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1.  CXCR4 drives the metastatic phenotype in breast cancer through induction of CXCR2 and activation of MEK and PI3K pathways 
Molecular Biology of the Cell  2014;25(5):566-582.
Continuous signaling of CXCR4 in MCF-7 cells results in epithelial-to-mesenchymal transition (EMT), up-regulation of metastasis-associated cytokines, cell migration, and metastasis. The EMT phenotype was reversed in 3D rBM with combined inhibition of CXCR4 and CXCR2 together or in combination with MEK or PI3K, supporting development for combinational therapy treatment in breast cancer.
Aberrant expression of CXCR4 in human breast cancer correlates with metastasis to tissues secreting CXCL12. To understand the mechanism by which CXCR4 mediates breast cancer metastasis, MCF-7 breast carcinoma cells were transduced to express wild-type CXCR4 (CXCR4WT) or constitutively active CXCR4 (CXCR4ΔCTD) and analyzed in two-dimensional (2D) cultures, three-dimensional reconstituted basement membrane (3D rBM) cultures, and mice using intravital imaging. Two-dimensional cultures of MCF-7 CXCR4ΔCTD cells, but not CXCR4WT, exhibited an epithelial-to-mesenchymal transition (EMT) characterized by up-regulation of zinc finger E box–binding homeobox 1, loss of E-cadherin, up-regulation of cadherin 11, p120 isoform switching, activation of extracellular signal-regulated kinase 1/2, and matrix metalloproteinase-2. In contrast to the 2D environment, MCF-7 CXCR4WT cells cultured in 3D rBM exhibited an EMT phenotype, accompanied by expression of CXCR2, CXCR7, CXCL1, CXCL8, CCL2, interleukin-6, and granulocyte–macrophage colony stimulating factor. Dual inhibition of CXCR2 with CXCR4, or inhibition of either receptor with inhibitors of mitogen-activated protein kinase 1 or phosphatidylinositol 3-kinase, reversed the aggressive phenotype of MCF-7 CXCR4-expressing or MDA-MB-231 cells in 3D rBM. Intravital imaging of CXCR4-expressing MCF-7 cells revealed that tumor cells migrate toward blood vessels and metastasize to lymph nodes. Thus CXCR4 can drive EMT along with an up-regulation of chemokine receptors and cytokines important in cell migration, lymphatic invasion, and tumor metastasis.
PMCID: PMC3937084  PMID: 24403602
2.  Genetic Interaction Screens Identify a Role for Hedgehog Signaling in Drosophila Border Cell Migration 
Cell motility is essential for embryonic development and physiological processes such as the immune response, but also contributes to pathological conditions such as tumor progression and inflammation. However, our understanding of the mechanisms underlying migratory processes is incomplete. Drosophila border cells provide a powerful genetic model to identify the roles of genes that contribute to cell migration.
Members of the Hedgehog signaling pathway were uncovered in two independent screens for interactions with the small GTPase Rac and the polarity protein Par-1 in border cell migration. Consistent with a role in migration, multiple Hh signaling components were enriched in the migratory border cells. Interference with Hh signaling by several different methods resulted in incomplete cell migration. Moreover, the polarized distribution of E-Cadherin and a marker of tyrosine kinase activity were altered when Hh signaling was disrupted. Conservation of Hh-Rac and Hh-Par-1 signaling was illustrated in the wing, in which Hh-dependent phenotypes were enhanced by loss of Rac or par-1.
We identified a pathway by which Hh signaling connects to Rac and Par-1 in cell migration. These results further highlight the importance of modifier screens in the identification of new genes that function in developmental pathways.
PMCID: PMC3721345  PMID: 23335293
cell migration; border cells; Drosophila; Hedgehog; Par-1; Rac
3.  Association between Phosphorylated AMP-Activated Protein Kinase and Acetyl-CoA Carboxylase Expression and Outcome in Patients with Squamous Cell Carcinoma of the Head and Neck 
PLoS ONE  2014;9(4):e96183.
Epidemiological studies have indicated that impaired glucose metabolism may increase the risk of squamous cell carcinoma of the head and neck (SCCHN). AMP-activated protein kinase (AMPK) regulates glucose and lipid metabolism via the phosphorylation and subsequent inactivation of its downstream target acetyl-CoA carboxylase (ACC).Thus, we analyzed the expression of pAMPK and its downstream target phosphorylated acetyl-CoA carboxylase (pACC), as well as their impact on the survival of patients with resected SCCHN.
