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1.  IL1RAP as a surface marker for leukemia stem cells is related to clinical phase of chronic myeloid leukemia patients 
Chronic myeloid leukemia (CML) is a clonal disease from hematopoietic stem cells. Surviving leukemia stem cells (LSCs) and progenitor cells are a potential source for CML relapse and progression. Recent data reported that IL-1 receptor accessory protein (IL1RAP) gene was differentially expressed in CML versus normal stem and progenitor cells. However, whether the level of IL1RAP is associated with clinical phases of CML, and correlations between IL1RAP expression and detections of diagnosis is still unclear. Here we demonstrated that IL1RAP was up-regulated in CD34+ and CD34+CD38- cells which highly enriched with stem cells. Furthermore, IL1RAP expression in CD34+CD38- cells was tightly consistent with the generation of BCR-ABL fusion gene and Philadelphia chromosome. Importantly, we found that the level of IL1RAP increased with disease progression from chronic phase (CP) into accelerated phase (AP) and blast phase (BP), which was investigated not only in new diagnosed CML patients but also in patients treated with tyrosine kinase inhibitors (TKI) and hydroxyurea. Negative correlation was detected between IL1RAP expression and neutrophil (NE), whereas no relation was found in white blood cell (WBC), lymphocyte (LY), red blood cell (RBC), platelet (PLT), age or gender of CML patients. In conclusion, we identified IL1RAP as a surface marker of LSCs may be a potential indicator for CML clinical phases.
PMCID: PMC4307422  PMID: 25663975
Chronic myeloid leukemia; leukemia stem cells; IL-1 receptor accessory protein
2.  Absence of Gamma-Interferon-Inducible Lysosomal Thiol Reductase (GILT) Is Associated with Poor Disease-Free Survival in Breast Cancer Patients 
PLoS ONE  2014;9(10):e109449.
Tumor immunosurveillance is known to be of critical importance in controlling tumorigenesis and progression in various cancers. The role of gamma-interferon-inducible lysosomal thiol reductase (GILT) in tumor immunosurveillance has recently been studied in several malignant diseases, but its role in breast cancer remains to be elucidated. In the present study, we found GILT as a significant different expressed gene by cDNA microarray analysis. To further determine the role of GILT in breast cancer, we examined GILT expression in breast cancers as well as noncancerous breast tissues by immunohistochemistry and real-time PCR, and assessed its association with clinicopathologic characteristics and patient outcome. The absence of GILT expression increased significantly from 2.02% (2/99) in noncancerous breast tissues to 15.6% (34/218) in breast cancer tissues (P<0.001). In accordance with its proliferation inhibiting function, GILT expression was inversely correlated with Ki67 index (P<0.05). In addition, absence of GILT was positively correlated with adverse characteristics of breast cancers, such as histological type, tumor size, lymph nodes status, and pTNM stage (P<0.05). Consistently, breast cancers with reduced GILT expression had poorer disease-free survival (P<0.005). Moreover, significantly decreased expression of GILT was found in both primary and metastatic breast cancer cells, in contrast to normal epithelial cells. These findings indicate that GILT may act as a tumor suppressor in breast cancer, in line with its previously suggested role in anti-tumor immunity. Thus, GILT has the potential to be a novel independent prognostic factor in breast cancer and further studies are needed to illustrate the underlying mechanism of this relationship.
doi:10.1371/journal.pone.0109449
PMCID: PMC4204821  PMID: 25333930
3.  Sensitization of neurons in the central nucleus of the amygdala via the decreased GABAergic inhibition contributes to the development of neuropathic pain-related anxiety-like behaviors in rats 
Molecular Brain  2014;7(1):72.
Background
Despite high prevalence of anxiety accompanying with chronic pain, the mechanisms underlying pain-related anxiety are largely unknown. With its well-documented role in pain and emotion processing, the amygdala may act as a key player in pathogenesis of neuropathic pain-related anxiety. Pain-related plasticity and sensitization of CeA (central nucleus of the amygdala) neurons have been shown in several models of chronic pain. In addition, firing pattern of neurons with spike output can powerfully affect functional output of the brain nucleus, and GABAergic neurons are crucial in the modulation of neuronal excitability. In this study, we first investigated whether pain-related plasticity (e.g. alteration of neuronal firing patterns) and sensitization of CeA neurons contribute to nerve injury-evoked anxiety in neuropathic rats. Furthermore, we explored whether GABAergic disinhibition is responsible for regulating firing patterns and intrinsic excitabilities of CeA neurons as well as for pain-related anxiety in neuropathic rats.
Results
We discovered that spinal nerve ligation (SNL) produced neuropathic pain-related anxiety-like behaviors in rats, which could be specifically inhibited by intra-CeA administration of anti-anxiety drug diazepam. Moreover, we found potentiated plasticity and sensitization of CeA neurons in SNL-induced anxiety rats, of which including: 1) increased burst firing pattern and early-adapting firing pattern; 2) increased spike frequency and intrinsic excitability; 3) increased amplitude of both after-depolarized-potential (ADP) and sub-threshold membrane potential oscillation. In addition, we observed a remarkable reduction of GABAergic inhibition in CeA neurons in SNL-induced anxiety rats, which was proved to be important for altered firing patterns and hyperexcitability of CeA neurons, thereby greatly contributing to the development of neuropathic pain-related anxiety. Accordantly, activation of GABAergic inhibition by intra-CeA administration of muscimol, a selective GABAA receptors agonist, could inhibit SNL-induced anxiety-like behaviors in neuropathic rats. By contrast, suppression of GABAergic inhibition by intra-CeA administration of bicuculline, a selective GABAA receptors antagonist, produced anxiety-like behavior in normal rats.
