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1.  Differential methylation in CN-AML preferentially targets non-CGI regions and is dictated by DNMT3A mutational status and associated with predominant hypomethylation of HOX genes 
Epigenetics  2014;9(8):1108-1119.
The extent and role of aberrant DNA methylation in promoter CpG islands (CGIs) have been extensively studied in leukemia and other malignancies. Still, CGIs represent only a small fraction of the methylome. We aimed to characterize genome-wide differential methylation of cytogenetically normal AML (CN-AML) cells compared with normal CD34+ bone marrow cells using the Illumina® 450K methylation array. Differential methylation in CN-AML was most prominent in genomic areas far from CGIs, in so called open sea regions. Furthermore, differential methylation was specifically found in genes encoding transcription factors (TFs), with WT1 being the most differentially methylated TF. Among genetic mutations in AML, DNMT3A mutations showed the most prominent association with the DNA methylation pattern, characterized by hypomethylation of CGIs (as compared with DNMT3A wild type cases). The differential methylation in DNMT3A mutant cells vs. wild type cells was predominantly found in HOX genes, which were hypomethylated. These results were confirmed and validated in an independent CN-AML cohort. In conclusion, we show that, in CN-AML, the most pronounced changes in DNA methylation occur in non-CGI regions and that DNMT3A mutations confer a pattern of global hypomethylation that specifically targets HOX genes.
PMCID: PMC4164496  PMID: 24866170
acute myeloid leukemia; DNA methylation; non-CGI region; DNMT3A; Homeobox gene family
2.  Evaluation of MCF10A as a Reliable Model for Normal Human Mammary Epithelial Cells 
PLoS ONE  2015;10(7):e0131285.
Breast cancer is the most common cancer in women and a leading cause of cancer-related deaths for women worldwide. Various cell models have been developed to study breast cancer tumorigenesis, metastasis, and drug sensitivity. The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. However, there is limited knowledge about whether MCF10A cells reliably represent normal human mammary cells. MCF10A cells were grown in monolayer, suspension (mammosphere culture), three-dimensional (3D) “on-top” Matrigel, 3D “cell-embedded” Matrigel, or mixed Matrigel/collagen I gel. Suspension culture was performed with the MammoCult medium and low-attachment culture plates. Cells grown in 3D culture were fixed and subjected to either immunofluorescence staining or embedding and sectioning followed by immunohistochemistry and immunofluorescence staining. Cells or slides were stained for protein markers commonly used to identify mammary progenitor and epithelial cells. MCF10A cells expressed markers representing luminal, basal, and progenitor phenotypes in two-dimensional (2D) culture. When grown in suspension culture, MCF10A cells showed low mammosphere-forming ability. Cells in mammospheres and 3D culture expressed both luminal and basal markers. Surprisingly, the acinar structure formed by MCF10A cells in 3D culture was positive for both basal markers and the milk proteins β-casein and α-lactalbumin. MCF10A cells exhibit a unique differentiated phenotype in 3D culture which may not exist or be rare in normal human breast tissue. Our results raise a question as to whether the commonly used MCF10A cell line is a suitable model for human mammary cell studies.
PMCID: PMC4493126  PMID: 26147507
3.  microRNA-34b/c on chromosome 11q23 is aberrantly methylated in chronic lymphocytic leukemia 
Epigenetics  2014;9(6):910-917.
A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22–23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL.
PMCID: PMC4053441  PMID: 24686393
chronic lymphatic leukemia; micro-RNA; DNA methylation; epigenetics
4.  Biodegradable CSMA/PECA/Graphene Porous Hybrid Scaffold for Cartilage Tissue Engineering 
Scientific Reports  2015;5:9879.
Owing to the limited repair capacity of articular cartilage, it is essential to develop tissue-engineered cartilage for patients suffering from joint disease and trauma. Herein, we prepared a novel hybrid scaffold composed of methacrylated chondroitin sulfate (CSMA), poly(ethylene glycol) methyl ether-ε-caprolactone-acryloyl chloride (MPEG-PCL-AC, PECA was used as abbreviation for MPEG-PCL-AC) and graphene oxide (GO) and evaluated its potential application in cartilage tissue engineering. To mimic the natural extracellular matrix (ECM) of cartilage, the scaffold had an adequate pore size, porosity, swelling ability, compression modulus and conductivity. Cartilage cells contacted with the scaffold remained viable and showed growth potential. Furthermore, CSMA/PECA/GO scaffold was biocompatible and had a favorable degradation rate. In the cartilage tissue repair of rabbit, Micro-CT and histology observation showed the group of CSMA/PECA/GO scaffold with cellular supplementation had better chondrocyte morphology, integration, continuous subchondral bone, and much thicker newly formed cartilage compared with scaffold group and control group. Our results show that the CSMA/PECA/GO hybrid porous scaffold can be applied in articular cartilage tissue engineering and may have great potential to in other types of tissue engineering applications.
