U87 MG Research Articles

Enzalutamide Induces Apoptotic Insults to Human Drug-Resistant and -Sensitive Glioblastoma Cells via an Intrinsic Bax-Mitochondrion-Cytochrome C Caspase Cascade Activation Pathway.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor. Temozolomide (TMZ) is the first-line chemotherapeutic drug for treating GBM. However, drug resistance is still a challenging issue in GBM therapy. Our preliminary results showed upregulation of androgen receptor (AR) gene expression in human GBM tissues. This study was designed to evaluate the effects of enzalutamide, a specific inhibitor of the AR, on killing drug-resistant and -sensitive glioblastoma cells and the possible mechanisms. Data mining from The Cancer Genome Atlas (TCGA) database revealed upregulation of AR messenger (m)RNA and protein expressions in human GBM tissues, especially in male patients, compared to normal human brains. In addition, expressions of AR mRNA and protein in human TMZ-sensitive U87 MG and -resistant U87 MG-R glioblastoma cells were elevated compared to normal human astrocytes. Exposure of human U87 MG and U87 MG-R cells to enzalutamide concentration- and time-dependently decreased cell viability. As to the mechanism, enzalutamide killed these two types of glioblastoma cells via an apoptotic mechanism. Specifically, exposure to enzalutamide augmented enzyme activities of caspase-9 rather than those of caspase-8. Moreover, enzalutamide successively triggered an elevation in levels of the proapoptotic Bax protein, a reduction in the mitochondrial membrane potential, release of cytochrome c, cascade activation of caspases-3 and -6, DNA fragmentation, and cell apoptosis in human TMZ-sensitive and -resistant glioblastoma cells. Pretreatment with Z-VEID-FMK, an inhibitor of caspase-6, caused significant attenuations in enzalutamide-induced morphological shrinkage, DNA damage, and apoptotic death. Taken together, this study showed that enzalutamide could significantly induce apoptotic insults to human drug-resistant and -sensitive glioblastoma cells via an intrinsic Bax-mitochondrion-cytochrome c-caspase cascade activation pathway. Enzalutamide has the potential to be a drug candidate for treating GBM by targeting the AR signaling axis.

Hypericin, a potential new BH3 mimetic.

Many types of cancer such as prostate cancer, myeloid leukemia, breast cancer, glioblastoma display strong chemo resistance, which is supported by enhanced expression of multiple anti-apoptotic Bcl-2, Bcl-XL and Mcl-1 proteins. The viable anti-cancer strategies are based on developing anti-apoptotic Bcl-2 proteins inhibitors, BH3 mimetics. Our focus in past years has been on the investigating a new potential BH3 mimetic, Hypericin (Hyp). Hyp is a naturally occurring photosensitive compound used in photodynamic therapy and diagnosis. We have demonstrated that Hyp can cause substantial effects in cellular ultrastructure, mitochondria function and metabolism, and distribution of Bcl2 proteins in malignant and non-malignant cells. One of the possible mechanisms of Hyp action could be the direct interactions between Bcl-2 proteins and Hyp. We investigated this assumption by in silico computer modelling and in vitro fluorescent spectroscopy experiments with the small Bcl2 peptide segments designed to correspond to Bcl2 BH3 and BH1 domains. We show here that Hyp interacts with BH3 and BH1 peptides in concentration dependent manner, and shows the stronger interactions than known BH3 mimetics, Gossypol (Goss) and ABT-263. In addition, interactions of Hyp, Goss and ABT263, with whole purified proteins Bcl-2 and Mcl-1 by fluorescence spectroscopy show that Hyp interacts stronger with the Bcl-2 and less with Mcl-1 protein than Goss or ABT-263. This suggest that Hyp is comparable to other BH3 mimetics and could be explore as such. Hyp cytotoxicity was low in human U87 MG glioma, similar to that of ABT263, where Goss exerted sufficient cytotoxicity, suggesting that Hyp acts primarily on Bcl-2, but not on Mcl-1 protein. In combination therapy, low doses of Hyp with Goss effectively decreased U87 MG viability, suggesting a possible synergy effect. Overall, we can conclude that Hyp as BH3 mimetic acts primarily on Bcl-2 protein and can be explored to target cells with Bcl-2 over-expression, or in combination with other BH3 mimetics, that target Mcl-1 or Bcl-XL proteins, in dual therapy.

