Proliferation Of U251 Cells Research Articles

Synergistic effects of rmhTRAIL and 17-AAG on the proliferation and apoptosis of multiple myeloma cells

ABSTRACT Objective: This study aimed to investigate synergistic effects of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmhTRAIL) and heat-shock protein 90 (HSP90) inhibitor (geldanamycin derivative 17 -allylamino- 17-demethoxy -geldanamycin, 17-AAG) on the proliferation and apoptosis of multiple myeloma (MM) cells. Methods: MTT assays evaluated inhibitory effects of rmhTRAIL and 17-AAG in different concentrations and treatment durations on the proliferation of RPMI8226 and U266 cells. The half maximal inhibitory concentration was calculated using OriginPro7.5. Synergistic effects of rmhTRAIL and 17-AAG on apoptosis of MM cells were detected using flow cytometry at 24 and 48 h post-treatment. To evaluate synergistic effects of rmhTRAIL and 17-AAG, the Q-value was calculated using King's formula. Results: rmhTRAIL exhibited significant inhibitory effects on the proliferation of RPMI8226 cells in a dose- and time-dependent manner (>50%), whereas U266 cells were not sensitive to rmhTRAIL (<50%). 17-AAG inhibited the proliferation of RPMI8226 and U266 cells in a dose-dependent manner (>80%). Significant synergistic effects of rmhTRAIL and 17-AAG on the proliferation of RPMI8226 cells were revealed (Q-value > 1.15), whereas synergistic effects were not evident on the proliferation of U266 cells (Q-value < 1.15). rmhTRAIL and 17-AAG exhibited significant synergistic effects on apoptosis of RPMI8226 and U266 cells (Q-value > 1.15). Conclusion: The combined application of rmhTRAIL and 17-AAG revealed favorable synergistic effects in the treatment of MM.

The effect and mechanism of ellagic acid on the proliferation and invasion properties of human glioma U251 cells

Objective To investigate the anti-proliferation and anti-metastatic effect of ellagic acid (EA) on glioma cell line U251 and related mechanisms. Methods In this study, we employed cell counting kit-8 (CCK-8) assay to test the anti-proliferative effect of EA. Flow cytometer was used to monitor cell apoptosis and cell cycle. Wound healing assay and Transwell assay were used to test the anti-invasion effect of EA. Western blotting was employed to test the effect of EA on the expressions of B cell lymphoma/leukemia-2 (bcl-2), bcl-2 associated X protein (bax), Cysteinyl aspartate-specific protease (Caspase)-3, matrix metalloproteinase (MMP)-9 and MMP-2. Results EA could significantly inhibit the proliferation of U251 cells in concentration-dependent manner and time-dependent manner, and the concentration of half maximal inhibitory concentration (IC50) after treated 48 hours was 39.24 μg/ml. The apoptosis rate of 40 μg/ml was (34.07±0.28)%, compared with control group, the difference was significant (P=0.001). Flow cytometer test shows the percentage of cells in S phase treated by EA 40 μg/ml was (47.75±2.02)%, compared with control group (20.45±0.28)%, the difference was significant (P=0.000). The expressions of bax and Caspase-3 were increased significantly, while bcl-2 was decreased. In Wound healing assay, when treated 24 h by EA, the wound healing rate in control group, 5 μg/ml group, 10 μg/ml group were (53.88±0.30)%, (36.82±0.56)% and (31.33±0.24)%, the difference was significant (P=0.000, P=0.000); In Transwell assay, the number of passed cells were (168.2±7.1), (66.4±2.5) and (38.6±1.5) respectively, the difference was significant (P=0.001, P=0.000). The expressions of MMP-9 and MMP-2 were decreased significantly. Conclusion EA inhibits the proliferation and invasion of glioma cell line U251, which suggests that EA is a potential therapeutic agent for glioma. Key words: Glioma; Ellagic acid; Proliferation; Invasion; U251 cells

Oncogenic Role of MicroRNA-30b-5p in Glioblastoma Through Targeting Proline-Rich Transmembrane Protein 2.

