The abnormal fractional amplitude of low-frequency fluctuation changes in patients with diabetic optic neuropathy: a steady-state fMRI study.

The spontaneous changes in brain activity in patients with diabetic optic neuropathy using steady-state fMRI. The fractional amplitude of the low-frequency fluctuation method was applied to evaluate neural activity changes. The Hospital Anxiety and Depression Scale was used to assess the anxiety and depression status of participants. The independent sample t-test and chi-squared test were applied to analyze the demographics of diabetic optic neuropathy patients and healthy controls. Receiver operating characteristic curves were applied to analyze the variation in mean fractional amplitude of low-frequency fluctuation values between diabetic optic neuropathy patients and healthy controls. Pearson's correlation analysis analyzed the relationships between the fractional amplitude of low-frequency fluctuation values of brain regions and clinical behaviors in the diabetic optic neuropathy group. The fractional amplitude of low-frequency fluctuation value of diabetic optic neuropathy patients was significantly higher than healthy controls in the right precentral gyrus. However, the fractional amplitude of low-frequency fluctuation values in the right anterior cingulate gyrus and left middle cingulate gyrus were markedly decreased in diabetic optic neuropathy patients. The area under the curve of receiver operating characteristics for each brain region showed high accuracy. Pearson's correlation analysis showed that fractional amplitude of low-frequency fluctuation values of the right anterior cingulate gyrus and left middle cingulate gyrus was negatively correlated with Hospital Anxiety and Depression Scale scores. The fractional amplitude of low-frequency fluctuation values of the left middle cingulate gyrus was negatively correlated with diabetic optic neuropathy disease duration. In conclusion, we found abnormal spontaneous brain activities in regions related to cognitive and emotional dysfunction, eye movement disorder, and vision loss in patients with diabetic optic neuropathy. These results may indicate the underlying neuropathological mechanisms of diabetic optic neuropathy and show that fractional amplitude of low-frequency fluctuation may be an effective method to distinguish patients with diabetic optic neuropathy from healthy individuals.

Hematopoietic stem/progenitor cell differentiation towards myeloid lineage is modulated by LIGHT/LIGHT receptor signaling.

The cytokine LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) is a member of the tumor necrosis factor (TNF) superfamily. It is expressed primarily on activated T lymphocytes, and detectable on monocytes, granulocytes, and immune dendritic cells. It mainly plays a role in immune regulation including T cell activation and dendritic cell maturation. We recently reported its role as an inducer in embryonic stem cell differentiation, but its role in regulation of adult stem cell has not been defined. In the present study, we examined the expression of LIGHT receptor in Lin- c-kit+ Sca-1+ hematopoietic stem/progenitor cells (HSC/HPCs). We found that HSC express HVEM, a LIGHT receptor, on its surface. We further identified the role of LIGHT in promoting myeloid differentiation of HSCs driven by granulocyte-monocyte colony stimulating factor (GM-CSF). Further studies showed that LIGHT enhances both GM-CSF and GM-CSF receptor (GM-CSFR) expression in HSCs. LIGHT stimulation increases PU.1 expression in HSC/HPCs. In vivo administration of LIGHT increases the colony-forming unit-granulocyte/monocyte (CFU-GM) colony formation and plasma GM-CSF level. Altogether, the data suggest LIGHT promote myeloid differentiation of HSC/HPCs.

Hepatic regenerative potential of mouse bone marrow very small embryonic-like stem cells.

Very small embryonic-like stem cells (VSELs) are a Sca-1 (+) Lin(-) CD45(-) cell population that has been isolated from the bone marrow of mice. The similarities and differences between the mRNA profiles of VSELs and embryonic stem (ES) cells have not yet been defined. Here, we report the whole genome gene expression profile of VSELs and ES cells. We analyzed the global gene expression of VSELs and compared it with ES cells by microarray analysis. We observed that 9,521 genes are expressed in both VSELs and ES cells, 1,159 genes are expressed uniquely in VSELs, and 420 genes are expressed uniquely in ES cells. We found that although VSELs are similar to ES cells in their expression of genes associated with stem cell behavior and pluripotency, there are also differences in their mRNA expression. We further analyzed the expression of stem cell-associated genes in VSELs and ES cells, and found that there were differences in these genes. For instance, the Pkd2 and Yap1 gene were reduced in their expression in VSELs when compared with ES cells. But we also found Zfp54 gene expression was higher in VSELs compared with ES cells. More interestingly, we demonstrated that VSELs express c-kit, the stem cell factor (SCF) receptor. In vitro, SCF promoted VSEL differentiation into hepatic colonies in the presence of hepatocyte growth factor. In vivo, transplantation of VSELs directly into CCl4-induced injured livers significantly reduced serum ALT and AST levels. Therefore, these data suggest that VSELs play a role in the repair of injured livers.

