Apoptosis In U251 Glioma Cells Research Articles

Nerve Growth Factor, Antimicrobial Peptides and Chemotherapy: Glioblastoma Combination Therapy to Improve Their Efficacy.

Glioblastoma (GBM) is an aggressive and lethal malignancy of the central nervous system with a median survival rate of 15 months. We investigated the combined anticancer effects of nerve growth factor (NGF), cathelicidin (LL-37), and protegrin-1 (PG-1) with chemotherapy (temozolomide, doxorubicin, carboplatin, cisplatin, and etoposide) in the glioblastoma U251 cell line to overcome the limitations of conventional chemotherapy and to guarantee specific treatments to succeed. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to study cell viability and to determine the cytotoxic effects of NGF, LL-37, and PG-1 and their combination with chemotherapy in U251 cells. Synergism or antagonism was determined using the combination index (CI) method. Caspase-3 activity was evaluated spectrophotometrically using a caspase-3 activity assay kit. Apoptosis was analyzed with flow cytometry using propidium iodide (PI) and YO-PRO-1. NGF and the peptides showed a strong cytotoxic effect on U251 glioma cells in the MTT test (IC50 0.0214, 3.1, and 26.1 μM, respectively) compared to chemotherapy. The combination of PG-1 + etoposide had a synergistic effect on apoptosis of U251 glioma cells. It should be noted that the cells were in the early and late stages of apoptosis, respectively, compared with the control cells. The caspase-3 activation analysis revealed that the caspase-3 level was not significantly (p > 0.05) increased in U251 cells following PG-1 with etoposide treatment compared with that in the untreated cells, suggesting that the combination of PG-1 and etoposide may induce caspase-independent apoptosis in U251 cells. NGF, LL-37, and PG-1 represent promising drug candidates as the treatment regimen for GBM. Furthermore, the synergistic efficacy of the combined protocol using PG-1 and etoposide may overcome some of the typical limitations of the conventional therapeutic protocols, thus representing a promising approach for GBM therapy.

Using Hyperbaric Oxygen to Improve the Radiosensitivity of Human U251 Glioma Cells.

The aim of this study was to explore the use of hyperbaric oxygen to enhance the radiosensitivity of human glioma cells. Sub-cultured U251 human glioma cells were randomly divided into four groups: an untreated control group, cells treated with hyperbaric oxygen (HBO) only, cells treated with X-ray irradiation (X-ray) only, and cells treated with both HBO and X-ray. Cell morphology, cell proliferation activity, cell cycle distribution, and apoptosis were observed in these groups to evaluate the role of HBO in improving the radiosensitivity of glioma cells. With the increase in X-ray doses (0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy), the survival fraction (SF) of glioma cells gradually decreased. Significantly lower SF was observed for the cells treated with the HBO and X-ray together than in the X-ray group for each dose (all P < 0.05). The proliferation inhibition was significantly higher in the HBO combined with X-ray group than in the X-ray group for each dose (all P < 0.05) for the U251 cell line. The percentage of G2/M phase cells was significantly higher in the HBO combined with X-ray (2 Gy) group (26.70% ± 2.46%) and the HBO group (22.36% ± 0.91%) than in the control group (11.56% ± 2.01%) and X-ray (2 Gy) group (10.35% ± 2.69%) (all P < 0.05). U251 cell apoptosis was significantly higher in the HBO combined with X-ray (2 Gy) group than in the HBO group, the X-ray (2 Gy) group, and the control group (all P < 0.05). We conclude that HBO can enhance the proliferation inhibition and apoptosis of glioma U251 cells by blocking glioma cells in the G2/M phase and improve the radiosensitivity of U251 glioma cells.

