Human Brain Glioma Cell Research Articles

Construction of a novel blood brain barrier-glioma microfluidic chip model: Applications in the evaluation of permeability and anti-glioma activity of traditional Chinese medicine components

Since most anti-glioma drug candidates hardly permeate through the blood-brain barrier (BBB), preclinical models that can integrate the complexity of the tumor microenvironment and the structure and function of the BBB is urgently needed for the treatment of glioma. Herein, we constructed an in vitro BBB-glioma microfluidic chip model lined by primary human brain microvascular endothelial cells, pericytes, astrocytes and glioma cells, which could recapitulate the high level of barrier function of the in vivo human BBB and glioma microenvironment. The BBB unit in BBB-glioma microfluidic chip (BBB–U251 chip) displayed selective permeability to fluorescein isothiocyanate isomer-dextran (FITC-dextran) with different molecular weights and three model drugs with different permeability behavior across BBB, which indicated that this glioma model included a functional barrier. Six potential anti-glioma components in traditional Chinese medicine (TCM) were delivered into the blood channel and the permeated amount was quantified by high-performance liquid chromatography combined with ultraviolet (HPLC-UV). The permeated drugs then directly acted on 3D cultured glioma cells (U251) to evaluate the drug efficacy. The results of permeability coefficients of drugs showed that the data were closer to the in vivo data of traditional Transwell model. The effect of the drugs on U251 cells in the BBB-U251 chip was significantly lower due to the existence of BBB. Drug responses on glioma demonstrated the necessity to take BBB into account during the development of anti-glioma new drugs. Therefore, this 3D glioma microfluidic models integrating the BBB functionality can be a useful platform for screening the anticancer drug for brain tumors.

Allyl Isothiocyanate (AITC) Induces Apoptotic Cell Death In Vitro and Exhibits Anti-Tumor Activity in a Human Glioblastoma GBM8401/luc2 Model.

Some clinically used anti-cancer drugs are obtained from natural products. Allyl isothiocyanate (AITC), a plant-derived compound abundant in cruciferous vegetables, has been shown to possess an anti-cancer ability in human cancer cell lines in vitro, including human brain glioma cells. However, the anti-cancer effects of AITC in human glioblastoma (GBM) cells in vivo have not yet been examined. In the present study, we used GBM8401/luc2 human glioblastoma cells and a GBM8401/luc2-cell-bearing animal model to identify the treatment efficacy of AITC. Here, we confirm that AITC reduced total cell viability and induced cell apoptosis in GBM8401/luc2 cells in vitro. Furthermore, Western blotting also showed that AITC induced apoptotic cell death through decreased the anti-apoptotic protein BCL-2, MCL-1 expression, increased the pro-apoptotic protein BAX expression, and promoted the activities of caspase-3, -8, and -9. Therefore, we further investigated the anti-tumor effects of AITC on human GBM8401/luc2 cell xenograft mice. The human glioblastoma GBM8401/luc2 cancer cells were subcutaneously injected into the right flank of BALB/c nude mice to generate glioblastoma xenograft mice. The animals were randomly divided into three groups: group I was treated without AITC (control); group II with 0.1 mg/day of AITC; and group III with 0.2 mg/day of AITC every 3 days for 27 days. Bodyweight, and tumor volume (size) were recorded every 3 days. Tumors exhibiting Luc2 intensity were measured, and we quantified intensity using Living Image software on days 0, 12, and 24. After treatment, tumor weight from each mouse was recorded. Tumor tissues were examined for histopathological changes using H&E staining, and we analyzed the protein levels via immunohistochemical analysis. Our results indicate that AITC significantly inhibited tumor growth at both doses of AITC due to the reduction in tumor size and weight. H&E histopathology analysis of heart, liver, spleen, and kidney samples revealed that AITC did not significantly induce toxicity. Body weight did not show significant changes in any experiment group. AITC significantly downregulated the protein expression levels of MCL-1, XIAP, MMP-9, and VEGF; however, it increased apoptosis-associated proteins, such as cleaved caspase-3, -8, and -9, in the tumor tissues compared with the control group. Based on these observations, AITC exhibits potent anti-cancer activity in the human glioblastoma cell xenograft model via inhibiting tumor cell proliferation and the induction of cell apoptosis. AITC may be a potential anti-GBM cancer drug that could be used in the future.

