Berberine Photo-Activation Potentiates Cytotoxicity in Human Astrocytoma Cells through Apoptosis Induction

Photodynamic therapy (PDT) has recently attracted interest as an innovative and adjuvant treatment for different cancers including malignant gliomas. Among these, Glioblastoma (GBM) is the most prevalent neoplasm in the central nervous system. Despite conventional therapeutic approaches that include surgical removal, radiation, and chemotherapy, GBM is characterized by an extremely poor prognosis and a high rate of recurrence. PDT is a physical process that induces tumor cell death through the genesis and accumulation of reactive oxygen species (ROS) produced by light energy interaction with a photosensitizing agent. In this contribution, we explored the potentiality of the plant alkaloid berberine (BBR) as a photosensitizing and cytotoxic agent coupled with a PDT scheme using a blue light source in human established astrocytoma cell lines. Our data mainly indicated for the combined BBR-PDT scheme a potent activation of the apoptosis pathway, through a massive ROS production, a great extent of mitochondria depolarization, and the sub-sequent activation of caspases. Altogether, these results demonstrated that BBR is an efficient photosensitizer agent and that its association with PDT may be a potential anticancer strategy for high malignant gliomas.

Bioinformatics and Prediction Analysis of Long Non-coding RNAs in the Regulation of Glioblastoma Cell Migration and Invasion

Long non-coding RNA (lncRNA) has been identified in the regulation of cell-invasion via various cellular processes. Research on lncRNAs is however lacking in the context of Glioblastoma (GBM), which held-up identification of GBM-related lncRNAs via bioinformatic databases. This study intends to identify potential lncRNA candidates in the regulation of GBM cell-invasion based on target miRNA and mRNA/proteins. Microarray was carried out to identify upregulated lncRNA in GBM and astrocytoma cells, followed by bioinformatics-based prediction analysis to identify lncRNAs involved in cellular processes related to GBM cell-invasion. A total of 372 and 806 upregulated lncRNA were identified in GBM and astrocytoma cells. From these lncRNAs, 8 lncRNAs were predicted based on functions of target proteins; LINC00221 associated with EMT, LINC01564 and LINC00265 with angiogenesis, and LOC100240735 with cell-migration. Network analysis of lncRNA based on function of target miRNAs predicted LINC00999, ALOX12-AS1, CHKB-AS1, and LINC01588 as regulators of angiogenesis, with LINC00482, LINC00239, and LINC01003 for regulation of EMT. Based on the network analysis, three novel lncRNA-miRNA-mRNA interacting axis from NEAT1, CRNDE, and SNHG1 were predicted to regulate GBM invasion. These findings have allowed prediction of lncRNA as regulators of GBM cell-invasion which can be considered as potential candidates for experimental studies as a GBM prognostic biomarkers.

Glioma-associated microglia/macrophages augment tumorigenicity in canine astrocytoma, a naturally occurring model of human glioma

Background Glioma-associated microglia/macrophage (GAM) markedly influence glioma progression. Under the influence of transforming growth factor beta (TGFB), GAM are polarized toward a tumor-supportive phenotype. However, neither therapeutic targeting of GAM recruitment, nor TGF Beta (TGFB) signaling demonstrated efficacy in glioma patients despite efficacy in preclinical models, underscoring the need for a comprehensive understanding of the TGFB/GAM axis. Spontaneously occurring canine gliomas share many features with human glioma and provide a complementary translational animal model for further study. Given the importance of GAM and TGFB in human glioma, the aims of this study were to further define the GAM-associated molecular profile and the relevance of TGFB signaling in canine glioma that may serve as the basis for future translational studies. Methods GAM morphometry, levels of GAM-associated molecules, and the canonical TGFB signaling axis were compared in archived samples of canine astrocytomas versus normal canine brain. Further, the effect of TGFB on the malignant phenotype of canine astrocytoma cells was evaluated. Results GAMs diffusely infiltrated canine astrocytomas. GAM density was increased in high-grade tumors that correlated with a pro-tumorigenic molecular signature and up-regulation of the canonical TGFB signaling axis. Moreover, TGFB1 enhanced migration of canine astrocytoma cells in vitro. Conclusions Canine astrocytomas share a similar GAM-associated immune landscape with human adult glioma. Our data also support a contributing role for TGFB1 signaling in the malignant phenotype of canine astrocytoma. These data further support naturally occurring canine glioma as a valid model for investigation of GAM-associated therapeutic strategies for human malignant glioma.

