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1.  RhoC, vascular endothelial growth factor and microvascular density in esophageal squamous cell carcinoma 
AIM: To investigate the expression of Ras homolog (Rho)C, vascular endothelial growth factor (VEGF) and CD105 in esophageal squamous cell carcinoma.
METHODS: Semi-quantitative reverse transcriptase polymerase chain reaction, in situ hybridization and immunohistochemical streptavidin-biotin- peroxidase methods were used to detect expression of RhoC mRNA and protein, and VEGF protein in 62 cases with esophageal squamous cell carcinoma, 31 cases with adjacent atypical hyperplastic tissues, and 62 cases with normal esophageal mucosa. CD105 antibody labeling was used to measure microvascular density. Expression levels were compared according to clinicopathologic and patient parameters.
RESULTS: Expression of RhoC mRNA showed a positive correlation with the protein level in esophageal squamous cell carcinoma, as well as with VEGF protein levels. RhoC mRNA expression was mainly located within the cytoplasm of the tumor cells, appearing as blue to purple particles by in situ hybridization. The differences in RhoC mRNA expression in esophageal squamous cell carcinoma, adjacent atypical hyperplasia and normal esophageal mucosa were significant (P < 0.05). The relative expression of RhoC mRNA in cancer tissues with lymph node metastasis was significantly higher than in the tissues without lymph node metastasis (P < 0.05). VEGF protein expression was consistent with microvascular density (t = 25.52, P < 0.05). Positive expression of VEGF protein in esophageal squamous cell carcinoma of different histologic gradings did not differ significantly. Positive expression of VEGF protein in carcinoma tissues with deep infiltration was significantly higher than in tissues with only superficial infiltration (P < 0.05). The positive expression of VEGF protein in cancer tissues with lymph node metastasis was significantly higher than in the tissues without lymph node metastasis (P < 0.05).
CONCLUSION: RhoC protein may upregulate VEGF expression, thereby promoting tumor angiogenesis. RhoC mRNA and protein expression was correlated with metastasis.
doi:10.3748/wjg.v21.i3.905
PMCID: PMC4299343  PMID: 25624724
Esophageal squamous cell carcinoma; Gene silencing; Ras homolog C; Vascular endothelial growth factor
2.  Analysis of Potato virus Y Coat Protein Epitopes Recognized by Three Commercial Monoclonal Antibodies 
PLoS ONE  2014;9(12):e115766.
Background
Potato virus Y (PVY, genus Potyvirus) causes substantial economic losses in solanaceous plants. Routine screening for PVY is an essential part of seed potato certification, and serological assays are often used. The commercial, commonly used monoclonal antibodies, MAb1128, MAb1129, and MAb1130, recognize the viral coat protein (CP) of PVY and distinguish PVYN strains from PVYO and PVYC strains, or detect all PVY strains, respectively. However, the minimal epitopes recognized by these antibodies have not been identified.
Methodology/Principal Findings
SPOT peptide array was used to map the epitopes in CP recognized by MAb1128, MAb1129, and MAb1130. Then alanine replacement as well as N- and C-terminal deletion analysis of the identified peptide epitopes was done to determine critical amino acids for antibody recognition and the respective minimal epitopes. The epitopes of all antibodies were located within the 30 N-terminal-most residues. The minimal epitope of MAb1128 was 25NLNKEK30. Replacement of 25N or 27N with alanine weakened the recognition by MAb1128, and replacement of 26L, 29E, or 30K nearly precluded recognition. The minimal epitope for MAb1129 was 16RPEQGSIQSNP26 and the most critical residues for recognition were 22I and 23Q. The epitope of MAb1130 was defined by residues 5IDAGGS10. Mutation of residue 6D abrogated and mutation of 9G strongly reduced recognition of the peptide by MAb1130. Amino acid sequence alignment demonstrated that these epitopes are relatively conserved among PVY strains. Finally, recombinant CPs were produced to demonstrate that mutations in the variable positions of the epitope regions can affect detection with the MAbs.
Conclusions/Significance
The epitope data acquired can be compared with data on PVY CP-encoding sequences produced by laboratories worldwide and utilized to monitor how widely the new variants of PVY can be detected with current seed potato certification schemes or during the inspection of imported seed potatoes as conducted with these MAbs.
doi:10.1371/journal.pone.0115766
PMCID: PMC4277358  PMID: 25542005
3.  A novel synthetic derivative of the natural product berbamine inhibits cell viability and induces apoptosis of human osteosarcoma cells, associated with activation of JNK/AP-1 signaling 
Cancer Biology & Therapy  2013;14(11):1024-1031.
Osteosarcoma is the most common primary bone tumor in children and adolescents. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have antitumor effects in several cancers. Here, we report that a novel synthetic berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of G292, KHOS, and MG-63 human osteosarcoma cells. Induction of apoptosis in these tumor cells depends on activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Since pan-caspase inhibitor (Z-VAD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) could block the cleavage of PARP, the apoptosis induced by BBMD3 is through intrinsic signaling pathway. BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in increase of phosphorylated c-Jun and total c-Fos, the major components of transcriptional factor AP-1. JNK inhibitor could partially suppress antitumor effect of BBMD3 on osteosarcoma cells. BBMD3 increased the production of reactive oxygen species (ROS) and ROS scavenger, N-acetylcysteine (NAC), could block the phosphorylation of JNK and c-Jun induced by BBMD3. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1 and D2, the positive cell cycle regulators, which is correlated with growth inhibition in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of human osteosarcoma.
doi:10.4161/cbt.26045
PMCID: PMC3925657  PMID: 24025361
berbamine derivative; osteosarcoma; apoptosis; JNK; AP-1; natural product
4.  Alzheimer's disease progression model based on integrated biomarkers and clinical measures 
Acta Pharmacologica Sinica  2014;35(9):1111-1120.