Conclusions
This study suggests that reduction of GABAergic inhibition may be responsible for potentiated plasticity and sensitization of CeA neurons, which likely underlie the enhanced output of amygdala and neuropathic pain-related anxiety in SNL rats.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-014-0072-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-014-0072-z
PMCID: PMC4201706  PMID: 25277376
Anxiety; Neuropathic pain; Firing pattern; CeA; GABA
4.  Exercise Preconditioning Protects against Spinal Cord Injury in Rats by Upregulating Neuronal and Astroglial Heat Shock Protein 72 
The heat shock protein 72 (HSP 72) is a universal marker of stress protein whose expression can be induced by physical exercise. Here we report that, in a localized model of spinal cord injury (SCI), exercised rats (given pre-SCI exercise) had significantly higher levels of neuronal and astroglial HSP 72, a lower functional deficit, fewer spinal cord contusions, and fewer apoptotic cells than did non-exercised rats. pSUPER plasmid expressing HSP 72 small interfering RNA (SiRNA-HSP 72) was injected into the injured spinal cords. In addition to reducing neuronal and astroglial HSP 72, the (SiRNA-HSP 72) significantly attenuated the beneficial effects of exercise preconditioning in reducing functional deficits as well as spinal cord contusion and apoptosis. Because exercise preconditioning induces increased neuronal and astroglial levels of HSP 72 in the gray matter of normal spinal cord tissue, exercise preconditioning promoted functional recovery in rats after SCI by upregulating neuronal and astroglial HSP 72 in the gray matter of the injured spinal cord. We reveal an important function of neuronal and astroglial HSP 72 in protecting neuronal and astroglial apoptosis in the injured spinal cord. We conclude that HSP 72-mediated exercise preconditioning is a promising strategy for facilitating functional recovery from SCI.
doi:10.3390/ijms151019018
PMCID: PMC4227258  PMID: 25334068
spinal cord compression; neurological severity score; heat shock protein 72; exercise preconditioning
5.  Stromal responses among common carcinomas correlated with clinicopathologic features 
Purpose
We have previously characterized a tumor stroma expression signature in a subset of breast tumors that correlates with better clinical outcome. The purpose of this study is to determine whether this stromal signature, termed the ‘DTF fibroblast’ signature, is specific to breast cancer or is a common stromal response found in different types of cancer.
Experimental Designs
The DTF fibroblast signature was applied to gene expression profiles from five ovarian, five lung, two colon and three prostate cancer expression microarray datasets. Additionally, two different tissue microarrays of 204 ovarian tumors and 140 colon tumors were examined for the expression of previously characterized protein markers of DTF fibroblast signature. The DTF fibroblast stromal response was then correlated with clinicopathologic features.
Results
The DTF fibroblast signature is robustly present in ovarian, lung, and colon carcinomas. Both expression microarray data and immunohistochemistry show that the subset of ovarian tumors with strong DTF fibroblast signature expression has statistically significant worse survival outcomes. No reproducible survival differences were found in either the lung or the colon cancers. The prostate cancers failed to demonstrate a DTF fibroblast signature. Multivariant analysis showed that DTF fibroblast signature was significantly more prognostic than the proliferation status in ovarian carcinomas.
Conclusion
Our results suggest that the DTF fibroblast signature is a common tumor stroma signature in different types of cancer including ovarian, lung and colon carcinomas. Our findings provide further insight into the DTF fibroblast stromal responses across different types of carcinomas and their potential as prognostic and therapeutic targets.
doi:10.1158/1078-0432.CCR-12-3127
PMCID: PMC3778157  PMID: 23804424
6.  In vivo ossification of a scaffold combining β-tricalcium phosphate and platelet-rich plasma 
Tricalcium phosphate (TCP) and platelet-rich plasma (PRP) are commonly used in bone tissue engineering. The aim of the present study was to investigate a composite that combined TCP with PRP and assess its effectiveness in the treatment of bone defects. Cavity-shaped bone defects were established on the tibiae of 27 beagle dogs, and were repaired by pure β-TCP with bone marrow stromal cells (BMSCs), β-TCP/PRP with BMSCs and autogenic ilium. The samples were harvested at 4, 8 and 12 weeks, and bone regeneration was evaluated using X-ray radiography, immunocytochemical staining of osteocalcin (OCN), hematoxylin and eosin staining and reverse transcription-polymerase chain reaction analyses. Biomechanical tests of the scaffolds were performed at the 12th week after scaffold implantation. When using pure β-TCP as a scaffold, the scaffold-bone interface was clear and no material adsorption and bone healing was observed. Substantial bone regeneration was observed when the tibial defects were restored using β-TCP/PRP and autogenic ilium. Furthermore, the mRNA expression levels of OCN, alkaline phosphatase and collagen type I α1 were significantly higher in the animals with β-TCP/PRP scaffolds at 8 and 12 weeks following implantation compared with those in the animals with the pure β-TCP scaffolds. The maximum load and compressive strength of the β-TCP/PRP scaffolds were similar to those of the autogenic ilium; however, they were significantly higher than those of the pure β-TCP scaffold. Thus, the β-TCP/PRP composite may be used as a potential scaffold to carry in vitro cultured BMSCs to treat bone defects.
doi:10.3892/etm.2014.1969
PMCID: PMC4186334  PMID: 25289027
tricalcium phosphate; platelet-rich plasma; tissue engineering; bone defect; bone marrow stromal cells
7.  Passive Smoking Exposure from Partners as a Risk Factor for ER+/PR+ Double Positive Breast Cancer in Never-Smoking Chinese Urban Women: A Hospital-Based Matched Case Control Study 
PLoS ONE  2014;9(5):e97498.
