U87 Malignant Glioma Cells Research Articles

3D Microfluidic System for Evaluating Inhibitory Effect of Chinese Herbal Medicine Oldenlandia diffusa on Human Malignant Glioma Invasion Combined with Network Pharmacology Analysis.

To investigate the anti-invasion efficacy of the ethanol extract of Oldenlandia diffusa Will. (EEOD) on a three-dimensional (3D) human malignant glioma (MG) cell invasion and perfusion model based on microfluidic chip culture and the possible mechanism of action of Oldenlandia diffusa Will. (OD). The comprehensive pharmacodynamic analysis method in this study was based on microfluidic chip 3D cell perfusion culture technology, and the action mechanism of Chinese medicine (CM) on human MG cells was investigated through network pharmacology analysis. First, the components of EEOD were analyzed by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Then, cell viability and apoptosis were assessed to determine the optimum concentration of EEOD for invasion experiments, and two-dimensional (2D) migration and invasion abilities of U87 and U251 MG cells were evaluated using scratch wound and Transwell assays. The possible mechanism underlying the effects of EEOD on glioma was analyzed through a network pharmacology approach. Thirty-five compounds of EEOD were detected by UPLC-Q-TOF/MS. EEOD suppressed the viability of MG cells, promoted their apoptosis, and inhibited their migratory and invasive potentials (all P<0.05). Network pharmacology analysis showed that OD inhibited the invasion of MG cells by directly regulating MAPK and Wnt pathways through MAPK, EGFR, MYC, GSK3B, and other targets. The anti-invasion effect of OD was also found to be related to the indirect regulation of microtubule cytoskeleton organization. ]EEOD could inhibit the invasion of human MG cells, and the anti-invasion mechanism of OD might be regulating MAPK and Wnt signaling pathways and microtubule cytoskeleton organization.

Chimeric Antigen Receptor T Cells With Modified Interleukin-13 Preferentially Recognize IL13Rα2 and Suppress Malignant Glioma: A Preclinical Study.

BackgroundInterleukin-13 receptor α 2 (IL13Rα2) is a promising tumor-directed antigen of malignant glioma (MG). Here, we examine the efficacy and safety of T cells containing a YYB-103 chimeric antigen receptor (CAR) that can preferentially bind to IL13Rα2 on MG cells.MethodsIL13 was modified on the extracellular domain by substitution of amino acids with E13K, R66D, S69D, and R109K and stably transfected into human T cells using a retroviral vector. The in vitro efficacy of YYB-103 CAR T cells was tested in cell lines with differing IL13Rα1 and IL13Rα2 expression. The in vivo efficacy of intracerebroventricular (i.c.v.) and intravenous (i.v.) routes of YYB-103 CAR T-cell administration were tested in orthotopic MG mouse models. Immunohistochemical staining of MG was performed using WHO grade 3/4 surgical specimens from 53 patients. IL13Rα2 expression was quantified by H-score calculated from staining intensity and percentage of positive cells.ResultsBinding affinity assay of YYB-103 verified apparently nil binding to IL13Rα1, which was more selective than previously reported IL13 modification (E13Y). YYB-103 CAR T cells showed selective toxicity toward co-cultured U87MG (IL13Rα1+/IL13Rα2+) cells but not A431 (IL13Rα1+/IL13Rα2−) cells. Consistently, YYB-103 CAR T cells suppressed tumor growth in nude mice receiving orthotopic injection of U87 MG cells. Both i.c.v. and i.v. injections of YYB-103 CAR T cells reduced tumor volume and prolonged overall survival of tumor-bearing mice. The median H-score for IL13Rα2 in patient-derived MG tissue was 5 (mean, 57.5; SD, 87.2; range, 0 to 300).ConclusionThis preclinical study demonstrates the efficacy of IL13Rα2-targeted YYB-103 CAR T cells against MG cells. The use of modified IL13 to construct a CAR facilitated the selective targeting of IL13Rα2-expressing MG cells while sparing IL13Rα1-expressing cells. Notably, YYB-103 CAR T cells exhibited effective blood–brain barrier crossing, suggesting compatibility with i.v. administration rather than intracranial injection. Additionally, the high H-score for IL13Rα2 in glioblastoma, especially in conjunction with the poor prognostic markers of wild-type isocitrate dehydrogenase-1 (IDH-1) and unmethylated O 6-methyl guanine methyl-transferase (MGMT), could be used to determine the eligibility of patients with recurrent glioblastoma for a future clinical trial of YYB-103 CAR T cells.

