5-Fluorouracil dose escalation generated desensitized colorectal cancer cells with reduced expression of protein methyltransferases and no epithelial-to-mesenchymal transition potential.

Background: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers. In many cases, the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil (5-FU). The epithelial-to-mesenchymal transition (EMT) and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers. This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC. Materials and Methods: HCT-116, Caco-2, and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU. The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays. This was followed by a Western blot which analyzed the protein expressions of the epithelial marker E-cadherin, mesenchymal marker vimentin, and the EMT transcription factor (EMT-TF), the snail family transcriptional repressor 1 (Snail) in the parental and desensitized cells. Western blotting was also conducted to study the protein expressions of the protein methyltransferases (PMTs), Euchromatic histone lysine methyltransferase 2 (EHMT2/G9A), protein arginine methyltransferase (PRMT5), and SET domain containing 7/9 (SETD7/9) along with the global lysine and arginine methylation profiles. Results: The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU. The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells. This was reflected in the observed reduction in E-cadherin, vimentin, and Snail in the desensitized cell lines. Additionally, the protein expressions of EHMT2/G9A, PRMT5, and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment. Conclusion: This study showed that continuous, dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.

Down-regulating Effect of a Standardized Extract of Cultured Lentinula edodes mycelia on Cortactin in Prostate Cancer Cells Is Dependent on Malignant Potential.

BACKGROUND/AIM: The incidence and mortality rates of prostate cancer have been increasing worldwide. Although prostate cancer cells grow slowly in the local original site, once the cancer cells spread to distant organs they grow rapidly and show very aggressive features. Cortactin is a protein that regulates the actin cytoskeleton and plays crucial roles in cancer metastasis. Up-regulated cortactin is correlated with the metastatic capacity of prostate cancer cells. AHCC®, a standardized extract of cultured Lentinula edodes mycelia, has been previously reported to have cortactin-down-regulating effects on human pancreatic cancer cells. In the present study, the effects of AHCC® treatment on cortactin levels in prostate cancer cells was evaluated. MATERIALS AND METHODS: LNCaP.FGC, DU145, and PC-3 are human prostate cancer cell lines. LNCaP.FGC is well differentiated, androgen-dependent, and poorly metastatic. DU145 is less differentiated, androgen-independent, and moderate metastatic. PC-3 is less differentiated, androgen-independent, and highly metastatic. The effects of AHCC® treatment on cortactin levels in prostate cancer cells was evaluated by western blot. RESULTS: In vitro AHCC® treatment decreased cortactin levels in LNCaP.FGC and DU145 cells but did not change those in PC-3 cells. CONCLUSION: AHCC® treatment down-regulated cortactin expression in poor and moderate metastatic LNCaP.FGC and DU145 cells but showed no effect on cortactin expression in the highly metastatic PC-3 cells. Further studies are required to elucidate the mechanism of the resistance to AHCC® treatment in highly metastatic PC-3 cells.

A Standardized Extract of Cultured Lentinula edodes mycelia Up-regulates COX-2 in Inflammation-related Malignant Progressive Fibrosarcoma Cell Clone QRsP-11.

BACKGROUND/AIM: Cyclooxygenase is an enzyme that transforms arachidonic acid to prostaglandins. Cyclooxygenase-2 (COX-2) is an isoform of cyclooxygenase. There exist many reports on the expression levels of COX-2 in cancer tissues, and prognosis of cancer patients has been reported to be related to COX-2 up-regulation. In the present study we assessed the suppressive effect of AHCC® on the expression of COX-2 in QRsP-11cells. MATERIALS AND METHODS: QR-32 is a clone which was derived from murine fibrosarcoma BMT-11 cells by treatment with quercetin. These clone cells regress spontaneously after injection into C57BL/6 mice. QRsP-11 is a clone derived from QR-32, showing very aggressive tumorigenicity. AHCC® is a standardized extract of cultured Lentinula edodes mycelia and has been reported to exert suppressive effects on various tumor-associated proteins including HSP27. The protein levels of COX-2 in QR-32 and QRsP-11 cells were compared by using western blotting. Furthermore, the expression levels of COX-2 were assessed in QRsP-11 cells after AHCC®-treatment. RESULTS: Western blot analysis showed a significant up-regulation of COX-2 in QRsP-11 cells compared to QR-32 cells. In vitro AHCC®-treatment increased COX-2 expression levels in QRsP-11 cells contrary to expectations. CONCLUSION: When using AHCC® in cancer treatment, it might be important to decrease COX-2 expression by means of non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin. Further studies are required to clarify the mechanism of up-regulation of COX-2 through AHCC®-treatment.

