U87 MG Research Articles

ACT001 modulates the NF-κB/MnSOD/ROS axis by targeting IKKβ to inhibit glioblastoma cell growth.

Glioblastomas are high-grade brain tumors with poor prognoses, and new therapeutic approaches for these tumors are critically needed. This study revealed the underlying mechanisms of a new orphan drug, ACT001, that is currently in clinical trials for the treatment of advanced glioblastoma in Australia and China. ACT001 significantly suppressed glioma cell proliferation and induced apoptosis and cell cycle arrest in vitro, as determined by Cell Counting Kit-8 assays and flow cytometry. In addition, U-118 MG cells with high expression of p-IKKβ were sensitive to ACT001. Changes in the oxidative stress pathway in U-118 MG cells were detected with the isobaric tags for relative and absolute quantitation (iTRAQ) method. We further verified that ACT001 elevated the levels of reactive oxygen species (ROS) by regulating NF-κB-targeted MnSOD. ACT001 markedly inhibited NF-κB activation by directly binding IKKβ and inhibiting its phosphorylation. Overexpression of IKKβ markedly attenuated the changes in MnSOD and NOX1, indicating that ACT001 increased the levels of ROS by reducing the protein expression of p-IKKβ. Furthermore, ACT001 reduced cyclin B1/CDC2 expression and triggered G2/M phase arrest by increasing ROS production. ACT001 also upregulated the expression of Bax and Bim and induced apoptosis in a ROS-dependent manner. ACT001 effectively suppressed the growth of U-118 MG tumors in BALB/c nude mice and GL-261-luciferase tumors in C57BL/6 J mice. Finally, ACT001 downregulated the expression of p-p65, MnSOD, cyclin B1, CDC2, and Ki67 in U-118 MG tumor tissues. Patients with activated NF-κB signaling should thus be given priority for enrollment in future phase II clinical trials. KEY MESSAGES: ACT001 directly bind to IKKβ and inhibited its phosphorylation. The inhibition of p-IKKβ induced the generation of ROS. ACT001 promoted the generation of ROS by regulating MnSOD expression to induce G2/M phase arrest.

Identification of functionally distinct and interacting cancer cell subpopulations from glioblastoma with intratumoral genetic heterogeneity.

BackgroundGlioblastomas display a high level of intratumoral heterogeneity with regard to both genetic and histological features. Within single tumors, subclones have been shown to communicate with each other to affect overall tumor growth. The aim of this study was to broaden the understanding of interclonal communication in glioblastoma.MethodsWe have used the U-343 model, consisting of U-343 MG, U-343 MGa, U-343 MGa 31L, and U-343 MGa Cl2:6, a set of distinct glioblastoma cell lines that have been derived from the same tumor. We characterized these with regard to temozolomide sensitivity, protein secretome, gene expression, DNA copy number, and cancer cell phenotypic traits. Furthermore, we performed coculture and conditioned media-based experiments to model cell-to-cell signaling in a setting of intratumoral heterogeneity.ResultsTemozolomide treatment of a coculture composed of all 4 U-343 cell lines presents a tumor relapse model where the least sensitive population, U-343 MGa 31L, outlives the others. Interestingly, the U-343 cell lines were shown to have distinct gene expression signatures and phenotypes although they were derived from a single tumor. The DNA copy number analysis revealed both common and unique alterations, indicating the evolutionary relationship between the cells. Moreover, these cells were found to communicate and affect each other’s proliferation, both via contact-dependent and -independent interactions, where NOTCH1, TGFBI, and ADAMTS1 signaling effects were involved, respectively.ConclusionsThese results provide insight into how complex the signaling events may prove to be in a setting of intratumoral heterogeneity in glioblastoma and provide a map for future studies.