Paclitaxel inhibits the migration of CD133+ U251 malignant glioma cells by reducing the expression of glycolytic enzymes.

Energy metabolic reprogramming (EMR) allows for the rearrangement of a series of metabolic genes and proteins when tumor cells adapt to their microenvironment. EMR is characterized by changes in the metabolic pattern and metabolic intermediates to meet the needs of tumor cells for their malignant proliferation and infiltrative growth. The present study investigated the role of low-dose paclitaxel (PTX) in changing the expression levels of key genes and proteins during glycolysis in CD133+ U251 glioma cells and explored the relevant regulatory mechanisms of action at the molecular level. CD133 immunomagnetic beads were applied to malignant CD133+ U251 glioma cells, which were then divided into a negative control and an experimental group treated with 1, 2, 4 or 8 µM PTX for 72 h. Cell Counting Kit-8 (CCK-8) was used to measure U251 cell proliferation. RNA and protein were extracted from the malignant glioma cells in all groups to observe changes in the expression levels of key glycolytic enzymes, such as glucose transporter 1 (GLUT1), pyruvate kinase M (PKM) and lactate dehydrogenase A (LDHA), using reverse transcription-quantitative PCR and western blot assays. Transwell migration assays were performed to quantify the effects of PTX solution on U251 cells. CD133+ U251 glioma cells were isolated successfully. CD1133+ cells had a higher rate of proliferation compared with CD1133- cells. In CD1133+ cells treated with PTX, a dose-dependent reduction in the expression levels of the key glycolytic enzymes GLUT1, PKM and LDHA was observed at both the mRNA and protein levels. PTX solution also inhibited cell migration. Differences between the control and experimental groups were statistically significant (P<0.05). Since glycolysis plays an indispensable role in the proliferation and migration of stem cell-like glioma cells, PTX may inhibit tumor cell growth by downregulating the gene and protein expression levels of glycolytic enzymes in CD133+ glioma cells.

Modification of graphene oxide by angiopep-2 enhances anti-glioma efficiency of the nanoscaled delivery system for doxorubicin.

Objective: This study aimed to evaluate the efficacy of the improved nanoscaled delivery system for doxorubicin (Dox) based on angiopep (ANG)-2 modified graphene oxide (GO), the so-called ANG-Dox-GO, in suppressing the growth and and metastasis of glioma cells.Results: Modification of GO by angiopep-2 significantly increased the cellular uptake of Dox. In addition, ANG-Dox-GO treatment of U87 MG cells significantly inhibited cell viability, decreased clone number, cell migration and invasion andinduced cell apoptosis, with superior efficiency over that of Dox-GO and free Dox. Similar results were observed in in vivo experiments—tumor size and weight of glioma xenograft mice were obviously decreased after treatments with ANG-Dox-GO, Dox-GO and Dox, respectively, as compared with control group, and the efficiency was the highest in ANG-Dox-GO, followed by Dox-Go and Dox.Conclusions: ANG-Dox-GO exhibited superior anti-glioma effects over Dox-GO both in vitro and in vivo experiments.Methods: The morphology of ANG-Dox-GO was analyzed by UV visible absorption spectroscopy and atomic force microscopy and its in vitro cellular uptake was measured using confocal imaging analysis. The antitumor effects of GO, unbound Dox, Dox-GO and ANG-Dox-GO were evaluated by MTT assay, colony-forming assay, cell apoptosis assay and Transwell assay in U87 malignant glioma (MG) cells.

