T98G Cells Research Articles

The autophagy inducer trehalose stimulates macropinocytosis in NF1-deficient glioblastoma cells

BackgroundGlioblastoma is a highly aggressive brain tumor. A big effort is required to find novel molecules which can cross the blood–brain barrier and efficiently kill these tumor cells. In this perspective, trehalose (α-glucopyranosyl‐[1→1]‐α‐d‐glucopyranoside), found in various dietary sources and used as a safe nutrient supplement, attracted our attention for its pleiotropic effects against tumor cells.MethodsHuman glioblastoma cell lines U373-MG and T98G were exposed to trehalose and analyzed at different time points. Cell proliferation was evaluated at medium term, and clonogenic capacity and cell morphology were evaluated at long term. Western blot was used to evaluate biochemical markers of autophagy (also measured in cells co-treated with EIPA or chloroquine), and mTOR, AMPK and ERK 1/2 signalling. Macropinocytosis was evaluated morphologically by bright-field microscopy; in cells loaded with the fluorescein-conjugated fluid-phase tracer dextran, macropinocytic vacuoles were also visualized by fluorescence microscopy, and the extent of macropinocytosis was quantified by flow cytometry.ResultsThe long-term effect of trehalose on U373-MG and T98G cell lines was impressive, as indicated by a dramatic reduction in clonogenic efficiency. Mechanistically, trehalose proved to be an efficient autophagy inducer in macropinocytosis-deficient T98G cells and an efficient inducer of macropinocytosis and eventual cell death by methuosis in U373-MG glioblastoma cells, proved to be poorly responsive to induction of autophagy. These two processes appeared to act in a mutually exclusive manner; indeed, co-treatment of U373-MG cells with the macropinocytosis inhibitor, EIPA, significantly increased the autophagic response. mTOR activation and AMPK inhibition occurred in a similar way in the two trehalose-treated cell lines. Interestingly, ERK 1/2 was activated only in macropinocytosis-proficient U373-MG cells harbouring loss-of-function mutations in the negative RAS regulator, NF1, suggesting a key role of RAS signalling.ConclusionsOur results indicate that trehalose is worthy of further study as a candidate molecule for glioblastoma therapy, due to its capacity to induce a sustained autophagic response, ultimately leading to loss of clonogenic potential, and more interestingly, to force macropinocytosis, eventually leading to cell death by methuosis, particularly in tumor cells with RAS hyperactivity. As a further anticancer strategy, stimulation of macropinocytosis may be exploited to increase intracellular delivery of anticancer drugs.

High levels of NRF2 sensitize temozolomide-resistant glioblastoma cells to ferroptosis via ABCC1/MRP1 upregulation

Glioblastoma patients have a poor prognosis mainly due to temozolomide (TMZ) resistance. NRF2 is an important transcript factor involved in chemotherapy resistance due to its protective role in the transcription of genes involved in cellular detoxification and prevention of cell death processes, such as ferroptosis. However, the relation between NRF2 and iron-dependent cell death in glioma is still poorly understood. Therefore, in this study, we analyzed the role of NRF2 in ferroptosis modulation in glioblastoma cells. Two human glioblastoma cell lines (U251MG and T98G) were examined after treatment with TMZ, ferroptosis inducers (Erastin, RSL3), and ferroptosis inhibitor (Ferrostatin-1). Our results demonstrated that T98G was more resistant to chemotherapy compared to U251MG and showed elevated levels of NRF2 expression. Interestingly, T98G revealed higher sensitivity to ferroptosis, and significant GSH depletion upon system xc− blockage. NRF2 silencing in T98G cells (T98G-shNRF2) significantly reduced the viability upon TMZ treatment. On the other hand, T98G-shNRF2 was resistant to ferroptosis and reverted intracellular GSH levels, indicating that NRF2 plays a key role in ferroptosis induction through GSH modulation. Moreover, silencing of ABCC1, a well-known NRF2 target that diminishes GSH levels, has demonstrated a similar collateral sensitivity. T98G-siABCC1 cells were more sensitive to TMZ and resistant to Erastin. Furthermore, we found that NRF2 positively correlates with ABCC1 expression in tumor tissues of glioma patients, which can be associated with tumor aggressiveness, drug resistance, and poor overall survival. Altogether, our data indicate that high levels of NRF2 result in collateral sensitivity on glioblastoma via the expression of its pro-ferroptotic target ABCC1, which contributes to GSH depletion when the system xc− is blocked by Erastin. Thus, ferroptosis induction could be an important therapeutic strategy to reverse drug resistance in gliomas with high NRF2 and ABCC1 expression.

