Cellular and molecular mechanisms of glioblastoma malignancy: Implications in resistance and therapeutic strategies.

Glioblastoma (GB) is the more frequent and malignant brain tumour. In spite of all efforts, the median overall survival of GB patients remains approximately 15 months under therapy. The molecular biology underlying GB is complex, which highlight the need of specific treatment strategies. In fact, the deregulation of several molecular signalling pathways, the existence of the blood-brain barrier (BBB), that makes almost all the chemotherapeutic agents inaccessible to the tumour site, and the existence of a population of stem-like cells known to be responsible for tumour recurrence after therapy, can contribute to GB chemoresistance. In the present review, we summarize the reliable factors responsible for the failure of the most important chemotherapeutic agents in GB. Specifically, we describe the utmost important characteristics of the BBB, as well as the genetic, molecular and transcription factors alterations that lead to tumour malignancy, and ultimately their impact on stem-like cell plasticity modulation. Recently, nanocarriers have attracted increasing attention in brain- and tumour-targeted drug-delivery systems, owing to their potential ability to target cell surface specific molecules and to cross the BBB delivering the drug specifically to the tumour cells, improving efficacy and thus reducing non-specific toxicity. In this sense, we will lastly highlight the therapeutic challenges and improvements regarding GB treatment.

Nucleolin is expressed in patient-derived samples and glioblastoma cells, enabling improved intracellular drug delivery and cytotoxicity.

One of the major challenges in Glioblastoma (GBM) therapy relates with the existence of glioma stem-like cells (GSCs), known to be chemo- and radio-resistant. GSCs and non-stem GBM cells have the ability to interchange, emphasizing the importance of identifying common molecular targets among those cell sub-populations. Nucleolin overexpression has been recently associated with breast cancer sub-populations with different stem-like phenotype. The goal of this work was to evaluate the potential of cell surface nucleolin as a target in GBM cells. Different levels of nucleolin expression resulted in a 3.4-fold higher association of liposomes targeting nucleolin (functionalized with the nucleolin-binding F3 peptide) in U87, relative to GBM11 glioblastoma cells. Moreover, nucleolin was suggested as a potential marker in OCT4-, NANOG-positive GSC, and in the corresponding non-stem GBM cells, as well as in SOX2-positive GSC. Doxorubicin delivered by liposomes targeting nucleolin enabled a level of cytotoxicity that was 2.5- or 4.6-fold higher compared to the non-targeted counterparts. Importantly, an overexpression of nucleolin was also observed in cells of patient-derived samples, as compared with normal brain. Overall, these results suggested nucleolin as a therapeutic target in GBM.

Tamoxifen in combination with temozolomide induce a synergistic inhibition of PKC-pan in GBM cell lines.

BACKGROUND: Glioblastoma (GBM) is a highly proliferative, angiogenic grade IV astrocytoma that develops resistance to the alkylating agents used in chemotherapy, such as temozolomide (TMZ), which is considered the gold standard. The mean survival time for GBM patients is approximately 12 months, increasing to 14.6 months after TMZ treatment. The resistance of GBM to chemotherapy seems to be associated to genetic alterations and to the constitutive activation of several signaling pathways. Therefore, the combination of different drugs with different mechanisms of action may contribute to circumvent the chemoresistance of glioma cells. Here we describe the potential synergistic behavior of the therapeutic combination of tamoxifen (TMX), a known inhibitor of PKC, and TMZ in GBM. METHODS: We used two GBM cell lines incubated in absence and presence of TMX and/or TMZ and measured cell viability, proliferation, apoptosis, cell cycle, migration ability, cytoskeletal organization and the phosphorylated amount of the p-PKC-pan. RESULTS: The combination of low doses of TMX with increasing doses of TMZ shows an increased antiproliferative and apoptotic effect compared to the effect with TMX alone. CONCLUSIONS: The combination of TMX and TMZ seems to potentiate the effect of each other. These alterations seem to be associated to a decrease in the phosphorylation status of PKC. GENERAL SIGNIFICANCE: We emphasize that TMX is an inhibitor of the p-PKC-pan and that these combination is more effective in the reduction of proliferation and in the increase of apoptosis than each drug alone, which presents a new therapeutic strategy in GBM treatment.

