U87MG Human Glioma Research Articles

Bioactive Alkaloids from the Actinomycete Actinoalloteichus sp. ZZ1866.

The new alkaloids marinacarbolines E-Q (1-10, 12-14), caerulomycin N (15), and actinoallonaphthyridine A (16), together with the known marinacarboline C (11) and cyanogramide (17), were isolated from the actinomycete Actinoalloteichus sp. ZZ1866. The structures of the isolated compounds were elucidated based on their HRESIMS data, extensive NMR spectroscopic analyses, Mosher's method, ECD calculations, single-crystal X-ray diffraction analysis, and chemical degradation studies. Marinacarbolines E-L (1-8) share an indole-pyridone-imidazole tetracyclic skeleton, which is the first example of this kind of skeleton. Caerulomycin N (15) and cyanogramide (17) exhibited cytotoxic activity against both human glioma U251 and U87MG cells with IC50 values of 2.0-7.2 μM. Marinacarbolines E (1), G (3), I (5), and M (9) showed cytotoxic activity against U87MG cells with IC50 values of 2.3-8.9 μM.

Subtipyrrolines A–C, novel bioactive alkaloids from the Mariana Trench-associated bacterium Bacillus subtilis SY2101

Abstract The new subtipyrrolines A–C were discovered from the hadal bacterium Bacillus subtilis SY2101 isolated from a sample of sediment collected at the Mariana Trench. Their structures were elucidated based on the HRESIMS data, extensive NMR spectroscopic analyses, ECD and 13C NMR calculations, and single crystal X-ray diffraction experiment. Subtipyrrolines A–C possessed an unprecedented pyrrole-pyrrole-dihydropyridine tricyclic skeleton. Subtipyrrolines A and C showed weak antiproliferative activities against human glioma U251 and U87MG cells, respectively, and subtipyrrolines A–C had weak antimicrobial activities in inhibiting the growth of Escherichia coli and Candida albicans. The plausible biosynthetic pathway of subtipyrrolines A–C was also proposed.

Abstract 2187: Kinetics and potency of T Cell-mediated cytolysis of glioblastoma

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer that has no effective treatments and a prognosis of only 12-15 months. Immune system effector T cells are a promising therapy due to their high specificity and innate cytotoxicity. The ability to assess the efficacy and potency of T cell therapies in vitro and with high throughput is vital for the further development of this promising therapy. Cellular impedance-based assays offer a sensitive, label-free, and nondestructive method to continuously monitor cancer cell proliferation and immune cell-mediated cytotoxicity in real-time and at high throughputs, revealing not only the potency but also the kinetics of T cell therapies. Here, we investigated the targeting potency of activated human T cells on human U87MG glioma cell monolayers across multiple cell densities. U87MG cells were plated at 10k, 25k, and 50k cells per well on a PDL-coated CytoView-Z 96-well plate (n=12 wells per density), and their impedance was continuously monitored on the Maestro Z impedance platform. After 24 hours, activated patient-derived T Cells (ImmunoCult CD3/CD28 activation media) were added in a 10:1 effector:target ratio. Impedance and cytolysis were subsequently monitored for another 120 hours. As expected, higher densities proliferated faster and reached a higher impedance level at 24 hours. The addition of activated human T cells resulted in a decrease of the impedance signal consistent with T cell-mediated lysis of the glioma cells. The highest density of T cells (500k) exhibited the fastest rate of U87 cytolysis, with a Kill Time 50 of 35 hours. All densities reached 100% cytolysis by 100 hours. Overall, activated human T cells were highly effective for cytolysis of U87MG glioma cell monolayers of varying densities. The cellular impedance assay revealed high potency, with kinetics that varied across cell densities despite the same effector:target ratios. Citation Format: Stacie A. Chvatal, Meghan T. Logun, Heather B. Hayes, Daniel C. Millard, Lohitash Karumbaiah. Kinetics and potency of T Cell-mediated cytolysis of glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2187.

