Human U87 Research Articles

Preparation of transferrin-conjugated poly-ε-caprolactone nanoparticles and delivery of paclitaxel to treat glioblastoma across blood–brain barrier

The delivery of anticancer drug to the brain remains a major challenge due to the existence of blood–brain barrier (BBB). Receptor-mediated transcytosis across BBB is a useful way to transport the therapeutics into the brain. In this study, transport of paclitaxel-loaded poly-e-caprolactone (PCL) nanoparticles was aided by tuning the amount of transferrin (Tf) on the surface of nanoparticles. The PCL nanoparticles (NPs) were synthesized using oil-in-emulsion technique and these were further conjugated with Tf of different concentrations. The characterization of PCL and physicochemical properties of the developed nanoparticles were analyzed using different techniques. The developed nanoparticles were spherical in shape with a diameter of ~ 112 nm as determined by scanning electron microscopy and dynamic light scattering techniques. The cytotoxicity study was performed with a MTT assay at 48 and 96 h against human primary glioblastoma cells (U87). Experimental results revealed that the developed Tf-conjugated PCL nanoparticles were biocompatible with cell viability of > 90%. The efficacy of the drug-loaded nanocarrier system for crossing the BBB was assessed using an in vitro model developed with human brain microvascular endothelial cells (HBMEC) and U87 cells. The results suggest that the in vitro cell uptake of the nanoparticles was dependent on optimal concentration of Tf. Among the nanocarriers developed, the PCL-C6-80Tf conjugates showed a significant increase in the fluorescence intensity as compared to other nanocarriers. Finally, it is concluded that the nanoparticles with specific avidity of Tf could effectively cross the BBB and thus, the developed nanocarriers could be a promising strategy for delivering the anticancer drugs into the brain.

Hesperetin Induces Apoptosis in Human Glioblastoma Cells via p38 MAPK Activation

Glioblastoma (GBM) is the most common and aggressive form of malignant brain tumor, with poor prognosis and a lack of effective treatment. Hesperetin, a natural product found in citrus fruits, displayed bioactivities including antioxidant, anti-inflammatory, and anticancer, while its effects on GBM cells were largely unknown. Here, we explored the anticancer effect of hesperetin on human GBM cells in vitro, as well as the underlying signaling mechanisms. By CCK-8 assay and live/dead assay, hesperetin presented significant inhibitory effect on human GBM U-251 and U-87 cell viability. By DAPI staining and Annexin V-FITC/PI assay, apoptotic death was proved to contribute to the cell viability reduction, and it was verified by the increased Bax/Bcl-2 ratio in western blotting results. Furthermore, by cell cycle analysis and western blotting for cyclin B1, CDK1, and p21, hesperetin was found to induce cell-cycle arrest at G2/M phase. For signaling mechanism, the western blotting results showed elevated p38 MAPK activation, and the reduced Bcl-2 and enhanced Bax upon hesperetin treatment were partly reversed by p38 MAPK inhibitor SB203580. In summary, we have discovered hesperetin as a natural product candidate for the treatment of GBM, and that it could induce GBM cell apoptosis via p38 MAPK activation.

Absent in melanoma 2 regulates tumor cell proliferation in glioblastoma multiforme.

Inflammation is a key aspect of glioblastoma multiforme (GBM) although it remains unclear how it contributes to GBM pathogenesis. Inflammasomes are intracellular multi-protein complexes that are involved in innate immunity and are activated by cellular stress, principally in macrophages. This study examined the expression of inflammasome-associated genes in GBM, particularly absent in melanoma 2 (AIM2). Tissue samples from surgically-resected GBM tumors (n = 10) were compared to resected brain specimens from patients with epilepsy (age- and sex-matched Other Disease Controls (ODC, n=5)) by qRT-PCR, western blotting and immunofluorescence. Gene expression studies in human astrocytoma U251 cells were performed and the effects of deleting the absent in melanoma 2 (AIM2) gene using the CRISPR-Cas9 system were analyzed. GBM tissues showed significantly elevated expression of multiple immune (CD3E, CD163, CD68, MX1, ARG1) and inflammasome (AIM2, NLRP1, IL18, CASP1, and IL-33) genes compared to ODC tissues, without induction of IL1B, IFNG or TNFA. An insert-containing AIM2 variant transcript was highly expressed in GBM tissues and in U251 cells. AIM2 immunoreactivity was concentrated in the tumor core in the absence of PCNA immunodetection and showed a predominant 52 kDa immunoreactive band on western blot. Deletion of AIM2 resulted in significantly enhanced proliferation of U251 cells, which also displayed increased resistance to temozolomide treatment. GBM tumors express a distinct profile of inflammasome-associated genes in a tumor-specific manner. AIM2 expression in tumor cells suppressed cell proliferation while also conferring increased susceptibility to contemporary GBM therapy.

