Human Epithelial Cell Adhesion Research Articles

Comparison of Adhesion, Invasion, Motility, and Toxin Production ofCampylobacterStrains and Their Resistant Mutants

The objectives of this study were to compare the in vitro adhesion and invasion of human epithelial cells, motility, and toxin production characteristics of Campylobacter-susceptible strains and their fluoroquinolone- or macrolide-resistant mutants. Susceptible strains and resistant mutants demonstrated similar adhesion capacities to epithelial cells. For Campylobacter coli, fluoroquinolone-resistant mutants with Thr86Ile or Asp90Asn substitutions showed a higher rate of invasion of Caco-2 cells than their isogenic parental strain. Fluoroquinolone resistance did not impact C. coli motility. Mutants harboring Asp90Asn had greater cytotoxic activity than the parental strain. Macrolide resistance had no impact on the studied characteristics of C. coli. For Campylobacter jejuni, fluoroquinolone-resistant mutants had slightly different invasiveness levels and significantly lower motility than the isogenic parental strain. C. jejuni macrolide-resistant mutants with A2074G substitution in the 23S rRNA gene had a higher invasiveness level than its parental strain, but mutants with A2074C in 23S rRNA and G221A in rplD showed reduced motility and similar invasion levels to the susceptible strains. Neither fluoroquinolone nor macrolide resistance appears to affect C. jejuni cytotoxicity. In conclusion, mutations that are frequently encountered in Campylobacter-resistant strains can enhance the invasiveness in Caco-2 cells.

The influence of biomimetic topographical features and the extracellular matrix peptide RGD on human corneal epithelial contact guidance

A major focus in the field of tissue engineering is the regulation of essential cell behaviors through biophysical and biochemical cues from the local extracellular environment. The impact of nanotopographical cues on human corneal epithelial cell (HCEC) contact guidance, proliferation, migration and adhesion have previously been demonstrated. In the current report we have expanded our study of HCEC responses to include both biophysical and controlled biochemical extracellular cues. By exploiting methods for the layer-by-layer coating of substrates with reactive poly(ethylene imine)/poly(2-vinyl-4,4-dimethylazlactone)-based multilayer thin films we have incorporated a single adhesion peptide motif, Arg–Gly–Asp (RGD), on topographically patterned substrates. This strategy eliminates protein adsorption onto the surface, thus decoupling the effects of the HCEC response to topographical cues from adsorbed proteins and soluble media proteins. The direction of cell alignment was dependent on the scale of the topographical cues and, to less of an extent, the culture medium. In EpiLife® medium cell alignment to unmodified-NOA81 topographical features, which allowed protein adsorption, differed significantly from cell alignment on RGD-modified features. These results demonstrate that the surface chemical composition significantly affects how HCECs respond to topographical cues. In summary, we have demonstrated modulation of the HCEC response to environmental cues through critical substrate and soluble parameters.

Effects of exogenous secreted protein acidic and rich in cysteine on human lens epithelial cell proliferation and adhesion

Many factors can impact human lens epithelium biological behavior. Secreted protein acidic and rich in cysteine (SPARC) is a newly discovered protein that can impact cell activity and may potentially affect human lens epithelial cells (HLECs). HLEC (SRA01/04) proliferation was detected by methyl thiazolyl tetrazolium (MTT) and the proliferation index was assessed by flow cytometry with SPARC at concentrations of 0, 0.2, 1 and 5 μg/ml. SPARC’s effect on cell cycle regulatory genes CCND1 and P21Waf1 was analyzed by PCR and SPARC’s effect on cell adhesion was detected by an enzyme labeling assay. SPARC displayed an inhibitory effect on HLEC proliferation. The amount of inhibition was related to SPARC concentrations and cell culture times. The inhibition rates were 9.67, 48.11 and 68.12% at concentrations of 0.2, 1 and 5 μg/ml, respectively, after 72 h of cell culture. With a MTT method, SPARC showed counter-adhesion of HLECs and this counter-adhesion was related to the concentration of SPARC. SPARC decreased the number of S+G2/M cells and caused G0/G1 phase arrest. With a 5 μg/ml SPARC treatment, CCND1 mRNA levels were significantly decreased and P21Waf1 mRNA levels were increased in comparison with the control group. SPARC inhibits HLEC proliferation and adhesion, which is related to the SPARC concentration and cell culture time. Therefore, SPARC seems to influence HLEC biological behavior. Key words: Secreted protein acidic and rich in cysteine (SPARC), human lens epithelial cells (HLEC), adhesion, proliferation, cell cycle regulatory gene.

