Oncomeric Profiles of microRNAs as New Therapeutic Targets for Treatment of Ewing's Sarcoma: A Composite Review.

Ewing's sarcoma is a rare type of cancer that forms in bones and soft tissues in the body, affecting mostly children and young adults. Current treatments for ES are limited to chemotherapy and/or radiation, followed by surgery. Recently, microRNAs have shown favourable results as latent diagnostic and prognostic biomarkers in various cancers. Furthermore, microRNAs have shown to be a good therapeutic agent due to their involvement in the dysregulation of various molecular pathways linked to tumour progression, invasion, angiogenesis, and metastasis. In this review, comprehensive data mining was employed to explore various microRNAs that might have therapeutic potential as target molecules in the treatment of ES.

Regulation of the unfolded protein response during dehydration stress in African clawed frogs, Xenopus laevis.

The unfolded protein response (UPR) is a wide-ranging cellular response to accumulation of malfolded proteins in the endoplasmic reticulum (ER) and acts as a quality control mechanism to halt protein processing and repair/destroy malfolded proteins under stress conditions of many kinds. Among vertebrate species, amphibians experience the greatest challenges in maintaining water and osmotic balance, the high permeability of their skin making them very susceptible to dehydration and challenging their ability to maintain cellular homeostasis. The present study evaluates the involvement of the UPR in dealing with dehydration-mediated disruption of protein processing in the tissues of African clawed frogs, Xenopus laevis. This primarily aquatic frog must deal with seasonal drought conditions in its native southern Africa environment. Key markers of cellular stress that impact protein processing were identified in six tissues of frogs that had lost 28% of total body water, as compared with fully hydrated controls. This included upregulation of glucose-regulated proteins (GRPs) that are resident chaperones in the ER, particularly 2-ninefold increases in GRP58, GRP75, and/or GRP94 in the lung and skin. Activating transcription factors (ATF3, ATF4, ATF6) that mediate UPR responses also responded to dehydration stress, particularly in skeletal muscle where both ATF3 and ATF4 rose strongly in the nucleus. Other protein markers of the UPR including GADD34, GADD153, EDEM, and XBP-1 also showed selective upregulation in frog tissues in response to dehydration and nuclear levels of the transcription factors XBP-1 and P-CREB rose indicating up-regulation of genes under their control.

Increased transcript levels and kinetic function of pyruvate kinase during severe dehydration in aestivating African clawed frogs, Xenopus laevis.

The African clawed frog, Xenopus laevis, can withstand extremely arid conditions through aestivation, resulting in dehydration and urea accumulation. Aestivating X. laevis reduce their metabolic rate, and rely on anaerobic glycolysis to meet reduced ATP demands. The present study investigated how severe dehydration affected the transcript levels, kinetic profile, and phosphorylation state of the key glycolytic enzyme pyruvate kinase (PK) in the liver and skeletal muscle of X. laevis. Compared to control frogs, severely dehydrated frogs showed an increase in the transcript abundance of both liver and muscle isoforms of PK. While the kinetics of muscle PK did not differ between dehydrated and control frogs, PK from the liver of dehydrated frogs had a lower Km for phosphoenolpyruvate (PEP) (38%), a lower Ka for fructose-1,6-bisphosphate (F1,6P2) (32%), and a greater activation of PK via F1,6P2 (1.56-fold). PK from dehydrated frogs also had a lower phosphorylation-state (25%) in comparison to the enzyme from control frogs in the liver. Experimental manipulation of the phosphorylation-state of liver PK taken from control frogs by endogenous protein phosphatases resulted in decreased phosphorylation, and a similar kinetic profile as seen in dehydrated frogs. The physiological consequence of dehydration-induced PK modification appears to adjust PK function to remain active during a metabolically depressed state. This study provides evidence for the maintenance of PK activity through elevated mRNA levels and a dephosphorylation event which activates frog liver PK in the dehydrated state in order to facilitate the production of ATP via anaerobic glycolysis.

The regulation of heat shock proteins in response to dehydration in Xenopus laevis.

