RTA404, an Activator of Nrf2, Activates the Checkpoint Kinases and Induces Apoptosis through Intrinsic Apoptotic Pathway in Malignant Glioma

Background: Malignant glioma (MG) is an aggressive malignant brain tumor. Despite advances in multidisciplinary treatment, overall survival rates remain low. A trifluoroethyl amide derivative of 2-cyano-3-,12-dioxoolean-1,9-dien-28-oic acid (CDDO), CDDO–trifluoroethyl amide (CDDO–TFEA) is a nuclear erythroid 2-related factor 2/antioxidant response element pathway activator. RTA404 is used to inhibit proliferation and induce apoptosis in cancer cells. However, its effect on tumorigenesis in glioma is unclear. Methods: This in vitro study evaluated the effects of RTA404 on MG cells. We treated U87MG cell lines with RTA404 and performed assessments of apoptosis and cell cycle distributions. DNA content and apoptosis induction were subjected to flow cytometry analysis. The mitotic index was assessed based on MPM-2 expression. Protein expression was analyzed through Western blotting. Results: RTA404 significantly inhibited the cell viability and induced cell apoptosis on the U87MG cell line. The Annexin-FITC/PI assay revealed significant changes in the percentage of apoptotic cells. Treatment with RTA404 led to a significant reduction in the U87MG cells’ mitochondrial membrane potential. A significant rise in the percentage of caspase-3 activity was detected in the treated cells. In addition, these results suggest that cells pass the G2 checkpoint without cell cycle arrest by RTA404 treatment in the MPM-2 staining. An analysis of CHK1, CHK2, and p-CHK2 expression suggested that the DNA damage checkpoint system seems also to be activated by RTA404 treatment in established U87MG cells. Therefore, RTA404 may not only activate the DNA damage checkpoint system, it may also exert apoptosis in established U87MG cells. Conclusions: RTA404 inhibits the cell viability of gliomas and induces cancer cell apoptosis through intrinsic apoptotic pathway in Malignant glioma. In addition, the DNA damage checkpoint system seems also to be activated by RTA404. Taken together, RTA404 activated the DNA damage checkpoint system and induced apoptosis through intrinsic apoptotic pathways in established U87MG cells.

Anti-Proliferative Effects of E2F1 Suppression in Glioblastoma Cells

Glioblastoma (GBM) is an aggressive malignant brain tumor; surgery, radiation, and temozolomide still remain the main treatments. There is evidence that E2F1 is overexpressed in various types of cancer, including GBM. E2F1 is a transcription factor that controls the cell cycle progression and regulates DNA damage responses and the proliferation of pluripotent and neural stem cells. To test the potentiality of E2F1 as molecular target for GBM treatment, we suppressed the <i>E2F1</i> gene (siRNA) in the U87MG cell line, aiming to inhibit cellular proliferation and modulate the radioresistance of these cells. Following E2F1 suppression, associated or not with gamma-irradiation, several assays (cell proliferation, cell cycle analysis, neurosphere counting, and protein expression) were performed in U87MG cells grown as monolayer or neurospheres. We found that siE2F1-suppressed cells showed reduced cell proliferation and increased cell death (sub-G1 fraction) in monolayer cultures, and also a significant reduction in the number of neurospheres. In addition, in irradiated cells, E2F1 suppression caused similar effects, with reduction of the number of neurospheres and neurosphere cell numbers relative to controls; these results suggest that E2F1 plays a role in the maintenance of GBM stem cells, and our results obtained in neurospheres are relevant within the context of radiation resistance. Furthermore, E2F1 suppression inhibited or delayed GBM cell differentiation by maintaining a reasonable proportion of CD133+ cells when grown at differentiation condition. Therefore, E2F1 proved to be an interesting molecular target for therapeutic intervention in U87MG cells.