MicroRNAs (miRs) have been found to play promoting or suppressive roles in different human cancers. However, the exact regulatory mechanism of miR-30b in glioblastoma remains unknown. Here we have shown that the expression of miR-30b is significantly increased in glioblastoma tissues and cell lines. Moreover, a high expression of miR-30b is significantly associated with a shorter survival time for glioblastoma patients. Knockdown of miR-30b caused a significant reduction in the proliferation, migration, and invasion of U87 and A172 cells. Proline-rich transmembrane protein 2 (PRRT2) was further identified as a novel target gene of miR-30b, and its protein expression is negatively regulated by miR-30b in U87 and A172 cells. Furthermore, PRRT2 is significantly downregulated in glioblastoma tissues and cell lines, and we found an inverse correlation between miR-30b and PRRT2 expression in glioblastoma tissues. In addition, inhibition of PRRT2 reversed the suppressive effect of miR-30b downregulation on the malignant phenotypes of U87 and A172 cells. Accordingly, we demonstrated that miR-30b promotes glioblastoma cell proliferation, migration, and invasion via targeting PRRT2. Therefore, miR-30b may be used as a promising therapeutic target for glioblastoma.

Histone deacetylase SIRT6 inhibits glioma cell growth through down-regulating NOTCH3 expression.

Gliomas are the most common brain tumors of the central nervous system. In this study, we investigated the molecular mechanisms and biological function of SIRT6 in human gliomas. The expression levels of SIRT6 in glioma tissues and cells were analyzed by qRT-PCR and western blot analysis. CCK8 and clonogenicity assays were performed to detect the cell proliferation. Furthermore, the migration and invasion of glioma cells were examined by transwell assays. It was found that the expression of SIRT6 was significantly lower in human glioma tissues or cell lines compared with the normal brain tissue or NHA. Up-regulated SIRT6 significantly decreased cell proliferation, migration and invasion of U87 and U251 cells. By contrast, knockdown of SIRT6 dramatically increased cell proliferation, migration and invasion of U87 and U251 cells. Moreover, over expression of NOTCH3 significantly increased the cell proliferation, migration, and invasion of U87 and U251 cells. However, these effects were abolished after overexpression of SIRT6. These results suggest that SIRT6 may suppress cell proliferation, migration, and invasion via inhibition of the NOTCH3 signaling pathway in glioma.

CEP55 promotes cell proliferation and inhibits apoptosis via the PI3K/Akt/p21 signaling pathway in human glioma U251 cells.

Human glioma is one of the major malignancies worldwide with an increased mortality rate. Centrosomal protein of 55 kDa (CEP55) is an essential component of the CEP family and has been identified as a prognostic marker for multiple types of cancer. However, the function of CEP55 during glioma tumorigenesis remains unclear. In the present study, the data derived from the Oncomine database indicated that the expression of CEP55 is increased in glioma tissues compared with normal tissues. Furthermore, the expression of CEP55 was also increased at the level of mRNA and protein in glioma cell lines compared with normal human astrocytes. The knockdown of CEP55 expression inhibited the proliferation of glioma U251 cells, whereas overexpression of CEP55 induced the proliferation of U251 cells. Flow cytometric analysis indicated that the knockdown of CEP55 resulted in an increased number of cells arrested at G2/M phase, and apoptosis was promoted. Further investigations revealed that the overexpression of CEP55 increased the phosphorylation of Akt and inhibited the activity of p21. By contrast, the knockdown of CEP55 resulted in the opposite effects. Taken together, the results of the present study suggested that CEP55 regulated the proliferation of glioma cells, further attributing to the carcinogenesis and progression of glioma via the PI3K/Akt/p21 signaling pathway. Therefore, CEP55 may be a novel therapeutic target for the treatment of glioma.

Allicin induces apoptosis through activation of both intrinsic and extrinsic pathways in glioma cells.

Allicin is an extract purified from Alliumsativum (garlic), and previous research has indicated that Allicin has an inhibitory effect on many kinds of tumor cells. The aim of the present study was to explore the anticancer activity of Allicin on human glioma cells and investigate the underlying mechanism. MTT and colony-formation assays were performed to detect glioma cell proliferation, and explore the effect of Allicin at various doses and time-points. The apoptosis of glioma cells was measured by fluorescence microscopy with Hoechst33258 staining, and then flow cytometry was used to analyzed changes in glioma cell apoptosis. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis were used to detect the effect of Allicin on the expression levels of Fas/Fas ligand (FasL), caspase‑3, B‑cell lymphoma2 and Bcl‑2‑associatedX protein. Allicin suppressed the proliferation and colony formation ability of U251cells in a dose‑and time‑dependent manner. A cytotoxic effect of Allicin was observed in glioma cells in a dose‑dependent manner. Changes in nuclear morphology were observed in U251 cells with Hoechst33258 staining. The activity of caspases were significantly elevated and Fas/FasL expression levels were increased following treatment with Allicin, at both the mRNA and protein level. These results demonstrated that Allicin suppresses proliferation and induces glioma cell apoptosis invitro. Both intrinsic mitochondrial and extrinsic Fas/FasL‑mediated pathways react in glioma cell after treating with Allicin, which then activate major apoptotic cascades. These results implicate Allicin as a novel antitumor agent in treating glioma.