AT-hook DNA-binding motif-containing protein one knockdown downregulates EWS-FLI1 transcriptional activity in Ewing's sarcoma cells.

Ewing's sarcoma is the second most common bone malignancy in children or young adults and is caused by an oncogenic transcription factor by a chromosomal translocation between the EWSR1 gene and the ETS transcription factor family. However, the transcriptional mechanism of EWS-ETS fusion proteins is still unclear. To identify the transcriptional complexes of EWS-ETS fusion transcription factors, we applied a proximal labeling system called BioID in Ewing's sarcoma cells. We identified AHDC1 as a proximal protein of EWS-ETS fusion proteins. AHDC1 knockdown showed a reduced cell growth and transcriptional activity of EWS-FLI1. AHDC1 knockdown also reduced BRD4 and BRG1 protein levels, both known as interacting proteins of EWS-FLI1. Our results suggest that AHDC1 supports cell growth through EWS-FLI1.

A comparative profile of total protein and six angiogenically-active growth factors in three platelet products.

Objectives: Platelet-derived products have been shown as promising novel therapeutic agents for chronic wounds. However, their clinical use requires a greater degree of method standardisation, part of which involved more extensive cataloguing of their biochemical composition. This study aimed to quantify and compare total protein and 6 angiogenically-active growth factors in three distinct platelet products. Methods: Platelet Lysate (PL, n=5), Calcium-activated Platelet Rich Plasma (Ca-PRP, n=5) and Platelet-Rich Fibrin (PRF, n=5) were prepared from pooled platelet apheresis products (n=10). Ca-PRP and PRF were prepared from the same units (n=5) by activation with 20 mmolL-1 calcium chloride. PL was prepared from the remaining (n=5) units using an established lysate. Total protein was quantified with the Bradford Assay. Sandwich enzyme-linked immunosorbent assay was used to quantify six growth factors: epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), stromal cell derived growth factor-1α (SDF-1α), endostatin, and transforming growth factor-ß1 (TGF-ß1). Results: Protein retrieval differed significantly (p<0.05) between the three products: PL (11.35±0.80 mg/mL) < Ca-PRP (20.44±8.17 mg/mL) < PRF (40.67±3.13 mg/mL). Growth factor yield was considerable in all three products and differed significantly for: VEGF (PL

A standardized extract of cultured Lentinula edodes mycelia downregulates cortactin in gemcitabine-resistant pancreatic cancer cells.

AHCC®, a standardized extract of cultured Lentinula edodes mycelia, enhances the therapeutic effects and reduces the adverse effects of chemotherapy. Our previous study reported that treatment with AHCC® downregulated the expression levels of tumor-associated proteins in the gemcitabine-resistant pancreatic cancer cell line, KLM1-R. However, to the best of our knowledge, the role of AHCC® in the inhibition of cell migration remains unexplored. Cortactin (CTTN), an actin nucleation-promoting factor, has been reported to be upregulated and correlated with migration, invasion and metastasis in pancreatic cancer cells. The present study aimed to investigate the effects of AHCC® on cell migration and the protein expression level of CTTN in KLM1-R cells. The Gene Expression Profiling Interactive Analysis (GEPIA2), an online bioinformatics platform, was used to analyze CTTN mRNA expression levels in pancreatic cancer tissues compared with normal pancreatic tissues. CTTN mRNA expression and its association with clinicopathological characteristics were assessed by using the GEPIA2 platform. Next, the effects of AHCC® on KLM1-R cell migration were investigated by in vitro wound-healing assay. The KLM1-R cells were treated with AHCC® at a concentration of 10 mg/ml for 48 h. Western blotting was performed on of cell lysates with anti-CTTN or anti-actin antibodies to assess the protein expression levels of CTTN. Bioinformatics analysis indicated that the mRNA expression level of CTTN increased in pancreatic cancer tissues. The increased mRNA expression levels of CTTN were inversely associated with clinicopathological characteristics, including disease stages and prolonged patient survival times. The administration of 10 mg/ml AHCC® significantly inhibited KLM1-R cells migration compared with controls. The protein expression levels of CTTN were significantly reduced in AHCC®-treated KLM1-R cells, whereas actin expression was not affected. The downregulation of CTTN indicated the anti-metastatic potential of AHCC® in pancreatic cancer cells. Overall, AHCC® may have the potential to be a complementary and alternative therapeutic approach in treating pancreatic cancer.

