Hepatoma cell functions modulated by NEK2 are associated with liver cancer progression.

NEK2 (NIMA-related expressed kinase 2) is a serine/threonine centrosomal kinase that acts as a critical regulator of centrosome structure and function. Aberrant NEK2 activities lead to failure in regulating centrosome duplication. NEK2 overexpression promotes tumorigenesis and is associated with poor prognosis in several cancers. Increased NEK2 expression during the late pathological stage has been detected in the Oncomine liver dataset and hepatocellular carcinoma (HCC) specimens. Elevated NEK2 protein is associated with poor overall survival in patients with HCC. However, the precise roles and mechanisms of NEK2 in liver cancer progression remain largely unknown. An earlier functional study revealed that NEK2 mediates drug resistance (cisplatin or lipo-doxorubicin) via expression of an ABCC10 transporter. Active angiogenesis and metastasis underlie the rapid recurrence and poor survival of HCC. Results from the current study showed that NEK2 mediates tumor growth, metastasis and angiogenesis in vivo. NEK2-mediated drug resistance was blocked by a specific PI3K or AKT inhibitor. Moreover, NEK2 mediated liver cancer cell migration via pAKT/NF-κB signaling and matrix metalloproteinase (MMP) activation. Angiogenesis was induced via the same signaling pathway and IL-8 stimulation. Our findings collectively indicate that NEK2 modulates hepatoma cell functions, including growth, drug resistance, metastasis and angiogenesis via downstream genes activation.

Negative modulation of the epigenetic regulator, UHRF1, by thyroid hormone receptors suppresses liver cancer cell growth.

The thyroid hormone, 3,3',5-triiodo-l-thyronine (T3 ), mediates several physiological processes, including embryonic development, cellular differentiation, metabolism and regulation of cell proliferation. Thyroid hormone (T3 ) and its receptor (TR) are involved in metabolism and growth. In addition to their developmental and metabolic functions, TRs play a tumor suppressor role, and therefore, their aberrant expression can lead to tumor transformation. Aberrant epigenetic silencing of tumor suppressor genes promotes cancer progression. The epigenetic regulator, Ubiquitin-like with PHD and ring finger domains 1 (UHRF1), is overexpressed in various cancers. In our study, we demonstrated that T3 negatively regulates UHRF1 expression, both in vitro and in vivo. Our results further indicate that UHRF1 regulation by T3 is indirect and mediated by Sp1. Sp1-binding elements of UHRF1 were identified at positions -664/-505 of the promoter region using the luciferase and chromatin immunoprecipitation assays. Notably, UHRF1 and Sp1 levels were elevated in subgroups of hepatocellular carcinoma patients and inversely correlated with TRα1 expression. Knockdown of UHRF1 expression should therefore provide a means to inhibit hepatoma cell proliferation. Expression of UHRF1 was downregulated by TRs, in turn, relieving silencing of the UHRF1 target gene, p21. Based on the collective findings, we propose that T3 /TR signaling induces hepatoma cell growth inhibition via UHRF1 repression.

Repression of microRNA-130b by thyroid hormone enhances cell motility.