LIGHT regulates the adipogenic differentiation of mesenchymal stem cells.

LIGHT is a cytokine belonging to the TNF family. This cytokine has been extensively defined in its role on T-cell regulation and dendritic cell maturation. It also exhibits the role in liver regeneration. We recently identified its role in regulation of hematopoietic stem cell differentiation. However, the question whether this cytokine regulates mesenchymal stem cells (MSCs) proliferation and/or differentiation remains unknown. In this study, we observed that MSCs express LT-ßR but not HVEM. PCR analysis show LIGHT mRNA is undectable in MSCs. LIGHT did promote neither MSCs proliferation nor migration. However, LIGHT promoted MSCs differentiation into adipocyte which was confirmed by Oil Red O Staining Assay. Since either MSCs or adipocytes are the major cell population in bone marrow niche, we then suggest that LIGHT regulate bone marrow niche, such as MSCs differentiation.

Anticancer effects and mechanisms of astragaloside­IV (Review).

Astragalus membranaceus Bunge is widely used in Traditional Chinese Medicine to treat various cancers. Astragaloside­IV (AS­IV) is one of the major compounds isolated from A. membranaceus Bunge and has been demonstrated to have antitumor effects by inhibiting cell proliferation, invasion and metastasis in various cancer types. Numerous studies have used in vitro cell culture and in vivo animal models of cancer to explore the antitumor activities of AS­IV. In the present study, the antitumor effects and mechanisms of AS­IV reported in studies recorded in the PubMed database were reviewed. First, the antitumor effects of AS­IV on proliferation, cell cycle, apoptosis, autophagy, invasion, migration, metastasis and epithelial­mesenchymal transition processes in cancer cells and the tumor microenvironment, including angiogenesis, tumor immunity and macrophage­related immune responses to cancer cells, were comprehensively discussed. Subsequently, the molecular mechanisms and related signaling pathways associated with antitumor effects of AS­IV as indicated by in vitro and in vivo studies were summarized, including the Wnt/AKT/GSK-3ß (glycogen synthase kinase­3ß)/ß­catenin, TGF­ß/PI3K/AKT/mTOR, PI3K/MAPK/mTOR, PI3K/AKT/NF­κB, Rac family small GTPase 1/RAS/MAPK/ERK, TNF­α/protein kinase C/ERK1/2­NF­κB and Tregs (T­regulatory cells)/IL­11/STAT3 signaling pathways. Of note, several novel mechanisms of Toll­like receptor 4 (TLR4)/NF­κB/STAT3, pSmad3C/3L, nuclear factor erythroid 2­related factor (NrF2)/heme oxygenase 1, circDLST/microRNA­489­3p/eukaryotic translation initiation factor 4A1 and macrophage­related high­mobility group box 1­TLR4 signaling pathways associated with the anticancer activity of AS­IV were also included. Finally, the limitations of current studies that must be addressed in future studies were pointed out to facilitate the establishment of AS­IV as a potent therapeutic drug in cancer treatment.

