U87 Tumor Research Articles

Use of FLOSEAL® as a scaffold and its impact on induced neural stem cell phenotype, persistence, and efficacy.

Induced neural stem cells (iNSCs) have emerged as a promising therapeutic platform for glioblastoma (GBM). iNSCs have the innate ability to home to tumor foci, making them ideal carriers for antitumor payloads. However, the in vivo persistence of iNSCs limits their therapeutic potential. We hypothesized that by encapsulating iNSCs in the FDA‐approved, hemostatic matrix FLOSEAL®, we could increase their persistence and, as a result, therapeutic durability. Encapsulated iNSCs persisted for 95 days, whereas iNSCs injected into the brain parenchyma persisted only 2 weeks in mice. Two orthotopic GBM tumor models were used to test the efficacy of encapsulated iNSCs. In the GBM8 tumor model, mice that received therapeutic iNSCs encapsulated in FLOSEAL® survived 30 to 60 days longer than mice that received nonencapsulated cells. However, the U87 tumor model showed no significant differences in survival between these two groups, likely due to the more solid and dense nature of the tumor. Interestingly, the interaction of iNSCs with FLOSEAL® appears to downregulate some markers of proliferation, anti‐apoptosis, migration, and therapy which could also play a role in treatment efficacy and durability. Our results demonstrate that while FLOSEAL® significantly improves iNSC persistence, this alone is insufficient to enhance therapeutic durability.

Cannabidiol inhibits RAD51 and sensitizes glioblastoma to temozolomide in multiple orthotopic tumor models.

BackgroundCannabidiol (CBD), a nonpsychoactive cannabinoid with a low toxicity profile, has been shown to produce antitumor activity across cancers in part through selective production of reactive oxygen species (ROS) in tumor cells. The alkylating agent, temozolomide (TMZ), is standard of care for treatment of glioblastoma (GBM). It can trigger increased ROS to induce DNA damage. It has also been reported that downregulating the expression of RAD51, an important DNA damage repair protein, leads to sensitization of GBM to TMZ.MethodsWe determined the extent to which CBD enhanced the antitumor activity of TMZ in multiple orthotopic models of GBM. In addition, we investigated the potential for CBD to enhance the antitumor activity of TMZ through production of ROS and modulation of DNA repair pathways.ResultsCBD enhanced the activity of TMZ in U87 MG and U251 GBM cell lines and in patient-derived primary GBM163 cells leading to stimulation of ROS, activation of the ROS sensor AMP-activated protein kinase (AMPK), and upregulation of the autophagy marker LC3A. CBD produced a sensitization of U87 and GBM163-derived intracranial (i.c.) tumors to TMZ and significantly increased survival of tumor-bearing mice. However, these effects were not observed in orthotopic models derived from GBM with intact methylguanine methyltransferase (MGMT) expression. We further demonstrate that CBD inhibited RAD51 expression in MGMT-methylated models of GBM, providing a potential mechanism for tumor sensitization to TMZ by CBD.ConclusionThese data support the potential therapeutic benefits of using CBD to enhance the antitumor activity of TMZ in GBM patients.

RGD Peptide-Conjugated Selenium Nanocomposite Inhibits Human Glioma Growth by Triggering Mitochondrial Dysfunction and ROS-Dependent MAPKs Activation

Chemotherapy is still one of the most common ways to treat human glioblastoma in clinic. However, severe side effects limited its clinic application. Design of cancer-targeted drugs with high efficiency and low side effect is urgently needed. Herein, silver nanoparticles (Ag NPs) and nano-selenium (Se NPs) conjugated with RGD peptides (Ag@Se@RGD NPs) to target integrin high-expressed glioma were designed. The results found that Ag@Se@RGD NPs displayed stable particle size and morphology in physiological condition, and induced significant integrin-targeted intracellular uptake. Ag@Se@RGD NPs in vitro dose-dependently inhibited U251 human glioma cells growth by induction of cells apoptosis through triggering the loss of mitochondrial membrane potential, overproduction of reactive oxygen species (ROS), and MAPKs activation. However, ROS inhibition dramatically attenuated Ag@Se@RGD NPs-induced MAPKs activation, indicating the significant role of ROS as an early apoptotic event. Importantly, Ag@Se@RGD NPs administration in vivov effectively inhibited U251 tumor xenografts growth by induction of apoptosis through regulation MAPKs activation. Taken together, our findings validated the rational design that Ag-Se NPs conjugated with RGD peptides was a promising strategy to combat human glioma by induction of apoptosis through triggering mitochondrial dysfunction and ROS-dependent MAPKs activation.