Bongardia chrysogonum (L.) Spach as a potential medicinal plant against cancer and Alzheimer’s disease management

Background and Aims: Bongardia chrysogonum (L.) Spach (Berberidaceae), an ancient medicinal plant in Eastern Mediterranean countries, is traditionally used for the treatment of epilepsy, hemorrhoids, urinary system infections and gastrointestinal disorders. This study was designed to evaluate potential neuroprotective and anticancer effects of different parts from B. chrysogonum. Material and Methods: Leaf, stem, and tuber parts of the plant were extracted and subjected to determine cholinesterase enzyme inhibitory potentials and anticancer activities. Enzyme inhibition assays were carried out to screen neuroprotective potentials, while the MTT assay was performed for evaluating anticancer activities of the extracts towards human lung carcinoma (A549), breast adenocarcinoma (MCF-7), brain glioma (C6), and non-tumorous human umbilical vein endothelial cell (HUVEC) lines. Results: Among the tested extracts, the highest enzyme inhibitory activity was exhibited by tuber-water extract (83.81±0.33% and 62.14±0.60% inhibition on AChE and BChE at 400 µg/mL p<0.01, respectively), whilst the lowest enzyme inhibition was exerted by the tuber-chloroform extract. Moreover, the tuber part of the plant was found to have the most cytotoxic activity against all the cancer cells, and the best anticancer activity was determined in tuber ethanol extract against MCF-7 cells (IC50= 28.36±0.04, p<0.01). Conclusion: To the best of our knowledge, this research is the first study that assessed the in vitro neuroprotective effect of the aerial parts and tuber extracts of the plant through inhibition of cholinesterase enzymes alongside an anticancer capacity towards human cancer cells. The results revealed that this plant is a good candidate for performing further clinical studies in cancer and neurodegenerative diseases.

Silencing lncRNA GIHCG increases radiosensitivity of glioma cells by up-regulating miR-146a-3p

Objective To investigate the effect of lncRNA GIHCG on the radiosensitivity of glioma cells and its mechanism. Methods The expression levels of GIHCG and miR-146a-3p in human brain normal glial cells HEB and glioma cell lines U251, A172, SHG139 and U87 were quantitatively measured by qRT-PCR assay. U251 and SHG139 cells were used for subsequent experiment. After silencing the expression of GIHCG or overexpressing miR-146a-3p in U251 and SHG139 cells, cell proliferation was detected by MTT assay, cell apoptosis was detected by flow cytometry, cell radiosensitivity was detected by colony formation assay and the expression levels of CDK1, CyclinD1, Bcl-2 and Bax proteins were measured by Western blot. The bioinformatics software predicted the presence of a binding site for GIHCG and miR-146a-3p. Dual luciferase reporter gene assay and qRT-PCR assay were adopted to verify the targeting relationship between GIHCG and miR-146a-3p. Results Compared with HEB cells, the expression of GIHCG was significantly up-regulated in glioma U87, U251, A172 and SHG139 cells (all P<0.05), whereas that of miR-146a-3p was remarkably down-regulated (P<0.05). Silencing GIHCG expression or overexpression of miR-146a-3p significantly decreased the U251 and SHG139 cell survival rate, survival fraction and the expression of CDK1, CyclinD1 and Bcl-2 proteins (all P<0.05), whereas considerably increased the apoptotic rate and expression of Bax protein (both P<0.05). GIHCG performed targeted negative regulation of miR-146a-3p expression in U251 and SHG139 cells and inhibition of miR-146a-3p expression reversed the effect of silencing GIHCG on proliferation, apoptosis and radiosensitivity of glioma cells. Conclusion Silencing GIHCG expression up-regulates the expression of miR-146a-3p, thereby enhancing the radiosensitivity of glioma cells. Key words: Glioma; lncRNA GIHCG; miR-146a-3p gene; Radiosensitivity

Anti-proliferative Activities of Some Bivalent Symmetrical 5-Substituted Hydantoin Derivatives towards Human Brain Glioma U251 Cells (U251) and Human Carcinoma Cells (KB3-1).