Efficiently targeted therapy of glioblastoma xenograft via multifunctional biomimetic nanodrugs

BackgroundGlioblastoma multiforme (GBM) is a fatal malignant primary brain tumor in adults. The therapeutic efficacy of chemotherapeutic drugs is limited due to the blood-brain barrier (BBB), poor drug targeting, and short biological half-lives. Multifunctional biomimetic nanodrugs have great potential to overcome these limitations of chemotherapeutic drugs.MethodsWe synthesized and characterized a biomimetic nanodrug CMS/PEG-DOX-M. The CMS/PEG-DOX-M effectively and rapidly released DOX in U87 MG cells. Cell proliferation and apoptosis assays were examined by the MTT and TUNEL assays. The penetration of nanodrugs through the BBB and anti-tumor efficacy were investigated in the orthotopic glioblastoma xenograft models.ResultsWe showed that CMS/PEG-DOX-M inhibited cell proliferation of U87 MG cells and effectively induced cell apoptosis of U87 MG cells. Intracranial antitumor experiments showed that free DOX hardly penetrated the BBB, but CMS/PEG-DOX-M effectively reached the orthotopic intracranial tumor through the BBB and significantly inhibited tumor growth. Immunofluorescence staining of orthotopic tumor tissue sections confirmed that nanodrugs promoted apoptosis of tumor cells. This study developed a multimodal nanodrug treatment system with the enhanced abilities of tumor-targeting, BBB penetration, and cancer-specific accumulation of chemotherapeutic drugs by combining chemotherapy and photothermal therapy. It can be used as a flexible and effective GBM treatment system and it may also be used for the treatment of other central nervous systems (CNS) tumors and extracranial tumors.

Brain targeted delivery of carmustine using chitosan coated nanoparticles via nasal route for glioblastoma treatment

This study aims to develop chitosan-coated PLGA nanoparticles intended for nose-to-brain delivery of carmustine. Formulations were prepared by the double emulsion solvent evaporation method and optimized by using Box-Behnken Design. The optimized nanoparticles were obtained to satisfactory levels in terms of particle size, PDI, entrapment efficiency, and drug loading. In vitro drug release and ex-vivo permeation showed sustained release and enhanced permeability (approx. 2 fold) of carmustine compared to drug suspension. The AUC0-t of brain obtained with carmustine-loaded nanoparticles via nasal administration in Albino Wistar rats was 2.8 and 14.7 times that of intranasal carmustine suspension and intravenous carmustine, respectively. The MTT assay on U87 MG cell line showed a significant decrease (P < 0.05) in the IC50 value of the formulation (71.23 μg ml−1) as compared to drug suspension (90.02 μg ml−1).These findings suggest chitosan coated nanoparticles could be used to deliver carmustine via intranasal administration to treat Glioblastoma multiforme.

Photobiomodulation and photodynamic therapy-induced switching of autophagy and apoptosis in human dermal fibroblasts.

Nowadays, photobiomodulation (PBM) in combination with chemotherapy or other therapeutic approaches is an attractive adjuvant modality for cancer treatment. Targeted destruction of cancer cells is one of the main advantages of photodynamic therapy (PDT). We have shown in previous studies that the combination of PBM at 808nm and hypericin-mediated PDT increases PDT efficacy in human glioblastoma cells U87 MG. The study presented here shows significant differences between U87 MG and non-cancerous human dermal fibroblasts (HDF) cells treated by PBM and PDT. This study focuses on mitochondria because PBM mainly affects these organelles. We demonstrated that an interplay between mitochondrial and autophagic proteins plays a crucial role in the response of HDF cells to PBM and PDT. Fluorescence microscopy, flow cytometry, and Western blot analysis were used to examine the autophagic profile of HDF cells after these treatments. An increase in ubiquitin, SQSTM1, LC3BII, and cytochrome c was accompanied by a decrease in M6PR, ATG16L1, and Opa1 in HDF cells exposed to PBM and PDT. Overall, we observed that the switching of autophagy and apoptosis is dose-dependent and also occurs independently of PBM in HDF cells after hypericin-mediated PDT. However, PBM might preferentially induce autophagy in noncancer cells, which might escape apoptosis under certain conditions.