Molecular and Genomic Alterations in Glioblastoma Multiforme.

In recent years, important advances have been achieved in the understanding of the molecular biology of glioblastoma multiforme (GBM); thus, complex genetic alterations and genomic profiles, which recurrently involve multiple signaling pathways, have been defined, leading to the first molecular/genetic classification of the disease. In this regard, different genetic alterations and genetic pathways appear to distinguish primary (eg, EGFR amplification) versus secondary (eg, IDH1/2 or TP53 mutation) GBM. Such genetic alterations target distinct combinations of the growth factor receptor-ras signaling pathways, as well as the phosphatidylinositol 3-kinase/phosphatase and tensin homolog/AKT, retinoblastoma/cyclin-dependent kinase (CDK) N2A-p16(INK4A), and TP53/mouse double minute (MDM) 2/MDM4/CDKN2A-p14(ARF) pathways, in cells that present features associated with key stages of normal neurogenesis and (normal) central nervous system cell types. This translates into well-defined genomic profiles that have been recently classified by The Cancer Genome Atlas Consortium into four subtypes: classic, mesenchymal, proneural, and neural GBM. Herein, we review the most relevant genetic alterations of primary versus secondary GBM, the specific signaling pathways involved, and the overall genomic profile of this genetically heterogeneous group of malignant tumors.

Tumor infiltrating immune cells in gliomas and meningiomas.

Tumor-infiltrating immune cells are part of a complex microenvironment that promotes and/or regulates tumor development and growth. Depending on the type of cells and their functional interactions, immune cells may play a key role in suppressing the tumor or in providing support for tumor growth, with relevant effects on patient behavior. In recent years, important advances have been achieved in the characterization of immune cell infiltrates in central nervous system (CNS) tumors, but their role in tumorigenesis and patient behavior still remain poorly understood. Overall, these studies have shown significant but variable levels of infiltration of CNS tumors by macrophage/microglial cells (TAM) and to a less extent also lymphocytes (particularly T-cells and NK cells, and less frequently also B-cells). Of note, TAM infiltrate gliomas at moderate numbers where they frequently show an immune suppressive phenotype and functional behavior; in contrast, infiltration by TAM may be very pronounced in meningiomas, particularly in cases that carry isolated monosomy 22, where the immune infiltrates also contain greater numbers of cytotoxic T and NK-cells associated with an enhanced anti-tumoral immune response. In line with this, the presence of regulatory T cells, is usually limited to a small fraction of all meningiomas, while frequently found in gliomas. Despite these differences between gliomas and meningiomas, both tumors show heterogeneous levels of infiltration by immune cells with variable functionality. In this review we summarize current knowledge about tumor-infiltrating immune cells in the two most common types of CNS tumors-gliomas and meningiomas-, as well as the role that such immune cells may play in the tumor microenvironment in controlling and/or promoting tumor development, growth and control.

The protein expression profile of meningioma cells is associated with distinct cytogenetic tumour subgroups.

AIMS: Limited information exists about the impact of cytogenetic alterations on the protein expression profiles of individual meningioma cells and their association with the clinicohistopathological characteristics of the disease. The aim of this study is to investigate the potential association between the immunophenotypic profile of single meningioma cells and the most relevant features of the tumour. METHODS: Multiparameter flow cytometry (MFC) was used to evaluate the immunophenotypic profile of tumour cells (n = 51 patients) and the Affymetrix U133A chip was applied for the analysis of the gene expression profile (n = 40) of meningioma samples, cytogenetically characterized by interphase fluorescence in situ hybridization. RESULTS: Overall, a close association between the pattern of protein expression and the cytogenetic profile of tumour cells was found. Thus, diploid tumours displayed higher levels of expression of the CD55 complement regulatory protein, tumours carrying isolated monosomy 22/del(22q) showed greater levels of bcl2 and PDGFRß and meningiomas carrying complex karyotypes displayed a greater proliferation index and decreased expression of the CD13 ectoenzyme, the CD9 and CD81 tetraspanins, and the Her2/neu growth factor receptor. From the clinical point of view, higher expression of CD53 and CD44 was associated with a poorer outcome. CONCLUSIONS: Here we show that the protein expression profile of individual meningioma cells is closely associated with tumour cytogenetics, which may reflect the involvement of different signalling pathways in the distinct cytogenetic subgroups of meningiomas, with specific immunophenotypic profiles also translating into a different tumour clinical behaviour.

