Quercetin exerts anti-inflammatory effects via inhibiting tumor necrosis factor-α-induced matrix metalloproteinase-9 expression in normal human gastric epithelial cells

BACKGROUND Gastric injury is the most common digestive system disease worldwide and involves inflammation,which can lead to gastric ulcer or gastric cancer(GC).Matrix metallopeptidase-9[MMP-9(gelatinase-B)]plays an important role in inflammation and GC progression.Quercetin and quercetin-rich diets represent potential food supplements and a source of medications for treating gastric injury given their anti-inflammatory activities.However,the effects and mechanisms of action of quercetin on human chronic gastritis and whether quercetin can relieve symptoms remain unclear.AIM To assess whether tumor necrosis factor-α(TNF-α)-induced MMP-9 expression mediates the anti-inflammatory effects of quercetin in normal human gastric mucosal epithelial cells.METHODS The normal human gastric mucosa epithelial cell line GES-1 was used to establish a normal human gastric epithelial cell model of TNF-α-induced MMP-9 protein overexpression to evaluate the antiinflammatory effects of quercetin.The cell counting Kit-8 assay was used to evaluate the effects of varying quercetin doses on cell viability in the normal GES-1 cell line.Cell migration was measured using Transwell assay.The expression of proto-oncogene tyrosine-protein kinase Src(cSrc),phospho(p)-c-Src,extracellular-signal-regulated kinase 2(ERK2),p-ERK1/2,c-Fos,p-c-Fos,nuclear factor kappa B(NF-κB/p65),and p-p65 and the effects of their inhibitors were examined using Western blot analysis and measurement of luciferase activity.p65 expression was detected by immunofluorescence.MMP-9 m RNA and protein levels were measured by quantitative reverse transcription polymerase chain reaction(q RT–PCR)and gelatin zymography,respectively.RESULTS q RT-PCR and gelatin zymography showed that TNF-αinduced MMP-9 m RNA and protein expression in a dose-and time-dependent manner.These effects were reduced by the pretreatment of GES-1 cells with quercetin or a TNF-αantagonist(TNFR inhibitor)in a dose-and timedependent manner.Quercetin and TNF-αantagonists decreased the TNF-α-induced phosphorylation of c-Src,ERK1/2,c-Fos,and p65 in a dose-and time-dependent manner.Quercetin,TNF-αantagonist,PP1,U0126,and tanshinone IIA(TSIIA)reduced TNF-α-induced c-Fos phosphorylation and AP-1–Luciferase(Luc)activity in a dose-and time-dependent manner.Pretreatment with quercetin,TNF-αantagonist,PP1,U0126,or Bay 11-7082 reduced TNF-α-induced p65 phosphorylation and translocation and p65–Luc activity in a dose-and timedependent manner.TNF-αsignificantly increased GES-1 cell migration,and these results were reduced by pretreatment with quercetin or a TNF-αantagonist.CONCLUSION Quercetin significantly downregulates TNF-α-induced MMP-9 expression in GES-1 cells via the TNFR-c-Src–ERK1/2 and c-Fos or NF-κB pathways.

Basic Investigations EXPRESSION OF GAP JUNCTION PROTEIN Cx43 IN CULTURED HUMAN NORMAL AND MALIGNANT LUNG CELLS

Gap junctional intercellular communicationexchange of small molecules and ions between contiguous cells through membranous gap junctional channelsis essential for growth control and tissue homecotasis. This work concerns the functional expression of gap junction protein connexin 43 (Cx43) in normal human lung cells and the changes in lung carcinoma cells. By. using Northern blot hybridization analysis and Cx43 immunocytochemical methods, it was otherved that cultured normal human embryonic lung cells expressed a high level of Cx43 in both mRNA and protein levels.The Cx43 immunofluorescence was localized at cell membrane regions corresponding to the location of gap junctions. These normal lung cells were competent of intercellular communication function as detected by Lucifer yellow dye transfer. In contrast to normal celis, Cx43 mRNA and protein was not detectable in the carcinoma PG cell line. These tumor cells were defective of intercellular communication function. These results demonstrate that Cx43 is expressed in normal cultured human embryonic lung cells but not in lung tumor cells. The lack of intercellular communication in the lung tumor cell line correlates with dysfunctional intercellular communication. The suggestive role of Cx as a tumor suppersor gene is discussed.