Antitumor activity of copper(II) complexes with Schiff bases derived from N′-tosylbenzene-1,2-diamine

The electrochemical oxidation of anodic metal copper in a solution of the ligands N-[(5-tert-butyl-2-hydroxyphenyl)methylidine]-N′-tosylbenzene-1,2-diamine [H2L1] and N-[(3,5-di-tert-butyl-2-hydroxyphenyl)methylidine]-N′-tosylbenzene-1,2-diamine, [H2L2] afforded homoleptic [CuL] compounds or solvate [CuLS] complexes. The addition to the electrochemical cell of coligands (L') such as 2,2′-bipyridine (2-bpy), 4,4′-bipyridine(4-bpy) or 1,10-phenanthroline (phen) allowed the synthesis, in one step, of heteroleptic [CuLL'] compounds, namely [CuL1(H2O)] (1), [CuL1(2,2′-bpy)]⋅CH3CN (2), [CuL1(phen)]·H2O (3), [Cu2L12(4,4′-bpy)] (4), [CuL2(CH3OH)] (5), [CuL2(2,2′-bpy)] (6), [CuL2(phen)] (7) and [Cu2L22(4,4′-bpy)] (8). The crystal structures of both ligands, H2L1, H2L2, and those of the complexes (2), (4), (5), (6) and (7) have been determined by X-ray diffraction techniques. Coordination polyhedron around metal atom is square planar for [CuL2(CH3OH)] (5) and [Cu2L12(4,4′-bpy)] (4) and square pyramid for the other complexes with additional chelating ligands. The cytotoxic activity of this new series of copper(II) complexes against the SH-SY5Y neuroblastoma cell line and U87-MG and U373-MG glioblastoma cell lines has been investigated. Most of the test compounds showed higher activity than cisplatin in the three cell lines. Among this series, compound [CuL1(phen)] (3) displayed the highest activity with IC50 equal to 1.77 μM on SH-SY5Y whereas compound [Cu2L12(4.4′-bpy)] (4) resulted the most potent compounds on U87 MG and U373 MG glioblastoma cell lines. Studies on the cytotoxic activity of these derivatives suggest that these compounds induce cell death by a mechanism other than apoptosis.

Synthesis, X-ray diffraction and anti-proliferative biological activity of hispolon derivatives and their (η6-p-cymene)(Hispolonato)Ruthenium[II] chloride complexes

Hispolon is a natural product extracted from Phellinus igniarius and Phellinus linteus fungi that has previously shown antitumor activity. In this study we present the synthesis, chemical characterization and in vitro anti-proliferative activity of three [(η6-p-cymene)Ru(L)Cl] neutral complexes, where L = hispolon derivatives. The single crystal X-ray structures of the three Ru complexes all have the expected piano stool geometry with the p-cymene ligand at the apex of the piano stool and occupying three of the sites in a distorted-octahedral arrangement. Completing the octahedral coordination sphere are two β-diketone oxygen atoms of the deprotonated hispolon derivatives which are coordinated in a bidentate fashionand a chlorine atom. The cytotoxicity of the three hispolon derivatives along with their corresponding complexes was studied for A549 lung, HCT116 colon and U87 glioblastoma cell lines. In the glioblastoma cell line, increase of biological activity (lower IC50) is seen for all three hispolons after arene-ruthenium complexation: 5.1 times higher for 2,3,4-trimethoxy-hispolon (Hisp8), and 1.5 times for both 3-methoxy,4-hydroxy-hispolon (Hisp1) and 3,4-dimethoxy-hispolon (Hisp4). This effect was less pronounced in the colon HCT116 cell line with complexation having 1.2–1.4 times higher biological activity. For the lung cell line A549, there was no such effect and complex formation slightly decreased the biological activity. Regardless, in the glioblastoma cell line, the presence of Hisp8, the most liposoluble agent, either as the pure compound or as ligand in the η6-p-cymene-Ru complex, proved effective as an antitumor agent. More specifically, the [(η6-p-cymene)Ru(Hisp8)Cl] complex showed a decrease in IC50 value for all 3 cell lines, when compared to Hisp8. We also performed molecular mechanics, DFT and docking calculations to determine the inhibitory ability of hispolon analogs towards aldehyde dehydrogenase, which targets glioblastoma stem cells. All three [(η6-p-cymene)Ru(Hisp)Cl] coordination complexes of hispolon derivatives are more effective than the related neutral hispolons in the U87 MG human glioblastoma cell line. Our computational studies clearly indicate an important role for aldehyde dehydrogenase Arg139 amino acid. In addition, computational results indicate hispolonato anions are much better inhibitors of aldehyde dehydrogenase and improve the anti-glioblastoma biological activity, consistent with IC50 data.