PMCID: PMC4426702  PMID: 25961959
5.  In vitro expression of Streptococcus pneumoniae ply gene in human monocytes and pneumocytes 
Streptococcus pneumoniae is one major cause of pneumonia in human and contains various virulence factors that contribute to pathogenesis of pneumococcal disease. This study investigated the role of pneumolysin, Ply, in facilitating S. pneumoniae invasion into the host blood stream.
S. pneumoniae strains were isolated from clinical blood and sputum samples and confirmed by PCR. Expression of ply gene was assessed by infecting human monocytes and pneumocytes.
A total of 23 strains of S. pneumoniae isolated from blood (n = 11) and sputum (n = 12) along with S. pneumoniae ATCC49619 were used to infect human monocyte (THP-1) and Type II pneumocyte (A549) cell lines. All clinical strains of S. pneumoniae showed higher expression of ply mRNA than the American Type Culture Collection (ATCC) strain. Among the clinical strains, blood isolates showed higher expression of ply genes than sputum isolates, i.e., 21.5- to 21.6-folds in THP-1 and 20.4- to 24.9-folds in A549 cell lines.
The data from the current study demonstrated that ply gene of blood- and sputum-derived S. pneumoniae is differentially expressed in two different cell lines. Under survival pressure, ply is highly expressed in these two cell lines for blood-derived S. pneumoniae, indicating that ply gene may facilitate S. pneumoniae invasion into the host blood system.
PMCID: PMC4426643  PMID: 25943628
Streptococcus pneumoniae; Infection; Virulence gene; Pneumolysin (ply)
6.  Growth inhibition and apoptosis induction by alternol in pancreatic carcinoma cells 
AIM: To investigate the effect of alternol on pancreatic cancer cells.
METHODS: Pancreatic cancer cells PANC-1 and BxPC3 were treated with various concentrations of alternol for 24, 48 and 72 h. Cell proliferation was measured by cell counting. Cell cycle distribution and mitochondrial membrane potential were determined by flow cytometry. Apoptosis was determined by a TdT-mediated dUTP nick end labeling assay and Hoechst staining. Expression of caspase 3, Bcl-2, p53 and p21 was measured by western blotting.
RESULTS: Alternol showed dose- and time-dependent inhibition of the proliferation of PANC-1 and BxPC3 cells in vitro. Alternol induced apoptosis and cell cycle arrest at S phase and decreased mitochondrial membrane potential. Alternol activated caspase 3, upregulated p53 and p21 expression, and downregulated Bcl-2 expression in a dose-dependent manner.
CONCLUSION: Our results suggested that alternol is a candidate for treatment of pancreatic cancer.
PMCID: PMC4402299  PMID: 25914461
Pancreatic cancer; Alternol; Chemotherapy; Cell cycle; Apoptosis
7.  Effects of 18α-glycyrrhizin on TGF-β1/Smad signaling pathway in rats with carbon tetrachloride-induced liver fibrosis 
Background: Glycyrrhizin has various pharmacological effects including hepato-protection. This study aimed to investigate the potential mechanism underlying the protective effects of 18α-glycyrrhizin (18α-GL) in rats with carbon tetrachloride (CCl4) induced liver fibrosis. Methods: Male Sprague-Dawley (SD) rats were randomly divided into control group, fibrosis group, 25 mg/kg 18α-GL group and 12.5 mg/kg 18α-GL group. Rats in experimental groups were subcutaneously injected with 40% CCl4 twice weekly for 8 weeks. Immunohistochemical examination was carried out to detect the protein expressions of collagen I, collagen III, TGF-β1, p-Smad2, p-Smad3, Smad 7 and SP-1, in the liver, and the mRNA and protein expressions of these genes were determined in the liver by real time PCR and Western blot assay, respectively. Results: 18α-GL ameliorated histological changes and significantly suppressed collagen deposition. 18α-GL significantly decreased the mRNA expressions of TGF-β1, Smad2, Smad3 and SP-1 in the liver. Immunohistochemical staining revealed that TGF-β1, p-Smad2, p-Smad3 and SP-1 expressions reduced following 18α-GL therapy. Western blot assay showed p-Smad2, p-Smad3, smad2 and smad3 expressions decreased after 18α-GL treatment. The mRNA and protein expression of Smad7 remained unchanged. Conclusion: 18α-GL is able to attenuate CCl4 induced liver fibrosis in rat.
