Treatment Of Human Glioblastoma Research Articles

Rhizochalinin Exhibits Anticancer Activity and Synergizes with EGFR Inhibitors in Glioblastoma In Vitro Models.

Rhizochalinin (Rhiz) is a recently discovered cytotoxic sphingolipid synthesized from the marine natural compound rhizochalin. Previously, Rhiz demonstrated high in vitro and in vivo efficacy in various cancer models. Here, we report Rhiz to be highly active in human glioblastoma cell lines as well as in patient-derived glioma-stem like neurosphere models. Rhiz counteracted glioblastoma cell proliferation by inducing apoptosis, G2/M-phase cell cycle arrest, and inhibition of autophagy. Proteomic profiling followed by bioinformatic analysis suggested suppression of the Akt pathway as one of the major biological effects of Rhiz. Suppression of Akt as well as IGF-1R and MEK1/2 kinase was confirmed in Rhiz-treated GBM cells. In addition, Rhiz pretreatment resulted in a more pronounced inhibitory effect of γ-irradiation on the growth of patient-derived glioma-spheres, an effect to which the Akt inhibition may also contribute decisively. In contrast, EGFR upregulation, observed in all GBM neurospheres under Rhiz treatment, was postulated to be a possible sign of incipient resistance. In line with this, combinational therapy with EGFR-targeted tyrosine kinase inhibitors synergistically increased the efficacy of Rhiz resulting in dramatic inhibition of GBM cell viability as well as a significant reduction of neurosphere size in the case of combination with lapatinib. Preliminary in vitro data generated using a parallel artificial membrane permeability (PAMPA) assay suggested that Rhiz cannot cross the blood brain barrier and therefore alternative drug delivery methods should be used in the further in vivo studies. In conclusion, Rhiz is a promising new candidate for the treatment of human glioblastoma, which should be further developed in combination with EGFR inhibitors.

Ursolic acid nanoparticles for glioblastoma therapy

BackgroundGlioblastoma multiforme (GBM) is the most common and fatal primary tumor in the central nervous system (CNS). The effect of chemotherapy of GBM is limited due to the existence of blood-brain barrier (BBB). The aim of this study is to develop self-assembled nanoparticles (NPs) of ursolic acid (UA) for GBM treatment. MethodsUA NPs were synthesized by solvent volatilization method. Western blot analysis fluorescent staining and flow cytometry were launched to explore the anti-glioblastoma mechanism of UA NPs. The antitumor effects of UA NPs were further confirmed in vivo using intracranial xenograft models. ResultsUA were successfully prepared. In vitro, UA NPs could significantly increase the protein levels of cleaved-caspase 3 and LC3-II to strongly eliminate glioblastoma cells through autophagy and apoptosis. In the intracranial xenograft models, UA NPs could further effectively enter the BBB, and greatly improve the survival time of the mice. ConclusionsWe successfully synthesized UA NPs which could effectively enter the BBB and show strong anti-tumor effect which may have great potential in the treatment of human glioblastoma.

The in vitro and in vivo anticancer activities of Antrodia salmonea through inhibition of metastasis and induction of ROS-mediated apoptotic and autophagic cell death in human glioblastoma cells