Influence of single walled carbon nanotubes at subtoxical concentrations on cell adhesion and other cell parameters of human epithelial cells

The production of SWCNT has increased tremendously in the last decade, due to high expectations of their benefits. Despite all precautions SWCNT may come into contact with organisms and may be taken up by them. The aim of the present study was to assess and elucidate the effects of SWCNT bundles (purified SWCNT) and SWCNT raw material on the behaviour of human epithelial cells (A549). When cells were exposed to SWCNT (bundles and raw material) cell adhesion was significantly affected. The effects of SWCNT were dependent on the SWCNT purity (purified SWCNT or SWCNT raw material), the SWCNT concentration and the incubation period. SWCNT raw material affected cell adhesion more than the SWCNT bundles did. Beside effects on cell adhesion, cell spreading and the development of focal adhesion points and the actin skeleton were affected, however to a much lesser extent. Cells grown in the presence of SWCNT showed an elevated amount of reactive oxygen species (ROS). The effect was concentration dependent.Other cell parameters, such as cell viability, cell death (apoptosis and necrosis), activation of cell signalling proteins, such as focal adhesion kinase (FAK) and mitogen activated protein kinase (MAPK-Erk1/2), as well as the gap junctional intercellular communication proteins (connexin-43 and β-catenin) were not seriously affected when cells were grown in the presence of SWCNT.

Inhibition of Prostaglandin E2 Receptors EP2 and EP4 Inhibits Adhesion of Human Endometriotic Epithelial and Stromal Cells Through Deregulated Integrin-Mediated Mechanisms.

Inhibition of Prostaglandin E2 Receptors EP2 and EP4 Inhibits Adhesion of Human Endometriotic Epithelial and Stromal Cells Through Deregulated Integrin-Mediated Mechanisms.

CTNNAL1 promotes the proliferation and adhesion of human bronchial epithelial cells

CTNNAL1 promotes the proliferation and adhesion of human bronchial epithelial cells

High interstitial fluid pressure is associated with low tumour penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes.

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via “wick-in-needle” technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.

Down‐regulation of dysferlin and myoferlin in human airway epithelium: differential effects on cell morphology and adhesion

Dysferlin and myoferlin belong to Ferlin protein family known to mediate endomembrane fusion events. Mutations of dysferlin lead to muscular dystrophy. Patients with dysferlinopathy show cardiopulmonary complications, suggesting that dysferlin may have functional roles in the pulmonary system. The high homology of myoferlin to dysferlin proposes that myoferlin may also play a role in lung pathology.ObjectivesTo examine dysferlin and myoferlin expression in human lungs and investigate the effects of silencing these genes in human airway epithelial cells (HAEC).MethodsDysferlin and myoferlin expression was evaluated by immunohistochemistry in human airway sections and by Western blot analysis in HAEC. Localization of dysfelrin and myoferlin in HAEC was studied using immunofluorescent staining. Effects of dysferlin and myoferlin knockdown on the morphology and adhesion of HAEC were also assessed.ResultsBoth dysferlin and myoferlin were present in the epithelium of human airway tissues and cultured HAEC. Dysferlin and myoferlin expression localized at the membrane and cytoplasm of HAEC. Knockdown of myoferlin, but not dysferlin, resulted in cell morphological changes, deficient cell adhesion and decreased the expression of zonular occludens 1 in HAEC.ConclusionPresence of dysferlin and myoferlin suggests they play potential roles in the adhesion of human airway epithelial cells.

