MicroRNA 128a Increases Intracellular ROS Level by Targeting Bmi-1 and Inhibits Medulloblastoma Cancer Cell Growth by Promoting Senescence

BACKGROUND: MicroRNAs (miRNAs) are a class of short non-coding RNAs that regulate cell homeostasis by inhibiting translation or degrading mRNA of target genes, and thereby can act as tumor suppressor genes or oncogenes. The role of microRNAs in medulloblastoma has only recently been addressed. We hypothesized that microRNAs differentially expressed during normal CNS development might be abnormally regulated in medulloblastoma and are functionally important for medulloblastoma cell growth. METHODOLOGY AND PRINCIPAL FINDINGS: We examined the expression of microRNAs in medulloblastoma and then investigated the functional role of one specific one, miR-128a, in regulating medulloblastoma cell growth. We found that many microRNAs associated with normal neuronal differentiation are significantly down regulated in medulloblastoma. One of these, miR-128a, inhibits growth of medulloblastoma cells by targeting the Bmi-1 oncogene. In addition, miR-128a alters the intracellular redox state of the tumor cells and promotes cellular senescence. CONCLUSIONS AND SIGNIFICANCE: Here we report the novel regulation of reactive oxygen species (ROS) by microRNA 128a via the specific inhibition of the Bmi-1 oncogene. We demonstrate that miR-128a has growth suppressive activity in medulloblastoma and that this activity is partially mediated by targeting Bmi-1. This data has implications for the modulation of redox states in cancer stem cells, which are thought to be resistant to therapy due to their low ROS states

The influence of sentinel lymph node tumour burden on additional lymph node involvement and disease-free survival in cutaneous melanoma – a retrospective analysis of 392 cases

Twenty per cent of sentinel lymph node (SLN)-positive melanoma patients have positive non-SLN lymph nodes in completion lymph node dissection (CLND). We investigated SLN tumour load, non-sentinel positivity and disease-free survival (DFS) to assess whether certain patients could be spared CLND. Sentinel lymph node biopsy was performed on 392 patients between 1999 and 2005. Median observation period was 38.8 months. Sentinel lymph node tumour load did not predict non-SLN positivity: 30.8% of patients with SLN macrometastases (⩾2 mm) and 16.4% with micrometastases (⩽2 mm) had non-SLN positivity (P=0.09). Tumour recurrences after positive SLNs were more than twice as frequent for SLN macrometastases (51.3%) than for micrometastases (24.6%) (P=0.005). For patients with SLN micrometastases, the DFS analysis was worse (P=0.003) when comparing those with positive non-SLNs (60% recurrences) to those without (17.6% recurrences). This difference did not translate into significant differences in DFS: patients with SLN micrometastasis, either with (P=0.022) or without additional positive non-SLNs (P<0.0001), fared worse than patients with tumour-free SLNs. The 2-mm cutoff for SLN tumour load accurately predicts differences in DFS. Non-SLN positivity in CLND, however, cannot be predicted. Therefore, contrary to other studies, no recommendations concerning discontinuation of CLND based on SLN tumour load can be deduced

Synergistic Anticancer Effects of the 9.2.27PE Immunotoxin and ABT-737 in Melanoma

In cancer, combinations of drugs targeting different cellular functions is well accepted to improve tumor control. We studied the effects of a Pseudomonas exotoxin A (PE) - based immunotoxin, the 9.2.27PE, and the BH-3 mimetic compound ABT-737 in a panel of melanoma cell lines. The drug combination resulted in synergistic cytotoxicity, and the cell death observed was associated with apoptosis, as activation of caspase-3, inactivation of Poly (ADP-ribose) polymerase (PARP) and increased DNA fragmentation could be prevented by pre-treatment with caspase and cathepsin inhibitors. We further show that ABT-737 caused endoplasmic reticulum (ER) stress with increased GRP78 and phosphorylated eIF2α protein levels. Moreover, treatment with ABT-737 increased the intracellular calcium levels, an effect which was enhanced by 9.2.27PE, which as a single entity drug had minimal effect on calcium release from the ER. In addition, silencing of Mcl-1 by short hairpin RNA (shRNA) enhanced the intracellular calcium levels and cytotoxicity caused by ABT-737. Notably, the combination of 9.2.27PE and ABT-737 caused growth delay in a human melanoma xenograft mice model, supporting further investigations of this particular drug combination