Glioblastoma cell adhesion properties through bacterial cellulose nanocrystals in polycaprolactone/gelatin electrospun nanofibers

Glioblastoma (GBM), the most common and extremely lethal type of brain tumor, is resistant to treatment and shows high recurrence rates. In the last decades, it is indicated that standard two-dimensional (2D) cell culture is inadequate to improve new therapeutic strategies and drug development. Hence, well-mimicked three-dimensional (3D) tumor platforms are needed to bridge the gap between in vitro and in vivo cancer models. In this study, bacterial cellulose nano-crystal (BCNC) containing polycaprolactone (PCL) /gelatin (Gel) nanofibrous composite scaffolds were successfully fabricated by electrospinning for mimicking the extracellular matrix of GBM tumor. The fiber diameters in the nanofibrous matrix were increased with an increased concentration of BCNC. Moreover, fiber morphology changed from the smooth formation to the beaded formation by increasing the concentration of the BCNC suspension. In-vitro biocompatibilities of nanofibrous scaffolds were tested with U251 MG glioblastoma cells and improved cell adhesion and proliferation was compared with PCL/Gel. PCL/Gel/BCNC were found suitable for enhancing axon growth and elongation supporting communication between tumor cells and the microenvironment, triggering the process of tumor recurrence. Based on these results, PCL/Gel/BCNC composite scaffolds are a good candidate for biomimetic GBM tumor platform.

ET-02 EFFICACY OF THE SALVAGE THERAPY VIA LOMUSTINE AND NIMUSTINE FOR RECURRENT GLIOBLASTOMA WITH TEMOZOLOMIDE RESISTANCE

Temozolomide (TMZ) is widely used as a part of the standard treatment of glioblastomas (GBMs). However, GBM often acquires resistance for TMZ after continuous treatment with TMZ and recurs as TMZ resistance GBM (TMZ-R GBM). Other alkylating agents such as lomustine (CCNU) or nimustine (ACNU) have been sometimes used as a salvage therapy for those TMZ-R GBMs, however, their efficacy against TMZ-R GBMs has not been thoroughly investigated yet. In this study, we investigated anti-tumor effects of CCNU and ACNU for TMZ-R GBM cell lines in vitro to examine whether these agents could become alternative to TMZ in the therapy of TMZ-R GBMs. TMZ resistant clones of human GBM cell lines U87 MG (U87-R cells) and U251 MG (U251-R cells) were established as the TMZ-R GBM cell lines by cultivating U87 MG cells and U251 MG cells under continuous TMZ treatment for at least 1 year. Induction of growth arrest and apoptosis by TMZ, CCNU or ACNU against these cells were analyzed by dye exclusion assay, vital dye staining assay, and immunoblotting. The results showed that growth arrest and apoptosis were triggered upon these cells after administration of each drugs. As expected, the anti-tumor effects of TMZ for U87-R cells or U251-R cells were significantly reduced compared with those for parental U87 MG cells or U251 MG cells, respectively. On the other hand, CCNU and ACNU showed similar growth suppressive effect upon U87-R cells or U251-R cells as compared with U87 MG cells or U251 MG cells. Throughout these experiments, CCNU demonstrated strongest anti-tumor effects for all cell lines, both parental and TMZ-R GBM cell lines, and ACNU also demonstrated stronger effects than TMZ. These results suggest that CCNU or ACNU may serve as a drug of choice for salvage treatment of TMZ-R GBM.

Novel arginase inhibitor alone and in combination with an immune check point inhibitor reduces tumour growth in murine experimental gliomas