Gestational Tissue-Derived Human Mesenchymal Stem Cells Use Distinct Combinations of Bioactive Molecules to Suppress the Proliferation of Human Hepatoblastoma and Colorectal Cancer Cells.

Background Cancer has been considered a serious global health problem and a leading cause of morbidity and mortality worldwide. Despite recent advances in cancer therapy, treatments of advance stage cancers are mostly ineffective resulting in poor survival of patients. Recent evidences suggest that multipotent human mesenchymal stem cells (hMSCs) play important roles in growth and metastasis of several cancers by enhancing their engraftment and inducing tumor neovascularization. However, the effect of hMSCs on cancer cells is still controversial because there are also evidences demonstrating that hMSCs inhibited growth and metastasis of some cancers. Methods In this study, we investigated the effects of bioactive molecules released from bone marrow and gestational tissue-derived hMSCs on the proliferation of various human cancer cells, including C3A, HT29, A549, Saos-2, and U251. We also characterized the hMSC-derived factors that inhibit cancer cell proliferation by protein fractionation and mass spectrometry analysis. Results We herein make a direct comparison and show that the effects of hMSCs on cancer cell proliferation and migration depend on both hMSC sources and cancer cell types and cancer-derived bioactive molecules did not affect the cancer suppressive capacity of hMSCs. Moreover, hMSCs use distinct combination of bioactive molecules to suppress the proliferation of human hepatoblastoma and colorectal cancer cells. Using protein fractionation and mass spectrometry analysis, we have identified several novel hMSC-derived factors that might be able to suppress cancer cell proliferation. Conclusion We believe that the procedure developed in this study could be used to discover other therapeutically useful molecules released by various hMSC sources for a future in vivo study.

Abstract 3608: BPM31510 exploits differential redox vulnerabilities between normal and glioblastoma cells to mediate its anti-cancer effect

Abstract Glioblastoma is an aggressive cancer, the proliferative capacity of which is correlated with glycolytic metabolism. BPM31510 is a novel formulation for delivery of supraphysiological levels of ubidecarenone to the mitochondria, enabling cancer specific metabolic switches. It is being studied in Phase I clinical trials versus a number of tumors, including glioma. Here, the effects of ubidecarenone on viability and redox homeostasis of glioma and non-tumorigenic cells was assessed using in vitro monoculture and coculture systems and an in vivo preclinical model. BPM31510 administration (50 mg/kg bid i.p., beginning 4-8 days post-inoculation) resulted in over a 20% long term survival rate in C6 tumor-bearing rats. We next compared BPM31510 effects in vitro between glioma lines (rat C6, human U251) and murine NIH3T3 fibroblasts, as a stromal control. In monocultures, decreased growth was observed in glioma lines and NIH3T3 with increasing BPM31510 doses; however, glioma lines were 2-fold more sensitive to BPM31510 compared to NIH3T3 cells (IC50 glioma lines: 230 µM vs IC50 NIH3T3: >460 µM). To investigate the differential sensitivity to BPM31510, a coculture system was developed by coincubating 2 x 105 C6-GFP labeled cells and NIH3T3 cells. After 6 days of coculture, the percentage of C6 relative to NIH3T3 cells was lowest at doses of BPM31510 between 115 µM and 230 µM, evidence of greater sensitivity to BPM31510-induced cytotoxicity in the C6 glioma cells than the non-tumorigenic component. At higher doses, differential effects on cell viability were less apparent. The level of superoxide, a central reactive oxygen species important in redox homeostasis, was also assessed using Mitosox in cocultures. At a BPM31510 dose which resulted in maximal differential viability between C6 and NIH3T3 cells (230 μM), the maximal differential superoxide level was likewise greatest. The basal differential in Mitosox signal was 9-fold between C6 and NIH3T3 cells, and it increased to over 50-fold upon treatment with BPM31510 (230 μM), implying that BPM31510 exploits differential redox vulnerabilities between C6 and NIH3T3 to mediate its anti-cancer activity. At high doses of BPM31510, differential effects on superoxide levels were less apparent. In summary, BPM31510 has marked anti-cancer activity in rats implanted with C6 glioma, and its differential effects on the viability of normal and transformed cells are associated with maximal differences in BPM31510-induced superoxide production. Together, these data suggest that differential redox vulnerabilities between tumorigenic and non-tumorigenic cells may underpin the anti-cancer activity of BPM31510, and identification of in vivo correlates of redox indices may represent an avenue to improved measurement of anti-cancer efficacy as well as define patient populations responsive to BPM31510. Citation Format: Jiaxin Sun, Seema Nagpal, Chirag Patel, Milton Merchant, Tiachang Jang, Anne R. Diers, Shiva Kazerounian, Stephane Gesta, Niven R. Narain, Rangaprasad Sarangarajan, Lawrence Recht. BPM31510 exploits differential redox vulnerabilities between normal and glioblastoma cells to mediate its anti-cancer effect [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 3608.