Abstract 2778: ADD3-loss in glioblastoma is associated with increased angiogenesis and malignancy

Abstract Introduction: Adducin 3 (ADD3) is one of the major functional component in the cytoskeleton. Dysregulation of ADD3 has been implicated in various diseases including cancer and participate in different signaling transductions. However, little is known about the functional properties of ADD3 in cancer, its role in cancer biology remains unclear and controversial. We aim to delineate the role of ADD3 dysregulation in GBM pathogenesis and to unravel the underlying molecular mechanisms. Methods: Microarray datasets were used to analyze the gene expression of ADD3 in normal tissue and grade II, III, IV gliomas. Gene expression levels were verified by western blot, qPCR and immuno-histological analyses by using human clinical specimens. Luciferase-expressing ADD3 stable knockdown U87 malignant glioma cells were injected to immunocompromised mice subcutaneously and orthotopically for xenograft models. ADD3 stable knockdown U87 cells were co-cultured with human endothelial cells (HUVEC) to assess the functional changes. Findings: Our study demonstrated significant downregulation of ADD3 in human GBM when compared with lower-grade gliomas. Downregulation of ADD3 was only shown in GBM (i.e., WHO grade IV lesion) but not in grade II and grade III tumor. Importantly, we found that ADD3 downregulation is associated with poor clinical outcome in GBM patients. And that ADD3 knock-down enhanced tumor growth in vivo, possibly mediated by promoting angiogenesis. Conclusions: These findings suggest that ADD3 is associated with glioma malignancy, implying that cancer progression may occur along with the loss of ADD3. It may contribute towards tumor growth by enhancing angiogenesis. Citation Format: Mei-Yee Kiang, Gilberto Ka-Kit Leung. ADD3-loss in glioblastoma is associated with increased angiogenesis and malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2778.

Synergistic Action of Gefitinib and GSK41364A Simultaneously Loaded in Ratiometrically-Engineered Polymeric Nanoparticles for Glioblastoma Multiforme.

Glioblastoma Multiforme is a deadly cancer of glial cells with very low survival rates. Current treatment options are invasive and have serious side effects. Single drug treatments make the tumor refractory after a certain period. Combination therapies have shown improvements in treatment responses against aggressive forms of cancer and are becoming a mainstay in the management of cancer. The purpose of this study is to design a combinatorial treatment regimen by engineering desired ratios of two different small molecule drugs (gefitinib and GSK461364A) in a single carrier that can reduce off-target effects and increase their bioavailability. Synergistic effects were observed with our formulation when optimal ratios of gefitinib and GSK461364A were loaded in poly (lactic-co-glycolic) acid and polyethylene glycol (PLGA-PEG) nanoparticles and tested for efficacy in U87-malignant glioma (U87-MG) cells. Combination nanoparticles proved to be more effective compared to single drug encapsulated nanoparticles, free drug combinations, and the mixture of two single loaded nanoparticles, with statistically significant values at certain ratios and drug concentrations. We also observed drastically reduced clonogenic potential of the cells that were treated with free drugs and nanoparticle combinations in a colony forming assay. From our findings, we conclude that the combination of GSK461364A and higher concentrations of gefitinib when encapsulated in nanoparticles yield synergistic killing of glioma cells. This study could form the basis for designing new combination treatments using nanoparticles to deliver multiple drugs to cancer cells for synergistic effects.

Photodynamic therapy with talaporfin sodium induces dose- and time-dependent apoptotic cell death in malignant meningioma HKBMM cells.