USP7 Enables Immune Escape of Glioma Cells by Regulating PD-L1 Expression

ABSTRACT Background Glioma is the most common primary intracranial tumor. This study investigated the mechanism of ubiquitin-specific processing protease 7 (USP7) on the immune escape of glioma cells via the regulation of programmed cell death ligand-1 (PD-L1). Methods USP7 and PD-L1 expressions in glioma and normal brain tissues were detected using quantitative reverse transcriptase polymerase chain reaction and Western blot. The glioma cells U-251 MG were transfected with si-USP7 and pcDNA3.1-PD-L1, or treated with anti-PD-L1, after which the cell viability, colony-forming ability, and apoptosis rate were evaluated using cell counting kit-8, colony formation, and TdT-mediated dUTP nick-end labeling assays. Then, CD8+ T cells were purified, extracted, then co-cultured with U-251 MG cells. CD8+ T cell proliferation and the concentrations of interferon (IFN-γ), tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, interleukin (IL)-1β, and IL-10 were verified using carboxyfluorescein succinimidyl amino ester proliferation assay and enzyme-linked immunosorbent assay. Afterwards, the interaction between USP7 and PD-L1 and the ubiquitination level of PD-L1 were also assessed. Results USP7 was highly-expressed and PD-L1 mRNA levels did not change, while PD-L1 protein levels were up-regulated in the glioma cells. Silencing USP7 in U-251 MG cells limited the growth of the glioma cells, promoted glioma cell apoptosis, and facilitated the proliferation of CD8+ T cells, thus inhibiting immune escape. USP7 stabilized PD-L1 expression through deubiquitination. PD-L1 overexpression reversed the inhibitory effect of silencing USP7 on the immune escape of glioma cells. Conclusion USP7 stabilized PD-L1 through deubiquitination and accelerated the immune escape of glioma cells.

MicroRNA-219 loaded chitosan nanoparticles for treatment of glioblastoma

Recent evidence has implicated microRNA-219 (miR-219) in regulation of gene contributed in glioblastoma (GBM) pathogenesis. This study aimed to prepare miR-219 in chitosan (CS) nanoparticles (NPs), characterize and investigate their efficacy on human GBM cell line (U87 MG). NPs were prepared using ionic gelation method. The influence of process parameters on physicochemical characteristics of NPs was investigated. Apoptotic effect of miR-219 was examined on U87 MG cells. Formulated NPs showed particle size of 109 ± 2.18 nm, with poly dispersity index equal to 0.2 ± 0.05, and zeta potential of +20.5 ± 0.7 mV. Entrapment efficiency of miR-219 in loaded NP has reached 95%. The in vitro release study demonstrated sustained release pattern of miR-219 from CS-NPs. Gel retardation assay has confirmed the integrity of miR-219 after production process. The fabricated NPs reduced the survival of U87 MG cells to 78% after 24 h of post-transfection, and into 67.5% after 48 h. However, fibroblasts were not affected by the NPs, revealing their specificity for GBM cells. Given the tumour suppressing function of miR-219, and advantage of CS-NPs for gene delivery to the central nervous system, the presented NPs have a great potential for treatment of GBM.

Abstract 893: GD2 is a potential biomarker for glioblastoma cancer stem cells

Abstract Introduction: Treatment resistance and tumor recurrence of Glioblastoma (GBM) have been associated with cancer stem cells (CSCs), a subpopulation of cancer cells defined by capabilities of sphere-formation in 3D culture, self-renewal, migration, and survival in serum-deprived conditions. Therefore, therapies specifically targeting CSCs are urgently needed. Previous works have identified Ganglioside GD2 as a biomarker of triple negative breast cancer CSCs and suggested that GD2 is highly expressed in GBM cells. Here, we hypothesize that GD2 is also a potential biomarker of GBM CSCs and is involved with cancer stemness. Methods: GBM cells U-87 MG and U-118 MG were sorted as GD2- and GD2+ cells via FACS. Cells were cultured in 2D and 3D. 3D neurosphere size was measured and GD2 in 2D and 3D cultures was quantified via flow cytometry. Transwell migration assay was used to examine the migration capability of GD2- and GD2+ cells. Unsorted cells were cultured in either complete (10% serum) or deprived (1% serum) media and their GD2 was quantified via flow cytometry. Results: To test our hypothesis, we determined whether GD2+ cells express CSCs capabilities of sphere-formation in 3D culture, migration, and survival in serum-deprived conditions. First, we examined the sphere-forming capability by culturing GD2-, GD2+, and unsorted cells in 3D and found that they all formed neurospheres. However, GD2+ neurosphere size was significantly larger compared with unsorted and GD2- neurospheres for day 1 and 2. Interestingly, the significant difference was no longer seen on day 6. Analysis for GD2 expression in the spheres revealed that GD2- spheres contained GD2+ cells, suggesting that GD2- cells could spontaneously dedifferentiate to generate CSCs. To test this, we sorted GD2- and GD2+ cells into 2D conditions and quantified daily GD2 expression via flow cytometry. We found that GD2- seeded cells spontaneously expressed GD2 over six days, explaining the presence of GD2 in GD2- neurospheres. GD2+ cells also demonstrated the enhanced capability to migrate of CSCs with a significantly higher number of migrated cells compared with GD2- cells in the transwell migration assay. To examine the ability to survive in serum-deprived conditions, we cultured unsorted cells in either complete or deprived media. We found that these cells proliferated in the serum-deprived condition at approximately the same level as complete media. GD2 analysis by flow cytometry also showed a remarkably higher percentage of GD2+ cells in deprived media, suggesting that GD2 is involved in driving cell survival and maintaining cancer stemness in serum-deprived conditions. Conclusion: Our data suggests that GD2+ cells demonstrate characteristics of CSCs and GD2 is a potential biomarker of CSCs in GBM. Therefore, GD2 could be utilized as a differential therapeutic target for GBM. Further mechanistic studies are necessary to elucidate how GD2 is synthesized and maintains the stemness of GBM CSCs. Citation Format: Khoa Nguyen, Minh Tran, Thomas Cheng, Kurtis Willingham, Reza Izadpanah, Matthew Burow. GD2 is a potential biomarker for glioblastoma cancer stem cells [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 893.