Aim:
Biomarkers and image markers of Alzheimer's disease (AD), such as cerebrospinal fluid Aβ42 and p-tau, are effective predictors of cognitive decline or dementia. The aim of this study was to integrate these markers with a disease progression model and to identify their abnormal ranges.
Methods:
The data of 395 participants, including 86 normal subjects, 108 early mild cognitive impairment (EMCI) subjects, 120 late mild cognitive impairment (LMCI) subjects, and 81 AD subjects were obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database. For the participants, baseline and long-term data on cerebrospinal fluid Aβ42 and p-tau, hippocampal volume, and ADAS-cog were available. Various linear and nonlinear models were tested to determine the associations among the ratio of Aβ42 to p-tau (the Ratio), hippocampal volume and ADAS-cog.
Results:
The most likely models for the Ratio, hippocampal volume, and ADAS-cog (logistic, Emax, and linear models, respectively) were used to construct the final model. Baseline disease state had an impact on all the 3 endpoints (the Ratio, hippocampal volume, and ADAS-cog), while APOEε4 genotype and age only influence the Ratio and hippocampal volume.
Conclusion:
The Ratio can be used to identify the disease stage for an individual, and clinical measures integrated with the Ratio improve the accuracy of mild cognitive impairment (MCI) to AD conversion forecasting.
doi:10.1038/aps.2014.57
PMCID: PMC4155529  PMID: 25088003
Alzheimer's disease; mild cognitive impairment; Aβ42; p-tau; hippocampus; ADAS-cog; disease progression model; NONMEM
5.  Dopamine D1 receptor activation induces dehydroepiandrosterone sulfotransferase (SULT2A1) in HepG2 cells 
Acta Pharmacologica Sinica  2014;35(7):889-898.
Aim:
Dopamine receptors are present in the nervous system and also widely distributed in the periphery. The aim of this study was to investigate the role of D1 subtype dopamine receptors (DRD1) in the regulation of dehydroepiandrosterone sulfotransferase (SULT2A1) in HepG2 cells.
Methods:
HepG2 cells were treated with DRD1 agonists with or without DRD1 antagonist for 9 d. DRD1 and SULT2A1 mRNA expression, protein expression, and SULT2A1 activity were detected using RT-PCR, Western blotting and HPLC, respectively. The level of cAMP was measured using a commercial kit.
Results:
All the 5 DR subtypes (DRD1–DRD5) were found to be expressed in HepG2 cells. Treatment of HepG2 cells with the specific DRD1 agonists SKF82958 (2.5 μmol/L) or SKF38393 (5 and 50 μmol/L) significantly increased the mRNA and protein expression of both DRD1 and SULT2A1, and increased SULT2A1 activity and cAMP levels. These effects were partially blocked by co-treatment with the specific DRD1 antagonist SCH23390 (2.5 μmol/L). In addition, transfection of HepG2 cells with DRD1-specific siRNAs decreased DRD1 mRNA expression by 40%, which resulted in the reduction of SULT2A1 mRNA expression by 60%, protein expression by 40%, and enzyme activity by 20%.
Conclusion:
DRD1 activation upregulates DRD1 and SULT2A1 expression and SULT2A1 activity in HepG2 cells, suggesting that the DRD1 subtype may be involved in the metabolism of drugs and xenobiotics through regulating SULT2A1.
doi:10.1038/aps.2014.19
PMCID: PMC4088277  PMID: 24909515
dopamine; D1 receptor; dehydroepiandrosterone sulfotransferase (SULT2A1); drug-metabolizing enzyme; SKF82958; SKF38393; SCH23390; siRNA; HepG2 cell
6.  C-Reactive Protein and Preterm Delivery 
Reproductive Sciences  2013;20(6):715-722.
To study the association between maternal C-reactive protein (CRP) and preterm delivery (PTD) pathways, CRP was measured in maternal plasma collected at mid-pregnancy (n = 1310). PTD was subdivided into spontaneous (sPTD) or medically indicated (MI-PTD). Histologic chorioamnionitis (HCA) was determined by placental histopathology (n = 1076). Adjusted CRP levels were elevated for sPTD (5.5 µg/mL) versus term deliveries (4.8 µg/mL) and higher in sPTD with HCA (6.3 µg/mL). After removing HCA, an interaction between body mass index (BMI) and sPTD in relation to CRP was noted. In BMI-stratified models, an association between CRP and sPTD among women with prepregnancy BMI >25 (8.9 µg/mL for sPTD; 7.2 µg/mL for term) was absent among women with lower BMI. We propose that this remaining association in overweight/obese women suggests that CRP may mark an obesity/inflammation PTD pathway that is distinct from the pathway indicated by HCA.
doi:10.1177/1933719112466302
PMCID: PMC3713547  PMID: 23221172
preterm birth; C-reactive protein; prepregnancy body mass index; placentas; chorioamnionitis
7.  Xanthoceraside Ameliorates Mitochondrial Dysfunction Contributing to the Improvement of Learning and Memory Impairment in Mice with Intracerebroventricular Injection of Aβ1-42 
The effects of xanthoceraside on learning and memory impairment were investigated and the possible mechanism associated with the protection of mitochondria was also preliminarily explored in Alzheimer's disease (AD) mice model induced by intracerebroventricular (i.c.v.) injection of Aβ1-42. The results indicated that xanthoceraside (0.08–0.32 mg/kg) significantly improved learning and memory impairment in Morris water maze test and Y-maze test. Xanthoceraside significantly reversed the aberrant decrease of ATP levels and attenuated the abnormal increase of ROS levels both in the cerebral cortex and hippocampus in mice injected with Aβ1-42. Moreover, xanthoceraside dose dependently reversed the decrease of COX, PDHC, and KGDHC activity in isolated cerebral cortex mitochondria of the mice compared with Aβ1-42 injected model mice. In conclusion, xanthoceraside could improve learning and memory impairment, promote the function of mitochondria, decrease the production of ROS, and inhibit oxidative stress. The improvement effects on mitochondria may be through withstanding the damage of Aβ to mitochondrial respiratory chain and the key enzymes in Kreb's cycle. Therefore, the results from present study and previous study indicate that xanthoceraside could be a competitive candidate for the treatment of AD.