Identification of circRNA-associated ceRNA networks in peripheral blood mononuclear cells as potential biomarkers for chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD), which is a common respiratory disorder with high morbidity and mortality globally, has a complex pathogenesis that is not fully understood. Some circular RNAs (circRNAs) have been recognized to serve as miRNA sponges for regulating target RNA transcripts during the processes of human diseases. In the present study, we aimed to investigate novel circRNA-associated biomarkers for COPD, 245 differentially expressed circRNAs were identified, including 111 up-regulated and 134 down-regulated circRNAs. These candidate circRNAs were enriched in inflammation-associated pathways (such as mTOR, B-cell receptor, and NF-κB signaling pathways) via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. A combination of two circRNAs (up-regulated hsa_circ_0067209 and down-regulated hsa_circ_0000673) demonstrated good diagnostic value (area under the receiver operating characteristic curve [AUC] = 0.866) for COPD by receiver operating characteristic curve (ROC) analysis and qRT-PCR validation. Subsequently, hsa-miR-8082 and hsa-miR-1248 were identified as targets for hsa_circ_0067209 and hsa_circ_0000673, respectively, via bioinformatics analysis and a dual-luciferase reporter assay, and the combination of these two miRNAs displayed better diagnosis potential for COPD (AUC = 0.967) than each other. Evaluation of COPD-related mRNA profiles revealed that the up-regulated genes ABR and TRPM6 were predicted downstream targets for hsa_circ_0067209/hsa-miR-8082, whereas the down-regulated gene RORC was a predicted downstream target for hsa_circ_0000673/hsa-miR-1248. In summary, hsa_circ_0067209 and hsa_circ_0000673 have potential as novel diagnostic biomarkers of COPD. In addition, competing endogenous RNA networks of hsa_circ_0067209/hsa-miR-8082/ABR/TRPM6 and hsa_circ_0000673/hsa-miR-1248/RORC may play critical regulation roles for COPD pathogenesis.

A direct pancreatic cancer xenograft model as a platform for cancer stem cell therapeutic development.

There is an enormous gap between the antiproliferative and in vivo antitumor efficacy of gemcitabine in cell line-based models and its clinical efficacy. This may be due to insensitiveness of the precursor, cancer stem cell (CSC) compartment to cytotoxic agents. The hedgehog pathway is associated with CSC signaling and control. We used a direct xenograft model of pancreatic cancer and a two-stage approach was used to test the hypotheses that targeting CSC could increase the efficacy of gemcitabine. Tumors from a gemcitabine-sensitive xenograft were treated with gemcitabine first, and randomized, after tumor regression to continuing treatment with gemcitabine, a hedgehog inhibitor alone or in combination with gemcitabine. We tested markers described as associated with CSC such as CD24, CD44, ALDH, nestin, and the hedgehog pathway. After induction with gemcitabine, treated tumor showed an enrichment in CSC markers such as ALDH and CD24. Subsequently, a release from gemcitabine prompted a repopulation of proliferating cells and a decrease in such markers to equilibrate from pretreatment levels. Combined treatment with gemcitabine and cyclopamine induced tumor regression and decrease in CSC markers and hedgehog signaling. Cytoplasmic CD24 and ALDH were inversely and strongly associated with growth and were expressed in a minority of cells that we propose constitute the CSC compartment. Hedgehog inhibitors as part of a dual compartment therapeutic approach were able to further reduce tumor growth and decreased both static and dynamic markers of CSC. Direct tumor xenografts are a valid platform to test multicompartment therapeutic approaches in pancreatic cancer.

The Ape-1/Ref-1 redox antagonist E3330 inhibits the growth of tumor endothelium and endothelial progenitor cells: therapeutic implications in tumor angiogenesis.

The apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape-1/Ref-1) is a multi-functional protein, involved in DNA repair and the activation of redox-sensitive transcription factors. The Ape-1/Ref-1 redox domain acts as a cytoprotective element in normal endothelial cells, mitigating the deleterious effects of apoptotic stimuli through induction of survival signals. We explored the role of the Ape-1/Ref-1 redox domain in the maintenance of tumor-associated endothelium, and of endothelial progenitor cells (EPCs), which contribute to tumor angiogenesis. We demonstrate that E3330, a small molecule inhibitor of the Ape-1/Ref-1 redox domain, blocks the in vitro growth of pancreatic cancer-associated endothelial cells (PCECs) and EPCs, which is recapitulated by stable expression of a dominant-negative redox domain mutant. Further, E3330 blocks the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into CD31(+) endothelial progeny. Exposure of PCECs to E3330 results in a reduction of H-ras expression and intracellular nitric oxide (NO) levels, as well as decreased DNA-binding activity of the hypoxia-inducible transcription factor, HIF-1alpha. E3330 also reduces secreted and intracellular vascular endothelial growth factor expression by pancreatic cancer cells, while concomitantly downregulating the cognate receptor Flk-1/KDR on PCECs. Inhibition of the Ape-1/Ref-1 redox domain with E3330 or comparable angiogenesis inhibitors might be a potent therapeutic strategy in solid tumors.