1060 SURVIVIN EXPRESSING CIRCULATING TUMOR CELLS IN NON-MUSCHE INVASIVE BLADDER CANCER A CELLECTION™ DYNABEADS ANALYSIS

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20121060 SURVIVIN EXPRESSING CIRCULATING TUMOR CELLS IN NON-MUSCHE INVASIVE BLADDER CANCER A CELLECTION™ DYNABEADS ANALYSIS Gian Maria Busetto, Arianna Petracca, Chiara Nicolazzo, Paola Gazzaniga, Vincenzo Gentile, and Ettore De Berardinis Gian Maria BusettoGian Maria Busetto Roma, Italy More articles by this author , Arianna PetraccaArianna Petracca Roma, Italy More articles by this author , Chiara NicolazzoChiara Nicolazzo Roma, Italy More articles by this author , Paola GazzanigaPaola Gazzaniga Roma, Italy More articles by this author , Vincenzo GentileVincenzo Gentile Roma, Italy More articles by this author , and Ettore De BerardinisEttore De Berardinis Roma, Italy More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1166AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The prognosis of T1G3 bladder cancer is highly ariable and not predictable basing upon clinical and pathological prognostic factors. There is need for improvement in risk stratification in this population; understanding the molecular profile of individual patients could provide a more personalized and tailored treatment. Main objective was to evaluate the prognostic significance of Survivin in tumor tissues and that of Survivin expressing circulating tumor cells (CTCs) in T1G3 tumors. METHODS 161 patients with T1G3 non muscle invasive bladder cancer (NMIBC), including CIS associated, were enrolled. Planned follow up was 24 mo. Survivin was evaluated by RT-PCR in tumoral tissues. CTCs were isolated from blood by CELLection™ dynabeads coated with the monoclonal antibody towards the human Epithelial Cell Adhesion Molecule. Cells were lysed and Dynabeads Oligo(dT) was used to capture poly A+ mRNA. cDNA was synthesised and analysed for the expression of CD45, CK8 and Survivin. The primary end point was disease free survival (DFS); the favourable group at 24 mo was defined as that without any clinical evidence of disease (NED); the unfavourable group was that with evidence of recurrent disease (RD) or progressive disease (PD). Tumour survivin expression and presence of CTC were correlated with DFS. Multivariate analysis was used to investigate whether the presence of CTC was an independent indicator of DFS. RESULTS Survivin was found in about 50% of tumors. Survivin-patients showed a longer DFS than Survivin + tumors (X2=6.222, p=0.019). CTCs were found in 84/161 patients (52%); 96% of CTC were Survivin expressing. The difference in DFS between CTC - and CTC + patients was statistically significant (X2=29.565; p=0.001). CTC presence was found an independent prognostic factor of DFS (p=0.001). CONCLUSIONS To date, for bladder cancer, there is the lack of a realible marker able to predict the prognosis and with the capability to remark the biological bahaviour of hign-risk NMIBC. The presence of CTC is an independent prognostic factor in patients with T1G3 bladder cancer and in the future could be an important parameter to evaluate. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e430 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gian Maria Busetto Roma, Italy More articles by this author Arianna Petracca Roma, Italy More articles by this author Chiara Nicolazzo Roma, Italy More articles by this author Paola Gazzaniga Roma, Italy More articles by this author Vincenzo Gentile Roma, Italy More articles by this author Ettore De Berardinis Roma, Italy More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Therapeutic Potential of Amanitin-Conjugated Anti-Epithelial Cell Adhesion Molecule Monoclonal Antibody Against Pancreatic Carcinoma