Polish and New Zealand Propolis as Sources of Antioxidant Compounds Inhibit Glioblastoma (T98G, LN-18) Cell Lines and Astrocytoma Cells Derived from Patient.

Gliomas, including glioblastoma multiforme and astrocytoma, are common brain cancers in adults. Propolis is a natural product containing many active ingredients. The aim of this study was to compare the chemical composition, total phenolic content and concentration of toxic elements as well as the anticancer potential of Polish (PPE) and New Zealand (Manuka—MPE) propolis extracts on diffuse astrocytoma derived from patient (DASC) and glioblastoma (T98G, LN-18) cell lines. The antioxidants such as flavonoids and chalcones (pinocembrin, pinobanksin, pinobanksin 3-acetate and chrysin) were the main components in both types of propolis. The content of arsenic (As) and lead (Pb) in MPE was higher than PPE. The anti-proliferative study showed strong activity of PPE and MPE propolis on DASC, T98G, and LN-18 cells by apoptosis induction, cell cycle arrest and attenuated migration. These findings suggest that despite their different geographic origins, Polish and New Zealand propolis are sources of antioxidant compounds and show similar activity and a promising anti-glioma potential in in vitro study. However, further in vivo studies are required in order to assess therapeutic potential of propolis.

Theranostic Potential of Adaptive Cold Atmospheric Plasma with Temozolomide to Checkmate Glioblastoma: An In Vitro Study

Simple SummaryGlioblastoma (GBM) is an aggressive form of brain cancer. Here, we present a combination therapy of cold atmospheric plasma (CAP) and temozolomide (TMZ) to treat GBM in vitro. We analyze the effects of the co-treatment in two GBM (TMZ-resistant and -sensitive) cell lines. The aim of this study is mainly to sensitize these cells using CAP so that they respond well to TMZ. We further found that the removal of cell culture media after CAP treatment does not affect the sensitivity of CAP to cancer cells but enhances the effects of TMZ. However, it was observed in our study that keeping the CAP-treated media for a shorter time did not significantly inhibit T98G cells. Interestingly, keeping the same plasma-treated media for a longer duration resulted in a decrease in cell viability. On the contrary, TMZ-sensitive cell A172 responded well to the co-treatment. This could be a potential reason for the sensitization of the combination therapy.Cold atmospheric plasma (CAP) has been used for the treatment of various cancers. The anti-cancer properties of CAP are mainly due to the reactive species generated from it. Here, we analyze the efficacy of CAP in combination with temozolomide (TMZ) in two different human glioblastoma cell lines, T98G and A172, in vitro using various conditions. We also establish an optimized dose of the co-treatment to study potential sensitization in TMZ-resistant cells. The removal of cell culture media after CAP treatment did not affect the sensitivity of CAP to cancer cells. However, keeping the CAP-treated media for a shorter time helped in the slight proliferation of T98G cells, while keeping the same media for longer durations resulted in a decrease in its survivability. This could be a potential reason for the sensitization of the cells in combination treatment. Co-treatment effectively increased the lactate dehydrogenase (LDH) activity, indicating cytotoxicity. Furthermore, apoptosis and caspase-3 activity also significantly increased in both cell lines, implying the anticancer nature of the combination. The microscopic analysis of the cells post-treatment indicated nuclear fragmentation, and caspase activity demonstrated apoptosis. Therefore, a combination treatment of CAP and TMZ may be a potent therapeutic modality to treat glioblastoma. This could also indicate that a pre-treatment with CAP causes the cells to be more sensitive to chemotherapy treatment.

