNA Augments Taxol Mediated Apoptotic Death in Human Glioblastoma U138MG and U251MG Cells

The anti-neoplastic drug taxol binds to β-tubulin to prevent tumor cell division, promoting cell death. However, high dose taxol treatment may induce cell death in normal cells too. The anti-apoptotic molecule Bcl-2 is upregulated in many cancer cells to protect them from apoptosis. In the current study, we knocked down Bcl-2 expression using cognate siRNA during low-dose taxol treatment to induce apoptosis in two human glioblastoma U138MG and U251MG cell lines. The cells were treated with either 100 nM taxol or 100 nM Bcl-2 siRNA or both for 72 h. Immunofluorescent stainings for calpain and active caspase-3 showed increases in expression and co-localization of these proteases in apoptotic cells. Fluorometric assays demonstrated increases in intracellular free [Ca²⁺], calpain, and caspase-3 indicating augmentation of apoptosis. Western blotting demonstrated dramatic increases in the levels of Bax, Bak, tBid, active caspases, DNA fragmentation factor-40 (DFF40), cleaved fragments of lamin, fodrin, and poly(ADP-ribose) polymerase (PARP) during apoptosis. The events related to apoptosis were prominent more in combination therapy than in either treatment alone. Our current study demonstrated that Bcl-2 siRNA significantly augmented taxol mediated apoptosis in different human glioblastoma cells through induction of calpain and caspase proteolytic activities. Thus, combination of taxol and Bcl-2 siRNA offers a novel therapeutic strategy for controlling the malignant growth of human glioblastoma cells. Special issue article in honor of Dr. George DeVries.     

miR-135b Contributes to the Radioresistance by Targeting GSK3β in Human Glioblastoma Multiforme Cells

Radioresistance remains a major challenge in the treatment of glioblastoma multiforme (GBM). Recent data strongly suggests the important role of miRNAs in cancer progression and therapeutic response. Here, we have established a radioresistant human GBM cell line U87R derived from parental U87 and found miR-135b expression was upregulated in U87R cells. miR-135b knockdown reversed radioresistance of U87R cells, and miR-135b overexpression enhanced radioresistance of U87 cells. Mechanically, bioinformatics analysis combined with experimental analysis demonstrated GSK3β (Glycogen synthase kinase 3 beta) was a novel direct target of miR-135b. Moreover, GSK3β protein expression was downregulated in U87R cells and restored expression of GSK3β increased radiosensitivity of U87R cells. In addition, clinical data indicated that the expression of miR-135b or GSK3β was significantly association with IR resistance of GBM samples. Our findings suggest miR-135b is involved in the radioresistance of human GBM cells and miR-135b-GSK3β axis may be a novel candidate for developing rational therapeutic strategies for human GBM treatment.     

Up-Regulation of microRNA-183 Promotes Cell Proliferation and Invasion in Glioma By Directly Targeting NEFL

Glioblastoma multiforme (GBM) is the most common and lethal type of primary malignant brain tumor. In recent years, increasing reports suggest that discovery of microRNAs (miRNAs) might provide a novel therapeutical target for human cancers, including GBM. The expression and roles of microRNA-183 (miR-183) has been explored in several types of human cancers, including in GBM, and plays important roles in tumor initiation and progression. However, its biological functions in GBM remain largely unknown. In this study, we demonstrated that miR-183 was significantly up-regulated in astrocytoma tissues and glioblastoma cell lines. Introduction of miR-183 mimics into U251 cells could promoted, while its antisense oligos inhibited cell proliferation and invasion. Moreover, we identified neurofilament light polypeptide (NEFL) as a novel target gene of miR-183. The expression levels of NEFL are inversely correlated with that of miR-183 in human astrocytoma clinical specimens. In addition, NEFL-siRNA could significantly attenuate the inhibitory effects of knockdown miR-183 on the proliferation and invasion of U251 cells via mTOR signaling pathway. Overall, This study revealed that miR-183 promotes glioma cell proliferation by targeting NEFL, and also demonstrated that miR-183 could be a potential target for GBM treatment.     