Study on South African Indigenous Teas-Antioxidant Potential, Nutritional Content, and Hypoxia-Induced Cyclooxygenase Inhibition on U87 MG Cell Line.

Background: This study comparatively assessed seven indigenous traditional tea plants on several attributes that included antioxidant, nutritional, caffeine contents, and cyclooxygenase activity. Methodology: Nutritional content of all tea plants were determined for energy, fat, carbohydrates, total sugars, dietary fiber and amino acids. Antioxidant potential and the antioxidant potentiating secondary metabolites were also measured and compared. Further, we investigated the tea plants for any role they would have on cyclooxygenase (COX) activity on cobalt chloride (CoCl2) induced human glioma cell lines (U87MG). Results: The tea plants were found non-cytotoxic at concentrations tested against the human Chang liver and HeK 293 kidney cells and were found to be naturally caffeine free. The lowest and highest extraction yield among the tea plants was 7.1% for B. saligna and 15.48% for L. scaberrimma respectively. On average, the flavonol content was 12 to 8 QE/g, ORAC 800 µmol TE/g, TEAC 150 µmol TE/g, FRAP 155 µmol AAE/g, polyphenols 40 mg GAE/g, flavanols 0.35 mg CE/g, flavonols 12 mg QE/g and total flavonoid content (TFC) 180 µg QE/mg. The COX activity has been found to be inhibited by a dose-dependent manner by L. scaberrimma, B. saligna and L. javanica. Conclusion: The results further support competitive value of tea plants and need for improved and further development.

The Protein L-Isoaspartyl (D-Aspartyl) Methyltransferase Regulates Glial-to-Mesenchymal Transition and Migration Induced by TGF-β1 in Human U-87 MG Glioma Cells.

The enzyme PIMT methylates abnormal aspartyl residues in proteins. U-87 MG cells are commonly used to study the most frequent brain tumor, glioblastoma. Previously, we reported that PIMT isoform I possessed oncogenic features when overexpressed in U-87 MG and U-251 MG glioma cells. Higher levels of wild-type PIMT stimulated migration and invasion in both glioma cell lines. Conversely, PIMT silencing reduced these migratory abilities of both cell lines. These results indicate that PIMT could play a critical role in glioblastoma growth. Here, we investigated for the first time, molecular mechanisms involving PIMT in the regulation of epithelial to mesenchymal transition (EMT) upon TGF-β1 treatments. Gene array analyses indicated that EMT genes but not PIMT gene were regulated in U-87 MG cells treated with TGF-β1. Importantly, PIMT silencing by siRNA inhibited in vitro migration in U-87 MG cells induced by TGF-β1. In contrast, overexpressed wild-type PIMT and TGF-β1 had additive effects on cell migration. When PIMT was inhibited by siRNA, this prevented Slug induction by TGF-β1, while Snail stimulation by TGF-β1 was increased. Indeed, overexpression of wild-type PIMT led to the opposite effects on Slug and Snail expression dependent on TGF-β1. These data highlighted the importance of PIMT in the EMT response dependent on TGF-β1 in U-87 MG glioma cells by an antagonist regulation in the expression of transcription factors Slug and Snail, which are critical players in EMT.