Expression and functional analysis of TCF4 isoforms in human glioma cells.

Transcription factor 4 (TCF4) is a member of the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor family in the Wnt/β‑catenin signaling pathway. The alternative splicing of TCF4 has been reported to exhibit potential carcinogenic properties in various cancer types. In the present study, TCF4 isoforms were cloned and identified in three human glioma cell lines, with the majority of splicing regions being exons 4, 5, 14, 15, and 16. Using MTT assays, it was demonstrated that the overexpression of TCF4 isoforms inhibits the proliferation of U251 cells. Flow cytometry and wound healing analyses revealed that the overexpression of TCF4 isoforms induced cell apoptosis and migration. Taken together, the β‑catenin binding domain of the TCF4 isoforms inhibited cell proliferation, and induced cell apoptosis and migration in glioma. Furthermore, all the isoforms identified contained the N‑terminal part of TCF4 including the β‑catenin binding domain. This implied that a high expression of TCF4 isoforms may lead to Wnt/β‑catenin signal activation and potentially promote malignant glioma development.

High expression of lnc-CRNDE presents as a biomarker for acute myeloid leukemia and promotes the malignant progression in acute myeloid leukemia cell line U937.

To detect the expression of long non-coding RNA-CRNDE in patients with acute myeloid leukemia and its effect on proliferation and apoptosis in acute myeloid leukemia cell line U937. 81 cases of newly diagnosed acute myeloid leukemia (AML) were enrolled, and 35 non-malignant hematological patients were selected as controls. Quantitative RT-PCR (qRT-PCR) was performed to detect the expression of lncRNA-CRNDE in the bone marrow specimens of the subjects, and the difference between the two groups was also compared. The correlation between the expression of lncRNA-CRNDE and the sex, age, classification and total survival of clinical patients was analyzed according to the clinical data. U937 cells and monocytes isolated from normal people were cultured, and the expression of lncRNA-CRNDE in acute myeloid leukemia cell line U937 and normal monocytes was compared. SiRNA-CRNDE and pcDNA-CRNDE were transfected into U937 cells, and cell counting kit-8 (CCK-8) assay was performed to detect proliferation of U937 cells, Annexin V/PI flow cytometry was carried out to detect cell apoptosis. Cell cycle was measured by flow cytometry. The expression of lncRNA-CRNDE in patients with AML and U937 cells was significantly higher than that in non-malignant hematological controls. Results of clinical data showed that the expression of lncRNA-CRNDE was associated with the classification and total survival of myeloid leukemia in clinical patients. After transfection of siRNA-CRNDE, the proliferation and cloning ability of U937 cells decreased, while the apoptosis increased (p < 0.01) and cells were arrested in G0-G1 phase. Meanwhile, after transfection of pcDNA-CRNDE, the proliferation ability of U937 cells increased significantly, which indicated that the expression of lncRNA-CRNDE might play an essential role in promoting the proliferation of U937 cells. LncRNA-CRNDE is highly expressed in the bone marrow tissues of AML patients, and the expression level is negatively correlated with the total survival of those clinical patients. Meanwhile, the expression is higher in FAB type M4 and M5 than that in M1, M2 and M3. LncRNA-CRNDE promotes the proliferation and cell cycle of U937 cells, and inhibits cell apoptosis, which is expected to become a molecular marker for predicting and treating AML.