Potential Neurotoxic Effects of Glioblastoma-Derived Exosomes in Primary Cultures of Cerebellar Neurons via Oxidant Stress and Glutathione Depletion

High-grade gliomas are the most fatal brain tumors. Grade 4 gliomas are called glioblastoma multiforme (GBM), which are associated with the poorest survival and a 5-year survival rate of less than 4%. Many patients with GBM developed concomitant cognitive dysfunctions and epilepsy. Although the cognitive decline is well defined in glioblastomas, the neurotoxic factors underlying this pathology are not well understood in GBM patients. In this study, we aimed to investigate whether GBM-derived exosomes play a role in neuronal toxicity. For this purpose, exosomes obtained from T98G and U373 GBM cells were applied to primary neuron culture at different concentrations. Subsequently, MTT, LDH, GSH, TAS, and TOS tests were performed. Both GBM-derived exosomes induced a dose-dependent and statistically significant increase of LDH release in cerebellar neurons. MTT assay revealed as both T98G and U373 GBM-derived exosomes induced dose-dependent neurotoxic effects in cerebellar neurons. To the best of our knowledge, this study is the first study demonstrating the toxic potential of GBM-derived exosomes to primary neurons, which may explain the peritumoral edema and cognitive decline in GBM patients.

Abstract 5360: Temozolomide-resistant glioblastoma cells are collaterally sensitive to ferroptosis through NRF2 high expression

Abstract Glioblastoma patients have a poor prognosis with a low median survival rate mainly due to temozolomide (TMZ) resistance. NRF2 is an important transcript factor involved in chemotherapy resistance due to its ability to regulate genes related to the antioxidant response and to prevent cell death processes, such as ferroptosis. However, the relation between NRF2 and iron-dependent cell death is contradictory and poorly understood. This study aimed to analyze the role of NRF2 in ferroptosis modulation in glioblastoma cells. To this end, it was analyzed two human glioblastoma cell lines (U251MG and T98G) after treatment with TMZ, ferroptosis inducers (Erastin, RSL3, and Sorafenib), and ferroptosis inhibitor (Ferrostatin-1). Also, we performed gene expression analysis of glioma patients. Our results demonstrated that T98G compared to the U251MG was more resistant to chemotherapy and showed elevated levels of NRF2 expression and its targets xCT, HMOX1, and ABCC1. Interestingly, T98G cells revealed higher sensitivity and lipoperoxidation levels after ferroptotic treatment. Next, we established T98G NRF2 silenced cells and we observed a significant reduction in cellular viability after TMZ treatment when compared to wild-type cells. On the other hand, T98G-shNRF2 was more resistant to ferroptosis induction, indicating that NRF2 plays a key role in the modulation of chemoresistance and ferroptosis. After, we showed that NRF2 controls levels of ABCC1/MRP1 in glioblastoma cells, and ABCC1 silencing promotes sensitivity to TMZ and resistance to Erastin. These results support a possible mechanism of ferroptosis modulation by NRF2 on TMZ-resistant gliomas through ABCC1, which has been recently associated with ferroptosis induction by promoting efflux of glutathione out of the cell. Furthermore, we confirmed that NRF2 has a positive correlation with ABCC1 in glioma patients, and higher ABCC1 expression was associated with tumor aggressiveness. Also, we validated ABCC1 as an independent prognostic factor for poor overall survival on glioma. Finally, we observed that T98G cells have sensitivity to the ferroptosis inducer FDA-approved, sorafenib. Altogether our data suggest that high levels of NRF2 may result in ferroptosis sensitivity on glioblastoma through the high levels of expression of its pro-ferroptotic target ABCC1, once the xCT system is blocked by Erastin. Thus, glioblastoma cell vulnerability to ferroptosis by NRF2 and ABCC1 high expression can be an Achilles’ heel to reverse drug resistance on glioblastoma through the treatment with ferroptosis inducers. Citation Format: Izadora de Souza, Linda Karolynne Monteiro, Camila Banca Guedes, Marcela Latancia, Marina Tomaz Andrade, Matheus Molina Silva, Bruna Contieri, Bruna Felício Milazzotto Ribeiro, Mariana Lazarini, Luciana Gomes, Clarissa Rocha. Temozolomide-resistant glioblastoma cells are collaterally sensitive to ferroptosis through NRF2 high expression [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 5360.

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.