doi:10.1155/2014/969342
PMCID: PMC4058193  PMID: 24976855
8.  Long-term effects of three Tiao-Bu Fei-Shen therapies on NF-κB/TGF-β1/smad2 signaling in rats with chronic obstructive pulmonary disease 
Background
The three Tiao-Bu Fei-Shen (Bufei Jianpi, Bufei Yishen, Yiqi Zishen) granules have been confirmed for their beneficial clinical efficacy in chronic obstructive pulmonary disease (COPD) patients on reducing frequency and duration of acute exacerbation, improving syndromes, pulmonary function and exercise capacity. But the short- or long-term mechanism of them is not fully clear. Nuclear factor (NF)-κB/transforming growth factor (TGF)-β1/smad2 signaling pathway is involved in the progress of inflammation and remodeling in chronic obstructive pulmonary disease COPD. This study aimed to explore the long-term effects mechanism of Tiao-Bu Fei-Shen granules by regulating NF-κB/TGF-β/Smads signaling in rats with COPD.
Methods
Sprague Dawley rats were randomized into control, model, Bufei Jianpi, Bufei Yishen, Yiqi Zishen and aminophylline groups. COPD rats, induced by cigarette smoke and bacterial infections exposures, were administrated intragastricly by normal saline, Bufei Jianpi, Bufei Yishen, Yiqi Zishen granules or aminophylline from week 9 through 20, respectively. At week 20 and 32, lung tissues were harvested. Immunohistochemistry was used to detect interleukin (IL)-1β and tumor necrosis factor (TNF)-α, quantitative real-time polymerase chain reaction (qRT-PCR) was used for TGF-β1 and Smad2 mRNA analysis, western blotting was used to determine the phosphorylation of NF-κB (p-NF-κB) and IκBα (p-IκBα).
Results
COPD rats had marked airway injury, such as chronic airway inflammation and remodeling, emphysema, which were improved in the three traditional Chinese medicines (TCM)-treated animals. The levels of IL-1β, TNF-α, p-NF-κB, p-IκBα, TGF-β1 and Smad2 were significantly higher in COPD rats than in controls, while they were dramatically reduced in the three TCM- and aminophylline-treated groups. At the meantime, all these endpoints were significantly lower in three TCM-treated groups than in aminophylline group, especially in Bufei Jianpi and Bufei Yishen groups. Compared to week 20, all endpoints decreased significantly in three TCM groups at week 32.
Conclusion
The three Tiao-Bu Fei-Shen therapies can reduce pulmonary inflammation and remodeling in COPD and have significant long-term effects. NF-κB/TGF-β1/smad2 signaling might be involved in the mechanism.
doi:10.1186/1472-6882-14-140
PMCID: PMC4006455  PMID: 24766819
Chronic obstructive pulmonary disease; Bufei jianpi; Bufei yishen; Yiqi zishen; Traditional Chinese medicine
9.  Tumors of the angle of Treitz: A single-center experience 
AIM: To explore the feasibility and oncologic outcomes of segmental jejunal resection on the left side of the mesenteric vessels in patients with tumors of the angle of Treitz using data from a single center.
METHODS: Thirteen patients with tumors of the angle of Treitz who underwent surgery at our institution were prospectively followed. A segmental jejunal resection on the left side of the mesenteric vessels was performed in all patients. Formalin-fixed and paraffin-embedded tumor samples were examined. The primary end point of this analysis was disease-free survival.
RESULTS: In this study, there were 8 males and 5 females (mean age, 50.1 years; range, 36-74 years). The mean tumor size was 8.1 cm (range, 3.2-15 cm). Histologic examination showed 11 gastrointestinal stromal tumors (GISTs) and 2 adenocarcinomas. Five of the GIST patients presented with potential low risk, and 6 presented with intermediate and high risk, according to the National Institutes of Health criteria. One potentially high-risk patient showed tumor progression at 46 mo and died 52 mo after surgery. One patient with locally advanced adenocarcinoma received neoadjuvant chemotherapy and adjuvant radiotherapy, but the disease progressed, and the patient died 9 mo after surgery. One GIST patient without progression died 16 mo after surgery because of a postoperative intestinal obstruction. The median overall survival rate was 84.6 mo, and the median disease-free survival rate was 94.5 mo.
CONCLUSION: The overall survival of patients with tumors of the angle of Treitz was encouraging even when the tumor size was relatively large. A segmental resection on the left side of the mesenteric vessels is considered to be a reliable and curative option for tumors of the angle of Treitz.
doi:10.3748/wjg.v20.i13.3628
PMCID: PMC3974531  PMID: 24707147
Gastrointestinal stromal tumor; Adenocarcinoma; Angle of Treitz; Surgical treatment; Prognosis
10.  Inflammation biomarkers in vaginal fluid and preterm delivery 
STUDY QUESTION
Which inflammation biomarkers detected in the vaginal fluid are most informative for identifying preterm delivery (PTD) risk?
SUMMARY ANSWER
Elevated interleukin (IL)-6 at mid-trimester was associated with increased odds of spontaneous PTD at <35 weeks and with PTD plus histologic chorioamnionitis (HCA), and had the greatest sensitivity for detecting these two PTD subtypes.
WHAT IS KNOWN ALREADY
Maternal and/or fetal inflammation play a role in some preterm deliveries, therefore inflammation biomarkers might help to identify women at greater risk.
STUDY DESIGN, SIZE, DURATION
We examined 1115 women from the Pregnancy Outcomes and Community Health Study, a cohort study conducted from September 1998 through June 2004, for whom data were available on mid-pregnancy inflammatory biomarkers.