ITGA2, LAMB3, and LAMC2 may be the potential therapeutic targets in pancreatic ductal adenocarcinoma: an integrated bioinformatics analysis.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer with an abysmal prognosis rate over the last few decades. Early diagnosis and prevention could effectively combat this malignancy. Therefore, it is crucial to discover potential biomarkers to identify asymptomatic premalignant or early malignant tumors of PDAC. Gene expression analysis is a powerful technique to identify candidate biomarkers involved in disease progression. In the present study, five independent gene expression datasets, including 321 PDAC tissues and 208 adjacent non-cancerous tissue samples, were subjected to statistical and bioinformatics analysis. A total of 20 differentially expressed genes (DEGs) were identified in PDAC tissues compared to non-cancerous tissue samples. Gene ontology and pathway enrichment analysis showed that DEGs were mainly enriched in extracellular matrix (ECM), cell adhesion, ECM-receptor interaction, and focal adhesion signaling. The protein-protein interaction network was constructed, and the hub genes were evaluated. Collagen type XII alpha 1 chain (COL12A1), fibronectin 1 (FN1), integrin subunit alpha 2 (ITGA2), laminin subunit beta 3 (LAMB3), laminin subunit gamma 2 (LAMC2), thrombospondin 2 (THBS2), and versican (VCAN) were identified as hub genes. The correlation analysis revealed that identified hub genes were significantly interconnected. Wherein COL12A1, FN1, ITGA2, LAMB3, LAMC2, and THBS2 were significantly associated with PDAC pathological stages. The Kaplan-Meier survival plots revealed that ITGA2, LAMB3, and LAMC2 expression were inversely correlated with a prolonged patient survival period. Furthermore, the Human Protein Atlas database was used to validate the expression and cellular origins of hub genes encoded proteins. The protein expression of hub genes was higher in pancreatic cancer tissue than in normal pancreatic tissue samples, wherein ITGA2, LAMB3, and LAMC2 were exclusively expressed in pancreatic cancer cells. Pancreatic cancer cell-specific expression of these three proteins may play pleiotropic roles in cancer progression. Our results collectively suggest that ITGA2, LAMB3, and LAMC2 could provide deep insights into pancreatic carcinogenesis molecular mechanisms and provide attractive therapeutic targets.

The Mechanistic Roles of ncRNAs in Promoting and Supporting Chemoresistance of Colorectal Cancer.

Colorectal Cancer (CRC) is one of the most common gastrointestinal malignancies which has quite a high mortality rate. Despite the advances made in CRC treatment, effective therapy is still quite challenging, particularly due to resistance arising throughout the treatment regimen. Several studies have been carried out to identify CRC chemoresistance mechanisms, with research showing different signalling pathways, certain ATP binding cassette (ABC) transporters and epithelial mesenchymal transition (EMT), among others to be responsible for the failure of CRC chemotherapies. In the last decade, it has become increasingly evident that certain non-coding RNA (ncRNA) families are involved in chemoresistance. Research investigations have demonstrated that dysregulation of microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) contribute towards promoting resistance in CRC via different mechanisms. Considering the currently available data on this phenomenon, a better understanding of how these ncRNAs participate in chemoresistance can lead to suitable solutions to overcome this problem in CRC. This review will first focus on discussing the different mechanisms of CRC resistance identified so far. The focus will then shift onto the roles of miRNAs, lncRNAs and circRNAs in promoting 5-fluorouracil (5-FU), oxaliplatin (OXA), cisplatin and doxorubicin (DOX) resistance in CRC, specifically using ncRNAs which have been recently identified and validated under in vivo or in vitro conditions.