Long noncoding RNA SNHG7 promotes the progression and growth of glioblastoma via inhibition of miR-5095

The long non-coding RNA SNHG7 (small nucleolar RNA host gene 7) has been reported to be involved in various cancers as a potential oncogene. However, the functions and molecular mechanisms of SNHG7 in glioblastoma (GBM) are largely unknown. In the present study, we showed that the expression of SNHG7 was significantly upregulated in GBM tissues and cell lines compared with non-cancerous brain tissues. Furthermore, we found that SNHG7 knockdown remarkably suppressed the proliferation, migration and invasion of A172 and U87 cells while inducing their apoptosis. Subsequently, we showed that SNHG7 knockdown significantly inhibited tumor growth and metastasis in vivo by using xenograft experiments in nude mice. In terms of mechanism, we found that SNHG7 directly inhibited miR-5095, which targeted the 3′ UTR of CTNNB1 mRNA and subsequently downregulated the Wnt/β-catenin signaling pathway in GBM. Using rescue experiments, we demonstrated that SNHG7 promoted the proliferation, migration and invasion of GBM cells through the inhibition of miR-5095 and concomitant activation of Wnt/β-catenin signaling pathway. Taken together, the SNHG7/miR-5095 axis might be a potential target for the development of effective GBM therapy.

RK1, the first very short peptide from Buthus occitanus tunetanus inhibits tumor cell migration, proliferation and angiogenesis

Scorpion toxins have been the subject of many studies which explore their pharmacological potential toward diverse molecular targets, known to monitor key mechanisms in cancer such as proliferation, migration and angiogenesis. The few peptides from scorpion venom that have an anti-tumor effect are generally cytotoxic. Herein, we present the first description of a short 14 amino acid peptide (called RK1), purified from the venom of Buthus occitanus tunetanus, with the particular capabilities, among different other scorpion peptides, to inhibit cell proliferation, migration and angiogenesis of U87 (Glioblastoma) and IGR39 (Melanoma). Moreover, RK1 is a first peptide derived from scorpion venom exhibiting a potential anti-tumoral activity with no manifest toxicity. Our results suggest that, in terms of its primary structure, RK1 is unique compared to a variety of known peptides purified from scorpion venoms. In addition, RK1 is the first natural peptide able to abolish completely the proliferation of cancer cells. The Chicken chorioallantoic membrane model revealed that RK1 strongly inhibits ex-vivo vascular growth.RK1 could open new perspective for the pharmaceutical application of short scorpion venom peptides in anticancer activity and may represent the first member of a new group of scorpion peptides.

A synthetic cell-penetrating peptide derived from nuclear localization signal of EPS8 exerts anticancer activity against acute myeloid leukemia

BackgroundOncogenic roles of epidermal growth factor receptor pathway substrate no.8 (EPS8) have been widely reported in various tumors, making targeting of EPS8 an appealing prospect. Here, we describe the role of EPS8 in acute myeloid leukemia (AML) and consider the potential of EPS8 as an anti-AML target. Nuclear localization signal (NLS) residues of tumor-associated proteins are crucial for cell cycle progression, and specific inhibitors derived from the NLS have inhibitory effect on cancer cells. The NLS in EPS8 has potential as a specific anti-AML target.MethodsGene Expression Omnibus expression profiles of AML patients were used to test associations between EPS8 expression and AML patient outcome. The biological characteristics of AML cells after EPS8 knockdown were analyzed in vitro and in vivo. A specific peptide (CP-EPS8-NLS) derived from the NLS of EPS8 (amino acids 298–310) was synthesized, and the anti-AML effects of CP-EPS8-NLS were analyzed in cancer cells and in xenograft models. Mutated CP-EPS8-NLS and penetratin served as controls.ResultsWe observed that elevated EPS8 expression in AML patients is associated with poor outcome. Knockdown of EPS8 significantly suppressed the survival of AML cells in vitro and in vivo. CP-EPS8-NLS interfered with EPS8-associated signaling and consequently exerted anti-AML activity. Importantly, CP-EPS8-NLS displayed anti-AML activity in various AML cell types, with diminished activity in PBMCs. CP-ESP8-NLS suppressed U937 cell proliferation, and injection of CP-EPS8-NLS exerted potent antitumor activity in the xenograft tumor models. A synergistic effect of CP-EPS8-NLS and chemotherapeutic agents was also observed in vitro and in vivo. Mechanistically, treatment of various AML cells with CP-EPS8-NLS downregulated the expression of EPS8 and its downstream pathways.ConclusionsThe function of CP-EPS8-NLS is explained by the presence of a NLS in EPS8, which has been shown to induce nuclear translocation, consequently resulting in EPS8 overexpression. These results indicate that EPS8 is a potential target for AML treatment.