Abstract Background Glioblastoma (GBM) is a common and aggressive brain tumour, which is resistant to standard therapies. Clinical and experimental data demonstrated upregulation of Arginase 1 (ARG1) gene and protein in malignant gliomas, particularly in tumour infiltrating microglia and macrophages. Elevated Arginase 1 expression leads to depletion of L-arginine required for T cell and natural killer cell proliferation. A novel, selective small molecule arginase inhibitor - OAT-1746- was developed by OncoArendi Therapeutic SA and we evaluated its antitumour efficacy as a monotherapy and in combination with antibody against PD-1. Methods Efficacy of the inhibitor in blocking microglia dependent glioma invasion has been determined in Matrigel invasion assays. Its potential cytotoxicity towards microglia and glioma cells in vitro has been evaluated with a MTT metabolism test. Antitumour activity of targeting drugs was assessed in a murine Gl261 glioma model. Tumour volume was measured using an in-vivo imaging system after 21 and 28 days. Immune heterogeneity of glioma microenvironment was analysed by flow cytometry. Results The co-culture of microglial cells with glioma cells increased the invasiveness of U87 MG and LN18 glioma cells. This microglia dependent invasion was reduced significantly and in a dose dependent manner in the presence of inhibitor. OAT-1746 orally administrated (twice a day) to animals implanted intracranially with GL-261 glioma cells reduced tumour volumes at day 21st and 28th as determined using an in vivo-imaging X-treme. While tumour growth was reduced with OAT-1746 or anti-PD-1 as monotherapies, the inhibition was higher when combination of OAT-1746 or anti-PD-1 was provided. OAT-1746 effectively crossed the BBB and increased arginine levels in brains of mice bearing gliomas. arginine levels in brains of mice bearing gliomas. Conclusion These results demonstrate that arginase is a key mediator of immuno suppression in gliomas, and inhibiting Arg1 shifts the immune response toward a pro-inflammatory environment. Our results suggest that combining OAT-1746 with other immunotherapies may improve responses in glioblastoma. Legal entity responsible for the study Molecular Neurobiology, Nencki Institute of Experimental Biology. Funding Project DIMUNO: Development of new cancer therapies based on selective antitumour immunomodulators, co-financed by the National Centre for Research and Development. Disclosure All authors have declared no conflicts of interest.

Biological Functions of TNKS1 and Its Relationship with Wnt/β-Catenin Pathway in Astrocytoma.

BackgroundTankyrase1 (TNKS1), which often shows abnormal expression in many malignant tumor cells, plays an important role in tumor progression. In our previous study, we found that TNKS1 is also closely related to pathologic grade in human astrocytoma and its expression level is positively correlated with the Wnt/β-catenin pathway. This study is aimed to further elucidate the biological functions of TNKS1 as well as its relationship with the Wnt/β-catenin pathway.MethodsTNSK1 overexpression and knockdown vectors were constructed and transfected into glioblastoma cell lines U251 MG and U87, respectively. Viability, apoptosis, cell cycle and cell invasiveness in the treated cells were investigated.ResultsIn comparison with untreated cells, U251 and U87 cells overexpressing TNSK1 showed significantly increased cell viability and decreased apoptosis, while the TNKS1 knockdown U251 and U87 cells had reduced cell invasive ability and increased apoptosis, respectively. In addition, immunoprecipitation study showed that TNKS1 could be detected by β-catenin antibody after pull-down, indicating that TNKS1 directly interacts with β-catenin, further indicating that TNKS1 could be regarded as a positive regulator of the Wnt/β-catenin pathway in astrocytoma. Moreover, knockdown of TNKS1 in U251 and U87 cells also leads to suppressed Wnt/β-catenin signaling, and subsequent decrease of cell growth and proliferation, reduced invasion ability and increased apoptosis.ConclusionOur findings suggest that TNKS1 might be a potential new therapeutic target for human astrocytoma in gene therapy.

Glucoheptoamidated polyamidoamine PAMAM G3 dendrimer as a vehicle for succinate linked doxorubicin; enhanced toxicity of DOX against grade IV glioblastoma U-118 MG cells

Glioblastoma multiforme (GBM), known as astrocytoma grade IV, is the most common primary malignant brain tumor and one of the most lethal forms of cancer with a median survival rate of approximately 15 months after standard treatment. Currently, the chance of increasing the survival rate is found in therapy with doxorubicin (DOX) delivered to the central nervous system by polymeric carriers.In this study, we synthesized and characterized by NMR spectroscopy, a vehicle for DOX based on glucoheptoamidated poly (amidoamine) dendrimers of third generation (G3gh) and its derivative substituted with 4 residues of DOX attached via succinate linkers (G3gh4DOX). The pH dependent DOX release profile from the well soluble G3gh4DOX indicates a slow release that is significantly higher in an acidic environment due to hydrolytic cleavage of an amide bond between DOX and the succinate linker, not the ester bond between succinate linker and the glucoheptoamide substituent. Biological studies demonstrate efficient grade IV human U-118 MG glioma cells damage by G3gh4DOX conjugates at very low concentrations, with IC50 < 1 μM after 24–72 h of incubation. The conjugate studied was about 2.7–4.5 fold more toxic than DOX alone. G3gh4DOX killed glioma cells through a more desirable apoptosis pathway and inhibited their proliferation with cell cycle-arrest at the G2/M phase. The effect of G3gh4DOX was probably the result of action of the entire conjugate, but not DOX alone. Therefore, G3gh4DOX seems to be a promising candidate for local glioma therapy.