Some plant metabolites from Petasítes sp. and their effect on cancer cells motility in vitro.

e15077 Background: Plant metabolites are traditionally a valuable source of anticancer drugs. The huge variety of molecular structures found in plants makes it possible to isolate substances that possess not only antimitotic, but also anti-migratory properties on cancer cells. The objective of this study was to evaluate the anti-migratory activity of selected metabolites from Petasítes sp. plants. Methods: Cells of cancer cell cultures PC3 (prostate cancer), A431 (skin cancer), CaCo-2 (colorectal cancer), HeLa (cervical cancer) and T98G (glioma) were seeded in an amount of 15*104 cells per well of a 24-well plate (Biofil, China) in DMEM medium (Gibco, USA) supplemented with 10% FBS (HyClone, USA). After cell adhesion the test substances at a concentration of 40 µM were added, and a wound healing assay was performed according to the standard procedure. The study included the following metabolites: 1) substance 1 (S1) - 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one; 2) substance 2 (S2) - 5-(hydroxymethyl)furan-2-carbaldehyde; 3) substance 3 (S3) - corynan. The wound area was measured using a Lionheart FX imager (BioTek, USA) at starting time point and after 48 hours of cultivation. Data are presented as mean±95% confidence interval (n = 12). Results: The studied cultures were found to be differently sensitive to the tested substances. Cells of CaCo-2 and PC3 cultures did not demonstrate a significant decrease in mobility, as the degree of scratch overgrowth did not decrease compared to the control without the addition of substances (α = 0.05, df = 22) in all variants of the experiment. However, the A431 skin cancer culture showed reduced motility in all three trials compared to the no-substance control. Namely, the wound area reduction was 81.52 ± 9.6% in the control, 63.77 ± 8.4% in S1 variant, 62.42 ± 6.3% in S2 variant, and 12.05 ± 7.1% in S3 variant. Also, a slowdown in cell motility was observed in HeLa (28.92±5.1% compared to 37.0±6.3% in control) and T98G cells (82.41±9.1% compared to with 97.35±1.9% in control) when incubated with S1. Taking into account the correction for multiple comparisons, only the results for A431 turned out to be significant, but the results obtained for HeLa and T98G cultures were not significant at a corrected significance level (α = 0.003, df = 22). Conclusions: Further studies of 2,4-dihydroxy-2,5-dimethylfuran-3(2H)-one anti-migratory properties are of particular interest, since it showed promising results in three of the five studied cancer cell cultures.

Effect of berberine on cancer cell motility in glioma, lung cancer, and prostate cancer cell cultures.

e15079 Background: Initially, berberine was used as an antimicrobial agent in traditional medicine, but its anticancer properties were later discovered. In addition to its cytotoxic effect, berberine also has the ability to inhibit cell motility, which has been demonstrated in some permanent cancer cell lines. The objective of this study was to assess berberine anti-migratory activity in permanent cell cultures compared to primary cell cultures which are generally thought to better reflect tumor characteristics. Methods: H1299 lung cancer, PC3 prostate cancer, and T98G glioma cells, as well as primary cell cultures of the corresponding cancer obtained in our laboratory, were planted in an amount of 15*104 cells per well of a 24-well plate (Biofil, China) in DMEM medium (Gibco, USA) supplemented with 10% FBS (HyClone, USA). After cell adhesion berberine was added in concentration 5 µM, and a wound healing assay was performed according to the standard procedure. Cell plates were continuously incubated and photographed in Lionheart FX imager (BioTek, USA) at 37°C and 5.0% CO2. The extent of cell migration was measured as the percentage reduction in wound area after 48 hours of incubation relative to baseline. Data are presented as Mean ± 95% confidence interval (n = 12). Results: The use of berberine at a concentration of 5 µM led to a significant decrease in cell motility in permanent cultures of lung cancer, glioma and prostate cancer. Namely, the reduction in the wound area after 48 hours of incubation with bereberine was 74.52±12.3% (compared with 94.56±6.2% in the control) in H1299 cell culture, 38.22±10.6% (compared with 83.89±15.5% in the control) in T98G cell culture, and 48.6±7.5% (compared with 69.56±8.1% in the control) in PC3 cell culture. The resulting difference between the control and experimental groups in permanent cell cultures was statistically significant at a significance level of 5% (df = 22). At the same time, the values of wound area reduction for primary cultures of the same cancers did not differ significantly in the control and under the influence of 5 μM berberine at the accepted level of significance. Namely, the reduction in the wound area after 48 hours of incubation with bereberine was 84.79±11.2% (compared with 81.47±15.3% in the control) in lung cancer primary cell culture, 94.64±5.1% (compared with 91.73±6.8% in the control) in glioma primary culture, and 62.63±5.8% (compared with 61.1±8.9% in the control) in prostate cancer primary cell culture. Conclusions: Permanent cell lines are more sensitive to berberine anti-migratory activity than primary cancer cell lines of the same localization.