Human epithelial cell adhesion molecule (EpCAM) is overexpressed in many cancers. Anti-EpCAM antibodies have shown promise in preclinical studies, but showed no tumor regression in a recent phase II clinical trial. Therefore, we generated a novel anti-EpCAM antibody-drug conjugate and assessed whether it showed enhanced antitumor effects. Chemical cross-linking was conducted to covalently conjugate α-amanitin, a toxin known to inhibit DNA transcription, with chiHEA125, a chimerized anti-EpCAM monoclonal antibody, to generate the antibody-drug conjugate α-amanitin-glutarate-chiHEA125 (chiHEA125-Ama). Antiproliferative activity of chiHEA125-Ama was tested in human pancreatic (BxPc-3 and Capan-1), colorectal (Colo205), breast (MCF-7), and bile duct (OZ) cancer cell lines in vitro using [(3)H]-thymidine incorporation assay. Antitumor activity of chiHEA125-Ama was assessed in vivo in immunocompromised mice bearing subcutaneous human BxPc-3 pancreatic carcinoma xenograft tumors (n = 66 mice). Cell proliferation and apoptosis were evaluated in xenograft tumors by immunohistochemistry. All statistical tests were two-sided. In all cell lines, chiHEA125-Ama reduced cell proliferation (mean half maximal inhibitory concentration [IC(50)] = 2.5 × 10(-10) to 5.4 × 10(-12) M). A single dose of chiHEA125-Ama inhibited BxPc-3 xenograft tumor growth (chiHEA125 [control, n = 4 mice] vs. chiHEA125-Ama [n = 6 mice], dose of 15 mg/kg with respect to IgG and 50 μg/kg with respect to α-amanitin, mean relative increase in tumor volume on day 16 = 884% vs. -79%, difference = 963%, 95% CI = 582% to 1344%, P = .019). Two higher doses of chiHEA125-Ama (100 μg/kg with respect to α-amanitin), administered 1 week apart (n = 10 mice per group), led to complete tumor regression in nine of 10 (90%) mice compared with chiHEA125, during the observation period of 16 days; increased apoptosis and reduced cell proliferation were observed in mice treated with chiHEA125-Ama. This preclinical study suggests that anti-EpCAM antibody conjugates with α-amanitin have the potential to be highly effective therapeutic agents for pancreatic carcinomas and various EpCAM-expressing malignancies.

Expression, crystallization and preliminary X-ray characterization of the human epithelial cell-adhesion molecule ectodomain

The epithelial cell-adhesion molecule (EpCAM; CD326) is a transmembrane glycoprotein involved in epithelial cell-cell adhesion, cell proliferation and differentiation. Its elevated expression level in various carcinomas is exploited by several antitumour therapies that are at various stages of clinical development. The 35 kDa polypeptide chain of EpCAM is divided into a large extracellular part, a transmembrane helix and a short cytoplasmic tail. The modular extracellular part of human EpCAM was cloned and mutated to prevent N-linked glycosylation. After expression in insect cells and purification using standard chromatographic techniques, the extracellular part was crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 86.83, b = 50.16, c = 66.56 Å, β = 127.9°. The crystal diffracted to 1.95 Å resolution and contained one molecule in the asymmetric unit.

Utilization of in vitro methods to determine the biocompatibility of intraocular lens materials

In vitro methods for measuring the adhesion and viability of lens epithelial cells on implant devices are needed to assess material biocompatibility. We investigated whether the use of confocal microscopy and spectrophotometric methods could determine the viability and adhesion of cells on a silicone biomaterial. Human lens epithelial cells adhered to silicone were treated with 0.01% benzalkonium chloride (cationic surfactant), 0.1% sodium dodecyl sulfate (anionic surfactant), and 10% Tween 20 (nonionic surfactant). Cell viability was then assessed using two fluorescent dyes (calcein and ethidium homodimer-1). Adhesion was determined directly by measuring the number of attached cells after surfactant treatment and by an indirect method that utilized the colorimetric agent crystal violet. The number of viable cells remaining on the biomaterial was determined both immediately after exposure and after the cells were allowed to grow for 1day following surfactant exposure. The measurements for adhesion showed that the anionic surfactant weakened cell surface binding more than the cationic or nonionic surfactant. This study demonstrated that confocal microscopy in conjunction with crystal violet as an indirect colorimetric indicator can quantify the viability and adhesion of human lens epithelial cells attached to a material surface.