The age of adult pilocytic astrocytoma cells

Adult pilocytic astrocytomas (PAs) have been regarded as indistinguishable from pediatric PAs in terms of genome-wide expression and methylation patterns. It has been unclear whether adult PAs arise early in life and remain asymptomatic until adulthood, or whether they develop during adulthood. We sought to determine the age and origin of adult human PAs using two types of “marks” in the genomic DNA. First, we analyzed the DNA methylation patterns of adult and pediatric PAs to distinguish between PAs of different anatomic locations (n = 257 PA and control brain tissues). Second, we measured the concentration of nuclear bomb test-derived 14C in genomic DNA (n = 14 cases), which indicates the time point of the formation of human cell populations. Our data suggest that adult and pediatric PAs developing in the infratentorial brain are closely related and potentially develop from precursor cells early in life, whereas supratentorial PAs might show age and location-specific differences.

Intracellular nickel accumulation induces apoptosis and cell cycle arrest in human astrocytic cells

Nickel, a heavy metal found in electronic wastes and fume from electronic cigarettes, induces neuronal cell death and is associated with neurocognitive impairment. Astrocytes are the first line of defense against nickel after entering the brain; however, the effects of nickel on astrocytes remain unknown. Herein, we investigated the effect of nickel exposure on cell survival and proliferation and the underlying mechanisms in U-87 MG human astrocytoma cells and primary human astrocytes. Intracellular nickel levels were elevated in U-87 MG cells in both a dose- and time-dependent manner after exposure to nickel chloride. The median toxic concentrations of nickel in astrocytoma cells and primary human astrocytes were 600.60 μM and > 1,000 μM at 48 h post-exposure, respectively. Nickel exposure triggered apoptosis in concomitant with the decreased expression of anti-apoptotic B-cell lymphoma protein (Bcl-2), and increased caspase-3/7 activity. Nickel induced reactive oxygen species formation. Additionally, nickel suppressed astrocyte proliferation in a dose- and time-dependent manner by delaying G2 to M phase transition through the upregulation of cyclin B1 and p27 protein expression. These results indicate that nickel-induced cytotoxicity of astrocytes is mediated by the activation of apoptotic pathway and disruption of cell cycle regulation.