PMCID: PMC4396252  PMID: 25973013
18α-glycyrrhizin; liver fibrosis; collagen; TGF-β1; Smad
8.  Claudin-1 enhances tumor proliferation and metastasis by regulating cell anoikis in gastric cancer 
Oncotarget  2014;6(3):1652-1665.
Claudin-1 (CLDN1) is overexpressed in gastric cancer and correlated with tumor invasion, metastasis and poor outcome. Here, we both down and up regulated CLDN1 expression in gastric cancer cells to elucidate its role in gastric carcinogenesis and tumor progression. We found that deficiency of CLDN1 inhibited cells migration, invasion, and colony formation in vitro and tumorigenicity, metastasis in vivo. Also, CLDN1 promoted cell aggregation and increased anoikis resistance. Down or up regulation of CLDN1 was accompanied with changes of membrane β-catenin expression as well as Akt and Src activities. When β-catenin was up-regulated in CLDN1-KD cells, cell aggregation and anoikis resistance were restored, and Akt and Src signal pathways were re-activated. Taken together, these findings suggest that CLDN1 is oncogenic in gastric cancer and its malignant potential may be attributed in part to regulation of anoikis, by mediating membrane β-catenin-regulated cell-cell adhesion and cell survival.
PMCID: PMC4359322  PMID: 25544763
Claudin-1; Anoikis; β-catenin; Gastric cancer
9.  Chikungunya Virus Glycoproteins Pseudotype with Lentiviral Vectors and Reveal a Broad Spectrum of Cellular Tropism 
PLoS ONE  2014;9(10):e110893.
Outbreaks of the Chikungunya virus (CHIKV) infection has been documented in over 40 countries, resulting in clinical symptoms characterized by fever and joint pain. Diagnosing CHIKV in a clinical lab setting is often omitted because of the high lab safety requirement. An infection system that mimics CHIKV infection will permit clinical evaluation of the production of neutralizing antibody for both disease diagnostics and treatment.
Methodology/Principal Findings
We generated a CHIKV construct expressing CHIKV structural proteins. This construct permits the production of CHIKV pseudo-viral particles with a luciferase reporter. The pseudo-virus was able to infect a wide range of cell lines. The pseudovirus could be neutralized by the addition of neutralizing antibodies from patients.
Taken together, we have developed a powerful system that can be handled at biosafety level 2 laboratories for evaluation of existence of CHIKV neutralizing antibodies.
PMCID: PMC4205015  PMID: 25333782
10.  Maternal embryonic leucine zipper kinase enhances gastric cancer progression via the FAK/Paxillin pathway 
Molecular Cancer  2014;13:100.
Elevated MELK expression is featured in multiple tumors and correlated with tumorigenesis and tumor development. This study is aimed to investigate the mechanisms of MELK-mediated development of gastric cancer.
MELK expression levels in human gastric cancer were determined by quantitative-PCR and immunohistochemistry. The effect of MELK on cell activity was explored by knockdown and overexpression experiments. Cell growth was measured using the CCK-8 assay. Apoptosis and cell cycle distributions were analyzed by flow cytometry. Migration and invasion were tested using a transwell migration assay. Cytoskeletal changes were analyzed by immunofluorescence. To explore the molecular mechanism and effect of MELK on migration and invasion, Western blotting was used to analyze the FAK/Paxillin pathway and pull down assays for the activity of small Rho GTPases. In vivo tumorigenicity and peritoneal metastasis experiments were performed by tumor cell engraftment into nude mice.
MELK mRNA and protein expression were both elevated in human gastric cancer, and this was associated with chemoresistance to 5-fluorouracil (5-FU). Knockdown of MELK significantly suppressed cell proliferation, migration and invasion of gastric cancer both in vitro and in vivo, decreased the percentages of cells in the G1/G0 phase and increased those in the G2/M and S phases. Moreover, knockdown of MELK decreased the amount of actin stress fibers and inhibited RhoA activity. Finally, knockdown of MELK decreased the phosphorylation of the FAK and paxillin, and prevented gastrin-stimulated FAK/paxillin phosphorylation. By contrast, MELK overexpression had the opposite effect.
MELK promotes cell migration and invasion via the FAK/Paxillin pathway, and plays an important role in the occurrence and development of gastric cancer. MELK may be a potential target for treatment against gastric cancer.
PMCID: PMC4113179  PMID: 24885567
MELK; Gastric cancer; Tumor migration; Tumor invasion; FAK; Paxillin
11.  The metastasis suppressor SOX11 is an independent prognostic factor for improved survival in gastric cancer 
International Journal of Oncology  2014;44(5):1512-1520.