The Anti-Tumorigenic Activity of Sema3C in the Chick Embryo Chorioallantoic Membrane Model.

Sema3C protein, a member of the class 3 family of secreted semaphorins, play an important role in tumor development by regulating cell proliferation, migration, invasion, and angiogenesis processes. Depending on the type and malignancy grade of the tumor, Sema3C function remains controversial. In this study, we constructed a stably overexpressing Sema3C glioblastoma cell line U87 MG and tested it on the chicken embryo chorioallantoic membrane (CAM) model with the aim to reveal Sema3C protein function on angiogenesis process in ovo. Our experiments showed that Sema3C not only affects angiogenesis of CAM by inhibiting neovascularization but also acts as an anti-tumorigenic molecule by hampering U87 MG cell invasion into mesenchyme. The effects of Sema3C on CAM were similar to the effects of anti-epileptic drug sodium valproate (NaVP). Both, anti-angiogenic and anti-tumorigenic activities of Sema3C were enhanced by the treatment of NaVP and, importantly, were not attributed to the cytotoxic effects. Our studies suggest that Sema3C could be a promising target for glioblastoma treatment.

ANGI-07. NOVEL FUSION GENE PTPRZ1-MET PROMOTES GLIOMAS ANGIOGENESIS BY UP-REGULATING EXOCRINE PROTEIN IGF2BP3

Abstract We previously reported a novel fusion gene PTPRZ1-MET in 15% of secondary GBM (sGBM). Subsequent studies showed that ZM fusion indicates a significantly worse prognosis and promotes glioma progression by increasing MET activity. We have demonstrated hyper-activation of MAPK and STAT3 signaling and elevated angiogenesis activities in patients and subcutaneous tumors with ZM fusion. However, the angiogenesis mechanism regulated by ZM is still unclear. In this study, 66 up-regulated and 66 down-regulated genes were identified on 8 sGBMs with ZM fusion comparing gene expression with 135 negative sGBMs (fold-change &gt;2.5 &amp; p.value &lt; 0.05). ZM-harboring U87 MG xenograft models were conducted and RNA sequencing was performed in 3 subcutaneous tumors with ZM-harboring U87 MG and 3 negative control. 384 up-regulated and 698 down-regulated genes were identified on 3 ZM-harboring samples comparing gene expression with 3 negative control samples (fold-change &gt;2.5 &amp; p.value &lt; 0.05. Three subcutaneous tumors with ZM-harboring U87 MG were treated by a MET kinase inhibitor, PLB-1001. Finally, the expression of 13 up-regulated gene decreased and 10 down-regulated genes increased after treatment by PLB-1001. We overlapped the candidate genes between patient samples and subcutaneous tumors and 5 up-regulated and 4 down-regulated genes was appeared and most associated with ZM fusion. Prognostic analysis of 9 candidate genes on sGBM revealed that a higher expression of IGF2BP3 indicates a worse prognosis. In further study, we found that IGF2BP3 can be secreted out of the gliomas stem cells (GSCs) and promote proliferation of vascular endothelial cells and the expression of CD31. IGF2BP3 siRNA can block this pathway and inhibit the angiogenesis. Conclusion, our study demonstrated a new pathway promoting angiogenesis in gliomas. But the regulating mechanism between ZM fusion gene and IGF2BP3 deserves to do further research.

Overexpression of Tripartite Motif-Containing 48 (TRIM48) Inhibits Growth of Human Glioblastoma Cells by Suppressing Extracellular Signal Regulated Kinase 1/2 (ERK1/2) Pathway.