BACKGROUND & AIMS: Thyroid hormone (T3) and its receptor (TR) are involved in cell growth and cancer progression. Although deregulation of microRNA (miRNA) expression has been detected in many tumor types, the mechanisms underlying functional impairment and specific involvement of miRNAs in tumor metastasis remain unclear. In the current study, we aimed to elucidate the involvement of deregulated miRNA-130b (miR-130b) and its target genes mediated by T3/TR in cancer progression. METHODS: Quantitative reverse transcription-PCR, luciferase and chromatin immunoprecipitation assays were performed to identify the miR-130b transcript and the mechanisms implicated in its regulation. The effects of miR-130b on hepatocellular carcinoma (HCC) invasion were further examined in vitro and in vivo. Clinical correlations among miR-130b, TRs and interferon regulatory factor 1 (IRF1) were examined in HCC samples using Spearman correlation analysis. RESULTS: Our experiments disclosed negative regulation of miR-130b expression by T3/TR. Overexpression of miR-130b led to marked inhibition of cell migration and invasion, which was mediated via suppression of IRF1. Cell migration ability was promoted by T3, but partially suppressed upon miR-130b overexpression. Furthermore, miR-130b suppressed expression of epithelial-mesenchymal transition (EMT)-related genes, matrix metalloproteinase-9, phosphorylated mammalian target of rapamycin (mTOR), p-ERK1/2, p-AKT and p-signal transducer and activator of transcription (STAT)-3. Notably, miR-130b was downregulated in hepatoma samples and its expression patterns were inversely correlated with those of TRα1 and IRF1. CONCLUSIONS: Our data collectively highlight a novel pathway interlinking T3/TR, miR-130b, IRF1, the EMT-related genes, p-mTOR, p-STAT3 and the p-AKT cascade, which regulates the motility and invasion of hepatoma cells.

Glucose-regulated protein 58 modulates ß-catenin protein stability in a cervical adenocarcinoma cell line.

BACKGROUND: Cervical cancer continues to threaten women's health worldwide, and the incidence of cervical adenocarcinoma (AD) is rising in the developed countries. Previously, we showed that glucose-regulated protein 58 (Grp58) served as an independent factor predictive of poor prognosis of patients with cervical AD. However, the molecular mechanism underlying the involvement of Grp58 in cervical carcinogenesis is currently unknown. METHODS: DNA microarray and enrichment analysis were used to identify the pathways disrupted by knockdown of Grp58 expression. RESULTS: Among the pathway identified, the WNT signaling pathway was one of those that were significantly associated with knockdown of Grp58 expression in HeLa cells. Our experiments showed that ß-catenin, a critical effector of WNT signaling, was stabilized thereby accumulated in stable Grp58 knockdown cells. Membrane localization of ß-catenin was observed in Grp58 knockdown, but not control cells. Using a transwell assay, we found that accumulated ß-catenin induced by Grp58 knockdown or lithium chloride treatment inhibited the migration ability of HeLa cells. Furthermore, an inverse expression pattern of Grp58 and ß-catenin was observed in cervical tissues. CONCLUSIONS: Our results demonstrate that ß-catenin stability is negatively regulated by Grp58 in HeLa cells. Overexpression of Grp58 may be responsible for the loss of or decrease in membranous ß-catenin expression in cervical AD.

Thyroid hormone receptor inhibits hepatoma cell migration through transcriptional activation of Dickkopf 4.

Triiodothyronine (T3) is a potent form of thyroid hormone mediates several physiological processes including cellular growth, development, and differentiation via binding to the nuclear thyroid hormone receptor (TR). Recent studies have demonstrated critical roles of T3/TR in tumor progression. Moreover, long-term hypothyroidism appears to be associated with the incidence of human hepatocellular carcinoma (HCC), independent of other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein that antagonizes the canonical Wnt signaling pathway, is induced by T3 at both mRNA and protein levels in HCC cell lines. However, the mechanism underlying T3-mediated regulation of DKK4 remains unknown. In the present study, the 5' promoter region of DKK4 was serially deleted, and the reporter assay performed to localize the T3 response element (TRE). Consequently, we identified an atypical direct repeat TRE between nucleotides -1645 and -1629 conferring T3 responsiveness to the DKK4 gene. This region was further validated using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Stable DKK4 overexpression in SK-Hep-1 cells suppressed cell invasion and metastatic potential, both in vivo andin vitro, via reduction of matrix metalloproteinase-2 (MMP-2) expression. Our findings collectively suggest that DKK4 upregulated by T3/TR antagonizes the Wnt signal pathway to suppress tumor cell progression, thus providing new insights into the molecular mechanism underlying thyroid hormone activity in HCC.