Proteomic Analysis of Hepatocellular Carcinoma Tissues With Encapsulation Shows Up-regulation of Leucine Aminopeptidase 3 and Phosphoenolpyruvate Carboxykinase 2.

BACKGROUND/AIM: Cancer is the most fatal disease worldwide whose most lethal characteristics are invasion and metastasis. Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. HCC often shows encapsulation, which is related to better prognosis. In this study, proteomic analysis of HCC tissues with and without encapsulation was performed, in order to elucidate the factors which play important roles in encapsulation. MATERIALS AND METHODS: Five HCC tissues surrounded by a capsule and five HCC tissues which broke the capsule were obtained from patients diagnosed with HCC who underwent surgical liver resection. Protein samples from these tissues were separated by two-dimensional gel electrophoresis (2-DE), and the protein spots whose expression was different between encapsulated and non-encapsulated HCC tissues were identified through gel imaging analysis software. The selected protein spots were analyzed and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Two-DE analysis showed 14 spots whose expression was different between encapsulated and non-encapsulated HCC tissues. Of these, 9 were up-regulated and 5 were down-regulated in HCC tissues without encapsulation. The validation by Western blot confirmed that leucine aminopeptidase 3 (LAP3) and phosphoenolpyruvate carboxykinase mitochondrial (PCK2) were up-regulated significantly in HCC tissues with a capsule, compared to HCC tissues that broke the capsule. CONCLUSION: These findings suggest that LAP3 and PCK2 could be factors responsible for the maintenance of encapsulation in HCC tissues.

NEAT1 and Paraspeckles in Cancer Development and Chemoresistance.

Non-coding RNA were previously thought to be biologically useless molecules arising from simple transcriptional noise. These are now known to be an integral part of cellular biology and pathology. The wide range of RNA molecules have a diverse range of structures, functions, and mechanisms of action. However, structural long non-coding RNAs (lncRNAs) are a particular class of ncRNA that are proving themselves more and more important in cellular biology, as the exact structures that such RNAs form and stabilise become more understood. Nuclear Enriched Abundant Transcript 1 (NEAT1) is a specific structural RNA emerging as a critical component in the progress and development of cancer. NEAT1 forms part of multiple biological pathways, acting through a diverse group of mechanisms. The most important of these is the formation of the paraspeckle, through which it can influence the stability of a tumour to develop resistance to drugs. This review will thus cover the range of effects by which NEAT1 interacts with cancer progression in order to describe the various roles of NEAT1 in chemoresistance, as well as to identify drug targets that protein research alone could not provide.

Redirection of transfusion waste and by-products for xeno-free research applications.

BACKGROUND AND AIM: Whole blood is processed to derive a red cell concentrate, plasma, and buffy coat (BC) (from which platelets can be further extracted). Unused plasma and BCs are common in most blood establishments and considered a liability. The redirection of these products to xeno-free applications is not complicated or time-consuming and cannot only benefit the research recipients but also the blood establishment suppliers interested in research collaboration. The aim of this study is to describe a diverse yet by no means an exhaustive list of options for preparing blood products for research applications. MATERIALS AND METHODS: Plasma and BCs from healthy donors were processed using basic laboratory techniques under aseptic conditions and tested for their ability to support the culture of mesenchymal stem cells in both 2D and 3D cultures using fibrin clots. The white blood cells (WBC) from the BCs were induced by phytohemagglutinin and CD marker expression was monitored using quantitative polymerase chain reaction. RESULTS: All the methods tested for preparing blood products were successful but the applicability to different settings varied greatly with the most successful being the supplementation of Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 with 20% cryodepleted plasma and 0.1 mg/mL platelet lysate, the formation of fibrin clots using a ratio of 3:1 (medium: plasma) and the culturing of WBCs with 5 µg/mL phytohemagglutinin. CONCLUSIONS: Using the wastes and by-products of blood establishments for xeno-free cell culturing of stem cells will reduce the reliance on commercially available, ready-made products, and increasing the potential for therapeutic stem cell research. Despite the benefits presented both in terms of cost and applications, further characterization and optimization of each blood product for reproducibility of results is required. RELEVANCE FOR PATIENTS: The availability of low-cost xeno-free reagents will speed up therapeutic stem cell research and allow patients to receive treatments of the expected high standards at lower costs.