To investigate the effect of photodynamic therapy (PDT) with the talaporfin sodium (mono-L-asparthyl chlorine e6: NPe-6) on human malignant meningioma cell line HKBMM cells in vitro. After incubation with NPe6 for 4 h, cells underwent PDT (diode laser irradiation: 3.4 mW/cm2 and 1 J/cm2. Cell viability was determined in 2 malignant meningioma cell lines (human origin; HKBMM cells and rat origin; KMY-J cells) and human malignant glioma U251 cells with Cell Counting Kit-8 assay. The HKBMM cells were examined for caspase-3 activity, annexin V or propidium iodide (PI) staining, and lactate dehydrogenase leakage. Morphological change was also investigated with phase-contrast microscopy. In human malignant meningioma HKBMM cells, viability showed a dose- and time-dependent decrease. After 24 h of laser irradiation, NPe6 at 20 μg/ml or more induced a significant decrease in cell viability in both HKBMM cells and KMY-J cells, although they more resistance than the malignant glioma cell line U251 cells. Two kinds of morphological change were also observed in the HKBMM cells, shrinkage of the cell body, indicating apoptosis, and swelling of the cell body, indicating necrosis. In addition, both caspase-3 activity and DNA fragmentation, biochemical markers indicative of apoptosis, showed a dose-dependent increase. The percentage of necrotic cells showing positive staining for annexin V or PI was greater than that of apoptotic cells at a high concentration of NPe6. Lactate dehydrogenase leakage, a biochemical marker of necrosis, also showed a marked increase at a high concentration of NPe6. Photodynamic therapy with NPe6 induced dose- and time-dependent apoptosis in human malignant meningioma HKBMM cells. At a high concentration of NPe6, however, it induced necrosis.

Cross-link regulation of precursor N-cadherin and FGFR1 by GDNF increases U251MG cell viability.

Glial cell line-derived neurotrophic factor (GDNF) is considered to be involved in the development of glioma. However, uncovering the underlying mechanism of the proliferation of glioma cells is a challenging work in progress. We have identified the binding of the precursor of N-cadherin (proN-cadherin) and GDNF on the cell membrane in previous studies. In the present study, we observed increased U251 Malignant glioma (U251MG) cell viability by exogenous GDNF (50ng/ml). We also confirmed that the high expression of the proN-cadherin was stimulated by exogenous GDNF. Concurrently, we affirmed that lower expression of proN-cadherin correlated with reduced glioma cell viability. Additionally, we observed glioma cell U251MG viability as the phosphorylation level of FGFR1 at Y653 and Y654 was increased after exogenous GDNF treatment, which led to increased interaction between proN-cadherin and FGFR1 (pY653+Y654). Our experiments presented a new mechanism adopted by GDNF supporting glioma development and indicated a possible therapeutic potential via the inhibition of proN-cadherin/FGFR1 interaction.

BmK CT enhances the sensitivity of temozolomide-induced apoptosis of malignant glioma U251 cells in vitro through blocking the AKT signaling pathway.

Temozolomide (TMZ) is a drug that has been demonstrated to improve the survival time of patients with glioblastoma multiforme (GBM) when administered with concomitant radiotherapy. However, chemoresistance is one of the major obstacles in the treatment of GBM. In the present study, an MTT assay and flow cytometry were used to demonstrate that chlorotoxin-like toxin in the venom of the scorpion Buthus martensii Kirsch (BmK CT) markedly inhibited cell proliferation and induced apoptosis in U251 cells when combined with TMZ. In combination with TMZ, BmK CT exhibited a significant and synergistic anti-tumor effect by inhibiting protein kinase B (AKT) independently and triggering the apoptosis signaling cascade in vitro. Furthermore, BmK CT increased the expression of phosphatase and tensin homolog at the transcriptional level, which is a key negative regulator of the AKT signaling pathway. The results of the present study demonstrated that BmK CT enhanced the sensitivity of TMZ-induced U251 cell apoptosis through the downregulation of phosphorylated AKT levels, suggesting that BmK CT and TMZ combination therapy may be a novel approach for glioma therapy.

Up-regulation of ANKDR49, a poor prognostic factor, regulates cell proliferation of gliomas.