PARTICIPANTS/MATERIALS, SETTING, METHODS
At enrollment at 16–27 weeks gestation, vaginal fluid samples were collected from a swab and 15 eluted biomarkers were measured using the Meso Scale Discovery multiplex electrochemiluminescence platform. Associations of biomarkers with PTD were examined, according to clinical circumstance, week at delivery and presence/absence of HCA. Weighted logistic regression was used to determine odds ratios (OR) and 95% confidence intervals (CI) adjusted for race. Sensitivity and specificity were compared between individual and multiple biomarkers, identified by a bootstrapping method.
MAIN RESULTS AND THE ROLE OF CHANCE
Elevated IL-6 (>75th percentile) displayed the strongest association with spontaneous PTD <35 weeks (OR 2.3; CI 1.3–4.0) and PTD with HCA (OR 2.8; CI 1.4–6.0). The sensitivity of IL-6 to detect spontaneous PTD <35 weeks or PTD with HCA was 0.43 and 0.51, respectively, while specificity was 0.74 and 0.75, respectively. IL-6 plus IL1β, IL-6r, tumor necrosis factor-alpha or granulocyte-macrophage colony-stimulating factor increased specificity (range 0.84–0.88), but decreased sensitivity (range 0.28–0.34) to detect both PTD subtypes. Results were similar when a combination of IL-6 and bacterial vaginosis (BV) was explored. Thus, the use of multiple biomarkers did not detect PTD subtypes with a greater sensitivity than IL-6 alone, and IL-6 is a specific but non-sensitive marker for the detection of spontaneous PTD.
LIMITATIONS, REASONS FOR CAUTION
Our ability to find small effect size associations between PTD and inflammation biomarkers (OR <2.0) might have been limited by the modest number of less common PTD subtypes in our population (e.g. spontaneous delivery <35 weeks, PTD accompanied by HCA) and by relatively higher variability for some cytokines, for example tumor necrosis factor-α, IL-12p70, IL-10 and granulocyte-macrophage colony-stimulating factor, that are less stable and commonly undetectable or detectable at low levels in human vaginal secretions.
WIDER IMPLICATIONS OF THE FINDINGS
Larger studies are needed to further explore a role of inflammation biomarkers in combination with other risk factors, including specific BV-associated organisms, for the prediction of PTD subtypes.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by the National Institute of Child Health and Human Development, National Institute of Nursing, March of Dimes Foundation, Thrasher Research Foundation and Centers for Disease Control and Prevention. The authors have no conflicts of interest.
doi:10.1093/humrep/det019
PMCID: PMC3600841  PMID: 23416276
biomarker; histologic chorioamnionitis; inflammation; preterm birth
11.  Malignant extra-gastrointestinal stromal tumor of the pancreas: Report of two cases and review of the literature 
Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that arise from the gastrointestinal tract. In rare cases, these tumors are found in intra-abdominal sites unrelated to the gastrointestinal tract, such as the mesentery, omentum and retroperitoneum. However, pancreatic extra-gastrointestinal stromal tumors are extremely rare, with only 14 previous cases reported. A 61-year-old man with no clinical symptoms had a routine check-up, during which an abdominal mass located in the pancreas tail was detected. Abdominal surgery was performed with resection of the pancreas tail and the spleen, and he was diagnosed with low-risk GISTs. Another 60-year-old man with no clinical symptoms underwent Computed tomography which revealed a well-demarcated tumor, 6 cm in diameter, in the head of the pancreas. He was diagnosed with pancreatic GISTs. Here, we describe two rare cases of pancreatic GISTs and review the cases previously reported in the literature.
doi:10.3748/wjg.v20.i3.863
PMCID: PMC3921496  PMID: 24574760
Gastrointestinal Stromal tumors; Extra-gastrointestinal Stromal Tumors; Pancreatic gastrointestinal stromal tumors
12.  Ultrasonography of lower limb vascular angiopathy and plaque formation in type 2 diabetes patients and finding its relevance to the carotid atherosclerotic formation 
Objective: One of the major complications of diabetes is blood vessel disease, termed angiopathy, which is characterized by abnormal angiogenesis. The objective of this study was to discuss the characteristics of lower limb vascular angiopathy and plaque formation in type 2 diabetes patients and finding its relevance to the carotid atherosclerotic plaque formation, thus directing the clinical diagnosis and treatment.
Methods: The ultrasonography was used to monitor the patients with carotid artery and lower limb artery.
Results: Compared with the control group, decreased blood flow to lower limb and lower limb angiopathy occurred more obviously in dorsal artery of foot than in popliteal artery. The study revealed that the detection rate of the prevalence of carotid atherosclerosis plaque and lower limb arterial plaque and the combination of plaque both carotid and lower limb arteries in diabetic patients was 369:342:296 (about 1.25:1.15:1) and that the prevalence of carotid plaque and lower limb arterial plaque in all subjects with plaque was 71.3%. The risk of plaque formation also had positive correlation with patient’s age. Color Doppler ultrasound had a clinical significance in the early diagnosis and curative effect observation in type 2 diabetes with lower limb angiopathy. The risk of simultaneous plaque formation in both carotid artery and lower extremity artery was greater in type 2 diabetes than that of control subjects, but they were not necessarily to occur simultaneously. The symptoms were inconspicuous in the early course of diabetes.
Conclusion: The application of ultrasound monitoring in patients with carotid artery and lower limb artery might play a role in early warning, delaying the occurrence of macrovascular diseases, and slowing down the development of macroangiopathy such as cerebral infarction and diabetic foot and so on, thus providing a significant basis for clinical diagnosis and treatment.
doi:10.12669/pjms.301.3907
PMCID: PMC3955542  PMID: 24639831
Ultrasonography; Color Doppler; Lower extremity artery; Diabetic vascular disease
13.  Pharmacokinetic-pharmacodynamic modeling of the anticancer effect of erlotinib in a human non-small cell lung cancer xenograft mouse model 
Acta Pharmacologica Sinica  2013;34(11):1427-1436.