Characterization of a fluorescent 1,8-naphthalimide-functionalized PAMAM dendrimer and its Cu(ii) complexes as cytotoxic drugs: EPR and biological studies in myeloid tumor cells

The activity and interacting ability of a polyamidoamine (PAMAM) dendrimer modified with 4-N-methylpiperazine-1,8-naphthalimide units (termed D) and complexed by Cu(ii) ions, towards healthy and cancer cells were studied. Comparative electron paramagnetic resonance (EPR) studies of the Cu(ii)-D complex are presented: coordination mode, chemical structure, flexibility and stability of these complexes, in the absence and presence of myeloid cancer cells and peripheral blood mononuclear cells (PBMC). The interactions of Cu(ii) ions in the biological media at different equilibrium times were studied, highlighting different stability and interacting conditions with the cells. Furthermore, flow cytometry and confocal analysis, trace the peculiar properties of the dendrimers in PBMC and U937 cells. Indeed, a new probe (Fly) was used as a potential fluorescent tool for biological imaging of Cu(ii). The study highlights that dendrimer and, mainly, the Cu(ii) metallodendrimer are cytotoxic agents for the cells, specifically for U937 tumor cells, inducing mitochondrial dysfunction, ROS increase and lysosome involvement. The metallodendrimer shows antitumor selectivity, fewer affecting healthy PBMC, inducing a massive apoptotic cell death on U937 cells, in line with the high stability of this complex, as verified by EPR studies. The results underline the potentiality of this metallodendrimer to be used as anticancer drug.

EXTH-35. INCREASING THERAPEUTIC INDUCED NEURAL STEM CELL PERSISTENCE IN THE GLIOBLASTOMA TUMOR RESECTION CAVITY

Abstract BACKGROUND Induced neural stem cells (iNSCs) have emerged as a promising therapeutic platform for glioblastoma (GBM). iNSCs have the innate ability to home to tumor foci, making them ideal carriers for anti-tumor payloads. However, iNSC persist for only two weeks in the murine GBM tumor resection cavity. We hypothesized that by encapsulating iNSCs in a scaffold matrix, we could increase both the persistence of the cells the therapeutic durability. METHODS iNSCs expressing TRAIL were encapsulated in a gelatin-thrombin matrix; fibrinogen was used to polymerize the matrix. SEM was used to explore interactions between iNSCs and the scaffold matrix. To evaluate persistence, iNSCs encapsulated in the matrix were implanted into mock resection cavities of athymic nude mice and followed via BLI. To study the impacts of encapsulation on iNSC efficacy, athymic nude mice were implanted with U87 or GBM8 tumors. Tumors were then resected, and iNSCs encapsulated in the matrix were implanted; tumor volume was monitored via BLI. RESULTS SEM images showed homogeneous distribution of iNSCs throughout the matrix; iNSCs were completed encased in the fibrin clot component of the matrix and did not adhere to gelatin. In vivo, encapsulated iNSCs persisted for nearly 100 days whereas iNSCs directly injected into the brain parenchyma persisted < 20 days. Using mice bearing GBM8 tumors, animals treated with a high dose of therapeutic encapsulated iNSCs survived ~60 days longer than animals treated with non-therapeutic cells. A similar trend was observed in animals inoculated with U87 tumors. While not statistically significant, 25% of mice treated with iNSCs encapsulated in the gelatin-thrombin matrix survived longer than those treated with iNSCs encapsulated in a fibrin-only matrix, suggesting additional benefit due to the gelatin component. FUTURE DIRECTIONS: Prospective experiments will explore the impact of the scaffold on iNSC phenotype, including proliferation, differentiation, and migration markers.

