NFIA and NFIB function as tumour suppressors in high-grade glioma in mice

Nuclear factor one (NFI) transcription factors are implicated in both brain development and cancer in mice and humans and play an essential role in glial differentiation. NFI expression is reduced in human astrocytoma samples, particularly those of higher grade, whereas over-expression of NFI protein can induce the differentiation of glioblastoma cells within human tumour xenografts and in glioblastoma cell lines in vitro. These data indicate that NFI proteins may act as tumour suppressors in glioma. To test this hypothesis, we generated complex mouse genetic crosses involving six alleles to target gene deletion of known tumour suppressor genes that induce endogenous high-grade glioma in mice, and overlaid this with loss of function Nfi mutant alleles, Nfia and Nfib, a reporter transgene and an inducible Cre allele. Deletion of Nfi resulted in reduced survival time of the mice, increased tumour load and a more aggressive tumour phenotype than observed in glioma mice with normal expression of NFI. Together, these data indicate that NFI genes represent a credible target for both diagnostic analyses and therapeutic strategies to combat high-grade glioma.

Molecular and Cellular Mechanisms of Human Astrocytoma Progression: Advances in Knowledge to Reach Therapeutic Horizons

Human astrocytic tumors are primary central nervous system (CNS) tumors that arise either from astrocytes or from precursor cells. A growing number of epidemiological and incidence studies in different countries underlined that, in addition to increasing economic costs for health systems, these cancers are still representing one of the main hurdles in developing a successful therapeutic goal for patients. On the other hand, new-omics technologies are offering customized instruments and more and more advantageous results toward personalized medicine approaches, underlining the concept that each tumor mass undergoes a peculiar transformation process under the control of specific genes’ and proteins’ functional signatures. The main aim of this Special Issue was to collect novel contributions in the wide field of human tumor astrocytic basic and translational research, to suggest further potential therapeutic targets/strategies that might interfere, possibly at the earliest stage of transformation, with the tumor progression, and to increase the molecular-based arsenal to counteract the prognostic poverty of high-grade astrocytic tumors.

Rapha Myr®, a Blend of Sulforaphane and Myrosinase, Exerts Antitumor and Anoikis-Sensitizing Effects on Human Astrocytoma Cells Modulating Sirtuins and DNA Methylation

Brain and other nervous system cancers are the 10th leading cause of death worldwide. Genome instability, cell cycle deregulation, epigenetic mechanisms, cytoarchitecture disassembly, redox homeostasis as well as apoptosis are involved in carcinogenesis. A diet rich in fruits and vegetables is inversely related with the risk of developing cancer. Several studies report that cruciferous vegetables exhibited antiproliferative effects due to the multi-pharmacological functions of their secondary metabolites such as isothiocyanate sulforaphane deriving from the enzymatic hydrolysis of glucosinolates. We treated human astrocytoma 1321N1 cells for 24 h with different concentrations (0.5, 1.25 and 2.5% v/v) of sulforaphane plus active myrosinase (Rapha Myr®) aqueous extract (10 mg/mL). Cell viability, DNA fragmentation, PARP-1 and γH2AX expression were examined to evaluate genotoxic effects of the treatment. Cell cycle progression, p53 and p21 expression, apoptosis, cytoskeleton morphology and cell migration were also investigated. In addition, global DNA methylation, DNMT1 mRNA levels and nuclear/mitochondrial sirtuins were studied as epigenetic biomarkers. Rapha Myr® exhibited low antioxidant capability and exerted antiproliferative and genotoxic effects on 1321N1 cells by blocking the cell cycle, disarranging cytoskeleton structure and focal adhesions, decreasing the integrin α5 expression, renewing anoikis and modulating some important epigenetic pathways independently of the cellular p53 status. In addition, Rapha Myr® suppresses the expression of the oncogenic p53 mutant protein. These findings promote Rapha Myr® as a promising chemotherapeutic agent for integrated cancer therapy of human astrocytoma.