Aim:
Erlotinib is used to treat non-small-cell lung cancer (NSCLC), which targets epidermal growth factor receptor (EGFR) tyrosine kinase. The aim of this study was to investigate the relationship between erlotinib plasma concentrations and phosphorylated EGFR (pEGFR) levels, as well as the relationship between pEGFR levels and tumor growth inhibition in a human non-small-cell lung cancer xenograft mouse model.
Methods:
Female BALB/c nude mice were implanted with the human NSCLC cell line SPC-A-1. The animals were given via gavage a single dose of erlotinib (4, 12.5, or 50 mg/kg). Pharmacokinetics of erlotinib was determined using LC-MS/MS. Tumor volume and pEGFR levels in tumor tissues were measured at different time points after erlotinib administration. The levels of pEGFR in tumor tissues was detected using Western blotting and ELISA assays.
Results:
The pharmacokinetics of erlotinib was described by a two-compartment model with first order extravascular absorption kinetics. There was a time delay of approximately 2 h between erlotinib plasma concentrations and pEGFR degradation. The time course of pEGFR degradation was reasonably fit by the indirect response model with a calculated IC50 value of 1.80 μg/mL. The relationship between pEGFR levels and tumor volume was characterized by the integrated model with a Kbio value of 0.507 cm3/week, which described the impact of pEGFR degradation on tumor growth.
Conclusion:
The pharmacokinetic/pharmacodynamic properties of erlotinib in a human tumor xenograft model were described by the indirect response model and integrated model, which will be helpful in understanding the detailed processes of erlotinib activity and determining an appropriate dosing regimen in clinical studies.
doi:10.1038/aps.2013.101
PMCID: PMC4006468  PMID: 24096601
non-small-cell lung cancer; anticancer drug; erlotinib; epidermal growth factor receptor; pharmacokinetic-pharmacodynamic modeling; human tumor xenograft mouse model
14.  Combined Administration of a Novel Mutant TGF-β1/Fc and Rapamycin Promotes Induction of Regulatory T Cells and Islet Allograft Tolerance1 
The critical roles of TGF-β in the reciprocal differentiation of tolerance-promoting CD4+Foxp3+ regulatory T cells (Treg) and pro-inflammatory Th17 effector cells impact alloimmune reactivity and transplant outcome. We reasoned that a strategy that harnessed TGF-β and blocked pro-inflammatory cytokines would inhibit the differentiation of Th17 cells and strengthen the cadre of Treg to promote tolerance induction and long-term allograft survival. Herein we report the development of a novel, long-lasting auto-active human mutant TGF-β1/Fc fusion protein that acts in conjunction with rapamycin to inhibit T cell proliferation and induce the de novo generation of Foxp3+ Treg in the periphery, while at the same time inhibiting IL-6-mediated Th17 cell differentiation. Short-term combined treatment with TGF-β1/Fc and rapamycin achieved long-term pancreatic islet allograft survival and donor-specific tolerance in a mouse model. This effect was accompanied by expansion of Foxp3+ Treg, enhanced alloantigen-specific Treg function, and modulation of transcript levels of Foxp3, IL-6 and IL-17. Our strategy of combined TGF-β1/Fc and rapamycin to target the IL-6-related Treg and Th17 signaling pathways provides a promising approach for inducing transplant tolerance and its clinical application.
doi:10.4049/jimmunol.1000769
PMCID: PMC3766970  PMID: 20844194
15.  Preclinical evaluation of herpes simplex virus armed with granulocyte-macrophage colony-stimulating factor in pancreatic carcinoma 
AIM: To investigate the therapeutic efficacy and mechanisms of action of oncolytic-herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor (HSVGM-CSF) in pancreatic carcinoma.
METHODS: Tumor blocks were homogenized in a sterile grinder in saline. The homogenate was injected into the right armpit of each mouse. After vaccination, the mice were randomly assigned into four groups: a control group, a high dose HSVGM-CSF group [1 × 107 plaque forming units (pfu)/tumor], a medium dose HSVGM-CSF group (5 × 106 pfu/tumor) and a low dose HSVGM-CSF group (5 × 105 pfu/tumor). After initiation of drug administration, body weights and tumor diameters were measured every 3 d. Fifteen days later, after decapitation of the animal by cervical dislocation, each tumor was isolated, weighed and stored in 10% formaldehyde solution. The drug effectiveness was evaluated according to the weight, volume and relative volume change of each tumor. Furthermore, GM-CSF protein levels in serum were assayed by enzyme-linked immunosorbent assays at 1, 2, 3 and 4 d after injection of HSVGM-CSF.
RESULTS: Injection of the recombinant mouse HSV encoding GM-CSF resulted in a significant reduction in tumor growth compared to the control group, and dose-dependent effects were observed: the relative tumor proliferation rates of the low dose, medium dose and high dose groups on 15 d after injection were 45.5%, 55.2% and 65.5%, respectively. The inhibition rates of the tumor weights of the low, middle, and high dose groups were 41.4%, 46.7% and 50.5%, respectively. Furthermore, the production of GM-CSF was significantly increased in the mice infected with HSVGM-CSF. The increase in the GM-CSF level was more pronounced in the high dose group compared to the other two dose groups.
CONCLUSION: Our study provides the first evidence that HSVGM-CSF could inhibit the growth of pancreatic cancer. The enhanced GM-CSF expression might be responsible for the phenomenon.
doi:10.3748/wjg.v19.i31.5138
PMCID: PMC3746387  PMID: 23964149
Pancreatic carcinoma; Gene therapy; Animal test; Herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor
16.  Overexpression of WDR62 is associated with centrosome amplification in human ovarian cancer 
Purpose
To assess the clinical significance of WD40 repeat containing 62 (WDR62), a novel centrosome abnormalities-associated gene, in ovarian cancer.
Materials and methods
In this study, WDR62 expression was assessed by western blot (6 ovarian cancer cell lines) and immunohistochemistry (primary epithelial ovarian cancer clinical specimens), and clinical variables were collected by retrospective chart review. Centrosome amplification was assessed by immunofluorescence staining in ovarian cancer cell lines, and by immunohistochemistry staining in ovarian cancer samples.