DDRE-30. CELL-PENETRATING PEPTIDE ANTAGONIST OF CCAAT/ENHANCER BINDING PROTEIN ß DISPLAYS POTENT ANTI-GLIOBLASTOMA ACTIVITY

Abstract CCAAT/Enhancer Binding Protein Beta (C/EBPß) is a transcription factor overexpressed in glioblastoma (GBM). Mechanistically, C/EBPß is a master regulator of mesenchymal transition in GBM, and its increased expression correlates with mesenchymal differentiation and predicts poor clinical outcome. C/EBPß activity requires dimerization with co-factors such as CREB/ATF family members via leucine zipper interactions. ST101 is a novel peptide antagonist of C/EBPß currently being evaluated in a Phase 1/2 clinical study in patients with advanced unresectable and metastatic solid tumors. ST101 binds to the C/EBPß leucine zipper, thereby preventing dimer formation and inhibiting its transcriptional activity, resulting in selective tumor cell cytotoxicity. Here, we describe ST101 non-clinical anti-tumor activity against GBM. In vitro studies in T98G and U251 cells demonstrate ST101 dose-dependent impact of cell viability (EC50 of 2.2 and 1.2 μM, respectively), accompanied by significant impact on C/EBPß-mediated gene expression as determined by qPCR analysis. In contrast, normal human mononuclear and epithelial cells were not sensitive to ST101 (EC50 > 80 μM). In vivo, ST101 displayed significant anti-tumor activity in a U251 GBM subcutaneous xenograft model, resulting in 81.4% tumor growth inhibition (TGI) vs. control and undetectable tumors in 50% of animals. Following ST101 exposure tumors displayed reduced BIRC3 and ID2 gene expression, and significantly increased cleaved caspase 3 immunostaining indicative of cell death induction. In U251 tumors, subtherapeutic ST101 (< 5% TGI) in combination with temozolomide (< 5% TGI) resulted in 52.8% TGI, significantly greater than either single-agent alone. Similarly, in a temozolomide-refractory T98G GBM subcutaneous xenograft model, ST101 (41.6% TGI) in combination with TMZ (< 5% TGI) resulted in significant anti-GBM response (72.4% TGI). These data emphasize the potential of ST101 as a potent peptide therapeutic for GBM.

EXTH-07. TEMPORAL ASSESSMENT OF ALTERED PERI-ABLATION BLOOD-BRAIN BARRIER PERMEABILITY FOLLOWING TUMOR CYTOREDUCTION BY THERMAL THERAPY IN A RAT ORTHOTOPIC GLIOBLASTOMA MODEL

Abstract Laser interstitial thermal therapy (LITT) is a minimally invasive tumor cytoreductive treatment for recurrent gliomas, brain tumors in eloquent regions and/or otherwise inaccessible. Following reports of persistent peri-ablation blood-brain barrier (BBB) opening in humans, we examined this phenomenon using a rat glioblastoma model. Athymic female rats were implanted with U251 tumor cells in one brain hemisphere. Tumor growth was monitored using magnetic resonance imaging (MRI) and dynamic contrast enhanced (DCE)-MRI. When tumors reached about 4 mm in diameter, they were ablated under supervision of diffusion-weighted MRI using Visualase®, a clinical LITT system. Four rats were used as controls. Longitudinal MRI data were obtained before LITT, and at post-LITT 2 (n=9), 3 (n=3) and 4 (n=9) weeks. After the terminal MRI at each time point, rats were injected intravenously with fluorescent isothiocyanate dextran (FITC-dextran; 2000 kDa) and Evans Blue (68 kDa after binding to plasma albumin) and the brains immersion fixed in 10% paraformaldehyde. The brains were cut into 100 μM thick slices in a vibratome and examined for the distribution of the two fluorophores. All rats survived the LITT procedure. The sham controls showed increased tumor burden by 2 weeks and were sacrificed. DCE-MRI data and fluorescent data showed elevated BBB permeability in peri-ablation regions, with leakage of a gadolinium contrast on DCE-MRI and of Evans Blue, but not of FITC-dextran. Histology showed little tumor tissue at 2 weeks, but evidence of recurrence at ablation margins at later times. These data demonstrate that LITT is adaptable to rat glioma models and can be performed under MRI monitoring. Peri-ablation regions showed selective increase in BBB permeability acutely due to sublethal heating, but later increases in permeability may be due to tumor recurrence. We suggest this model is useful for examining the temporal and spatial development of peri-ablation BBB opening following LITT.