One hundred eighteen patients with surgically resected SCCHN were enrolled. Immunohistochemical (IHC) staining for pAMPK and pACC was performed using tissue microarrays of operative specimens of SCCHN. The expression was divided into two or three groups according to the IHC score [pAMPK: negative (0), positive (1–3); pACC: negative (0), low expression (1, 2), and high expression (3)]. Statistical analysis was performed to determine the association of pAMPK expression with clinicopathological features and pACC and pErk expression.
The positive rates of pAMPK and pACC expression were 64.4% (76/118) and 68.6% (81/118), respectively. pAMPK was significantly higher in patients aged younger than 60 years (P = 0.024; χ2test) and those with early-stage (T1/T2; P = 0.02; χ2 test) and oral cavity (P = 0.026; Fisher’s exact test) tumors. In multivariate analysis, pAMPK expression was not significantly correlated with overall survival (OS) (adjusted hazard ratio [HR]: 0.66; 95% confidence interval [CI]: 0.35–1.23), whereas high pACC expression was independently associated with worse OS in node-positive patients (adjusted HR: 17.58; 95% CI: 3.50–88.18).
Strong expression of pACC was found to be an independent prognostic marker for patients with node-positive SCCHN. Our results suggest that pACC may play a role in tumor progression of SCCHN and may help to identify patient subgroups at high risk for poor disease outcome.
PMCID: PMC4000216  PMID: 24769813
4.  Free Glycine Accelerates the Autoproteolytic Activation of Human Asparaginase 
Chemistry & biology  2013;20(4):533-540.
Human asparaginase 3 (hASNase3), which belongs to the N-terminal nucleophile (Ntn) hydrolase superfamily, is synthesized as a single polypeptide that is devoid of asparaginase activity. Intramolecular autoproteolytic processing releases the amino group of Thr168, a moiety required for catalyzing asparagine hydrolysis. Recombinant hASNase3 purifies as the uncleaved, asparaginase-inactive form, and undergoes self-cleavage to the active form at a very slow rate. Here we show that the free amino acid glycine selectively acts to accelerate hASNase3 cleavage both in vitro and in human cells. Other small amino acids such as alanine, serine, or the substrate asparagine are not capable of promoting autoproteolysis. Crystal structures of hASNase3 in complex with glycine in the uncleaved and cleaved enzyme states reveal the mechanism of glycine-accelerated post-translational processing, and explain why no other amino acid can substitute for glycine.
PMCID: PMC3633088  PMID: 23601642
5.  Impact of a Breastfeeding-Friendly Workplace on an Employed Mother's Intention to Continue Breastfeeding After Returning to Work 
Breastfeeding Medicine  2013;8(2):210-216.
Ever-increasing populations of women in their childbearing years are choosing to become employed. Breastfeeding provides unique health advantages to both the infant and mother. A breastfeeding-friendly workplace might be an important factor for predicting breastfeeding rates among working women. To explore the impact of breastfeeding-friendly support on the intention of working mothers to continue breastfeeding, we conducted a survey at a female labor-intensive electronics manufacturer in Taiwan.
Subjects and Methods
A structured questionnaire survey was administered to 715 working mothers employed in an electronics manufacturing plant in Tainan Science Park in Southern Taiwan. Questionnaire content included female employee demographics, employment characteristics, continued breastfeeding behavior after returning to work, access to lactation rooms, and employee perception of the breastfeeding policy and support when raising their most recently born child.
A higher education level (odds ratio [OR]=2.66), lower work load (8 work hours/day) (OR=2.66), lactation room with dedicated space (OR=2.38), use of breast pumping breaks (OR=61.6), and encouragement from colleagues (OR=2.78) and supervisors (OR=2.44) to use breast pumping breaks were significant predictors of continued breastfeeding for more than 6 months after returning to work.
The findings of the present study suggest that to encourage and increase the rate of continued breastfeeding, workplaces should establish dedicated breastfeeding rooms and maintain a comfortable and clean environment. Furthermore, employers should provide encouragement and support for working mothers to continue breastfeeding after returning to work.
PMCID: PMC3616406  PMID: 23390987
6.  Synthesis and SAR study of modulators inhibiting tRXRα-dependent AKT activation 
RXRα represents an intriguing and unique target for pharmacologic interventions. We recently showed that Sulindac and a designed analog could bind to RXRα and modulate its biological activity, including inhibition of the interaction of an N-terminally truncated RXRα (tRXRα) with the p85α regulatory subunit of phosphatidylinositol-3-OH kinase (PI3K). Here we report the synthesis, testing and SAR of a series of novel analogs of Sulindac as potential modulators for inhibiting tRXRα-dependent AKT activation. A new compound 30 was identified to have improved biological activity.