Cymbopogon citratus industrial waste as a potential source of bioactive compounds.

BACKGROUND: Cymbopogon citratus (Cc), commonly known as lemongrass, is a very important crop worldwide, being grown in tropical countries. It is widely used in the food, pharmaceutical, cosmetic and perfumery industries for its essential oil. Cc aqueous extracts are also used in traditional medicine. They contain high levels of polyphenols, which are known for their antioxidant and anti-inflammatory properties. Hydrodistillation of lemongrass essential oil produces an aqueous waste (CcHD) which is discarded. Therefore a comparative study between CcHD and Cc infusion (CcI) was performed to characterize its phytochemical profile and to research its antioxidant and anti-inflammatory potential. RESULTS: HPLC-PDA/ESI-MS(n) analysis showed that CcI and CcHD have similar phenolic profiles, with CcHD presenting a higher amount of polyphenols. Additionally, both CcI and CcHD showed antioxidant activity against DPPH (EC50 of 41.72 ± 0.05 and 42.29 ± 0.05 µg mL(-1) respectively) and strong anti-inflammatory properties, by reducing NO production and iNOS expression in macrophages and through their NO-scavenging activity, in a dose-dependent manner. Furthermore, no cytotoxicity was observed. CONCLUSION: The data of this study encourage considering the aqueous solution from Cc leaf hydrodistillation as a source of bioactive compounds, which may add great industrial value to this crop.

Development of an in vitro dendritic cell-based test for skin sensitizer identification.

The sensitizing potential of chemicals is currently assessed using animal models. However, ethical and economic concerns and the recent European legislative framework triggered intensive research efforts in the development and validation of alternative methods. Therefore, the aim of this study was to develop an in vitro predictive test based on the analysis and integration of gene expression and intracellular signaling profiles of chemical-exposed skin-derived dendritic cells. Cells were treated with four known sensitizers and two nonsensitizers, and the effects on the expression of 20 candidate genes and the activation of MAPK, PI3K/Akt, and NF-κB signaling pathways were analyzed by real-time reverse transcription polymerase chain reaction and Western blotting, respectively. Genes Trxr1, Hmox1, Nqo1, and Cxcl10 and the p38 MAPK and JNK signaling pathways were identified as good predictor variables and used to construct a dichotomous classifier. For validation of the model, 12 new chemicals were then analyzed in a blind assay, and from these, 11 were correctly classified. Considering the total of 18 compounds tested here, 17 were correctly classified, representing a concordance of 94%, with a sensitivity of 92% (12 of 13 sensitizers identified) and a specificity of 100% (5 of 5 nonsensitizers identified). Additionally, we tested the ability of our model to discriminate sensitizers from nonallergenic but immunogenic compounds such as lipopolysaccharide (LPS). LPS was correctly classified as a nonsensitizer. Overall, our results indicate that the analysis of proposed gene and signaling pathway signatures in a mouse fetal skin-derived dendritic cell line represents a valuable model to be integrated in a future in vitro test platform.

Activation of phosphatidylinositol 3-kinase/Akt and impairment of nuclear factor-kappaB: molecular mechanisms behind the arrested maturation/activation state of Leishmania infantum-infected dendritic cells.

Understanding the complex interactions between Leishmania and dendritic cells (DCs) is central to the modulation of the outcome of this infection, given that an effective immune response against Leishmania is dependent on the successful activation and maturation of DCs. We report here that Leishmania infantum promastigotes successfully infect mouse bone marrow-derived DCs without triggering maturation, as shown by a failure in the up-regulation of CD40 and CD86 expression, and that parasites strongly counteract the lipopolysaccharide-triggered maturation of DCs. A small increase in interleukin (IL)-12 and IL-10 transcription and secretion and a decrease in IL-6 were observed in infected cells. This arrested DC maturation state is actively promoted by parasites because heat-killed or fixed parasites increased cytokine and costimulatory molecule expression. At a molecular level, L. infantum rapidly induced activation of phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2, whereas no effect was observed in the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase proinflammatory pathways. Moreover, parasites actively promoted cleavage of the nuclear factor-κB p65(RelA) subunit, causing its impairment. The blockade of phosphatidylinositol 3-kinase/Akt by either treatment of bone marrow-derived DCs with wortmannin or transfection with an Akt dominant-negative mutant resulted in a strong decrease in infection rates, revealing for the first time a crucial role of this pathway on Leishmania engulfment by DCs. Overall, our data indicate that activation of Akt and impairment of nuclear factor-κB are responsible for immunogenicity subversion of L. infantum-infected DCs.