The Ankyrin repeat domain 49 (ANKRD49) is an evolutionarily conserved protein, which is related to mediate protein–protein interaction. However, the function of ANKRD49 in human glioma remains elusive. Mining through The Cancer Genome Atlas (TCGA) database, we found that the expression of ANKRD49 was increased in glioma tissues and that high expression of ANKRD49 was strongly associated with high disease grade and poor overall survival. To investigate the role of ANKRD49 in malignant glioma, lentivirus expressing shRNA targetting ANKRD49 was constructed in U251 and U87 malignant glioma cells. We demonstrated that ANKRD49 knockdown reduced the proliferation rate of U251 and U87 cells. Further mechanism analysis indicated that depletion of ANKRD49 led to the cell-cycle arrest and induced apoptosis in U251 and U87 cells. ANKRD49 knockdown also changed the expression of key effectors that are involved in stress response, cell cycle, and apoptosis, including p-HSP27 (heat shock protein 27), p-Smad2 (SMAD family member 2), p-p53, p-p38, p-MAPK (mitogen-activated protein kinase), p-SAPK/JNK (stress-activated protein kinase/c-jun n-terminal kinase), cleveagated Caspase-7, p-Chk1 (checkpoint kinase 1), and p-eIF2a (eukaryotic translation initiation factor 2a). Taken together, our findings implicate that ANKRD49 promotes the proliferation of human malignant glioma cells. ANKRD49 maybe an attractive target for malignant glioma therapy.

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.

Fibronectin-adherent peripheral blood derived mononuclear cells as Paclitaxel carriers for glioblastoma treatment: An in vitro study

BackgroundGlioblastoma (GBM) represents the most aggressive malignant brain tumor in adults, with a risible median life expectancy despite gold standard treatment. Novel drug-delivery methods have been explored. Here we evaluated the possibility to use mononuclear cells (MCs) belonging to the monocytic-dendritic lineage as drug-carrier. MethodsMCs were obtained from 10 patients harboring a GBM, and from healthy volunteers, considered as controls. GBM tissue was also obtained from patients. MCs were cultured and the adherent population on fibronectin (FN-MCs), after immunocytochemistry and flow cytometry characterization, was loaded with Paclitaxel (FN-MCs-PTX). Antiproliferative and migration activity of FN-MCs-PTX was evaluated in two-dimensional (2D) and three-dimensional (3D) co-culture assays with red fluorescent U87 Malignant Glioma cells and primary GBM cells. Antiangiogenic properties of FN-MCs-PTX were tested on cultures with endothelial cells. ResultsPhenotypical characterization showed a high expression of monocytic-dendritic markers in GBM cells and FN-MCs. FN-MCs demonstrated to effectively uptake PTX and to strongly inhibit GBM growth in vitro (P < 0.01). Moreover, tumor-induced migration of MCs, although partially affected by the PTX cargo, remained statistically significant when compared with unprimed cells and this was confirmed in a 3D Matrigel model (P < 0.01) and in a Trans-well assay (P < 0.01). FN-MCs-PTX also disclosed considerable antiangiogenic properties. DiscussionOur results suggest that the fibronectin-adherent population of MCs isolated from peripheral blood can be an effective tool to inhibit GBM growth. Given the relative facility to obtain such cells and the short time needed for their culture and drug loading this approach may have potential as an adjuvant therapy for GBM.

A hydrogen sulfide probe activates Nrf2, inhibits cancer cell growth and induces cell apoptosis

Application of a probe for hydrogen sulfide, HF-NBD.

Inhibition of PTTG1 expression by microRNA suppresses proliferation and induces apoptosis of malignant glioma cells.