African clawed frogs (Xenopus laevis) endure bouts of severe drought in their natural habitats and survive the loss of approximately 30% of total body water due to dehydration. To investigate molecular mechanisms employed by X. laevis during periods of dehydration, the heat shock protein response, a vital component of the cytoprotective stress response, was characterized. Using western immunoblotting and multiplex technology, the protein levels of HSP27, HSP40, HSP60, HSP70, HSC70, and HSP90 were quantified in the liver, skeletal muscle, kidney, lung, and testes from control frogs and those that underwent medium or high dehydration (~16 or ~30% loss of total body water). Dehydration increased HSP27 (1.45-1.65-fold) in the kidneys and lungs, and HSP40 (1.39-2.50-fold) in the liver, testes, and skeletal muscle. HSP60 decreased in response to dehydration (0.43-0.64 of control) in the kidneys and lungs. HSP70 increased in the liver, lungs, and testes (1.39-1.70-fold) during dehydration, but had a dynamic response in the kidneys (levels increased 1.57-fold with medium dehydration, but decreased to 0.56 of control during high dehydration). HSC70 increased in the liver and kidneys (1.20-1.36-fold), but decreased in skeletal muscle (0.27-0.55 of control) during dehydration. Lastly, HSP90 was reduced in the kidney, lung, and skeletal muscle (0.39-0.69 of control) in response to dehydration, but rose in the testes (1.30-fold). Overall, the results suggest a dynamic tissue-specific heat shock protein response to whole body dehydration in X. laevis.

Integrating toxicogenomics into human health risk assessment: lessons learned from the benzo[a]pyrene case study.

The use of short-term toxicogenomic tests to predict cancer (or other health effects) offers considerable advantages relative to traditional toxicity testing methods. The advantages include increased throughput, increased mechanistic data, and significantly reduced costs. However, precisely how toxicogenomics data can be used to support human health risk assessment (RA) is unclear. In a companion paper ( Moffat et al. 2014 ), we present a case study evaluating the utility of toxicogenomics in the RA of benzo[a]pyrene (BaP), a known human carcinogen. The case study is meant as a proof-of-principle exercise using a well-established mode of action (MOA) that impacts multiple tissues, which should provide a best case example. We found that toxicogenomics provided rich mechanistic data applicable to hazard identification, dose-response analysis, and quantitative RA of BaP. Based on this work, here we share some useful lessons for both research and RA, and outline our perspective on how toxicogenomics can benefit RA in the short- and long-term. Specifically, we focus on (1) obtaining biologically relevant data that are readily suitable for establishing an MOA for toxicants, (2) examining the human relevance of an MOA from animal testing, and (3) proposing appropriate quantitative values for RA. We describe our envisioned strategy on how toxicogenomics can become a tool in RA, especially when anchored to other short-term toxicity tests (apical endpoints) to increase confidence in the proposed MOA, and emphasize the need for additional studies on other MOAs to define the best practices in the application of toxicogenomics in RA.

Comparison of toxicogenomics and traditional approaches to inform mode of action and points of departure in human health risk assessment of benzo[a]pyrene in drinking water.

Toxicogenomics is proposed to be a useful tool in human health risk assessment. However, a systematic comparison of traditional risk assessment approaches with those applying toxicogenomics has never been done. We conducted a case study to evaluate the utility of toxicogenomics in the risk assessment of benzo[a]pyrene (BaP), a well-studied carcinogen, for drinking water exposures. Our study was intended to compare methodologies, not to evaluate drinking water safety. We compared traditional (RA1), genomics-informed (RA2) and genomics-only (RA3) approaches. RA2 and RA3 applied toxicogenomics data from human cell cultures and mice exposed to BaP to determine if these data could provide insight into BaP's mode of action (MOA) and derive tissue-specific points of departure (POD). Our global gene expression analysis supported that BaP is genotoxic in mice and allowed the development of a detailed MOA. Toxicogenomics analysis in human lymphoblastoid TK6 cells demonstrated a high degree of consistency in perturbed pathways with animal tissues. Quantitatively, the PODs for traditional and transcriptional approaches were similar (liver 1.2 vs. 1.0 mg/kg-bw/day; lungs 0.8 vs. 3.7 mg/kg-bw/day; forestomach 0.5 vs. 7.4 mg/kg-bw/day). RA3, which applied toxicogenomics in the absence of apical toxicology data, demonstrates that this approach provides useful information in data-poor situations. Overall, our study supports the use of toxicogenomics as a relatively fast and cost-effective tool for hazard identification, preliminary evaluation of potential carcinogens, and carcinogenic potency, in addition to identifying current limitations and practical questions for future work.

Hepatic genotoxicity and toxicogenomic responses in Muta™Mouse males treated with dibenz[a,h]anthracene.