PMCID: PMC3738195  PMID: 23434637
RXRα; αtRXR modulator; AKT activation; Sulindac analogs
7.  Using Surgical Microscope for Sclera Buckling and Transscleral Cryopexy: An Alternative Procedure of Treatment for Rhegmatogenous Retinal Detachment 
BioMed Research International  2014;2014:364961.
Purpose. To observe the long-term effectiveness of scleral buckling and transscleral cryopexy conducted under a surgical microscope in the treatment of uncomplicated rhegmatogenous retinal detachment. Methods. This was a retrospective analysis in a total of 227 consecutive patients (244 eyes) with uncomplicated rhegmatogenous retinal detachment (proliferative vitreoretinopathy ≤ C2). All patients underwent scleral buckling and transscleral cryopexy under a surgical microscope without using a binocular indirect ophthalmoscope or a contact lens. Results. After initial surgery, complete retinal reattachment was achieved in 226 eyes (92.6%), and retinal redetachment developed in 18 eyes (7.4%). The causes of retinal redetachment included presence of new breaks in eight eyes (44%), failure to completely seal the breaks in five eyes (28%), missed retinal breaks in four eyes (22%), and iatrogenic retinal breaks in one eye (6%). Scleral buckling surgery was performed again in 12 eyes (66%). Four eyes (22%) developed proliferative vitreoretinopathy and then were treated by vitrectomy. The sealing of retinal breaks and complete retinal reattachment were achieved in 241 eyes (98.8%). Conclusion. Probably because of clear visualization of retinal breaks and being controllable under a surgical microscope, the microsurgery of scleral buckling and transscleral cryopexy for uncomplicated retinal detachment exhibits advisable effectiveness.
PMCID: PMC3984779  PMID: 24790997
8.  The Expression of Ribonucleotide Reductase M2 in the Carcinogenesis of Uterine Cervix and Its Relationship with Clinicopathological Characteristics and Prognosis of Cancer Patients 
PLoS ONE  2014;9(3):e91644.
To investigate the implication of ribonucleotide reductase M2 (RRM2) in the carcinogenesis of uterine cervix and its relationship with clinicopathological characteristics and prognosis of cancer patients.
Methodology and Principal Findings
The impact of RRM2 on cell viability was investigated in SiHa cervical cancer cells after RRM2 knockdown and the addition of cisplatin, which induces inter- and intra-strand DNA crosslinks. RRM2 immunoreactivity was evaluated by semi-quantitative H score among 29 normal, 30 low-grade dysplasia, 30 high-grade dysplasia and 103 invasive cancer tissue specimens of the uterine cervix, using tissue microarrays. RRM2 was then correlated with the clinicopathological variables of cervical cancer and patient survival. A greater toxic effect on cell viability using cisplatin was reflected by the greater reduction in RRM2 protein expression in SiHa cells. The RRM2 expression in cancer tissues was higher than that in high-grade dysplasia, low-grade dysplasia or normal cervical tissues. RRM2 upregulation was correlated with deep stromal invasion, large tumors and parametrial invasion and predicted poor survival.
RRM2 is a new molecular marker for the diagnosis and clinical outcomes of cervical cancer. It is involved in cervical carcinogenesis and predicts poor survival, and may be a potential therapeutic target including in cisplatin treatment.
PMCID: PMC3956764  PMID: 24637958
9.  The economic value of personalized medicine tests: what we know and what we need to know 
There is uncertainty about when personalized medicine tests provide economic value. We assessed evidence on the economic value of personalized medicine tests and gaps in the evidence base.
We created a unique evidence base by linking data on published cost–utility analyses from the Tufts Cost-Effectiveness Analysis Registry with data measuring test characteristics and reflecting where value analyses may be most needed: (i) tests currently available or in advanced development, (ii) tests for drugs with Food and Drug Administration labels with genetic information, (iii) tests with demonstrated or likely clinical utility, (iv) tests for conditions with high mortality, and (v) tests for conditions with high expenditures.
We identified 59 cost–utility analyses studies that examined personalized medicine tests (1998–2011). A majority (72%) of the cost/quality-adjusted life year ratios indicate that testing provides better health although at higher cost, with almost half of the ratios falling below $50,000 per quality-adjusted life year gained. One-fifth of the results indicate that tests may save money.
Many personalized medicine tests have been found to be relatively cost-effective, although fewer have been found to be cost saving, and many available or emerging medicine tests have not been evaluated. More evidence on value will be needed to inform decision making and assessment of genomic priorities.