Background
The relationship between passive smoking exposure (PSE) and breast cancer risk is of major interest.
Objective
To evaluate the relationship between PSE from partners and breast cancer risk stratified by hormone-receptor (HR) status in Chinese urban women population.
Design
Hospital-based matched case control study.
Setting
Chinese urban breast cancer patients without current or previous active smoking history in China Medical University 1st Hospital, Liaoning Province, China between Jan 2009 and Nov 2009.
Patients
Each breast cancer patient was matched 1∶1 with healthy controls by gender and age (±2 years) from the same hospital.
Measurements
The authors used unconditional logistic regression analyses to estimate odds ratio for women with PSE from partners and breast cancer risk.
Results
312 pairs were included in the study. Women who endured PSE had significantly increased risk of breast cancer (adjusted OR: 1.46; 95% CI: 1.05–2.03; P = 0.027), comparing with unexposed women. Women who exposed to >5 cigarettes/day also had significant increased risk (adjusted OR: 1.99; 95% CI: 1.28–3.10; P = 0.002), as were women exposed to passive smoke for 16–25 years (adjusted OR: 1.87 95% CI: 1.22–2.86; P = 0.004), and those exposed to > 4 pack-years (adjusted OR: 1.71 95% CI: 1.17–2.50; P = 0.004). Similar trends were significant for estrogen receptor (ER)/progesterone receptor (PR) double positive subgroup(adjusted OR: 1.71; 2.20; 1.99; 1.92, respectively), but not for ER+/PR−, ER−/PR+, or ER−/PR− subgroups.
Limitations
limitations of the hospital-based retrospective study, lack of information on entire lifetime PSE and low statistical power.
Conclusions
Our findings provide further evidence that PSE from partners contributes to increased risk of breast cancer, especially for ER/PR double positive breast cancer, in Chinese urban women.
doi:10.1371/journal.pone.0097498
PMCID: PMC4035255  PMID: 24866166
8.  Strong anisotropic lifetime orientation distributions of a two-level quantum emitter around a plasmonic nanorod 
Nanoscale Research Letters  2014;9(1):194.
Spontaneous emission lifetime orientation distributions of a two-level quantum emitter in metallic nanorod structures are theoretically investigated by the rigorous electromagnetic Green function method. It was found that spontaneous emission lifetime strongly depended on the transition dipole orientation and the position of the emitter. The anisotropic factor defined as the ratio between the maximum and minimum values of the lifetimes along different dipole orientations can reach up to 103. It is much larger than those in dielectric structures which are only several times usually. Our results show that the localized plasmonic resonance effect provides a new degree of freedom to effectively control spontaneous emission by the dipole orientation of the quantum emitters.
doi:10.1186/1556-276X-9-194
PMCID: PMC4006315  PMID: 24808801
Surface plasmons; Spontaneous emission; Lifetime distribution; Nanorod; 78.67.Qa; 73.20.Mf; 42.50.-p
9.  Acquisition of Tense Marking in English-Speaking Children with Cochlear Implants: A Longitudinal Study 
This study investigated the development of tense markers (e.g., past tense –ed) in children with cochlear implants (CIs) over a 3-year span. Nine children who received CIs before 30 months of age participated in this study at three, four, and five years postimplantation. Nine typical 3-, 4-, and 5-year- olds served as control groups. All children participated in a story-retell task. Percent correct of tense marking in the task was computed. Within the groups, percent correct of tense marking changed significantly in children with CIs and in typical children who had more hearing experience. Across the groups, children with CIs were significantly less accurate in tense marking than typical children at four and five years postimplantation. In addition, the performance of tense marking in children with CIs was correlated with their speech perception skills at earlier time points. Errors of tense marking tended to be omission rather than commission errors in typical children as well as in children with CIs. The findings suggested that despite the perceptual and processing constraints, children who received CIs may learn tense marking albeit with a delayed pattern.
doi:10.1093/deafed/ens069
PMCID: PMC3697805  PMID: 23288713
10.  Gene expression changes in adipose tissue with diet- and/or exercise-induced weight loss 
Adipose tissue plays a role in obesity-related cancers via increased production of inflammatory factors, steroid hormones, and altered adipokines. The impact of weight loss on adipose-tissue gene expression may provide insights into pathways linking obesity with cancer risk. We conducted an ancillary study within a randomized trial of diet, exercise, or combined diet+exercise vs. control among overweight/obese postmenopausal women. In 45 women, subcutaneous adipose-tissue biopsies were performed at baseline and after 6 months and changes in adipose-tissue gene expression were determined by microarray with an emphasis on pre-specified candidate pathways, as well as by unsupervised clustering of >37,000 transcripts (Illumina). Analyses were conducted first by randomization group, and then by degree of weight change at 6-months in all women combined. At 6 months, diet, exercise and diet+exercise participants lost a mean of 8.8 kg, 2.5 kg, and 7.9 kg (all p<0.05 vs. no change in controls). There was no significant change in candidate-gene expression by intervention group. In analysis by weight-change category, greater weight loss was associated a decrease in 17β-hydroxysteroid dehydrogenase-1 (HSD17B1, p-trend<0.01) and leptin (LEP, p-trend<0.01) expression, and marginally significant increased expression of estrogen receptor-1 (ESR1, p-trend=0.08) and insulin-like growth factor binding protein-3 (IGFBP3, p-trend=0.08). Unsupervised clustering revealed 83 transcripts with statistically significant changes. Multiple gene-expression changes correlated with changes in associated serum biomarkers. Weight-loss was associated with changes in adipose-tissue gene expression after 6 months, particularly in two pathways postulated to link obesity and cancer, i.e., steroid-hormone metabolism and IGF signaling.