Novel bivalent twin-drug type hydantoin derivatives were evaluated in vitro using a human brain glioma cell line (U251) and a human carcinoma cell line (KB3-1). Among the 5-substituted hydantoin derivatives (1a-b and 2a-d) examined in this study, bivalent symmetrical 5-substituted hydantoin derivative 1b showed the highest anti-proliferative activity towards both U251 and KB3-1 cells. The values of anti-proliferative activity (IC50) of this hydantoin derivative against the two cell lines (U251 and KB3-1) were 0.46 and 5.21 µM, respectively. The anti-proliferative activity of all of the compounds except for compounds 2a and 2d against U251 cells was higher than that of cisplatin. Bivalent symmetrical compound 1b had a biphenylmethane linker in the molecule. All of the tested bivalent hydantoin derivatives showed higher activity against U251 cells than against KB3-1 cells. For twin-drug type hydantoin derivatives 2a-d, which have a linear methylene linker in the molecules, it was found that methylene linker length in these molecules have an effect on the anti-proliferative activity against U251 and KB3-1 cells.

Anti-proliferative Activities towards Human Brain Glioma U251 Cells and Human Carcinoma Cells (KB3-1) of Some Twin-Drug Type Bivalent C2-Symmetrical Phenylboronic Acid Derivatives.

Derivatives of C2-symmetrical bivalent phenylboronic acid exhibit several remarkable biological activities such as anti-herpes simplex virus (HSV)-1 and cytotoxic activities against Vero cells and they can reverse the effect of anticancer drugs. Novel symmetrical bivalent molecules were synthesized and their biological activities were evaluated in vitro using a human brain glioma cell line (U251) and a human carcinoma cell line (KB3-1). Among the tested compounds (1a-i), bivalent C2-symmetrical phenylboronic acid derivative 1g showed the highest anti-proliferative activity towards both U251 and KB3-1 cells. The values of 50% anti-proliferative activity (IC50) of this compound against the two cell lines (U251 and KB3-1) were 19.0 and 3.78 µM, respectively. The anti-proliferative activity of compound 1g towards KB3-1 cells was higher than that of cisplatin. The bivalent C2-symmetrical compound 1g had a linear methylene linker in the molecule.

Overexpression of Hsc70 promotes proliferation, migration, and invasion of human glioma cells.

Migration and invasion are often recognized as the main reasons for the high recurrence and death rates of glioma and limit the efficacy of surgery and other antitumor therapies. In this study, we found over activation of heat shock cognate protein 70 (Hsc70) in human glioma specimens, which was closely related to glioma grade. We investigated whether Hsc70 induced the migration and invasion of glioma cells. Wound healing and transwell migration assay were used to determine the migration and invasion ability of human glioma U251 and U87 cells, in which the expression of Hsc70 was knocked down by small interfering RNA. Western blot analysis was performed to determine the expression of FAK-Src signaling in malignant glioma cells. The results showed that Hsc70 deficiency significantly retarded migration and invasion and reduced the phosphorylation of FAK, Src, and Pyk2 in U251 and U87 cells. Overall, our results indicate that the migration and invasion capacity of human brain glioma cells is at least partly induced by Hsc70-dependent activation of FAK-Src signaling.