Dickkopf 4 positively regulated by the thyroid hormone receptor suppresses cell invasion in human hepatoma cells.

UNLABELLED: Thyroid hormone (T(3)) mediates cellular growth, development, and differentiation by binding to the nuclear thyroid hormone receptor (TR). Recent studies suggest that long-term hypothyroidism is associated with human hepatocellular carcinoma (HCC) independent from other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein, antagonizes the Wnt signal pathway. In this study, we demonstrate that T(3) may play a suppressor role by inducing DKK4 expression in HCC cells at both the messenger RNA (mRNA) and protein levels. DKK4 was down-regulated in 67.5% of HCC cancerous tissues. The decrease in DKK4 levels was accompanied by a concomitant decrease in TR protein levels in the matched cancerous tissues in 31% of tissues compared by immunoblotting with the adjacent noncancerous tissues. Further, TR and DKK4 expression levels were positively correlated in both normal and cancerous specimens by tissue array analysis. In function assays, stable DKK4 transfected into J7 or HepG2 cells decreased cell invasion in vitro. Conversely, knocking down DKK4 restores cell invasiveness. DKK4-expressing J7 clones showed increased degradation of ß-catenin, but down-regulation of CD44, cyclin D1, and c-Jun. To investigate the effect of DKK4 and TR on tumor growth in vivo, we established a xenograft of J7 cells in nude mice. J7-DKK4 and J7-TRα1 overexpressing mice, which displayed growth arrest, lower lung colony formation index, and smaller tumor size than in control mice, supporting an inhibitory role of DKK4 in tumor progression. CONCLUSION: Taken together, these data suggest that the TR/DKK4/Wnt/ß-catenin cascade influences the proliferation and migration of hepatoma cells during the metastasis process and support a tumor suppressor role of the TR.

Evaluation and Application of Drug Resistance by Biomarkers in the Clinical Treatment of Liver Cancer.

Liver cancer is one of the most lethal cancers in the world, mainly owing to the lack of effective means for early monitoring and treatment. Accordingly, there is considerable research interest in various clinically applicable methods for addressing these unmet needs. At present, the most commonly used biomarker for the early diagnosis of liver cancer is alpha-fetoprotein (AFP), but AFP is sensitive to interference from other factors and cannot really be used as the basis for determining liver cancer. Treatment options in addition to liver surgery (resection, transplantation) include radiation therapy, chemotherapy, and targeted therapy. However, even more expensive targeted drug therapies have a limited impact on the clinical outcome of liver cancer. One of the big reasons is the rapid emergence of drug resistance. Therefore, in addition to finding effective biomarkers for early diagnosis, an important focus of current discussions is on how to effectively adjust and select drug strategies and guidelines for the treatment of liver cancer patients. In this review, we bring this thought process to the drug resistance problem faced by different treatment strategies, approaching it from the perspective of gene expression and molecular biology and the possibility of finding effective solutions.

The Utilization of Optically Induced Dielectrophoresis (ODEP)-Based Cell Manipulation in a Microfluidic System for the Purification and Sorting of Circulating Tumor Cells (CTCs) with Different Sizes.

The analysis of circulating tumor cells (CTCs) at the molecular level holds great promise for several clinical applications. For this goal, the harvest of high-purity, size-sorted CTCs with different subtypes from a blood sample are important. For this purpose, a two-step CTC isolation protocol was proposed, by which the immunomagnetic beads-based cell separation was first utilized to remove the majority of blood cells. After that, an optically induced dielectrophoresis (ODEP) microfluidic system was developed to (1) purify the CTCs from the remaining magnetic microbeads-bound blood cells and to (2) sort and separate the CTCs with different sizes. In this study, the ODEP microfluidic system was designed and fabricated. Moreover, its optimum operation conditions and performance were explored. The results exhibited that the presented technique was able to purify and sort the cancer cells with two different sizes from a tested cell suspension in a high-purity (93.5% and 90.1% for the OECM 1 and HA22T cancer cells, respectively) manner. Overall, this study presented a technique for the purification and sorting of cancer cells with different sizes. Apart from this application, the technique is also useful for other applications in which the high-purity and label-free purification and sorting of cells with different sizes is required.