MicroRNA-4731-5p delivered by AD-mesenchymal stem cells induces cell cycle arrest and apoptosis in glioblastoma

Glioblastoma multiforme (GBM) exhibits the most malignant brain tumor with very poor prognosis. MicroRNAs (miRNAs) are regulatory factors that can downregulate the expression of multiple genes. Several miRNAs acting as tumor-suppressor genes have been identified so far. The delivery of miRNA by mesenchymal stem cell (MSC) due to their ability to specifically target tumors is a new, hopeful therapeutic approach for glioblastoma. The objective of our study is the investigation of the effect of lentivirus-mediated microRNA-4731 (miR-4731) genetic manipulated adipose-derived (AD)-MSC on GBM. The downregulation of miR-4731 in human GBM tumor was detected using the GEO dataset. To evaluate the function of miR-4731, we overexpressed miR-4731 using lentiviral vectors in U-87 and U-251 GBM cell lines. The effects of miR-4731 on cell proliferation and cell cycle of glioma cells were analyzed by wound test and flow-cytometry assay. miR-4731 inhibited the proliferation of GBM cancer cells. Coculturing was used to study the antiproliferative effect of miR-4731-AD-MSCs on GBM cell lines. Direct and indirect coculture of GBM cell lines with miR-4731-AD-MSCs induced cell cycle arrest and apoptosis. Our findings suggest that AD-MSCs expressing miR-4731 have favorable antitumor characteristics and should be further explored in future glioma therapy.     

MiR-18a regulates the proliferation,migration and invasion of human glioblastoma cell by targeting neogenin

MiR-17-92 cluster has recently been reported as an oncogene in some tumors. However, the association of miR-18a, an important member of this cluster, with glioblastoma remains unknown. Therefore, this study aims to investigate the expression of miR-18a in glioblastoma and its role in biological behavior of U87 and U251 human glioblastoma cell lines. Quantitative RT-PCR results showed that miR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines compared with that in human brain tissues and primary normal human astrocytes, and the expression levels were increased along with the rising pathological grades of glioblastoma. Neogenin was identified as the target gene of miR-18a by dual-luciferase reporter assays. RT-PCR and western blot results showed that its expression levels were decreased along with the rising pathological grades of glioblastoma. Inhibition of miR-18a expression was established by transfecting exogenous miR-18a inhibitor into U87 and U251 cells, and its effects on the biological behavior of glioblastoma cells were studied using CCK-8 assay, transwell assay and flow cytometry. Inhibition of miR-18a expression in U87 and U251 cells significantly up-regulated neogenin, and dramatically suppressed the abilities of cell proliferation, migration and invasion, induced cell cycle arrest and promoted cellular apoptosis. Collectively, these results suggest that miR-18a may regulate biological behavior of human glioblastoma cells by targeting neogenin, and miR-18a can serve as a potential target in the treatment of glioblastoma.     

Nuclear factor-κB signaling pathway is involved in phospholipase Cε-regulated proliferation in human renal cell carcinoma cells

Glioblastoma is the most aggressive cerebral gliomas. Despite advances in therapies, the prognosis is still very poor. Therefore, novel therapeutic strategies are required. As a proteasome inhibitor, bortezomib has shown its efficacy as an active antitumor agent against a variety of tumors. However, inhibition of proteasome activity leads to cell death and also induces cell autophagy, and due to the dual roles of autophagy in the survival and death of tumor cells, the effect of inhibition of autophagy on glioblastoma cells remains to be explored. We therefore assessed whether bortezomib is capable of inducing autophagy, and investigated the antitumor effect of bortezomib combined with autophagy inhibitors on human glioblastoma U251 and U87 cells. Cell viability was measured by MTT assay. The expressions of autophagy and apoptosis-related proteins were determined by Western blot analysis. U251 and U87 cells proliferation was inhibited in a dose-dependent manner. Both apoptosis and autophagy induced by bortezomib were observed in human glioblastoma U87 and U251 cells. However, when U251 and U87 cells were co-treated with bortezomib and autophagy inhibitors 3-MA or Atg7 siRNA, the autophagy inhibitors blocked the autophagy in the cells and resulted in a further inhibition of cell proliferation and a further increase in cell apoptosis as compared with that treated with bortezomib alone. These findings indicated that combination of bortezomib and autophagy inhibitors may shed new light on glioblastoma treatment.     