The present study aimed to investigate the role of pituitary tumor-transforming gene 1 (PTTG1) in the proliferation, invasion and apoptosis of human malignant glioma U251 cells. Firstly, 2 microRNAs (miRNAs) targeting PTTG1 messenger (m)RNA were ligated into a pcDNA6.2-GW/EmGFP-miR expression vector. The recombinant plasmids, miRNA-1 and miRNA-2 (miR-2), were transfected into U251 cells using the liposome method. PTTG1 mRNA and protein levels were evaluated using quantitative polymerase chain reaction and western blot analysis. The proliferation and invasion abilities of U251 cells were determined using methylthiazol tetrazolium and Matrigel assays. Flow cytometry analysis with Annexin V/propidium iodide double staining was used to determine the percentage of apoptotic cells. PTTG1 expression was effectively suppressed by miR-2. U251 cell growth was inhibited between 10.7 and 34.7% in the miR-2 group compared with the blank group. The Matrigel assay demonstrated that the percentage of infiltrating U251 cells was significantly lower in the miR-2 group (12.3±1.0%) compared to the blank group (24.7±1.4%; P<0.001) and the negative control group (24.0±2.0%; P<0.05). A higher percentage of apoptotic U251 cells were observed in the miR-2 group compared with the blank group (53.6 vs. 32.4%) using flow cytometry due to cycle arrests at the G2/M phase. The miR-2-transfected U251 cells were subcutaneously injected into nude mice, and these mice possessed a decreased tumor tissue growth rate and higher percentage of apoptotic cells compared with the blank and negative control groups. In conclusion, PTTG1 gene expression in human malignant glioma U251 cells was effectively suppressed by exogenous miR-2. The downregulation of PTTG1 induced glioma cell apoptosis and cell cycle arrest at the G2/M phase, which inhibited cell proliferation, reverse invasion and infiltration of glioma cells.

Nanostructured lipid carriers, solid lipid nanoparticles, and polymeric nanoparticles: which kind of drug delivery system is better for glioblastoma chemotherapy?

Context: Glioblastoma is a malignant brain tumor originating in the central nervous system. Successfully therapy of this disease required the efficient delivery of therapeutic agents to the tumor cells and tissues. Delivery of anticancer drugs using novel nanocarriers is promising in glioma treatment.Objective: Polymeric nanoparticles (PNPs), solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs) were constructed for the delivery of temozolomide (TMZ). The anti-tumor effects of the three kinds of nanocarriers were compared to provide the optimum choice for gliomatosis cerebri treatment.Methods: TMZ-loaded PNPs (T-PNPs), SLNs (T-SLNs), and NLCs (T-NLCs) were formulated. Their particle size, zeta potential, drug encapsulation efficiency (EE), and drug loading (DL) capacity were evaluated. Anti-tumor efficacies of the three kinds of nanocarriers were evaluated on U87 malignant glioma cells (U87 MG cells) and mice-bearing malignant glioma model.Results: T-NLCs displayed the best anti-tumor activity than other formulations in vivo and in vitro. The most significantly glioma inhibition was observed on NLCs formulations than PNPs and SLNs.Conclusion: This work demonstrates that NLCs can deliver TMZ into U87MG cells more efficiently, with higher inhibition efficacy than PNPs and SLNs. T-NLCs could be an excellent drug delivery system for glioblastoma chemotherapy.

Novel RGD containing, temozolomide-loading nanostructured lipid carriers for glioblastoma multiforme chemotherapy

Context: Glioblastoma multiforme (GBM) is the most common malignant brain tumor originating in the central nervous system. Efficient delivery of therapeutic molecules to the cells and tissues is a difficult challenge.Objective: Arginine-glycine-aspartic acid peptide (RGD)-modified nanostructured lipid carriers (NLCs) were used for the delivery of temozolomide (TMZ) into the GBM to provide a new paradigm in gliomatosis cerebri treatment.Methods: RGD-conjugated polyethylene glycol-b-distearoylphosphatidylethanolamine (PEG-DSPE) was synthesized. RGD containing, TMZ-loaded NLCs (RGD-TMZ/NLCs) were prepared. Their particle size, zeta potential, drug encapsulation efficiency (EE) and drug release behavior were evaluated. In vitro cytotoxicity study of TMZ/NLCs was tested in U87 malignant glioma cells (U87MG cells). In vivo antitumor efficacy of the carriers was evaluated on mice bearing GBM model.Results: The U87MG cells were successfully inhibited by RGD-TMZ/NLCs in vitro. RGD-TMZ/NLCs also displayed the highest antitumor efficacy in vivo than all the other formulations used for comparison.Conclusion: RGD-TMZ/NLCs were efficient in selective delivery of TMZ into U87MG cells, and inhibition efficacy is high. These RGD-modified vectors could be a superior drug delivery nano-system to achieve therapeutic efficacy, and this research could be a new promising strategy for treatment in malignant gliomatosis cerebri.

