Integrin β3 down-regulates invasive features of ovarian cancer cells in SKOV3 cell subclones

Purpose  To investigate the role of integrin β3 in invasive features of ovarian cancer SKOV3 cells, by comparing different metastatic subclones. Methods  In the present study, two cell subclones, termed as S1 and S21, which possessed high and low metastatic potential, respectively, were isolated and established from human ovarian cancer parental cell line SKOV3 by the limited dilution method. The expressions of integrin αv, integrin αvβ3, integrin β3, E-cadherin, FAK and ILK in the two cell subclones were compared by means of real-time RT-PCR or flow cytometry. Subsequently, S21 was transfected with siRNA for integrin β3 and the effects of transfection were examined by methyl thiazolyl tetrazolium (MTT) assay, colony formation assay, Matrigel invasion assay and cell migration assay. Results  The expressions of integrin αvβ3, integrin β3 and E-cadherin were markedly down-regulated in S1; however, there were no significant differences in the expressions of integrin αv, FAK and ILK β. Of note, more than 70% knockdown of integrin β3 expression was obtained by siRNA technique. The integrin β3-siRNA-transfected cells showed significant increases in cell proliferation, cell migration and invasive activity in contrast with the mock-transfected cells. The expressions of integrin αvβ3 and E-cadherin were lower in the integrin β3-siRNA-transfected cells compared to the mock control. Conclusion  Integrin β3, like E-cadherin, may be also a suppressor gene down-regulating invasive features of ovarian cancer cells in SKOV3 cell subclones.     

Expression of c-kit receptor in human cholangiocarcinoma and in vivo treatment with imatinib mesilate in chimeric mice

AIM: To investigate the c-kit expression in biliary tract cancer cell lines and histological sections from patients with extrahepatic cholangiocarcinoma (CC) and to evaluate the efficacy of in vitro and in vitro treatment with imatinib mesilate. METHODS: The protein expression of c-kit in the human biliary tract cancer cell lines Mz-ChA-2 and EGI-1 and histological sections from 19 patients with extrahepatic CC was assessed by immunoblotting, immunocytochemistry, and immunohistochemistry. The anti-proliferative effect of imatinib mesilate on biliary tract cancer cell lines Mz-ChA-2 and EGI-1 was studied in vitro by automated cell counting. In addition, immunodeficient NMRI mice (TaconicTM) were subcutaneously injected with 5×106 cells of cell lines MzChA-2 and EGI-1. After having reached a tumour volume of 200 mm3, daily treatment was started intra peritonea I ly with imatinib mesilate at a dose of 50 mg/kg or normal saline (NS). Tumor volume was calculated with a Vernier caliper. After 14 d, mice were sacrificed with tumors excised and tumor mass determined. RESULTS: Immunoblotting revealed presence of c-kit in Mz-ChA-2 and absence in EGI-1 cells. Immunocytochemistry with c-kit antibodies displayed a cytoplasmatic and membraneous localization of receptor protein in Mz-ChA-2 cells and absence of c-kit in EGI-1 cells, c-kit was expressed in 7 of 19 (37%) extrahepatic human CC tissue samples, 2 showed a moderate and 5 a rather weak immunostaining. Imatinib mesilate at a low concentration of 5μmol/L caused a significant growth inhibition in the c-kit positive cell line Mz-ChA-2 (31%), but not in the c-kit negative cell line EGI-1 (0%) (P<0.05). Imatinib mesilate at an intermediate concentration of 10μmol/L inhibited cellular growth of both cell lines (51% vs 57%). Imatinib mesilate at a higher concentration of 20μmol/L seemed to have a general toxic effect on both cell lines. The IC50 values were 9.7μmol/L and 11μmol/L, respectively. After 14 d of in vitro treatment with imatinib mesilate, using the chimeric mouse model, c-kit positive Mz-ChA-2 tumors had a significantly reduced volume and mass as compared to NS treatment (P< 0.05). In contrast to that, treatment of mice bearing c-kit negative EGI-1 tumors did not result in any change of tumor volume and mass as compared to NS treatment. CONCLUSION: c-kit expression is detectable at a moderate to low protein level in biliary tract cancer. Imatinib mesilate exerts marked effects on tumor growth in vitro and in vitro dependent on the level of c-kit expression.     