Dibenz[a,h]anthracene (DB[a,h]A) is a polycyclic aromatic hydrocarbon that is a by-product of combustion and a potent carcinogen. Few studies have investigated the effects of DB[a,h]A on mRNA and microRNA expression to dissect the mechanisms involved in carcinogenesis. In this study, mature male mice (Muta(™)Mouse) were exposed to 6.25, 12.5 and 25mg/kg/day DB[a,h]A by oral gavage for 28 consecutive days. Results were compared with mice similarly exposed to benzo[a]pyrene (B[a]P) in our previous work. Liver DNA adduct levels and lacZ mutant frequency increased dose dependently for both chemicals. Aryl hydrocarbon receptor (AhR) potency was greater for DB[a,h]A than B[a]P using the chemical-activated luciferase expression assay. Microarray analysis revealed 19 up-regulated and 22 down-regulated genes (false discovery rate-adjusted P ≤ 0.05; fold change ≥ 1.5) following treatment with 6.25 mg/kg/day DB[a,h]A. Thirteen transcripts were up-regulated and 32 down-regulated in the 12.5mg/kg/day group. The 25mg/kg/day dose had major effects on mRNA expression with 135 up-regulated and 104 down-regulated genes. Overall, perturbations were greater for DB[a,h]A than for B[a]P; in vitro chemical-activated luciferase expression supports that this may be driven by the AhR. Many of the DB[a,h]A-affected genes are implicated in cancer and are essential in vital biological functions including circadian rhythm, glucose metabolism, lipid metabolism, immune response, cell cycle and apoptosis. Although a number of functional groups were similarly affected by B[a]P and DB[a,h]A, in general the responses generated by each chemical were quite distinct. Commonalities included a DNA damage response leading to induction of cell cycle arrest and apoptosis in both Tp53-dependent and Tp53-independent manners. MicroRNA expression was identical for both chemicals, with only miR-34a showing a dose-dependent increase in treated mice.

Hepatic mRNA, microRNA, and miR-34a-target responses in mice after 28 days exposure to doses of benzo(a)pyrene that elicit DNA damage and mutation.

Benzo(a)pyrene (BaP) is a mutagenic carcinogen that is ubiquitous in our environment. To better understand the toxic effects of BaP and to explore the relationship between toxicity and toxicogenomics profiles, we assessed global mRNA and microRNA (miRNA) expression in Muta™Mouse. Adult male mice were exposed by oral gavage to 25, 50, and 75 mg/kg/day BaP for 28 days. Liver tissue was collected 3 days following the last treatment. Initially, we established that exposure to BaP led to the formation of hepatic DNA adducts and mutations in the lacZ transgene of the Muta™Mouse. We then analyzed hepatic gene expression profiles. Microarray analysis of liver samples revealed 134 differentially expressed transcripts (adjusted P < 0.05; fold changes > 1.5). The mRNAs most affected were involved in xenobiotic metabolism, immune response, and the downstream targets of p53. In this study, we found a significant 2.0 and 3.6-fold increase following exposure to 50 and 75 mg/kg/day BaP, respectively, relative to controls for miR-34a. This miRNA is involved in p53 response. No other significant changes in miRNAs were observed. The protein levels of five experimentally confirmed miR-34a targets were examined, and no major down-regulation was present. The results suggest that liver miRNAs are largely unresponsive to BaP doses that cause both DNA adducts and mutations. In summary, the validated miRNA and mRNA expression profiles following 28 day BaP exposure reflect a DNA damage response and effects on the cell cycle, consistent with the observed increases in DNA adducts and mutations.

Transcriptional regulation of antioxidant enzymes by FoxO1 under dehydration stress.

Antioxidant defenses are an important part of adaptation to environmental stress for many organisms. This study analyzed responses to dehydration stress by manganese-dependent superoxide dismutase (MnSOD) and catalase, and the role of forkhead box class O type 1 (FoxO1) transcription factor in regulating their up-regulation, in selected tissues of the African clawed frogs, Xenopus laevis. Protein and mRNA levels of MnSOD and catalase were analyzed by immunoblotting and PCR. Analysis of FoxO1 included protein and phosphoprotein (FoxO1(ser245)) levels, nuclear versus cytoplasmic distribution, and FoxO1 binding to DNA. MnSOD protein increased significantly in the liver during dehydration whereas catalase rose in the liver and skeletal muscle. This was supported in liver by 1.5-2.2 fold increases in MnSOD and catalase mRNA levels. FoxO1 transcriptional activity was enhanced in liver of dehydrated versus control frogs as evidenced by: (a) 1.8-fold increase in FoxO1 protein in the nucleus, (b) strong decreases in inactive phosphorylated FoxO1(ser245) in total and nuclear extracts, and (c) a 57% increase in FoxO1 binding to DNA in nuclear extracts. The study documents up-regulation of MnSOD and catalase in frog organs during dehydration and indicates a role for FoxO1 in controlling expression of these genes in liver. Dehydration-rehydration has components of an ischemia-reperfusion event and the oxidative stress that this generates appears to be effectively addressed, at least in X. laevis liver, by enhanced production of antioxidant enzymes under FoxO1 regulation.