Signal transduction profile of chemical sensitisers in dendritic cells: an endpoint to be included in a cell-based in vitro alternative approach to hazard identification?

The development of non-animal testing methods for the assessment of skin sensitisation potential is an urgent challenge within the framework of existing and forthcoming legislation. Efforts have been made to replace current animal tests, but so far no alternative methods have been developed. It is widely recognised that alternatives to animal testing cannot be accomplished with a single approach, but rather will require the integration of results obtained from different in vitro and in silico assays. The argument subjacent to the development of in vitro dendritic cell (DC)-based assays is that sensitiser-induced changes in the DC phenotype can be differentiated from those induced by irritants. This assumption is derived from the unique capacity of DC to convert environmental signals encountered at the skin into a receptor expression pattern (MHC class II molecules, co-stimulatory molecules, chemokine receptors) and a soluble mediator release profile that will stimulate T lymphocytes. Since signal transduction cascades precede changes in surface marker expression and cytokine/chemokine secretion, these phenotypic modifications are a consequence of a signal transduction profile that is specifically triggered by sensitisers and not by irritants. A limited number of studies have addressed this subject and the present review attempts to summarise and highlight all of the signalling pathways modulated by skin sensitisers and irritants. Furthermore, we conclude this review by focusing on the most promising strategies suitable for inclusion into a cell-based in vitro alternative approach to hazard identification.

Intratumoral patterns of clonal evolution in gliomas.

Few studies have explored the patterns of clonal evolution in gliomas. Here, we investigate the cytogenetic patterns of intratumoral clonal evolution of gliomas and their impact on tumor histopathology and patient survival. Cytogenetic analysis of 90 gliomas was performed in individual tumor cells (>200 cells/tumor) using multicolor (N = 16 probes) interphase-FISH. Overall, chromosome gains were more frequent than chromosome losses. Gains of chromosome 7 and/or EGFR amplification were detected in 91% of the cases, whereas del(9p21) (77%) and del(10q23) (78%) were the most frequent chromosome losses. Virtually, all cases (99%) showed >or=2 tumor cell clones, with higher numbers among high- versus low-grade gliomas (p = 0.001). Nine different cytogenetic patterns were found in the ancestral tumor clones. In most gliomas, ancestral clones showed abnormalities of chromosome 7, 9p, and/or 10q and cytogenetic evolution consisted of acquisition of additional abnormalities followed by tetraploidization. Conversely, early tetraploidization was associated with low-grade astrocytomas-2/3 pilocytic and 3/6 grade II diffuse astrocytomas-and combined loss of 1p36/19q13 with oligodendrogliomas, respectively; both aberrations were associated with a better patient outcome (p = 0.03). Overall, our results support the existence of different pathways of intratumoral evolution in gliomas.

Screening of five essential oils for identification of potential inhibitors of IL-1-induced Nf-kappaB activation and NO production in human chondrocytes: characterization of the inhibitory activity of alpha-pinene.