Novel bexarotene derivatives: Synthesis and cytotoxicity evaluation for glioma cells in 2D and 3D in vitro models

Glioblastoma (GBM) is an aggressive and lethal form of brain cancer with a high invasion capacity and a lack of effective chemotherapeutics. Retinoid bexarotene (BXR) inhibits the neurospheroidal colony formation and migration of primary glioblastoma cells but has side effects. To enhance the BXR glioblastoma selectivity and cytotoxicity, we chemically modified it at the carboxyl group with either nitroethanolamine (NEA) bearing a NO-donating group (a well-known bioactivity enhancer; BXR-NEA) or with a dopamine (DA) moiety (to represent the highly toxic for various tumor cells N-acyldopamine family; BXR-DA). These two novel compounds were tested in the 2D (monolayer culture) and 3D (multicellular tumor spheroids) in vitro models. Both BXR-DA and BXR-NEA were found to be more toxic for rat C6 and human U-87MG glioma cells than the initial BXR. After 24 h incubation of the cells (monolayer culture) with the drugs, the IC50 values were in the range of 28–42, and 122–152 μM for BXR derivatives and BXR, respectively. The cell death occurred via apoptosis according to the annexin staining and caspase activation. The tumor spheroids demonstrated higher resistance to the treatment compared to that one of the monolayer cultures. BXR-DA and BXR-NEA were more specific against tumor cells than the parental drug, in particular the selectivity index was 1.8–2.7 vs. 1.3–1.5, respectively. Moreover, they inhibited cell migration more effectively than parental BXR according to a scratch assay. Cell spreading from the tumor spheroids was also inhibited. Thus, the obtained BXR derivatives could be promising for glioblastoma treatment.

Design, synthesis, DNA binding studies and evaluation of anticancer potential of novel substituted biscarbazole derivatives against human glioma U87 MG cell line.

In this research paper, we report the design and synthesis of novel substituted biscarbazole derivatives which were characterized by 1H and 13C NMR, high resolution mass spectroscopy (HRMS). The SAR study of the compounds is reported based on different substituents and their positions in the biscarbazole scaffold. In vitro cytotoxicity of the compounds was evaluated against human glioma U87 MG cell line by MTT assay for 24h. The IC50 values of the compounds (30-35, 48-53 and 54-62) were calculated at the concentration range from 1.00µM to 500µM. The compound 34 showed the most significant in vitro cytotoxicity (IC50=3.9µM) against human glioma U87 MG cell line and was found to be better than standard drugs used for the treatment of brain tumors such as temozolomide (IC50=100µM) and carmustine (IC50=18.2µM) respectively. To determine the mode of binding of compound 34 with CT-DNA, various biophysical techniques like UV-spectrophotometer, fluorescence, circular dichroism, viscosity, topoisomerase assay and molecular docking analysis, were used. Our results demonstrated groove binding mode of interaction of the compound 34 with CT-DNA with a plausible static bio-molecular quenching rate constant (Kq) 1.7×1012 M-1s-1. The studies of biscarbazole derivatives are anticipated to develop potential novel anticancer agents against brain tumors.

Kinetics and potency of T Cell-mediated cytolysis of glioblastoma

Abstract Glioblastoma is an aggressive brain cancer with no effective treatments and a prognosis of 12–15 months. Immune system effector T cells are a promising therapy due to their high specificity and innate cytotoxicity. Assessing the efficacy and potency of T cell therapies in vitro and with high throughput is vital for development of this promising therapy. Cellular impedance-based assays offer sensitive, label-free, and continuous monitoring of cell proliferation and immune cell-mediated cytotoxicity, revealing both the potency and kinetics of T cell killing. Here, we investigated the targeting potency of activated human T cells on human U87MG glioma and patient-derived glioma cells. U87MG and patient-derived glioma cells were plated at densities ranging from 10k to 50k cells per well on a PDL-coated CytoView-Z 96-well plate (n=12 wells per density). After 24 hours, activated patient-derived T Cells (ImmunoCult CD3/CD28 activation media) were added in a 10:1 effector:target ratio. Impedance and cytolysis were continuously monitored for over 120 hours. As expected, higher densities of glioma cells proliferated faster and reached a higher impedance level at 24 hours. Addition of activated human T cells resulted in a decrease in impedance consistent with T cell-mediated lysis of the glioma cells. The highest density of T cells (500k) exhibited the fastest rate of U87 cytolysis, with a Kill Time 50 of 35 hours. All densities reached 100% cytolysis by 100 hours. Overall, activated human T cells were highly effective for cytolysis of U87MG and patient-derived glioma cells. The cellular impedance assay revealed high potency, with kinetics that varied across cell densities despite the same effector:target ratios.