Hybrid Self-Assembling Nanoparticles Encapsulating Zoledronic Acid: A Strategy for Fostering Their Clinical Use.

Self-assembling nanoparticles (SANPs) promise an effective delivery of bisphosphonates or microRNAs in the treatment of glioblastoma (GBM) and are obtained through the sequential mixing of four components immediately before use. The self-assembling approach facilitates technology transfer, but the complexity of the SANP preparation protocol raises significant concerns in the clinical setting due to the high risk of human errors during the procedure. In this work, it was hypothesized that the SANP preparation protocol could be simplified by using freeze-dried formulations. An in-depth thermodynamic study was conducted on solutions of different cryoprotectants, namely sucrose, mannitol and trehalose, to test their ability to stabilize the produced SANPs. In addition, the ability of SANPs to deliver drugs after lyophilization was assessed on selected formulations encapsulating zoledronic acid in vitro in the T98G GBM cell line and in vivo in an orthotopic mouse model. Results showed that, after lyophilization optimization, freeze-dried SANPs encapsulating zoledronic acid could retain their delivery ability, showing a significant inhibition of T98G cell growth both in vitro and in vivo. Overall, these results suggest that freeze-drying may help boost the industrial development of SANPs for the delivery of drugs to the brain.

Allopregnanolone Promotes Migration and Invasion of Human Glioblastoma Cells through the Protein Tyrosine Kinase c-Src Activation.

Glioblastomas (GBs) are the most aggressive and common primary malignant brain tumors. Steroid hormone progesterone (P4) and its neuroactive metabolites, such as allopregnanolone (3α-THP) are synthesized by neural, glial, and malignant GB cells. P4 promotes cellular proliferation, migration, and invasion of human GB cells at physiological concentrations. It has been reported that 3α-THP promotes GB cell proliferation. Here we investigated the effects of 3α-THP on GB cell migration and invasion, the participation of the enzymes involved in its metabolism (AKR1C1-4), and the role of the c-Src kinase in 3α-THP effects in GBs. 3α-THP 100 nM promoted migration and invasion of U251, U87, and LN229 human-derived GB cell lines. We observed that U251, LN229, and T98G cell lines exhibited a higher protein content of AKR1C1-4 than normal human astrocytes. AKR1C1-4 silencing did not modify 3α-THP effects on migration and invasion. 3α-THP activated c-Src protein at 10 min (U251 cells) and 15 min (U87 and LN229 cells). Interestingly, the pharmacological inhibition of c-Src decreases the promoting effects of 3α-THP on cell migration and invasion. Together, these data indicate that 3α-THP promotes GB migration and invasion through c-Src activation.

The mesoionic compound MI-D changes energy metabolism and induces apoptosis in T98G glioma cells

The mesoionic compound 4-phenyl-5-(4-nitro-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride (MI-D) impairs mitochondrial oxidative phosphorylation and has a significant antitumour effect against hepatocarcinoma and melanoma. This study evaluated the cytotoxic effect of MI-D on T98G glioblastoma cells and investigated whether the impairment of oxidative phosphorylation promoted by MI-D is relevant to its cytotoxic effect. The effects of MI-D on T98G cells cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) HG (glycolysis-dependent) and galactose plus glutamine-supplemented Dulbecco's modified Eagle's medium (DMEM) GAL (oxidative phosphorylation-dependent) were compared. T98G cells grown in DMEM GAL medium exhibited higher respiration rates and citrate synthase activity and lower lactate levels, confirming the metabolic shift to oxidative phosphorylation in these cells. MI-D significantly decreased the cell viability in a dose-dependent manner in both media; however, T98G cells cultured in DMEM GAL medium were more susceptible. The mesoionic significantly inhibited mitochondrial oxidative phosphorylation of glioma cells in both media. At the same time, lactate levels were not altered, indicating an absence of compensatory glycolysis activation. Additionally, MI-D increased the citrate synthase activity of cells in both media, which in DMEM HG-cultivated cells was followed by citrate accumulation. Apoptosis dependent on caspase-3 mediated the toxicity of MI-D on T98G cells. The higher susceptibility of glioma cells cultured in DMEM GAL medium to MI-D indicates that the impairment of mitochondrial functions is involved in mesoionic cytotoxicity. The results of this study indicate the potential use of MI-D for glioblastoma treatment.