Effects of miRNA-130a on the proliferation and apoptosis of glioma cell lines

Regulatory ability of micro-ribose nucleic acid-130a (miRNA-130a) in the proliferation and invasive growth of human brain glioma cells and its mechanism were investigated. RT-qPCR was used to analyze expression of miRNA-130a in U-87MG glioma specimens; lipidosome was used to mediate miRNA-130a mimic transfecting glioma cells and the expression of miRNA-130a was detected by using RT-qPCR after transfection; methyl thiazolyl tetrazolium (MTT) assay and flow cytometry (FCM) were adopted to evaluate the changes in biological characteristics of cell growth and proliferation; the migration and invasion abilities of tumor cells were measured through scratch assay and Transwell in vitro cell migration assay. In miRNA-130a mimic-transfected U-87MG cells, RT-qPCR showed that the expression of miRNA-130a was upregulated; MTT assay and FCM revealed that the cell growth was strengthened; scratch assay and Transwell in vitro cell migration assay verified that the migration and invasion abilities of cells were enhanced. In conclusion, the high expression of miRNA-130a can promote growth and invasion, indicating that miRNA-130a can be considered as a candidate target of gene therapy for glioma.

The Long Noncoding RNA TP73-AS1 Interacted with miR-124 to Modulate Glioma Growth by Targeting Inhibitor of Apoptosis-Stimulating Protein of p53.

P73 antisense RNA 1T (non-protein coding), known as TP73-AS1 or PDAM, is a long noncoding RNA (lncRNA), which may regulate apoptosis by regulation of p53-dependent antiapoptotic genes. An abnormal change of TP73-AS1 expression was noticed in cancers. The effects of TP73-AS1 in brain glioma growth and the underlying mechanism remain unclear so far. In this study, the effect of TP73-AS1 in human brain glioma cell lines and clinical tumor samples was detected so as to reveal its role and function. In this study, TP73-AS1 was specifically upregulated in brain glioma cell lines and promoted glioma cell growth through targeting miR-124. TP73-AS1 knocking down suppressed human brain glioma cell proliferation, invasion, and metastasis in vitro. The inhibitory effect of TP73-AS1 knocking down on glioma cell proliferation and invasion could partly be restored by miR-124 inhibition. In addition, miR-124-dependent inhibitor of apoptosis-stimulating protein of p53 (iASPP) regulation was required in TP73-AS1-induced brain glioma cell growth. Data from this study revealed that TP73-AS1 inhibited the brain glioma growth and metastasis as a competing endogenous RNA (ceRNA) through miR-124-dependent iASPP regulation. In conclusion, we regarded TP73-AS1 as an oncogenic lncRNA promoting brain glioma proliferation and metastasis and a potential target for human brain glioma treatment.

Effect of Twist on proliferation and apoptosis of human brain glioma cells and underlying mechanism

Objective To investigate the effect of Twist on the growth and apoptosis of human brain glioma cells and the underlying mechanism. Methods Human glioma U251 cells were transfected with Twist small interfering RNA (siRNA) and siRNA control respectively. The expression levels of Twist mRNA and protein in transfected cells were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting respectively. The cell proliferation was measured by methyl thiazol tetrazolium (MTT) assay, apoptosis was examined by flow cytometry, and the levels of cleaved cysteinyl aspartate specific proteinase 3 (Cleaved Caspase-3), signal transducer and activators of transcription 3 (STAT3) and phosphorylated STAT3 (p-STAT3) proteins were detected by Western blotting. Results In U251 cells transfected with siRNA control, the cell proliferation activity (0.97±0.12, P=0.406), apoptosis rate [(11.92±1.10)%, P=0.788] and Cleaved Caspase-3 (0.23±0.06, P=0.198), STAT3 (0.96±0.12, P=0.165), p-STAT3 (0.15±0.05, P=0.890) and Twist levels in the cells (for mRNA and protein: 0.98±0.07 and 0.86±0.06; P=0.152, 0.671) showed no significant change as compared with the U251 cells without transfection. In U251 cells transfected with Twist siRNA, cell proliferation activity (0.62±0.05, P=0.004) and p-STAT3 levels (0.05±0.01, P=0.010) were decreased, the apoptosis rate [(45.62±4.36)%, P=0.000] and the level of Cleaved Caspase-3 (0.84±0.07, P=0.001) increased as compared with U251 cells without transfection. Conclusion After down-regulation of Twist, the proliferation of human glioma cells decreased and apoptosis increased, which might be related to the inhibition of STAT3 phosphorylation. Key words: Glioma; Signal transducer and activators of transcription 3; Twist; Apoptosis; Proliferation

Human sodium iodide transporter gene-mediated imaging and therapy of mouse glioma, comparison between 188Re and 131I.