doi:10.1158/1940-6207.CAPR-12-0212
PMCID: PMC3738189  PMID: 23341572
Adiposity; gene expression; obesity; weight-loss; exercise; diet; leptin; sex hormones; inflammation; adipokines; human; randomized-controlled trial
11.  Consequences, measurement, and evaluation of the costs associated with adverse drug reactions among hospitalized patients in China 
Background
Adverse drug reactions (ADRs) are a leading cause of morbidity in developed countries and represent a substantial burden on health-care resources. Many countries spent 15% to 20% of their hospital budgets to treat drug complications. However, few studies have measured the pharmacoeconomic effects of ADRs on hospitalized patients in China. The study estimates the costs of ADRs as identified from the spontaneous voluntary reports completed from healthcare professionals. To do so, we calculate these costs, determine the sum of Medicare payments and their proportion of total healthcare spending, and evaluate the incidence of ADRs, characteristics of hospitalized ADR patients, and outcomes of ADRs in China.
Methods
This retrospective survey studied patients who experienced ADRs during their hospitalization at a Chinese tertiary-care teaching hospital. The patients were divided into group A and group B according to general ADRs and serious ADRs in Provisions for Adverse Drug Reaction Monitoring and Reporting. The direct costs included treatment fees, inspection fees, laboratory fees, materials fees, bed charges, drug charges, nursing care, meals, and other expenses and the sunk-cost losses were calculated according to the hospital information system (HIS). Indirect costs of ADR treatment were calculated according to the human capital approach. The epidemiological characteristics of ADRs were evaluated.
Results
2739 were diagnosed with ADR during the study period, which translates to an ADR rate of 0.81%. The total socioeconomic loss from 2739 cases of ADR was estimated at ¥817401.69, consisting of direct costs of ¥603252.81 and indirect costs of ¥214148.88. On average, the costs per patient amounted to ¥196.10 in group A, ¥7032.29 in group B. The sum of medicare payment and proportion were ¥219061.13 (65.23%) and ¥105422.02 (39.42%) in group A and B. The ADR incidence in old-age patients was significantly higher than in other age groups (P < 0.0001). The most common drug class associated with ADRs represented antibiotics (957 patients, 34.94%).
Conclusions
The costs of especially severe ADRs could not be ignored, and in this hospital 0.13% of patients were diagnosed with ADRs associated with relatively higher direct costs than who suffered from mild ADRs, largely due to extended hospitalization.
doi:10.1186/1472-6963-14-73
PMCID: PMC3931293  PMID: 24533894
Adverse drug reaction; Direct medical cost; Hospitalized patients; Tertiary care teaching hospital; Pharmacoeconomic
12.  Consonant Development in Pediatric Cochlear Implant Users Who Were Implanted Before 30 Months of Age 
This study provided a yearly record of consonant development for the initial 4 years of cochlear implant (CI) use and established a precedent for using a standardized articulation test, the Goldman–Fristoe Test of Articulation—2 (Goldman, R., & Fristoe, M. [2000]. Goldman–Fristoe Test of Articulation—2. Circle Pines, MN: American Guidance Services). The study used CI age as a referent for 32 children who received their CI before 30 months of age. Consonants produced by 70% of the children were listed, as were the most common error types, which were consonant omissions and substitutions. Using consonant repertoire lists and standard scores, the study revealed that children with CIs had acquisition patterns that were similar to their peers when the duration of CI experience was similar to the chronological age norms of typically developing children. The results revealed that CI users need time to coordinate their articulatory organizing principles with the input they receive from their CI. It is appropriate to use length of CI use as a proxy for chronological age during the first 4 years when comparing articulation development with hearing peers.
doi:10.1093/deafed/ens038
PMCID: PMC3521776  PMID: 23143855
13.  Acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress 
Cancer & Metabolism  2013;1:23.
Background
A variety of oncogenic and environmental factors alter tumor metabolism to serve the distinct cellular biosynthetic and bioenergetic needs present during oncogenesis. Extracellular acidosis is a common microenvironmental stress in solid tumors, but little is known about its metabolic influence, particularly when present in the absence of hypoxia. In order to characterize the extent of tumor cell metabolic adaptations to acidosis, we employed stable isotope tracers to examine how acidosis impacts glucose, glutamine, and palmitate metabolism in breast cancer cells exposed to extracellular acidosis.
Results
Acidosis increased both glutaminolysis and fatty acid β-oxidation, which contribute metabolic intermediates to drive the tricarboxylic acid cycle (TCA cycle) and ATP generation. Acidosis also led to a decoupling of glutaminolysis and novel glutathione (GSH) synthesis by repressing GCLC/GCLM expression. We further found that acidosis redirects glucose away from lactate production and towards the oxidative branch of the pentose phosphate pathway (PPP). These changes all serve to increase nicotinamide adenine dinucleotide phosphate (NADPH) production and counter the increase in reactive oxygen species (ROS) present under acidosis. The reduced novel GSH synthesis under acidosis may explain the increased demand for NADPH to recycle existing pools of GSH. Interestingly, acidosis also disconnected novel ribose synthesis from the oxidative PPP, seemingly to reroute PPP metabolites to the TCA cycle. Finally, we found that acidosis activates p53, which contributes to both the enhanced PPP and increased glutaminolysis, at least in part, through the induction of G6PD and GLS2 genes.