Vincristine and temozolomide combined chemotherapy for the treatment of glioma: a comparison of solid lipid nanoparticles and nanostructured lipid carriers for dual drugs delivery

Context: Glioma is a common malignant brain tumor originating in the central nervous system. Efficient delivery of therapeutic agents to the cells and tissues is a difficult challenge. Co-delivery of anticancer drugs into the cancer cells or tissues by multifunctional nanocarriers may provide a new paradigm in cancer treatment.Objective: In this study, solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) were constructed for co-delivery of vincristine (VCR) and temozolomide (TMZ) to develop the synergetic therapeutic action of the two drugs. The antitumor effects of these two systems were compared to provide a better choice for gliomatosis cerebri treatment.Methods: VCR- and TMZ-loaded SLNs (VT-SLNs) and NLCs (VT-NLCs) were formulated. Their particle size, zeta potential, drug encapsulation efficiency (EE) and drug loading capacity were evaluated. The single TMZ-loaded SLNs and NLCs were also prepared as contrast. Anti-tumor efficacies of the two kinds of carriers were evaluated on U87 malignant glioma cells and mice bearing malignant glioma model.Results: Significantly better glioma inhibition was observed on NLCs formulations than SLNs, and dual drugs displayed the highest antitumor efficacy in vivo and in vitro than all the other formulations used.Conclusion: VT-NLCs can deliver VCR and TMZ into U87MG cells more efficiently, and inhibition efficacy is higher than VT-SLNs. This dual drugs-loaded NLCs could be an outstanding drug delivery system to achieve excellent therapeutic efficiency for the treatment of malignant gliomatosis cerebri.

Nanostructured lipid carriers based temozolomide and gene co-encapsulated nanomedicine for gliomatosis cerebri combination therapy

Background: Co-delivery of gene and anticancer drug into the same cancer cells or tissues by multifunctional nanocarriers may provide a new paradigm in cancer treatment. In this study, nanostructured lipid carriers (NLCs) were constructed as multifunctional nanomedicine for co-delivery of enhanced green fluorescence protein plasmid (DNA) and temozolomide (TMZ).Methods: TMZ- and DNA-loaded NLCs (TMZ/DNA-NLCs) were prepared. Their particle size, zeta potential, gene-loading capacity (GL) and drug encapsulation efficiency (EE) were evaluated. In vitro cytotoxicity study TMZ/DNA-NLCs was tested in U87 malignant glioma cells (U87 MG cells). In vivo gene transfection and anti-tumor efficacy of the carriers were evaluated on mice bearing malignant glioma model.Results: The optimum TMZ/DNA-NLCs formulations with the particle size of 179 nm and with a +23 mV surface charge; got 91% of GL and 83% of EE. The growth of U87 MG cells in vitro was obviously inhibited. TMZ/DNA-NLCs also displayed the highest gene transfection efficiency and the best antitumor activity than other formulations in vivo.Conclusion: The results demonstrated that TMZ/DNA-NLCs were efficient in selective delivery to malignant glioma cells. Also TMZ/DNA-NLCs transfer both drug and gene to the gliomatosis cerebri, enhance the antitumor capacity and gene transfection efficacy. Thus, TMZ/DNA-NLCs could prove to be a superior co-delivery nanomedicine to achieve therapeutic efficacy and this report could be a new promising strategy for treatment in malignant gliomatosis cerebri.

Study of the biodistribution of fluorescein in glioma-infiltrated mouse brain and histopathological correlation of intraoperative findings in high-grade gliomas resected under fluorescein fluorescence guidance.