Background:Antrodia salmonea (AS) exhibits anticancer activities against various cancers. ObjectiveThis study investigated the anticancer activities of AS on human glioblastoma (GBM8401 and U87MG) cells both in vitro and in vivo and explained the underlying molecular mechanism. MethodsMTT, colony formation, migration/invasion assay, immunoblotting, immunofluorescence, TUNEL, Annexin V/PI staining, AO staining, GFP-LC3 transfection, TEM, qPCR, siLC3, DCFH2-DA assay, and xenografted-nude mice were used to assess the potential of AS therapy. ResultsAS treatment retarded growth and suppressed colony formation in glioblastoma cells. AS attenuates EMT by suppressing invasion and migration, increasing E-cadherin expression, decreasing Twist, Snail, and N-cadherin expression, and inhibiting Wnt/β-catenin pathways in GBM8401 and U87MG cells. Furthermore, AS induced apoptosis by activating caspase-3, cleaving PARP, and dysregulating Bax and Bcl-2 in both cell lines. TUNEL assay and Annexin V/PI staining indicated AS-mediated late apoptosis. Interestingly, AS induced autophagic cell death by LC3-II accumulation, AVO formation, autophagosome GFP-LC3 puncta, p62/SQSTM1 expression, and ATG4B inhibition in GBM8401 and U87MG cells. TEM data revealed that AS favored autophagosome and autolysosome formation. The autophagy inhibitors 3-MA/CQ and LC3 knockdown suppressed AS-induced apoptosis in glioblastoma cells, indicating that the inhibition of autophagy decreased AS-induced apoptosis. Notably, the antioxidant N-acetylcysteine (NAC) inhibited AS-mediated ROS production and AS-induced apoptotic and autophagic cell death. Furthermore, AS induced ROS-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. AS reduced the tumor burden in GBM8401-xenografted nude mice and significantly modulated tumor xenografts by inducing anti-EMT, apoptosis, and autophagy. AS could be a potential antitumor agent in human glioblastoma treatment.

Potent anti-glioblastoma effect of 4-methylthio-3-butenyl isothiocyanate from Raphanus sativus and antioxidant activity

ABSTRACT Several scientific researchers have focused on biological activities of edible plants such as radish. However, there are no researches on the anticancer effect of this plant in human glioblastoma. Thus, aims of this study were to evaluate the phytochemical composition, antioxidant and anticancer activities of Raphanus sativus bulbs aqueous and methanol extracts against U-87 MG cells. Five phenol compounds were found in aqueous extract using HPLC-DAD with syringic acid followed by protocatechuic acid, gallic acid, p-coumaric acid and cirsilineol. The DPPH, ABTS, OH radical, iron (II) chelation and FRAP assays exhibited that aqueous extract shows high antioxidant activity. The chemical composition analysis of the methanol extract using GC-MS showed that 4-methylthio-3-butenyl isothiocyanate (raphasatin) was the most abundant compound (20%). Cytotoxicity assessment by MTT method showed that methanol extract exhibited a significant cytotoxicity to U-87 MG cells with IC50 at 241.35 ± 15.21 µg/mL after 72 h while no effect was noted for aqueous extract. Dis-adhesion assay of the extracts as well as of raphasatin was assessed using different protein matrices (fibronectin, fibrinogen and poly-L-lysine) and the anti-proliferative effect was performed using doxorubicin as positive control. Methanol extract showed a high ability to inhibit cell adhesion to the used protein matrices with IC50 of 20.46 ± 2.52; 12.23 ± 1.41 and 16.21 ± 1.92 µg/mL, respectively. Moreover, raphasatin strongly inhibited tumor cell adhesion with IC50 of 0.63 ± 0.09; 1.27 ± 0.18 and 0.95 ± 0.12 µg/mL, respectively. In addition, it totally blocked the proliferation of cancer cells at a dose of 0.5 µg/mL after 4 days of incubation. These data demonstrate, for the first time, the protective effect of R. sativus methanol extract and its major compound, 4-methylthio-3-butenyl isothiocyanate against U-87 MG cancer cells. Raphasatin may represent an innovative and promising tool for human glioblastoma treatment.