Nuclear factor-kappaB is a key transcription factor activated by pro-inflammatory signals, like interleukin-1beta (IL-1), being required for the expression of many inflammatory and catabolic mediators, such as nitric oxide (NO), that play an important role in arthritic diseases. This work aimed at screening and identifying natural inhibitors of IL-induced NF-kappaB activation and NO production in human articular chondrocytes. Five essential oils obtained from four plants of the Iberian flora, Mentha x piperita L. (Lamiaceae), Origanum virens L. (Lamiaceae), Lavandula luiseri L. (Lamiaceae), and Juniperus oxycedrus L. subsp. oxycedrus (Cupressaceae), were screened for their ability to prevent IL-1-induced NO production. The oil showing higher inhibitory activity was fractionated, concentrated, analyzed for composition elucidation and prepared for further assays. For this purpose, the human chondrocytic cell line C-28/I2 was used to evaluate NF-kappaB activation by determining the cytoplasmic levels of the total and phosphorylated forms of the inhibitory protein, I kappaB-alpha, and the NF-kappaB-DNA binding activity. The essential oil from the leaves of J. oxycedrus in a concentration of 0.02 % (v/v) achieved the greatest inhibition (80 +/- 8%) of IL-1-induced NO production. Chemical analysis showed that this essential oil is predominantly composed of monoterpene hydrocabons, being alpha-pinene [2,6,6-trimethyl-bicyclo(3.1.1)hept-3-ene] the major constituent (76 %). Similarly to the effect of the whole oil, a fraction containing 93% alpha-pinene reduced significantly IL-1-induced I kappaB-alpha degradation. Moreover, alpha-pinene also decreased I kappaB-alpha phosphorylation, NF-kappaB-DNA binding activity, and NO production. Another fraction containing oxygenated mono- and sesquiterpenes was nearly as effective as alpha-pinene. The ability of the alpha-pinene-containing fraction to reduce IL-1-induced NF-kappaB activation and NO production warrants further studies to demonstrate the usefulness of alpha-pinene in the treatment of arthritic diseases and other conditions in which NF-kappaB and NO play pathological roles.

Claspin: From replication stress and DNA damage responses to cancer therapy.

Cancer is still one of the major causes of death worldwide. Radiation therapy and chemotherapy remain the main treatment modalities in cancer. These therapies exert their effect mainly through interference with DNA replication and induction of DNA damage. It is believed that one way of improving the efficacy of cancer treatment will be to inhibit the replication stress and DNA damage responses and promote mitotic catastrophe of cancer cells. So far, the majority of the efforts have focused central players of checkpoint responses, such as ATR and CHK1, and DNA damage repair, such as PARPs. Being a key player in the replication stress response, checkpoint activation, and the DNA damage response, Claspin constitutes an attractive therapeutic target in cancer, namely for radio- and chemo-sensitization. In this review, we will go through Claspin functions in the replication stress and DNA damage responses and will discuss how Claspin can be targeted in cancer treatment, as well as the effects of Claspin inhibition.

GBM-Derived Wnt3a Induces M2-Like Phenotype in Microglial Cells Through Wnt/ß-Catenin Signaling.

Glioblastoma is an extremely aggressive and deadly brain tumor known for its striking cellular heterogeneity and capability to communicate with microenvironment components, such as microglia. Microglia-glioblastoma interaction contributes to an increase in tumor invasiveness, and Wnt signaling pathway is one of the main cascades related to tumor progression through changes in cell migration and invasion. However, very little is known about the role of canonical Wnt signaling during microglia-glioblastoma crosstalk. Here, we show for the first time that Wnt3a is one of the factors that regulate interactions between microglia and glioblastoma cells. Wnt3a activates the Wnt/ß-catenin signaling of both glioblastoma and microglial cells. Glioblastoma-conditioned medium not only induces nuclear translocation of microglial ß-catenin but also increases microglia viability and proliferation as well as Wnt3a, cyclin-D1, and c-myc expression. Moreover, glioblastoma-derived Wnt3a increases microglial ARG-1 and STI1 expression, followed by an upregulation of IL-10 mRNA levels, and a decrease in IL1ß gene expression. The presence of Wnt3a in microglia-glioblastoma co-cultures increases the formation of membrane nanotubes accompanied by changes in migration capability. In vivo, tumors formed from Wnt3a-stimulated glioblastoma cells presented greater microglial infiltration and more aggressive characteristics such as growth rate than untreated tumors. Thus, we propose that Wnt3a belongs to the arsenal of factors capable of stimulating the induction of M2-like phenotype on microglial cells, which contributes to the poor prognostic of glioblastoma, reinforcing that Wnt/ß-catenin pathway can be a potential therapeutic target to attenuate glioblastoma progression.

Differential modulation of CXCR4 and CD40 protein levels by skin sensitizers and irritants in the FSDC cell line.