PMCID: PMC3949119  PMID: 24232413
cost-effectiveness; economic value; ethical/legal/social implications (ELSI); genetic tests; personalized medicine
10.  Development of a simple score to predict outcome for unresponsive wakefulness syndrome 
Critical Care  2014;18(1):R37.
Accurate assessment of prognosis for patients with unresponsive wakefulness syndrome (UWS; formerly vegetative state) may help clinicians and families guide the type and intensity of therapy; however, there is no suitable and accurate means to predict the outcome so far. We aimed to develop a simple bedside scoring system to predict the likelihood of awareness recovery in patients with UWS.
We prospectively enrolled 56 patients (age range 10 to 73 years) with UWS 3 to 12 weeks post-onset. We collected demographic data and performed neurological, serological and neurophysiological tests at study entry. Each patient received a one year follow-up, during which awareness recovery was assessed by experienced physicians on the basis of clinical criteria. Univariate and multivariable analyses were employed to assess the relationships between predictors and awareness recovery.
A total of 56 participants were included in the study; of these, 24 patients recovered awareness, 3 with moderate disabilities, 8 with severe disabilities, 12 were in a minimally conscious state, and 1 died after recovery. During the study, 23 patients remained in UWS and 9 died in UWS. Motor response, type of brain injury, electroencephalogram reactivity, sleep spindles and N20 were shown to be independent predictors for awareness recovery. Based on their coefficients in the model, we assigned these predictors with 1 point each and created a 5-point score for prediction of awareness recovery. The resulting score showed good predictive accuracy in the derivation cohort. The area under the receiver operating characteristic curve for the score was 0.918 with 87.50% sensitivity.
This simple bedside prognostic score can be used to predict the probability of awareness recovery in UWS, thus provide families and clinicians with useful outcome information.
PMCID: PMC4056750  PMID: 24571596
11.  (Z)-N-[(Z)-3-(2,5-Di­methyl­phenyl­imino)­butan-2-yl­idene]-2,5-di­methyl­aniline 
The asymmetric unit of the title compound, C20H24N2, contains one half-mol­ecule, with the single C—C bond of the 1,4-di­aza­butadiene fragment situated on a centre of symmetry. The benzene rings are inclined to the 1,4-di­aza­butadiene mean plane by 59.5 (1)°.
PMCID: PMC3998329  PMID: 24764890
12.  Deletion of Gpr128 results in weight loss and increased intestinal contraction frequency 
AIM: To generate a Gpr128 gene knockout mouse model and to investigate its phenotypes and the biological function of the Gpr128 gene.
METHODS: Bacterial artificial chromosome-retrieval methods were used for constructing the targeting vector. Using homologous recombination and microinjection technology, a Gpr128 knockout mouse model on a mixed 129/BL6 background was generated. The mice were genotyped by polymerase chain reaction (PCR) analysis of tail DNA and fed a standard laboratory chow diet. Animals of both sexes were used, and the phenotypes were assessed by histological, biochemical, molecular and physiological analyses. Semi-quantitative reverse transcription-PCR and Northern blotting were used to determine the tissue distribution of Gpr128 mRNA. Beginning at the age of 4 wk, body weights were recorded every 4 wk. Food, feces, blood and organ samples were collected to analyze food consumption, fecal quantity, organ weight and constituents of the blood and plasma. A Trendelenburg preparation was utilized to examine intestinal motility in wild-type (WT) and Gpr128-/- mice at the age of 8 and 32 wk.
RESULTS: Gpr128 mRNA was highly and exclusively detected in the intestinal tissues. Targeted deletion of Gpr128 in adult mice resulted in reduced body weight gain, and mutant mice exhibited an increased frequency of peristaltic contraction and slow wave potential of the small intestine. The Gpr128+/+ mice gained more weight on average than the Gpr128-/- mice since 24 wk, being 30.81 ± 2.84 g and 25.74 ± 4.50 g, respectively (n = 10, P < 0.01). The frequency of small intestinal peristaltic contraction was increased in Gpr128-/- mice. At the age of 8 wk, the frequency of peristalsis with an intraluminal pressure of 3 cmH2O was 6.6 ± 2.3 peristalsis/15 min in Gpr128-/- intestine (n = 5) vs 2.6 ± 1.7 peristalsis/15 min in WT intestine (n = 5, P < 0.05). At the age of 32 wk, the frequency of peristaltic contraction with an intraluminal pressure of 2 and 3 cmH2O was 4.6 ± 2.3 and 3.1 ± 0.8 peristalsis/15 min in WT mice (n = 8), whereas in Gpr128-/- mice (n = 8) the frequency of contraction was 8.3 ± 3.0 and 7.4 ± 3.1 peristalsis/15 min, respectively (2 cmH2O: P < 0.05 vs WT; 3 cmH2O: P < 0.01 vs WT). The frequency of slow wave potential in Gpr128-/- intestine (35.8 ± 4.3, 36.4 ± 4.2 and 37.1 ± 4.8/min with an intraluminal pressure of 1, 2 and 3 cmH2O, n = 8) was also higher than in WT intestine (30.6 ± 4.2, 31.4 ± 3.9 and 31.9 ± 4.5/min, n = 8, P < 0.05).