Oocytes of baboon fetal primordial ovarian follicles express estrogen receptor beta mRNA

In fetal ovaries of estrogen-suppressed baboons, we have previously shown that follicle numbers were 50% lower than in estrogen-replete animals and contained oocytes with a reduced number of microvilli. In the baboon fetal ovary, although estrogen receptor (ER)α and β have been detected by immunocytochemistry in granulosa cells, it is not known whether oocytes express ER. Because the actions of estrogen are mediated by interaction with cell-specific receptors, the current study determined whether ERα/β mRNA were expressed in oocytes of baboon fetal ovaries obtained on day 165 (term = day 184) of gestation. Oocyte nuclei and cytoplasm from primordial follicles were isolated by laser capture microdissection and ERα, ERβ, GATA-4 (granulosa cell specific marker) mRNAs, and 18S rRNA determined by RT-PCR and products verified by sequencing. ERβ mRNA was expressed in oocytes of 5 of 5 fetuses. In contrast, fetal oocytes did not express ERα mRNA. Although 18S rRNA was expressed in all oocytes, GATA-4 mRNA was not detected in oocytes and only detected in granulosa cells confirming purity of oocytes sampled. We conclude that oocytes of the fetal baboon ovary express ERβ mRNA, thereby providing a mechanism by which estrogen regulates oocyte function, e.g. microvillus development.     

Integrin alpha-2 and beta-3 gene polymorphisms and colorectal cancer risk

Background and aims  Integrins such as α₂β₁, α_IIbβ₃, and α_vβ₃ have been suggested as key players for cancer development and progression. Several polymorphisms affecting these molecules, two in integrin α₂ (ITGA2 807C>T and 1648G>A) and one in β₃ (ITGB3 176T>C), influence their levels, structure, and possibly their function. To analyze the role of ITGA2 and ITGB3 polymorphisms for colorectal cancer risk and clinical presentation, we performed a case–control study. Materials and methods  Four hundred thirty-three colorectal cancer patients and 433 healthy sex- and age-matched control subjects were investigated. ITGA2 and ITGB3 polymorphisms were determined by 5′-nuclease assays. Results/findings  The ITGA2 807C>T polymorphism was associated with reduced colorectal cancer risk. In a codominant model, the odds ratio for each additional 807-T allele for colorectal cancer was 0.77 (95% confidence interval 0.64–0.94; p = 0.011). The ITGA2 1648G> and the ITGB3 176T>C polymorphism were not associated with colorectal cancer. None of the three polymorphisms investigated was associated with tumor size, histological grade, presence of primary lymph node metastases, tumor stage, or age at diagnosis. Interpretation/conclusion  We conclude that the ITGA2 807C>T polymorphism may be associated with reduced colorectal cancer risk. Armin Gerger and Günter Hofmann contributed equally to this work.     

Molecular assays to profile 10 estrogen receptor beta isoform mRNA copy numbers in ovary, breast, uterus, and bone tissues

Estrogens regulate various biological processes in a diverse range of reproductive and nonreproductive tissues through two genetically distinct but structurally related high affinity nuclear receptors, the estrogen receptor alpha and beta (ERα and ERβ). The physiological significance of the presence of two ERs that have redundant functions is not known. Several unique properties of ERβ together with its distinct expression patterns are considered to be, in part, the basis for diverse functional actions of estrogens and opposing actions of selective estrogen receptor modulators (SERMs) in different tissues. To understand how relative expression levels of two ERs correlate to seemingly dissimilar actions of estrogens and SERMs, quantitative methods are required that can precisely measure the levels of every isoform. Previously, methods to quantify eight ERα isoforms have been described [Poola I. (2003) Anal. Biochem. 314, 217–226]. In this article, real-time PCR-based molecular assays are described that can distinguish and quantify as low as 100 copies of 10 ERβ isoform mRNAs, the ERβ1, ERβ2, ERβ4, ERβ5, and ERβ exon 2Δ, exon 3Δ, exon 4Δ, exon 5Δ, exon 6Δ, and exons 5–6Δ. Each isoform mRNA is quantified using a specific primer pair and a 5′FAM (carboxy-fluorescein)- and 3′TAMARA (6-carboxy tetraethyl-rhodamine)-labeled probe and in comparison with a standard curve constructed with known copy numbers of its respective reverse transcribed cRNA. The devised assays were applied to profile 10 ERβ isoforms in four estrogen-sensitive tissues—ovary, breast, uterus, and bone. The sensitivity of detection of each isoform in these tissues varied from picograms to nanograms of reverse-transcribed total RNA depending on the isoform and the tissue. The results presented also show that each tissue has a distinct profile of 10 isoform mRNAs. Interestingly, ERα-negative breast cancer cell lines and tumors expressed significant amounts of ERβ isoforms suggesting that mitogenic stimulation by estrogen exists in these tissues. Bone tissues expressed several isoforms, although wild type was not present. In addition to the assay development, evidence is presented to demonstrate for the first time that ERβ4 and ERβ5 are full length receptors, contrary to previous reports that they are short receptors of exon 7–8. It is expected that the methods described here will significantly contribute to delineating the functional roles of various ERβ isoforms and in conjunction with ERα isoform profiling, will highly facilitate designing of individualized tissue specific therapies to treat estrogen-related pathologies.     