SOX11 is involved in gastrulation and in malignant diseases. The aim of this study was to investigate the role of SOX11 in gastric cancer and its expression pattern and clinical significance. SOX11 overexpression cell model was used to examine in vitro and in vivo the role of SOX11 in cell growth and metastasis. Cell cycle analysis and Annexin V/PI double staining were used to investigate the effect of SOX11 on cell cycle progression and apoptosis. The expression of SOX11 in human gastric cancer was examined by immunohistochemistry. The correlation of SOX11 expression with clinicopathological characteristics and survival of patients was analyzed by Pearson’s χ2 and Kaplan-Meier analyses, respectively. Cox’s proportional hazard model was employed in multivariate analysis. SOX11 overexpression did not inhibit cell growth but strongly suppressed cell migration/invasion in vitro and in vivo. We found a significant correlation between high SOX11 protein levels and Lauren’s classification (intestinal type), differentiation status (high and medium), and early TNM stage. SOX11 is an independent prognostic factor for improved survival in gastric cancer patients. SOX11 was a potential tumor-suppressor and an independent positive prognostic factor in gastric cancer patients with less advanced clinicopathological features.
PMCID: PMC4027874  PMID: 24604109
SOX11; gastric cancer; prognostic factor; metastasis
12.  MiR-133b is frequently decreased in gastric cancer and its overexpression reduces the metastatic potential of gastric cancer cells 
BMC Cancer  2014;14:34.
Emerging evidence has shown that microRNAs are involved in gastric cancer development and progression. Here we examine the role of miR-133b in gastric cancer.
Quantitative real-time PCR analysis was performed in 140 patient gastric cancer tissues and 8 gastric cancer cell lines. The effects of miR-133b in gastric cancer cells metastasis were examined by scratch assay, transwell migration and matrigel invasion. In vivo effects of miR-133b were examined in an intraperitoneal mouse tumor model. Targets of miR-133b were predicted by bioinformatics tools and validated by luciferase reporter analyses, western blot, and quantitative real-time PCR.
MiR-133b was significantly downregulated in 70% (98/140) of gastric cancer patients. Expression of miR-133b was negatively correlated with lymph node metastasis of gastric cancer in patients. Similarly, the expression of miR-133b was significantly lower in seven tested gastric cancer cell lines than in the immortalized non-cancerous GES-1 gastric epithelial cells. Overexpression of miR-133b markedly inhibited metastasis of gastric cancer cells in vitro and in vivo. Moreover, the transcriptional factor Gli1 was identified as a direct target for miR-133b. Level of Gli1 protein but not mRNA was decreased by miR-133b. Activity of luciferase with Gli1 3′-untranslated region was markedly decreased by miR-133b in gastric cancer cells. Gli1 target genes, OPN and Zeb2, were also inhibited by miR133b.
MiR-133b is frequently decreased in gastric cancer. Overexpression of miR-133b inhibits cell metastasis in vitro and in vivo partly by directly suppressing expression of Gli1 protein. These results suggested that miR-133b plays an important role in gastric cancer metastasis.
PMCID: PMC3925791  PMID: 24443799
MicroRNA; miR-133b; Gastric cancer; Metastasis
13.  Mesoporous Magnetic Gold “Nanoclusters” as Theranostic Carrier for Chemo-Photothermal Co-therapy of Breast Cancer 
Theranostics  2014;4(7):678-692.
Photothermal therapy (PTT) is proved to be an efficient manner for superficial tumor therapy in preclinical studying. The tumor suppression of chemotherapy can be enhanced by combining with PTT. In this study, we reported a mesoporous magnetic gold “nanoclusters” (MMGNCs) structure as theranostic carrier for chemo-photothermal co-therapy. MMGNCs were successfully prepared and they exhibited efficient photo-thermal effect for PTT. The mesoporous structure provided MMGNCs with high drug loading capacity. By in vitro cytotoxicity testing, we revealed that the combination of PTT and chemotherapy could cause more damage than chemotherapy or PTT did alone. By topically targeting mediated by the extra-magnetic field (MF), MMGNCs can be targeted to the tumor site efficiently. In vivo chemo-photothermal co-therapy of 4T1 breast cancer, under the combinational treatments of chemo-photothermal co-therapy and extra-MF targeting, the tumor growth has been efficiently inhibited, and the pulmonary and mediastinal metastasis have also been prevented. The survival of the cancer bearing mice was prolonged. The bio-imaging applications of this system and the mechanism of the metastasis prevention are ongoing.
PMCID: PMC4038750  PMID: 24883118
Multifunctional nanoparticles; Photothermal Therapy; Multidrug Resistance; Co-therapy.