Conclusions
Acidosis alters the cellular metabolism of several major metabolites, which induces a significant degree of metabolic inflexibility. Cells exposed to acidosis largely rely upon mitochondrial metabolism for energy generation to the extent that metabolic intermediates are redirected away from several other critical metabolic processes, including ribose and glutathione synthesis. These alterations lead to both a decrease in cellular proliferation and increased sensitivity to ROS. Collectively, these data reveal a role for p53 in cellular metabolic reprogramming under acidosis, in order to permit increased bioenergetic capacity and ROS neutralization. Understanding the metabolic adaptations that cancer cells make under acidosis may present opportunities to generate anti-tumor therapeutic agents that are more tumor-specific.
doi:10.1186/2049-3002-1-23
PMCID: PMC4178214  PMID: 24359630
14.  Characterizing and quantifying leukocyte populations in human adipose tissue: impact of enzymatic tissue processing 
Journal of immunological methods  2012;386(1-2):50-59.
Adipose tissue inflammation is a major mechanistic link between obesity and chronic disease. To isolate and characterize specific leukocyte populations, e.g. by flow cytometry, tissue needs to be processed to digest the extracellular matrix. We have systematically compared the impact of different commonly used collagenase preparations, digestion times, and normalization strategies on the reproducibility of flow cytometric phenotyping of adipose tissue leukocyte populations. Subcutaneous adipose tissue was obtained from 11 anonymous donors undergoing elective procedures at a plastic surgery clinic in Seattle, WA. We found that collagenase alone consistently produced better cell yields (p=0.007) than when combined with additional proteases such as the commercially available liberases. Moreover, liberase significantly degraded the cell surface expression of CD4 (p<0.001) on T cells and to a lesser extent CD16 (p=0.058) on neutrophils. Extension of the digestion interval from 30 to 120 min did not significantly impact cell viability (p=0.319) or yield (p=0.247). Normalization by either ‘live-gate’, or percentage of CD45pos leukocytes exhibited the lowest coefficient of variation for tissue digests between 60 and 75 min, compared to normalization per gram of tissue, which consistently exhibited the greatest variability. Our data suggest that digestion of adipose tissue using pure collagenase for 60 to 75 min provides the best cell yield and viability, with minimal degradation of cell surface markers used to identify immune cell subpopulations, and best reproducibility independent of the normalization strategy.
doi:10.1016/j.jim.2012.08.018
PMCID: PMC3478958  PMID: 22974837
adipose tissue; leukocytes; enzymatic digestion; normalization
15.  Involvement of the Serine Protease Inhibitor, SERPINE2, and the Urokinase Plasminogen Activator in Cumulus Expansion and Oocyte Maturation 
PLoS ONE  2013;8(8):e74602.
The serpin peptidase inhibitor, clade E, member 2 (SERPINE2) inhibits urokinase-type plasminogen activator (PLAU) and tissue-type plasminogen activator. Higher SERPINE2 expression levels were detected in cumulus cells of human immature oocytes than in those of mature oocytes. The objective of this study was to evaluate whether high SERPINE2 levels in cumulus cells are associated with oocyte immaturity. Using the mouse cumulus–oocyte complex as an experimental model, the effects of elimination and overexpression of SERPINE2 in cumulus cells on cumulus expansion and oocyte maturation were assayed by in vitro maturation. Serpine2 and PLAU transcripts were the most highly expressed serpins and plasminogen activators, respectively. Their expression was coordinately regulated in cumulus cells during gonadotropin-induced oocyte maturation. Silencing of Serpine2 expression using small interfering RNAs or blockage of SERPINE2 protein using a specific antibody had no effect on oocyte maturation. However, overexpression of Serpine2 or exogenous supplementation with high levels of SERPINE2 impaired cumulus expansion and oocyte maturation, probably by decreasing hyaluronan synthase 2 (Has2) and versican (Vcan) mRNA expression. Amiloride, a specific PLAU inhibitor, also suppressed these processes. PLAU supplementation of the oocyte in vitro maturation medium caused earlier and more extensive expansion of cumulus cells and oocyte maturation that may be mediated by increased Has2 mRNA expression. However, these effects were neutralized by coincubation with SERPINE2 or amiloride and PLAU. In conclusion, SERPINE2 and PLAU are involved in cumulus expansion and oocyte maturation. High SERPINE2 levels impair these processes, probably by decreasing cumulus matrix gene expression as well as reducing cumulus hyaluronan contents and inhibiting PLAU activity. These findings may explain why cumulus cells surrounding immature human oocytes express high SERPINE2 levels.
doi:10.1371/journal.pone.0074602
PMCID: PMC3758271  PMID: 24023701
16.  Nicotine Promotes Mammary Tumor Migration via A Signaling Cascade Involving PKC and cdc42 
Cancer research  2008;68(20):8473-8481.