Abstract 6307: The botanical drug PBI-05204, a supercritical CO₂ extract of Nerium oleander, augments the antitumor efficacy of radiotherapy in treatment of human glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common as well as one of the most malignant types of brain cancer. Despite progress in development of novel therapies for the treatment of GBM, it remains largely incurable with a poor prognosis and a very low life expectancy. Recent studies have shown that oleandrin, a unique cardiac glycoside from Nerium oleander, as well as defined extract (PBI-05204) that contains this molecule, inhibit growth of human glioblastoma, and modulate glioblastoma patient-derived stem cell-renewal properties. The present study aimed to investigate the radiosensitization of PBI-05204 in glioblastoma using both in vitro and in vivo cancer models as well as to explore the potential mechanism of actions in GBM. The radiosensitizing effect of PBI-05204 was assessed against human GBM U87MG, U251, T98G and A172 cell lines as well as their relevant xenograft and orthotopic models. The induction of apoptosis, DNA damage and repair of DNA double strand breaks were assessed with determination of caspase 3 and 9 protein expression, DNA laddering, protein expression of rH2AX, Ku70, DNA-PKcs, and RAD51 as well as a Comet Assay. PBI-05204 treatment leads to an increased in vitro sensitivity of GBM cells, including U87MG, U251, T98G and A172 cells, to radiotherapy (RT) in which the main mechanisms are the transition from autophagy to apoptosis and enhanced DNA damage evidenced by increased expression of γH2AX. Additionally, relative increased expression of Ku70, DNA-PKcs and RAD51 due to RT were reduced by PBI-05204 in U87MG and U251 cells, suggesting PBI-05204 lessened RT mediated DNA repair. PBI-05204 significantly enhanced the RT mediated inhibition of tumor growth by 4.7-, 2.1- and 2.2-fold in U87MG, U251 and T98G xenograft models, respectively. The combination of RT and PBI-05204 showed a significantly enhancement of disease-free survival to 103.0 ± 63.2 days compared to the control group (p < 0.001) which was 3-fold longer than that of RT only group. Collectively, these results reveal that PBI-05204 enhances antitumor activity of RT in preclinical/murine models of human GBM. Given the fact that PBI-05204 has already been examined in Phase I and II clinical trials for cancer patients, its efficacy when combined with standard-of-care radiotherapy regimens in GBM should be explored in future clinical trials of this difficult to treat brain cancer. Citation Format: Alessandro Colapietro, Peiying Yang, Alessandra Rosetti, Andrea Mancini, Flora Vital, Stefano Martellucci, Francesco Marampon, Vincenzo Mattei, Giovanni Luca Gravina, Robert Newman, Claudio Festuccia. The botanical drug PBI-05204, a supercritical CO₂ extract of Nerium oleander, augments the antitumor efficacy of radiotherapy in treatment of human glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6307.

Capsaicin induces redox imbalance and ferroptosis through ACSL4/GPx4 signaling pathways in U87-MG and U251 glioblastoma cells.

Glioblastoma is one of the deadliest malignant gliomas. Capsaicin is a homovanillic acid derivative that can show anti-cancer effects by regulating various signaling pathways. The aim of this study is to investigate the effects of capsaicin on cell proliferation via ferroptosis in human U87-MG and U251 glioblastoma cells. Firstly, effects of capsaicin treatment on cell viability were determined by MTT analysis. Next, cellular-proliferation and cytotoxicity assays were determined by analyzing bromodeoxyuridine (BrdU) and lactate dehydrogenase (LDH) activity, respectively. Following, acyl-CoA synthetase long chain family member 4 (ACSL4), glutathione peroxidase 4 (GPx4), 5-hydroxyeicosatetraenoic acid (5-HETE), total oxidant status (TOS), malondialdehyde (MDA), total antioxidant status (TAS) and reduced glutathione (GSH) levels were determined by ELISA. Additionally, ACSL4 and GPx4 mRNA and protein levels were analyzed. Capsaicin showed a concentration-dependent anti-proliferative effects in U87-MG and U251 cells. Cell viability was decreased in the both cell lines treated with capsaicin concentrations above 50μM, while LDH activity increased. Treatment of 121.6, 188.5, and 237.2μM capsaicin concentrations for 24h indicated an increase in ACSL4, 5-HETE, TOS and MDA levels in U87-MG and U251 cells (p < 0.05). On the other hand, we found that capsaicin administration caused a decrease in BrdU, GPx4, TAS and GSH levels in U87-MG and U251 cells (p < 0.05). Besides, ACSL4 mRNA and protein levels were increased in the glioblastoma cells treated with capsaicin, whereas GPx4 mRNA and protein levels were decreased. Finally, capsaicin might be used as a potential anticancer agent with ferroptosis-induced anti-proliferative effects in the treatment of human glioblastoma.

Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform.

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical treatment, computed tomography (CT) and magnetic resonance imaging (MRI) were combined during the treatment-planning process. Positron emission tomography (PET) imaging was subsequently added to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI, and PET) using a micro-irradiation system proved to be labor-intensive because multimodal imaging, treatment planning, and dose delivery have to be completed sequentially in the preclinical setting. This also results in a workflow that is more prone to human error. Therefore, a user-friendly algorithm to further optimize preclinical multimodal imaging-based radiation treatment planning was implemented. This software tool was used to evaluate the accuracy and efficiency of dose painting radiation therapy with micro-irradiation by using an in silico study design. The new methodology for dose painting radiation therapy is superior to the previously described method in terms of accuracy, time efficiency, and intra- and inter-user variability. It is also an important step towards the implementation of inverse treatment planning on micro-irradiators, where forward planning is still commonly used, in contrast to clinical systems.

P13.21 PET-based dose painting radiation therapy strategy in a glioblastoma rat model using the small animal radiation research platform

Abstract BACKGROUND Previously, a rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was established. Similarly to the clinic, CT and MRI were combined during the treatment planning process. PET imaging was subsequently added which allowed us to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI and PET) using a micro-irradiation system, proved to be labour intensive because multimodal imaging, treatment planning and dose delivery have to be completed sequentially in the preclinical setting. MATERIAL AND METHODS Two different methodologies were compared in silico for performing preclinical [18F]FET PET based radiation therapy (20 Gy based on MRI, 8 Gy boost based on PET) based on three different cases. Method 1 is based on the previously published methods1,2. However, the process is automated using an in-house developed MATLAB code. Method 2 consists of a more sophisticated method where a series of isocenters and jaw dimensions for the motorised variable collimator were determined based on the [18F]FET PET uptake. Both methods were evaluated by means of the dose volume histograms (DVH) and Q-volume histograms. RESULTS The setup parameters for both methods were calculated. The DVHs for method 2 are systematically closer to the ideal dose distribution compared to method 1. These findings are confirmed by the D90 and D50 values which are considerably lower for method 1. When observing the Q-factor, method 2 always results in dose distributions that are closer to the dose objectives (method 1: 0.141±0.046; method 2: 0.064±0.011). CONCLUSION The described novel method to optimize the preclinical treatment planning process has many advantages in terms of dose delivery, time efficiency and variability, when compared to the previously used methods1,2. These improvements are important to narrow the gap between clinical and preclinical radiation research and for the development of new therapeutics and/or radiation therapy procedures for glioblastoma. 1. Bolcaen, J., Descamps, B., Boterberg, T., Vanhove, C. &amp; Goethals, I. PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator. J. Vis. Exp. 1–10 (2017) doi:10.3791/56601. 2. Verhoeven, J. et al. Technical feasibility of [18F]FET and [18F]FAZA PET guided radiotherapy in a F98 glioblastoma rat model. Radiat. Oncol. 14, (2019).

Nanoemulsion containing a synergistic combination of curcumin and quercetin for nose-to-brain delivery: In vitro and in vivo studies

Objective: To characterize a nanoemulsion containing a synergistic combination of curcumin (CUR) and quercetin (QUE) for brain targeting through the nose. Methods: The synergistic activity of the combination of two phytoconstituents, curcumin and quercetin (CUR-QUE) was determined at a 2:1 ratio. Nanoemulsions containing the synergistic combination were prepared using high-pressure homogenization technique. Nanoemulsion formulation was characterized for globule size, drug content, thermodynamic stability, zeta potential measurement, and drug release. Histological studies were performed using isolated nasal mucosa of sheep. Moreover, in vivo studies using allograft model were performed. Results: CUR-QUE in a 2:1 ratio combination showed the lowest combination index values 0.99 (combination index Conclusions: This study demonstrates that synergistic combination of CUR-QUE was optimal for treatment of human glioblastoma. Moreover, significant site-specific delivery to the brain was achieved by administering nanoemulsion through the nose. The study also reveals that intranasal delivery of nanoemulsion containing synergistic combination of CUR and QUE could be a promising strategy for brain tumor treatment.