Nucleolar and spindle-associated protein 1 is a tumor grade correlated prognosis marker for glioma patients.

Despite therapeutic advances in glioma management including surgery, radiation, and chemotherapy, the improvement of patient outcome is far from satisfactory. Nucleolar and spindle-associated protein 1 (NUSAP1) is an important functional protein during mitosis, and its abnormal expression is implicated in progression of different types of tumors. However, the role of NUSAP1 in gliomas remains unclear. NUSAP1 expression in gliomas with different grades was investigated based on GEO glioma datasets. Kaplan-Meier survival analysis was used to evaluate its prognostic significance. In vitro assays were also performed to evaluate effects of NUSAP1 on malignant phenotypes of glioma cells by silencing NUSAP1. NUSAP1 expression was correlated not only with glioma grade but also with prognosis of glioma patients. NUSAP1 depletion suppressed proliferation of U251 cells by inducing cell cycle arrest at G2/M phase and apoptosis. NUSAP1 depletion rendered U251 cells impaired migratory ability as well. NUSAP1 is a potential prognosis marker for glioma patients and therapeutic strategies targeting NUSAP1 might hold promise in improving glioma treatment.

Long non-coding RNA SPRY4-IT1 promotes the proliferation and invasion of U251 cells through upregulation of SKA2.

The long non-coding RNA SPRY4-intronic transcript 1 (SPRY4-IT1) has been shown to promote the progression of cancer; however, the role of SPRY4-IT1 in glioma remains unclear. The present study demonstrated that SPRY4-IT1 expression was markedly increased in glioma tissues and cells compared with normal brain tissues, whereas knockdown of SPRY4-IT1 inhibited cell proliferation, migration, and invasion in U251 cells. Spindle and kinetochore associated complex subunit 2 (SKA2) was found to be a target of SPRY4-IT1 and was downregulated by SPRY4-IT1-knockdown. Additionally, SPRY4-IT1 expression was positively correlated with SKA2 in glioma tissues. To the best of our knowledge, the present study provides the first demonstration that SKA2 may have an oncogenic role in U251 cells. These results indicate that SPRY4-IT1 may serve a notable role in the molecular etiology of glioma and represents a potential target in glioma therapy.

Knockdown of immature colon carcinoma transcript1 induces suppression of proliferation, S-phase arrest and apoptosis in leukemia cells.

Immature colon carcinoma transcript1 (ICT1), a human mitochondrial translation release factor, is a ribosome-dependent codon-independent peptidyl-tRNA hydrolase. ICT1-deficiency has been recognized as a cell growth inhibitor of hepatoblastoma and glioblastoma multiforme. To explore the role of ICT1 in human leukemia, 2short hairpin RNAs (shRNAs) targeting ICT1 sequences were designed in leukemia U937 cells. The successful infection of ICT1 in the U937 cells was observed under a fluorescence microscope and further quantified by western blotting and quantitative real-time PCR (qRT-PCR) analysis. Tetrazolium dye (MTT) assay revealed a significant decrease in proliferation of ICT1-knockdown U937 cells on the fourth and fifth day as compared with the control. Depletion of ICT1 resulted in an increase in Sphase and sub-G1 (representing cell apoptosis) fractions. AnnexinV-APC/7-AAD staining assay confirmed that knockdown of ICT1 played a crucial role in boosting early and late apoptotic programs in U937 cells. Downregulation of ICT1 also altered cyclinA2 transcription expression, caspase-3 activity and p21 protein expression. Additionally, decreased levels of heat shock protein27 (HSP27) phosphorylation at Ser78 was correlated with knockdown of ICT1 in U937 cells. Thus, we concluded that the regulatory role of ICT1 in leukemia may be used as a potential therapeutic target for the treatment of leukemia.