Up-regulation of the pentose phosphate pathway and HIF-1α expression during neural progenitor cell induction following glutamate treatment in rat ex vivo retina.

The metabolic state influences the regulation of neural stem/progenitor cells. The pentose phosphate pathway (PPP), an alternative metabolic pathway that operates parallel to glycolysis, not only provides key intermediates for biosynthetic reactions but also controls the fate of neural stem/progenitor cells. We have previously shown that glutamate application leads to the induction of neural progenitor cells in mature ex vivo rat retina. In this study, we investigated whether regulation of the PPP might be changed following glutamate treatment of the retina. Immunoblot analysis revealed that the amount of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP as well as that of 6-phosphogluconate dehydrogenase (6PGD), another enzyme in this pathway, increased in the glutamate-treated retina. Consistent with the fact that both these enzymes generate reduced nicotinamide adenine dinucleotide phosphate (NADPH), the amount of NAPDH in the treated retina was significantly higher compared with that in the untreated retina. We also found that both DNA synthesis as well as the expression of fatty acid synthase (FASN) increased significantly in the glutamate-treated retina. Furthermore, hypoxia-inducible factor 1-α (HIF-1α), a positive transcriptional regulator of PPP enzymes, was up-regulated at both messenger RNA (mRNA) and protein levels. Finally, we found the interaction of HIF-1α with the M2 isozyme of pyruvate kinase (PKM2), with this interaction having been shown to contribute to a positive feedback loop in the control of glycolysis. Our results thus show that specific metabolic change in the PPP occurs in the process of neural progenitor cell induction in the mature rat retina.

Nuclear paraspeckles function in mediating gene regulatory and apoptotic pathways.

The nucleus is an essential hub for the regulation of gene expression in both spatial and temporal contexts. The complexity required to manage such a feat has resulted in the evolution of multiple sub-structures in the nucleus such as the nucleolus, small cajal bodies and nuclear stress bodies. The paraspeckle is another membraneless structure composed of RNA elements, primarily the long non-coding RNA (lncRNA) Nuclear Enriched Abundant Transcript 1 (NEAT1), associated with RNA binding proteins (RBPs). The paraspeckle is showing signs of being involved in various aspects of gene regulation and its role in many pathologies from cancer to viral infection is beginning to be addressed. Research into paraspeckle-directed gene regulation highlights the increase in the appreciation of the biological significance of non-coding RNA (ncRNA). This review will thus cover the basis of how a structure as large as a paraspeckle forms along with its functions. It will also explore how it effects pathological conditions and can be used in clinical intervention, with special emphasis on the multitude of methods utilised by paraspeckles for apoptotic regulation.

MiRNA influences in mesenchymal stem cell commitment to neuroblast lineage development.