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.

Cardiac Glycosides Increase Temozolomide Anticancer Activity in Therapy Resistant Glioblastoma Cells

Glioblastomas (GBMs) are a form of malignant gliomas characterized by a dismal prognosis. Standard treatment for glioblastoma patients is combined maximal surgical removal of the tumor with postoperative radiotherapy and concomitant chemotherapy with Temozolomide (TMZ). Among the histological characteristics that contribute to GBM progression are the rapid proliferation and neo-angiogenetic processes. The Na+/K+-ATPase is a transporter that promotes the migration of cancer cells, and its aberrant expression and activity have been associated with several cancers, including GBM. Using cardiac glycosides, we examined the effects of direct inhibition of the Na+/K+-ATPase in glioblastoma cells in vitro. We found that cardiac glycoside Digoxin is an effective anticancer agent on several glioma cell lines via Na+/K+-ATPase inhibition. Drug cytotoxicity assays showed that Digoxin as monotherapy significantly increased cell death and increased the efficacy of Temozolomide (TMZ) in the glioma cell lines T98G, U-97 MG, and primary GBM cells BNC-6. Additionally, Digoxin exhibited important anti-migratory effects on the highly aggressive and chemotherapy-resistant T98G glioma cell-line, demonstrating a potential therapeutic role for cardiac glycosides.

Compound cellular stress maximizes apoptosis independently of p53 in glioblastoma

ABSTRACT We examined the apoptotic response of two glioblastoma cells, p53 wild type U87 and p53 mutated T98G, to doxorubicin, bortezomib, and vorinostat, which respectively target DNA, 26S proteasome and histone deacetylase, to clarify p53ʹs function in apoptosis. We demonstrated that doxorubicin induced apoptosis in U87 cells but not in T98G cells. The level of p53 was definitively correlated to the extent of DNA damage and apoptosis initiation. Dominant-negative p53 reduced p21 expression, but did not affect doxorubicin-induced apoptosis, so the transcriptional activity of p53 seemed not to participate in doxorubicin-induced apoptosis. However, p53 concentrated into the nucleus during heavy apoptosis. Bortezomib could induce apoptosis in U87 with high sensitivity and T98G cells with low sensitivity. In contrast, vorinostat promoted apoptosis in both U87 and T98G cells and reduced the basal level of p53 in U87 cells, indicating that p53 played no role in the vorinostat-induced apoptosis. To clearly define the role of p53 in bortezomib- and doxorubicin-induced apoptosis, we combined doxorubicin with bortezomib to treat U87 cells to assess this combination’s effect on apoptosis and p53 status. Interestingly, the combination of doxorubicin with bortezomib engendered compound stress, resulting in a synergistic outcome for apoptosis in U87 cells. However, the amounts of p53 in the total count and in the nucleus were much lower with the combination than with doxorubicin alone, suggesting that p53 played no role in either the compound stress, doxorubicin-only or bortezomib-induced apoptosis.

NCAPG Promotes Tumor Progression and Modulates Immune Cell Infiltration in Glioma.

Glioma is one of the most deadly types of brain cancer. As it is highly invasive, the prognosis for glioma patients remains dismal, with median survival rarely exceeding 16 months. Thus, developing a new prognostic biomarker for glioma and investigating its molecular mechanisms is necessary for the development of an efficient treatment strategy. In this study, we analyzed a cohort of 1,131 glioma patients using RNA-seq data from The Cancer Genome Atlas (TCGA project) and Gene Expression Omnibus (GSE4290 and GSE16011 datasets), and validated the results using the RNA-seq data of 1,018 gliomas from the Chinese Glioma Genome Atlas (CGGA project). We used the R language as the main tool for statistical analysis and data visualization. We found that NCAPG, a mitosis-associated chromosomal condensing protein, is highly expressed in glioma tissues. Furthermore, the expression of NCAPG increased significantly with the increase in tumor grade, and high NCAPG expression was found to be a predictor of poor overall survival in glioma patients (P < 0.001). This result shows that NCAPG expression could be an independent prognostic factor. Importantly, when the expression of NCAPG was knocked down, the CCK-8 assay revealed that the proliferation of glioma cells (LN-229 and T98G cell lines) decreased significantly compared with the control group. In addition, the healing rates of these cells were significantly lower in the si-NCAPG group than in the control group (P < 0.001). We then used the CIBERSORT algorithm to analyze the expression levels of 22 subpopulations of immune cells and found that NCAPG was significantly negatively correlated with natural killer cell activation. In addition, it was positively correlated with MHC-I molecules and ADAM17. Our study is first in comprehensively describing the high expression of NCAPG in glioma. It also shows that NCAPG can function as an independent prognostic predictor of glioma, and that targeting NCAPG can be a new strategy for the treatment of glioma patients.