A Novel Hydroxamic Acid-Based Curcumin Derivative as Potent Histone Deacetylase Inhibitor for the Treatment of Glioblastoma.

Glioblastoma (GBM) is one of the most common primary and deadliest malignant brain tumor with chemoresistance and poor prognosis. There is a lack of effective chemotherapeutic drug for the treatment of GBM. In this work, we reported the preparation of a histone deacetylase (HDAC) inhibitor, DMC-HA, from the structural modification of natural product curcumin. DMC-HAs were tested in an HDAC inhibition assay and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cytotoxicity. It showed potent inhibition of HDAC1–2 and HDAC6 with IC50 values in the submicromolar concentration range. DMC-HA significantly inhibited the proliferation of human glioblastoma U87 cells and mediated apoptosis of U87 cells in a dose- and time-dependent manner. In addition, DMC-HA elevated the acetylation level of histone H3 in U87 cells. Pharmacokinetic studies showed that DMC-HA possessed acceptable pharmacokinetic profiles, accompanied with certain brain permeability. Lastly, we showed that DMC-HA suppressed the growth of tumor in U87 tumor xenograft model in vivo with no obvious toxicity. These results demonstrate that DMC-HA has the potential to be developed as a chemotherapeutic drug for GBM patients.

LncRNA CDKN2B-AS1 contributes to glioma development by regulating the miR-199a-5p/DDR1 axis.

Although cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) is upregulated in glioma, its function and potential mechanism in glioma remain unclear. CDKN2B-AS1 level in glioma tissues and cell lines LN229, U251, and U87 was measured by qRT-PCR. Loss-of-function assays using short hairpin RNA for CDKN2B-AS1 (sh-CDKN2B-AS1) were performed to evaluate the effect of CDKN2B-AS1 on cell invasion, migration, proliferation, and apoptosis. The relationship among CDKN2B-AS1, miR-199a-5p, and DDR1 was determined by bioinformatics analysis and luciferase reporter assay. Rescue experiments were conducted to explore the function of CDKN2B-AS1 and miR-199a-5p in glioma. An in vivo animal model of lentivirally transduced U87 glioma xenografts in mice was established to confirm the role of CDKN2B-AS1. CDKN2B-AS1 is significantly upregulated in glioma tissues and cell lines. CDKN2B-AS1 knockdown significantly inhibits cell proliferation, invasion, and migration, while promoting apoptosis of glioma cell lines U251 and U87. Further, a miR-199a-5p inhibitor attenuates the inhibitory effects of sh-CDKN2B-AS1 on these cell phenotypes. CDKN2B-AS1 positively regulates DDR1 expression by directly sponging miR-199a-5p. Moreover, CDKN2B-AS1 knockdown efficiently inhibits U87 tumor xenograft growth in mice. Our study reveals that CDKN2B-AS1 promotes glioma development by regulating the miR-199a-5p/DDR1 axis, suggesting that this lncRNA might be a potential therapeutic target.

Prolonged sub-lethal exposure to galaxolide (HHCB) and tonalide (AHTN) promotes the metastatic potential of glioblastoma tumor spheroids

Galaxolide and tonalide are well-known polycyclic musks whose intensive use without limitations in numerous cleaning, hygiene, and personal care products has resulted in widespread direct human exposure via absorption, inhalation, and oral ingestion. Latest data shows that long-term, low-dose exposure to toxic chemicals can induce unpredictable harmful effects in a variety of living systems, however, interactions between synthetic musks and brain tumours remain largely unexplored. Glioblastoma (GB) accounts for nearly half of all tumours of the central nervous system and is characterized by very poor prognosis. The aims of this study were (1) to investigate the potential effect of long-term (20-generation) single and combined application of galaxolide and tonalide at sub-lethal doses (5–2.5 u M) on the angiogenesis, invasion, and migration of human U87 cells or tumour spheroids, and (2) to explore the underlying molecular mechanisms. Random amplified polymorphic DNA assays revealed significant DNA damage and increased total mutation load in galaxolide- and/or tonalide-treated U87 cells. In those same groups, we also detected remarkable tumour spheroid invasion and up-regulation of both HIF1-α/VEGF/MMP9 and IL6/JAK2/STAT3 signals, known to have important roles in hypoxia-related angiogenesis and/or proliferation. Prolonged musk treatment further altered angio-miRNA expression in a manner consistent with poor prognosis in GB. We also detected significant over-expression of the genes Slug, Snail, ZEB1, and Vimentin, which are biomarkers of epithelial to mesenchymal transition. In addition, matrigel, transwell, and wound healing assays clearly showed that long-term sub-lethal exposure to galaxolide and/or tonalide induced invasion and migration proposing a high metastatic potential. Our results suggest that assessing expression of HIF-1a, VEGF, STAT3, and the miR-17-92 cluster in biopsy samples of GB patients who have a history of possible long-term exposure to galaxolide or tonalide could be beneficial for deciding a therapy regime. Additionally, we recommend that extensively-used hygiene and cleaning materials be selected from synthetic musk-free products, especially when used in palliative care processes for GB patients.