Abstract 1300: Novel thyrointegrin αvβ3 antagonist in the treatment of glioblastoma multiforme

Abstract Background: The proliferative and pro-angiogenic actions mediated by L-thyroxine, pro-inflammatory and all known growth factors in glioblastoma multiforme (GBM) are initiated at cell surface thyrointegrin αvβ3 receptors for thyroid hormone on the extracellular domain of integrin αvβ3. Thyrointegrin αvβ3 receptors are over-expressed on cancer and rapidly dividing blood vessel cells, but quiescent on normal cells. A macromolecule Polyethylene glycol-conjugated bi-TriAzole Tetraiodothyroacetic acid (P-bi-TAT) acts with high affinity (Ki 3.1 nM) and specificity for the thyrointegrin αvβ3 receptors without any significant nuclear translocation. Methods: In the present studies, three primary human GBM cells and U87 glioma cell line were implanted orthotopically or subcutaneously (s.c.) into nude mice that were treated daily for up to 21 days with P-bi-TAT at different doses. Additionally, preclinical Pharmacokinetic and Safety assessments were carried out in multiple species. Results: Pharmacokinetic profiles indicated once-a-day dosing, and safety assessment studies demonstrated high safety and tolerability at 100-fold over the anticipated human equivalent dose. Fluorescence labeled P-bi-TAT administered s.c. demonstrated high biodistribution to GBM tumor in the brain that was comparable to peripherally implanted tumor. P-bi-TAT administered daily for 10-21 days s.c. at 0.3-10 mg/kg resulted in a dose-dependent suppression of GBM tumor growth and viability monitored with IVIS imaging (p <0.001). Histopathological analysis of tumors revealed 95% loss of the vascularity of treated tumors at 10 days (p <0.001) along with extensive cellular necrosis and apoptosis, without intratumoral hemorrhage. GBM tumors had a 97% volume loss and maximal loss of GBM cell viability during a subsequent 22 days off-treatment period (p < 0.001). Genomic micro-array studies with human primary GBM revealed that over 30 pathways relevant to the progression of GBM are modulated by P-bi-TAT. Conclusions: P-bi-TAT is a promising lead clinical candidate effective in the treatment of human GBM. Citation Format: Shaker A. Mousa, Thangirala Sudha, Kavitha Godugu, Mehdi Rajabi, Stewart Sell, Paul J. Davis. Novel thyrointegrin αvβ3 antagonist in the treatment of glioblastoma multiforme [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 1300.

Abstract 3089: Evaluation of highly potent and selective single-molecule dual EGFR/PI3K inhibitors in preclinical models of adult and pediatric high-grade glioma