Nicotine, one of major components in tobacco, has been shown to be at high concentrations in the blood stream of cigarette smokers. However, the mechanisms of how nicotine affects tumor development and whether nicotine is a potential carcinogen for malignancies induced by second-hand smoking are not fully understood yet. Here, we investigate the signaling pathways by which nicotine potentiates tumorigenesis in human mammary epithelial-like MCF10A or cancerous MCF7 cells. We demonstrate that human MCF10A and MCF7 cells both express four subunits of nAChR. The treatment of these cells with nicotine enhances the activity of protein kinase C (PKC) α without altering the expression level of this kinase. Nicotine also stimulates [3H]thymidine incorporation into the genome of these cells as well as forces serum-starved cells to enter S phase of the cell cycle, resulting of growth promotion. Importantly, upon nicotine treatment, the mobility of MCF10A and MCF7 cells are enhanced, which can be blocked by the addition of nAChR or PKC inhibitor. Experiments using siRNA knockdown or ectopic expression of cdc42 demonstrated that cdc42 functions as a downstream effector of PKC and is crucial in the regulation of nicotine-mediated migratory activity in the cells. Together, our findings suggest that nicotine, through interacting with its receptor, initiates a signaling cascade that involves PKC and cdc42, and consequently promotes migration in mammary epithelial or tumor cells.
doi:10.1158/0008-5472.CAN-08-0131
PMCID: PMC3698481  PMID: 18922921
17.  A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro 
The epidermal growth factor receptor (EGFR) serves an important function in the proliferation of tumors in humans and is an effective target for the treatment of cancer. In this paper, we studied the targeting characteristics of small peptides (AEYLR, EYINQ, and PDYQQD) that were derived from three major autophosphorylation sites of the EGFR C-terminus domain in vitro. These small peptides were labeled with fluorescein isothiocyanate (FITC) and used the peptide LARLLT as a positive control, which bound to putative EGFR selected from a virtual peptide library by computer-aided design, and the independent peptide RALEL as a negative control. Analyses with flow cytometry and an internalization assay using NCI-H1299 and K562 with high EGFR and no EGFR expression, respectively, indicated that FITC-AEYLR had high EGFR targeting activity. Biotin-AEYLR that was specifically bound to human EGFR proteins demonstrated a high affinity for human non-small-cell lung tumors. We found that AEYLR peptide-conjugated, nanostructured lipid carriers enhanced specific cellular uptake in vitro during a process that was apparently mediated by tumor cells with high-expression EGFR. Analysis of the MTT assay indicated that the AEYLR peptide did not significantly stimulate or inhibit the growth activity of the cells. These findings suggest that, when mediated by EGFR, AEYLR may be a potentially safe and efficient delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy.
doi:10.2147/IJN.S43627
PMCID: PMC3632632  PMID: 23626467
EGFR; small peptide; tumor targeting; lung cancer; NLC
18.  Dual control of Shuanghuang Shengbai granule on upstream and downstream signal modulators of CyclinD-CDK4/6 signaling pathway of cell cycle in Lewis-bearing mice with cyclophosphamide-induced myelosuppression 
OncoTargets and therapy  2013;6:199-209.
Background
This study investigated the dual control mechanism of the Shuanghuang Shengbai granule in modulating the cell cycle in Lewis-bearing mice with cyclophosphamide induced myelosuppression.
Methods
Thirty Lewis-bearing mice were randomly grouped into an untreated group, control group, and treated group. Both treated and untreated groups were intraperitoneally injected with cyclophosphamide to produce a myelosuppression model. Mice in the treated group were fed with the Shuanghuang Shengbai granule (40 g/day) for 6 consecutive days. Standard blood tests and the count of bone marrow nuclear cells were performed, and the cell reproductive cycles of bone marrow and tumors were measured in these mice. In addition, the western blot approach was used to measure the upstream activating signals of CyclinD-CDK4/6 such as c-Myc and CDC25A, the upstream suppression signals such as p16INK4a, and the expression of downstream activated signals such as Rb, pRB, and E2F. All of the tested results were validated by reverse transcription quantitative real-time polymerase chain reaction.
Results
The results showed that the Shuanghuang Shengbai granule could elevate the count of leukocyte and bone marrow nuclear cells of Lewis-bearing mice with cyclophosphamide induced myelosuppression. It could also stimulate bone marrow cells to move from G0/G1 phases to S phase, accelerating the progress of the cell reproductive cycle and increasing the cell proliferation index. Simultaneously, the Shuanghuang Shengbai granule could also suppress cancer cells moving from G0/G1 phase to S phase, reducing the proliferation index. The tumor weight of Lewis-bearing mice in the treated group was much less than those of the control group. Expression levels of c-Myc, CDC25A, Rb, pRb, and E2F of bone marrow in Shuanghuang Shengbai granule-treated mice was higher compared to the control group, whereas they were lower in the cancer cells.
Conclusion
The experimental results demonstrate that the Shuanghuang Shengbai granule has dual control on the cell reproductive cycles in cancer cells and bone marrow nuclear cells in Lewis-bearing mice.
doi:10.2147/OTT.S37407
PMCID: PMC3615883  PMID: 23569384
Shuanghuang Shengbai granule; CyclinD-CDK4/6; c-Myc; Rb; pRb; E2F
19.  Prone breast forward intensity-modulated radiotherapy for Asian women with early left breast cancer: factors for cardiac sparing and clinical outcomes 
Journal of Radiation Research  2013;54(5):899-908.