CONCLUSION: We have generated a mouse model with a targeted deletion of Gpr128 and found reduced body weight and increased intestinal contraction frequency in this animal model.
PMCID: PMC3923024  PMID: 24574718
G-protein-coupled receptors; Gpr128; Knockout mouse; Weight loss; Intestinal contraction frequency
13.  Targeting Truncated Retinoid X Receptor-α by CF31 induces TNFα-dependent apoptosis 
Cancer research  2012;73(1):307-318.
A truncated version of retinoid X receptor-α, tRXRα, promotes cancer cell survival by activating the PI3K/AKT pathway. However, targeting the tRXRα-mediated survival pathway for cancer treatment remains to be explored. We report here our identification of a new natural product molecule, CF31, a xanthone isolated from Cratoxylum formosum ssp. Pruniflorum, and the biological evaluation of its regulation of the tRXRα-mediated PI3K/AKT pathway. CF31 binds RXRα and its binding results in inhibition of RXRα transactivation. Through RXRα mutational analysis and computational studies, we show that Arg316 of RXRα, known to form salt bridges with certain RXRα ligands such as 9-cis-retinoic acid (9-cis-RA), is not required for the antagonist effect of CF31, demonstrating a distinct binding mode. Evaluation of several CF31 analogs suggests that the antagonist effect is mainly attributed to an interference with Leu451 of helix H12 in RXRα. CF31 is a potent inhibitor of AKT activation in various cancer cell lines. When combined with TNFα, it suppresses TNFα activation of AKT by inhibiting TNFα-induced tRXRα interaction with the p85α regulatory subunit of PI3K. CF31 inhibition of TNFα activation of AKT also results in TNFα-dependent activation of caspase-8 and apoptosis. Together, our results demonstrate that CF31 is an effective converter of TNFα signaling from survival to death by targeting tRXRα in a unique mode and suggest that identification of a natural product that targets an RXR-mediated cell survival pathway that regulates PI3K/Akt may offer a new therapeutic strategy to kill cancer cells.
PMCID: PMC3537848  PMID: 23151904
retinoid X receptor; CF31; AKT; TNFα; cancer; apoptosis
14.  Novel peptide isomer strategy for stable inhibition of catecholamine release: Application to hypertension 
Hypertension  2012;60(6):1552-1559.
Although hypertension remains the most potent and widespread cardiovascular risk factor, its pharmacological treatment has achieved only limited success. The chromogranin A derived fragment catestatin inhibits catecholamine release by acting as an endogenous nicotinic cholinergic antagonist, and can “rescue” hypertension in the setting of CHGA targeted ablation. Here we undertook novel peptide chemistry to synthesize isomers of catestatin: normal/wild-type (W-T) as well as a retro-inverso (R-I) version, with not only inversion of chirality (L→D amino acids) but also reversal of sequence (carboxyl→amino). The R-I peptide was entirely resistant to proteolytic digestion, and displayed enhanced potency as well as preserved specificity of action towards nicotinic cholinergic events: catecholamine secretion, agonist desensitization, secretory protein transcription, and cationic signal transduction. Structural modeling suggested similar side chain orientations of the W-T and R-I isomers, while CD spectroscopy documented inversion of chirality. In vivo, the R-I peptide “rescued” hypertension in two mouse models of the human trait: monogenic Chga targeted ablation, with prolonged efficacy of the R-I version; and a polygenic model, with magnified efficacy of the R-I version. These results may have general implications for generation of metabolically stable mimics of biologically active peptides for cardiovascular pathways. The findings also point the way toward a potential new class of drug therapeutics for an important risk trait, and more generally open the door to broader applications of the retro-inverso strategy in other pathways involved in cardiovascular biology, with the potential for synthesis of diagnostic and therapeutic probes for both physiology and disease.
PMCID: PMC3523723  PMID: 23129699
Hypertension; peptides; cardiovascular diseases
15.  Safety of in vitro amplified HLA-haploidentical donor immune cell infusions for childhood malignancies 
Chinese Journal of Cancer  2013;32(12):661-666.