14.  Serum dihydroxyacetone kinase peptide m/z 520.3 as predictor of disease severity in patients with compensated chronic hepatitis B 
Background & aim
Due to known limitations of liver biopsy, reliable non-invasive serum biomarkers for chronic liver diseases are needed. We performed serum peptidomics for such investigation in compensated chronic hepatitis B (CHB) patients.
Liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed peptides in sera from 40 CHB patients (20 with S0G0-S1G1 and 20 with S3G3-S4G4). Ion pair quantification from differentially expressed peptides in a validation set of sera from 86 CHB patients was done with multiple reaction monitoring (MRM).
21 differentially represented peptide peaks were found through LC-MS/MS. Ion pairs generated from eleven of these peptides (m/z < 800) were quantified by MRM. Summed peak area ratios of 6 ion pairs from peptide m/z 520.3 (176.1, 353.7, 459.8, 503.3, 351.3, 593.1), which was identified as dihydroxyacetone kinase (DAK) fragment, decreased from mild to advanced stages of fibrosis or inflammation. Area Under Receiver Operating Characteristic Curves (AUROCs) of five ion models discriminating fibrosis degrees were 0.871 ~ 0.915 (S2-4 versus S0-1) and 0.804 ~ 0.924 (S3-4 versus S0-2). AUROCs discriminating inflammation grades were 0.840 ~ 0.902 (G2-4 versus G0-1) and 0.787 ~ 0.888 (G3-4 versus G0-2). The diagnostic power of these models provides improved sensitivity and specificity for predicting disease progression as compared to aspartate aminotransferase to platelet ratio index (APRI), FIB-4, Forn’s index and serum DAK protein.
The peptide fragment (m/z 520.3) of DAK is a promising biomarker to guide timing of antiviral treatment and to avoid liver biopsy in compensated CHB patients.
PMCID: PMC3851457  PMID: 24289155
Peptidome; Dihydroxyacetone kinase; Chronic hepatitis B; Multiple reaction monitoring; Liquid chromatography combined with tandem mass spectrometry
15.  Decrease of miR-202-3p Expression, a Novel Tumor Suppressor, in Gastric Cancer 
PLoS ONE  2013;8(7):e69756.
Emerging studies have indicated that microRNAs are involved in the development and progression of cancer. Here we found that miR-202-3p was frequently down-regulated in gastric cancer tissues. Overexpression of miR-202-3p in gastric cancer cells MKN-28 and BGC-823, markedly suppressed cell proliferation and induced cell apoptosis both in vitro and in vivo. Furthermore, Gli1 expression was frequently positive in gastric cancer tissues and inversely correlated with miR-133b expression. We demonstrate that the transcriptional factor Gli1 was a target of miR-202-3p and plays an essential role as a mediator of the biological effects of miR-202-3p in gastric cancer. MiR-202-3p also inhibited the expression of γ-catenin and BCL-2. Taken together, these findings suggest that miR-202-3p may function as a novel tumor suppressor in gastric cancer and its anti-tumor activity may attribute the direct targeting and inhibition of Gli1.
PMCID: PMC3723650  PMID: 23936094
16.  Roles of virulence genes (PsaA and CpsA) on the invasion of Streptococcus pneumoniae into blood system 
Streptococcus pneumoniae (SP) is the major cause of childhood mortality worldwide, we need to understand virulence genes of SP so can better target the treatment.
We investigated the expression of virulence genes PsaA and CpsA in different strains of SP interacting with monocyte cell line (THP-1) or pneumocyte cell line (A549) and the possible mechanism of SP invasion of the blood system.
A total of 23 strains of SP were collected from hospitalized patients (blood-derived and sputum-derived) in the Second Affiliated Hospital of Wenzhou Medical College. The strains and ATCC 49619 were cultured, and RNAs were extracted. THP-1 and A549 cells were stimulated by different SP and ATCC 49619 for 4 h and 8 h, respectively. Quantitative real-time PCR was used to analyze the mRNA expression of PsaA and CpsA. The data were analyzed by SPSS 17.0.
The mRNA level of PsaA and CpsA were all significantly increased in clinical SP strains when compared to ATCC49619 after tedTHP-1 and A549 cells were stimulated. Clinical SPs showed higher virulence compared with ATCC49619.
The expression of CpsA is the basis of the pathogenicity of SP. The expression of virulence gene PsaA may be helpful to the invasion of SP to the blood system.
PMCID: PMC3695859  PMID: 23683724
Streptococcus pneumonia; Real-time PCR; Virulence gene; PsaA; CpsA
17.  Stromal fibroblasts in the microenvironment of gastric carcinomas promote tumor metastasis via upregulating TAGLN expression 
BMC Cell Biology  2013;14:17.