Exploring the underlying mechanism of oleanolic acid treating glioma by transcriptome and molecular docking

ObjectiveOleanolic acid is a promising drug for treating gliomas, but its underlying mechanism is unclear. This study aimed to determine the potential effect of oleanolic acid on glioma and its mechanism. MethodsFirstly, the effects of oleanolic acid on the proliferation, invasion, and apoptosis of glioma U251 cells were detected by in vitro experiments such as MTT assay, cell cloning, and flow cytometry. The transcriptome data of U251 cells treated with oleanolic acid and untreated were sequenced by mRNA, and then the differentially expressed genes were analyzed by gene ontology (GO), genomic encyclopedia (KEGG) pathway enrichment analysis, and protein interaction topology analysis. The underlying mechanism of oleanolic acid was predicted, and the related protein interaction network was constructed. Finally, Western blotting and molecular docking techniques verified the mRNA sequencing results. ResultsOleanolic acid could effectively inhibit the proliferation, colony formation, and invasion of U251 cells and induce apoptosis. A total of 446 differentially expressed genes were detected by mRNA sequencing, of which 96 genes were up-regulated and 350 down-regulated. Oleanolic acid induces the TNF signal pathway and NOD-like receptor signal pathway at the intracellular level. In addition, OAS2, OASL, IFIT3, RSAD2, and IRF1 may be the core targets of oleanolic acid in treating glioma. ConclusionTranscriptome combined with molecular docking technique is used to predict the possible mechanism of oleanolic acid in the treatment of glioma, which provides new ideas and insights for developing and researching antitumor drugs.

Momordica charantia-derived extracellular vesicles-like nanovesicles inhibited glioma proliferation, migration, and invasion by regulating the PI3K/AKT signaling pathway

• MCELNs were similar to animal cell-derived extracellular vesicles. • MCELNs penetrated the BBB. • MCELNs inhibited glioma growth via reguating PI3K/AKT pathway. • MCELNs suppressed metastasis via downregulating MMP9. Momordica.charantia has antioxidant and neuroprotective effects, but its anti-glioma capacity remains unclear. Plant-derived nanovesicles have shown therapeutic potential in inflammation and cancer. This study explored the therapeutic effects of Momordica.charantia -derived extracellular vesicles-like nanovesicles (MCELNs) in treating glioma and its underlying mechanisms. The MCELNs were successfully isolated and characterized similarly to animal cell-derived extracellular vesicles. The in vitro assays identified that MCELNs inhibited the proliferation but did not promote apoptosis of U251 glioma cells. MCELNs repressed U251 glioma cells migration and invasion via proceeding the epithelial-mesenchymal transition process and downregulating MMP9. MCELNs penetrated the BBB and suppressed the glioma growth and metastasis in vivo . The ratios of p-AKT/AKT and p-PI3K/PI3K were downregulated in MCELNs treated U251 glioma cells compared with the control. Highly expressed miR5813 in MCELNs might be involved in the PI3K/AKT pathway that needs further investigation. MCELNs could serve as a novel therapeutic strategy for glioma treatment.

Proapoptotic effect of Ginkgo biloba extract on U87 glioma cells

Objective To study the pro-apoptotic effect of Ginkgo biloba extract on U87 glioma cells. Methods After 24 hours of Ginkgo biloba extract intervention, the proliferation of U87 glioma cells was observed by CCK-8 with different concentrations of Ginkgo biloba extract at 100, 150, 200 and 250mg/L. The effect of Ginkgo biloba extract on apoptosis of U87 glioma cells was analyzed by flow cytometry. AnnexinV-FITC and PI double staining were used to detect the effect of Ginkgo biloba extract on apoptosis of U87 glioma cells. Results Ginkgo biloba extract could inhibit the proliferation of U87 glioma cells in a concentration-dependent manner. After 24 hours of Ginkgo biloba extract intervention, flow cytometry showed that the apoptosis rate of U87 glioma cells increased significantly with the increase of Ginkgo biloba extract concentration. Conclusion Ginkgo biloba extract can induce apoptosis of U87 glioma cells.

Corilagin induces human glioblastoma U251cell apoptosis by impeding activity of (immuno)proteasome.

Glioma is a type of common primary intracranial tumor, which is difficult to treat. It has been confirmed by research that corilagin (the primary active constituent of the matsumura leafflower herb) has significant antitumor effect. In particular, our previous research demonstrated that corilagin effectively promotes apoptosis of glioma U251 cells and has a synergistic effect when used with temozolomide. However, the mechanism by which corilagin causes apoptosis in U251 cells has yet to be investigated. Proteasomes are catalytic centers of the ubiquitin-proteasome system, which is the major protein degradation pathway in eukaryotic cells; they are primarily responsible for the degradation of signal molecules, tumor suppressors, cyclins and apoptosis inhibitors and serve an important role in tumor cell proliferation and apoptosis. The present study investigated the pro-apoptotic effect of corilagin on glioma U251 cells and confirmed that decreased proteasome activity and expression levels serve an important role in corilagin-induced U251 cell apoptosis.