Kinetics and potency of T Cell-mediated cytolysis of glioblastoma

Background & Aim Glioblastoma is an aggressive brain cancer with no effective treatments and a prognosis of 12-15 months. Immune system effector T cells are a promising therapy due to their high specificity and innate cytotoxicity. Assessing the efficacy and potency of T cell therapies in vitro and with high throughput is vital for development of this promising therapy. Cellular impedance-based assays offer sensitive, label-free, and continuous monitoring of cell proliferation and immune cell-mediated cytotoxicity, revealing both the potency and kinetics of T cell killing. Here, we investigated the targeting potency of activated human T cells on human U87MG glioma and patient-derived glioma cells. Methods, Results & Conclusion U87MG and patient-derived glioma cells were plated at densities ranging from 10k to 50k cells per well on a PDL-coated CytoView-Z 96-well plate (n=12 wells per density). After 24 hours, activated patient-derived T Cells (ImmunoCult CD3/CD28 activation media) were added in a 10:1 effector:target ratio. Impedance and cytolysis were continuously monitored for over 120 hours. As expected, higher densities of glioma cells proliferated faster and reached a higher impedance level at 24 hours. Addition of activated human T cells resulted in a decrease in impedance consistent with T cell-mediated lysis of the glioma cells. The highest density of T cells (500k) exhibited the fastest rate of U87 cytolysis, with a Kill Time 50 of 35 hours. All densities reached 100% cytolysis by 100 hours. Overall, activated human T cells were highly effective for cytolysis of U87MG and patient-derived glioma cells. The cellular impedance assay revealed high potency, with kinetics that varied across cell densities despite the same effector:target ratios.

Tanshinone I induces apoptosis and protective autophagy in human glioblastoma cells via a reactive oxygen species‑dependent pathway.

Glioma is the most common primary malignancy of the central nervous system and is associated with high mortality rates. Despite the available treatment options including surgery, radiotherapy and chemotherapy, the median patient survival rate is low. Therefore, the development of novel anticancer agents for the treatment of glioma is urgently required. Tanshinone I (TS I) is a tanshinone compound that is isolated from Danshen. Accumulating evidence indicates that TS I exhibits antiproliferative activity in a variety of cancer types. However, the role of TS I and its mechanism of action in human glioma remain to be elucidated. In the present study, the anticancer potential of TS I against human glioma U87 MG cells was investigated. The results indicated that TS I exerted a potential cytotoxic effect on human glioma U87 MG cells. TS I was found to induce cell proliferation, inhibition, cell cycle arrest, apoptosis and autophagy in U87 MG cells. Mechanistic experiments indicated that TS I activated endoplasmic reticulum (ER) stress and inhibited AKT signaling and apoptosis in human glioma U87 MG cells. Furthermore, the present study demonstrated that TS I induced protective autophagy in U87 MG cells. Additionally, ER stress and AKT signal-mediated apoptosis and protective autophagy were found to be induced by TS I via intracellular reactive oxygen species accumulation. The results of the present study demonstrated that TS I may be a potential anticancer drug candidate that may be of value in the treatment of human glioma.