Overexpression of ADP-ribosylation factor 1 in human gastric carcinoma and its clinicopathological significance.

Gastric cancer is the sixth leading cause of cancer-related death in Taiwan, and the identification of related factors is essential to increase patient survival. ADP-ribosylation factor 1 (ARF1) was initially identified using 2-D electrophoresis combined with MALDI-time-of-flight mass spectrometry. ADP-ribosylation factor 1 belongs to the Ras superfamily or GTP-binding protein family and has been shown to enhance cell proliferation. In the current study, we evaluated the potential of ARF1 as a biomarker for gastric cancer detection. ADP-ribosylation factor 1 mRNA was upregulated in tumor tissues (compared with adjacent non-tumor tissues, n = 55) in approximately 67.2% of gastric cancer patients. Expression of ARF1 protein was additionally observed using Western blot and immunohistochemistry (IHC) analyses. The clinicopathological correlations of ARF1 were further evaluated. Elevated ARF1 expression was strongly correlated with lymph node metastasis (P = 0.008), serosal invasion (P = 0.046), lymphatic invasion (P = 0.035), and pathological staging (P = 0.010). Moreover, the 5-year survival rate for the lower ARF1 expression group (n = 50; IHC score < 90) was higher than that of the higher expression group (n = 60; IHC score ≥ 90) (P = 0.0228, log-rank test). To establish the specific function of ARF1 in human gastric cancer, isogenic ARF1-overexpressing cell lines were prepared. Our results showed that ARF1-overexpressing clones display enhanced cell proliferation, migration, and invasion. Furthermore, ARF1-overexpression might contribute to poor prognosis of patients. These findings collectively support the utility of ARF1 as a novel prognostic marker for gastric cancer and its role in cell invasion.

Effects of Thyroid Hormones on Lipid Metabolism Pathologies in Non-Alcoholic Fatty Liver Disease.

The typical modern lifestyle contributes to the development of many metabolic-related disorders, as exemplified by metabolic syndrome. How to prevent, resolve, or avoid subsequent deterioration of metabolic disturbances and the development of more serious diseases has become an important and much-discussed health issue. Thus, the question of the physiological and pathological roles of thyroid hormones (THs) in metabolism has never gone out of fashion. Although THs influence almost all organs, the liver is one of the most important targets as well as the hub of metabolic homeostasis. When this homeostasis is out of balance, diseases may result. In the current review, we summarize the common features and actions of THs, first focusing on their effects on lipid metabolism in the liver. In the second half of the review, we turn to a consideration of non-alcoholic fatty liver disease (NAFLD), a disease characterized by excessive accumulation of fat in the liver that is independent of heavy alcohol consumption. NAFLD is a growing health problem that currently affects ~25% of the world's population. Unfortunately, there are currently no approved therapies specific for NAFLD, which, if left uncontrolled, may progress to more serious diseases, such as cirrhosis or liver cancer. This absence of effective treatment can also result in the development of non-alcoholic steatohepatitis (NASH), an aggressive form of NAFLD that is the leading cause of liver transplantation in the United States. Because THs play a clear role in hepatic fat metabolism, their potential application in the prevention and treatment of NAFLD has attracted considerable research attention. Studies that have investigated the use of TH-related compounds in the management of NAFLD are also summarized in the latter part of this review. An important take-home point of this review is that a comprehensive understanding of the physiological and pathological roles of THs in liver fat metabolism is possible, despite the complexities of this regulatory axis-an understanding that has clinical value for the specific management of NAFLD.