Results
Six ovarian cancer cell lines exhibited significant WDR62 protein overexpression, and amplification of centrosome. High-grade ovarian cancer specimens exhibited significantly stronger nuclear staining of WDR62 than low-grade ovarian carcinoma specimens (80.4% vs 41.3%; P<0.012). High WDR62 expression was strongly associated with supernumerary centrosome count in tumor cells (P < 0.001).
Conclusion
Our findings suggest that WDR62 overexpression is related to centrosome amplification in ovarian cancer. It may be a novel useful differentiation biomarker and a potential therapy target for OC. Further assessment of WDR62 expression is highly warranted in large, prospective studies.
doi:10.1186/1757-2215-6-55
PMCID: PMC3737014  PMID: 23898938
Ovarian cancer; WDR62; Centrosome
17.  Restoring the Treg cell to Th17 cell ratio may alleviate HBV-related acute-on-chronic liver failure 
AIM: To investigate the role of T helper 17 cells (Th17) and regulatory T cells (Treg) in hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF).
METHODS: We enrolled 79 patients with HBV infection into the study, 50 patients with HBV-related ACLF and 29 patients with chronic hepatitis B (CHB), from the First Affiliated Hospital of Medical College from January 2009 to June 2012. The ACLF patients were diagnosed according to the criteria recommended by The 19th Conference of the Asian Pacific Association for the Study of the Liver in 2009. Twenty healthy individuals with a similar gender and age structures to the two patient groups were also included as the normal controls (NC). Of the 50 ACLF patients, 28 were subsequently classified as non-survivors: 19 patients died from multi-organ failure, 3 underwent liver transplantation, and 6 discontinued therapy during follow-up because of financial reasons. The remaining 22 ACLF patients whose liver and anticoagulation function recovered to nearly normal levels within the next 6 mo were classified as survivors. The number of circulating Treg and Th17 cells was determined upon diagnosis and during the 8th week of follow-up through flow cytometry.
RESULTS: The percentage of circulating Treg cells in the ACLF group was significantly higher than that in the CHB group (5.50% ± 1.15% vs 3.30% ± 1.13%, P < 0.01). The percentages of circulating Th17 cells in the ACLF and the CHB groups were significantly higher than that in the NC group (6.32% ± 2.22% vs 1.56% ± 0.44%, P < 0.01; 3.53% ± 1.65% vs 1.56% ± 0.44%, P < 0.01). No significant difference in Treg cell to Th17 cell ratio was observed between the ACLF group and the CHB group (0.98 ± 0.44 vs 1.12 ± 0.64, P = 0.991), whereas those in the two HBV infection groups were significantly lower than that in the NC group (1.85 ± 1.22; both P < 0.01). The percentage of Treg cells in the survivors during the 8th week of follow-up was significantly lower than that during peak ACLF severity [total bilirubin (TBIL) peak] (3.45% ± 0.97% vs 5.18% ± 1.02%, P < 0.01). The percentage of Th17 cells in survivors during the 8th week of follow-up was significantly lower than that during the peak TBIL (2.89% ± 0.60% vs 5.24% ± 1.46%; P < 0.01). The Treg cell to Th17 cell ratio during the 8th week of follow-up was significantly higher than that during the TBIL peak (1.22 ± 0.36 vs 1.10 ± 0.54; P < 0.05).
CONCLUSION: Restoring the Treg cell to Th17 cell ratio during the follow-up phase of ACLF could maintain the immune system at a steady state, which favours good prognosis.
doi:10.3748/wjg.v19.i26.4146
PMCID: PMC3710416  PMID: 23864777
Hepatitis B virus; Acute-on-chronic liver failure; Regulatory T cells; T helper 17 cells; Treg cell to Th17 cell ratio
18.  Methotrexate and 5-aminoimidazole-4-carboxamide riboside exert synergistic anticancer action against human breast cancer and hepatocellular carcinoma 
Acta Pharmacologica Sinica  2013;34(7):951-959.
Aim:
To investigate the influences of methotrexate (MTX) on the anticancer actions and pharmacokinetics of 5-aminoimidazole-4-carboxamide riboside (AICA riboside) in human breast cancer and hepatocellular carcinoma.
Methods:
Human breast cancer cell line MCF-7 and human hepatocellular carcinoma cell line HepG2 were examined. The cell proliferation was assessed using a sulforhodamine B assay. Western blotting and radioactivity assays were used to analyze the phosphorylation of AMPK. The DNA synthesis was analyzed with BrdU incorporation. Nude mice bearing MCF-7 cell xenografts were used to for in vivo study. MTX (50 mg/kg, ip, per week) and AICA riboside (200 mg/kg, ip, every other day) were administered the animals for 2 weeks. The concentrations of AICA riboside and its active metabolite AICA ribotide in the plasma and tumors were measured with HPLC.
Results:
Synergistic cytotoxicity in vitro was observed with MTX (0.1, 0.5, and 1 μmol/L) combined with AICA riboside (0.25–1 mmol/L) in MCF-7 cells, and with MTX (0.5 and 1 μmol/L) combined with AICA riboside (0.5 and 1 mmol/L) in HepG2 cells. MTX (1 μmol/L) significantly enhanced the AICA riboside-induced AMPK activation and BrdU incorporation in both MCF-7 and HepG2 cells. Co-treatment with MTX and AICA riboside exerted more potent inhibition on the tumor growth in nude mice than either drug alone. After injection of AICA riboside (200 mg/kg, iv) in nude mice bearing MCF-7 xenografts, MTX (50 mg/kg, iv) significantly increased the concentrations of AICA riboside and its active metabolite AICA ribotide in tumors.