Oxidized extracellular DNA affects DNA damage and transcription of genes regulating DNA repair and apoptosis in human astrocytoma cells

В работе исследовали влияние окисленной внеклеточной ДНК (овкДНК) на повреждение геномной ДНК и активацию транскрипции генов, регулирующих репарацию ДНК и процессы апоптоза в клетках астроцитомы человека линии 1321NI. Фрагменты овкДНК добавляли в среду культивирования клеток в концентрации 50-100 нг/мл на 0,5-24 часа. Уровни окислительных повреждений и двунитевых разрывов ДНК ядер, экспрессии белков BCL2, BAX определяли с помощью антител к 8-окси-7,8-дигидрогуанозину (8oxodG), фосфорилированной форме гистона H2AX (γH2AX), белкам BCL2 и BAX, соответственно, используя метод проточной цитофлуориметрии. Уровень экспрессии генов BCL2, ВАХ, BRCA1, BRCA2, ТВР (контроль) оценивали методом ПЦР в реальном времени. Воздействие на клетки 1321NI 50-100 нг/мл овкДНК в течение 30-60 мин приводило к 2-4-кратному повышению уровня 8-охоdG (р < 0,01, уровень двунитевых разрывов увеличивался в 3-3,5 раза (р < 0,01), а экспрессия генов репарации повреждений BRCA1 и BRCA2 возрастала в 3-5 раз (р<0,001). Позже, через 5-24 часа после воздействия на клетки овкДНК, в 3,5-5 раз увеличивалось отношение экспрессии генов BCL2/BAX по сравнению с нестимулированными культурами (p<0,001). Таким образом, в клетках астроцитомы овкДНК индуцирует и перестройку генома, и адаптивные реакции транскриптома, c последующей активацией анти-апоптотического процесса в клетках. Предполагается, что тем самым овкДНК может способствовать накоплению резистентных к лечению мутантных клонов в резидуальной опухоли, провоцируя рецидивы. The effects of oxidized extracellular DNA (oecDNA) on genomic DNA damage and activation of transcription of genes regulating DNA repair and apoptosis in human astrocytoma 1321NI cells were studied. OecDNA fragments, 50-100 ng/ml, were added to 1321NI cells for 0.5-24 hours. The levels of oxidative damage and double stranded DNA breaks, BCL and BAX protein expression were determined using antibodies to 8-hydroxy-7,8-dihydroguanosine (8-ohdG), phosphorylated histone γH2AX, BCL2 and BAX proteins and flow cytofluorimetry. The levels of gene expression of BCL2, BAC, BRCA1, BRCA2, TBP (control) were evaluated by real-time PCR. The exposure of 1321NI cells to oecDNA resulted in a 2-4-fold increased levels of 8-ohdG (p <0.01) and 3-3.5-fold increases in double-strand breaks (p < 0.01), whereas the expression of damage repair genes BRCA1 and BRCA2 was enchanced by 3-5 times (p <0.001). In 5-24 hours after exposure to oecDNA cells, the ratio of BCL2/BAX gene expression increased by 3.5-5 times vs. unexposed cell cultures (p <0.001). Therefore, in astrocytoma cells, oecDNA induces both genome rearrangement and adaptive transcriptome reactions followed by activation of an anti-apoptotic path in malignant cells. It is suggested that oecDNA contributes to the accumulation in a residual tumor of mutant clones resistant to treatment thus provoking relapse.

Pathways of 4-Hydroxy-2-Nonenal Detoxification in a Human Astrocytoma Cell Line

One of the consequences of the increased level of oxidative stress that often characterizes the cancer cell environment is the abnormal generation of lipid peroxidation products, above all 4-hydroxynonenal. The contribution of this aldehyde to the pathogenesis of several diseases is well known. In this study, we characterized the ADF astrocytoma cell line both in terms of its pattern of enzymatic activities devoted to 4-hydroxynonenal removal and its resistance to oxidative stress induced by exposure to hydrogen peroxide. A comparison with lens cell lines, which, due to the ocular function, are normally exposed to oxidative conditions is reported. Our results show that, overall, ADF cells counteract oxidative stress conditions better than normal cells, thus confirming the redox adaptation demonstrated for several cancer cells. In addition, the markedly high level of NADP+-dependent dehydrogenase activity acting on the glutahionyl-hydroxynonanal adduct detected in ADF cells may promote, at the same time, the detoxification and recovery of cell-reducing power in these cells.