Opa-Interacting Protein 5 Expression in Human Glioma Tissues Is Essential to the Biological Function of U251 Human Malignant Glioma Cells.

Opa-interacting protein 5 (OIP5) is a member of the cancer-testis antigen (CTA) family that elicits a spontaneous antitumor immune response. The failure of current immunotherapies for glioma has prompted the search for novel biomarkers that may be utilized as therapeutic targets. This study aimed to investigate whether OIP5 serves as a target for malignant glioma immunotherapy. Glioma specimens from 53 adult patients were evaluated for OIP5 expression by immunohistochemical (IHC) staining, and the correlation of OIP5 expression with World Health Organization (WHO) tumor grade was analyzed. Endogenous expression of OIP5 in glioma cell lines was determined via real-time polymerase chain reaction (RT-PCR). Using lentiviral siOIP5, the effect of OIP5 gene knockdown on proliferation, cell cycle, and apoptosis in U251 glioma cells was studied. The results show that OIP5 is overexpressed in glioma tissues and is correlated with WHO tumor grade (P < 0.001). However, OIP5 protein expression is barely detectable in normal adult brain tissues. MTT assays and analysis using the Celigo Imaging Cytometry System reveal that the silencing of OIP5 inhibits U251 cell growth. Cell cycle assays and Annexin V staining show that OIP5 silencing disrupts the balance of the cell cycle and increases U251 cell death. These results indicate that OIP5 is upregulated in malignant glioma specimens but barely detected in normal brain tissues. OIP5 knockdown inhibits the biological function of glioma cells, reinforcing that OIP5 may serve as an immunotherapeutic target for malignant glioma.

Effect of hnRNPA2/B1 on the proliferation and apoptosis of glioma U251 cells via the regulation of AKT and STAT3 pathways.

Glioma is the most common malignant tumor in the human central nervous system. Although heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) was previously presumed to be a tumor-promoting gene, the relationship between hnRNPA2/B1 and glioma is unclear. Targeting hnRNPA2/B1 interference in glioma cells can significantly inhibit proliferation and increase apoptosis of human glioma cells in vitro. In a tumor xenograft model, knockdown of hnRNPA2/B1 suppressed tumor growth in glioma cells in vivo. In terms of a mechanism, the knockdown of hnRNPA2/B1 led to inactivation of the AKT and STAT3 signaling pathways, which ultimately reduced the expression of B-cell lymphoma-2 (Bcl-2), CyclinD1 and proliferating cell nuclear antigen (PCNA). Collectively, these data suggest that the inhibition of hnRNPA2/B1 can reduce the growth of gliomas through STAT3 and AKT signaling pathways, and this inhibition is expected to be a therapeutic target for gliomas.

Increased RLIP76 expression in IDH1 wild‑type glioblastoma multiforme is associated with worse prognosis.

Mutation of the isocitrate dehydrogenase (IDH) gene is regarded a novel indicator for the prognosis of patients with glioma. However, the role of the IDH1 gene mutations in carcinogenesis and the mechanisms underlying their function in glioblastoma multiforme (GBM) remain unknown. The present study aimed to determine whether the association of RLIP76 with the different IDH1 mutational status could serve as a putative biomarker for improving disease prognosis. Quantitative PCR, western blotting and immunohistochemical staining assays were used to investigate the expression levels of RLIP76 in 124 patients with GBM with different IDH1 mutational status. In addition, the association between RLIP76 expression, IDH1 mutational status and clinicopathological characteristics was investigated. The effects of RLIP76 expression and IDH1 mutational status on cell proliferation, cell apoptosis, and cell signaling were examined by Cell Counting Kit-8, flow cytometry and western blot assays, respectively. The data demonstrated that IDH1 wild-type (IDH1Wt) patients with low RLIP76 expression exhibited improved overall and progression-free survival. This effect was not observed in patients with IDH1 mutant (IDH1Mut) GBM. In vitro assays demonstrated that knockdown of IDH1 or overexpression of the IDH1 R132H mutation suppressed cell proliferation and promoted cell apoptosis in U87 glioma cells. Mechanistic studies further indicated that although the IDH1 R132H mutant phenotype exhibited similar antitumor effects on GBM cells as those observed with the IDH1 knockdown, it acted via a different mechanism with regard to the regulation of the apoptosis signaling pathway. IDH1 R132H mutant cells promoted p53-induced apoptosis, while the IDH1 knockdown inhibited the RLIP76-dependent apoptotic pathway in glioma cells. The findings of the present study provided insight to the contribution of IDH1 mutation in the development of GBM and indicated that RLIP76 may be considered as a prognostic biomarker of IDH1Wt GBM.