Conclusion:
MTX and AICA riboside exert synergistic anticancer action against MCF-7 and HepG2 cells in vitro and in vivo. MTX increases the concentration of AICA riboside and its active metabolite AICA ribotide in tumors in vivo.
doi:10.1038/aps.2013.16
PMCID: PMC4002606  PMID: 23603981
methotrexate; 5-aminoimidazole-4-carboxamide riboside; antitumor agents; human breast cancer; human hepatocellular carcinoma; drug synergism; pharmacokinetics; AMP-activated protein kinase; DNA synthesis
19.  ROCK is Involved in Vimentin Phosphorylation and Rearrangement Induced by Dengue Virus 
Cell Biochemistry and Biophysics  2013;67(3):1333-1342.
Our previous study showed that dengue virus 2 (DENV2) infection induces rearrangement of vimentin into dense structures at the perinuclear area. However, the underlying mechanism of this phenomenon is poorly characterized. In the present work, we found that vimentin and Ser71 phosphorylated vimentin display similar distributions in DENV2-infected cells. DENV2 infection also induced ROCK activation and phosphorylation of vimentin at Ser71 as the DENV2 infection progressed. Furthermore, Ser71 phosphorylation and vimentin rearrangement induced by DENV2 infection were blocked by the ROCK inhibitor Y-27632. In addition, DENV2 led to endoplasmic reticulum (ER) redistribution in the perinuclear region of the host cells, which was partially blocked by pretreatment with Y-27632. Together, these data support indicate that ROCK may have a role in governing regulating vimentin and ER rearrangement during DENV2 infection. We hypothesize that DENV2 infection, via ROCK activation, induces both vimentin rearrangement and ER redistribution around the perinuclear region, which may play a structural role in anchoring DENV2 to replication sites.
doi:10.1007/s12013-013-9665-x
PMCID: PMC3838595  PMID: 23737341
Dengue virus; Vimentin; Rearrangement; Phosphorylation; ROCK; Endoplasmic reticulum
20.  A Novel Perspective and Approach to Intestinal Octreotide Absorption: Sinomenine-Mediated Reversible Tight Junction Opening and Its Molecular Mechanism 
In this work, we assessed the effects of sinomenine (SN) on intestinal octreotide (OCT) absorption both in Caco-2 cell monolayers and in rats. We also investigated the molecular mechanisms of tight junction (TJ) disruption and recovery by SN-mediated changes in the claudin-1 and protein kinase C (PKC) signaling pathway. The data showed that exposure to SN resulted in a significant decrease in the expression of claudin-1, which represented TJ weakening and paracellular permeability enhancement. Then, the recovery of TJ after SN removal required an increase in claudin-1, which demonstrated the transient and reversible opening for TJ. Meanwhile, the SN-mediated translocation of PKC-α from the cytosol to the membrane was found to prove PKC activation. Finally, SN significantly improved the absolute OCT bioavailability in rats and the transport rate in Caco-2 cell monolayers. We conclude that SN has the ability to enhance intestinal OCT absorption and that these mechanisms are related at least in part to the important role of claudin-1 in SN-mediated, reversible TJ opening via PKC activation.
doi:10.3390/ijms140612873
PMCID: PMC3709818  PMID: 23787475
octreotide; sinomenine; intestinal absorption; tight junction
21.  Bortezomib induces apoptosis and growth suppression in human medulloblastoma cells, associated with inhibition of AKT and NF-ĸB signaling, and synergizes with an ERK inhibitor 
Cancer Biology & Therapy  2012;13(6):349-357.
Medulloblastoma is the most common brain tumor in children. Here, we report that bortezomib, a proteasome inhibitor, induced apoptosis and inhibited cell proliferation in two established cell lines and a primary culture of human medulloblastomas. Bortezomib increased the release of cytochrome c to cytosol and activated caspase-9 and caspase-3, resulting in cleavage of PARP. Caspase inhibitor (Z-VAD-FMK) could rescue medulloblastoma cells from the cytotoxicity of bortezomib. Phosphorylation of AKT and its upstream regulator mTOR were reduced by bortezomib treatment in medulloblastoma cells. Bortezomib increased the expression of Bad and Bak, pro-apoptotic proteins, and p21Cip1 and p27Kip1, negative regulators of cell cycle progression, which are associated with the growth suppression and induction of apoptosis in these tumor cells. Bortezomib also increased the accumulation of phosphorylated IĸBα, and decreased nuclear translocation of NF-ĸB. Thus, NF-ĸB signaling and activation of its downstream targets are suppressed. Moreover, ERK inhibitors or downregulating ERK with ERK siRNA synergized with bortezomib on anticancer effects in medulloblastoma cells. Bortezomib also inhibited the growth of human medulloblastoma cells in a mouse xenograft model. These findings suggest that proteasome inhibitors are potentially promising drugs for treatment of pediatric medulloblastomas.
doi:10.4161/cbt.19239
PMCID: PMC3341212  PMID: 22313636
Apoptosis; brain tumor; JAK2; neuroblastoma; NFκB; proliferation; Sorafenib; STAT3
22.  Risk factors for hepatic decompensation in patients with primary biliary cirrhosis 
AIM: To examine the clinical features and analyze prognostic factors in a prospective study of primary biliary cirrhosis (PBC) patients.
METHODS: From 1995 to 2010, PBC patients without hepatic decompensation seen at the Peking Union Medical College Hospital were enrolled. Clinical signs and manifestations (pruritus, persistent fatigue, jaundice and pain in the right hypochondrium), laboratory parameters (auto-antibodies for autoimmune hepatic disease, biliary and hepatic enzymes, immunoglobulin, bilirubin, and albumin) and imaging findings were recorded at entry and at specific time points during follow-up. Cox regression and Kaplan-Meier analyses, respectively, assessed the risk factors for hepatic decompensation and survival.