Abstract High grade gliomas (HGG), including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG), carry poor prognosis with median survival rates under 15 months post diagnosis. Due to dysregulation of the RTK/PI3K pathways within these tumors, including RTK pathway amplifications, targeted kinase inhibition has been considered a promising strategy to improve patient outcomes. However, many single-agent inhibitors of EGFR or PI3K have shown limited response in gliomas due to poor blood-brain barrier (BBB) penetrance and activation of compensatory signaling. The design of BBB-penetrant novel therapeutics that target multiple kinases is crucial for overcoming these obstacles. Single-molecule multikinase inhibitors may decrease resistance, present a single pharmacokinetic dosage profile, and reduce risks of multi-agent toxicities, supporting this strategy over dual drug approaches. A panel of inhibitors exploiting known binding modes of structurally-related ATP binding site inhibitors of EGFR/PI3K were synthesized and characterized, of which six promising candidates were chosen for in vitro study. The interaction of the inhibitors with common drug efflux proteins were probed to predict BBB penetrance. Of the compounds, MTX-241 was least likely to act as a substrate for efflux proteins such as P-glycoprotein. Treatment of MTX-241 in three human U87 GBM lines, some of which overexpress wild-type or vIII EGFR, three patient-derived DIPG lines, and two glioma stem cell (GSC) lines, one of which is radioresistant, showed strong cytotoxic potency measured by growth inhibitory activity, with IC50s in the <10 µM range. MTX-241 was significantly more potent than clinically relevant inhibitors targeting EGFR/RTKs (gefitinib, lapatinib, dasatinib, imatinib) or PI3K (alpelisib, idelalisib). Notably, selectivity of MTX-241 towards these HGG cell lines in comparison to normal human astrocytes (NHA) was observed, with NHA nearly insensitive to MTX-241 even at >100 µM. We found significant inhibition of p-EGFR and p-Akt in these cell lines compared to DMSO controls. Further, we identified a unique glycolytic-suppressing activity of MTX-241 in the U87/DIPG lines, to a greater extent than treatment with gefitinib or alpelisib. Additionally, as the clinically relevant HDAC inhibitor vorinostat upregulated EGFR activity, we examined the combination of MTX-241 and vorinostat, which significantly reduced proliferative abilities, achieving synergistic combination indices. MTX-241 and vorinostat treatment in the NHA model was not synergistic, highlighting the selectivity for HGG tumor models. Our data suggests that a dual inhibitor of EGFR and PI3K, alone or in combination with an HDAC inhibitor, represents a viable therapeutic strategy in adult/pediatric HGG. Future studies will focus on evaluation of in vivo efficacy in tumor-bearing mouse models. Citation Format: Yusha Y. Sun, Cavan P. Bailey, Trever R. Carter, Christopher E. Whitehead, Judith S. Sebolt-Leopold, Joya Chandra. Evaluation of highly potent and selective single-molecule dual EGFR/PI3K inhibitors in preclinical models of adult and pediatric high-grade glioma [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 3089.

Abstract 1951: Increased glioma collagen is associated with greater blood flow and peri-tumoral fluid flux, but less infiltration

Abstract Introduction: Composition of the glioma microenvironment (TME) determines tumor growth rate, invasion, metastasis and resistance to treatment. Compared to normal brain, tumor extracellular matrix (ECM) has a higher concentration of structural proteins including collagen, laminin, tenascin, and vinculin. We compared collagen expression in two rat models of glioma, human U251 grown in immune-compromised rats and 9L in syngeneic Fischer-344 rats, representing primary and recurrent glioma features, respectively. Experimental procedures: The U251 and 9L cells were implanted in the right brain hemisphere of female RNU rats (n=10) and Fischer-344 rats (n=7), respectively. Rats were imaged 2 to 3 weeks after implantation by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Tumor size, blood flow, apparent diffusion coefficient (ADC), blood-to-tumor forward volumetric transfer constant (Ktrans), peri-tumoral contrast flux (Flux), hydraulic conductivity (K), and extracellular volume fraction (VD) in the tumor (VD-tumor), tumor rim (VD-rim) and its periphery (VD-peri) along with tumor interstitial fluid pressure (TIFP) were measured. Immediately after imaging, brains were processed for paraffin embedding and histopathology. Brain sections (6 µm) containing the tumor were stained for collagen using Picrosirius red and adjacent brain sections with hematoxylin and eosin (H&E). MRI data were compared by t-tests and significance inferred at p≤0.05. Digital images of Picrosirius red staining were analyzed using ImageJ for collagen expression expressed as % fraction of total tumor area. It was compared to the MRI biomarkers, and to the patterns of tumor cell dispersion into normal brain observed on H&E stained images. Results: Tumor diameters averaged 4 mm for both models at the time of imaging. The U251 tumors showed greater Flux (p=0.03), higher blood flow (p=0.02), smaller VD_tumor (p=0.01) and VD_rim (p=0.02) values than the 9L tumors. TIFP also tended to be higher in the U251 model (p=0.06). Collagen expression in U251 tumors was significantly higher than in 9L tumors (p=0.002). On H&E stained sections, the U251 tumors showed a well delineated tumor margin with very few cells invading the host tissue. In contrast, the 9L tumors showed cancer cells infiltrating singly and in clusters, along white matter tracts as well as in perivascular spaces. Conclusions: Increased collagen content was associated with elevated blood flow, Flux and TIFP, but less tumor cell infiltration in the U251 glioma. Observations of low collagen content in conjunction with increased tumor cell invasion in the 9L model suggest that treatment with collagenase, while increasing drug penetration may also make the tumors more infiltrative. A closer examination of TME to determine the composition of ECM proteins that regulate glioma aggressiveness and modulate its response to treatments is warranted. Note: This abstract was not presented at the meeting. Citation Format: Tavarekere N. Nagaraja, Rasha Elmghirbi, Stephen L. Brown, Ian Y. Lee, Glauber Cabral, Swayamprava Panda, Robert A. Knight, James R. Ewing. Increased glioma collagen is associated with greater blood flow and peri-tumoral fluid flux, but less infiltration [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 1951.