Valproic Acid (VPA) in Combination with Knockdown of AKT3 and PI3KCA Genes Inhibits Proliferation, Induces Apoptosis and Autophagy in T98G and U87MG Glioblastoma Multiforme Cells

Purpose: Glioblastoma Multiforme (GBM) is a heterogenous and highly vascularized brain tumor that avoid apoptosis due to P-glycoprotein (P-gp) mediated multi-drug resistance. Therefore, development of new therapeutic strategies that induce apoptosis, inhibit proliferation, and overcome multi-drug resistance is urgently warranted. We examined the efficacy of combination of Valproic Acid (VPA) and knockdown of AKT3 and PI3KCA genes in human glioblastoma T98G and U87MG cell lines. Material and Methods: T98G and U87MG cells were transfected with AKT3 or PI3KCA siRNAs. Transfection efficiency was assessed using Flow Cytometry (FC) and fluorescence microscopy. The influence of AKT3 and PI3KCA siRNAs in combination with VPA on T98G and U87MG cell viability, proliferation, apoptosis and autophagy was evaluated as well. Alterations in the mRNA expression of apoptosis-related genes (CASP3 and Bid) were analyzed using QRT-PCR. Results: The transfection of T98G and U87MG cells with AKT3 or PI3KCA siRNAs and exposition on VPA led to a significant reduction in cell viability, the accumulation of subG1-phase cells and a reduced fraction of cells in the S and G2/M phases, apoptosis or necrosis induction and induction of autophagy. Conclusions: The siRNA-induced AKT3 and PI3KCA mRNA knockdown in combination with VPA may offer a novel therapeutic strategy to more effective control the growth of human GBM cells. Thus, knockdown of these genes in combination with valproic acid inhibits proliferation, induces apoptosis and autophagy in T98G and U87MG cells, but further studies are necessary to confirm a positive phenomenon for the treatment of GBM.

Generation of dendritic cells using viral lysate of tumor cells in vitro.

e15202 Background: The purpose of the study was to investigate the effect of a new unclassified rotavirus on the effectiveness of maturation of dendritic cells (DC) and their activation of lymphocytes in vitro. Methods: Strain No.100 of the RVK virus was isolated by S.A. Kolpakov and characterized as rotavirus. U87MG cell lysate (glioblastoma) was obtained by incubation with 108 RVK particles in the DMEM medium with L-glutamine for 72 hours; a cytopathic effect was observed. Immature DCs were cultured for 7 days in the presence of IL-4 and GM-CSF. For antigen loading of DCs, we used the following options for 48 h: a) U87MG lysate obtained by repeated freeze-thaw cycles (TL); b) U87MG lysate obtained by co-cultivation with RVK (VTL); c) RVK. To assess the DC ability to activate autologous lymphocytes, they were co-cultured for 5 days in a 3:1 ratio. Results: The use of VTL culture for DC loading caused an increase in the expression of mature DC (mDC) markers compared to TL: the number of CD83+/CD86+ cells increased by more than 2 times, CD83+/CD80+ by 1.2 times, CD83+HLA-DR - by 5.5 times. With VTL, expression of markers of immature DCs (CD1a, CD14) was minimal. The use of RVK as an antigen induced the generation of DCs from monocytes, but their maturation was much less pronounced: a significant increase in the membrane expression of CD86, but not CD83, was determined. These DCs demonstrated a higher expression of markers of immature DCs, compared to stimulation with cell lysates (both TL and VTL): CD1a and CD14; CD80+/CD86+ level was the highest among all options. Analysis of the DC effect on co-cultivated lymphocytes showed that DCs loaded with RVK, both alone and as part of VTL, stimulated predominantly the NKT subpopulation. The same samples contained more T lymphocytes, activated CD4+ and CD8+ compared to samples stimulated by TL. However, the samples co-cultivated with VTL contained the maximal amount of CD4+/CD25+/CD127dim phenotypically corresponding to Tregs. Conclusions: The antigen loading of immature DCs with RVK alone causes their activation, but not maturation, which is not realized in typical terms of the DC generation from blood monocytes. The presence of RVK, including in VTL, has a stimulating effect on NKT lymphocytes suggesting the possible generation of specific highly active cytotoxic lymphocytes.