Efficacy of Temozolomide-Conjugated Gold Nanoparticle Photothermal Therapy of Drug-Resistant Glioblastoma and Its Mechanism Study.

Temozolomide (TMZ) is a standard-of-care chemotherapeutic drug for the treatment of glioblastoma (GBM), but TMZ-acquired resistance limits its therapeutic effect. In this study, TMZ-loaded gold nanoparticles (TMZ@GNPs) with anti-EphA3 modification on the surface (anti-EphA3-TMZ@GNPs) were synthesized for chemical and auxiliary plasma photothermal treatment (GNPs-PPTT), aiming to overcome the problem of glioma resistance to TMZ and improve the therapeutic effects of GBM. The prepared anti-EphA3-TMZ@GNPs were spherical with a particle size of 45.88 ± 1.9 nm, and the drug loading was 7.31 ± 0.38%. In vitro, cell-culture-based experiments showed that anti-EphA3 increased the cellular uptake of GNPs in T98G cells. Upon laser irradiation, the cytotoxicity and apoptosis rate in the anti-EphA3-TMZ@GNPs-treated group were significantly higher than those in the GNPs and nonphotothermal groups (p < 0.001). The Western blot analysis showed that the GNPs-PPTT-mediated killing of tumor cells induced apoptosis by regulating the apoptotic signaling molecules and cell cycle inhibitors; the expression of MGMT significantly decreased upon p53 induction, thereby reversing drug resistance. After photothermal treatment, the survival time of the subcutaneous GBM model of nude mice in the anti-EphA3-TMZ@GNPs group was prolonged to 46 days, 1.64-fold longer as compared to that in the TMZ group. Based on H&E and TUNEL staining, GNPs-PPTT could elevate apoptosis in T98G cells. In vivo thermal imaging results showed that GNPs could enter the brain via intranasal administration and be eliminated in 2 days, indicating that GNPs are safe for brain. In conclusion, GNPs-PPTT could effectively induce apoptosis in glioma cells and reverse TMZ resistance, thereby, indicative of a promising treatment strategy for GBM.

Quercetin Inhibits Glioblastoma Growth and Prolongs Survival Rate through Inhibiting Glycolytic Metabolism

Introduction: Quercetin has been reported to have antitumor activity of a wide range of cancers, including breast, lung, colon, prostate. Here, we investigated the protective role of quercetin in glioblastoma (GBM), which causes a higher risk of morbidity and mortality, and explored the antitumor effects of quercetin on GBM using the U87MG and T98G cells and GBM mouse models. Methods: Cell viability and colony formation assays were performed by CCK-8 and clone-formation assays. GBM xenograft mouse model was established to evaluate the tumor burden of mice treated with or without quercetin. To investigate spontaneous locomotor activity and survival rate of mice, orthotopic transplantation was performed through brain stereotaxic injection of U87 cells. Seahorse and Western blot were performed to examine the alteration of glycolytic metabolism GBM. Results: We found that quercetin administration inhibited GBM cell proliferation and promoted cell apoptosis in vitro. Quercetin suppressed GBM growth, restored spontaneous locomotor activity, and improved survival rate without toxicity to peripheral organs in vivo. Moreover, quercetin inhibited glycolytic metabolism in tumor tissue. Discussion/Conclusion: Mechanistically, quercetin inhibited proliferation and angiogenesis, promoted cancer cell apoptosis, and finally improved locomotor activity and survival by inhibiting the glycolytic metabolism in GBM tissues, suggesting that quercetin is a potential drug for the treatment of GBM.