Bioactive drimane sesquiterpenoids and isocoumarins from the marine-derived fungus Penicillium minioluteum ZZ1657

Four new compounds, including three drimane sesquiterpenoids purpurides E–G (1–3) and one isocoumarin peniisocoumarin H (4), together with 15 known compounds, were isolated from the marine-sourced fungus Penicillium minioluteum ZZ1657. Their structures were elucidated by a combination of the HRESIMS data, extensive NMR spectroscopic analyses, and single crystal X-ray diffractions. Purpurides E (1) and F (2) are two N-acetyl-l-valine-conjugated drimane sesquiterpenoids and showed antimicrobial activities against methicillin-resistant Staphylococcus aureus, Escherichia coli, and Candida albicans with MIC values of 6–12 and 3–6 μg/mL, respectively. Purpuride G (3) showed antiproliferative activities against human glioma U251 and U87MG cells with IC50 values of 4.49 and 10.9 μM, respectively.

A new antimicrobial indoloditerpene from a marine-sourced fungus aspergillus versicolor ZZ761

A new indoloditerpene (1) and fifteen known compounds (2–16) were isolated from a marine-derived fungus Aspergillus versicolor ZZ761. Structure of the new compound was elucidated as (3 R,9S,12R,13S,17S,18S)-2-carbonyl-3-hydroxylemeniveol based on its HRESIMS data, NMR spectroscopic analyses, the Mosher′s method, and ECD calculation. This new indoloditerpene (1) showed antimicrobial activities with MIC values of 20.6 μM against Escherichia coli and 22.8 μM against Candida albicans. Diorcinol (2) and versicolorin B (6) had activities in inhibiting the proliferation of human glioma U87MG and U251 cells with IC50 values of 4.4 and 6.2 μM and 11.3 and 30.5 μM, respectively.

Bioactive Streptoglutarimides A-J from the Marine-Derived Streptomyces sp. ZZ741.

The new streptoglutarimides A-J (1-10) and the known streptovitacin A (11) were isolated from a marine-derived actinomycete, Streptomyces sp. ZZ741. Structures of the isolated compounds were elucidated based on their HRESIMS data, extensive NMR spectroscopic analyses, ECD calculations, Mosher's method, and a single-crystal X-ray diffraction experiment. Streptoglutarimide H (8) and streptovitacin A (11) showed potent antiproliferative activity against human glioma U87MG and U251 cells with IC50 values of 1.5-3.8 μM for 8 and 0.05-0.22 μM for 11. All isolated compounds exhibited antimicrobial activity with MIC values of 9-11 μg/mL against methicillin-resistant Staphylococcus aureus, 8-12 μg/mL against Escherichia coli, and 8-20 μg/mL against Candida albicans.

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

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

Global extracellular vesicle proteomic signature defines U87-MG glioma cell hypoxic status with potential implications for non-invasive diagnostics

PurposeGlioblastoma multiforme (GBM) is the most common and lethal of primary malignant brain tumors. Hypoxia constitutes a major determining factor for the poor prognosis of high-grade glioma patients, and is known to contribute to the development of treatment resistance. Therefore, new strategies to comprehensively profile and monitor the hypoxic status of gliomas are of high clinical relevance. Here, we have explored how the proteome of secreted extracellular vesicles (EVs) at the global level may reflect hypoxic glioma cells.MethodsWe have employed shotgun proteomics and label free quantification to profile EVs isolated from human high-grade glioma U87-MG cells cultured at normoxia or hypoxia. Parallel reaction monitoring was used to quantify the identified, hypoxia-associated EV proteins. To determine the potential biological significance of hypoxia-associated proteins, the cumulative Z score of identified EV proteins was compared with GBM subtypes from HGCC and TCGA databases.ResultsIn total, 2928 proteins were identified in EVs, out of which 1654 proteins overlapped with the ExoCarta EV-specific database. We found 1034 proteins in EVs that were unique to the hypoxic status of U87-MG cells. We subsequently identified an EV protein signature, “HYPSIGNATURE”, encompassing nine proteins that strongly represented the hypoxic situation and exhibited close proximity to the mesenchymal GBM subtype.ConclusionsWe propose, for the first time, an EV protein signature that could comprehensively reflect the hypoxic status of high-grade glioma cells. The presented data provide proof-of-concept for targeted proteomic profiling of glioma derived EVs, which should motivate future studies exploring its utility in non-invasive diagnosis and monitoring of brain tumor patients.