Small-molecule inhibitor of the AP endonuclease 1/REF-1 E3330 inhibits pancreatic cancer cell growth and migration.

AP endonuclease 1 (APE1; also known as REF-1) contains a DNA repair domain and a redox regulation domain. APE1 is overexpressed in several human cancers, and disruption of APE1 function has detrimental effects on cancer cell viability. However, the selective contribution of the redox and the DNA repair domains to maintenance of cellular homeostasis in cancer has not been elucidated. In the present study, we used E3330, a small-molecule inhibitor of APE1 redox domain function, to interrogate the functional relevance of sustained redox function in pancreatic cancer. We show that E3330 significantly reduces the growth of human pancreatic cancer cells in vitro. This phenomenon was further confirmed by a small interfering RNA experiment to knockdown APE1 expression in pancreatic cancer cells. Further, the growth-inhibitory effects of E3330 are accentuated by hypoxia, and this is accompanied by striking inhibition in the DNA-binding ability of hypoxia-inducible factor-1alpha, a hypoxia-induced transcription factor. E3330 exposure promotes endogenous reactive oxygen species formation in pancreatic cancer cells, and the resulting oxidative stress is associated with higher levels of oxidized, and hence inactive, SHP-2, an essential protein tyrosine phosphatase that promotes cancer cell proliferation in its active state. Finally, E3330 treatment inhibits pancreatic cancer cell migration as assessed by in vitro chemokine assays. E3330 shows anticancer properties at multiple functional levels in pancreatic cancer, such as inhibition of cancer cell growth and migration. Inhibition of the APE1 redox function through pharmacologic means has the potential to become a promising therapeutic strategy in this disease.

Cancer initiating cells or cancer stem cells in the gastrointestinal tract and liver.

It has been suggested that cancer stem cells population within the solid tumor with indefinite proliferation potential drives the growth and metastasis of cancer. In literature, these malignant stem cells also named Cancer initiating cells. Cancer stem cells exhibit low rate of division and proliferation in their niche that help them to avoid chemotherapy and radiation. Epithelial cancers are believed to originate from transformation of tissue stem cells. Bone marrow-derived cells, which are frequently recruited to sites of tissue injury and inflammation, might also represent a potential source of malignancy in the gastrointestinal tract. Pancreatic cancer is one of most common cause of cancer-related death. Pancreatic cancer stem cells have been characterized recently through serial transplantation of human pancreatic cancer cells. The phenotype of Pancreatic cancer stem cells has been defined as CD24(+)CD44(+)CD326 (ESA)(+). CD133 antigen has been also suggested as a potential marker for cancer stem cell in gastrointestinal tract but recently there is also debate in this regard. More recently, other cancer stem cells in gastrointestinal tract, such as colon cancer stem cells, liver cancer stem cells, have been also characterized in their phenotype. These advances clearly will bring the new strategy in cancer treatment and control in the gastrointestinal tract. In this review, the author will discuss the current status and progress about cancer stem cell research in gastrointestinal tract and liver.

TNF family molecule LIGHT regulates chemokine CCL27 expression on mouse embryonic stem cell-derived dendritic cells through NF-kappaB activation.

Cytokine LIGHT is a type II transmembrane protein belonging to the TNF family that was originally identified as a weak inducer of apoptosis. It plays a role in inducing maturation of dendritic cells, such as upregulating CD80, CD86 expression on dendritic cells. However, whether LIGHT induces CC chemokine expression in DC and promotes their migration remains unknown. In this study, we found that esDC express CCR7 and CCR10 (the receptor of CCL27) upon the LIGHT stimulation. LIGHT also upregulates CCL27, but not CCL19 and CCL21 expression in esDC. The esDC migration potential has been increased in LIGHT activated DCs compared with control cells. LIGHT activated DCs autocrine CCL27 which regulate their migration as Blockage of CCL27 on esDC using neutralizing antibody reduces migration potential. In signaling study, we identified that LIGHT activated NF-kappaB in esDC and inhibition of NF-kappaB activation by specific inhibitor can partly attenuate the effect of LIGHT in regulation of CCL27 expression. Moreover, Shp-2 is required in LIGHT activated NF-kappaB because Knockdown of Shp-2 affects the NF-kappaB activation induced by LIGHT and consequently influences LIGHT mediated CCL27 expression. TRAF6 is critical in DC maturation in recent reports; however, knockdown of TRAF6 expression using siRNA did not alter CCL27 expression in LIGHT matured DCs. Our study demonstrates that LIGHT stimulation enhances CCL27 expression through activation of NF-kappaB in DCs.