BackgroundHerein, we found that tripartite motif-containing 48 (TRIM48) was reduced in human glioblastoma (GBM) cell lines. We investigated whether and how TRIM48 functions in human GBM in vitro.Material/MethodsHuman GBM cells (U87 MG and U138 MG) were infected with lentivirus to overexpress TRIM48, and 1 human GBM cell line (T98G) was infected with siRNAs to knock down TRIM48 expression. Techniques used included cell proliferation assay, measured by CCK-8 and BrdU-ELISA method, and cell cycle assay, determined using flow cytometry. Curcumin, a specific activator of extracellular signal regulated kinases (ERK1/2), or PD98059, a specific inhibitor of ERK1/2, was used to activate or block the ERK1/2 pathway, respectively. Expression of phosphorylated (p)-ERK1/2, and its downstream targets (Cyclin D1) were measured to assess the mechanism.ResultsOur data suggest that overexpression of TRIM48 reduces the viability of U87 MG and U138 MG and leads to cell cycle arrest (in G0-G1 phase), which is associated with blockade of the ERK1/2 pathway and reduction of Cyclin D1. In contrast, knockdown of TRIM48 resulted in the opposite effects. Interestingly, the inhibitory effect of TRIM48 overexpression on human GBM cell growth and the inactivation of ERK1/2 were significantly alleviated with additional curcumin treatment, while it the promoted the effect of siTRIM48 on human GBM cell growth, and the activation of ERK1/2 was significantly alleviated with additional PD98059 treatment.ConclusionsTRIM48 suppressed the growth of human GBM cell via the prevention of ERK1/2 activation.

DJ-1 Contributes to Self-renewal of Stem Cells in the U87-MG Glioblastoma Cell Line.

DJ-1, an oncogenic molecule, helps to maintain somatic stem cells by reducing the intracellular level of reactive oxygen species (ROS). This study investigated the role of DJ-1 in glioma stem cells (GSCs). U87-MG (U87) and U251-MG (U251) glioblastoma cell lines that express wild-type and mutant p53, respectively, were used. These were cultured with DJ-1-targeting siRNA and subjected to a variety of in vitro experiments or intracranial transplantation into nude mice. Knockdown of DJ-1 reduced clonogenicity only in U87 cells, which was rescued by p53 depletion. ROS accumulated in DJ-1-depleted cells, although treatment with N-acetyl cysteine, which quenches ROS, did not affect exhaustion of CSCs among U87 cells by DJ-1 knockdown. In a serial transplantation study, DJ-1 knockdown prolonged the survival of mice in secondary transplantation. DJ-1 plays a pivotal role in maintenance of stem cell self-renewal in the U87 cell line.

The Effect of Biotinylated PAMAM G3 Dendrimers Conjugated with COX-2 Inhibitor (celecoxib) and PPARγ Agonist (Fmoc-L-Leucine) on Human Normal Fibroblasts, Immortalized Keratinocytes and Glioma Cells in Vitro.

Glioblastoma multiforme (GBM) is the most malignant type of central nervous system tumor that is resistant to all currently used forms of therapy. Thus, more effective GBM treatment strategies are being investigated, including combined therapies with drugs that may cross the blood brain barrier (BBB). Another important issue considers the decrease of deleterious side effects of therapy. It has been shown that nanocarrier conjugates with biotin can penetrate BBB. In this study, biotinylated PAMAM G3 dendrimers substituted with the recognized anticancer agents cyclooxygenase-2 (COX-2) inhibitor celecoxib and peroxisome proliferator-activated receptor γ (PPARγ) agonist Fmoc-L-Leucine (G3-BCL) were tested in vitro on human cell lines with different p53 status: glioblastoma (U-118 MG), normal fibroblasts (BJ) and immortalized keratinocytes (HaCaT). G3-BCL penetrated efficiently into the lysosomal and mitochondrial compartments of U-118 MG cells and induced death of U-118 MG cells via apoptosis and inhibited proliferation and migration at low IC50 = 1.25 µM concentration, considerably lower than either drug applied alone. Comparison of the effects of G3-BCL on expression of COX-2 and PPARγ protein and PGE2 production of three different investigated cell line phenotypes revealed that the anti-glioma effect of the conjugate was realized by other mechanisms other than influencing PPAR-γ expression and regardless of p53 cell status, it was dependent on COX-2 protein level and high PGE2 production. Similar G3-BCL cytotoxicity was seen in normal fibroblasts (IC50 = 1.29 µM) and higher resistance in HaCaT cells (IC50 = 4.49 µM). Thus, G3-BCL might be a good candidate for the targeted, local glioma therapy with limited site effects.