Novel treatment options are urgently required for patients with glioma who are not effectively treated through standard therapy. Human sodium iodide symporter (hNIS) is a molecular target of certain tumors types. Compared with 131I, 188Re possesses a higher energy and shorter half-life; therefore, the effects of 188Re and 131I were compared in hNIS-mediated gene imaging and therapy in the present study. Recombinant human brain glioma cell line U87 was transfected with a recombinant lentiviral vector containing hNIS (U87-hNIS). U87-0 cell line transfected with blank lentivirus was prepared as a control. In vitro, the 188Re and 131I uptake of U87-hNIS cells were 21.3-times and 25.9-times that of the control groups, however the excretion rate of the two nuclides was very rapid, and the half-life was only ~4 min. Sodium perchlorate inhibited hNIS-mediated 188Re and 131I uptake to levels observed in the control groups. 188Re and 131I were able to kill U87-hNIS cells selectively, with a survival of only 21.6 and 36.2%, respectively. U87-hNIS nude mice appeared to accumulate 188Re, with a ratio of radioactivity counts between tumor and non-tumor sites of ~13.5 compared with 10.3 of 131I 1 h after radionuclide injection. In contrast with in vitro studies, U87-hNIS cells demonstrated a notable increase in 188Re retention in vivo, even 24 h after 188Re injection. U87-hNIS cells also exhibited increased 131I retention in vivo; however, as the time increased, 131I was rapidly released with the tumor no longer able to be imaged 24 h after 131I injection. Following treatment, U87-hNIS tumors experienced a volume reduction of 24.1%, whereas U87-0 cells demonstrated an increase of 28.8%. 188Re and 131I were revealed to be effective at decreasing tumor volume compared with the control. However, 188Re was significantly more potent compared with 131I (P<0.01). The present study indicated that the U87-hNIS cell line is sufficient to induce specific 188Re and 131I uptake, which may kill cells in vitro and in vivo. 188Re exhibited an increased retention time in vivo compared with 131I, which facilitates the imaging and therapy of U87-hNIS tumors.

The Long Non-Coding RNA TP73-AS1 Interacted With miR-142 to Modulate Brain Glioma Growth Through HMGB1/RAGE Pathway.

P73 antisense RNA 1T (non-protein coding), also known as TP73-AS1 or PDAM, is a long non-coding RNA which may regulate apoptosis via regulation of p53-dependent anti-apoptotic genes. An abnormal change of TP73-AS1 expression was noticed in cancers. The effects of TP73-AS1 in brain glioma growth and the underlying mechanism remain unclear so far. In the present study, TP73-AS1 was specifically upregulated in brain glioma tissues and cell lines, and was associated with poorer prognosis in patients with glioma. TP73-AS1 knocking down suppressed human brain glioma cell proliferation and invasion in vitro, as well as HMGB1 protein. MiR-142 has been reported to play a pivotal role in cancers; here we observed that TP73-AS1 and miR-142 could negatively regulate each other. Results from luciferase assays suggested that TP73-AS1 might compete with HMGB1 for miR-142 binding. Further, HMGB1/RAGE was involved in TP73-AS1/miR-142 regulation of glioma cell proliferation and invasion. In glioma tissues, TP73-AS1 and HMGB1 expression was up-regulated, whereas miR-142 expression was down-regulated. Data from the present study revealed that TP73-AS1 promoted the brain glioma growth and invasion through acting as a competing endogenous RNA (ceRNA) to promote HMGB1 expression by sponging miR-142. In conclusion, we regarded TP73-AS1 as an oncogenic lncRNA promoting brain glioma proliferation and invasion, and a potential target for human brain glioma treatment. J. Cell. Biochem. 119: 3007-3016, 2018. © 2017 Wiley Periodicals, Inc.