Since December 2009, after breast-conserving surgery for Stage 0–I cancer of the left breast, 21 women with relatively pendulous breasts underwent computed tomography prone and supine simulations. The adjuvant radiotherapy was 50 Gy in 25 fractions to the left breast alone. Four plans—conventional wedged tangents and forward intensity-modulated radiotherapy (fIMRT) in supine and prone positions—were generated. fIMRT generated better homogeneity in both positions. Prone position centralized the breast tissue by gravity and also shortened the breast width which led to better conformity in both planning techniques. Prone fIMRT significantly reduced doses to left lung, Level I and Level II axilla. The mean cardiac doses did not differ between positions. Among the four plans, prone fIMRT produced the best target dosimetry and normal organ sparing. In subgroup analysis, patients with absolute breast depth > 7 cm in the prone position or breast depth difference > 3 cm between positions had significant cardiac sparing with prone fIMRT. Sixteen patients with significant cardiac sparing in prone position were treated using prone fIMRT and the others using supine fIMRT. All patients received a supine electron tumor bed boost of 10 Gy in 5 fractions. No patients developed Grade 2 or worse acute or late toxicities. There was no difference in the number of segments or beams, monitor units, treatment time, or positioning reproducibility between prone and supine positions. At a median follow-up time of 26.8 months, no locoregional or distant recurrence or death was noted.
doi:10.1093/jrr/rrt019
PMCID: PMC3766291  PMID: 23504450
breast cancer; prone breast radiotherapy; intensity-modulated radiotherapy; breast tangents; Asian women
20.  Male germ cell-associated kinase is overexpressed in prostate cancer cells and causes mitotic defects via deregulation of APC/C-CDH1 
Oncogene  2011;31(24):2907-2918.
Male germ cell-Associated Kinase (MAK), a direct transcriptional target of androgen receptor (AR), is a coactivator of AR. In this study, we determined the activating mechanism of MAK and identified a previously unknown AR-independent role of MAK in mitosis. We found that MAK kinase activity requires dual phosphorylation of the conserved TDY motif and that the phosphorylation is dynamic during cell cycle. MAK associates with CDH1 (FZR1, fizzy/cell division cycle 20 related 1) and phosphorylates CDH1 at sites phosphorylated by CDK. When MAK is overexpressed, the binding of CDH1 to Anaphase Promoting Complex/Cyclosome decreased, resulting in an attenuation of APC/C ubiquitin ligase activity and the consequential stabilization of the CDH1 targets such as Aurora kinase A and PLK1. As such, overexpression of MAK leads to mitotic defects such as centrosome amplification and lagging chromosomes. Our immunohistochemistry result showed that MAK is overexpressed in prostate tumor tissues, suggesting a role of MAK in prostate carcinogenesis. Taken with our previous results, our data implicate MAK in both AR activation and chromosomal instability, acting in both early and late prostate cancer (PCA) development.
doi:10.1038/onc.2011.464
PMCID: PMC3566783  PMID: 21986944
APC/C complex; CDH1/FZR1; MAK; Mitosis; Prostate Cancer
21.  Functional interaction between responses to lactic acidosis and hypoxia regulates genomic transcriptional outputs 
Cancer Research  2011;72(2):491-502.
Within solid tumor microenvironments, lactic acidosis and hypoxia each have powerful effects on cancer pathophysiology. However, the influence that these processes exert on each other is unknown. Here we report that a significant portion of the transcriptional response to hypoxia elicited in cancer cells is abolished by simultaneous exposure to lactic acidosis. In particular, lactic acidosis abolished stabilization of HIF-1α protein which occurs normally under hypoxic conditions. In contrast, lactic acidosis strongly synergized with hypoxia to activate the unfolded protein response (UPR) and an inflammatory response, displaying a strong similarity to ATF4-driven amino acid deprivation responses (AAR). In certain breast tumors and breast tumor cells examined, an integrative analysis of gene expression and array CGH data revealed DNA copy number alterations at the ATF4 locus, an important activator of the UPR/AAR pathway. In this setting, varying ATF4 levels influenced the survival of cells after exposure to hypoxia and lactic acidosis. Our findings reveal that the condition of lactic acidosis present in solid tumors inhibits canonical hypoxia responses and activates UPR and inflammation responses. Further, they suggest that ATF4 status may be a critical determinant of the ability of cancer cells to adapt to oxygen and acidity fluctuations in the tumor microenvironment, perhaps linking short-term transcriptional responses to long-term selection for copy number alterations in cancer cells.
doi:10.1158/0008-5472.CAN-11-2076
PMCID: PMC3261313  PMID: 22135092
22.  PI3K Synergizes With Loss of PKC to Elicit Apoptosis via the UPR 
Journal of cellular biochemistry  2009;107(1):76-85.
It is known that Ras mutations, together with loss of PKC, are apoptotic in various types of mammalian cells. The mechanism of how aberrant Ras transmits this apoptotic signaling remains unclear. Using three V12-Ha-ras loop mutants that preferentially bind to and activate one of Ras effectors, we tested the role of Ras downstream pathways in the induction of apoptosis in rat lung epithelia, human lung or prostate cancer cells. After PKC inhibition, the activation of PI3K/Akt renders the susceptibility of cells to apoptosis. We also demonstrate that the amount of ROS is moderately increased in the cells ectopically expressing V12C40 and dramatically elevated by suppression of PKC, which leads to apoptosis through the activation of UPR. Thus, our study suggests that after PKC abrogation, PI3K functions downstream of Ras to perturb the state of cellular redox and signals to ER stress-regulated apoptotic machinery.
doi:10.1002/jcb.22102
PMCID: PMC3545399  PMID: 19241442
apoptosis; Ras; PI3K/Akt; JNK; ROS; ER; UPR
23.  CDK-associated Cullin 1 can promote cell proliferation and inhibit cisplatin-induced apoptosis in the AGS gastric cancer cell line 
Background
Gastric cancer is a common and highly lethal malignancy in the world, but its pathogenesis remains elusive. In this study, we focus on the biological functions of CDK-associated Cullin1 (CAC1), a novel gene of the cullin family, in gastric cancer, which may help us to further understand the origin of this malignancy.