TMBIM1 promotes proliferation and attenuates apoptosis in glioblastoma cells by targeting the p38 MAPK signalling pathway

Glioblastoma multiforme (GBM) is the most common and most fatal primary malignant brain tumour in adults. The average survival time of patients after diagnosis is only 12–15 months. And its characteristics of excessive proliferation and apoptosis evasion play a crucial role in the poor prognosis of patients. Therefore, it is worth investigating the molecular mechanism of GBM to find an effective therapeutic target to overcome the dilemma. In the current study, Transmembrane BAX inhibitor motif containing 1 (TMBIM1) was highly expressed in GBM tissues and high TMBIM1 expression in GBM cell lines (U87 and U251) could promote cell proliferation and inhibit cell cycle arrest. In addition, TMBIM1 could significantly attenuate GBM cell apoptosis and decrease the sensitivity of GBM cells to temozolomide (TMZ). In terms of the molecular mechanism, we revealed that TMBIM1 interferes with the p38/MAPK pathway by inhibiting p38 phosphorylation to promote cell proliferation and attenuate cell apoptosis. In vivo experiments showed that the survival time of mice in TMBIM1 knockdown group was significantly prolonged. Our discovery provided an important basis for future intensive molecular mechanism research in GBM and presented a potential target for the treatment of GBM.     

miR-373 Inhibits Glioma Cell U251 Migration and Invasion by Down-Regulating CD44 and TGFBR2

Glioblastoma multiforme (GBM) is the most malignant glioma, unveiling the underlying mechanisms of its aggressiveness could promote the discovery of potential targets for effective treatment. MicroRNAs (miRNAs) are important participants in both development and disease, its involvement in cancers has long been recognized. In this study, we investigated the role of miRNA-373 (miR-373) in GBM cell line U251, demonstrated that although miR-373 does not affect cell growth of U251, it inhibits migration and invasion of U251. Forced expression of miR-373 down-regulates the expressions CD44 and TGFBR2, while knockdown of CD44 and TGFBR2 presents the similar phenotype as miR-373 overexpression, suggesting that CD44 and TGFBR2 are functional targets of miR-373, down-regulation of CD44 and TGFBR2 by miR-373 are partly responsible for the migration, and invasion suppressive role of miR-373 in U251.     

MiR-155 promotes proliferation of human breast cancer MCF-7 cells through targeting tumor protein 53-induced nuclear protein 1

Background

MiR-155 has emerged as an “oncomiR”, which is the most significantly up-regulated miRNA in breast cancer. However, the mechanisms of miR-155 functions as an oncomiR are mainly unknown. In this study, the aims were to investigate the effects of miR-155 on cell proliferation, cell cycle, and cell apoptosis of ERalpha (+) breast cancer cells and to verify whether TP53INP1 (tumor protein 53-induced nuclear protein 1) is a target of miR-155, and tried to explore the mechanisms of miR-155 in this process.

Results

The expression of miR-155 is significantly higher in MCF-7 cells compared with MDA-MB-231 cells. Ectopic expression of TP53INP1 inhibits growth of MCF-7 cells by inducing cell apoptosis and inhibiting cell cycle progression. Overexpression of miR-155 increases cell proliferation and suppress cell apoptosis, whereas abrogating expression of miR-155 suppress cell proliferation and promotes cell apoptosis of MCF-7 cells. In addition, miR-155 negatively regulates TP53INP1 mRNA expression and the protein expression of TP53INP1, cleaved-caspase-3, -8, -9, and p21, and luciferase reporter reveals that TP53INP1 is targeted by miR-155.

Conclusions

TP53INP1 is the direct target of miR-155. MiR-155, which is overexpressed in MCF-7 cells, contributes to proliferation of MCF-7 cells possibly through down-regulating target TP53INP1.     

Swelling‐induced chloride current in glioblastoma proliferation,migration, and invasion

Glioblastoma (GBM) remains as the most common and aggressive brain tumor. The survival of GBM has been linked to the aberrant activation of swelling‐induced chloride current I_Cl,swell. In this study, we investigated the effects of I_Cl,swell on cell viability, proliferation, and migration in the human GBM cell lines, U251 and U87, using a combination of patch clamp electrophysiology, MTT, colony formation, wound healing assays and Western immunoblotting. First, we showed that the specific inhibitor of I_Cl,swell, DCPIB, potently reduced the I_Cl,swell in U87 cells. Next, in both U87 and U251 cells, we found that DCPIB reduced GBM viability, proliferation, colony formation, migration, and invasion. In addition, our Western immunoblot assay showed that DCPIB‐treated U251 cells had a reduction in JAK2, STAT3, and Akt phosphorylation, thus, suggesting that DCPIB potentially suppresses GBM functions through inhibition of the JAK2/STAT3 and PI3K/Akt signaling pathways. Therefore, the I_Cl,swell may be a potential drug target for GBM.     