Homogeneous MGMT Immunoreactivity Correlates with an Unmethylated MGMT Promoter Status in Brain Metastases of Various Solid Tumors

The O6-methylguanine-methyltransferase (MGMT) promoter methylation status is a predictive parameter for the response of malignant gliomas to alkylating agents such as temozolomide. First clinical reports on treating brain metastases with temozolomide describe varying effects. This may be due to the fact that MGMT promoter methylation of brain metastases has not yet been explored in depth. Therefore, we assessed MGMT promoter methylation of various brain metastases including those derived from lung (n = 91), breast (n = 72) kidney (n = 49) and from malignant melanomas (n = 113) by methylation-specific polymerase chain reaction (MS-PCR) and MGMT immunoreactivity. Fifty-nine of 199 brain metastases (29.6%) revealed a methylated MGMT promoter. The methylation rate was the highest in brain metastases derived from lung carcinomas (46.5%) followed by those from breast carcinoma (28.8%), malignant melanoma (24.7%) and from renal carcinoma (20%). A significant correlation of homogeneous MGMT-immunoreactivity (>95% MGMT positive tumor cells) and an unmethylated MGMT promoter was found. Promoter methylation was detected in 26 of 61 (43%) tumors lacking MGMT immunoreactivity, in 17 of 63 (27%) metastases with heterogeneous MGMT expression, but only in 5 of 54 brain metastases (9%) showing a homogeneous MGMT immunoreactivity. Our results demonstrate that a significant number of brain metastases reveal a methylated MGMT-promoter. Based on an obvious correlation between homogeneous MGMT immunoreactivity and unmethylated MGMT promoter, we hypothesize that immunohistochemistry for MGMT may be a helpful diagnostic tool to identify those tumors that probably will not benefit from the use of alkylating agents. The discrepancy between promoter methylation and a lack of MGMT immunoreactivity argues for assessing MGMT promoter methylation both by immunohistochemical as well as by molecular approaches for diagnostic purposes

Memantine increases NMDA receptor level in the prefrontal cortex but fails to reverse apomorphine-induced conditioned place preference in rats

Studies have shown that inflammation and neurodegeneration may accompany the development of addiction to apomorphine and that the glutamate NMDA receptor antagonist, memantine, may be neuroprotective. The similarity between apomorphine and dopamine with regard to their chemical, pharmacological and toxicological properties provided a basis for investigating the mechanism of action of the former agent. In this study, we investigated whether memantine would suppress apomorphine-seeking behavior in rats subjected to apomorphine-induced place preference conditioning, through modulation of NMDA receptors in the prefrontal cortex. Repeated apomorphine (1 mg/kg) treatment induced conditioned place preference (CPP) and had no significant effect on NMDA receptor levels in the prefrontal cortex. Prior treatment with memantine (5 mg/kg or 10 mg/kg) increased the levels of NMDA receptors in the prefrontal cortex but did not suppress CPP induced by apomorphine. These data give further support to the addictive effect of apomorphine and demonstrate that blockade of NMDA receptors by memantine is unable to suppress apomorphine-seeking behavior

Anaesthesia and PET of the Brain

Although drugs have been used to administer general anaesthesia for more than a century and a half, relatively little was known until recently about the molecular and cellular effects of the anaesthetic agents and the neurobiology of anaesthesia. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) studies have played a valuable role in improving this knowledge. PET studies using 11C-flumazenil binding have been used to demonstrate that the molecular action of some, but not all, of the current anaesthetic agents is mediated via the GABAA receptor. Using different tracers labelled with 18F, 11C and 15O, PET studies have shown the patterns of changes in cerebral metabolism and blood flow associated with different intravenous and volatile anaesthetic agents. Within classes of volatile agents, there are minor variations in patterns. More profound differences are found between classes of agents. Interestingly, all agents cause alterations in the blood flow and metabolism of the thalamus, providing strong support for the hypothesis that the anaesthetic agents interfere with consciousness by interfering with thalamocortical communication.</p