Four new cucurbitane-type triterpenes from Momordica charantia L. with their cytotoxic activities and protective effects on H2O2-damaged pancreatic cells.

Four new cucurbitane-type triterpenes were isolated from the fruit of Momordica charantia L. The structures of the new compounds were identified based on HR-ESI-MS and 1D- and 2D-NMR spectroscopic methods. The cytotoxicity of the isolated compounds was evaluated using three human cancer cell lines, HeLa, Caco2, and U87. Compound 3 exhibited significant cytotoxic activity against HeLa cells with an IC50 value of 11.18μM. Additionally, the cytoprotective activity of these compounds was determined in vitro against H2O2-induced pancreatic injury. The results revealed that all the compounds obtained possess cytoprotective effects against H2O2-induced injury in MIN6 β-cells at a concentration of 10μM.

Zika virus NS5 protein inhibits cell growth and invasion of glioma

Glioma is the most common primary brain tumor with high mortality. Given the poor outcomes with standard-of-care treatments, novel treatment strategies are needed. Oncolytic viral therapy for glioma has developed as an exciting therapeutic method in recent years. Zika virus, a member of flavivirus family, has oncolytic activity against glioma cells but the mechanism is unknown. Here, we aimed to determine which viral protein might play a critical role in mitigating glioma cell growth. We examined the tumor suppressor function of four nonstructural proteins NS1, NS3, NS4B and NS5 in human glioma cell line U87. As a result, we found that only NS5 significantly inhibited proliferation, migration and invasion of U87 cells. Moreover, expression of NS5 suppressed tumorigenicity of mouse GL261 glioma cell in vivo. Our findings provide some clues for further exploration of oncolytic Zika virus in the treatment of glioma.

Targeting VPAC1 Receptors for Imaging Glioblastoma.

Scintigraphic imaging of malignant glioblastoma (MG) continues to be challenging. We hypothesized that VPAC1 cell surface receptors can be targeted for positron emission tomography (PET) imaging of orthotopically implanted MG in a mouse model, using a VPAC1-specific peptide [64Cu]TP3805. The expression of VPAC1 in mouse GL261 and human U87 glioma cell lines was determined by western blot. The ability of [64Cu]TP3805 to bind to GL261 and U87 cells was studied by cell-binding. Receptor-blocking studies were performed to validate receptor specificity. GL261 tumors were implanted orthotopically in syngeneic T-bet knockout C57BL/6 mouse brain (N = 15) and allowed to grow for 2-3weeks. Mice were injected i.v., first with ~ 150μCi of 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) then 24h later with ~ 200μCi of [64Cu]TP3805. In another set of tumor-bearing mice, (N = 5), ionic [64Cu]Cl2 was injected as a control. Mice were imaged at a 2-h post-injection using an Inveon micro-PET/CT, sacrificed and % ID/g of [64Cu]TP3805 and [64Cu]Cl2 were calculated in a tumor, normal brain, and other tissues. For histologic tissue examination, 3-μm thick sections of the tumors and normal brain were prepared, digital autoradiography (DAR) was performed, and then the sections were H&E stained for histologic examination. Western blots showed a strong signal for VPAC1 on both cell lines. [64Cu]TP3805 cell-binding was 87 ± 1.5%. Receptor-blocking reduced cell-binding to 24.3 ± 1.5% (P < 0.01). PET imaging revealed remarkable accumulation of [64Cu]TP3805 in GL261 MG with a negligible background in the normal brain, as compared to [18F]FDG. Micro-PET/CT image analyses and tissue distribution showed that the brain tumor uptake for [64Cu]TP3805 was 8.2 ± 1.7% ID/g and for [64Cu]Cl2 2.1 ± 0.5% ID/g as compared to 1.0 ± 0.3% ID/g and 1.4 ± 0.3% ID/g for normal mouse brains, respectively. The high tumor/normal brain ratio for [64Cu]TP3805 (8.1 ± 1.1) allowed tumors to be visualized unequivocally. Histology and [64Cu]TP3805 DAR differentiated malignant tumors from healthy brain and confirmed PET findings. Targeting VPAC1 receptors using [64Cu]TP3805 for PET imaging of MG is a promising novel approach and calls for further investigation.