NOS2 inhibitor 1400W Induces Autophagic Flux and Influences Extracellular Vesicle Profile in Human Glioblastoma U87MG Cell Line

The relevance of nitric oxide synthase 2 (NOS2) as a prognostic factor in Glioblastoma Multiforme (GBM) malignancy is emerging. We analyzed the effect of NOS2 inhibitor 1400W on the autophagic flux and extracellular vesicle (EV) secretion in U87MG glioma cells. The effects of glioma stem cells (GSC)-derived EVs on adherent U87MG were evaluated. Cell proliferation and migration were examined while using Cell Counting Kit-8 assay (CCK-8) and scratch wound healing assay. Cell cycle profile and apoptosis were analyzed by flow cytometry. Autophagy-associated acidic vesicular organelles were detected and quantified by acridine orange staining. The number and size of EVs were assessed by nanoparticle tracking analysis. EV ultrastructure was verified by transmission electron microscopy (TEM). WB was used to analyze protein expression and acid sphingomyelinase was determined through ceramide levels. 1400W induced autophagy and EV secretion in both adherent U87MG and GSCs. EVs secreted by 1400W-treated GSC, but not those from untreated cells, were able to inhibit adherent U87MG cell growth and migration while also inducing a relevant level of autophagy. The hypothesis of NOS2 expression as GBM profile marker or interesting therapeutic target is supported by our findings. Autophagy and EV release following treatment with the NOS2 inhibitor could represent useful elements to better understand the complex biomolecular frame of GBM.

NEUROPROTECTIVE AND COGNITIVE ENHANCING EFFECT OF METHANOLIC MORUS ALBA LEAF FRACTION IN U87MG CELL LINES AND EXPERIMENTAL RAT MODEL

Objective: The present study aims to investigate the protective effect of methanol fraction of Morus alba (MEMA) leaves against hydrogen peroxide (H2O2)-induced U87MG cell toxicity and aluminum fluoride (ALF)-induced rat toxicity. Methods: The study was divided into in vitro and in vivo sections. U87MG cell lines were pre-treated with different fractions of MEMA for 20 h and further tested against 1000 ϻM of H2O2. The best fraction from in vitro studies was used to study the protective effects against ALF-induced neurotoxicity. Rats were divided i nto five different groups, and MEMA (200 and 400 mg/kg p.o) was administered for 14 days to the animals with α-tocopherol as the standard drug treatment. Behavioral studies were assessed using Barnes maze. The major biochemical measurements included catalase, superoxide dismutase and glutathione reductase, lipid peroxidation (LPO), and acetylcholinesterase (AchE) levels. Results: In vitro studies indicated MEMA as a potential candidate followed by AQMA and ethyl acetate. The MEMA fraction was able to ameliorate ALF-induced neurotoxicity in the behavioral assessment. The higher antioxidant content in the fraction decreased the LPO levels from 250±4.07 to 115±3.22 as well as elevated the levels of most of the endogenous antioxidant enzyme levels. AchE levels were also decreased to 33.89±0.71 from 38.94±0.64. Conclusion: Although the results obtained indicate that MEMA could significantly suppress oxidative stress-induced central neuronal damage both in vitro and in vivo, further mechanistic studies are required to delineate its neuroprotective pathway.

Development of a novel protein identification approach to define mitochondrial proteomic signatures in glioblastoma oncogenesis: T98G vs U87MG cell lines model

Glioblastoma Multiforme is a cancer type with an important mitochondrial component. Here was used mitochondrial proteome Random Sampling in 2D gels from T98G (oxidative metabolism) and U87MG (glycolytic metabolism) cell lines to obtain and analyze representative spots (regardless of their intensity, size, or difference in abundance between cell lines) by Principal Component Analysis for protein identification. Identified proteins were ordered into specific Protein-Protein Interaction networks, to each cell line, showing mitochondrial processes related to metabolic change, invasion, and metastasis; and other nonmitochondrial processes such as DNA translation, chaperone response, and autophagy in gliomas. T98G and U87MG cell lines were used as glioblastoma transition model; representative proteomic signatures, with the most important biological processes in each cell line, were defined. This pipeline analysis describes the metabolic status of each line and defines clear mitochondria performance differences for distinct glioblastoma stages, introducing a new useful strategy for the understanding of glioblastoma carcinogenesis formation.Biological significanceThis study defines the mitochondria as an organelle that follows and senses the carcinogenesis process by an original proteomic approach, a random sampling in 2DE gels to obtain a representative spots sample and analyzing their relative abundance by Principal Components Analysis; to faithfully describe glioblastoma cells biology.