Mesenchymal Stem Cells (MSCs) are widely used in therapeutic applications. Their plasticity and predisposition to differentiate into a variety of cell types, including those of the neuronal lineage, makes them ideal to study whether a selection of miRNAs may direct the differentiation of MSCs into neuroblasts or neuroblastoma to mature neurons. Following a short-listing, miR-107, 124 and 381 were selected as the most promising candidates for this differentiation. MSCs differentiated into cells of the neural lineage (Conditioned Cells) upon addition of conditioned medium (rich in microvesicles containing miRNAs) obtained from cultured SH-SY5Y neuroblastoma cells. Characterisation of stemness (including SOX2, OCT4, Nanog and HCG) and neural markers (including Nestin, MASH1, TUBB3 and NeuN1) provided insight regarding the neuronal state of each cell type. This was followed by transfection of the three miRNA antagonists and mimics, and quantification of their respective target genes. MiRNA target gene expression following transfection of MSCs with miRNA inhibitors and mimics demonstrated that these three miRNAs were not sufficient to induce differentiation. In conditioned cells the marginal changes in the miRNA target expression levels reflected potential for the modulation of intermediate neural progenitors and immature neuron cell types. Transfection of various combinations of miRNA inhibitors and/or mimics revealed more promise. Undoubtedly, a mix of biomolecules is being released by the SH-SY5Y in culture that induce MSCs to differentiate. Screening for those biomolecules acting synergistically with specific miRNAs will allow further combinatorial testing to elucidate the role of miRNA modulation.

CUB Domain-containing Protein 1 (CDCP1) Is Down-regulated by Active Hexose-correlated Compound in Human Pancreatic Cancer Cells.

BACKGROUND/AIM: We have previously reported that treatment of pancreatic cancer cells with active hexose-correlated compound (AHCC), an extract of a basidiomycete mushroom, decreases the levels of tumor-associated proteins including heat-shock protein 27 (HSP27), heat shock factor 1 (HSF1) and sex-determining region Y-box 2 (SOX2). The transmembrane glycoprotein, CUB domain-containing protein 1 (CDCP1) has been reported to be up-regulated in various cancers, and be associated with invasion and metastasis. The aim of this study was to examine the effect of AHCC on the expression of CDCP1 in KLM1-R cells. MATERIALS AND METHODS: Gemcitabine-resistant pancreatic cancer cells (KLM1-R) were treated with AHCC (10 mg/ml) for 48 h. Western blot analysis of cell extracts with anti-CDCP1 or anti-actin antibodies was performed to assess the expression of CDCP1. RESULTS: Expression of CDCP1 was reduced by AHCC treatment of KLM1-R cells, whereas expression of actin was not affected. The ratio of intensities of CDCP1/actin in AHCC-treated KLM1-R cells was significantly suppressed (p<0.05) compared to untreated cells. CONCLUSION: AHCC down-regulated CDCP1 expression and inhibited the malignant progression of pancreatic cancer cells.

Clinical Presentation of Preeclampsia and the Diagnostic Value of Proteins and Their Methylation Products as Biomarkers in Pregnant Women with Preeclampsia and Their Newborns.

Preeclampsia (PE) is a disorder which affects 1-10% of pregnant women worldwide. It is characterised by hypertension and proteinuria in the later stages of gestation and can lead to maternal and perinatal morbidity and mortality. Other than the delivery of the foetus and the removal of the placenta, to date there are no therapeutic approaches to treat or prevent PE. It is thus only possible to reduce PE-related mortality through early detection, careful monitoring, and treatment of the symptoms. For these reasons the search for noninvasive, blood-borne, or urinary biochemical markers that could be used for the screening, presymptomatic diagnosis, and prediction of the development of PE is of great urgency. So far, a number of biomarkers have been proposed for predicting PE, based on pathophysiological observations, but these have mostly proven to be unreliable and inconsistent between different studies. The clinical presentation of PE and data gathered for the biochemical markers placental growth factor (PlGF), soluble Feline McDonough Sarcoma- (fms-) like tyrosine kinase-1 (sFlt-1), asymmetric dimethylarginine (ADMA), and methyl-lysine is being reviewed with the aim of providing both a clinical and biochemical understanding of how these biomarkers might assist in the diagnosis of PE or indicate its severity.

Enzyme-treated Asparagus Extract Down-regulates Heat Shock Protein 27 of Pancreatic Cancer Cells.