RNAi knockdown of transcription factor Pu.1 in the differentiation of mouse embryonic stem cells.

Murine embryonic stem (mES) cells are pluripotent cells derived from the inner cell mass of the preimplantation blastocyst. These cells are primitive and undifferentiated and have the potential to become a wide variety of specialized cell types. Mouse ES cells can be regarded as a versatile biological tool that has led to major advances in our understanding of cell and developmental biology. To study specific gene function in early developmental events, gene knockout approaches have been traditionally used, however, this is a time-consuming and expensive approach. Recently, we have shown that small interfering RNA is an effective strategy to knockdown target gene expression, during ES cell differentiation, and consequently, one can alter cell fates in ES-derived differentiated cells. This method will be useful to test the function of a wide variety of gene products using the ES cell differentiation system.

RNAi Technique in Stem Cell Research: Current Status and Future Perspectives.

RNAi is a mechanism displayed by most eukaryotic cells to rid themselves of foreign double-strand RNA molecules. In the 18 years since the initial report, RNAi has now been demonstrated to function in mammalian cells to alter gene expression and has been used as a means for genetic discovery as well as a possible strategy for genetic correction and genetic therapy in cancer and other disease. The aim of this review is to provide a general overview of how RNAi suppresses gene expression and to examine some published RNAi approaches that have resulted in changes in stem cell function and suggest the possible clinical relevance of this work in cancer therapy through targeting cancer stem cells.

RNAi Knockdown of Ape1 Gene in the Differentiation of Mouse Embryonic Stem Cells.

Murine embryonic stem cells (ES) are pluripotent cells and have the potential to become a wide variety of specialized cell types. Mouse ES cell differentiation can be regarded as a valuable biological tool that has led to major advances in our understanding of cell and developmental biology. In vitro differentiation of mouse ES cells can be directed to a specific lineage formation, such as hematopoietic lineage, by appropriate cytokine and/or growth factor stimulation. To study specific gene function in early developmental events, gene knockout approaches have been traditionally used, however, this is a time-consuming and expensive approach. Recently, we have shown that siRNA is an effective strategy to knock down target gene expression, such as Ape1, during ES cell differentiation, and consequently, one can alter cell fates in ES-derived differentiated cells. This approach will be applicable to test the function of a wide variety of gene products using the ES cell differentiation system.

The use of SHP-2 gene transduced bone marrow mesenchymal stem cells to promote osteogenic differentiation and bone defect repair in rat.

Bone tissue engineering is a promising approach for bone regeneration, in which growth factors play an important role. The tyrosine phosphatase Src-homology region 2-containing protein tyrosine phosphatase 2 (SHP2), encoded by the PTPN11 gene, is essential for the differentiation, proliferation and metabolism of osteoblasts. However, SHP-2 has never been systematically studied for its effect in osteogenesis. We predicted that overexpression of SHP-2 could promote bone marrow-derived mesenchymal stem cell (BMSC)osteogenic differentiation and SHP-2 transduced BMSCs could enhance new bone formation, determined using the following study groups: (1) BMSCs transduced with SHP-2 and induced with osteoblast-inducing liquid (BMSCs/SHP-2/OL); (2) BMSCs transduced with SHP-2 (BMSCs/-SHP-2); (3) BMSCs induced with osteoblast-inducing liquid (BMSCs/OL) and (4) pure BMSCs. Cells were assessed for osteogenic differentiation by quantitative real-time polymerase chain reaction analysis, western blot analysis, alkaline phosphatase activity and alizarin red S staining. For in vivo assessment, cells were combined with beta-tricalcium phosphate scaffolds and transplanted into rat calvarial defects for 8 weeks. Following euthanasia, skull samples were explanted for osteogenic evaluation, including micro-computed tomography measurement, histology and immunohistochemistry staining. SHP-2 and upregulation of its gene promoted BMSC osteogenic differentiation and therefore represents a potential new therapeutic approach to bone repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1871-1881, 2016.