Rnd3 induces apoptosis of glioma cells through promoting the ubiquitination of p65

Objective Explore the role and mechanism of Rnd3 during the process of apoptosis in glioma cells. Methods Rnd3 plasmid was transfected to regulate the expression of Rnd3 in U251 glioma cells, Myc plasmid was transfected as control. Flow cytometry was used to detect apoptosis. We detected the expression of Rnd3, p65, B cell lymphoma/leukemia-2 (bcl-2) and cleaved-Caspase-3 in glioma cells by Western blotting. Flag-p65 plasmid was used to restore p65 expression, then detected the expression of bcl-2 and cleaved-Caspase-3 by Western blotting, Flag plasmid was transfected as control. MG-132 was applied to inhibit the ubiquitination of protein after Rnd3 plasmid transfected, then detected the expression of p65 by Western blotting. Results After transfection with eukaryotic expression plasmid, the expression of Rnd3 protein in Myc-Rnd3 group was significantly higher than that in Myc group (P<0.05); the apoptotic rate of Myc-Rnd3 group increased from 7.28% (5.36±0.10, 1.92±0.12) in U251-Myc group to 19.93% (13.51±0.32, 6.42±0.21) in U251-Myc-Rnd3 group, and the apoptotic rate was significantly higher than that in Myc group (P<0.05); compared with U251-Myc group, the expression of p65 protein in U251-Myc-Rnd3 group decreased, and the expression of Rnd3 in U251-Myc group decreased. The expression of p65 was down-regulated, which resulted in the down-regulation of bcl-2 and the up-regulation of cleaved-Caspase-3 (P<0.05). After reversing the function of p65 protein (p65+ , Rnd3-), the expression of p65 was increased (P<0.05), the expression of bcl-2 was increased (P<0.05), the expression of cleaved-Caspase-3 was decreased (P<0.05); (p65+ , Rnd3+ ) the expression of p65+ , Rnd3-) was higher than that of (p65+ , Rnd3-). The expression of p65-, Rnd3-) was higher than that of (p65-, Rnd3-) Rnd3 (P<0.05), p65 expression was lower (P<0.05), bcl-2 expression was lower (P<0.05), cleaved-Caspase-3 expression was higher (P<0.05), and MG-132 inhibited ubiquitination pathway of intracellular protein, which showed that the expression of p65 decreased with the increase of Rnd3 protein expression. In MG-132 treatment group, with the increase of Rnd3 protein expression, there was no significant difference in p65 protein expression. Conclusion Rnd3 induces apoptosis of U251 glioma cells may through promoting the ubiquitination of p65. Key words: Rnd3; p65; Glioma; Ubiquitination

Bone marrow mesenchymal stem cells suppress growth and promote the apoptosis of glioma U251 cells through downregulation of the PI3K/AKT signaling pathway