Fibroblasts play a critical role in tumorigenesis, tumor progression and metastasis. However, their detailed molecular characteristics and clinical significance are still elusive. TAGLN is an actin-binding protein that plays an important role in tumorigenesis.
We investigated the interaction between cancer cells and the tumor microenvironment to determine how the fibroblasts from human gastric carcinoma facilitate tumorigenesis through TAGLN. QRT-PCR and Western blot indicated that TAGLN expression was upregulated in gastric carcinoma-associated fibroblasts (CAFs) that promote gastric cancer cell migration and invasion. Using small interfering RNA (siRNA), we found that CAFs enhanced tumor metastasis through upregulated TAGLN in vitro and in vivo. The expression of matrix metalloproteinase-2 (MMP-2) was significantly lower after TAGLN knock-down by siRNA. TAGLN levels were elevated in human gastric cancer stroma than normal gastric stroma and associated with differentiation and lymph node metastasis of gastric cancer.
CAFs may promote gastric cancer cell migration and invasion via upregulating TAGLN and TAGLN induced MMP-2 production.
PMCID: PMC3610155  PMID: 23510049
TAGLN; Fibroblast; Microenvironment; Tumor metastasis; Gastric carcinoma
18.  Quinelorane, a dopamine D3/D2 receptor agonist, reduces prepulse inhibition of startle and ventral pallidal GABA efflux: Time course studies 
Startle is inhibited when the startling stimulus is preceded 30–300 ms by a weak prepulse. Prepulse inhibition (PPI), an operational measure of sensorimotor gating, is deficient in schizophrenia patients, and reduced in rats and humans by dopamine agonists. The neural basis for the PPI-disruptive effects of dopamine agonists in rats is studied to understand neural circuitry regulating PPI and its deficits in schizophrenia. Existing data suggest that ventral pallidal (VP) GABAergic transmission regulates PPI and its disruption by dopamine agonists. We measured changes in VP GABA efflux and PPI in rats in response to the D2/D3 agonist, quinelorane. Wistar rats were administered quinelorane (vehicle, 0.003 or 0.01 mg/kg). In some rats, VP dialysate was analyzed for GABA content. In others, PPI was assessed using 120 dB(A) startle pulses and prepulses 10 dB over a 70 dB(A) background. Quinelorane reduced GABA efflux, with significant effects for 0.01 but not 0.003 mg/kg, persisting for at least 100 min. Quinelorane reduced PPI for 50 min, an effect significant for both the 0.003 (p<0.05) and 0.01 mg/kg doses (p<0.015). Differences in time course and dose sensitivity of quinelorane effects on VP GABA efflux and PPI are discussed.
PMCID: PMC3250091  PMID: 18579193
gamma amino butyric acid (GABA); microdialysis; capillary electrophoresis-coupled laser-induced fluorescence (CE-LIF); dopamine; quinelorane; prepulse inhibition; schizophrenia; startle
19.  18α-Glycyrrhetinic Acid Down-Regulates Expression of Type I and III Collagen via TGF-Β1/Smad Signaling Pathway in Human and Rat Hepatic Stellate Cells 
Objective: To investigate the effects of 18α-glycyrrhetinic acid (18α-GA) on the expression of type I and III collagen in human and rat hepatic stellate cells (HSC) and to explore the role of TGF-β1/Smad signaling pathway involved.
Methods: Following 18α-GA treatment, the cell viability and cell growth were detected to determine the optimal concentration of 18α-GA. The expressions of TGF-β1/Smad signaling-related genes including type I and III collagen in human and rat HSCs before and after 18α-GA treatment were measured by real time PCR. The expression of related proteins was verified by western blot assay. The phosphorylation level of Smad2 and Smad3 was detected by immunocytochemistry. The DNA binding activities of SP-1, AP-1 and NF-κB were measured by both EMSA and ArrayStar transcription factor activity assay.
Results: 18α-GA could decrease the mRNA and protein expression of Smad3, type I and III collagen, increase the Smad7 expression in human and rat HSCs (P<0.05), and reduce phosphorylation level of Smad3 at 24 h and 48 h after treatment. The DNA binding activities of transcription factors were suppressed by 18α-GA in human and rat HSCs at 24 h, and the activities reduced in a time dependent manner with the lowest activities at 48 h, especially for SP-1.
Conclusion: 18α-GA could inhibit the mRNA and protein expression of type I and III collagen in human and rat HSCs, which may be attributed to down-regulation of Smad3, up-regulation of Smad7, and inhibition of DNA binding activities of SP-1, AP-1 and NF-κB.