Intravenous fluorescein sodium has been used during resection of high-grade gliomas to help the surgeon visualize tumor margins. Several studies have reported improved rates of gross-total resection (GTR) using high doses of fluorescein sodium under white light. The recent introduction of a fluorescein-specific camera that allows for high-quality intraoperative imaging and use of very low dose fluorescein has drawn new attention to this fluorophore. However, the ability of fluorescein to specifically stain glioma cells is not yet well understood. The authors designed an in vitro model to assess fluorescein uptake in normal human astrocytes and U251 malignant glioma cells. An in vivo experiment was also subsequently designed to study fluorescein uptake by intracranial U87 malignant glioma xenografts in male nonobese diabetic/severe combined immunodeficient mice. A genetically induced mouse glioma model was used to adjust for the possible confounding effect of an inflammatory response in the xenograft model. To assess the intraoperative application of this technology, the authors prospectively enrolled 12 patients who underwent fluorescein-guided resection of their high-grade gliomas using low-dose intravenous fluorescein and a microscope-integrated fluorescence module. Intraoperative fluorescent and nonfluorescent specimens at the tumor margins were randomly analyzed for histopathological correlation. The in vitro and in vivo models suggest that fluorescein demarcation of glioma-invaded brain is the result of distribution of fluorescein into the extracellular space, most likely as a result of an abnormal blood-brain barrier. Glioblastoma tumor cell-specific uptake of fluorescein was not observed, and tumor cells appeared to mostly exclude fluorescein. For the 12 patients who underwent resection of their high-grade gliomas, the histopathological analysis of the resected specimens at the tumor margin confirmed the intraoperative fluorescent findings. Fluorescein fluorescence was highly specific (up to 90.9%) while its sensitivity was 82.2%. False negatives occurred due to lack of fluorescence in areas of diffuse, low-density cellular infiltration. Margins of contrast enhancement based on intraoperative MRI-guided StealthStation neuronavigation correlated well with fluorescent tumor margins. GTR of the contrast-enhancing area as guided by the fluorescent signal was achieved in 100% of cases based on postoperative MRI. Fluorescein sodium does not appear to selectively accumulate in astrocytoma cells but in extracellular tumor cell-rich locations, suggesting that fluorescein is a marker for areas of compromised blood-brain barrier within high-grade astrocytoma. Fluorescein fluorescence appears to correlate intraoperatively with the areas of MR enhancement, thus representing a practical tool to help the surgeon achieve GTR of the enhancing tumor regions.

Abstract 4398: MicroRNA-203 enhances radiosensitivity of human malignant glioma cells

Abstract Glioblastoma multiform (GBM) is the most common, lethal and aggressive type of primary brain tumor. Despite the improvement of surgical and chemo/radiotherapeutic techniques, the prognosis for GBM is still poor, with median survival of 9-12 months. The occurrence of radio-resistance is one of the major obstacles to treat the GBM. Aberrant expression of miRNAs has been demonstrated to be involved in the initiation, progression, and metastasis of cancers. miRNA expression profiling studies revealed that a number of miRNA were dysregulated in GBM. Here, we investigated the potential role of miR-203, which is one of the downregulated miRNAs, in the modulation of radio-sensitivity in GBM. Transiently overexpressed miR-203 sensitized U251 malignant glioma cells to radiation concurrent with the downregulation of AKT activity. Overexpression of miR-203 prolonged radiation-induced γH2AX foci formation and downregulation of DNA-dependent protein kinases, suppressed invasion, migration potential and vasculogenic mimicry formation. In contrast, antisense-mediated inhibition of miR-203 expression results in decreased radio-sensitivity and features of Epithelial-Mesenchymal Transition (EMT). Taken together, our findings suggest that the miR-203 could be a useful target for overcoming the radio-resistance of GBM. Citation Format: Yeohyeon Hwang, David J. Lee, Eun Jung Choi, Bong Jun Cho, In-Ah Kim. MicroRNA-203 enhances radiosensitivity of human malignant glioma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4398. doi:10.1158/1538-7445.AM2014-4398