Mesenchymal stem cells (MSCs), with the capacity for self-renewal and differentiation into multiple cell types, exhibit the property of homing towards tumor sites and immunosuppression and have been used as tumor-tropic vectors for tumor therapy. However, few studies have investigated the underlying molecular mechanisms that link MSCs to targeted tumor cells. In this study, we elucidated the inhibitory effects and mechanisms of human bone marrow mesenchymal stem cells (hBMSCs) on human glioma U251 cells using a co-culture system in vitro. The anti-tumor activity of co-cultured hBMSCs was assessed by morphological changes, the MTT assay, and Hoechst 33258 staining. Cell apoptosis and cell cycle distribution were evaluated by flow cytometry. Cell migration and invasion were evaluated using a 24-well Transwell chamber. A proteomics approach was used to identify differentially expressed proteins after hBMSCs treatment in U251 cells, and quantitative polymerase chain reaction was used to validate the results. Bioinformatics analyses were also implemented to better understand the identified proteins, and Western blotting analyses were used to analyze the associated proteins. The results showed that hBMSCs could inhibit cell proliferation and induce cell cycle arrest in the G1 phase, resulting in apoptosis of U251 cells. Transwell and Matrigel invasion assays showed that hBMSCs reduced the migration and invasion of U251 cells. Using proteomics, 11 differentially expressed proteins were identified and observed. Bioinformatics analyses indicated that the identified proteins participated in several biological processes and exhibited various molecular functions, mainly related to the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. Moreover, hBMSCs regulated changes in proteins linked to cell apoptosis and cell cycle progression and inhibited the epithelial-mesenchymal transition (EMT)-like and PI3K/AKT pathway. Taken together, the findings in our study suggest that hBMSCs inhibit U251 cells proliferation and the EMT-like by downregulating the PI3K/AKT signaling pathway, which indicates that hBMSCs have a potential antitumor characteristics and should be further explored in future glioma therapy.

Effects of curcumin on glioma cell U87 and its effects on cysteinyl aspartate-specific protease-3, cysteinyl aspartate-specific protease-9, B cell lymphoma/leukemia-2 and B cell lymphoma/leukemia-2 associated X protein

Objective To explore the effects of curcumin on glioma cell U87 and its effects on cysteinyl aspartate-specific protease (Caspase)-3, Caspase-9, B cell lymphoma/leukemia-2 (bcl-2) and bcl-2 associated X protein (bax). Methods The U87 cells were treated with curcumin solution. Methyl thiazol tetrazolium (MTT) assay was used to measure cell proliferation. Flow cytometry was used to examine cell apoptosis. Enzyme-linked immunosorbent assay was used to detect Caspase 3 and Caspase 8 levels in cell culture medium. Western blotting was used to detect the expression of bcl-2 and bax proteins. Results The proliferative rate of U87 cells at 24 h and 48 h in low dose curcumin group [(8.48±1.19)% and (11.89±1.39)%] and high dose curcumin group [(16.04±1.58)% and (25.63±1.66)%] was significantly lower than that in blank control group [(1.47±0.10)% and (1.39±0.19)%] (P<0.05). The proliferative rate of U87 cells at 24 h and 48 h cells in high dose curcumin group was significantly lower than that in low dose curcumin group (P<0.05). The proliferative rate of U87 cells at 48 h in high dose curcumin group and low dose curcumin group was significantly lower than that at 24 h in the corresponding groups (P<0.05). The apoptosis rate of U87 cells in low dose curcumin group and high dose curcumin group [(9.53±1.21)% and (20.52±2.35)%] was significantly higher than that in blank control group (2.22±0.14)% (P<0.05). The apoptosis rate of U87 cells in high dose curcumin group was significantly higher than that in low dose curcumin group (P<0.05). Caspase-3 and Casepase-8 levels in cell culture supernatant of low dose curcumin group [(156.35±29.63) and (158.35±25.36) ng/ml], and high dose curcumin group [(252.52±35.21) and (225.61±30.66) ng/ml] were significantly higher than those of blank control group [(50.63±10.36) and (78.23±11.35) ng/ml, P<0.05]. Caspase-3 and Casepase-8 levels in cell culture supernatant of high dose curcumin group were significantly higher than those of low dose curcumin group (P<0.05). The expression level of bcl-2 protein in U87 cells of low dose curcumin group (0.61±0.10 and 0.67±0.09) and high dose curcumin group (0.42±0.08 and 0.88±0.11) was significantly lower than that of blank control group (0.88±0.16 and 0.33±0.04), and that of bax protein in low dose curcumin group and high dose curcumin group was significantly higher than that of blank control group (P<0.05). The expression level of bcl-2 protein in U87 cells of high dose curcumin group was significantly higher than that of low dose curcumin group, and that of bax protein was significantly higher in low dose curcumin group and high dose curcumin group than that in blank control group (P<0.05). Conclusion Curcumin can inhibit the proliferation and promote the apoptosis of U87 glioma cells by promoting the expression of Caspase-3 and Caspase-8 and altering the expression of bcl-2 and bax. Key words: Curcumin; Glioma; Cysteinyl aspartate-specific protease-3; Cysteinyl aspartate-specific protease-9; B cell lymphoma/leukemia-2; B cell lymphoma/leukemia-2 associated X protein