Niche‐specific contribution to streptococcal virulence of a MalR‐regulated carbohydrate binding protein

Low G+C Gram-positive bacteria typically contain multiple LacI/GalR regulator family members, which often have highly similar amino-terminal DNA binding domains, suggesting significant overlap in target DNA sequences. The LacI/GalR family regulator catabolite control protein A (CcpA) is a global regulator of the Group A Streptococcus (GAS) transcriptome and contributes to GAS virulence in diverse infection sites. Herein, we studied the role of the maltose repressor (MalR), another LacI/GalR family member, in GAS global gene expression and virulence. MalR inactivation reduced GAS colonization of the mouse oropharynx but did not detrimentally affect invasive infection. The MalR transcriptome was limited to only 25 genes, and a highly conserved MalR DNA-binding sequence was identified. Variation of the MalR binding sequence significantly reduced MalR binding in vitro. In contrast, CcpA bound to the same DNA sequences as MalR but tolerated variation in the promoter sequences with minimal change in binding affinity. Inactivation of pulA, a MalR regulated gene which encodes a cell surface carbohydrate binding protein, significantly reduced GAS human epithelial cell adhesion and mouse oropharyngeal colonization but did not affect GAS invasive disease. These data delineate a molecular mechanism by which hierarchical regulation of carbon source utilization influences bacterial pathogenesis in a site-specific fashion.

507 HIGH-RISK NMIBC (NON-MUSCLE INVASIVE BLADDER CANCER): PROGNOSTIC VALUE OF CIRCULATING TUMOR CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research1 Apr 2011507 HIGH-RISK NMIBC (NON-MUSCLE INVASIVE BLADDER CANCER): PROGNOSTIC VALUE OF CIRCULATING TUMOR CELLS Ettore De Berardinis, Gian Maria Busetto, Arianna Petracce, Chiara Nicolazzo, Paola Gazzaniga, and Vincenzo Gentile Ettore De BerardinisEttore De Berardinis Roma, Italy More articles by this author , Gian Maria BusettoGian Maria Busetto Roma, Italy More articles by this author , Arianna PetracceArianna Petracce Roma, Italy More articles by this author , Chiara NicolazzoChiara Nicolazzo Roma, Italy More articles by this author , Paola GazzanigaPaola Gazzaniga Roma, Italy More articles by this author , and Vincenzo GentileVincenzo Gentile Roma, Italy More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1203AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The prognosis of T1G3 bladder cancer is highly variable and not predictable basing upon clinical and pathological prognostic factors. There is need for improvement in risk stratification in this population; understanding the molecular profile of individual patients could provide a more personalized and tailored treatment. Main objective was to evaluate the prognostic significance of Survivin in tumor tissues and that of Survivin expressing circulating tumor cells (CTCs) in T1G3 tumors. METHODS 141 patients with T1G3 non muscle invasive ladder cancer (NMIBC) were enrolled. Additional inclusion criteria were: absence of CIS and multifocality. Planned follow up was 24 mo. Survivin was evaluated by RT-PCR in tumoral tissues. CTCs were isolated from blood by CELLection™ Dynabeads coated with the monoclonal antibody towards the human Epithelial Cell Adhesion Molecule. Cells were lysed and Dynabeads Oligo(dT) was used to capture poly A_mRNA. cDNA was synthesised and analysed for the expression of CD45, CK8 and Survivin. The primary end point was disease free survival (DFS); the favourable group at 24 mo was defined as that without any clinical evidence of disease (NED); the unfavourable group was that with evidence of recurrent disease (RD) or progressive disease (PD). Tumoral Survivin expression and presence of CTC were correlated to DFS. Multivariate nalysis was used to investigate whether CTC presence was independent indicator of DFS. RESULTS Survivin was found in about 50% of tumors. Survivin - patients showed a longer DFS than Survivin + (/2:4.572; p=0.029). CTCs were found in 51/141 patients (47%); 94% of CTC were Survivin expressing. The difference in DFS between CTC - and CTC + patients was statistically significant (/2: 28.098; p<0.001). CTC presence was found an independent prognostic factor of DFS (p<0.001). CONCLUSIONS CTC presence is an independent prognostic factor in high risk NMIBC patients. In the future will be important the count of CTC and this will allow us to establish the correlation between CTC number and prognosis. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e205-e206 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ettore De Berardinis Roma, Italy More articles by this author Gian Maria Busetto Roma, Italy More articles by this author Arianna Petracce Roma, Italy More articles by this author Chiara Nicolazzo Roma, Italy More articles by this author Paola Gazzaniga Roma, Italy More articles by this author Vincenzo Gentile Roma, Italy More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Gene expression profile of circulating tumor cells (CTCs) in clear cell metastatic renal cell carcinoma (mRCC).