CRNDE acts as an epigenetic modulator of the p300/YY1 complex to promote HCC progression and therapeutic resistance.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common primary liver malignancies worldwide. The long-term prognosis for HCC remains extremely poor, with drug resistance being the major underlying cause of recurrence and mortality. The lncRNA colorectal neoplasia differentially expressed (CRNDE) is an epigenetic mediator and plays an important role to drive proliferation and drug resistance in HCC. However, CRNDE as an epigenetic regulator with influences sorafenib resistance in HCC is unclear. Thus, we explore the potential of targeting the CRNDE/p300/YY1 axis as a novel therapeutic strategy to overcome sorafenib resistance of HCC. METHOD: Detection of the expression level of CRNDE and EGFR in clinical specimens of HCC. CRNDE, EGFR, p300, and YY1expression were altered in HCC cells through transfection with different plasmids, and cell proliferation, migration, invasion, and sorafenib resistance were subsequently observed. Immunoprecipitation, chromatin immunoprecipitation, re-chromatin immunoprecipitation, site-directed mutagenesis, RNA Immunoprecipitation, immune fluorescence, qRT-PCR, and western blotting were performed to uncover the mechanisms of CRNDE regulation. The xenograft nude mice model was used to investigate the tumor growth and sorafenib resistance. RESULTS: In this study, we showed that CRNDE expression is significantly positively correlated with that of epidermal growth factor receptor (EGFR) in clinical specimens of HCC and induces proliferation and sorafenib resistance of HCC via EGFR-mediated signaling. Mechanistically, CRNDE stabilized the p300/YY1 complex at the EGFR promoter and simultaneously enhanced histone H3K9 and H3K27 acetylation, which serve as markers of relaxed chromatin. EGFR was positively upregulated by the epigenetic complex, p300/YY1, in a manner dependent on CRNDE expression, leading to enhanced tumor cell proliferation and sorafenib resistance. Furthermore, C646, a p300 inhibitor, suppressed EGFR transcriptional activity by decreasing chromatin relaxation and YY1 binding, which effectively reduced proliferation/sorafenib resistance and prolonged overall survival. CONCLUSION: Our collective findings support the potential of targeting the CRNDE/p300/YY1 axis as a novel therapeutic strategy to overcome sorafenib resistance of HCC.

Glucose-regulated protein 58 modulates cell invasiveness and serves as a prognostic marker for cervical cancer.

Human papilloma virus infection is critical but not sufficient to cause cervical cancer. Molecular markers of cervical carcinogenesis are essential. The aim of this study was to identify aberrantly expressed proteins in cervical cancer and determine their clinical significance. A two-dimensional polyacrylamide gel electrophoresis (2-DE) proteomic strategy was used for screening candidate proteins. Immunoblotting and immunohistochemical (IHC) analyses were performed to confirm the results of 2-DE, and the clinical significance was estimated. Glucose-regulated protein 58 (Grp58) was overexpressed in 73% of cancers. The IHC staining showed that the Grp58 histoscore was significantly higher in patients with adenocarcinoma (AD) compared with squamous cell carcinoma (P < 0.05). Grp58 staining was intense in AD with a penetration depth greater than half of the cervical stroma (P = 0.033). High Grp58 expression was associated with low overall survival and recurrence-free survival (RFS) rates (P = 0.007 and P = 0.013, respectively). In multivariate analysis, high Grp58 expression (P = 0.042) and lymph node metastasis (P = 0.026) were determined as independent prognostic factors for RFS. Patients exhibiting both high Grp58 expression and lymph node metastasis displayed poorer outcomes than the other patient groups. In functional studies, knockdown of Grp58 in HeLa cells led to decreased cell invasiveness and inhibition of lung metastasis in a xenograft mouse model. In conclusion, Grp58 serves as a potent prognostic factor of cervical AD. Estimation of the Grp58 index in conjunction with the lymph node metastasis status might aid in predicting the prognosis of cervical AD.

Overexpression of gelsolin in human cervical carcinoma and its clinicopathological significance.