Inhibitory effects of novel isothiocyanate derivative on proliferation and invasion of glioma U251 cells and regulation of circRNAs and lncRNAs

// Yu Zhu 1 and Ping Yang 1 1 Department of Clinical Laboratory, Tianjin Huanhu Hospital, Tianjin Key Laboratory of Cerebral Vessels and Neural Degeneration, Tianjin 300350, P.R. China Correspondence to: Yu Zhu, email: zhuyutj@126.com Ping Yang, email: tjhhhospitallab@126.com Keywords: glioma; isothiocyanate; lncRNAs; circRNAs; proliferation Received: November 08, 2017 Accepted: December 26, 2017 Published: January 02, 2018 ABSTRACT Gliomas proliferation and invasion plays the critical roles in the tumor progression. Isothiocyanate is the hydrolysis products of Brassica vegetables and suppress many types of tumor including gliomas. We design and synthesized an isothiocyanate derivative by computer-aided drug design base on alkylglycerone phosphate synthase (AGPS) to improve targeting and explore its effect on the proliferation, invasion, apoptosis, cell cycle, and the expression of circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) in U251 cell line. The isothiocyanate derivative suppressed proliferation and invasion by inducing apoptosis with improving the activity of caspase-3/8 and blocked the cell cycle at G2/M phase and regulating the expression of circRNAs and lncRNAs involved in the above biological behavior in human glioma U251 cells. Our results showed the effect of isothiocyanate derivative on the anti-tumor activity, which shows potential for application as a anti-glioma drug.

MicroRNA-375 regulates proliferation and apoptosis of glioma cancer cells by inhibiting CTGF-EGFR signaling pathway.

To evaluate the correlation between miRNA-375 and cell proliferation and apoptosis in glioma cancer cell. Collecting 30 cases of glioma cancer patients and 30 cases of cerebral infarction patients. The miRNA-375 and CTGF protein expressions were evaluated by ISH and IHC methods. In the cell experiment, the U87 cells were divided into 3 groups: NC group (the cells were treated with normal method); BL group (the cells were transfected with empty vector) and miRNA group (the cells were transfected with miRNA-375). The U87 cell proliferation and apoptosis rates and cell cycle of the different groups were measured by MTT and flow cytometry. The relative proteins (CTGF, EGFR, AKT, Erk and P21) expressions were measured by WB assay. The miRNA-375 and CTGF expressions of glioma cancer tissues were significantly different compared with those of no-cancer tissues (p < 0.05, respectively). In the cell experiments, the cell proliferation of miRNA group was significantly decreased compared with that of NC group (p < 0.05); the cell apoptosis and G1 phase rate of miRNA group was significantly decreased compared with NC group (p < 0.05, respectively). Depending on the WB assay, the CTGF, EGFR, AKT, Erk and P21 proteins expressions of miRNA group were significantly different compared with proteins expressions of NC group (p < 0.05, respectively). miRNA-375 over-expression suppresses glioma cancer cells development via CTGF-EGFR pathway (Fig. 3, Ref. 30).

Sinomenine Induces G1-Phase Cell Cycle Arrest and Apoptosis in Malignant Glioma Cells Via Downregulation of Sirtuin 1 and Induction of p53 Acetylation.

Sinomenine, a bioactive alkaloid isolated from the traditional Chinese herb Sinomenium acutum, possesses antiinflammatory, antinociceptive, antifibrotic, and antitumorigenic properties. In this work, we sought to explore the biological effects of sinomenine on glioma cells. It was found that sinomenine caused a concentration-dependent inhibition of viability in both U87 and U251 glioma cells. Sinomenine at 16 μmol/L caused 55% to 60% reduction in the proliferation of U87 and U251 cells. Moreover, sinomenine treatment induced a G0/G1 cell cycle arrest and apoptosis. Mechanistically, sinomenine promoted p53 expression and acetylation and reduced the expression of sirtuin 1. Ectopic expression of sirtuin 1 significantly prevented sinomenine-induced p53 acetylation and growth suppression in glioma cells. Moreover, sinomenine inhibited the growth of U87 xenograft tumors in vivo and raised the p53 protein expression. Collectively, sinomenine shows antiproliferative effects against glioma cells which is mediated through downregulation of sirtuin 1 and induction of p53 activity.

Berberine inhibits angiogenesis in glioblastoma xenografts by targeting the VEGFR2/ERK pathway