Development of transferrin-modified poly(lactic-co-glycolic acid) nanoparticles for glioma therapy.

Glioma is a primary intracranial malignant tumor with poor prognosis. In this study, we aimed to develop transferrin (Tf)-modified poly(lactic-co-glycolic acid) (PLGA) nanoparticles to deliver temozolomide (TMZ) to glioma and evaluate their efficacy to kill glioma. TMZ-loaded nanoparticles were prepared by nanoprecipitation technique and targeted by Tf. Tf-PLGA-TMZ and PLGA-TMZ were characterized for average particle sizes and zeta potentials, cellular uptake and cytotoxicity as well as in-vitro drug release of these nanoparticles were evaluated in human glioma U87MG cells. In-vivo antiglioma efficacy of Tf-PLGA-TMZ was evaluated in nude mice. Polydispersity ratio increased from 0.132 to 0.150, while encapsulation efficiency decreased from 69.4 to 55.8% after Tf modification of PLGA-TMZ. High performance liquid chromatography test showed that Tf-targeted nanoparticles were better internalized into U87MG cells than nontargeted nanoparticles. Moreover, Tf-PLGA-TMZ significantly decreased the viability of U87MG cells compared with nontargeted nanoparticles (P<0.05). In addition, Tf-PLGA-TMZ significantly decreased tumor volume and improved the survival of nude mice injected with U87MG cells. Tf-modified PLGA nanoparticles could be used for effective delivery of TMZ and have promise for the treatment of glioma.

Novel Bioactive Penicipyrroether A and Pyrrospirone J from the Marine-Derived Penicillium sp. ZZ380.

The marine-sourced fungus Penicillium sp. ZZ380 was previously reported to have the ability to produce a series of new pyrrospirone alkaloids. Further investigation on this strain resulted in the isolation and identification of novel penicipyrroether A and pyrrospirone J. Each of them represents the first example of its structural type, with a unique 6/5/6/5 polycyclic fusion that is different from the 6/5/6/6 fused ring system for the reported pyrrospirones. Their structures were elucidated by extensive nuclear magnetic resonance (NMR) and high resolution electrospray ionization mass spectroscopy (HRESIMS) spectroscopic analyses, electronic circular dichroism (ECD) and 13C NMR calculations and X-ray single crystal diffraction. Penicipyrroether A showed potent antiproliferative activity against human glioma U87MG and U251 cells with half maximal inhibitory concentration (IC50) values of 1.64–5.50 μM and antibacterial inhibitory activity with minimum inhibitory concentration (MIC) values of 1.7 μg/mL against methicillin-resistant Staphylococcus aureus and 3.0 μg/mL against Escherichia coli.

Nano hydroxyapatite induces glioma cell apoptosis by suppressing NF-κB signaling pathway.