High Glucose Promotes Human Glioblastoma Cell Growth by Increasing the Expression and Function of Chemoattractant and Growth Factor Receptors

Diabetes mellitus, characterized by hyperglycemia, is considered as a risk factor of cancers including malignant gliomas. However, the direct effect of high glucose on cancer cell behavior is not clear. We therefore investigated the effect of hyperglycemia on the growth of human glioblastoma (GBM) cells. Our results revealed that high glucose (HG) promoted the proliferation and inhibited the apoptosis of a human GBM cell line U87. Mechanistically, HG upregulated the expression and function of a G-protein coupled chemoattractant receptor (GPCR) formyl peptide receptor 1 (FPR1) and epidermal growth factor receptor (EGFR) on GBM cells, which upon activation by their agonists, promoted cell migration and proliferation. In addition, the invasiveness and the production of VEGF by U87 cells were enhanced under HG conditions, the effects of which were mediated by FPR1 and EGFR agonists. The tumor promoting activity of HG was further substantiated by increased tumorigenicity and growth of xenograft tumors formed by GBM cells in nude mice with induced diabetes mellitus. Thus, our study demonstrates the capacity of HG to promote GBM progression via enhancement of the function of chemoattractant and growth factor receptors.

Impact of extremely low-frequency electromagnetic field (100 Hz, 100 G) exposure on human glioblastoma U87 cells during Temozolomide administration

ABSTRACTGlioblastoma multiforme (GBM) is a highly malignant brain tumor with an extremely dismal prognosis, a median survival is12 months. Temozolomide (TMZ) is an alkylating agent widely used to treat cancer, resistance to this drug is often found. One unexplored possibility for overcoming this resistance is a treatment based on concomitant exposure to electromagnetic fields (EMF) and TMZ. Indeed, many evidences show that EMF affects cancer cells and drug performance. Therefore, the present study was carried out to evaluate the potential synergistic effect of 100 µM TMZ and EMF (100 Hz, 100 G) on human glioma cell line U87 U87 cells with four experimental groups (I–IV) were exposed to ELF-EMF and TMZ for 120 and 144 h, as follows: (I) control; (II) ELF-EMF; (III) TMZ; (IV) ELF-PEMFs / TMZ. mRNA expression of genes such as (Nestin,CD133, Notch4 and GFAP) were investigated by Real-time PCR and western blot. We also evaluated, SOD activity, MDA and calcium concentration by ELISA assay. Co-treatment synergistically decreased the expression of Nestin,CD133, and Notch4 and increased the GFAP genes. We also observed an increase in Superoxide dismutase (SOD) activity, Malondialdehyde (MDA) and Ca2+concentration in comparison to controls.TMZ prevents cancer progression not only through the induction of cell death, but also by inducing differentiation in cancer cells. In addition, our data demonstrate ELF-EMF (100 Hz, 100 G) can significantly enhance the effects of TMZ on human glioblastoma U87 cell. These findings may open new window for future studies.