Ape1 regulates WNT/ß-catenin signaling through its redox functional domain in pancreatic cancer cells.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape1/Ref-1, Ape1) is a multifunctional protein that is upregulated in human pancreatic cancer. Ape1 redox domain plays an essential role in regulating the effects of reactive oxygen species (ROS) generated during physiological metabolism and pathological stress. In the present study, we explored whether Ape1 and ROS affect WNT/ß-catenin signaling. We used E3330, a small molecule inhibitor of the redox activity of Ape1, and a siRNA approach to knock down Ape1, in two human pancreatic cancer cell lines. Inhibition of Ape1 resulted in growth suppression of pancreatic cancer cells, increased ROS levels, upregulation of ß-catenin and c-myc and downregulation of cyclin D1. Consistent with these data, overexpression of Ape1 in pancreatic cancer cells reduced ROS and c-myc levels and increased cyclin D1 levels. Moreover, treatment of pancreatic cancer cells with H2O2 to induce oxidative stress resulted in upregulated ROS levels, decreased Ape1 at both the mRNA and protein level, and alterations in WNT/ß-catenin pathway components. Finally, treatment of pancreatic cancer cells with the WNT/ß-catenin inhibitor IWR-1 resulted in growth inhibition, which was greatly enhanced when combined with E3330 treatment. In summary, our results demonstrate that ROS is an important intracellular messenger that can modulate WNT/ß­catenin signaling. The present study provides interesting new insight into crosstalk between the redox function of Ape1 and WNT/ß-catenin signaling in cancer cells. Furthermore, our data show that the combination of Ape1 and WNT inhibitors enhanced the inhibition of pancreatic cell proliferation. These results provide a promising novel therapeutic strategy for treating pancreatic cancer in future.

LPS-stimulated inflammatory environment inhibits BMP-2-induced osteoblastic differentiation through crosstalk between TLR4/MyD88/NF-κB and BMP/Smad signaling.

Bone morphogenetic protein-2 (BMP-2) is a novel differentiation factor that is capable of inducing osteoblast differentiation and bone formation, making it an attractive option in treatment of bone defects, fractures, and spine fusions. Inflammation, which was a common situation during bone healing, is recognized to inhibit osteogenic differentiation and bone formation. However, the effect of inflammation on BMP-2-induced osteoblastic differentiation remains ambiguous. In this study, we showed that an inflammatory environment triggered by lipopolysaccharide (LPS) in vitro would suppress BMP-2-induced osteogenic differentiation of bone marrow mesenchymal stem cells, which represented by decreased alkaline phosphatase (ALPase) activity and down-regulated osteogenic genes. In addition, LPS activated nuclear factor-κB (NF-κB) via a TLR4/MyD88-dependent manner and inhibited BMP-2-induced phosphorylation and nuclear translocation of Smad1/5/8. The blocking of NF-κB signaling by pretreatment with specific inhibitors such as BAY-11-7082, TPCK and PDTC, or by transfection with plasmids encoding p65 siRNA or IκBα siRNA could significantly reverse the inhibitory effect of LPS on BMP-2-induced BMP/Smad signaling and osteogenic differentiation. By contrast, even without stimulation of LPS, overexpression of p65 gene showed obvious inhibitory effects on BMP-2-induced BMP/Smad signaling and ALPase activity. These data indicate that the LPS-mediated inflammatory environment inhibits BMP-2-induced osteogenic differentiation, and that the crosstalk between TLR4/MyD88/NF-κB and BMP/Smad signaling negatively modulates the osteoinductive capacity of BMP-2.

Aberrant expression of redox protein Ape1 in colon cancer stem cells.