Nano-sized hydroxyapatite (nHA) particles have been demonstrated to exert anti-cancer effects on multiple cancer cell lines and animal models of cancer biology. However, the molecular mechanism underlying the effects of nHA particles on glioma cells remains unclear. The present study aimed to examine the effects of nHA on the behavior of glioma cells and investigate its underlying molecular mechanism. Rat glioma C6 cells and human glioma U87MG ATCC cells were exposed to nHA (20–100 µg/ml), and its effects on cell morphology, viability, apoptosis, cell cycle, invasion and nuclear factor (NF)-κB signaling were analyzed. Exposure of C6 and U87MG ATCC cells to 20 µg/ml nHA for 24 h caused cell detachment. Viability of C6 and U87MG ATCC cells were significantly reduced by nHA in a dose-dependent manner (P<0.05). Nuclear staining with Hoechst 33258 exhibited clear chromatin condensation in C6 cells following 24 h exposure to ≥25 µg/ml nHA. Flow cytometry revealed that nHA (20–100 µg/ml) significantly induced apoptosis and cell cycle G2/M arrest in C6 and U87MG ATCC cells (P<0.05). Transwell invasion assay demonstrated that nHA (20–60 µg/ml) significantly inhibited invasion of U87MG ATCC cells (P<0.05). Furthermore, western blotting and confocal immunofluorescence microscopy revealed that nHA (20–100 µg/ml) decreased NF-κB p65 protein expression and blocked NF-κB p65 nuclear translocation in C6 cells. The protein expression of NF-κB target molecules, such as B cell lymphoma 2, cyclooxygenase-2 and survivin, were also significantly reduced by nHA in a dose-dependent manner in both C6 and U87MG ATCC cells (P<0.05). In conclusion, it was demonstrated that the inhibitory effect of nHA on glioma cells is likely associated with the downregulation of NF-κB signaling.

Lentivirus mediated γ-interferon-inducible lysosomal thiol reductase (GILT) knockdown suppresses human glioma U373MG cell proliferation

BackgroundGlioma is the most common malignancy in brain carcinoma with poor prognosis due to the lack of understanding of the mechanism underlying the disease. γ-interferon-inducible lysosomal thiol reductase (GILT) plays a critical role in the process of antigen processing. However, the role of GILT in the tumorigenesis of glioma remains unknown. Materials and methodsThe expression of GILT was analyzed by bioinformatics using the public database and by qPCR in three human glioma cell lines. Cell growth and viability were determined by Celigo and MTT assays, while cell cycle arrest and apoptosis were determined using flow cytometry. Giemsa staining was used to analyze the colony formation, while cell motility was assessed using transwell migration and invasion assays, as well as, using tumor growth in nude mice. ResultsGILT was highly expressed as observed in the public database on human gliomas and two human glioma cell lines, U373MG and U87MG cells. The downregulation of GILT by lentiviral-mediated silencing inhibits the cell growth, colony formation, and migration but promotes apoptosis and results in cell cycle arrest at the G0/G1 phase in the U373MG cells. Also, the knockdown of GILT inhibits tumor growth in vivo. ConclusionElevated GILT is positively associated with glioma progression. GILT silencing suppresses cell proliferation, colony formation, migration, and tumor growth, and induces apoptosis and cell cycle arrest. GILT may serve as a potential target for the treatment of glioma.

In vitro effect of chlorambucil on human glioma cell lines (SF767 and U87-MG), and human microvascular endothelial cell (HMVEC) and endothelial progenitor cells (ECFCs), in the context of plasma chlorambucil concentrations in tumor-bearing dogs.

The objective of this study was to investigate a possible mechanism of action of metronomic chlorambucil on glioma by studying the in vitro cytotoxicity and anti-angiogenic effects on glioma and endothelial cells, respectively. The in vitro LD50 and IC50 of chlorambucil were determined using human SF767 and U87-MG glioma cell lines, human microvascular endothelial cells (HMVECs) and human endothelial colony forming cells (ECFCs). Results were analyzed in the context of chlorambucil concentrations measured in the plasma of tumor-bearing dogs receiving 4 mg m-2 metronomic chlorambucil. The LD50 and IC50 of chlorambucil were 270 μM and 114 μM for SF767, and 390 μM and 96 μM for U87-MG, respectively. The IC50 of chlorambucil was 0.53 μM and 145 μM for the HMVECs and ECFCs, respectively. In pharmacokinetic studies, the mean plasma Cmax of chlorambucil was 0.06 μM. Results suggest that metronomic chlorambucil in dogs does not achieve plasma concentrations high enough to cause direct cytotoxic or growth inhibitory effects on either glioma or endothelial cells.