Activating transcription factor 6 regulates cystathionine to increase autophagy and restore memory in Alzheimer’ s disease model mice

Endoplasmic reticulum stress (ER stress) plays a crucial role in the process of Alzheimer's disease (AD). Activating transcription factor 6 (ATF6) is a crucial sensor of ER stress. In AD patients, the homeostasis of the endogenous signal H2S produced by cystathionine γ-lyase (CTH) is in disbalance. However, the role of ATF6 and CTH in AD is rarely reported. Herein, we found that ATF6 and CTH were reduced in AD patients and APP/PS1 mice by immunohistochemistry and western blots. In LN229 and U87 MG cells, knockdown of ATF6 attenuated CTH expression, whereas overexpression of ATF6 resulted in upregulation of CTH. Brain-specific ATF6 knockout mice expressed significantly down-regulated CTH in the hippocampus and cortex compared to wild-type mice. Mechanistically, ATF6 and CTH increased H2S generation and autophagy-related proteins. Further we observed that CTH promoted the sulfhydration of αSNAP. This is probably to be the specific mechanism by which AFT6 promotes autophagy. Through in vivo studies, we found that αSNAP sulfhydration expression was significantly lower in ATF6 knockout mice than in wild-type mice. Decreased ATF6 impaired spatial memory retention, while addition of CTH rescued memory loss. Together, we demonstrate that ATF6 positively regulates the expression of CTH, which is closely related to the rescue of AD. Targeting the ATF6/CTH signal pathway may provide a new strategy for the treatment of AD.

Combined treatment with niclosamide and camptothecin enhances anticancer effect in U87 MG human glioblastoma cells.

Glioblastoma multiforme (GBM) is one of the deadliest cancers of the brain. Its ability to infiltrate healthy brain tissues renders it difficult to remove surgically. Furthermore, it exhibits high rates of radio- and chemoresistance, making the survival rates of patients with GBM poor. Therefore, novel effective therapies for GBM remain urgently in demand. Niclosamide is an anti-helminthic drug and recently it has been receiving attention due to its reported anticancer effects in cancer models, including GBM. Furthermore, camptothecin (CPT) is a naturally-occurring alkaloid and has been previously reported to be a potential chemotherapeutic agent by targeting the nuclear topoisomerase I. In the present study, the possible combined chemotherapeutic effects of niclosamide and CPT on the human glioblastoma cell line U87 MG was investigated by MTT assay and western blot analysis. Niclosamide exhibited synergistic activities with CPT to suppress the proliferation of U87 MG cells. Additionally, niclosamide suppressed cell proliferation and induced cell death mainly by triggering ER stress and autophagy, whilst CPT induced cell apoptosis mainly through p53-mediated mitochondrial dysfunction and activation of the MAPK (ERK/JNK) pathways. Overall, these findings suggest that co-administration of niclosamide and CPT may provide a novel therapeutic treatment strategy for GBM.

GALNT12 is associated with the malignancy of glioma and promotes glioblastoma multiforme in vitro by activating Akt signaling

Abnormal expression of mucin-type O-glycosylation has been reported to be associated with a variety of human cancers including gliomas. However, little is known about its contribution to the malignancy of Glioblastoma Multiforme (GBM), the deadliest form of brain tumors. Here, we conducted a detailed analysis of the expression profiles of GALNT gene family, which encode polypeptide-N-acetyl-galactosaminyltransferases (GalNAc-Ts) and are responsible for initiating O-glycans, both in the Cancer Genome Atlas (TCGA) and in the Chinese Glioma Genome Atlas (CGGA) databases. We discovered that GALNT12 is the only member within the GALNT family, whose expression demonstrated significant correlation with a worse prognosis of GBM. Genetic knockdown (KD) and knockout (KO) of GALNT12 in U87 MG, a representative GBM cell line with high GALNT12 expression, confirmed that GALNT12 deficiency leads to decreased cell proliferation, migration and invasion. Mechanism study revealed that GALNT12 KD and KO decreased the level of epidermal growth factor (EGF) and consequently attenuated Akt signaling within the cell. In summary, our results indicated that GALNT12 facilitates the malignant characteristics of GBM by influencing the PI3K/Akt/mTOR axis and may serve as a novel prognosis biomarker and a potential therapeutic target of GBM.

Brain-Targeted Codelivery of Bcl-2/Bcl-xl and Mcl-1 Inhibitors by Biomimetic Nanoparticles for Orthotopic Glioblastoma Therapy.