Ape1 is an important redox protein, essential for specific cytokine-induced signal transduction. Ape1 signaling is also important in regulating the growth of cancer cells, including colon cancer cells. The present study investigated whether Ape1 signaling plays a role in the regulation of colon cancer stem cell (CCSC) growth. The results showed that Ape1 was aberrantly expressed in CCSCs, as determined by quantitative (q)PCR assay. A laser confocal microscopy assay demonstrated that the Ape1 protein was mainly distributed in the nuclei, but not the cytoplasm, of the CSCs. Treatment of CCSCs with Ape1 redox inhibitor (E3330) significantly affected growth in vitro. In colon cancer xenograft mice, in vivo administration of E3330 enhanced tumor responses to the chemotherapeutic drug, 5-fluorouracil (5-FU). Furthermore, the combination of E3330 and 5-FU evidently increased the cytotoxicity of 5-FU in CSC growth. In the qPCR assay, the CCSCs were demonstrated to express the dominant ATP-binding cassette sub-family G member 2 (ABC-G2), but not the multidrug resistance 1, genes. Thus, we hypothesized that drug resistance in CCSCs is mediated by ABC-G2. Since CSCs are involved in cancer metastasis, the Ape1 inhibitor may be a potential agent in the inhibition of colon cancer growth and metastasis.

Characterization of interstitial Cajal progenitors cells and their changes in Hirschsprung's disease.

Interstitial cells of Cajal (ICC) are critical to gastrointestinal motility. The phenotypes of ICC progenitors have been observed in the mouse gut, but whether they exist in the human colon and what abnormal changes in their quantity and ultrastructure are present in Hirschsprung's disease (HSCR) colon remains uncertain. In this study, we collected the surgical resection of colons, both proximal and narrow segments, from HSCR patients and normal controls. First, we identified the progenitor of ICC in normal adult colon using immunofluorescent localization techniques with laser confocal microscopy. Next, the progenitors were sorted to observe their morphology. We further applied flow cytometry to examine the content of ICC progenitors in these fresh samples. The ultrastructural changes in the narrow and proximal parts of the HSCR colon were observed using transmission electron microscopy (TEM) and were compared with the normal adult colon. The presumed early progenitor (c-Kit(low)CD34(+)Igf1r(+)) and committed progenitor (c-Kit(+)CD34(+)Igf1r(+)) of ICC exist in adult normal colon as well as in the narrow and proximal parts of the HSCR colon. However, the proportions of mature, early and committed progenitors of ICC were dramatically reduced in the narrow segment of the HSCR colon. The proportions of mature and committed progenitors of ICC in the proximal segment of the HSCR colon were lower than in the adult normal colon. Ultrastructurally, ICC, enteric nerves, and smooth muscle in the narrow segment of the HSCR colon showed severe injury, including swollen vacuola or ted mitochondria, disappearance of mitochondrial cristae, dilated rough endoplasmic reticulum, vesiculation and degranulation, and disappearance of the caveolae on the ICC membrane surface. The contents of ICC and its progenitors in the narrow part of the HSCR colon were significantly decreased than those of adult colon, which may be associated with HSCR pathogenesis.

Elevated bile acids in newborns with Biliary Atresia (BA).

Biliary Atresia (BA), a result from inflammatory destruction of the intrahepatic and extrahepatic bile ducts, is a severe hepatobiliary disorder unique to infancy. Early diagnosis and Kasai operation greatly improve the outcome of BA patients, which encourages the development of early screening methods. Using HPLC coupled tandem mass spectrometry, we detected primary bile acids content in dried blood spots obtained from 8 BA infants, 17 neonatal jaundice and 292 comparison infants at 3-4 days of life. Taurocholate (TC) was significantly elevated in biliary atresia infants (0.98 ± 0.62 µmol/L) compared to neonatal jaundice (0.47 ± 0.30 µmol/L) and comparison infants (0.43 ± 0.40 µmol/L), with p=0.0231 and p=0.0016 respectively. The area under receiver operating characteristic (ROC) curve for TC to discriminate BA and comparison infants was 0.82 (95% confidence interval: 0.72-0.92). A cutoff of 0.63 µmol/L produced a sensitivity of 79.1% and specificity of 62.5%. The concentrations of total bile acids were also raised significantly in BA compared to comparison infants (6.62 ± 3.89 µmol/L vs 3.81 ± 3.06 µmol/L, p=0.0162), with the area under ROC curve of 0.75 (95% confidence interval: 0.61-0.89). No significant difference was found between the bile acids of neonatal jaundice and that of comparison infants. The early increase of bile acids indicates the presentation of BA in the immediate newborn period and the possibility of TC as newborn screening marker.