PMCID: PMC3399217  PMID: 22811611
18α-glycyrrhetinic acid; hepatic stellate cell; TGF-β1/Smad; transcription factor
20.  18α-Glycyrrhizin induces apoptosis and suppresses activation of rat hepatic stellate cells 
To investigate the potential mechanisms underlying the protective effects of 18α Glycyrrhizin (GL) on rat hepatic stellate cells (HSCs) and hepatocytes in vivo and in vitro.
Sprague-Dawley (SD) rats were randomly divided into 5 groups: normal control group, liver fibrosis group, high-dose 18α GL group (25 mg/kg/d), intermediate-dose 18α GL group (12.5 mg/kg/d) and low-dose 18α GL group (6.25 mg/kg/d). The rat liver fibrosis model was induced by carbon tetrachloride (CCl4). The expressions of α-smooth muscle actin (αSMA) and NF-κB were determined by real-time PCR and immunohistochemistry.
18αGL dose-dependently inhibited the CCl4-induced liver fibrosis. There were significant differences in the mRNA and protein expressions of αSMA between the fibrosis group and 18α-GL treatment groups, suggesting that 18α GL can suppress the proliferation and activation of HSCs. Few HSCs were apoptotic in the portal area and fibrous septum in the liver fibrosis group. However, the double-color staining of a-SMA and TUNEL showed that 18α-GL treatment groups increased HSC apoptosis. NF-κB was mainly found in the nucleus in the fibrosis group, while cytoplasmic expression of NF-κB was noted in the 18αGL groups. In the in vitro experiments, 18α GL promoted the proliferation of hepatocytes, but inhibited that of HSCs. HSCs were arrested in the G2/M phase following 18α GL treatment and were largely apoptotic.
18α-GL can suppress the activation of HSCs and induce the apoptosis of HSCs by blocking the translocation of NF-κB into the nucleus, which plays an important role in the protective effect of 18α-GL on liver fibrosis.
PMCID: PMC3560665  PMID: 22207106
18α-glycyrrhizin; hepatocyte; hepatic stellate cell; proliferation; apoptosis
21.  Thioredoxin-like 2 regulates human cancer cell growth and metastasis via redox homeostasis and NF-κB signaling 
Cancer cells have an efficient antioxidant system to counteract their increased generation of ROS. However, whether this ability to survive high levels of ROS has an important role in the growth and metastasis of tumors is not well understood. Here, we demonstrate that the redox protein thioredoxin-like 2 (TXNL2) regulates the growth and metastasis of human breast cancer cells through a redox signaling mechanism. TXNL2 was found to be overexpressed in human cancers, including breast cancers. Knockdown of TXNL2 in human breast cancer cell lines increased ROS levels and reduced NF-κB activity, resulting in inhibition of in vitro proliferation, survival, and invasion. In addition, TXNL2 knockdown inhibited tumorigenesis and metastasis of these cells upon transplantation into immunodeficient mice. Furthermore, analysis of primary breast cancer samples demonstrated that enhanced TXNL2 expression correlated with metastasis to the lung and brain and with decreased overall patient survival. Our studies provided insight into redox-based mechanisms underlying tumor growth and metastasis and suggest that TXNL2 could be a target for treatment of breast cancer.
PMCID: PMC3007146  PMID: 21123948
22.  Realistic expectations of prepulse inhibition in translational models for schizophrenia research 
Psychopharmacology  2008;199(3):331-388.
Under specific conditions, a weak lead stimulus, or “prepulse”, can inhibit the startling effects of a subsequent intense abrupt stimulus. This startle-inhibiting effect of the prepulse, termed “prepulse inhibition” (PPI), is widely used in translational models to understand the biology of brain based inhibitory mechanisms and their deficiency in neuropsychiatric disorders. In 1981, four published reports with “prepulse inhibition” as an index term were listed on Medline; over the past 5 years, new published Medline reports with “prepulse inhibition” as an index term have appeared at a rate exceeding once every 2.7 days (n = 678). Most of these reports focus on the use of PPI in translational models of impaired sensorimotor gating in schizophrenia. This rapid expansion and broad application of PPI as a tool for understanding schizophrenia has, at times, outpaced critical thinking and falsifiable hypotheses about the relative strengths vs. limitations of this measure.
This review enumerates the realistic expectations for PPI in translational models for schizophrenia research, and provides cautionary notes for the future applications of this important research tool.
In humans, PPI is not “diagnostic”; levels of PPI do not predict clinical course, specific symptoms, or individual medication responses. In preclinical studies, PPI is valuable for evaluating models or model organisms relevant to schizophrenia, “mapping” neural substrates of deficient PPI in schizophrenia, and advancing the discovery and development of novel therapeutics. Across species, PPI is a reliable, robust quantitative phenotype that is useful for probing the neurobiology and genetics of gating deficits in schizophrenia.