Molecular analysis of a patient with type I Glanzmann thrombasthenia and clinical impact of the presence of anti-αIIbβ3 alloantibodies

The occurrence of transfusion-related alloimmunization against αIIbβ3 is a major concern in patients with Glanzmann thrombasthenia (GT). However, few data are available about molecular defects of GT patients with anti-αIIbβ3 alloantibodies as well as clinical impact of these antibodies on platelet transfusion. Here, we report a case of type I GT with anti-HLA and anti-αIIbβ3 alloantibodies, who underwent laparoscopic total gastrectomy due to gastric cancer. We found a novel β3 nonsense mutation, 892C > T (Arg272X), and the patient was homozygous for the mutation. Laparoscopic gastrectomy was successfully performed with continuous infusion of HLA-matched platelet concentrates and bolus injection of recombinant factor VIIa at 2 h intervals. Total bleeding was 370 mL and no red-cell transfusion was necessary. Flow cytometric analysis employing anti-αIIbβ3 monoclonal antibody revealed that the transfused platelet count was maintained around 20–30 × 10⁹/L during the operation and 10 × 10⁹/L on the following day. Flow cytometric analysis also showed that transfused platelets retained normal reactivity to ADP stimulation. These results indicate that flow cytometry is useful to assess survival and function of transfused platelets in GT patients with anti-αIIbβ3 antibodies.     

Functional expression of β1 and β2 integrins on tumor infiltrating lymphocytes (TILs) in colorectal cancer

Integrins play an important role in various lymphocyte functions. In this study, tumor-infiltrating lymphocytes (TIL) were isolated from colorectal cancer tissues and the expression of β1 and β2 integrins on the TIL was quantitatively examined with two-color flow cytometry. In comparison with peripheral blood lymphocytes (PBL), TIL expressed a lower level of common β1 chain (CD29) in both CD4 and CD8 sub-populations. Among the associated α chains, the expressions of α1 (CD49a) and α2 (CD49b) were slightly higher in TIL than in PBL, whereas α4 (CD49d) and α6 (CD49f) were markedly downregulated in TIL. Both αL (CD11a) and β2 (CD18) were reduced in CD8(+) TIL but not in CD4(+) TIL. TIL with the CD8(+) cytotoxic phenotype showed significantly decreased binding to purified intracellular adhesion molecules (ICAM)-1, and vascular adhesion cell molecule (VCAM)-1, and HT29 colon cancer cells, compared with the in counterparts in PBL. The peculiar expression pattern and functional down regulation of these integrins may explain why TIL in colorectal cancer cannot eradicate the malignant cells. Received: August 24, 1998/Accepted: November 27, 1998     

Type I collagen and divalent cation shifts disrupt cell-cell adhesion, increase migration, and decrease PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells

Background: We have shown in FG pancreatic cancer cells that α₂β₁ integrin-mediated type I collagen adhesion decreases parathyroid hormone-related protein (PTHrP), inerleukin-6 (IL-6), and interleukin-8 (IL-8) expression, decreases the localization of E-cadherin and β-catenin in cell-cell contacts, increases cell migration, and increases glycogen synthase kinase 3 (GSK3) and protein kinase B (PKB/Akt) phosphorylation states relative to α₅β₁ integrin-mediated fibronectin (Fn) adhesion. Aim of the Study: To extend our observations in FG cells to other pancreatic cancer cell lines, and to determine whether E-cadherin-mediated cell-cell adhesion and its downstream effectors were functionally involved in the ECM-mediated regulation of PTHrP, IL-6, and IL-8. Methods: We used standard biochemical techniques to determine ECM-specific differences in E-cadherin and β-catenin localization, GSK3 and PKB/Akt phosphorylation, haptokinetic cell migration, and cytokine expression in pancreatic cancer cells. We also conducted functional studies using pharmacological inhibitors for GSK3 and PKB/Akt, as well as elevated Mg²⁺/Ca²⁺ ratios similar to pancreatic juice, and examined their effects on cytokine expression. Results: Differences in E-cadherin and β-catenin localization along with GSK3 and PKB/Akt phosphorylation occur in multiple pancreatic cancer cell lines, resulting in differences in ECM-mediated haptokinesis and cytokine expression that are generally consistent with previous observations in FG cells. Our functional studies also suggest that E-cadherin-mediated cell-cell adhesion and downstream effectors are involved in PTHrP, IL-6, and IL-8 expression. Conclusions: These data indicate that α₂β₁ integrin-mediated type I collagen adhesion disrupts cell-cell adhesion architecture, resulting in increased migration and decreased PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells.     

Effects of the inhibition of cytosolic phospholipase A<Subscript>2</Subscript>α in non-small cell lung cancer cells

Purpose  

The aim of this study was to investigate the expression of cPLA₂α in non-small lung cancer cell lines and tissues, and we sought to determine the in vitro effects of the pyrrolidine-2 inhibitor on cPLA₂α sensitivity in three different non-small lung cancer cell lines.     

Stage-specific regulation of adhesion molecule expression segregates epithelial stem/progenitor cells in fetal and adult human livers

Purpose Regulated expression of cell adhesion molecules could be critical in the proliferation, sequestration, and maintenance of stem/progenitor cells. Therefore, we determined fetal and adult stage-specific roles of cell adhesion in liver cell compartments. Methods We performed immunostaining for the adhesion molecules, E-cadherin and Ep-CAM, associated proteins, β-catenin and α-actinin, hepatobiliary markers, albumin, α-fetoprotein, and cytokeratin-19, and the proliferation marker, Ki-67. Expression of albumin was verified by in situ mRNA hybridization. Results In the fetal liver, hepatoblasts showed extensive proliferation with wide expression of E-cadherin, β-catenin, and α-actinin, although Ep-CAM was expressed in these cells less intensely and focally in the cell membrane to indicate weak cell adhesion. Hepatoblasts in ductal plate and bile ducts showed less proliferation and Ep-CAM was intensely expressed in these cells throughout the cell membrane, indicating strong adhesion. In some ductal plate cells, β-catenin was additionally in the cytoplasm and nucleus, suggesting active cell signaling by adhesion molecules. In adult livers, cells were no longer proliferating and E-cadherin, β-catenin, and α-actinin were expressed in hepatocytes throughout, whereas Ep-CAM was expressed in only bile duct cells. Some cells in ductal structures of the adult liver with Ep-CAM coexpressed albumin and cytokeratin-19, indicating persistence of fetal-like stem/progenitor cells. Conclusions Regulated expression of Ep-CAM supported proliferation in fetal hepatoblasts through weak adhesion and helped in biliary morphogenesis by promoting stronger adhesion in hepatoblasts during this process. Restriction of Ep-CAM expression to bile ducts in the adult liver presumably facilitated sequestration of stem/progenitor cells. This stage-specific and cell compartment-related regulation of adhesion molecules should be relevant for defining how liver stem/progenitor cells enter, exit, and remain in hepatic niches during both health and disease.     