EVALUATING THE EFFECTIVENESS OF THE TUMOR-INITIATING STEM CELLS ERADICATION STRATEGY ON THE EXAMPLE OF HUMAN GLIOBLASTOMA CELL LINE U87

The effectiveness of the new therapeutic approach aimed at the destruction of the cancer cell community was evaluated on the immortalized human glioblastoma cell line U87. Initially, the reference elements of a new strategy for eradication of tumor-initiating stem cells were characterized. The main points of the strategy were as follows. 1) Evaluation of the TAMRA+ (tumor-initiating stem cells) presence in the population of U87 culture cells. 2) Determination of the reference time points of the DNA interstrand cross-links repair, induced by cross-linking cytostatics mitomycin C. 3) Determination of the day after initiation of therapy when TAMRA+ cells accumulate and are synchronously present in the G1/S sensitive for the treatment phase of the cell cycle. Based on the data obtained, a therapeutic regimen aimed at eradicating TAMRA+ cells (tumor-initiating stem cells) was identified. Treatment of the culture was carried out by cross-linking cytostatic mitomycin C and complex DNA preparation. Transplantation experiments showed that high experimental doses of mitomycin C (20 μg/ml) totally destroy transplantation potential of U87 cells. Diminution of mitomycin C to therapeutic doses (5 μg/ ml) clearly demonstrated effect of complex double-stranded DNA preparation to reducing of U87 tumorigenic potential. These fully confirmed the concept of developed cancer treatment technology.

The Volume-Regulated Anion Channel LRRC8/VRAC Is Dispensable for Cell Proliferation and Migration.

Cells possess the capability to adjust their volume for various physiological processes, presumably including cell proliferation and migration. The volume-regulated anion channel (VRAC), formed by LRRC8 heteromers, is critically involved in regulatory volume decrease of vertebrate cells. The VRAC has also been proposed to play a role in cell cycle progression and cellular motility. Indeed, recent reports corroborated this notion, with potentially important implications for the VRAC in cancer progression. In the present study, we examined the role of VRAC during cell proliferation and migration in several cell types, including C2C12 myoblasts, human colon cancer HCT116 cells, and U251 and U87 glioblastoma cells. Surprisingly, neither pharmacological inhibition of VRAC with 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB), carbenoxolone or 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), nor siRNA-mediated knockdown or gene knockout of the essential VRAC subunit LRRC8A affected cell growth and motility in any of the investigated cell lines. Additionally, we found no effect of the VRAC inhibition using siRNA treatment or DCPIB on PI3K/Akt signaling in glioblastoma cells. In summary, our work suggests that VRAC is dispensable for cell proliferation or migration.

Impact on U251 glioma cell radiosensitivity and CFL1 level via inhibiting cell motility regulator ROCKII of RhoA-RockII-CFL1 pathway.

e14630 Background: In response to ionizing radiation, cofilin1 (CFL1) is essential for actin dynamics and tumor metastasis. In our previous study, CFL1 was revealed up-regulated in radioresistant astrocytomas of surgical samples and positively correlated with glioma cell radioresistance. Methods: The present study aims to clarify the potential mechanism of CFL1-mediated radioresistance and to find new molecular targets to improve the radiosensitivity of U251 glioma cell. Radioresistant U251 (RR-U251) was established by serially irradiation with 5Gy until 60 Gy of irradiation was accumulated on human U251 glioma cell. Y-27632, PAK1-shRNA and PAK1-shRNA were respectively utilized to inhibit CFL1 upstream regulators in normal U251 and RR-U251 cells. CFL1 expression level and the alteration of radiosensitivity were evaluated; the cell radiosensitivity was assessed through cell vitality, invasion and migration experiments after irradiation. Results: 20 ¦Ìmol/L Y-27632 was screened and administrated to inhibit ROCKII in glioma cells followed by 5Gy radiation treatment. The CFL1 level was reduced in both normal U251 and RR-U251 cells. Meanwhile, cell viability, migration and invasion abilities significantly decreased in RR-U251 cells. After being transfected with PAK1-shRNA, cell radiosensitivity were significantly improved in both normal U251 and RR-U251 cells; while CFL1 level showed no significant difference after PAK1 silence . After inhibiting MRCK¦Á expression, cell viability, migration and invasion capabilities significantly reduced in both normal and radioresistant cells; while CFL1 levels showed no significant decrease. Conclusions: The results of the present study indicate that inhibiting ROCKII of RhoA-RockII-CFL1 pathway could improve radiosensitivity of radioresistant glioma cells and simultaneously down-regulate CFL1 expression; while the inhibiton of PAK1 or MRCK¦Á ameliorated the radiosensitivity with no significant regulation effect on CFL1. In conclusion, the present study emphasize the involvement of ROCKII in CFL1-mediated radioresistant phenotype and provide potential biomarkers for improvement of further clinical radiotherapy.