Mixed-Generation PAMAM G3-G0 Megamer as a Drug Delivery System for Nimesulide: Antitumor Activity of the Conjugate Against Human Squamous Carcinoma and Glioblastoma Cells.

Polyhydroxylated dendrimer was synthesized from poly(amidoamine) (PAMAM) dendrimer generation 3 by addition of glycidol (G3gl). G3gl megamer was further modified by binding PAMAM G0 dendrimers by activation of G3gl with p-nitrophenylchloroformate, followed by the addition of excess PAMAM G0 and purification using dialysis. The maximum G0 binding capacity of G3gl was 12 in the case when G0 was equipped with two covalently attached nimesulide equivalents. Nimesulide (N) was converted into N-(p-nitrophenyl) carbonate derivative and fully characterized using X-ray crystallography and spectral methods. Nimesulide was then attached to G0 via a urea bond to yield G02N. The mixed generation G3gl–G02N megamer was characterized using 1H NMR spectroscopy, and its molecular weight was estimated to be 22.4 kDa. The AFM image of G3gl–G02N deposited on mica demonstrated aggregation of nimesulide-covered megamer. The height of the deposited megamer was 8.5 nm. The megameric conjugate with nimesulide was tested in vitro on three human cell lines: squamous cell carcinoma (SCC-15) and glioblastoma (U-118 MG) overexpressing cyclooxygenase-2 (COX-2), and normal skin fibroblasts (BJ). The conjugate efficiently penetrated into all cells and was more cytotoxic against SCC-15 than against BJ. Moreover, the conjugate produced a strong and selective antiproliferative effect on both cancer cell lines (IC50 < 7.5 µM).

Adsorption properties, the pH-sensitive release of 5-fluorouracil and cytotoxicity studies of mesoporous silica drug delivery matrix

Mesoporous silica materials were investigated as the carriers for pH-sensitive drug delivery systems. Porous silica SBA-15 was first functionalized by anchoring N-[3-(trimethoxysilyl) propyl] aniline groups on the surface. After loading of an antineoplastic agent 5-fluorouracil, the pores were capped by β-cyclodextrin molecules. The studied samples were characterized by N2 adsorption/desorption measurements, thermal analysis, powder X-Ray Diffraction, and Transmission Electron Microscopy. Adsorption properties of 5-fluorouracil were explored via the construction of adsorption isotherms. The amount of 5-FU adsorbed on amine-functionalized SBA-15 was 60 mg per 1 g of solid. The adsorption of 5-fluorouracil on silica was also monitored by microcalorimetry, showing low adsorption enthalpies. Drug release properties from matrices were studied using UV–Visible spectroscopy with the un-blocked and blocked pores configuration to demonstrate the efficiency of the pH-responsive nanovalves and evaluated using different kinetic models. It was shown that no drug release occurred at neutral pH and that more than 80% of drug adsorbed amount was released at pH = 5. Working at the equilibrium, the initial burst of the drug from the silica surface, usually observed in other porous silica drug delivery systems, was avoided. The interaction between the β-cyclodextrin molecules and grafted amine functions were also studied as a function of pH. Finally, the cytotoxicity tests were performed using human glioma U87 MG cells.