Smarcd1 Inhibits the Malignant Phenotypes of Human Glioblastoma Cells via Crosstalk with Notch1

Smarcd1 is a component of an evolutionary conserved chromatin remodeling complex—SWI/SNF, which is involved in transcription factor recruitment, DNA replication, recombination, and repair. Suppression of the SWI/SNF complex required for cellular differentiation and gene regulation may be inducible for cell proliferation and tumorigenicity. However, the inhibitory role of Smarcd1 in human glioblastoma cells has not been well illustrated. Both U87 and U251 human glioblastoma cell lines were employed in the present study. The lentivirus-mediated gene knockdown and overexpression approach was conducted to determine the function of Smarcd1. The protein levels were tested by western blot, and the relative mRNA contents were detected by quantitative real-time PCR. Cell viability was tested by CCK-8 and colony-forming assay. Transwell assays were utilized to evaluate the motility and invasive ability. Flow cytometry was employed to analyze cell cycle and apoptosis. SPSS software was used for statistical analysis. Low expression of Smarcd1 was observed in glioblastoma cell lines and in patients with high-grade glioma. Importantly, the depletion of Smarcd1 promoted cell proliferation, invasion, and chemoresistance, whereas enhanced expression of Smarcd1 inhibited tumor-malignant phenotypes. Mechanistic research demonstrated that overexpression of Smarcd1 decreased the expression of Notch1, while knockdown of Notch1 increased the expression of Smarcd1 through Hes1 suppression. Hence, the crosstalk between Smarcd1 and Notch1, which formed a feedback loop, was crucial in regulation of glioblastoma malignant phenotypes. Furthermore, targeting Smarcd1 could be a potential strategy for human glioblastoma treatment.

Disulfiram, a Ferroptosis Inducer, Triggers Lysosomal Membrane Permeabilization by Up-Regulating ROS in Glioblastoma.

IntroductionDisulfiram (DSF), a drug used in the treatment of alcoholism since 1948, has been shown to have antitumor properties in various tumor types possibly due to the induction of a type cell death, ferroptosis, and the sensitization of cells to chemo- and radiotherapy. In this study, we explored the antitumor properties of DSF in glioblastoma (GBM) and investigated the underlying molecular mechanisms.MethodsGBM cell lines U251 and LN229 were treated with DSF to assess cytotoxicity and activity of the molecule in vitro. Response of cells to treatment was examined using cell viability, flow cytometry, LDH release assay, immunofluorescence and Western blot analysis.ResultsDSF inhibited cell growth of GBM U251 and LN229 cell lines in vitro in a concentration-dependent manner. Flow cytometry demonstrated that DSF caused G0-G1 growth arrest. DSF treatment led to increased ROS and lipid peroxidation levels relative to controls indicating the involvement of ferroptosis. Furthermore, DSF triggered lysosomal membrane permeabilization (LMP), a critical mechanism promoting cell death, in a ROS-dependent manner. Finally, DSF enhanced radiosensitivity of U251 and LN229 cells.DiscussionOur findings indicated that DSF induced ferroptosis and LMP and enhanced the radiosensitivity of GBM cells. Therefore, DSF might have efficient antitumor activity in the treatment of human GBM.