Imaging Hallmarks of the Tumor Microenvironment in Glioblastoma Progression.

Glioblastoma progression involves multifaceted changes in vascularity, cellularity, and metabolism. Capturing such complexities of the tumor niche, from the tumor core to the periphery, by magnetic resonance imaging (MRI) and spectroscopic imaging (MRSI) methods has translational impact. In human-derived glioblastoma models (U87, U251) we made simultaneous and longitudinal measurements of tumor perfusion (Fp), permeability (Ktrans), and volume fractions of extracellular (ve) and blood (vp) spaces from dynamic contrast enhanced (DCE) MRI, cellularity from apparent diffusion coefficient (ADC) MRI, and extracellular pH (pHe) from an MRSI method called Biosensor Imaging of Redundant Deviation in Shifts (BIRDS). Spatiotemporal patterns of these parameters during tumorigenesis were unique for each tumor. While U87 tumors grew faster, Fp, Ktrans, and vp increased with tumor growth in both tumors but these trends were more pronounced for U251 tumors. Perfused regions between tumor periphery and core with U87 tumors exhibited higher Fp, but Ktrans of U251 tumors remained lowest at the tumor margin, suggesting primitive vascularization. Tumor growth was uncorrelated with ve, ADC, and pHe. U87 tumors showed correlated regions of reduced ve and lower ADC (higher cellularity), suggesting ongoing proliferation. U251 tumors revealed that the tumor core had higher ve and elevated ADC (lower cellularity), suggesting necrosis development. The entire tumor was uniformly acidic (pHe 6.1-6.8) early and throughout progression, but U251 tumors were more acidic, suggesting lower aerobic glycolysis in U87 tumors. Characterizing these cancer hallmarks with DCE-MRI, ADC-MRI, and BIRDS-MRSI will be useful for exploring tumorigenesis as well as timely therapies targeted to specific vascular and metabolic aspects of the tumor microenvironment.

Magnetic and GRPR-targeted reduced graphene oxide/doxorubicin nanocomposite for dual-targeted chemo-photothermal cancer therapy

Herein, we design a rGO-based magnetic nanocomposite by decorating rGO with citrate-coated magnetic nanoparticles (CMNP). The magnetic rGO (mrGO) was modified by phospholipid-polyethylene glycol to prepare PEGylated mrGO, for conjugating with gastrin-releasing peptide receptor (GRPR)-binding peptide (mrGOG). The anticancer drug doxorubicin (DOX) was bound to mrGO (mrGOG) by π-π stacking for drug delivery triggered by the low pH value in the endosome. The mrGOG showed enhanced photothermal effect under NIR irradiation, endorsing its role for dual targeted DOX delivery. With efficient DOX release in the endosomal environment and heat generation from light absorption in the NIR range, mrGOG/DOX could be used for combination chemo-photothermal therapy after intracellular uptake by cancer cells. We characterized the physico-chemical as well as biological properties of the synthesized nanocomposites. The mrGOG is stable in biological buffer solution, showing high biocompatibility and minimum hemolytic properties. Using U87 glioblastoma cells, we confirmed the magnetic drug targeting effect in vitro for selective cancer cell killing. The peptide ligand-mediated targeted delivery increases the efficiency of intracellular uptake of both nanocomposite and DOX up to ~3 times due to the over-expressed GRPR on U87 surface, leading to higher cytotoxicity. The increased cytotoxicity using mrGOG over mrGO was shown from a decreased IC50 value (0.70 to 0.48 μg/mL) and an increased cell apoptosis rate (19.8% to 47.1%). The IC50 and apoptosis rate changed further to 0.19 μg/mL and 76.8% in combination with NIR laser irradiation, with the photothermal effect supported from upregulation of heat shock protein HSP70 expression. Using U87 tumor xenograft model created in nude mice, we demonstrated that magnetic guidance after intravenous delivery of mrGOG/DOX could significantly reduce tumor size and prolong animal survival over free DOX and non-magnetic guided groups. Augmented with NIR laser treatment for 5 min, the anti-cancer efficacy significantly improves with elevated cell apoptosis and reduced cell proliferation. Together with safety profiles from hematological as well as major organ histological analysis of treated animals, the mrGOG nanocomposite is an effective nanomaterial for combination chemo-photothermal cancer therapy.