OBJECTIVES: Cervical carcinoma is the second most common cause of death from gynecological cancers worldwide. Knowledge of the molecular mechanisms underlying the tumorigenesis of cervical cancer cell, except human papilloma virus infection, is limited. METHODS: A microarray was used to study the differential expression of genes in cancerous tissues to identify new molecular markers for diagnosis and prognosis. Their differential expression was confirmed with Western blotting and immunohistochemical analyses. The clinical correlations and prognostic significance of the aberrantly expressed proteins were evaluated to identify novel biomarkers of cervical cancer. RESULTS: The expression of gelsolin was significantly upregulated in 78% of patients with cervical cancer, and gelsolin was selected for further study. Gelsolin expression was stronger in cervical tumor tissues than in the surrounding noncancerous tissues (P<0.001). Gelsolin expression in the plasma of cervical cancer patients was increased 2.2-fold compared with that of healthy control subjects (P<0.001). The levels of plasma gelsolin in the early and late stages were significantly different (P=0.006). According to immunohistochemical analysis, increased gelsolin expression was associated with histological type and FIGO stage II. The 5-year overall survival and recurrence-free survival rates for the low-expression group (cut-off=115) were significantly higher than those of the high-expression group. Cancer cells with reduced gelsolin expression exhibited reduced migration and proliferation. CONCLUSIONS: These results provide strong evidence that gelsolin plays an important role in cellular proliferation and migration in cervical cancer and suggest that gelsolin is a promising marker for cervical cancer screening and prognosis.

Thyroid hormone receptor-mediated regulation of the methionine adenosyltransferase 1 gene is associated with cell invasion in hepatoma cell lines.

The thyroid hormone T(3) regulates differentiation, growth, and development. We demonstrated that methionine adenosyltransferase 1A (MAT1A) was positively regulated by T(3) identified by cDNA microarray previously. The expression of the MAT1A was upregulated by T(3) in hepatoma cell lines overexpressing thyroid hormone receptors (TRs). Additionally, these findings indicate that MAT1A may be regulated by CCAAT/enhancer binding protein (C/EBP). The critical role of the C/EBP binding sites was confirmed by the reporter or chromatin immuno-precipitation (ChIP) assay. In addition, C/EBP was upregulated in hepatoma cells after T(3) treatment and ectopic expression of MAT1A inhibited cell migration and invasion in J7 hepatoma cells. Conversely, knockdown of MAT1A expression increased cell migration. Together, these findings suggest that the expression of the MAT1A gene is mediated by C/EBP and is indirectly upregulated by T(3). Finally, TR was downregulated in a small subset of hepatocellular carcinoma cells concomitantly reduced the expression of C/EBPalpha and MAT1A.

Functional and Clinical Significance of Dysregulated microRNAs in Liver Cancer.

Liver cancer is the leading cause of cancer-related mortality in the world. This mainly reflects the lack of early diagnosis tools and effective treatment methods. MicroRNAs (miRNAs) are a class of non-transcribed RNAs, some of which play important regulatory roles in liver cancer. Here, we discuss microRNAs with key impacts on liver cancer, such as miR-122, miR-21, miR-214, and miR-199. These microRNAs participate in various physiological regulatory pathways of liver cancer cells, and their modulation can have non-negligible effects in the treatment of liver cancer. We discuss whether these microRNAs can be used for better clinical diagnosis and/or drug development. With the advent of novel technologies, fast, inexpensive, and non-invasive RNA-based biomarker research has become a new mainstream approach. However, the clinical application of microRNA-based markers has been limited by the high sequence similarity among them and the potential for off-target problems. Therefore, researchers particularly value microRNAs that are specific to or have special functions in liver cancer. These include miR-122, which is specifically expressed in the liver, and miR-34, which is necessary for the replication of the hepatitis C virus in liver cancer. Clinical treatment drugs have been developed based on miR-34 and miR-122 (MRX34 and Miravirsen, respectively), but their side effects have not yet been overcome. Future research is needed to address these weaknesses and establish a feasible microRNA-based treatment strategy for liver cancer.