Effects of norcantharidin on proliferation and apoptosis of glioma cells via mitogen-activated protein kinase/extracellular regulated protein kinase signaling pathway

Objective To investigate the effects of norcantharidin (NCTD) on the proliferation and apoptosis on glioma cells. Methods Glioma U87 cells were treated with various concentrations of NCTD (25, 50, 100, 200 μmol/L) for 24, 48 h. The effects of NCTD on the proliferation and apoptosis of glioma cells were measured by methyl thiazol tetrazolium (MTT) assay and flow cytometry. The Mitogen-activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) signaling pathway changes were detected by Western blotting. Results The results showed that NCTD effectively inhibited cell growth and induced apoptosis in U87 cells in a time- and dose-dependent manner. After incubation with NCTD for 24 h and 48 h, the average half maximal inhibitory concentration (IC50) was 147.7 μmol/L and 62.5 μmol/L, respectively. It could significantly inhibit proliferation at 24 h and 48 h (F=30.234, 42.106, P=0.000). As compared with the control group [(1.2±0.9)%], the cell apoptosis rate induced by 30, 50 μmol/L of NCTD for 10 h was (6.8±1.2)% and (17.2±2.0)%, respectively, which indicated that NCTD could induce apoptosis of cells in a dose-dependent manner (F=405.761, P=0.000). Western blotting results revealed that the phospho-MEK and phospho-ERK were down-regulated significantly in NCTD group as compared with control group (P=0.007). Conclusion Our findings demonstrated that NCTD inhibited proliferation and induced apoptosis of U87 glioma cells through MAPK/ERK signaling pathway. Key words: Glioma; Norcantharidin; Mitogen-activated protein kinase/extracellular regulated protein kinase signaling pathway; Apoptosis

The experimental research on anti-glioma with temozolomide, all-trans retinoic acid and radiotherapy

Objective To investigate whether the collaborative application of temozolomide (TMZ), all-trans retinoic acid (ATRA) and radiotherapy can induce the apoptosis of U251 glioma cells and the molecular mechanism. Methods We detected the cell apoptosis rate of control group (100 μl nutrient solution), radiotherapy group (radiation dosage of 8 Gy), medicine group (TMZ 200 μmol/L+ ATRA 20 μmol/L) and combined group (radiation dosage of 8 Gy+ TMZ 200 μmol/L+ ATRA 20 μmol/L) by flow cytometry method. The protein expression of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) was detected by Western blotting. Results The apoptosis rate was (9.96±3.78)%, (37.35±5.36)%, (59.89±7.15)% and (69.35±8.25)% in control group, radiotherapy group, medicine group and combined group respectively. The apoptosis rate in combined group was higher than that in other groupsThese differences were more obvious (χ2=7 471.175, P=0.000; χ2=2 057.235, P=0.000; χ2=195.717, P=0.000). Combined group can up-regulate the protein expression of p38MAPK in contrast to control guoup, radiation group and medicine group. These differences were more obvious (t=46.000, P=0.000; t=10.150, P=0.000; t=4.266, P=0.013). Combined group can down-regulate the protein expression of NF-κB in contrast to control guoup, radiation group and medicine group. These differences were more obvious (t=36.380, P=0.000; t=30.090, P=0.000; t=68.620, P=0.000). Conclusion The combined use of TMZ, ATRA and radiotherapy can induce the apoptosis of U251 glioma cells probably by the upregulation of p38MAPK and downregulation of NF-κB. Key words: Glioma; Apoptosis; Temozolomide; All-trans retinoic acid; Radiotherapy; p38 mitogen-activated protein kinase; Nuclear factor-κB

Underlying mechanism of the photodynamic activity of hematoporphyrin‑induced apoptosis in U87 glioma cells.