Regulation of autophagy inhibition and inflammatory response in glioma by Wnt signaling pathway.

The objective of this study was to investigate the mechanism of the function of Wnt signaling pathway in regulating autophagy and inflammatory response in glioma cells. Human brain glioma cells U118 were selected and divided into three groups: i) the Wnt signaling inhibitor IWR-1 group (the observation group); ii) the PBS negative control group (the PBS group) and iii) the blank control group. After 24 h culture, Wnt5a/β-catenin protein, autophagy marker, microtubule-associated-proteins-1A1B-light-chain-3C (LC-3) II and Beclin I, and inflammatory factors IL-6 and TNF-α protein expression levels were evaluated using western blotting. Compared with both control groups, Wnt5a/β-catenin, IL-6 and TNF-α protein expression levels were significantly lower, and LC-3II and Beclin I protein expression levels were significantly higher in the observation group. In conclusion, Wnt5a/β-catenin signaling pathway regulates autophagy and inflammatory response of glioma cells.

WITHDRAWN: Cold-water extract of Korean Red Ginseng exhibits potent inhibitory effects against cholesteryl ester transfer protein activity and fructose-mediated glycation along with lipid-lowering activity in hyperlipidemic zebrafish

Abstract Background Red ginseng (RG) is widely used to treat complications of diabetes and atherosclerosis because of its antioxidative and anti-inflammatory activities. Methods In order to investigate the antiatherosclerotic and antisenescence activities of water extracts of RG powder, the lyophilized extract was treated to human macrophage, dermal cells, and brain glioma cells, and supplemented to hyperlipidemic zebrafish. Results Aqueous extracts of RG showed potent inhibitory activity against fructose-mediated glycation and protective effect against low-density lipoprotein (LDL) oxidation. RG extracts also showed potent antiatherogenic activities, demonstrated inhibitory activity against cholesteryl ester transfer protein (CETP), and inhibited cellular uptake of oxidized LDL into macrophages. As in vivo test, zebrafish consumed a high cholesterol diet (HCD) [final, 4% in diet (wt/wt)] with or without RG [final, 10% or 20% (wt/wt)]. The 10% and 20% RG groups showed reductions of 5% and 3%, respectively, in weight and height compared to the HCD group. Plasma total cholesterol level was significantly reduced in both the 10% and 20% RG groups, and glutamic–pyruvic transaminase activity was reduced in the 20% RG group compared to the HCD group. Finally, plasma from zebrafish that consumed 20% RG showed the lowest plasma CETP activity with the least hepatic inflammation and fatty liver change. Conclusion Aqueous extracts of RG suppressed the incidence of atherosclerosis via strong antioxidant potential, prevention of apolipoprotein A-I glycation, uptake of oxLDL, and inhibition of CETP activity. Consumption of RG in hyperlipidemic zebrafish model exhibited potent lipid-lowering and atheroprotective activities with the least fatty liver change.

Establishment of human brain glioma cell SHG-44 subcutaneous model in nude mice and observation of growth characteristics