Nestin Is Required for Spindle Assembly and Cell-Cycle Progression in Glioblastoma Cells.

Nestin, a class IV intermediate filament protein, is generally considered as a putative marker of neural stem and progenitor cells in the central nervous system. Glioma is a common type of adult brain tumors, and glioblastoma (GBM) represents the most aggressive form of glioma. Here, we report that Nestin expression is significantly upregulated in human GBM, compared with other types of glioma. Nestin knockdown or deletion in U251 cells and tumor cells from GBM patients derived xenografts resulted in G2-M arrest, finally leading to apoptosis in tumor cells. Using proximity-dependent biotin identification method, we identified βII-tubulin as an interacting protein of Nestin in U251 cells. Nestin stabilized βII-tubulin in U251 cells through physical interaction. Knockdown of Nestin or βII-tubulin disrupted spindle morphology in tumor cells. Our studies further revealed that Nestin deficiency in U251 cells and GBM PDX cells repressed tumor growth upon transplantation. Finally, we found that Nestin deficiency sensitized GBM cells to microtubule-destabilizing drugs such as vinblastine and vincristine. Our studies demonstrate the essential functions and underlying mechanisms of Nestin in the growth and drug response of GBM cells. IMPLICATIONS: Through interaction with βII-tubulin, Nestin facilitates cell-cycle progression and spindle assembly of tumor cells in glioblastoma.

Detection of metabolic change in glioblastoma cells after radiotherapy using hyperpolarized 13 C-MRI.

Dynamic nuclear polarization (DNP) of 13C‐labeled substrates enables the use of magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors and offers an opportunity to investigate these differences. In this study, DNP‐MRI chemical shift imaging with hyperpolarized [1‐13C] pyruvate was conducted to evaluate the metabolic change in glycolytic profiles after radiation of two glioma stem‐like cell‐derived gliomas (GBMJ1 and NSC11) and an adherent human glioblastoma cell line (U251) in an orthotopic xenograft mouse model. The DNP‐MRI showed an increase in Lac/Pyr at 6 and 16 h after irradiation (18% ± 4% and 14% ± 3%, respectively; mean ± SEM) compared with unirradiated controls in GBMJ1 tumors, whereas no significant change was observed in U251 and NSC11 tumors. Metabolomic analysis likewise showed a significant increase in lactate in GBMJ1 tumors at 16 h. An immunoblot assay showed upregulation of lactate dehydrogenase‐A expression in GBMJ1 following radiation exposure, consistent with DNP‐MRI and metabolomic analysis. In conclusion, our preclinical study demonstrates that the DNP‐MRI technique has the potential to be a powerful diagnostic method with which to evaluate GBM tumor metabolism before and after radiation in the clinical setting.

Cannabinoid WIN 55,212-2 Inhibits Human Glioma Cell Growth by Triggering ROS-Mediated Signal Pathways.