Photodynamic therapy (PDT) is a relatively novel type of tumor therapy method with low toxicity and limited side‑effects. The aim of the present study was to investigate the underlying mechanism and potential microRNAs (miRNAs) involved in the treatment of glioma by PDT with hematoporphyrin, a clinical photosensitizer. The photodynamic activity of hematoporphyrin on the cell viability and apoptosis of gliomas was investigated by MTT, and flow cytometry and fluorescence microscopy, respectively. Alterations in singlet oxygen and mitochondrial membrane potential were detected. The differentially expressed miRNAs and proteins were evaluated by miRNA gene chip and apoptosis‑associated protein chip, respectively. The results demonstrated that cell viability significantly decreased with hematoporphyrin concentration. PDT with hematoporphyrin significantly increased cell apoptosis at a later stage, induced the content of reactive oxygen species (ROS) and decreased the mitochondrial membrane potential, indicating that PDT with hematoporphyrin inhibited cell growth via induction of radical oxygen, decreased the mitochondrial membrane potential and induced apoptosis. The upregulated miRNAs, including hsa‑miR‑7641, hsa‑miR‑9500, hsa‑miR‑4459, hsa‑miR‑21‑5p, hsa‑miR‑663a and hsa‑miR‑205‑5p may be important in PDT‑induced cell apoptosis in glioma. Transporter1, ATP binding cassette subfamilyB member‑and nuclear factor‑κB‑mediated apoptosis signaling pathways were the most significant pathways. Thus, the current study presents PDT as a potential therapeutic approach for the treatment of malignant glioma, and identified miRNAs for the molecular design and development of a third‑generation photosensitizer (PS).

The Role of miR-330-3p/PKC-α Signaling Pathway in Low-Dose Endothelial-Monocyte Activating Polypeptide-II Increasing the Permeability of Blood-Tumor Barrier.

This study was performed to determine whether EMAP II increases the permeability of the blood-tumor barrier (BTB) by affecting the expression of miR-330-3p as well as its possible mechanisms. We determined the over-expression of miR-330-3p in glioma microvascular endothelial cells (GECs) by Real-time PCR. Endothelial monocyte-activating polypeptide-II (EMAP-II) significantly decreased the expression of miR-330-3p in GECs. Pre-miR-330-3p markedly decreased the permeability of BTB and increased the expression of tight junction (TJ) related proteins ZO-1, occludin and claudin-5, however, anti-miR-330-3p had the opposite effects. Anti-miR-330-3p could enhance the effect of EMAP-II on increasing the permeability of BTB, however, pre-miR-330-3p partly reversed the effect of EMAP-II on that. Similarly, anti-miR-330-3p improved the effects of EMAP-II on increasing the expression levels of PKC-α and p-PKC-α in GECs and pre-miR-330-3p partly reversed the effects. MiR-330-3p could target bind to the 3′UTR of PKC-α. The results of in vivo experiments were similar to those of in vitro experiments. These suggested that EMAP-II could increase the permeability of BTB through inhibiting miR-330-3p which target negative regulation of PKC-α. Pre-miR-330-3p and PKC-α inhibitor decreased the BTB permeability and up-regulated the expression levels of ZO-1, occludin and claudin-5 while anti-miR-330-3p and PKC-α activator brought the reverse effects. Compared with EMAP-II, anti-miR-330-3p and PKC-α activator alone, the combination of the three combinations significantly increased the BTB permeability. EMAP-II combined with anti-miR-330-3p and PKCα activator could enhance the DOX’s effects on inhibiting the cell viabilities and increasing the apoptosis of U87 glioma cells. Our studies suggest that low-dose EMAP-II up-regulates the expression of PKC-α and increases the activity of PKC-α by inhibiting the expression of miR-330-3p, reduces the expression of ZO-1, occludin and claudin-5, and thereby increasing the permeability of BTB. The results can provide a new strategy for the comprehensive treatment of glioma.

Wnt/β-catenin signaling pathway inhibits the proliferation and apoptosis of U87 glioma cells via different mechanisms.

The Wnt signaling pathway is necessary for the development of the central nervous system and is associated with tumorigenesis in various cancers. However, the mechanism of the Wnt signaling pathway in glioma cells has yet to be elucidated. Small-molecule Wnt modulators such as ICG-001 and AZD2858 were used to inhibit and stimulate the Wnt/β-catenin signaling pathway. Techniques including cell proliferation assay, colony formation assay, Matrigel cell invasion assay, cell cycle assay and Genechip microarray were used. Gene Ontology Enrichment Analysis and Gene Set Enrichment Analysis have enriched many biological processes and signaling pathways. Both the inhibiting and stimulating Wnt/β-catenin signaling pathways could influence the cell cycle, moreover, reduce the proliferation and survival of U87 glioma cells. However, Affymetrix expression microarray indicated that biological processes and networks of signaling pathways between stimulating and inhibiting the Wnt/β-catenin signaling pathway largely differ. We propose that Wnt/β-catenin signaling pathway might prove to be a valuable therapeutic target for glioma.