S Phase Cell Percentage Normalized BrdU Incorporation Rate, a New Parameter for Determining S Arrest

In this study, a new parameter, S phase cell percentage （S fraction） normalized BrdU （SFN-BrdU） incorporation rate, was introduced to detect $ arrest. The results showed a positive linear correlation between the BrdU incorporation rate and the S fraction in unperturbed 16HBE cells. Theoretical analysis indicated that only S arrest could result in a decrease in the SFN-BrdU incorporation rate. Additionally, the decrease in SFN-BrdU incorporation rate and the activation of DNA damage checkpoints further demonstrated that S arrest was induced by diethyl sulfate treatment of 16HBE cells. In conclusion, $FN-BrdU incorporation rate can be used to detecting S arrest.

CHARACTERIZING FLUORESCENCE LIFETIME OF NAD(P)H IN HUMAN LEUKEMIC MYELOID CELLS AND MONONUCLEAR CELLS

The aim of this er vito study was to explore the potential of using the fluorescence lifetime of intraellular reduced nicotinamide adenine dinucleotide(phosphate)(NAD(P)H)as a label-free indicator to characterize the di ferencs between human leukemic myeloid cells and normal mononuclear cells(MNC).The steady-state and time-resolved autofuorescence of two human leukemic myeloid cell lines(K562,HL60)and MNC were measured by a spectrofuorimeter.According to excitation-enmission matrix(EEM)analysis,the optimal emission of NAD(P)H in these cells suspensions occurred at 445 nm.Furthermore,the fuorescence lifetimes of NAD(P)H in leukemic myeloid cells and MNC were determined by fitting the time-resolved autofuorescence data.The mean fuorescence lifetimes of NAD(P)H in K562,HL60,and MNC cells were 557±1.19,4.45±0.71,and 7.31±0.60 ns,respectively.There was a significant diference in the mean lifetime of NAD(P)H between leukemic myeloid cells and MNC(p<0.05).The difference was essentally caused by the change in relative concentration of free and protein-bound NAD(P)H.This study suggests that the mean fuorescence lifetime of NAD(P)H might be a potential label-free indicator for differentiating leukemic myeloid cells from MNC.

Differential Proteomics in Malignant and Normal Liver Cell Lines

Objective： To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods： Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 （Immobilized Mental Affinity Capture） and WCX2 （Weak Cation Exchange） chips on the SELDI-TOF-MS ProteinChip reader. Results： Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion： Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

Conditionally Reprogrammed Human Normal Airway Epithelial Cells at ALI: A Physiological Model for Emerging Viruses

Cancer cell lines have been used widely in cancer biology, and as biological or functional cell systems in many biomedical research fields. These cells are usually defective for many normal activities or functions due to significant genetic and epigenetic changes. Normal primary cell yields and viability from any original tissue specimens are usually relatively low or highly variable. These normal cells cease after a few passages or population doublings due to very limited proliferative capacity. Animal models(ferret, mouse, etc.) are often used to study virus-host interaction. However, viruses usually need to be adapted to the animals by several passages due to tropism restrictions including viral receptors and intracellular restrictions. Here we summarize applications of conditionally reprogrammed cells(CRCs), long-term cultures of normal airway epithelial cells from human nose to lung generated by conditional cell reprogramming(CR) technology, as an ex vivo model in studies of emerging viruses. CR allows to robustly propagate cells from non-invasive or minimally invasive specimens, for example, nasal or endobronchial brushing. This process is rapid(2 days) and conditional. The CRCs maintain their differentiation potential and lineage functions, and have been used for studies of adenovirus, rhinovirus, respiratory syncytial virus, influenza viruses, parvovirus, and SARS-CoV. The CRCs can be easily used for airliquid interface(ALI) polarized 3 D cultures, and these coupled CRC/ALI cultures mimic physiological conditions and are suitable for studies of viral entry including receptor binding and internalization, innate immune responses, viral replications, and drug discovery as an ex vivo model for emerging viruses.

Telomerase and hTERT: Can they serve as markers for gastric cancer diagnosis?

AIM: To investigate telomerase activity and human telomerase reverse transcriptase (hTERT) expression in normal human gastric mucosal epithelial cells (nhGMECs) and fibroblasts (nhGMFs).