The development of non-animal methods for skin sensitization testing is an urgent challenge. Some of the most promising in vitro approaches are based on the analysis of phenotypical and functional modifications induced by sensitizers in dendritic cell models. In this work, we evaluated, for the first time, a fetal skin-derived dendritic cell line (FSDC) as a model to discriminate between sensitizers and irritants, through analysis of their effects on CD40 and CXCR4 protein expression. The chemicals concentrations were chosen based on a slight cytotoxicity effect (up to 15%). Protein levels were evaluated by Western blot and immunocytochemistry, after stimulation with the skin sensitizers 2,4-dinitrofluorobenzene (DNFB), 1,4-phenylenediamine (PPD) and nickel sulphate (NiSO(4)), the non-sensitizer 2,4-dichloronitrobenzene (DCNB), and the irritants sodium dodecyl sulphate (SDS) and benzalkonium chloride (BC). All sensitizers tested increased CD40 and CXCR4 levels. In contrast, irritants decreased both proteins levels, with a more pronounced effect on CXCR4. In agreement with these results, dendritic cells derived from human peripheral blood monocytes-derived dendritic cells (MoDC) showed a similar response pattern to the skin sensitizer and irritant tested, PPD and SDS, respectively. In conclusion, evaluation of CD40 and CXCR4 proteins in chemical-treated FSDC may represent a useful tool in a future in vitro test for sensitizing assessment.

Essential oil of Daucus carota subsp. halophilus: composition, antifungal activity and cytotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Essential oils are known to possess antimicrobial activity against a wide spectrum of bacteria and fungi. Daucus carota L. is used since olden times in traditional medicine, due to recognized therapeutic properties, namely the antimicrobial activity of their essential oils. AIM OF THE STUDY: In the present study the composition and the antifungal activity of the oils of Daucus carota L. subsp. halophilus (Brot.) A. Pujadas (Apiaceae), an endemic plant from Portugal, were evaluated. Moreover, their cytotoxicity in mouse skin dendritic cells at concentration showing significant antifungal activity was also evaluated. MATERIAL AND METHODS: The oils were investigated by GC and GC-MS and the antifungal activity (MIC and MLC) were evaluated against yeasts, dermatophyte and Aspergillus strains. Assessment of cell viability was made by the MTT assay. RESULTS: The results showed large variations in the compositions during ontogenesis, particularly in the amounts of elemicin that increased significantly in the ripe umbels (5.9% vs. 31.0%). The results also demonstrated that the oil with high amounts of elemicin, which have stronger antifungal activity, showed no cytotoxic effect, at concentrations ranging from 0.16 to 0.64 microl/ml, for as long as 24h. CONCLUSION: It is possible to find appropriate doses of Daucus carota oil showing both antifungal activity and very low detrimental effect on mammalian cells.

Prognostic stratification of adult primary glioblastoma multiforme patients based on their tumor gene amplification profiles.

Several classification systems have been proposed to address genomic heterogeneity of glioblastoma multiforme, but they either showed limited prognostic value and/or are difficult to implement in routine diagnostics. Here we propose a prognostic stratification model for these primary tumors based on tumor gene amplification profiles, that might be easily implemented in routine diagnostics, and potentially improve the patients management. Gene amplification profiles were prospectively evaluated in 80 primary glioblastoma multiforme tumors using single-nucleotide polymorphism arrays and the results obtained validated in publicly available data from 267/347 cases. Gene amplification was detected in 45% of patients, and chromosome 7p11.2 including the EGFR gene, was the most frequently amplified chromosomal region - either alone (18%) or in combination with amplification of DNA sequences in other chromosomal regions (10% of cases). Other frequently amplified DNA sequences included regions in chromosomes 12q(10%), 4q12(7%) and 1q32.1(4%). Based on their gene amplification profiles, glioblastomas were subdivided into: i) tumors with no gene amplification (55%); ii) tumors with chromosome 7p/EGFR gene amplification (with or without amplification of other chromosomal regions) (38%); and iii) glioblastoma multiforme with a single (11%) or multiple (6%) amplified DNA sequences in chromosomal regions other than chromosome 7p. From the prognostic point of view, these amplification profiles showed a significant impact on overall survival of glioblastoma multiforme patients (p>0.001). Based on these gene amplification profiles, a risk-stratification scoring system was built for prognostic stratification of glioblastoma which might be easily implemented in routine diagnostics, and potentially contribute to improved patient management.