Tubeimoside-I sensitizes temozolomide-resistant glioblastoma cells to chemotherapy by reducing MGMT expression and suppressing EGFR induced PI3K/Akt/mTOR/NF-κB-mediated signaling pathway

BackgroundGlioblastoma multiforme (GBM, World Health Organization [WHO] grade IV) is one of the malignant Central Nerve System (CNS) tumors with high incidence rate and poor prognosis. The use of alkylating agents, such as temozolomide (TMZ), has been the main method of cytotoxic therapy for glioma patients for decades. However, TMZ resistance may be one of the major reasons for treatment failure, so far. In searching for effective agents to reverse TMZ resistance, we found that Tubeimoside-I (TBMS1), a saponin from traditional Chinese medicine, Bolbostemma paniculatum (Maxim.) Franquet, showed activities of reversing TMZ resistance of GBM. However, the ability of TBMS1 enhancing the chemosensitivity of GBM has been rarely studied, and its underlying mechanisms remain unclear. PurposeThis study purposes to reveal the synergistic effects and mechanism of TBMS1 and TMZ against TMZ-resistant GBM cells. MethodsCCK8 assay was used to investigate the anti-proliferative effects on grade IV glioblastoma human T98G and U118 MG cells. Cell proliferation was determined by EdU assay and clonogenic assay after TMZ plus TBMS1 treatment. Apoptosis was analyzed by flow cytometry. DNA damage and DNA Double Strand Break (DSB) were assessed by cleaved Poly (ADP-ribose) polymerase (PARP), γH2AX Foci Assay and Comet Assay, respectively. Expression of proteins associated with apoptosis and DNA repair enzymes were measured by Western blot analysis. The prognostic significance of key proteins of the epidermal growth factor receptor (EGFR) induced PI3K/Akt/mTOR/NF-κB signaling pathway was analyzed using GEPIA (http://gepia.cancer-pku.cn) and validated by Western blotting. ResultsHere we demonstrated that TBMS1 sensitized TMZ-resistant T98G and U118 MG glioblastoma cells to chemotherapy and exhibited promotion of apoptosis and inhibition on cell viability, proliferation and clone formation. Coefficient of drug in interaction (CDI) values showed a notable synergistic effect between TBMS1 and TMZ. Moreover, we observed that combination of TBMS1 and TMZ induced apoptosis was accompanied by robust DSB, γH2AX Foci formation and increasing cleaved PARP, as well as the heightened ratio of Bax/Bcl-2, cleavages of caspase-3 and caspase-9. In addition, the synergistic anti-glioma effect between TBMS1 and TMZ was intimately related to the reduction of MGMT expression in TMZ-resistant GBM cells. Moreover, it was also associated with attenuated expression of EGFR, p-PI3K-p85, p-Akt (Ser473), p-mTOR (Ser2481) and p-NF-κB p65(Ser536), which implying deactivation of the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway. ConclusionWe first demonstrated that synergistic effects of TBMS1 and TMZ induced apoptosis in GBM cells through reducing MGMT expression and inhibiting the EGFR induced PI3K/Akt/mTOR/NF-κB signaling pathway. This study provides a rationale for combined application of TMZ and TBMS1 as a potential chemotherapeutic treatment for MGMT+ GBM patients.

Biocatalytic Synthesis of Terpene Esters and their Biological Activity in Human Glioma Cells

Gliomas are highly malignant brain tumours with high resistance to chemotherapy. Therefore, investigations of new therapeutic molecules with high anti-glioma activity are of great importance. In this work, biocatalytic esterification of terpene alcohols with proven anti-cancer activity was performed to enhance their potency to induce cell death in human glioblastoma multiforme T98G and anaplastic astrocytoma MOGGCCM cell lines in vitro. We used primary terpene alcohols and carboxylic acids with a length of two to nine carbon atoms. The structure of the alcohols had an influence on the esterification efficiency, which decreased in the following order: monocyclic > linear > bicyclic. Terpene alcohols and their esters only induced apoptotic cell death, which is highly desirable from a therapeutic point of view, but did not induce autophagy and necrosis. The esterification of perillyl alcohol with butyric acid caused a 4-fold increase in cell death induction in the T98G line. Citronellol valerate, caprylate, and pelargonate and myrtenol butyrate, caprylate and pelargonate also showed higher activity than their alcohol precursors. We have herein shown that esterification of natural alcohols by biocatalysis can be used for improving the activity of other compounds investigated for their anti-glioma activity.