Glioblastoma (GBM) is among the most treatment-resistant solid tumors and often recurrs after resection. One of the mechanisms through which GBM escapes various treatment modalities is the overexpression of anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2, Bcl-xl, and Mcl-1) in tumor cells. Small-molecule inhibitors such as ABT-263 (ABT), which can promote mitochondrial-mediated cell apoptosis by selectively inhibiting the function of Bcl-2 and Bcl-xl, have been proven to be promising anticancer agents in clinical trials. However, the therapeutic prospects of ABT for GBM treatment are hampered by its limited blood-brain barrier (BBB) penetration, dose-dependent thrombocytopenia, and the drug resistance driven by Mcl-1, which is overexpressed in GBM cells and further upregulated upon treatment with ABT. Herein, we reported that the Mcl-1-specific inhibitor A-1210477 (A12) can act synergistically with ABT to induce potent cell apoptosis in U87 MG cells, drug-resistant U251 cells, and patient-derived GBM cancer stem cells. We further designed a biomimetic nanomedicine, based on the apolipoprotein E (ApoE) peptide-decorated red blood cell membrane and pH-sensitive dextran nanoparticles, for the brain-targeted delivery of ABT and A12. The synergistic anti-GBM effect was retained after encapsulation in the nanomedicine. Additionally, the obtained nanomedicine possessed good biocompatibility, exhibited efficient BBB penetration, and could effectively suppress tumor growth and prolong the survival time of mice bearing orthotopic GBM xenografts without inducing detectable adverse effects.

Extracellular Cardiolipin Modulates Select Immune Functions of Astrocytes in Toll-Like Receptor (TLR) 4-Dependent Manner.

Alzheimer's disease (AD) is characterized by chronic neuroinflammation, which is partially mediated by dysregulated functions of glial cells. Cardiolipin (CL) is a phospholipid normally confined to the inner mitochondrial membrane; however, it has been detected in human sera, indicating that it can exist in the extracellular space where it may interact with nearby cells. Although CL has been shown to modulate several functions of microglia in a toll-like receptor (TLR) 4-dependent manner, the effects of extracellular CL on astrocytes are unknown. In addition to their homeostatic functions, astrocytes participate in neuroimmune responses of the brain and express TLR 4. Therefore, we hypothesized that extracellular CL (1) modulates the secretion of cytokines and cytotoxins by astrocytes, as well as their phagocytic activity, and (2) acts by interacting with astrocyte TLR 4. We demonstrate that CL inhibits the lipopolysaccharide- (LPS-) induced secretion of cytotoxins and expression of glial fibrillary acidic protein (GFAP) by human U118 MG astrocytic cells. CL alone upregulates the phagocytic activity of human astrocytic cells and primary murine astrocytes. CL in combination with LPS upregulates secretion of interleukin (IL)-1β by astrocytic cells. Furthermore, CL alone increases the secretion of monocyte chemoattractant protein (MCP)-1 by astrocytic cells, which is blocked by the TLR 4-specific antagonist TAK-242. We demonstrate that CL upregulates MCP-1 secretion in the absence of its natural carrier protein, β2-glycoprotein 1, indicating that CL may be bioactive in the brain where this protein is not present. Lastly, we show that CL downregulates the expression of astrocytic TLR 4, implying that CL engages this receptor, as its activation has been shown to lead to its degradation. Overall, our study extends the list of cell type functions of which CL modulates and provides evidence that CL, or liposomes containing this phospholipid can be used to modulate specific neuroimmune functions of astrocytes.

Lichen Secondary Metabolites Inhibit the Wnt/β-Catenin Pathway in Glioblastoma Cells and Improve the Anticancer Effects of Temozolomide.

Lichens are a source of secondary metabolites with significant pharmacological potential. Data regarding their possible application in glioblastoma (GBM) treatment are, however, scarce. The study aimed at analyzing the mechanism of action of six lichen secondary metabolites: atranorin, caperatic acid, physodic acid, squamatic acid, salazinic acid, and lecanoric acid using two- and three-dimensional GBM cell line models. The parallel artificial membrane permeation assay was used to predict the blood-brain barrier penetration ability of the tested compounds. Their cytotoxicity was analyzed using the MTT test on A-172, T98G, and U-138 MG cells. Flow cytometry was applied to the analysis of oxidative stress, cell cycle distribution, and apoptosis, whereas qPCR and microarrays detected the induced transcriptomic changes. Our data confirm the ability of lichen secondary metabolites to cross the blood-brain barrier and exert cytotoxicity against GBM cells. Moreover, the compounds generated oxidative stress, interfered with the cell cycle, and induced apoptosis in T98G cells. They also inhibited the Wnt/β-catenin pathway, and this effect was even stronger in case of a co-treatment with temozolomide. Transcriptomic changes in cancer related genes induced by caperatic acid and temozolomide were the most pronounced. Lichen secondary metabolites, caperatic acid in particular, should be further analyzed as potential anti-GBM agents.