Circulating p16-Positive and p16-Negative Tumor Cells Serve as Independent Prognostic Indicators of Survival in Patients with Head and Neck Squamous Cell Carcinomas.

BACKGROUND: Decisions regarding the staging, prognosis, and treatment of patients with head and neck squamous cell carcinomas (HNSCCs) are made after determining their p16 expression levels and human papillomavirus (HPV) infection status. METHODS: We investigated the prognostic roles of p16-positive and p16-negative circulating tumor cells (CTCs) and their cell counts in HNSCC patients. We enrolled patients with locally advanced HNSCCs who received definitive concurrent chemoradiotherapy for final analysis. We performed CTC testing and p16 expression analysis before chemoradiotherapy. We analyzed the correlation between p16-positive and p16-negative CTCs and HPV genotyping, tissue p16 expression status, response to chemoradiotherapy, disease-free survival, and overall survival. RESULTS: Forty-one patients who fulfilled the study criteria were prospectively enrolled for final analysis. The detection rates of p16-positive (>0 cells/mL blood) and p16-negative (≥3 cells/mL blood) CTCs were 51.2% (n = 21/41) and 70.7%, respectively. The best responses of chemoradiotherapy and the p16 positivity of CTCs are independent prognostic factors of disease progression, with hazard ratios of 1.738 (95% confidence interval (CI): 1.031-2.927), 5.497 (95% CI: 1.818-16.615), and 0.176 (95% CI: 0.056-0.554), respectively. The p16 positivity of CTCs was a prognostic factor for cancer death, with a hazard ratio of 0.294 (95% CI: 0.102-0.852). CONCLUSIONS: The p16-positive and p16-negative CTCs could predict outcomes in HNSCC patients receiving definitive chemoradiotherapy. This non-invasive CTC test could help stratify the risk and prognosis before chemoradiotherapy in clinical practice and enable us to perform de-intensifying therapies.

Human testicular orphan receptor 4 enhances thyroid hormone receptor signaling.

The thyroid hormone receptor (TR) and human testicular orphan receptor 4 (TR4) belong to the nuclear hormone receptor superfamily. They are ligand-dependent transcription factors. TR and TR4 bind to a similar thyroid response element (TRE), known as a direct repeat with four nucleotide spacing (DR4). This study examined the possible interaction or cross-talking between those two receptors. We hypothesized that protein-protein interaction between TR4 and TR may promote TR-mediated transcriptional activity. Glutathione S-transferase pull-down and immunoprecipitation assays showed direct interaction between TR and TR4. Electrophoretic mobility-shift assay demonstrated that TR and TR4 could co-occupy the same TRE. The interaction between TR4 and TR may enhance regulation of genes targeted by TR, such as furin, fibrinogen, cdk2 and p21 expression. We found that TR4 function is similar with TR as TR4 alone could regulate expression of some TR target genes, and could increase cell migration or inhibit cell proliferation. Importantly, the TR-dependent inhibition of cell proliferation and stimulation of cell migration are more enhanced in the HepG2-TR cells stably over-expressing TR4. Overall, TR4 not only has modulation abilities similar to TR but also can cross-talk with TR and promote the TR signaling pathway.

Predisposing factors for hypoglycaemia in the emergency department.

OBJECTIVE: Hypoglycaemia is a common complication of diabetes mellitus. Previous studies suggest that hypoglycaemic episodes may occur with other comorbidities, influencing the outcome of recovery. Recognising the predisposing factors for hypoglycaemic episodes in the emergency department is important. Therefore, we investigated the characteristics of and predisposing factors for hypoglycaemia in the emergency department. METHODS: Data from 186 patients were retrospectively collected from a medical centre in northern Taiwan. We divided the patients into the advanced-age group (132 patients) and the younger group (54 patients). Associated data collected for statistical analysis included vital signs on arrival, first measured blood glucose level, laboratory results, related comorbidities, length of hospital stay, and survival to discharge. RESULTS: Hypoglycaemia was more frequently observed in women in the advanced-age group than in the younger group. Tachycardia and elevated systolic blood pressure were less predominant in the advanced-age group than younger group. More patients in the advanced-age group had concurrent infection, and more patients in the younger group had liver dysfunction, elevated liver enzymes, liver cirrhosis, and concurrent stroke. CONCLUSIONS: Closer attention should be paid to the possibility of infection in patients of advanced age. Liver disease and stroke need to be ruled out in younger patients.