Antitumor and anti-nematode activities of α-mangostin

α-Mangostin, one of the major xanthones isolated from pericarp of mangosteen (Garcinia mangostana Linn), exhibits a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial as well as anticancer, both in in vitro and in vivo studies. In the present study, α-mangostin' anti-cancer and anti-parasitic properties were tested in vitro against three human cell lines, including squamous carcinoma (SCC-15) and glioblastoma multiforme (U-118 MG), compared to normal skin fibroblasts (BJ), and in vivo against Caenorhabditis elegans. The drug showed cytotoxic activity, manifested by decrease of cell viability, inhibition of proliferation, induction of apoptosis and reduction of adhesion at concentrations lower than 10 μM (the IC50 values were 6.43, 9.59 and 8.97 μM for SCC-15, U-118 MG and BJ, respectively). The toxicity, causing cell membrane disruption and mitochondria impairment, was selective against squamous carcinoma with regard to normal cells. Moreover, for the first time anti-nematode activity of α-mangostin toward C. elegans was described (the LC50 = 3.8 ± 0.5 μM), with similar effect exerted by mebendazole, a well-known anthelmintic drug.

Sono-Polymerization of Poly(ethylene glycol)-Based Nanoparticles for Targeted Drug Delivery.

Engineering functional nanoparticles (NPs) with low nonspecific interactions and a high specific targeting property is highly desired for improved drug delivery. Herein, we report a targeted poly(ethylene glycol) (PEG)-based chemotherapy system synthesized via a catalyst-free sono-polymerization process for drug delivery. The polymerization process was fast (20 min), and different monomers were able to be polymerized to form NPs in a one-pot process. Glutathione (GSH)-responsive platinum prodrugs and fluorescent dyes could be encapsulated in NPs by amidation formation. Cyclic peptides containing Arg-Gly-Asp (RGD)-modified PEG-based NPs possessed a much higher cell targeting (∼90%) than the unmodified PEG-based NPs (∼10%) after a 12 h incubation with U87 MG cells, which could improve drug delivery efficacy. The IC50 (50% inhibitory concentration) could also be reduced more than 50% compared to the nontargeted PEG-based NPs. Importantly, these PEG-based NPs can be freeze-dried into a powder form and redispersed in an aqueous solution without aggregation, which may facilitate the storage and transportation of nanomedicine. This study establishes a green and efficient method to engineer targeted drug carriers for drug delivery.

Synthesis and Activity Investigation of Novel 1H-Purin-6(9H)-one and 3H-Imidazo[4,5-d][1,2,3]triazin-4(7H)-one Derivatives.

Novel 1H-purin-6(9H)-one (D) and 3H-imidazo[4,5-d][1,2,3]trazin-4(7H)-one (E) derivatives were designed, synthesized, and characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry spectra. Their herbicidal activity bioassay showed that compound 7d exhibited relatively good activity with 70.4% inhibition rate against Amaranthus retroflexus in postemergence treatments at 1500 g/ha. Antitumor activity indicated that most of the title compounds displayed potent antitumor activity at 20 μM, among all of the promising compounds possessing lower IC50 values than that of temozolomide, compound 7i demonstrated highest activity inhibiting both HepG-2 and U-118 MG cell lines with IC50 values of 2.0 and 3.8 μM, respectively. The structure–activity relationship analysis revealed that introduction of halogen atoms, a bulky bridging bond between benzene ring and nitrogen atom, longer R2 substituents could contribute to the improvement of antitumor activity. Analysis suggested that compound 7i might have potential as new highly active antitumor agent. Overall, D series had better anticancer activities than E series derivatives.

Nanostructuring Iridium Complexes into Crystalline Phosphorescent Nanoparticles: Structural Characterization, Photophysics, and Biological Applications.

One of the key challenges in materials science is to control the properties of a material by directing its supramolecular arrangement. Here we show that iridium complexes, such as FIrpic, Ir-PPY, and Ir-MDQ, can be organized into crystalline and phosphorescent nanoparticles through the nanoprecipitation method, which allows thorough modification of their functional properties. Moreover, we found that it is possible to combine different iridium complexes into a single multicomponent nanostructure, thus creating nanoparticles whose photonic properties derive from the close spatial proximity of the electronic excited states of the different Ir complexes. The morphology of all nanoparticles was fully characterized by microscopic and spectroscopic techniques, and their ordered arrangement was assessed by X-ray diffraction (XRD) and high-resolution transmission electron microscopy (TEM) measurements. We demonstrate that the nanostructuring of the complexes influences their optical and redox properties-by promoting a fine-tuning of emission, photoluminescence quantum yield, excited state lifetime, HOMO/LUMO energy levels, and energy-transfer processes-as well as their interaction with living cells. Investigations on glioblastoma U-251 MG cells demonstrate that nanostructuring represents an effective tool to regulate the efficiency of cell loading, cell viability, colocalization, and penetration in 3D spheroids.