Transcriptomic Profiling of 3D Glioblastoma Tumoroids for the Identification of Mechanisms Involved in Anticancer Drug Resistance

Among various types of brain tumors, glioblastoma is the most malignant and highly aggressive brain tumor that possesses a high resistance against anticancer drugs. To understand the underlined mechanisms of tumor drug resistance, a new and more effective research approach is required. The three dimensional (3D) in vitro cell culture models could be a potential approach to study cancer features and biology, as well as screen for anti-cancer agents due to the close mimicry of the 3D tumor microenvironments. With our developed 3D alginate scaffolds, Ilumina RNA-sequencing was used to transcriptomically analyze and compare the gene expression profiles between glioblastoma cells in traditional 2-dimensional (2D) monolayer and in 3D Ca-alginate scaffolds at day 14. To verify the reliability and accuracy of Illumina RNA-Sequencing data, ATP-binding cassette transporter genes were chosen for quantitative real-time polymerase chain reaction) verification. The results showed that 7,411 and 3,915 genes of the 3D glioblastoma were up-regulated and down-regulated, respectively, compared with the 2D-cultured glioblastoma. Furthermore, the Kyoto Encyclopaedia of Genes and Genomes pathway analysis revealed that genes related to the cell cycle and DNA replication were enriched in the group of down-regulated gene. On the other hand, the genes involved in mitogen-activated protein kinase signaling, autophagy, drug metabolism through cytochrome P450, and ATP-binding cassette transporter were found in the up-regulated gene collection. 3D glioblastoma tumoroids might potentially serve as a powerful platform for exploring glioblastoma biology. They can also be valuable in anti-glioblastoma drug screening, as well as the identification of novel molecular targets in clinical treatment of human glioblastoma.

Erratum for Chen et al., "Treatment of Human Glioblastoma with a Live Attenuated Zika Virus Vaccine Candidate".

Volume 9, issue 5, e01683-18, 2018, [https://doi.org/10.1128/mBio.01683-18][1]. The fourteenth author’s name was misspelled. The byline should appear as shown above. [1]: /lookup/doi/10.1128/mBio.01683-18

Inhibition of ATM kinase upregulates levels of cell death induced by cannabidiol and γ-irradiation in human glioblastoma cells.

Despite advances in glioblastoma (GBM) therapy, prognosis of the disease remains poor with a low survival rate. Cannabidiol (CBD) can induce cell death and enhance radiosensitivity of GBM but not normal astrocytes. Inhibition of ATM kinase is an alternative mechanism for radiosensitization of cancer cells. In this study, we increased the cytotoxic effects of the combination of CBD and γ-irradiation in GBM cells through additional inhibition of ATM kinase with KU60019, a small molecule inhibitor of ATM kinase. We observed in GBM cells treated by CBD, γ-irradiation and KU60019 high levels of apoptosis together with strong upregulation of the percentage of G2/M-arrested cells, blockade of cell proliferation and a massive production of pro-inflammatory cytokines. Overall, these changes caused both apoptotic and non-apoptotic inflammation-linked cell death. Furthermore, via JNK-AP1 activation in concert with active NF-κB, CBD upregulated gene and protein expression of DR5/TRAIL-R2 and sensitize GBM cells to TRAIL-induced apoptosis. In contrast, CBD notably decreased in GBM surface levels of PD-L1, a critical immune checkpoint agent for T-lymphocytes. We also used in the present study TS543 human proneural glioma cells that were grown as spheroid culture. TS543 neurospheres exhibited dramatic sensitivity to CBD-mediated killing that was additionally increased in combination with γ-irradiation and KU60019. In conclusion, treatment of human GBM by the triple combination (CBD, γ-irradiation and KU60019) could significantly increase cell death levels in vitro and potentially improve the therapeutic ratio of GBM.