The Integration of a Three-Dimensional Spheroid Cell Culture Operation in a Circulating Tumor Cell (CTC) Isolation and Purification Process: A Preliminary Study of the Clinical Significance and Prognostic Role of the CTCs Isolated from the Blood Samples of Head and Neck Cancer Patients.

Conventional positive and negative selection-based circulating tumor cell (CTC) isolation methods might generally ignore metastasis-relevant CTCs that underwent epithelial-to- mesenchymal transition and suffer from a low CTC purity problem, respectively. To address these issues, we previously proposed a 2-step CTC isolation method integrating a negative selection CTC isolation and subsequent spheroid cell culture. In addition to its ability to isolate CTCs, more importantly, the spheroid cell culture used could serve as a cell culture model mimicking the process of new tumor tissue formation during cancer metastasis. Therefore, it is promising not only to selectively isolate metastasis-relevant CTCs but also to test the potential of cancer metastasis and thus the prognosis of disease. To explore these issues, experiments were performed. The key findings of this study demonstrated that the method was able to harvest both epithelial (E)- and mesenchymal (M)-type CTCs without selection bias. Moreover, both the M-type CTC count and the information obtained from the multidrug resistance-associated protein 2 (MRP2) and MRP5 gene expression analysis of the CTCs isolated via the 2-step CTC isolation method might be able to serve as prognostic factors for progression-free survival in head and neck squamous cell carcinoma.

An Optically Induced Dielectrophoresis (ODEP)-Based Microfluidic System for the Isolation of High-Purity CD45neg/EpCAMneg Cells from the Blood Samples of Cancer Patients-Demonstration and Initial Exploration of the Clinical Significance of These Cells.

Circulating tumour cells (CTCs) in blood circulation play an important role in cancer metastasis. CTCs are generally defined as the cells in circulating blood expressing the surface antigen EpCAM (epithelial cell adhesion molecule). Nevertheless, CTCs with a highly metastatic nature might undergo an epithelial-to-mesenchymal transition (EMT), after which their EpCAM expression is downregulated. In current CTC-related studies, however, these clinically important CTCs with high relevance to cancer metastasis could be missed due to the use of the conventional CTC isolation methodologies. To precisely explore the clinical significance of these cells (i.e., CD45neg/EpCAMneg cells), the high-purity isolation of these cells from blood samples is required. To achieve this isolation, the integration of fluorescence microscopic imaging and optically induced dielectrophoresis (ODEP)-based cell manipulation in a microfluidic system was proposed. In this study, an ODEP microfluidic system was developed. The optimal ODEP operating conditions and the performance of live CD45neg/EpCAMneg cell isolation were evaluated. The results demonstrated that the proposed system was capable of isolating live CD45neg/EpCAMneg cells with a purity as high as 100%, which is greater than the purity attainable using the existing techniques for similar tasks. As a demonstration case, the cancer-related gene expression of CD45neg/EpCAMneg cells isolated from the blood samples of healthy donors and cancer patients was successfully compared. The initial results indicate that the CD45neg/EpCAMneg nucleated cell population in the blood samples of cancer patients might contain cancer-related cells, particularly EMT-transformed CTCs, as suggested by the high detection rate of vimentin gene expression. Overall, this study presents an ODEP microfluidic system capable of simply and effectively isolating a specific, rare cell species from a cell mixture.