The long non-coding RNA HOTAIR is transcriptionally activated by HOXA9 and is an independent prognostic marker in patients with malignant glioma.

The lncRNA HOTAIR has been implicated in several human cancers. Here, we evaluated the molecular alterations and upstream regulatory mechanisms of HOTAIR in glioma, the most common primary brain tumors, and its clinical relevance. HOTAIR gene expression, methylation, copy-number and prognostic value were investigated in human gliomas integrating data from online datasets and our cohorts. High levels of HOTAIR were associated with higher grades of glioma, particularly IDH wild-type cases. Mechanistically, HOTAIR was overexpressed in a gene dosage-independent manner, while DNA methylation levels of particular CpGs in HOTAIR locus were associated with HOTAIR expression levels in GBM clinical specimens and cell lines. Concordantly, the demethylating agent 5-Aza-2'-deoxycytidine affected HOTAIR transcriptional levels in a cell line-dependent manner. Importantly, HOTAIR was frequently co-expressed with HOXA9 in high-grade gliomas from TCGA, Oncomine, and our Portuguese and French datasets. Integrated in silico analyses, chromatin immunoprecipitation, and qPCR data showed that HOXA9 binds directly to the promoter of HOTAIR. Clinically, GBM patients with high HOTAIR expression had a significantly reduced overall survival, independently of other prognostic variables. In summary, this work reveals HOXA9 as a novel direct regulator of HOTAIR, and establishes HOTAIR as an independent prognostic marker, providing new therapeutic opportunities to treat this highly aggressive cancer.

Claspin functions in cell homeostasis-A link to cancer?

Cancer remains one of the leading causes of mortality worldwide. Most cancers present high degrees of genomic instability. DNA damage and replication checkpoints function as barriers to halt cell cycle progression until damage is resolved, preventing the perpetuation of errors. Activation of these checkpoints is critically dependent on Claspin, an adaptor protein that mediates the phosphorylation of the effector kinase Chk1 by ATR. However, Claspin also performs other roles related to the protection and maintenance of cell and genome integrity. For instance, following DNA damage and checkpoint activation, Claspin bridges checkpoint responses to DNA repair or to apoptosis. During DNA replication, Claspin acts a sensor and couples DNA unwinding to strand polymerization, and may also indirectly regulate replication initiation at firing origins. As Claspin participates in several processes that are vital to maintenance of cell homeostasis, its function is tightly regulated at multiple levels. Nevertheless, little is known about its role in cancer. Accumulating evidence suggests that Claspin inactivation could be an essential event during carcinogenesis, indicating that Claspin may function as a tumour suppressor. In this review, we will examine the functions of Claspin and how its deregulation may contribute to cancer initiation and progression. To conclude, we will discuss means by which Claspin can be targeted for cancer therapy.

Urtica spp.: Phenolic composition, safety, antioxidant and anti-inflammatory activities.

Urtica dioica and other less studied Urtica species (Urticaceae) are often used as a food ingredient. Fifteen hydroxycinnamic acid derivatives and sixteen flavonoids, flavone and flavonol-type glycosides were identified in hydroalcoholic extracts from aerial parts of Urtica dioica L., Urtica urens L. and Urtica membranacea using HPLC-PDA-ESI/MSn. Among them, the 4-caffeoyl-5-p-coumaroylquinic acid and three statin-like 3-hydroxy-3-methylglutaroyl flavone derivatives were identified for the first time in Urtica urens and U. membranacea respectively. Urtica membranacea showed the higher content of flavonoids, mainly luteolin and apigenin C-glycosides, which are almost absent in the other species studied. In vitro, Urtica dioica exhibited greater antioxidant activity but Urtica urens exhibited stronger anti-inflammatory potential. Interestingly, statin-like compounds detected in Urtica membranacea have been associated with hypocholesterolemic activity making this plant interesting for future investigations. None of the extracts were cytotoxic to macrophages and hepatocytes in bioactive concentrations (200 and 350µg/mL), suggesting their safety use in food applications.