Zika virus has an oncolytic activity against human glioblastoma U87 cells

Glioblastoma is a highly lethal brain cancer. Virotherapy with the use of oncolytic viruses has since recently been regarded as a promising approach for the clinic treatment of human glioblastomas. The purpose of this work was to perform a primary evaluation of the Zika virus as a potential oncolytic agent against glioblastomas.In vitroexperiments showed that the Zika virus strain MR 766 is able to selectively infect and lyse neoplastic cells of the human glioblastoma cell line U87 MG. The selectivity index (SI, the ratio of infectious titer for tumor cells to titer on normal untransformed cells) was 2·102. The selectivity of the replicative activity of Zika virus in relation to U87 MG glioblastoma cells was additionally confrmed by indirect immunofluorescence. Using the model of immunodefcient SCID mice with subcutaneous xenografts of human glioblastoma U87 MG, a strong antitumor activity of the Zika virus under a course (daily for 4 days) of intratumoral administration of 5·105 TCID50 of Zika virus was shown. Treatment with Zika virus resulted in more than a 10­fold reduction in mean volumes of tumors. The tumor growth inhibition index was 92.63 %. Recurrences (metastases) of tumor re­growth were not registered within 64 days of observation. This result demonstrated the prospect of further in­depth studies of the Zika virus as a potential oncolytic agent against human glioblastomas.

Treatment of Human Glioblastoma with a Live Attenuated Zika Virus Vaccine Candidate.

Glioblastoma (GBM) is the deadliest type of brain tumor, and glioma stem cells (GSCs) contribute to tumor recurrence and therapeutic resistance. Thus, an oncolytic virus targeting GSCs may be useful for improving GBM treatment. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we investigated the safety and efficacy of a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a GSC-derived orthotopic model. Intracerebral injection of ZIKV-LAV into mice caused no neurological symptoms or behavioral abnormalities. The neurovirulence of ZIKV-LAV was more attenuated than that of the licensed Japanese encephalitis virus LAV 14-14-2, underlining the superior safety of ZIKV-LAV for potential GBM treatment. Importantly, ZIKV-LAV significantly reduced intracerebral tumor growth and prolonged animal survival by selectively killing GSCs within the tumor. Mechanistically, ZIKV infection elicited antiviral immunity, inflammation, and GSC apoptosis. Together, these results further support the clinical development of ZIKV-LAV for GBM therapy.IMPORTANCE Glioblastoma (GBM), the deadliest type of brain tumor, is currently incurable because of its high recurrence rate after traditional treatments, including surgery to remove the main part of the tumor and radiation and chemotherapy to target residual tumor cells. These treatments fail mainly due to the presence of a cell subpopulation called glioma stem cells (GSCs), which are resistant to radiation and chemotherapy and capable of self-renewal and tumorigenicity. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we tested a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a human GSC-derived orthotopic model. Our results showed that ZIKV-LAV retained good efficacy against glioblastoma by selectively killing GSCs within the tumor. In addition, ZIKV-LAV exhibited an excellent safety profile upon intracerebral injection into the treated animals. The good balance between the safety of ZIKV-LAV and its efficacy against human GSCs suggests that it is a potential candidate for combination with the current treatment regimen for GBM therapy.

Sinomenine Hydrochloride Inhibits the Metastasis of Human Glioblastoma Cells by Suppressing the Expression of Matrix Metalloproteinase-2/-9 and Reversing the Endogenous and Exogenous Epithelial-Mesenchymal Transition.

As shown in our previous study, sinomenine hydrochloride (SH), the major bioactive alkaloid isolated from Sinomenium acutum Rehd. et Wils. (Fam. Menispermaceae), initiates the autophagy-mediated death of human glioblastoma cells by generating reactive oxygen species and activating the autophagy-lysosome pathway. However, its effects on the migration and invasion of human glioblastoma cells have not yet been elucidated. Therefore, human glioblastoma U87 and SF767 cells were treated with SH (0.125 and 0.25 mM) for 24 h, and cell migration and invasion were assessed using scratch wound healing, migration and invasion assays. SH promoted G0/G1 phase arrest, inhibited the migration and invasion of the two cell lines, suppressed the activation of nuclear factor kappa B (NFκB) and the expression of matrix metalloproteinase (MMP)-2/-9, triggered endoplasmic reticulum (ER) stress, reversed the exogenous epithelial-mesenchymal transition (EMT) induced by the inflammatory microenvironment and the endogenous EMT. Additionally, NFκB p65 overexpression blocked the SH-mediated inhibitory effects on MMP-2/-9 expression and cell invasion. SH-induced autophagy was reduced in CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) or autophagy-related 5 (ATG5)-silenced human glioblastoma cells and cells treated with 4-phenylbutyric acid (4-PBA) or 3-methyladenine (3-MA), as shown by the decreased levels of the microtubule-associated protein light chain 3B (LC3B)-II and autophagic vacuoles (AVs) stained with monodansylcadaverine (MDC), respectively. Moreover, knockdown of CHOP or ATG5 and treatment with 4-PBA or 3-MA abolished the SH-mediated inhibition of mesenchymal markers (vimentin, Snail and Slug) expression and cell invasion, respectively. Importantly, SH also regulated the above related pathways in nude mice. Based on these findings, SH inhibited cell proliferation by inducing cell cycle arrest, and attenuated the metastasis of U87 and SF767 cells by suppressing MMP-2/-9 expression and reversing the endogenous and exogenous EMT in vitro and/or in vivo. Thus, SH might be a new potential anti-metastasis agent for the treatment of human glioblastoma.