In vitro amplified human leukocyte antigen (HLA)-haploidentical donor immune cell infusion (HDICI) is not commonly used in children. Therefore, our study sought to evaluate its safety for treating childhood malignancies. Between September 2011 and September 2012, 12 patients with childhood malignancies underwent HDICI in Sun Yat-sen University Cancer Center. The median patient age was 5.1 years (range, 1.7-8.4 years). Of the 12 patients, 9 had high-risk neuroblastoma (NB) [7 showed complete response (CR), 1 showed partial response (PR), and 1 had progressive disease (PD) after multi-modal therapies], and 3 had Epstein-Barr virus (EBV)-positive lymphoproliferative disease (EBV-LPD). The 12 patients underwent a total of 92 HDICIs at a mean dose of 1.6×108 immune cells/kg body weight: 71 infusions with natural killer (NK) cells, 8 with cytokine-induced killer (CIK) cells, and 13 with cascade primed immune cells (CAPRIs); 83 infusions with immune cells from the mothers, whereas 9 with cells from the fathers. Twenty cases (21.7%) of fever, including 6 cases (6.5%) accompanied with chills and 1 (1.1%) with febrile convulsion, occurred during infusions and were alleviated after symptomatic treatments. Five cases (5.4%) of mild emotion changes were reported. No other adverse events occurred during and after the completion of HDIDIs. Neither acute nor chronic graft versus host disease (GVHD) was observed following HDICIs. After a median of 5.0 months (range, 1.0-11.5 months) of follow-up, the 2 NB patients with PR and PD developed PD during HDICIs. Of the other 7 NB patients in CR, 2 relapsed in the sixth month of HDICIs, and 5 maintained CR with disease-free survival (DFS) ranging from 4.5 to 11.5 months (median, 7.2 months). One EBV-LPD patient achieved PR, whereas 2 had stable disease (SD). Our results show that HDICI is a safe immunotherapy for childhood malignancies, thus warranting further studies.
PMCID: PMC3870850  PMID: 23706769
Childhood malignancies; HLA-haploidentical donor cells; immunotherapy; safety
16.  Recombinant adenovirus-p53 (Gendicine) sensitizes a pancreatic carcinoma cell line to radiation 
In this study, we examine the effects of recombinant adenovirus-p53 (rAd-p53) on the pancreatic carcinoma cell line SW1990. Specifically, we determine if expression of rAd-p53 sensitizes these cells to radiation.
Following transfection of SW1990 cells with rAd-p53, we measured expression of P53, P21 and Bax by immunocytochemistry. Both transfected and control cell lines were irradiated with a range of doses, and the survival fractions (SF) were calculated. Dose survival curves were constructed and modeled for comparison.
Transfection of SW1990 cells with rAd-p53 resulted in increased expression of P53, P21 and Bax in a time-dependent manner. At 96 h after transfection, 89.92% of cells expressed P53, 56.8% expressed P21, and 76.50% expressed Bax. The SF following radiation was lower in the rAd-p53 transfected cells compared to the control cells, suggesting that rAd-p53 sensitizes SW1990 cells to radiation (D0 for the experimental and control groups was 2.199 and 2.462, respectively).
Use of the adenoviral vector is an effective means of transfecting SW1990 cells with wild-type P53, and this sensitizes the cell line to irradiation. This work suggests that combining rAd-p53 with radiation therapy in pancreatic cancer may be therapeutically beneficial.
PMCID: PMC3872551  PMID: 24385699
Pancreatic carcinoma; recombinant adenovirus-p53 (rAd-p53); transfection; radiosensitization
17.  Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor 
The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ∼330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM). The synthetic methodology, development of structure-activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.
PMCID: PMC3729231  PMID: 23010269
GPCRs; HTS; APJ; Apelin; Cardiovascular disease; Antagonists; SAR
18.  Reduction of Decoy Receptor 3 Enhances TRAIL-Mediated Apoptosis in Pancreatic Cancer 
PLoS ONE  2013;8(10):e74272.
Most human pancreatic cancer cells are resistant to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. However, the mechanisms by which pancreatic cancer cells utilize their extracellular molecules to counteract the proapoptotic signaling mediated by the TNF family are largely unknown. In this study, we demonstrate for the first time that DcR3, a secreted decoy receptor that malignant pancreatic cancer cells express at a high level, acts as an extracellular antiapoptotic molecule by binding to TRAIL and counteracting its death-promoting function. The reduction of DcR3 with siRNA unmasked TRAIL and greatly enhanced TRAIL-induced apoptosis. Gemcitabine, a first-line drug for pancreatic cancer, also reduced the level of DcR3. The addition of DcR3 siRNA further enhanced gemcitabine-induced apoptosis. Notably, our in vivo study demonstrated that the therapeutic effect of gemcitabine could be enhanced via further reduction of DcR3, suggesting that downregulation of DcR3 in tumor cells could tip the balance of pancreatic cells towards apoptosis and potentially serve as a new strategy for pancreatic cancer therapy.