Dengue hemorrhagic fever outbreak in children in Port Sudan.

Dengue fever (DF)/dengue hemorrhagic fever (DHF) has emerged as a global public health problem with countries in Asia and the Pacific sharing more than 70% of the disease burden. In 2004-2005 a total of 312 cases admitted to Pediatric and Sea Port Hospitals in Port Sudan were clinically diagnosed as DHF. The mortality rate recorded was 3.8% (n=12). Of the cases 73.4% were patients 5-15 years of age. A total of 91.2% of cases were admitted during May and June 2005 with 49.4% residing in the eastern region of Port Sudan. Dengue shock syndrome was observed in 37 of 312 (11.9%). All patients had thrombocytopenia with platelets count ranged from <100,000 to <150,000 cell/mm³. Of the 40 sera tested using RAPID-cassette test in the Khartoum Central Public Health Lab, 36 (90%) were dengue IgM positive. A subset of these sera (n=23) were sent to NAMRU-3 and confirmed by IgM-capture ELISA; 9 of 23 were PCR positive for dengue serotype 3.

Activation of extracellular signal-regulated kinases during dehydration in the African clawed frog, Xenopus laevis.

In its native environment the African clawed frog, Xenopus laevis, can experience seasonally arid conditions that impose dehydration stress. Activation of intracellular signal transduction cascades can mediate and coordinate biochemical responses to ameliorate dehydration stress. This study examines the extracellular signal-regulated kinase (ERK) signaling cascade, analyzing responses of both upstream and downstream components in six tissues of X. laevis experiencing medium and high levels of dehydration, 16.6+/-1.59 and 28.0+/-1.6% of total body water lost, respectively. Immunoblotting was used to assess the three tiers in this mitogen-activated protein kinase (MAPK) cascade: the initiating MAPK kinase kinases (c-Raf, MEKK), the MAPK kinase (MEK1/2), and finally the MAPK (ERK1/2). The amount of active phosphorylated c-Raf(Ser338) rose by 2- to 2.5-fold under high dehydration in muscle, lung and skin whereas MEKK protein levels rose in these organs and also increased 4-fold in liver. As a result, phosphorylated active MEK1/2(Ser217/221) increased significantly by 2- to 6-fold during dehydration which, in turn, led to 2- to 6-fold increases in phospho-ERK(Thr202/Tyr204) content in all tissues except skin. Given this clear demonstration of ERK cascade activation, two downstream targets of ERK2 were then evaluated. The amount of phosphorylated active transcription factor, STAT3(Ser727) and p90 ribosomal S6 kinase (RSK(Ser380)) increased particularly in muscle, lung and kidney. Furthermore, RSK activation was correlated with a 5- to 8-fold increase in phosphorylation of its target, S6 ribosomal protein. Overall, the results show a strong conserved activation of the ERK cascade in X. laevis tissues in response to dehydration.

Activation of antioxidant defense during dehydration stress in the African clawed frog.

The glutathione S-transferase (GST) and aldo-keto reductase (AKR) families of proteins are major groups of detoxifying enzymes that are known to be regulated by the NF-E2 related factor 2 (Nrf2) transcription factor via the antioxidant response element that is present in the promoter regions of GST and AKR genes. Expression of Nrf2, GST and AKR proteins was analyzed in the African clawed frog, Xenopus laevis, focusing on their responses to dehydration stress. Dehydration/rehydration cycles can generate oxidative stress and this could be ameliorated by enhancing antioxidant defenses. Dehydration to 28% of total body water lost triggered organ-specific changes in nrf2 mRNA expression (a 2-fold increase in liver), total Nrf2 protein (2-4-fold elevation in lung, heart, skin and liver), and a 4.3-fold increase in the content of Nrf2 in the nucleus in muscle. Protein levels of six GST and three AKR family members were assessed and showed organ-specific patterns of expression during dehydration. In particular, GSTP1 was strongly induced in liver, heart and skin, levels rising by 9-, 2.6- and 1.7-fold, respectively, whereas GSTM1 and M3 rose in skeletal muscle, kidney and skin. Selective expression of GSTK1, A3 and T1 also occurred. Dehydration also stimulated organ-specific increases in the levels of AKR family members (AKR1B4, AKR1A3, AFAR1) by 1.5-2-fold. The results show that metabolic responses to dehydration include activation of the Nrf2 transcription factor and selective up-regulation of genes under Nrf2 control.