Role of RANTES and Its Receptor in Gastric Cancer Metastasis

This study examined the role of regulated upon activation normal T cell expressed and secreted（RANTES） and its receptor C-C chemokine receptor type 5（CCR5） in gastric cancer metastasis and the associated mechanism.The expression of RANTES and CCR5 was detected by using immunohistochemical staining and Western blotting in the gastric cancer tissues obtained from 60 gastric cancer patients with or without lymph node metastasis（n=30 in each）.The results showed that the expression levels of RANTES and CCR5 were higher in gastric cancer with lymph node metastasis than in that without metastasis（P0.05）.The expression levels of RANTES in 30 lymph nodes with cancerous invasion were higher than in 30 normal lymph nodes（P0.05）.Chemotactic test revealed that the number of migrating gastric cancer cells（n=295.0±54.6） induced by the protein of cancer-invading lymph nodes was greater than that by the protein mixture from cancer-invading lymph nodes and RANTES antibody（n=42.5±11.6）（P0.05）.RT-PCR showed that the expression levels of the main Th1 cytokines（IL-2,γ-IFN） were lower in gastric cancer with lymph node metastasis（2.22±0.90,3.26±1.15 respectively） than in that without metastasis（3.07±1.67,4.77±1.52 respectively）（P0.05）,but the expression level of the main Th 2 cytokine（IL-10） was higher in gastric cancer with lymph nodes metastasis（6.06±2.04） than in that without metastasis（4.88±1.87）（P0.05）.It was concluded that RANTES and its receptor CCR5 may contribute to gastric cancer metastasis through influencing the balance of Th1/Th2.RANTES and CCR5 may become a marker of gastric cancer metastasis.

Targeting tight junctions during epithelial to mesenchymal transition in human pancreatic cancer

Pancreatic cancer continues to be a leading cause of cancer-related death worldwide and there is an urgent need to develop novel diagnostic and therapeutic strategies to reduce the mortality of patients with this disease. In pancreatic cancer, some tight junction proteins, including claudins, are abnormally regulated and therefore are promising molecular targets for diagnosis, prognosis and therapy. Claudin-4 and-18 are overexpressed in human pancreatic cancer and its precursor lesions. Claudin-4 is a high affinity receptor of Clostridium perfringens enterotoxin(CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer. Claudin-18 could be a putative marker and therapeutic target with prognostic implications for patients with pancreatic cancer. Claudin-1,-7, tricellulin and marvelD3 are involved in epithelial to mesenchymal transition(EMT) of pancreatic cancer cells and thus might be useful as biomarkers during disease. Protein kinase C is closely related to EMT of pancreatic cancer and regulates tight junctions of normal human pancreatic duct epithelial cells and the cancer cells. This review focuses on the regulation of tight junctions via protein kinase C during EMT in human pancreatic cancer for the purpose of developing new diagnostic and therapeutic modalities for pancreatic cancer.

The evolving landscape of prostate cancer stem cell: Therapeutic implications and future challenges

Prostate cancer(PCa)is the most common cause of malignancy in males and the second leading cause of cancer mortality in United States.Current treatments for PCa include surgery,radiotherapy,and androgen-deprivation therapy.Eventually,PCa relapses to an advanced castration-resistant PCa(CRPC)that becomes a systematic disease and incurable.Therefore,identifying cellular components and molecular mechanisms that drive aggressive PCa at early stage is critical for disease prognosis and therapeutic intervention.One potential strategy for aggressive PCa is to target cancer stem cells(CSCs)that are identified by several unique characteristics such as immortal,self-renewal,and pluripotency.Also,CSC is believed to be a major factor contributing to resistance to radiotherapy and conventional chemotherapies.Moreover,CSCs are thought to be the critical cause of metastasis,tumor recurrence and cancer-related death of multiple cancer types,including PCa.In this review,we discuss recent progress made in understanding prostate cancer stem cells(PCSCs).We focus on the therapeutic strategies aimed at targeting specific surface markers of CSCs,the key signaling pathways in the maintenance of self-renewal capacity of CSCs,ATP-binding cassette(ABC)transporters that mediate the drug-resistance of CSCs,dysregulated microRNAs expression profiles in CSCs,and immunotherapeutic strategies developed against PCSCs surface markers.ª2016 Editorial Office of Asian Journal of Urology.Production and hosting by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Effects of polar cortical cytoskeleton and unbalanced cortical surface tension on intercellular bridge thinning during cytokinesis

To probe the contributions of polar cortical cytoskeleton and the surface tension of daughter cells to intercellular bridge thinning dynamics during cytokinesis,we applied cytochalasin D（CD） or colchicine（COLC） in a highly localized manner to polar regions of dividing normal rat kidney（NRK） cells.We observed cellular morphological changes and analyzed the intercellular bridge thinning trajectories of dividing cells with different polar cortical characteristics.Global blebbistatin（BS） application was used to obtain cells losing active contractile force groups.Our results show that locally released CD or colchicine at the polar region caused inhibition of cytokinesis before ingression.Similar treatment at phases after ingression allowed completion of cytokinesis but dramatically influenced the trajectories of intercellular bridge thinning.Disturbing single polar cortical actin induced transformation of the intercellular bridge thinning process,and polar cortical tension controlled deformation time of intercellular bridges.Our study provides a feasible framework to induce and analyze the effects of local changes in mechanical properties of cellular components on single cellular cytokinesis.