MiR-205 suppresses epithelial-mesenchymal transition and inhibits tumor growth of human glioma through down-regulation of HOXD9.

Epithelial–mesenchymal transition (EMT) plays a pivotal role in cancer progression. Hsa-miR-205 is considered one of the fundamental regulators of EMT. In the present study, we found that miR-205 was down-regulated in glioma tissues and human glioma cells U87 and U251. Meanwhile, miR-205 overexpression enhanced E-cadherin, reduced mesenchymal markers, and decreased cell proliferation, migration, and invasion in vitro. In vivo, miR-205 suppressed tumor growth. Additionally, HOXD9 was confirmed as a direct target of miR-205. Suppression of HOXD9 by miR-205 was demonstrated by luciferase reporter assay, quantitative real time-PCR analysis, and western blot. Moreover, we observed a negative correlation between miR-205 and HOXD9 in human glioma tissues. In summary, our findings demonstrated that miR-205 suppresses glioma tumor growth, invasion, and reverses EMT through down-regulating its target HOXD9.

Anti-tumor Effects of Chlorogenic Acid on Human Glioblastoma Cell Lines U251 and Related Pro-apoptotic Mechanism

Anti-tumor Effects of Chlorogenic Acid on Human Glioblastoma Cell Lines U251 and Related Pro-apoptotic Mechanism

MiR‑342 inhibits glioma cell proliferation by targeting GPRC5A.

Accumulating evidence suggests that microRNAs (miRNAs) play a key role in the biological behaviors and progression of glioma. However, the function and bio-molecular mechanisms of miR-342 in glioma remain largely unclear. In the present study, reverse transcription quantitative-polymerase chain reaction and western blotting were performed to determine the mRNA and protein expression levels of the factors investigated. MTT assay was performed to examine the proliferation rates. Luciferase reporter assay was performed to test the binding between miRNA-342 and its putative target. Data indicated that miR-342 expression was markedly decreased in human glioma tissues and cell line U87, and reduced miR-342 expression significantly promoted cell proliferation. In order to explore the mechanisms, G-protein coupled receptor family C group 5 member A (GPRC5A) was identified as a target of miR-342 and depletion of GPRC5A suppressed cell proliferation. Our findings demonstrated that miR-342 regulates the cell proliferation of glioma by targeting GPRC5A, which indicates that miR-342 is a target of interest regarding the treatment of refractory glioma, and it may provide a promising prognostic and therapeutic strategy for glioma treatment.

Knockdown of RAD18 inhibits glioblastoma development.

This study aimed at investigating the role of RAD18 in the regulation of glioblastoma development as well as the underlying mechanisms. The human glioblastoma U251 and U87MG cells were transfected with siRNAs specifically targeting RAD18, and the effects of knockdown of RAD18 on the viability, apoptosis, migration, and invasion of U251 and U87MG cells were investigated. Transcriptome sequencing of the siRNA-RAD18-tranfected and siRNA-NC-transfected U251 cells was performed, followed by bioinformatic analyses for sequencing data. The results showed that knockdown of RAD18 significantly inhibited cell viability, promoted apoptosis, and suppressed migration and invasion of U251 and U87MG cells. Bioinformatic analyses of sequencing data identified 1,051 differentially expressed genes (DEGs) (369 up- and 682 downregulated genes) in the siRNA-RAD18-transfected U251 cells compared with siRNA-NC-transfected U251 cells. Eleven DEGs, including nerve growth factor (NGF), colony-stimulating factor 2 (CSF2), matrix metallopeptidase 1 (MMP1), platelet-derived growth factor receptor α (PDGFRA), and heme oxygenase 1 (HMOX1), were identified as the hub nodes in protein-protein interaction (PPI) network. Moreover, the aforementioned 11 hub genes were significantly enriched in PI3K-Akt signaling pathway and GO functions associated with the extracellular region. Notably, quantitative real-time polymerase chain reaction further confirmed that the expression levels of NGF, CSF2, HMOX1, and MMP1 were significantly downregulated, while that of PDGFRA was markedly upregulated in the siRNA-RAD18-transfected U251 cells than in the siRNA-NC cells. In conclusion, the knockdown of RAD18 may inhibit glioblastoma development by regulating the expression of the aforementioned key DEGs.