miR-155-TP53INP1调节轴在结直肠癌化疗药物敏感性中的作用机制

摘要 目的：初步揭示miR-155通过靶向调节TP53INP1表达水平影响结直肠癌细胞对5-FU化疗敏感性。方法：将人结肠直肠癌细胞系HCT116进行培养,提取细胞总RNA后,采用miR-155逆转录特异性引物构建反转录体系进行PCR扩增,通过qRT-PCR检测miR-155在5-FU耐药细胞HCT116/FU及敏感细胞株HCT116中的表达情况;取对数生长期细胞,分别转染miR-155mimics、miR-155抑制剂、miR-155阴性对照后,采用CCK-8法检测miR-155对细胞5-FU药物敏感性的影响,双荧光素酶报告基因系统验证miR-155与TP53INP1的靶基因关系,Western blot检测miR-155对 TP53INP1表达的影响。结果：miR-155在HCT116 /Fu细胞中的表达量是HCT116细胞的7.25倍;在相同5-FU浓度时,HCT116+阴性对照的细胞生长抑制率均高于HCT116+mimics、半数抑制浓度显著低于HCT116+mimics,差异均具有统计学意义(P＜0.05);TP53INP1是miR-155的靶基因,能显著降低野生型TP53INP1 3''-UTR的荧光素酶活性;转染miR-155 mimics后,TP53INP1的相对表达量显著下降,转染miR-155抑制剂后,TP53INP1的相对表达量显著升高,差异均具有统计学意义(P＜0.05)。结论：miR-155水平升高使HCT116细胞对5-FU的敏感性降低,miR-155可能通过靶向调节TP53INP1的表达水平,从而影响结直肠癌细胞对5-FU的敏感性。     

CFTR activation suppresses glioblastoma cell proliferation,migration and invasion

The aim of this study was to investigate the function of Cystic fibrosis transmembrane conductance regulator (CFTR) in human glioblastoma (GBM) cells. Data dining results of the Human Protein Atlas showed that low CFTR expression was associated with poor prognosis for GBM patients. We found that CFTR protein expression was lower in U87 and U251 GBM cells than that in normal humane astrocyte cells. CFTR activation significantly reduced GBM cell proliferation. In addition, CFTR activation significantly abrogated migration and invasion of GBM cells. Besides, CFTR activator Forskolin treatment markedly reduced MMP-2 protein expression. These effects of CFTR activation were significantly inhibited by CFTR inhibitor CFTRinh-172 pretreatment. Our findings suggested that JAK2/STAT3 signaling was involved in the anti-glioblastoma effects of CFTR activation. Moreover, CFTR overexpression in combination with Forskolin induced a synergistic anti-proliferative response in U87?cells. Overall, our findings demonstrated that CFTR activation suppressed GBM cell proliferation, migration and invasion likely through the inhibition of JAK2/STAT3 signaling.     

Apoptosis related microRNAs and MGMT in glioblastoma cell lines submitted to treatments with ionizing radiation and temozolomide

AimTo evaluate the effect of radiotherapy and temozolomide on the expression of miRNAs apoptotic (miRNAs-21, -221, -222 (anti-apoptotic) and miRNAs-15a, -16 (pro-apoptotic)) and the gene MGMT in glioblastoma cell lines.BackgroundThe limited knowledge of the molecular biology of malignant gliomas may hinder the development of therapeutic modalities. In this scenario, one of the greatest advances of recent years was the identification of microRNAs. These molecules have an important role in biological processes involving cancer, including glioblastoma.Materials and methodsTrypan blue was used to verify the cell viability, and real time PCR to quantify the expression of microRNAs and gene 24, 48 and 120 h after exposure to treatments.ResultsThere was a statistically significant decrease of expression of miR-15a between 48 and 120 h in line T98 G treated with radiation, increased expression of miR-15a between 24 and 120 h in line U251 treated with radiation and temozolomide, and increased expression of miR-16 between 24 and 120 h in line U251 treated with radiation alone and when combined with temozolomide. There was a decrease in MGMT gene expression, between 24 and 48 h in U343 cells treated with temozolomide.ConclusionsIonizing radiation and temozolomide modified the expression of miRNAs studied and MGMT.     