CSIG-19. THE DEUBIQUITINASE BRCC3 LINKS ALT TELOMERES TO SUPPRESSION OF INNATE IMMUNITY IN IDH1-MUTANT GLIOMA

Approximately 10% of tumors including all IDH1-mutant lower-grade glioma resolve telomeric shortening using Alternative Lengthening of Telomere (ALT) mechanism. Although the ALT process lengthens telomeres, it also generates small bits of extrachromosomal, telomere-containing DNA (ECTRs). These ECTRs can bind and activate cyclic GMP-AMP synthase (cGAS), the major cytosolic sensor of double-stranded DNA, which in turn can activate expression of stimulator of IFN genes (STING) and the interferon-based innate immune response. To limit the immune response, ALT cells inactivate the cGAS-STING pathway, although the mechanism by which this occurs is unknown. Here we show that the deubiquitinase BRCC3 links ALT telomeres to suppression of the cGAS-STING pathway and the innate immune response. Astrocytoma cells dependent on the ALT-mechanism (IDH1-mutant and ATRX-deficient genetically-modified human astrocytes and MGG119 PDX) contained ECTR and had reduced expression of the cGAS and the downstream components of the cGAS-STING pathway (STING, and IFN-β) relative to matched non-ALT (isogenic ATRX WT astrocytes and MGG152 PDX) cells lacking ECTRs. Decreased levels of cGAS in ALT cells were in turn associated with deubiquitiantion and destabilization of cGAS. The telomere-derived ECTR in ALT-dependent cells lacked two proteins normally found in ALT telomeres (TRF2 or PARP), but retained two other proteins, Mre11 and its binding partner, normally nuclear deubiquitinase BRCC3. Furthermore, either pharmacologic inhibition or genetic suppression of BRCC3 levels had no effect on ECTR levels but stabilized cGAS and activated the cGAS-STING pathway. This cGAS-mediated activation could be blocked by exogenous expression of WT BRCC3, but not by expression of a mutant BRCC3 incapable of deubiquitination. These results show that BRCC3 translocated along with ECTRs to the cytoplasm degrades cGAS and protects ALT-dependent cells from activating the innate immune response. The BRCC3-controlled cGAS-STING pathway may therefore represent a therapeutically targetable means to enhance the immune response in IDH1-mutant lower grade glioma.

Yokukansan, a Japanese Herbal Medicine, Suppresses Substance P-Induced Production of Interleukin-6 and Interleukin-8 by Human U373 MG Glioblastoma Astrocytoma Cells

Context: Yokukansan is a traditional Japanese herbal medicine that has an antiallodynic effect in patients with chronic pain. However, the mechanisms by which yokukansan inhibits neuropathic pain are unclear. Objective: This study aimed to investigate the molecular effects of yokukansan on neuroinflammation in U373 MG glioblastoma astrocytoma cells, which express a functional high-affinity neurokinin 1 receptor (substance P receptor), and produce interleukin (IL)-6 and IL-8 in response to stimulation by substance P (SP). Methods: We assessed the effect of yokukansan on the expression of ERK1/2, P38 MAPK, nuclear factor (NF)-κB, and cyclooxygenase-2 (COX-2) in U373 cells by western blot assay. Levels of IL-6 and IL-8 in conditioned medium obtained after stimulation of cells with SP for 24 h were measured by enzyme-linked immunosorbent assay. All experiments were conducted in triplicate. Results were analyzed by one-way ANOVA, and significance was accepted at p < 0.05. Results: Yokukansan suppressed SP-induced production of IL-6 and IL-8 by U373 MG cells, and downregulated SP-induced COX-2 expression. Yokukansan also inhibited phosphorylation of ERK1/2 and p38 MAPK, as well as nuclear translocation of NF-κB, induced by SP stimulation of U373 MG cells. Conclusion: Yokukansan exhibits anti-inflammatory activity by suppressing SP-induced production of IL-6 and IL-8 and downregulating COX-2 expression in U373 MG cells, possibly via inhibition of the activation of signaling molecules, such as ERK1/2, p38 MAPK, and NF-κB.

Intracellular stiffening competes with cortical softening in glioblastoma cells

Cancer cell softening increases with the progression of the disease, suggesting that mechanical phenotyping could be used as a diagnostic or prognostic method. Here we investigate the cell mechanics of gliomas, brain tumors that originate from glial cells or glial progenitors. We use two microrheology techniques, a single cell parallel plates rheometer to probe whole cell mechanics and optical-tweezers to probe intracellular rheology. We show that cell mechanics discriminates human glioma cells of different grades. When probed globally, grade IV glioblastoma cells are softer than grade III astrocytoma cells, while they are surprisingly stiffer at the intracellular level. We explain this difference between global and local intracellular behaviours by changes in the composition and spatial organization of the cytoskeleton and by changes in nuclear mechanics. Our study highlights the need to combine rheology techniques for potential diagnostic or prognostic methods based on cancer cell mechanophenotyping.