MORPHOMETRIC ANALYSIS OF SPINOUS CELL IN ORAL SUBMUCOUS FIBROSIS COMPARISON WITH NORMAL MUCOSA, LEUKOPLAKIA AND SQUAMOUS CELL CARCINOMA

The morphometric analysis of the spinous cell in 16 specimens of oral submucous fibrosis (OSF) was made by using interactive image analysis system (IBAS-II). 19 parameters of the size and shape were chosen, and compared with normal mucosa, leukoplakia, dysplasia and carcinoma. The results indicated that the cell dimensions (area, perimeter, all kinds of diameter) and nuclear cytoplasmic ratio in OSF were between normal mucosa and dysplasia as well as carcinoma. The former showed a progressive decrease (P<0.01), and the latter showed a progressive increase (P<0.01). The dimensions of the nuclei did not show considerable differences among the groups (P>0.05). A series of discriminant functions had been developed with stepwise discriminant analysis, the agreement ratio for OSF was 93.75%. The decrease of cell area and the increase of nuclear cytoplasmic ratio could reflect a malignant progress. The cell morphometric model could discriminate OSF well from other groups, suggesting that the change of the epith

Water–soluble and polarity–sensitive near–infrared fluorescent probe for long–time specific cancer cell membranes imaging and C. Elegans label

A novel D–π –A structure and near–infrared fluorescent probe(DCITT) with high polarity sensitivity and membrane targeting was reported. The fluorescent spectra of DCITT were polarity dependent and Stokes shift was greater than 300 nm. Due to its high fluorescence quantum yield, low cytotoxicity and photostability, DCITT could be used as a labeling probe in multicellular organisms. In particular, DCITT effectively distinguished tumor cells from normal cells because it could specifically light up the cancer cells membrane based on strong red fluorescence for a long time. On this basis, a polar–sensitive cell membrane probe is developed to differentiate tumor cells from normal cells, which provides an idea and method for the early diagnosis of tumor at cellular level.

Manifesting viscosity changes in lipid droplets during iodined CT contrast media treatment by the real-time and in situ fluorescence imaging

Computed tomography(CT) is one of the most commonly used non-invasive clinical imaging modalities to predict, diagnose and treat the disease. Iodinated contrast media(ICM) is a form of intravenous radiocontrast agent containing iodine, which enhances the visibility of hollow tissue structures in medical CT imaging. ICM may cause allergic reactions, contrast-induced nephropathy, hyperthyroidism and possibly metformin accumulation. It is significant to find out the risk factors, pathogenesis, diagnosis, prevention, and treatment of adverse reactions caused by ICM. Revealing the changes of the lipid droplets(LDs)viscosity in pathophysiological processes such as cancer and iodined contrast media induced adverse reaction is not only important for monitoring the occurrence and development of some pathophysiological processes but also vital for the deep insight of the biological effects of LDs in these pathophysiological processes. A lipid droplets targeted fluorescent probe DN-1 was devised to sense cellular viscosity alteration with high selectivity and sensitivity, which was applied to distinguish cancer cells and normal cells and reveal viscosity changes during iodined CT contrast media treatment.

New design of a compact aero-robotic drilling end effector: An experimental analysis

This paper presents the development of a normal adjustment cell (NAC) in aero-robotic drilling to improve the quality of vertical drilling, by using an intelligent double-eccentric disk normal adjustment mechanism (2-EDNA), a spherical plain bearing and a floating compress module with sensors. After the surface normal vector is calculated based on the laser sensors' feedback, the 2-EDNA concept is conceived specifically to address the deviation of the spindle from the surface normal at the drilling point. Following the angle calculation, depending on the actual initial position, two precise eccentric disks (PEDs) with an identical eccentric radius are used to rotate with the appropriate angles using two high-resolution DC servomotors. The two PEDs will carry the spindle to coincide with the surface normal, keeping the vertex of the drill bit still to avoid repeated adjustment and position compensation. A series of experiments was conducted on an aeronautical drilling robot platform with a precise NAC. The effect of normal adjustment on bore diameter, drilling force, burr size, drilling heat, and tool wear was analyzed. The results validate that using the NAC in robotic drilling results in greatly improved vertical drilling quality and is attainable in terms of intelligence and accuracy. (C) 2015 The Authors. Production and hosting by Elsevier Ltd. on behalf of Chinese Society of Aeronautics and Astronautics.