MicroRNA-330 Is an Oncogenic Factor in Glioblastoma Cells by Regulating SH3GL2 Gene

MicroRNAs have recently emerged as key regulators of cancers. This study was therefore conducted to investigate the role of miR-330 in biological behaviors of human glioblastoma U87 and U251 cell lines and its molecular mechanism. SH3GL2 gene was identified as the target of miR-330. MiR-330 overexpression was established by transfecting miR-330 precursor into U87 and U251 cells, and its effects on proliferation, migration, invasion, cell cycle and apoptosis were studied. Overexpression of miR-330 can enhance cellular proliferation, promote migration and invasion, activate cell cycle and also inhibit apoptosis in U87 and U251 cells. Collectively, these above-mentioned results suggest that miRNA-330 plays an oncogenic role in human glioblastoma by regulating SH3GL2 gene and might be a new therapeutic target of human glioblastoma.     

Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line’s sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.     

Verbascoside inhibits progression of glioblastoma cells by promoting Let-7g-5p and down-regulating HMGA2 via Wnt/beta-catenin signalling blockade

Glioblastoma (GBM) continues to show a poor prognosis despite advances in diagnostic and therapeutic approaches. The discovery of reliable prognostic indicators may significantly improve treatment outcome of GBM. In this study, we aimed to explore the function of verbascoside (VB) in GBM and its effects on GBM cell biological processes via let-7g-5p and HMGA2. Differentially expressed GBM-related microRNAs (miRNAs) were initially screened. Different concentrations of VB were applied to U87 and U251 GBM cells, and 50 µmol/L of VB was selected for subsequent experiments. Cells were transfected with let-7g-5p inhibitor or mimic, and overexpression of HMGA2 or siRNA against HMGA2 was induced, followed by treatment with VB. The regulatory relationships between VB, let-7g-5p, HMGA2 and Wnt/β-catenin signalling pathway were determined. The results showed that HMGA2 was a direct target gene of let-7g-5p. VB treatment or let-7g-5p overexpression inhibited HMGA2 expression and the activation of Wnt/β-catenin signalling pathway, which further inhibited cell viability, invasion, migration, tumour growth and promoted GBM cell apoptosis and autophagy. On the contrary, HMGA2 overexpression promoted cell viability, invasion, migration, tumour growth while inhibiting GBM cell apoptosis and autophagy. We demonstrated that VB inhibits cell viability and promotes cell autophagy in GBM cells by up-regulating let-7g-5p and down-regulating HMGA2 via Wnt/β-catenin signalling blockade.     

BKM120 sensitizes glioblastoma to the PARP inhibitor rucaparib by suppressing homologous recombination repair

PARP inhibitors have been approved for the therapy of cancers with homologous recombination (HR) deficiency based on the concept of “synthetic lethality”. However, glioblastoma (GBM) patients have gained little benefit from PARP inhibitors due to a lack of BRCA mutations. Herein, we demonstrated that concurrent treatment with the PARP inhibitor rucaparib and the PI3K inhibitor BKM120 showed synergetic anticancer effects on GBM U251 and U87MG cells. Mechanistically, BKM120 decreased expression of HR molecules, including RAD51 and BRCA1/2, and reduced HR repair efficiency in GBM cells, therefore increasing levels of apoptosis induced by rucaparib. Furthermore, we discovered that the two compounds complemented each other in DNA damage response and drug accumulation. Notably, in the zebrafish U87MG-RFP orthotopic xenograft model, nude mouse U87MG subcutaneous xenograft model and U87MG-Luc orthotopic xenograft model, combination showed obviously increased antitumor efficacy compared to each monotherapy. Immunohistochemical analysis of tumor tissues indicated that the combination obviously reduced expression of HR repair molecules and increased the DNA damage biomarker γ-H2AX, consistent with the in vitro results. Collectively, our findings provide new insight into combined blockade of PI3K and PARP, which might represent a promising therapeutic approach for GBM.Subject terms: Targeted therapies, Drug development