RESULTS: Two hundred and sixty-two PBC patients were enrolled with a median follow-up of 75.2 mo (range, 21-201 mo). The 240 patients were aged 51.5 ± 10.2 years at diagnosis and 91.6% were female. Two hundred and forty-five (93.5%) were seropositive for anti-mitochondrial antibodies. At presentation, 170 patients (64.9%) were symptomatic, while 96 patients (36.6%) had extra-hepatic autoimmune disease. During the follow-up period, 62 (23.7%) patients developed hepatic decompensation of whom four underwent liver transplantation and 17 died. The cumulative survival rate and median survival time were 83.9% and 181.7 mo, respectively. Cox regression analysis revealed that an incomplete ursodeoxycholic acid (UDCA) response or inconsistent treatment [P < 0.001; hazard risk (HR) 95%CI = 2.423-7.541], anti-centromere antibodies (ACA) positivity (P < 0.001; HR 95%CI = 2.516-7.137), alanine aminotransferase ratio (AAR) elevations (P < 0.001; HR 95%CI = 1.357-2.678), and histological advanced liver disease (P = 0.006; HR 95%CI = 1.481-10.847) were predictors of hepatic decompensation. The clinical features and survival of PBC in China are consistent with those described in Western countries.
CONCLUSION: Incomplete UDCA response or inconsistent treatment, ACA positivity, AAR elevations, and advanced histological stage are predictors of decompensation.
doi:10.3748/wjg.v19.i7.1111
PMCID: PMC3582000  PMID: 23467321
Primary biliary cirrhosis; Risk factor; Hepatic decompensation; Survival; Ursodeoxycholic acid response; Anti-centromere antibodies; Histological stage
23.  Role of autoimmunity in primary biliary cirrhosis 
Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of serum autoantibodies and chronic nonsuppurative destructive cholangitis. The pathogenesis of PBC involves environmental factors, genetic predisposition and loss of immune tolerance. In recent years, it has become univocally accepted that an inappropriately activated immune response is one of the most important factors in PBC. In this study, the role of autoimmunity in PBC is summarized and a feasible research orientation is recommended.
doi:10.3748/wjg.v18.i48.7141
PMCID: PMC3544015  PMID: 23326118
Primary biliary cirrhosis; Autoimmunity; Humoral immunity; Cellular immunity; Nonspecific immunity
24.  Spirooxindole Derivative SOID-8 Induces Apoptosis Associated with Inhibition of JAK2/STAT3 Signaling in Melanoma Cells 
PLoS ONE  2012;7(11):e49306.
Melanoma is generally refractory to current chemotherapy, thus new treatment strategies are needed. In this study, we synthesized a series of spirooxindole derivatives (SOID-1 to SOID-12) and evaluated their antitumor effects on melanoma. Among the 12 spirooxindole derivatives, SOID-8 showed the strongest antitumor activity by viability screening. SOID-8 inhibited viability of A2058, A375, SK-MEL-5 and SK-MEL-28 human melanoma cells in a dose- and time-dependent manner. SOID-8 also induced apoptosis of these tumor cells, which was confirmed by positive Annexin V staining and an increase of poly(ADP-ribose) polymerase cleavage. The antiapoptotic protein Mcl-1, a member of the Bcl-2 family, was downregulated and correlated with SOID-8 induced apoptosis. In addition, SOID-8 reduced tyrosine phosphorylation of Signal Tansducer and Activator of Transcription 3 (STAT3) in both dose- and time-dependent manners. This inhibition was associated with decreased levels of phosphorylation of Janus-activated kinase-2 (JAK2), an upstream kinase that mediates STAT3 phosphorylation at Tyr705. Accordingly, SOID-8 inhibited IL-6-induced activation of STAT3 and JAK2 in melanoma cells. Finally, SOID-8 suppressed melanoma tumor growth in a mouse xenograft model, accompanied with a decrease of phosphorylation of JAK2 and STAT3. Our results indicate that the antitumor activity of SOID-8 is at least partially due to inhibition of JAK2/STAT3 signaling in melanoma cells. These findings suggest that the spirooxindole derivative SOID-8 is a promising lead compound for further development of new preventive and therapeutic agents for melanoma.
doi:10.1371/journal.pone.0049306
PMCID: PMC3500295  PMID: 23166634
25.  Population pharmacokinetics of modafinil acid and estimation of the metabolic conversion of modafinil into modafinil acid in 5 major ethnic groups of China 
Acta Pharmacologica Sinica  2012;33(11):1401-1408.
Aim:
To describe the population pharmacokinetic profile of modafinil acid and to compare the extent of metabolism of modafinil into modafinil acid in 5 major ethnic groups (Han, Mongolian, Korean, Uygur, and Hui) of China.
Methods:
In a multi-center, open-label, single dose clinical trial, 49 healthy volunteers from the 5 ethnic groups received 200 mg of modafinil orally. Blood samples for pharmacokinetic evaluation of modafinil and modafinil acid were drawn before and at different time after the administration. Systematic population pharmacokinetic (PopPK) modeling for modafinil acid was conducted, integrating with our previous PopPK model for modafinil. The influence of ethnicity, gender, height, body weight and body mass index (BMI) was estimated. The extent of metabolism of modafinil into modafinil acid, expressed as the relative conversion fraction, was estimated and compared among the 5 ethnic groups.
Results:
When combined with the PopPK model of modafinil, the concentration of modafinil acid versus time profile was best described with a one-compartment model. The typical clearance and volume of distribution for modafinil acid were 4.94 (l/h) and 2.73 (l), respectively. The Korean group had 25% higher clearance, and the Uygur and Hui groups had 12% higher clearance than the Han group. The median for the relative conversion fraction was 0.53 for Koreans, and 0.24 for the other 4 ethnicities.
Conclusion:
Ethnicity has significant influence on the clearance of modafinil acid. When patients in the 5 ethnic groups are administered drugs or prodrugs catalyzed by esterases and/or amidases, the variability in the extent of drug metabolism should be considered.
doi:10.1038/aps.2012.124
PMCID: PMC4011351  PMID: 23103618
modafinil; modafinil acid; ethnicity; ethnic group in China; population pharmacokinetics; NONMEM; drug metabolism; esterase; amidaset

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