PMCID: PMC3808375  PMID: 24204567
19.  Comparative Efficacy and Acceptability of Antidepressants in Parkinson's Disease: A Network Meta-Analysis 
PLoS ONE  2013;8(10):e76651.
Depression is a common non-motor symptom in patients with Parkinson's disease (PD). There are many kinds of antidepressants being used, such as tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), and Dopamine agonists which are suggested as alternative antidepressants for the treatment of depression in PD. Which one should we choose first? Literatures have shown inconsistent results.
We conducted a network meta-analysis of randomized controlled trials to compare the efficacy and acceptability of therapeutic methods for the treatment of depression in Parkinson's disease.
We used the odds ratios (OR) as effect size firstly and the results indicated no statistical significance between each compared intervention. Then we used the logarithm of the individual odds ratios as effect size. With efficacy of TCAs as the standard of comparison, the degree of incoherence (a measure of how closely the entire network fits together) was small (ω =  4.824827e-05). The logor were: SSRIs −0.69 (95% CI −1.28– −0.10); Pramipexole −0.73 (−1.71– −0.26); Pergolide −1.97 (−3.67– 0.27); SNRIs −0.86 (−1.86– 0.15); Placebo −1.24 (−1.99– −0.50). With Placebo as the standard of comparison, the logor were: TCAs 1.24 (0.50– 1.99); SSRIs 0.55 (−0.03– 1.13); Pramipexole 0.51 (−0.12– 1.15); Pergolide −0.73 (−2.25– 0.80); SNRIs 0.38 (−0.42– 1.19); TCAs, pramipexole, pergolide and SNRIs showed better profile of acceptability, leading to significant fewer discontinuations than that of SSRIs.
There is insufficient evidence to support antidepressant efficacy for SSRIs, pramipexole, pergolide and SNRIs. TCAs might be the best choice when starting antidepressant treatment in patients of Parkinson's disease because it has the most favorable balance between benefits and acceptability, followed by pramipexole and SNRIs, SSRIs might be the last choice.
PMCID: PMC3788746  PMID: 24098546
20.  Germinal-center type B-cell classification and clinical characteristics of Chinese pediatric diffuse large B-cell lymphoma: a report of 76 cases 
Chinese Journal of Cancer  2013;32(10):561-566.
Pediatric diffuse large B-cell lymphoma (DLBCL) is a highly aggressive disease with unique clinical characteristics. This study analyzed the germinal-center type B-cell (GCB) classification and clinical characteristics of Chinese pediatric DLBCL. A total of 76 patients with DLBCL newly diagnosed in Sun Yat-sen University Cancer Center between February 2000 and May 2011, with an age younger than 18 years, were included in the analysis. The male/female ratio was 3.47:1. The median age was 12 years (range, 2 to 18 years), and 47 (61.8%) patients were at least 10 years old. Of the 76 patients, 48 (63.2%) had stage III/IV disease, 9 (11.8%) had bone marrow involvement, 1 (1.3%) had central nervous system (CNS) involvement, and 5 (6.6%) had bone involvement. The GCB classification was assessed in 45 patients: 26 (57.8%) were classified as GCB subtype, and 19 (42.2%) were classified as non-GCB subtype. The modified B-NHL-BFM-90/95 regimen was administered to 50 patients, and the 4-year event-free survival (EFS) rate was 85.8%. Among these 50 patients, 31 were assessed for the GCB classification: 17 (54.8%) were classified as GCB subtype, with a 4-year EFS rate of 88.2%; 14 (45.2%) were classified as non-GCB subtype, with a 4-year EFS rate of 92.9%. Our data indicate that bone marrow involvement and stage III/IV disease are common in Chinese pediatric DLBCL patients, whereas the percentage of patients with the GCB subtype is similar to that of patients with the non-GCB subtype. The modified B-NHL-BFM-90/95 protocol is an active and effective treatment protocol for Chinese pediatric patients with DLBCL.
PMCID: PMC3845538  PMID: 23544447
Non-Hodgkin's lymphoma; diffuse large B-cell lymphoma; pediatric; chemotherapy; germinal-center type B-cell classification; clinical characteristics
22.  Structures of apo and product-bound human L-asparaginase: Insights into the mechanism of autoproteolysis and substrate hydrolysis 
Biochemistry  2012;51(34):6816-6826.
Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. Bacterial asparaginases are used in cancer chemotherapy to deplete asparagine from the blood, since several hematological malignancies depend on extracellular asparagine for growth. To avoid the immune response against the bacterial enzymes it would be beneficial to replace them with human asparaginases. However, unlike the bacterial asparaginases, the human enzymes have a millimolar Km value for asparagine, making them inefficient in depleting the amino acid from blood. To facilitate the development of human variants suitable for therapeutic use, we solved the structure of human L-asparaginase (hASNase3). This asparaginase is an N-terminal nucleophile (Ntn) family member that requires autocleavage between Gly167 and Thr168 to become catalytically competent. For most Ntn-hydrolases this autoproteolytic activation occurs efficiently. In contrast, hASNas3 is relatively stable in its uncleaved state, and this allowed us to observe the structure of the enzyme prior to cleavage. To determine the structure of the cleaved state we exploited our discovery that the free amino acid glycine promotes complete cleavage of hASNase3. Both enzyme states were elucidated in the absence and presence of the product aspartate. Together, these structures provide insight into the conformational changes required for cleavage, and on the precise enzyme-substrate interactions. The new understanding of hASNase3 will serve to guide the design of variants that possess a decreased Km value for asparagine, making the human enzyme a suitable replacement for the bacterial asparaginases in cancer therapy.
PMCID: PMC3448794  PMID: 22861376
23.  Resistance Studies of a Dithiazol Analogue, DBPR110, as a Potential Hepatitis C Virus NS5A Inhibitor in Replicon Systems 
Hepatitis C virus (HCV), a member of the Flaviviridae family, affects approximately 3% of the world's population and is becoming the leading cause of liver disease in the world. Therefore, the development of novel or more effective treatment strategies to treat chronic HCV infection is urgently needed. In our previous study, we identified a potential HCV NS5A inhibitor, BP008. After further systemic optimization, we discovered a more potent HCV inhibitor, DBPR110. DBPR110 reduced the reporter expression of the HCV1b replicon with a 50% effective concentration (EC50) and a selective index value of 3.9 ± 0.9 pM and >12,800,000, respectively. DBPR110 reduced HCV2a replicon activity with an EC50 and a selective index value of 228.8 ± 98.4 pM and >173,130, respectively. Sequencing analyses of several individual clones derived from the DBPR110-resistant RNAs purified from cells harboring genotype 1b and 2a HCV replicons revealed that amino acid substitutions mainly within the N-terminal region (domain I) of NS5A were associated with decreased inhibitor susceptibility. P58L/T and Y93H/N in genotype 1b and T24A, P58L, and Y93H in the genotype 2a replicon were the key substitutions for resistance selection. In the 1b replicon, V153M, M202L, and M265V play a compensatory role in replication and drug resistance. Moreover, DBPR110 displayed synergistic effects with alpha interferon (IFN-α), an NS3 protease inhibitor, and an NS5B polymerase inhibitor. In summary, our results present an effective small-molecule inhibitor, DBPR110, that potentially targets HCV NS5A. DBPR110 could be part of a more effective therapeutic strategy for HCV in the future.
PMCID: PMC3553732  PMID: 23165461
25.  Deficiency of Adiponectin Protects against Ovariectomy-Induced Osteoporosis in Mice 
PLoS ONE  2013;8(7):e68497.
Adipokine adiponectin (APN) has been recently reported to play a role in regulating bone mineral density (BMD). To explore the mechanism by which APN affects BMD, we investigated BMD and biomechanical strength properties of the femur and vertebra in sham-operated (Sham) and ovariectomized (OVX) APN knockout (KO) mice as compared to their operated wild-type (WT) littermates. The results show that APN deficiency has no effect on BMD but induces increased ALP activity and osteoclast cell number. While OVX indeed leads to significant bone loss in both femora and vertebras of WT mice with comparable osteogenic activity and a significant increase in osteoclast cell number when compared to that of sham control. However, no differences in BMD, ALP activity and osteoclast cell number were found between Sham and OVX mice deficient for APN. Further studies using bone marrow derived mesenchymal stem cells (MSCs) demonstrate an enhanced osteogenic differentiation and extracellular matrix calcification in APN KO mice. The possible mechanism for APN deletion induced acceleration of osteogenesis could involve increased proliferation of MSCs and higher expression of Runx2 and Osterix genes. These findings indicate that APN deficiency can protect against OVX-induced osteoporosis in mice, suggesting a potential role of APN in regulating the balance of bone formation and bone resorption, especially in the development of post-menopausal osteoporosis.
PMCID: PMC3699501  PMID: 23844209

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