302 Background: The presence of circulating tumor cells (CTCs) is described in 50% of patients affected by mRCC. The detection of CTC seems to be correlated with advanced tumor stage and more aggressive tumor phenotype. In some other solid tumors, such as breast, colorectal and prostate cancer, a growing body of evidence is emerging about the molecular portrait of these cells, whose nature is still largely unknown. The lack of information about CTCs characterization in RCC strongly hampers their application into the clinical practice. Aim of the study was to trace a gene expression profile of CTCs in mRCC patients. Methods: 18 metastatic clear cell mRCC patients were enrolled in the study. All patients provided informed consent. CTCs were isolated from 10cc of peripheral blood by CELLection Dynabeads coated with the monoclonal antibody towards the human Epithelial Cell Adhesion Molecule (EpCam). CELLection Epithelial Enrich is designed to optimally enrich bead-free, viable epithelial tumor cells. Tumor cells were eluted, subjected to RNA extraction and cDNA synthesis. PCR was then performed to investigate the expression of cytokeratin 8 (CK8) and vimentin as markers of clear cell RCC, multidrug resistance (MDR1), multidrug resistance related proteins 1–5–7 (MRP 1, MRP5, MRP 7) as drug resistance markers. Results: According with standard CTCs definition (CK+/CD45-), we found their presence in 9/18 samples (50%). Out of them, vimentin was found in 2/9 (22%), MDR1 in 3/9 (33%), MRP1 in 1/9 (11%), MRP5 in 6/9 (66%) and MRP7 in 9/9 (100%). Conclusions: To our knowledge this is the first attempt to describe a gene expression portrait in CTCs from clear cell mRCC. Of note is that vimentin, classically used as diagnostic marker for clear cell RCC is often lost in CTCs. This seems to further confirm the discrepancy between CTCs and primary tumor phenotype, already demonstrated in breast cancer. The expression profile of genes involved in multidrug resistance in CTCs seems to reflect the well known drug resistance observed in RCC. How these biological information obtainable from a gene expression profile performed on CTCs in clear cell mRCC may be translated into the clinic is currently under investigation. No significant financial relationships to disclose.

Influence of RGDRGD peptide on proliferation and adhesion of human lens epithelial cells in vitro