The influence of simulated microgravity on proliferation and apoptosis in U251 glioma cells.

Several studies have indicated that microgravity can influence cellular progression, proliferation, and apoptosis in tumor cell lines. In this study, we observed that simulated microgravity inhibited proliferation and induced apoptosis in U251 malignant glioma (U251MG) cells. Furthermore, expression of the apoptosis-associated proteins, p21 and insulin-like growth factor binding protein-2 (IGFBP-2), was upregulated and downregulated, respectively, following exposure to simulated microgravity. These findings indicate that simulated microgravity inhibits proliferation while inducing apoptosis of U251MG cells. The associated effects appear to be mediated by inhibition of IGFBP-2 expression and stimulation of p21 expression. This suggests that simulated microgravity might represent a promising method to discover new targets for glioma therapeutic strategy.

Angelica polysaccharides inhibit the growth and promote the apoptosis of U251 glioma cells in vitro and in vivo

BackgroundAngelica sinensis (Oliv) Diels (Apiaceae) is a traditional medicine that has been used for more than 2000 years in China. It exhibits various therapeutic effects including neuroprotective, anti-oxidant, anti-inflammatory, and immunomodulatory activities. Angelica polysaccharides (APs), bioactive constituents of Angelica have been shown to be responsible for these effects; however, the utility of APs for the treatment of glioma and their mechanism of action remain to be elucidated. PurposeIn this study, we investigated the inhibitory effects of APs on a glioma cell line and their molecular mechanism of action. Study designU251 cells were utilized to confirm the effects of APs on glioma. MethodsThe human glioblastoma cell line U251 was utilized for both in vitro and in vivo models, in which we tested the effects of APs. Flow cytometry, gene expression analysis, western blotting, and MTT assays were used to elucidate the effects of APs on cell proliferation, cell cycle, and apoptosis. ResultsThe results demonstrated that APs significantly inhibited the growth and proliferation of U251 cells and induced their apoptosis. Furthermore, APs effectively reduced the expression of several cell cycle regulators: cyclins D1, B, and E. The apoptosis suppressor protein Bcl-2 was also downregulated, and the expression of pro-apoptotic proteins Bax and cleaved-caspase-3 increased. Additionally, APs inhibited the transforming growth factor (TGF)-β signaling pathway and stimulated the expression of E-cadherin, thus prohibiting cell growth. ConclusionIn conclusion, the results indicate that APs attenuate the tumorigenicity of glioma cells and promote their apoptosis by suppressing the TGF-β signaling pathway. The present study therefore provides evidence of the inhibitory effects of APs against glioma progression, and proposes their potential application as alternative therapeutic agents for glioma.

Lentivirus‑mediated knockdown of chondroitin polymerizing factor inhibits glioma cell growth invitro.

Glioma is the most common primary tumor in the central nervous system, characterized by rapid progression, aggressive behavior, frequent recurrence and poor prognosis. In the present study we demonstrated that chondroitin polymerizing factor(CHPF) is highly expressed in human glioma tissues and 4glioma cell lines. To explore the role of CHPF in glioma, a lentiviral vector expressing CHPF shRNA was constructed and transfected into the glioma U251 cells, which stably downregulated the expression levels of the CHPF gene in U251 cells invitro. U251 cell proliferation inhibition rates were determined by MTT assay. The effect of survivin shRNA on U251 cell cycle distribution and cell apoptosis was determined by flow cytometry. Compared to the shRNA‑Ctrl group of cells, the shRNA-CHPF group of cells exhibited decreased proliferation and a significant increase in the proportion of cells in the G0/G1phase. In addition, we found that knockdown of the expression of CHPF increased apoptosis in glioma U251 cells. Therefore, our results confirmed that CHPF promotes growth and inhibits apoptosis in glioma U251 cells. Thus, by invivo and invitro data, the present study suggests that CHPF could be a new potential therapeutic target for glioma.