Objective To establish nude mouse model with human brain glioma SHG-44 and understand its growing characteristics in vivo. Methods The 4-week-old male mice were randomly divided into high density cell suspension inoculation group(n=10), low density cell suspension group(n=10), the tumor tissue mass vaccination group(n=10)and the blank control group with normal saline injection(n=10). The SHG-44 human brain glioma cell suspension was injected into the subcutaneous of the nude mice's armpit. The tumor tissue was cut into 1 mm3 after tumor tissue growth and formation, and re-inoculated into the subcutaneous of the new nude mice's armpits. Apart from daily observation, the long and short diameters of tumor were recorded every 5 days after graft. All the mice were sacrificed at 60 days and the tumor tissues were harvested for pathological examination. Results With a longer incubation period and slower growth rate, the tumor formation rate in high density cell suspension inoculation group and low density cell suspension group was lower compared with that in the tumor tissue mass vaccination group.Around day 20, grafted tumor appeared remarkably big((41.51±6.42)mm3) with good morphology.On day 50, the tumor derived from group the tumor tissue mass vaccination group((565.69±123.36)mm3) showed a bigger size in comparison with that from high density cell suspension inoculation group((203.85±104.63)mm3) and low density cell suspension group ((153.02±31.76)mm3, all P<0.05). The tumors in three groups were well defined with a rich vascularity and no apparent invasion was observed.The positive expression of GFAP and S-100 in a large body of tumor cells was observed under optical microscope. Conclusion With a shorter incubation period and faster growth, the mouse tumor models established with tissue pieces from the tumor-bearing mice are much better compared to those with cell suspension. Key words: Nude mice; Animal model; SHG-44 human brain glioma cell

Noninvasive In Vivo Diagnosis of Brain Glioma Using RGD-Decorated Fluorescent Carbonaceous Nanospheres.

Fluorescent carbonaceous nanospheres (CDs) have gained significant attention because of their promising applications, especially in biology and medicine, due to their unique properties. However, the application of CDs in the noninvasive imaging of diseased tissues has been restricted by the poor targeting efficiency of CDs. In this study, CDs were prepared from sucrose and glutamic acid with a particle size of 122.5 nm. Due to quantum confinement in the nanoparticles, CDs exhibited emission from 450 to 600 nm upon excitation at approximately 400 nm. This feature made it possible to use the CDs for low-background bioimaging of deep diseased tissues. RGD, a ligand that can target α(v)β3, which is highly expressed on most tumor and neovascular cells, was decorated onto the CDs after PEGylation. The product, RGD-PEG-CDs, possessed low cytotoxicity, as determined by MTT assay. In vitro, RGD-PEG-CDs targeted U87 (a human brain glioma cell line) cells with a higher cellular uptake intensity than CDs and PEGylated CDs (PEG-CDs), and endosomes were involved in the uptake procedure. The internalization of RGD-PEG-CDs, PEG-CDs and CDs all were primarily mediated by macropinocytosis and a clathrin-mediated pathway, which were energy-dependent. Additionally, the uptake of RGD-PEG-CDs could be significantly inhibited by free RGD, indicating that the uptake was mediated by the receptor of RGD. In vivo, RGD-PEG-CDs accumulated in U87 glioma at high intensity, at values that were 1.67- and 1.64-fold higher than those of PEG-CDs and CDs. Furthermore, RGD-PEG-CDs exhibited good colocalization with neovasculature. In conclusion, RGD-PEG-CDs could be successfully used for noninvasive U87 glioma imaging.

Impact of Mitochondria-Mediated Apoptosis in U251 Cell Cycle Arrest in G1 Stage and Caspase Activation.

BackgroundMost mitochondria-mediated apoptosis has some relevance to the cell cycle, but there is still a lack of investigations about U251 cell cycle in human brain glioma cells. In this study, we aimed to clarify the correlation of mitochondria-mediated apoptosis with the U251 cell cycle and its influence on apoptosis, through observing the impact of mitochondria-mediated apoptosis in U251cell specificity cycle arrest and Caspase activation.Material/MethodsAnnexinV/PI and API were used to label the brain glioma cells for flow cytometry analysis of U251 cell apoptosis and cell cycle. RT-PCR and Western blot were performed to detect Caspase-3 and Caspase-9 activation.ResultsPeripheral blood in stationary phase is not sensitive to apoptosis induction, but U251 cells have obvious apoptosis. Mitochondria-mediated apoptosis mainly occurs in the G1 phase of the cell cycle. Caspase-3 and Caspase-9 mRNAs and proteins expression increased significantly after the cells were treated by mitochondrial apoptosis-related gene Bax induction.ConclusionsMitochondria-mediated apoptosis is related to the U251 cell cycle with specific G1 stage arrest. Caspase activation occurs in the process of cell apoptosis.