Background RGD is a small molecular multiple peptide containing Arg-Gly-Asp with an important role in inhibiting the adhesion,migration and neovascularization of tumor.Our previous study determined that RGD can suppress the adhesion and proliferation of lens epithelial cells(LECs),and RGDRGD may be of a stronger effect. Objective Present study was to investigate and compare the effect of RGDRGD peptide on the proliferation and adhesion of immortalized human LECs(HLEB-3)in vitro. Methods Human LECs harvested by trpsin-EDTA were suspended in DMEM medium with serial dilutions of RGD peptide and RGDRGD peptide(from 1000 mg/L to 250 mg/L)at 37℃ for 15 minutes as experimental group,and the HLECs cultured by common culture medium were used as the control group.The cells were then seeded into the 96-well plates with precoated fibmnectin (FN)and I collagen at the density of 2×104/ml.MTT stainingcolorimetry was used to measure the adhesion rates of lens epithelial cells cultured in different concentrations RGDRGD and RGD peptides after 1 hour.Cells were seeded into the 96-well plates for 24 hours at 37℃ in 5% CO2.Medium was then replaced with DMEM overnight.Subsequently,the cells were treated with serial dilutions of RGD and RGDRGD(from 2000 mg/L to 250 ms/L)dissolved in DMEM medium plus 20% fetal bovine serum.The inhibition of RGDRGD and RGD on the adhesion and proliferation of Human LECs was analyzed by MTT aher 24,48 and 72 hours. Results The inhibition rate of RGD peptide on the adhesion of LECs was gradually enhanced with the increase of concentration with the significant difference among the different concentrations groups(F=1089.56,P＜0.01),and the statistically significant elevation in inhibitory rate was found in RGDRGD peptide compared with RGD peptide(P＜0.01).The inhibition rate of RGDRGD peptide on the proliferation of LECs wag gradually increased with the increase of concentration with the significant difference among the different concentrations groups with a strongest effect in 1000 ms/L group(F=127.31,P＜0.01),and the much stronger inhibition Wag Been in RGDRGD peptide(F=1589.85,P＜0.01).The suppression rate of RGDRGD on LECs proliferation Wag much stronger with the prolong of time(F=1606.43,P＜0.01). Conclusion RGDRGD peptide and RGD peptide have inhibitory effect on adhesion and proliferation of human LECs in a dose-and time-dependent manner.Effect of RGDRGD peptide is much stronger than RGD peptide.These results imply that RGDRGD peptide and RGD peptide have the important role for prevention of PCO. Key words: RGDRGD peptide; Lens epithelial cells; Posterior capsular opacity

Adhesion and Mechanical Properties of RSV Infected Human Epithelial Cells

Respiratory syncytial virus is the leading cause of lower respiratory tract infection in infants and currently lacks an effective vaccine or treatment beyond symptom relief. The atomic force microscope is particularly well suited for imaging biological samples including cells, DNA, viruses and proteins. Analysis of fixed HEP-2 cells with the AFM after infection and incubation with RSV for periods of 0.5–24 h reveals several physical changes within the cells that may lead to a better understanding of the viral effects on living cells. Analysis of force–distance curves reveals changes in the mechanical properties of the cells throughout the infection period as well as changes in the surface chemistry, specifically in the presence of hydrophobic domains, of the cell membrane as shown by variability in the measured adhesion force. Elastic moduli of the fixed cells did not show a statistically significant trend, but hydrophobic molecular expression on the cellular surface decreased with increasing infection period. This change in the chemical nature of the cell membrane might be explained by the rapid removal, repair and replacement of the lipid bilayer occurring as a result of the exocytosis of RSV virions.

Relationship between ganglioside expression and anti-cancer effects of the monoclonal antibody against epithelial cell adhesion molecule in colon cancer

The human colorectal carcinoma-associated GA733 antigen epithelial cell adhesion molecule (EpCAM) was initially described as a cell surface protein selectively expressed in some myeloid cancers. Gangliosides are sialic acid-containing glycosphingolipids involved in inflammation and oncogenesis. We have demonstrated that treatment with anti-EpCAM mAb and RAW264.7 cells significant inhibited the cell growth in SW620 cancer cells, but neither anti-EpCAM mAb nor RAW264.7 cells alone induced cytotoxicity. The relationship between ganglioside expression and the anti- cancer effects of anti-EpCAM mAb and RAW264.7 was investigated by high-performance thin-layer chromatography. The results demonstrated that expression of GM1 and GD1a significantly increased in the ability of anti-EpCAM to inhibit cell growth in SW620 cells. Anti-EpCAM mAb treatment increased the expression of anti-apoptotic proteins such as Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-α, caspase-3, cleaved caspase-3, and cleaved caspase-8 were unaltered. We observed that anti-EpCAM mAb significantly inhibited the growth of colon tumors, as determined by a decrease in tumor volume and weight. The expression of anti-apoptotic protein was inhibited by treatment with anti-EpCAM mAb, whereas the expression of pro-apoptotic proteins was increased. These results suggest that GD1a and GM1 were closely related to anticancer effects of anti-EpCAM mAb. In light of these results, further clinical investigation should be conducted on anti-EpCAM mAb to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.

Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits adhesion, migration, and invasion of human endometriotic cells

OBJECTIVE: Prostaglandin E2 plays an important role in the pathogenesis of endometriosis. The objective of the present study was to determine functional interaction between PGE2 signaling and adhesion, migration, and invasion of human endometriotic epithelial and stromal cells and to unravel the underlying molecular and cellular mechanisms. DESIGN: Human immortalized endometriotic epithelial and stromal cells were treated with EP2 and EP4 receptor inhibitors, and their effects on adhesion, migration, and invasion of cells were studied and the underlined molecular mechanisms were uncovered. MATERIALS AND METHODS: Adhesion of endometriotic cells with ECM substrate and invasion of endometriotic ells into matrigel were analyzed in vitro. Set of integrins, MMPs and TIMPS proteins were analyzed by western blot. RESULTS: Results of the present study indicated that selective inhibition of EP2 and EP4-mediated PGE2 signaling: (i) suppressed expression and/or activity specific integrin receptors a2b1, avb3, a5b1, and a3b1 and thereby decreased adhesion of human endometriotic epithelial and stromal cells to extracellular matrix substrates collagen I, collagen IV, vitronectin, fibronectin, and laminins; and (ii) suppressed expression and/or activity of MMP1, MPP2, MMP3, MMP7 and MMP9 and increased expression of TIMP1, TIMP2, TIMP3, and TIMP4 and thereby decreased migration and invasion of human endometriotic epithelial and stromal cells. CONCLUSION: These novel findings may provide an important molecular framework for further evaluation of selective inhibition of EP2 and EP4 as potential nonestrogen or nonsteroidal therapy to expand the spectrum of currently available treatment options for endometriosis in child-bearing age women. Further, our results suggest potential opportunities for translational studies that could lead to preclinical and clinical-phase trials.

Prognostic significance of survivin‐expressing circulating tumour cells in T1G3 bladder cancer

To evaluate the prognostic significance of survivin in tumour tissues and that of survivin-expressing circulating tumour cells (CTCs) in T1G3 bladder tumours, as the prognosis of T1G3 bladder cancer is highly variable and unpredictable from clinical and pathological prognostic factors. The study included 54 patients with T1G3 non-muscle-invasive bladder cancer. Additional inclusion criteria were: tumour size <3 cm, absence of carcinoma in situ and multifocality. The planned follow-up was 24 months. Survivin was evaluated by reverse transcription-polymerase chain reaction in tumour tissues. CTCs were isolated from blood by CELLection Dynabeads (Invitrogen, Carlsbad, CA, USA) coated with the monoclonal antibody towards the human epithelial cell adhesion molecule. Cells were lysed and Dynabeads Oligo(dT) was used to capture poly A + mRNA. cDNA was synthesized and analysed for the expression of CD45, CK8 and survivin. The primary endpoint was disease-free survival (DFS); the favourable group at 24 months was defined as that with no clinical evidence of disease; the unfavourable group was that with evidence of recurrent disease or progressive disease. Tumour survivin expression and presence of CTC were correlated with DFS. Multivariate analysis was used to investigate whether the presence of CTC was an independent indicator of DFS. Survivin was found in half of the tumours; patients with survivin-negative tumours had a longer DFS than those with survivin-positive tumours (chi-square, P = 0.029). CTCs were found in 24/54 patients (44%); 92% of CTC expressed survivin. The difference in DFS between CTC-ve and CTC+ve patients was statistically significant (chi-square, P < 0.001). The presence of CTC was an independent prognostic factor for DFS (P < 0.001). The presence of CTC is an independent prognostic factor in patients with T1G3 bladder cancer.