Magnetoelectric nanoparticles for delivery of antitumor peptides into glioblastoma cells by magnetic fields.

We studied externally controlled anticancer effects of binding tumor growth inhibiting synthetic peptides to magnetoelectric nanoparticles (MENs) on treatment of glioblastomas. Hydrothermally synthesized 30-nm MENs had the core-shell composition of CoFe2O4@BaTiO3. Molecules of growth hormone-releasing hormone antagonist of the MIA class (MIA690) were chemically bound to MENs. In vitro experiments utilized human glioblastoma cells (U-87MG) and human brain microvascular endothelial cells. The studies demonstrated externally controlled high-efficacy binding of MIA690 to MENs, targeted specificity to glioblastoma cells and on-demand release of the peptide by application of d.c. and a.c. magnetic fields, respectively. The results support the use of MENs as an effective drug delivery carrier for growth hormone-releasing hormone antagonists in the treatment of human glioblastomas.

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV). At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

Sinomenine Hydrochloride Inhibits Human Glioblastoma Cell Growth through Reactive Oxygen Species Generation and Autophagy-Lysosome Pathway Activation: An In Vitro and In Vivo Study.

Glioblastoma is the most common malignant primary brain tumor, and it is one of the causes of cancer fatality in both adult and pediatric populations. Patients with glioblastoma require chemotherapy after surgical resection and radiotherapy. Therefore, chemotherapy constitutes a viable approach for the eradication of glioblastoma cells. In this study, the anti-tumor activity of sinomenine hydrochloride (SH) was evaluated in U87 and SF767 cells in vitro and in vivo. The results showed that SH potently inhibited U87 and SF767 cell viability and did not cause caspase-dependent cell death, as demonstrated by the absence of significant early apoptosis and caspase-3 cleavage. Instead, SH activated an autophagy-mediated cell death pathway, as indicated by the accumulated microtubule-associated protein light chain 3B (LC3B)-II, triggered autophagic flux and enhanced cell viability after pretreatment with autophagy inhibitors. SH-mediated autophagy in the two cell lines was implicated in reactive oxygen species (ROS) generation, protein kinase B (Akt)-mammalian target of rapamycin (mTOR) pathway suppression and c-Jun NH2-terminal kinase (JNK) pathway activation. The ROS antioxidant N-acetylcysteine (NAC), the Akt-specific activator insulin-like growth factor-1 (IGF-1) and the JNK-specific inhibitor SP600125 attenuated SH-induced autophagy. Moreover, ROS activated autophagy via the Akt-mTOR and JNK pathways. Additionally, SH treatment may promote lysosome biogenesis through activating transcription factor EB (TFEB). The in vivo study found that SH effectively suppressed glioblastoma growth without exhibiting significant toxicity. In conclusion, our findings reveal a novel mechanism of action of SH in cancer cells via the induction of autophagy through ROS generation and autophagy-lysosome pathway activation; these findings also supply a new potential therapeutic agent for the treatment of human glioblastoma.