BACKGROUND/AIM: From the standpoint of cancer therapy, it is valuable to enhance the anticancer effects of chemotherapy. Our previous reports revealed that up-regulation of heat-shock protein 27 (HSP27) has been linked to gemcitabine resistance of pancreatic cancer cells. Enzyme-treated asparagus extract (ETAS) is an extract that is produced from asparagus. The purpose of this study was to investigate the effect of ETAS on the expression of HSP27 and other HSPs in the gemcitabine-resistant pancreatic cancer cell line KLM1-R. MATERIALS AND METHODS: KLM1-R cells were treated with ETAS, and expression levels of HSPs, including HSP27, were investigated by western blotting. RESULTS: ETAS down-regulated HSP27 and pHSP27 (serine 78) in KLM1-R cells, but, HSP70 and GRP78 levels were not altered. CONCLUSION: This study suggests the potential therapeutic benefit of ETAS in enhancing anticancer effects by its combination with gemcitabine for patients with pancreatic cancer.

Optimization of fixative solution for retinal morphology: a comparison with Davidson's fixative and other fixation solutions.

PURPOSE: Numerous fixative solutions are available but many are not amenable to the histomorphological preservation of retinae. The investigators specifically focused on retinal histological studies, which rather than 4% formaldehyde (FA), often use Davidson's fixative. However the latter has its limitations. The purpose of this study was to produce a new fixative which maintains retinae closer to the in vivo conditions. STUDY DESIGN: Experimental design. METHODS: Four fixative formulations (4% paraformaldehyde, Davidson's fixative, modified Davidson's fixative and an in-house fixative - TB-Fix) were tested on retinae and the outcomes on histomorphology and immunohistochemical staining for selected antigenic markers was compared. RESULTS: TB-Fix markedly improved morphological detail following hematoxylin and eosin staining, most importantly eliminating the spongiform appearance in the plexiform layer and the swelling of somata (including Müller cells), when compared to FA, Davidson's fixative and its modified version. Retinal samples fixed with TB-Fix or FA showed comparable results in immunohistological staining for neurons and glia in the retina. Importantly, while the whole eye fixed with FA collapsed in shape and induced artificial retinal detachment, the eye fixed with TB-Fix avoided deformation and detachment. Furthermore, we found that TB-Fix also prevented detachment from the culture plate when used to fix HEK293 cells, which are known to detach from the plate easily. CONCLUSION: It was demonstrated that TB-Fix provides an overall improvement in the preservation of retinal morphology and chemical composition.

MiRNA Influences in Neuroblast Modulation: An Introspective Analysis.

Neuroblastoma (NB) is the most common occurring solid paediatric cancer in children under the age of five years. Whether of familial or sporadic origin, chromosome abnormalities contribute to the development of NB and cause dysregulation of microRNAs (miRNAs). MiRNAs are small non-coding, single stranded RNAs that target messenger RNAs at the post-transcriptional levels by repressing translation within all facets of human physiology. Such gene 'silencing' activities by miRNAs allows the development of regulatory feedback loops affecting multiple functions within the cell, including the possible differentiation of neural stem cell (NSC) lineage selection. Neurogenesis includes stages of self-renewal and fate specification of NSCs, migration and maturation of young neurones, and functional integration of new neurones into the neural circuitry, all of which are regulated by miRNAs. The role of miRNAs and their interaction in cellular processes are recognised aspects of cancer genetics, and miRNAs are currently employed as biomarkers for prognosis and tumour characterisation in multiple cancer models. Consequently, thorough understanding of the mechanisms of how these miRNAs interplay at the transcriptomic level will definitely lead to the development of novel, bespoke and efficient therapeutic measures, with this review focusing on the influences of miRNAs on neuroblast modulations leading to neuroblastoma.

The Role of Toll-Like Receptors in Autoimmune Diseases through Failure of the Self-Recognition Mechanism.

Toll-like receptors (TLRs), part of the innate immune system that recognises molecular signatures, are important in the recognition of pathogenic components. However, when specific cellular contexts develop in which TLRs are inappropriately activated by self-components, this may lead to sterile inflammation and result in the occurrence of autoimmunity. This review analyses the available data regarding TLR biochemistry, the specific mechanisms which are brought about by TLR activation, and the importance of these mechanisms in the light of any existing and potential therapies in the field of autoimmunity.