Glioblastoma is a highly invasive primary malignant tumor of the central nervous system. Cannabinoid analogue WIN 55,212-2 (WIN) exhibited a novel anticancer effect against human tumors. However, the anticancer potential and underlying mechanism of WIN against human glioma remain unclear. Herein, the anticancer efficiency and mechanism of WIN in U251 human glioma cells were investigated. The results showed that WIN dose-dependently inhibited U251 cell proliferation, migration, and invasion in vitro. WIN treatment also effectively suppressed U251 tumor spheroids growth ex vivo. Further studies found that WIN induced significant apoptosis as convinced by the caspase-3 activation and release of cytochrome C. Mechanism investigation revealed that WIN triggered ROS-mediated DNA damage and caused dysfunction of VEGF-AKT/FAK signal axis. However, ROS inhibition effectively attenuated WIN-induced DNA damage and dysfunction of VEGF-AKT/FAK signal axis and eventually improved U251 cell proliferation, migration, and invasion. Taken together, our findings validated that WIN had the potential to inhibit U251 cell proliferation, migration, and invasion and induce apoptosis by triggering ROS-dependent DNA damage and dysfunction of VEGF-AKT/FAK signal axis.

Imaging 6-Phosphogluconolactonase Activity in Brain Tumors In Vivo Using Hyperpolarized δ-[1-13C]gluconolactone.

IntroductionThe pentose phosphate pathway (PPP) is essential for NADPH generation and redox homeostasis in cancer, including glioblastomas. However, the precise contribution to redox and tumor proliferation of the second PPP enzyme 6-phosphogluconolactonase (PGLS), which converts 6-phospho-δ-gluconolactone to 6-phosphogluconate (6PG), remains unclear. Furthermore, non-invasive methods of assessing PGLS activity are lacking. The goal of this study was to examine the role of PGLS in glioblastomas and assess the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive imaging.MethodsTo interrogate the function of PGLS in redox, PGLS expression was silenced in U87, U251 and GS2 glioblastoma cells by RNA interference and levels of NADPH and reduced glutathione (GSH) measured. Clonogenicity assays were used to assess the effect of PGLS silencing on glioblastoma proliferation. Hyperpolarized δ-[1-13C]gluconolactone metabolism to 6PG was assessed in live cells treated with the chemotherapeutic agent temozolomide (TMZ) or with vehicle control. 13C 2D echo-planar spectroscopic imaging (EPSI) studies of hyperpolarized δ-[1-13C]gluconolactone metabolism were performed on rats bearing orthotopic glioblastoma tumors or tumor-free controls on a 3T spectrometer. Longitudinal 2D EPSI studies of hyperpolarized δ-[1-13C]gluconolactone metabolism and T2-weighted magnetic resonance imaging (MRI) were performed in rats bearing orthotopic U251 tumors following treatment with TMZ to examine the ability of hyperpolarized δ-[1-13C]gluconolactone to report on treatment response.ResultsPGLS knockdown downregulated NADPH and GSH, elevated oxidative stress and inhibited clonogenicity in all models. Conversely, PGLS expression and activity and steady-state NADPH and GSH were higher in tumor tissues from rats bearing orthotopic glioblastoma xenografts relative to contralateral brain and tumor-free brain. Importantly, [1-13C]6PG production from hyperpolarized δ-[1-13C]gluconolactone was observed in live glioblastoma cells and was significantly reduced by treatment with TMZ. Furthermore, hyperpolarized δ-[1-13C]gluconolactone metabolism to [1-13C]6PG could differentiate tumor from contralateral normal brain in vivo. Notably, TMZ significantly reduced 6PG production from hyperpolarized δ-[1-13C]gluconolactone at an early timepoint prior to volumetric alterations as assessed by anatomical imaging.ConclusionsCollectively, we have, for the first time, identified a role for PGLS activity in glioblastoma proliferation and validated the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive in vivo imaging of glioblastomas and their response to therapy.

Imaging the transmembrane and transendothelial sodium gradients in gliomas

Under normal conditions, high sodium (Na+) in extracellular (Na+e) and blood (Na+b) compartments and low Na+ in intracellular milieu (Na+i) produce strong transmembrane (ΔNa+mem) and weak transendothelial (ΔNa+end) gradients respectively, and these manifest the cell membrane potential (Vm) as well as blood–brain barrier (BBB) integrity. We developed a sodium (23Na) magnetic resonance spectroscopic imaging (MRSI) method using an intravenously-administered paramagnetic polyanionic agent to measure ΔNa+mem and ΔNa+end. In vitro 23Na-MRSI established that the 23Na signal is intensely shifted by the agent compared to other biological factors (e.g., pH and temperature). In vivo 23Na-MRSI showed Na+i remained unshifted and Na+b was more shifted than Na+e, and these together revealed weakened ΔNa+mem and enhanced ΔNa+end in rat gliomas (vs. normal tissue). Compared to normal tissue, RG2 and U87 tumors maintained weakened ΔNa+mem (i.e., depolarized Vm) implying an aggressive state for proliferation, whereas RG2 tumors displayed elevated ∆Na+end suggesting altered BBB integrity. We anticipate that 23Na-MRSI will allow biomedical explorations of perturbed Na+ homeostasis in vivo.