Methods
The AGS and MGC803 gastric cancer cell lines and the GES-1 gastric mucosa cell line were selected for study. At first, CAC1 expressions of those cell lines were examined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blot examinations, then CAC1 small interfering RNA (CAC1-siRNA) were designed and transfected into the AGS cell line with a relatively high level of CAC1. Once CAC1 was silenced, a series of biological characteristics of AGS cells such as cell proliferation, cell cycle, apoptosis, and expressions of apoptosis-related genes (P53, BCL2 and BAX) were determined by MTT, flow cytometry, qRT-PCR and western blot, respectively.
Results
CAC1 expression of AGS or MGC803 was much higher than that of GES-1. After CAC1 expression was effectively depressed by RNA interference in AGS cells, significant cell growth inhibition occurred. Furthermore, the proportion of cells treated with CAC1-siRNA increased in the G1 phase and decreased in the S phase, indicative of G1 cell cycle arrest. More importantly, the proportions of early/late apoptosis in AGS cells were enhanced with cis-diaminedichloroplatinum (cisplatin, CDDP) treatment, but to a higher extent with cisplatin plus CAC1-siRNA. Interestingly, BCL2 mRNA copies showed about a 30% decrease in the cisplatin group, but dropped by around 60% in the cisplatin plus CAC1-siRNA group. Conversely, the P53 mRNA expressions obtained nearly a two-fold increase in the cisplatin group, in addition to a five-fold increase in the cisplatin plus CAC1-siRNA group, and the BAX mRNA levels had almost a two- and four-fold augmentation, respectively. Meanwhile, P53, BAX and BCL2 showed the same alteration patterns in western blot examinations.
Conclusions
CAC1 can promote cell proliferation in the AGS gastric cancer cell line. Moreover, it can prevent AGS cells from experiencing cisplatin-induced apoptosis via modulating expressions of P53, BCL2 and BAX.
doi:10.1186/1477-7819-11-5
PMCID: PMC3585504  PMID: 23311997
Gastric cancer; CDK-associated Cullin1; Proliferation; Cell cycle; Apoptosis
24.  The in vivo performance of small-caliber nanofibrous polyurethane vascular grafts 
Background
In a previous in vitro study, we confirmed that small-caliber nanofibrous polyurethane (PU) vascular grafts have favorable mechanical properties and biocompatibility. In the present study, we examined the in vivo biocompatibility and stability of these grafts.
Methods
Forty-eight adult male beagle dogs were randomly divided into two groups receiving, respectively, polyurethane (PU) or polytetrafluoroethylene (PTFE) grafts (n = 24 animals / group). Each group was studied at 4, 8, 12, and 24 weeks after graft implantation. Blood flow was analyzed by color Doppler ultrasound and computed tomography angiography. Patency rates were judged by animal survival rates. Coverage with endothelial and smooth muscle cells was characterized by hematoxylin-eosin and immunohistological staining, and scanning electron microscopy (SEM).
Results
Patency rates were significantly higher in the PU group (p = 0.02 vs. PTFE group). During the first 8 weeks, endothelial cells gradually formed a continuous layer on the internal surface of PU grafts, whereas coverage of PTFE graft by endothelial cells was inhomogeneous. After 12 weeks, neointimal thickness remained constant in the PU group, while PTFE group showed neointimal hyperplasia. At 24 weeks, some anastomotic sites of PTFE grafts became stenotic (p = 0.013 vs. PU group). Immunohistological staining revealed a continuous coverage by endothelial cells and an orderly arrangement of smooth muscle cells on PU grafts. Further, SEM showed smooth internal surfaces in PU grafts without thrombus or obvious neointimal hyperplasia.
Conclusions
Small-caliber nanofibrous PU vascular grafts facilitate the endothelialization process, prevent excessive neointimal hyperplasia, and improve patency rates.
doi:10.1186/1471-2261-12-115
PMCID: PMC3523977  PMID: 23206536
Nanofibers; Polyurethane; Small-caliber vascular grafts; Animal study
25.  Epidemiology and Predictors of End-stage Renal Disease in Taiwanese Children With Idiopathic Nephrotic Syndrome 
Journal of Epidemiology  2012;22(6):517-522.
Background
The incidence of idiopathic nephrotic syndrome (INS) varies among countries, with Asia reporting a higher incidence in comparison with Western countries. We investigated the epidemiologic features of INS and attempted to identify factors that predispose individuals to develop end-stage renal disease (ESRD).
Methods
Claims data from the Taiwanese National Health Insurance program from 1996 to 2008 were used to investigate the epidemiologic features and clinical variables of INS (International Classification of Diseases, Ninth Revision, Clinical Modification code, 581) in children younger than 18 years.
Results
We enrolled 4083 children (male-female ratio, 1.91:1). During the 13 years of observation, annual incidence decreased from 9.91 to 3.36 per 100 000 children. Annual number of hospital admissions progressively decreased during the first 3 years after diagnosis. At 3.14 ± 2.77 years after INS onset, ESRD had developed in 145 (3.6%) children. Independent predictors of ESRD included older age at onset, acute renal failure (ARF), hypertensive encephalopathy, and a histologic subtype with focal segmental glomerulosclerosis (FSGS).
Conclusions
Pediatric INS in Taiwan was more frequent in boys. Unlike India, the current incidence of pediatric INS in Taiwan is very similar to that reported in Western studies. Older age at disease onset, ARF, hypertensive encephalopathy, and FSGS on biopsy are important predictors of poor renal outcome.
doi:10.2188/jea.JE20120033
PMCID: PMC3798563  PMID: 22971550
idiopathic nephrotic syndrome; long-term outcome; children; end-stage renal disease

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