PMCID: PMC2771731  PMID: 18568339
Animal models; Antipsychotic; Dopamine; Prepulse inhibition; Schizophrenia; Sensorimotor gating; Startle
23.  Neural basis for a heritable phenotype: differences in the effects of apomorphine on startle gating and ventral pallidal GABA efflux in male Sprague–Dawley and Long–Evans rats 
Psychopharmacology  2009;207(2):271-280.
Prepulse inhibition (PPI) of startle is a measure of sensorimotor gating that is heritable and deficient in certain psychiatric disorders, including schizophrenia. Sprague–Dawley (SD) rats are more sensitive to PPI disruptive effects of dopamine (DA) agonists at long interstimulus intervals (60–120 ms) and less sensitive to their PPI-enhancing effects at short (10–30 ms), compared with Long–Evans (LE) rats. These heritable strain differences in sensitivity to the PPI disruptive effects of DA agonists must ultimately reflect neural changes "downstream" from forebrain DA receptors.
The current study evaluated the effects of the DA agonist, apomorphine (APO), on ventral pallidal (VP) gamma-aminobutyric acid (GABA) and glutamate efflux and PPI in SD and LE rats.
PPI was tested in SD and LE rats after vehicle or APO (0.5 mg/kg, subcutaneously (s.c.)) in a within-subject design. In different SD and LE rats, VP dialysate was collected every 10 min for 120 min after vehicle or APO (0.5 mg/kg, s.c.) and analyzed for GABA and glutamate content by capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF).
As predicted, SD rats exhibited greater APO-induced PPI deficits at long intervals and less APO-induced PPI enhancement at short intervals compared to LE rats. APO significantly reduced VP GABA efflux in SD but not in LE rats; glutamate efflux was unaffected in both strains.
Heritable strain differences in PPI APO sensitivity in SD vs LE rats parallel, and may be mediated by, strain differences in the VP GABA efflux.
PMCID: PMC2770636  PMID: 19756524
Gamma-aminobutyric acid (GABA); Microdialysis; Dopamine; Prepulse inhibition; Schizophrenia; Strain; CE-LIF
24.  Comparative proteomics analysis of human gastric cancer 
AIM: To isolate and identify differentially expressed proteins between cancer and normal tissues of gastric cancer by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).
METHODS: Soluble fraction proteins of gastric cancer tissues and paired normal tissues were separated by 2-DE. The differentially expressed proteins were selected and identified by MALDI-TOF-MS and database search.
RESULTS: 2-DE profiles with high resolution and reproducibility were obtained. Twenty-three protein spots were excised from sliver staining gel and digested in gel by trypsin, in which fifteen protein spots were identified successfully. Among the identified proteins, there were ten over-expressed and five under-expressed proteins in stomach cancer tissues compared with normal tissues.
CONCLUSION: In this study, the well-resolved, reproducible 2-DE patterns of human gastric cancer tissue and paired normal tissue were established and optimized and certain differentially-expressed proteins were identified. The combined use of 2-DE and MS provides an effective approach to screen for potential tumor markers.
PMCID: PMC2748199  PMID: 18837081
Proteome; Two-dimensional polyacrylamide gel electrophoresis; Mass spectrometry; Gastric cancer; Tumor markers
25.  Inhibitory effect of Polo-like kinase 1 depletion on mitosis and apoptosis of gastric cancer cells 
AIM: Polo-like kinase 1 (PLK1) serine/threonine kinase plays a vital role in multiple phases of mitosis in gastric cancer cells. To investigate the effect of PLK1 depletion on mitosis and apoptosis of gastric cancer cells.
METHODS: PLK1 expression was blocked by small RNA interference(siRNA). The expression levels of PLK1, cdc2, cyclin B and caspase 3 were detected by Western blotting. Then, PLK1 depletion, cdc2 activity, cell proliferation, cell cycle phase distribution, mitotic spindle structure, and the rate of apoptosis of the PLK1 knockdown cells were observed.
RESULTS: PLK1 gene knockdown was associated with increased cyclin B expression, increased cdc2 activity (but not with the expression levels), accumulation of gastric cancer cells at G2/M, improper mitotic spindle formation, delayed chromosome separation and delayed or arrested cytokinesis. Moreover, PLK1 depletion in gastric cancer cells was associated with decreased proliferation, attenuated pro-caspase 3 levels and increased apoptosis.
CONCLUSION: Blockage of PLK1 expression may lead to decreased mitosis or even apoptosis in gastric cancer cells, indicating that PLK1 may be a valuable therapeutic target for gastric cancer.
PMCID: PMC4077490  PMID: 16440413
Gastric cancer cells; PLK1 gene; Mitosis; Cell cycle; Apoptosis

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