Enhanced tumor accumulation and therapeutic efficacy of liposomal drugs through over-threshold dosing

BackgroundMost intravenously administered drug-loaded nanoparticles are taken up by liver Kupffer cells, and only a small portion can accumulate at the tumor, resulting in an unsatisfactory therapeutic efficacy and side effects for chemotherapeutic agents. Tumor-targeted drug delivery proves to be the best way to solve this problem; however, the complex synthesis, or surface modification process, together with the astonishing high cost make its clinical translation nearly impossible.MethodsReferring to Ouyang’s work and over-threshold dosing theory in general, blank PEGylated liposomes (PEG-Lipo) were prepared and used as tumor delivery enhancers to determine whether they could significantly enhance the tumor accumulation and in vivo antitumor efficacy of co-injected liposomal ACGs (PEG-ACGs-Lipo), a naturally resourced chemotherapeutic. Here, the phospholipid dose was used as an indicator of the number of liposomes particles with similar particle sizes, and the liposomes was labelled with DiR, a near-red fluorescent probe, to trace their in vivo biodistribution. Two mouse models, 4T1-bearing and U87-bearing, were employed for in vivo examination.ResultsPEG-Lipo and PEG-ACGs-Lipo had similar diameters. At a low-threshold dose (12 mg/kg equivalent phospholipids), PEG-Lipo was mainly distributed in the liver rather than in the tumor, with the relative tumor targeting index (RTTI) being ~ 0.38 at 72 h after administration. When over-threshold was administered (50 mg/kg or 80 mg/kg of equivalent phospholipids), a much higher and quicker drug accumulation in tumors and a much lower drug accumulation in the liver were observed, with the RTTI increasing to ~ 0.9. The in vivo antitumor study in 4T1 tumor-bearing mice showed that, compared to PEG-ACGs-Lipo alone (2.25 mg/kg phospholipids), the co-injection of a large dose of blank PEG-Lipo (50 mg/kg of phospholipids) significantly reduced the tumor volume of the mice by 22.6% (P < 0.05) and enhanced the RTTI from 0.41 to 1.34. The intravenous injection of a low drug loading content (LDLC) of liposomal ACGs (the same dose of ACGs at 50 mg/kg of equivalent phospholipids) achieved a similar tumor inhibition rate (TIR) to that of co-injection. In the U87 MG tumor-bearing mouse model, co-injection of the enhancer also significantly promoted the TIR (83.32% vs. 66.80%, P < 0.05) and survival time of PEG-ACGs-Lipo.ConclusionAn over-threshold dosing strategy proved to be a simple and feasible way to enhance the tumor delivery and antitumor efficacy of nanomedicines and was benefited to benefit their clinical result, especially for liposomal drugs.Graphical

Synthesis and Properties of α-Mangostin and Vadimezan Conjugates with Glucoheptoamidated and Biotinylated 3rd Generation Poly(amidoamine) Dendrimer, and Conjugation Effect on Their Anticancer and Anti-Nematode Activities.

α-Mangostin and vadimezan are widely studied potential anticancer agents. Their biological activities may be improved by covalent bonding by amide or ester bonds with the third generation poly(amidoamine) (PAMAM) dendrimer, substituted with α-D-glucoheptono-1,4-lactone and biotin. Thus, conjugates of either ester- (G3gh4B5V) or amide-linked (G32B12gh5V) vadimezan, and equivalents of α-mangostin (G3gh2B5M and G32B12gh5M, respectively), were synthesized, characterized and tested in vitro against cancer cells: U-118 MG glioma, SCC-15 squamous carcinoma, and BJ normal human fibroblasts growth, as well as against C. elegans development. α-Mangostin cytotoxicity, stronger than that of Vadimezan, was increased (by 2.5–9-fold) by conjugation with the PAMAM dendrimer (with the amide-linking being slightly more effective), and the strongest effect was observed with SCC-15 cells. Similar enhancement of toxicity resulting from the drug conjugation was observed with C. elegans. Vadimezan (up to 200 µM), as well as both its dendrimer conjugates, was not toxic against both the studied cells and nematodes. It showed an antiproliferative effect against cancer cells at concentrations ≥100 µM. This effect was significantly enhanced after conjugation of the drug with the dendrimer via the amide, but not the ester bond, with G32B12gh5V inhibiting the proliferation of SCC-15 and U-118 MG cells at concentrations ≥4 and ≥12 μM, respectively, without a visible effect in normal BJ cells. Thus, the drug delivery system based on the PAMAM G3 dendrimer containing amide bonds, partially-blocked amino groups on the surface, larger particle diameter and higher zeta potential can be a useful tool to improve the biological properties of transported drug molecules.