Honokiol enhances temozolomide-induced apoptotic insults to malignant glioma cells via an intrinsic mitochondrion-dependent pathway

BackgroundTemozolomide (TMZ) is a first-line chemotherapeutic drug for malignant gliomas. Nonetheless, TMZ-induced side effects and drug resistance remain challenges. Our previous study showed the suppressive effects of honokiol on growth of gliomas. PurposeThis study was further aimed to evaluate if honokiol could enhance TMZ-induced insults toward malignant glioma cells and its possible mechanisms. MethodsHuman U87 MG glioma cells were exposed to TMZ, honokiol, and a combination of TMZ and honokiol. Cell survival, apoptosis, necrosis, and proliferation were successively assayed. Fluorometric substrate assays were conducted to determine activities of caspase-3, -6, -8, and -9. Levels of Fas ligand, Bax, and cytochrome c were immunodetected. Translocation of Bax to mitochondria were examined using confocal microscopy. Mitochondrial function was evaluated by assaying the mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and complex I enzyme activity. Caspase-6 activity was suppressed using specific peptide inhibitors. The honokiol-induced effects were further confirmed using human U373 MG and murine GL261 cells. ResultsExposure of human U87 MG glioma cells to honokiol significantly increased TMZ-induced DNA fragmentation and cell apoptosis. Interestingly, honokiol enhanced intrinsic caspase-9 activity without affecting extrinsic Fas ligand levels and caspase-8 activity. Sequentially, TMZ-induced changes in Bax translocation, the MMP, mitochondrial complex I enzyme activity, intracellular ROS levels, and cytochrome c release were enhanced by honokiol. Consequently, honokiol amplified TMZ-induced activation of caspases-3 and -6 in human U87 MG cells. Fascinatingly, suppressing caspase-6 activity concurrently decreased honokiol-induced DNA fragmentation and cell apoptosis. The honokiol-involved improvement in TMZ-induced intrinsic apoptosis was also confirmed in human U373 MG and murine GL261 glioma cells. ConclusionsThis study showed that honokiol can enhance TMZ-induced apoptotic insults to glioma cells via an intrinsic mitochondrion-dependent mechanism. Our results suggest the therapeutic potential of honokiol to attenuate TMZ-induced side effects.

Proteasome mediated degradation of CDC25C and Cyclin B1 in Demethoxycurcumin treated human glioma U87 MG cells to trigger G2/M cell cycle arrest

Recently, we have reported that Demethoxycurcumin induced Reactive oxygen species via inhibition of Mitochondrial Superoxide Dismutase is an initial event to trigger apoptosis through caspase-8 and 9 activation and to inhibit Akt/NF-κB survival signaling in human glioma U87 MG cells (Kumar et al., 2018). Although cell-cycle disruption had been suggested to be the possible mechanism for DMC inhibitory effect on human glioma U87 MG cells, comprehensive mechanisms of cell-cycle arrest caused by DMC are not fully understood. The present study was designed to elucidate the DMC induced mechanism of cell cycle arrest in human glioma U87 MG cells. In this study, the results illustrated that DMC induced Reactive oxygen species (ROS) leads to reduced expression of CDC25C, Cyclin B1 and CDK1 (Thr161) triggers G2/M cell cycle arrest in U87 MG glioma cells. Moreover, the DMC induced ROS generation activates ubiquitination and proteasome degradation of CDC25C and Cyclin B1 in U87MG glioma cells. In addition, the immunoprecipitation results showed that significant dissociation of CDK1or CDC2-Cyclin B1 complex leads to G2/M cell cycle arrest. To explore the possibility of direct involvement of DMC in the dissociation of CDK1/Cyclin B1 complex, the molecular docking and MD simulation studies were carried. The results showed that DMC nicely fitted into the binding site of CDK1 and Cyclin B1 with minimum binding energy (ΔG) of -9.46 kcal/mol (Ki = 0.11 μM) and − 9.90 kcal/mol (Ki = 0.05 μM) respectively. Therefore, this is the first study demonstrating CDC25C and Cyclin B1 proteins could be used as potential target for anticancer therapy and DMC may be explored as new therapeutic agent in the cure of Glioblastoma (GBM).