General Anesthetics Predicted to Block the GLIC Pore with Micromolar Affinity

Although general anesthetics are known to modulate the activity of ligand-gated ion channels in the Cys-loop superfamily, there is at present neither consensus on the underlying mechanisms, nor predictive models of this modulation. Viable models need to offer quantitative assessment of the relative importance of several identified anesthetic binding sites. However, to date, precise affinity data for individual sites has been challenging to obtain by biophysical means. Here, the likely role of pore block inhibition by the general anesthetics isoflurane and propofol of the prokaryotic pentameric channel GLIC is investigated by molecular simulations. Microscopic affinities are calculated for both single and double occupancy binding of isoflurane and propofol to the GLIC pore. Computations are carried out for an open-pore conformation in which the pore is restrained to crystallographic radius, and a closed-pore conformation that results from unrestrained molecular dynamics equilibration of the structure. The GLIC pore is predicted to be blocked at the micromolar concentrations for which inhibition by isofluorane and propofol is observed experimentally. Calculated affinities suggest that pore block by propofol occurs at signifcantly lower concentrations than those for which inhibition is observed: we argue that this discrepancy may result from binding of propofol to an allosteric site recently identified by X-ray crystallography, which may cause a competing gain-of-function effect. Affinities of isoflurane and propofol to the allosteric site are also calculated, and shown to be 3 mM for isoflurane and for propofol; both anesthetics have a lower affinity for the allosteric site than for the unoccupied pore

Serologic and immunohistochemical prognostic biomarkers of cutaneous malignancies

Biomarkers are important tools in clinical diagnosis and prognostic classification of various cutaneous malignancies. Besides clinical and histopathological aspects (e.g. anatomic site and type of the primary tumour, tumour size and invasion depth, ulceration, vascular invasion), an increasing variety of molecular markers have been identified, providing the possibility of a more detailed diagnostic and prognostic subgrouping of tumour entities, up to even changing existing classification systems. Recently published gene expression or proteomic profiling data relate to new marker molecules involved in skin cancer pathogenesis, which may, after validation by suitable studies, represent future prognostic or predictive biomarkers in cutaneous malignancies. We, here, give an overview on currently known serologic and newer immunohistochemical biomarker molecules in the most common cutaneous malignancies, malignant melanoma, squamous cell carcinoma and cutaneous lymphoma, particularly emphasizing their prognostic and predictive significance

Impact of non-LTR retrotransposons in the differentiation and evolution of anatomically modern humans

Background: Transposable elements are biologically important components of eukaryote genomes. In particular, non-LTR retrotransposons (N-LTRrs) played a key role in shaping the human genome throughout evolution. In this study, we compared retrotransposon insertions differentially present in the genomes of Anatomically Modern Humans, Neanderthals, Denisovans and Chimpanzees, in order to assess the possible impact of retrotransposition in the differentiation of the human lineage. Results: We first identified species-specific N-LTRrs and established their distribution in present day human populations. These analyses shortlisted a group of N-LTRr insertions that were found exclusively in Anatomically Modern Humans. These insertions are associated with an increase in the number of transcriptional/splicing variants of those genes they inserted in. The analysis of the functionality of genes containing human-specific N-LTRr insertions reflects changes that occurred during human evolution. In particular, the expression of genes containing the most recent N-LTRr insertions is enriched in the brain, especially in undifferentiated neurons, and these genes associate in networks related to neuron maturation and migration. Additionally, we identified candidate N-LTRr insertions that have likely produced new functional variants exclusive to modern humans, whose genomic loci show traces of positive selection. Conclusions: Our results strongly suggest that N-LTRr impacted our differentiation as a species, most likely inducing an increase in neural complexity, and have been a constant source of genomic variability all throughout the evolution of the human lineage