Expression of plexinA1 and Ki-67 in tissue of patients with human glioma cells and their clinical significance

Objective To explore expression of plexinA1 and Ki-67 in tissue of human brain glioma cells and their clinical significance. Methods 43 specimens from patients with brain glioma were collected. Immunohistochemical(IHC) staining was used for detecting the expression of tissue plexinA1 and Ki-67 in human glioma cells of 43 cases of patients with brain glioma. The positive expression rate of plexinA1 and Ki-67 among the different pathological grade tissues and their clinical significance were analyzed. So did correlation studies about plexinA1 and Ki-67. Results The positive expression rates of plexinA1 in Ⅰ-Ⅱ grade group(18 cases) and Ⅲ-Ⅳ grade group (25 cases) were 22.22 % (4/18) and 72.00 % (18/25) (P<0.05). The positive expression rates of Ki-67 in Ⅰ-Ⅱ grade group and Ⅲ-Ⅳ grade group were 16.67 % (3/18) and 56.00 % (14/25), respectively (P<0.05). PlxinA1 and Ki-67 expression in the tissue of human brain glioma were positively correlated (r=0.997, P<0.05). Conclusions The positive expression rate of plexinA1 is higher in high malignancy human glioma group than that in low malignancy group which has an important reference value in the estimation of prognosis for human glioma. PlexinA1 and Ki-67 maybe synergism in occurrence and development of glioma. Key words: plexinA1; Ki-67; Glioma; Prognosis

TGF-β/Smad2/3 signal pathway involves in U251 cell proliferation and apoptosis

TGF-β/Smad2/3 signal pathway is regarded as a central regulator in various tumors, but its roles in brain cancer therapy remain unknown. In this study, we identify that the TGF-β/Smad2/3 signal pathway is activated in human brain glioma cells; inhibitor (SB203580) and siRNA against Smad2/3 quickly inhibited the phosphorylation of Smad2 and 3, expression of its major downstream gene, Ki-67, arrested cells in the G2/M phase and induced apoptosis of cells. The findings suggest that TGF-β/Smad2/3 pathway plays a key role in U251 cell growth and metastasis, which suggests its potential role in the molecular therapy of brain cancer.

A protease storm cleaves a cell-cell adhesion molecule in cancer: multiple proteases converge to regulate PTPmu in glioma cells.

Cleavage of the cell-cell adhesion molecule, PTPµ, occurs in human glioblastoma multiforme brain tumor tissue and glioma cell lines. PTPµ cleavage is linked to increased cell motility and growth factor independent survival of glioma cells in vitro. Previously, PTPµ was shown to be cleaved by furin in the endoplasmic reticulum to generate membrane associated E- (extracellular) and P- (phosphatase) subunits, and by ADAMs and the gamma secretase complex at the plasma membrane. We also identified the presence of additional extracellular and intracellular PTPµ fragments in brain tumors. We set out to biochemically analyze PTPµ cleavage in cancer cells. We determined that, in addition to the furin-processed form of PTPµ, a pool of 200 kDa full-length PTPµ exists at the plasma membrane that is cleaved directly by ADAM to generate a larger shed form of the PTPµ extracellular segment. Notably, in glioma cells, full-length PTPµ is also subject to calpain cleavage, which generates novel PTPµ fragments not found in other immortalized cells. We also observed glycosylation and phosphorylation differences in the cancer cells. Our data suggest that an additional serine protease also contributes to PTPµ shedding in glioma cells. We hypothesize that a "protease storm" occurs in cancer cells whereby multiple proteases converge to reduce the presence of cell-cell adhesion molecules at the plasma membrane and to generate protein fragments with unique biological functions. As a consequence, the "protease storm" could promote the migration and invasion of tumor cells.