Imaging of Fibroblast Activation Protein in Cancer Xenografts Using Novel (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine-Based Small Molecules.

Fibroblast activation protein (FAP) has become a favored target for imaging and therapy of malignancy. We have synthesized and characterized two new (4-quinolinoyl)-glycyl-2-cyanopyrrolidine-based small molecules for imaging of FAP, QCP01 and [111In]QCP02, using optical and single-photon computed tomography/CT, respectively. Binding of imaging agents to FAP was assessed in six human cancer cell lines of different cancer types: glioblastoma (U87), melanoma (SKMEL24), prostate (PC3), NSCLC (NCIH2228), colorectal carcinoma (HCT116), and lung squamous cell carcinoma (NCIH226). Mouse xenograft models were developed with FAP-positive U87 and FAP-negative PC3 cells to test pharmacokinetics and binding specificity in vivo. QCP01 and [111In]QCP02 demonstrated nanomolar inhibition of FAP at Ki values of 1.26 and 16.20 nM, respectively. Both were selective for FAP over DPP-IV, a related serine protease. Both enabled imaging of FAP-expressing tumors specifically in vivo. [111In]QCP02 showed high uptake at 18.2 percent injected dose per gram in the U87 tumor at 30 min post-administration.

Magnetic cobalt oxide nanosheets: green synthesis and in vitro cytotoxicity.

Cobalt oxide nanoparticles were prepared via green chemistry route and fully characterized by Field Emission Scanning Electron Microscope (FESEM), Energy-dispersive X-ray spectroscopy (EDAX), X-ray diffraction (XRD), High-resolution transmission electron microscopy (HRTEM) and Transmission electron microscopy (TEM) analyses; the CoO and Co3O4 nanoparticles, in sheet-shaped cobalt oxide form, ensued simultaneously in one step. The varying concentrations of NPs were analyzed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test on the cancer cell line (U87) which revealed that with increasing concentration of cobalt oxide nanoparticles, the survival rate of U87 tumor cells decreases; IC50 of nanoparticles being ~ 55µg/ml-1.

Elaborated study of Cu(II) carbosilane metallodendrimers bearing substituted iminopyridine moieties as antitumor agents.

Iminopyridine-decorated carbosilane metallodendrimers have recently emerged as a promising strategy in the treatment of cancer diseases. Their unique features such as the nanometric size, the multivalent nature and the structural perfection offer an extraordinary platform to explore structure-to-property relationships. Herein, we showcase the outstanding impact on the antitumor activity of a parameter not explored before: the iminopyridine substituents in meta position. New Cu(II) carbosilane metallodendrimers, bearing methyl or methoxy substituents in the pyridine ring, were synthesized and thoroughly characterized. Electron Paramagnetic Resonance (EPR) was exploited to unveil the properties of the metallodendrimers. This study confirmed the presence of different coordination modes of the Cu(II) ion (Cu-N2O2, Cu-N4 and Cu-O4), whose ratios were determined by the structural features of the dendritic molecules. These metallodendrimers exhibited IC50 values in the low micromolar range (<6μM) in tumor cell lines such as HeLa and MCF-7. The subsequent invitro assays on both healthy (PBMC) and tumor (U937) myeloid cells revealed two key facts which improved the cytotoxicity and selectivity of the metallodrug: First, maximizing the Cu-N2O2 coordination mode; second, adequately selecting the pair ring-substituent/metal-counterion. The most promising candidates, G1(-CH3)Cl (8) and G1(-OCH3)NO3(17), exhibited a substantial increase in the antitumor activity in U937 tumor cells, compared to the non-substituted counterparts, probably through two different ROS-production pathways.