Ecto-Calreticulin Expression After Particle Irradiation Versus Conventional Photon Irradiation in Human Cancer Cell Lines

Radiotherapy can activate anti-tumor immune responses by triggering immunogenic cell death (ICD) of tumor cells. ICD is characterized by the release of danger-associated molecular patterns (DAMPs). Calreticulin (CRT) is one of the most potent DAMPs. The cell surface translocation of CRT (ecto-CRT) serves as an “eat me” signal to antigen-presenting cells (APCs), which plays a pivotal role in activating anti-tumor immunity. However, there is limited information describing the impact of proton and carbon-ion irradiation on ecto-CRT expression. Thus, we investigated ecto-CRT expression in multiple human cancer cell lines irradiated by proton and carbon-ion in comparison to conventional photon irradiation. This study examined four human cancer cell lines: A549 (lung adenocarcinoma), U251 MG (glioma), Tca8113 (tongue squamous carcinoma) and CNE-2 (nasopharyngeal carcinoma). Cell lines were irradiated to 0,2,4,10Gy with proton, carbon ion, or photon. The expression levels of ecto-CRT were analyzed by flow cytometry at 12h, 24h and 48h post-irradiation. Mean Fluorescence Intensity (MFI) of all samples were calculated by cytometry analysis software. There was a dose-dependent increase of ecto-CRT expression after photon, proton or carbon-ion irradiation in all four cell lines. Higher dose irradiation induced more ecto-CRT expression (p<0.05). The expression levels of ecto-CRT were significantly increased at 48h post-irradiation rather than 12h or 24h post-irradiation (p<0.05). When comparing at the same relative biological effectiveness (RBE) dose, 10GyE, there was no significant difference of ecto-CRT expression changes at 48h post-irradiation with proton and carbon-ion in comparison to photon. The MFI fold changes of 10GyE dose at 48h post-irradiation compared to control group (0Gy) for A549, U251, Tca8113 and CNE-2 were 1.63-1.73, 1.29-1.79,1.54-1.69 and 2.21-2.59, respectively. Proton and carbon-ion as well as photon irradiation can enhance the ecto-CRT expression with a dose-dependent manner in human cancer cell lines.

Integral regulation of CHI3L1 gene and ERVW-1 locus expression by heparin in the gioblastoma cell lines U-87 MG and U-251 MG

Aim. To study the effect of heparin, as a factor of tumor microenvironment, on the expression of ERVW-1 locus and CHI3L1 gene, involved in increasing of cancer cells metastatic potential, in U-87 MG (U87) and U-251 MG (U251) gliotblastoma cells. Methods. U87 and U251 cells were cultured with or without the addition of heparin to the cultural medium. Total cellular RNA was isolated and used for cDNA synthesis and subsequent PCR with primers to different regions of ERVW-1 and CHI3L1. Results. Both U87 and U251 cells showed high level of expression of full-length ERVW-1 RNA. But only in U251 cells inhibition of the full-length transcript by heparin was revealed. Spliced isoform hasn’t been detected in any of variants. Though the solely env gene transcript expressed at low level in both lines and was inhibited similarly by heparin. Expression of CHI3L1 has been detected in both lines with primers towards exons 4-5 with some lowering in level under the heparin influence. Only in U87 cells the PCR-fragment with primers to exons 8-9 has been detected. In control U87 cells hypothetic spliced isoform of CHI3L1 transcript has been revealed with primers to exons 1-9. Conclusions. Heparin has complex and cell line-dependent regulation of expression of ERVW-1 locus and CHI3L1 gene in the glioblastoma cell lines U87 and U251.&#x0D; Keywords: glioblastoma, heparin, ERVW-1, CHI3L1.