Context: Berberine is used in traditional Chinese medicine for thousands of years with recent reports of its anticancer activity.Objective: To test antiangiogenic effects of berberine on human glioblastoma and clarify involvement of the VEGFR2/ERK pathway.Materials and methods: Cell viability, proliferation and migration assays were performed to determine in vitro antiangiogenic effects of berberine (6.25–200 μmol/L, 6–48 h). Ectopic and orthotopic xenograft models in BALB/c nude mice were induced to determine antitumour and antiangiogenic effects of berberine (50 mg/kg by oral gavage for 28 days) or vehicle control (carboxymethylcellulose sodium).Results: Berberine inhibited cell viability (IC50 of 42 and 32 μmol/L, respectively) and proliferation of U87 and U251 human glioblastoma cell lines. Berberine (50 μmol/L) inhibited cell migration of HUVEC by 67.50 ± 8.14% in the Transwell assay and tube formation of HUVEC by 73.00 ± 11.12% in the Matrigel assay. In the ectopic xenograft model, tumour weight was significantly decreased by 50 mg/kg of berberine (401.2 ± 71.5 mg vs. 860.7 ± 117.1 mg in vehicle group, p ˂ 0.001). Berberine significantly decreased haemoglobin content (28.81 ± 3.64 μg/mg vs. 40.84 ± 5.15 μg/mg in vehicle group, p ˂ 0.001) and CD31 mRNA expression in tumour tissue. In the orthotopic xenograft model, berberine (50 mg/kg) significantly improved the survival rate of mice (p = 0.0078). Berberine inhibited (p ˂ 0.001) the phosphorylation of VEGFR2 and ERK.Discussion and conclusions: Berberine inhibited angiogenesis in glioblastoma xenografts by targeting the VEGFR2/ERK pathway. Our work sheds new light on complementary and alternative therapy for glioblastoma.

Study on Therapeutic Action and Mechanism of TMZ Combined with RITA Against Glioblastoma

Background/Aims: Glioblastoma multiforme (GBM) is a malignant and aggressive central nervous system (CNS) tumor with high mortality and low survival rate. Effective treatment of GMB is a challenge worldwide. Temozolomide (TMZ) is a drug used to treat GBM, while the survival period of GBM patients with positive treatment remains less than 15 months. Reactivating p53 and Inducing Tumor Apoptosis (RITA) is a novel potential anti-cancer small molecular drug. Thus, it is essential to discover novel targets or develop effective drugs combination strategy to treat GBM. Methods: The U87 cells and U251 cells (p53 mutated) were treated with DMSO and 1, 5,10, 20 μM RITA, TMZ, RITA+TMA or PFT-α. The cell proliferation was measured using the MTS cell proliferation assay. The cell apoptosis was analyzed by Annexin V-FITC/PI Apoptosis Detection Kit. The key protein expression level was evaluated by WB. Molecular docking and molecular dynamics (MD) simulation methods were applied to simulate the interaction between RITA and ASK1. Results: Herein, we found that combination RITA and TMZ effectively inhibited the proliferation of U87 cells and promoted the apoptosis of U87 cells. Then the mechanism of RITA and TMZ treating GBM were further studied by detecting the expression of the proteins associating with p53 pathway, such as ASK1, Bax, and so on. RITA bound to the amino acids residues in the activation domain of the ASK1, then induced the conformation change of ASK1 receptor, activated ASK1 and caused a series of signal transduction, further resulted in the physiological effects. Conclusion: Taken together, the RITA suppressed the cell proliferation in glioblastoma via targeting ASK1.

Overexpression of centrosomal protein 55 regulates the proliferation of glioma cell and mediates proliferation promoted by EGFRvIII in glioblastoma U251 cells.

The epidermal growth factor receptor (EGFR) is often amplified in glioma, with the most common extracellular domain mutation being EGFR variant III (EGFRvIII). Abnormal EGFRvIII signaling has been shown to be important in driving tumor progression. Centrosomal protein 55 (CEP55), a member of the centrosomal relative proteins family, participates cytokinesis in the cell cycle. It exists in a few normal tissues and various tumor cells. The expression and function of CEP55 in human glioma cells need to investigate. In this study, the expression of CEP55 was detected in 40 cases of glioma tissues and 10 cases of non-tumor brain tissue. The proliferation of glioblastoma U251 cells was analyzed after transfection with EGFRvIII and CEP55 siRNA. We found that the expression of CEP55 was increased significantly in the glioma tissues than in normal brain tissue. The proliferation of U251 cells increased remarkably after transfection with EGFRvIII. Knockdown of CEP55 inhibited proliferation of U251 cells and was able to eliminate the effect of promoting proliferation induced by EGFRvIII in U251 cells. CEP55 played a key role in the proliferation of glioma cells and mediated EGFRvIII-stimulated proliferation in glioma cells. CEP55 might be a novel molecular therapeutic target in patients with gliomas expressing EGFRvIII.