The effect of infliximab on chemokines in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by infiltration of lymphocytes, macrophages, and plasma cells into synovial membrane. The chemokines family promotes chemotactic activity in various leukocyte cell types. Chemokines thus play an essential role in the pathological formation of RA. The aim of the present study was to evaluate the influence of infliximab on serum levels of various chemokines. Twenty-four RA patients were involved in this study, which took place between March 2003 and February 2006. Infliximab was administered by intravenous infusion at a dosage of 3 mg/kg. All patients underwent general and physical examinations and routine blood and urinary analysis at the baseline, at 14 weeks, and at 30 weeks after the initial treatment. To determine whether serum and synovial fluid from RA also contained significant levels of chemokines compared with osteoarthritis patients (OA), GRO-α, MIP-1α, MIP-1β and regulated on activation normal T cell expressed and secreted (RANTES) levels of serum and synovial fluid were measured by ELISA in 20 RA patients and 20 OA patients. GRO-α, MIP-1β, and RANTES levels were significantly higher in RA compared with normal volunteers, while MIP-1α levels showed no significant differences. The mean GRO-α levels in serum from RA patients treated with infliximab decreased significantly after the initial treatment. The mean RANTES and MIP-1β levels did not change significantly after the treatment. Infliximab treatment significantly lowered the serum GRO-α levels of RA patients. GRO-α is one of the crucial cytokines affected by infliximab treatment. The blocking therapy of RANTES and MIP-1β combined with infliximab treatment may have an additional effect without competition in the TNFα cascade.     

Alterations in plasma and ovarian immunoreactive inhibin during reproductive aging in the female rat

Previously, we showed that ovarian inhibin α- and β;_A-subunit mRNAs are elevated in middle-aged and old persistent-estrous (PE) female rats. To determine whether higher inhibin subunit mRNA expressions result in increased circulating inhibins during reproductive aging, plasma immunoreactive inhibin α (ir-inh α) and gonadotropins were measured in young, middle-aged and PE rats. Plasma LH profiles were distinctly divergent in the middle-aged rats with some showing LH surges indistinguishable from young rats and others showing significantly attenuated LH surges. Plasma ir-inh α in middle-aged rats with LH surges were similar to those of young rats. However, animals with attenuated LH surges had higher peak ir-inh α levels than young and middle-aged animals with LH surges. Immunohistochemistry revealed increased levels of ovarian inhibin α staining in those animals with attenuated LH surges. Overall, the highest plasma and ovarian inhibin α were found in PE rats which lack LH surges. However, significant decreases of plasma and ovarian inhibin α were seen after reinstatement of estrous cyclicity with progesterone implant treatment. Thus, increases in both plasma and ovarian inhibin α appear to be closely associated with attenuation or loss of the preovulatory gonadotropin surge that occurs during aging.     

Restoring TGFβ function in microsatellite unstable (MSI-H) colorectal cancer reduces tumourigenicity but increases metastasis formation

Background  TGFβ is an important cell growth regulator which may have a role in metastasis formation. Microsatellite unstable (MSI-H) colon cancer serves as a unique model to demonstrate this as most MSI-H colon cancers have a mutation in the transforming growth factor beta receptor II (TGFβRII) gene and a low metastatic rate. Aims  To demonstrate an increase in invasion and metastasis in a MSI-H colorectal cancer cell line with a known mutation in TGFβRII. Materials and methods  By restoring the wild-type TGFβRII gene in the KM12C MSI-H colorectal carcinoma cell line with a known mutation in TGFβRII, we have demonstrated that both invasion and metastasis in this cell line was significantly increased. A mouse metastatic model have shown that liver metastases were increased in mice inoculated with cells containing a wild-type TGFβRII gene (42% for the transfected group compared with 15% for the control group; p = 0.0379), despite a reduction in the size of primary tumours. Conclusions  This study highlights an important mechanism which may contribute to the low metastatic rate of MSI-H colon cancers and demonstrates the importance of TGFβ signalling in metastasis formation. Previous studies involving breast cancer cell lines have shown that blocking TGFβ signalling results in a reduction in metastasis formation. This study is the first study to use a cell line with a low metastatic rate and TGFβRII mutations to demonstrate that restoring TGFβ signalling increases the metastatic rate.     

Changes in expression, and/or mutations in TGF-β receptors (TGF-β RI and TGF-β RII) and Smad 4 in human ovarian tumors

Purpose  

Loss of sensitivity to transforming growth factor β (TGF-β) signaling typically occurs in human ovarian cancer cells, but there is paucity of information regarding this in human ovarian tumors. Thus the association of inactivating mutations and/or variations in expression levels of TGF-β signaling components with human ovarian tumors was evaluated.     

Overexpression of osteopontin and integrin αv in laryngeal and hypopharyngeal carcinomas associated with differentiation and metastasis

Purpose  

Osteopontin (OPN) is a secreted phosphoprotein, which functions as a cell attachment protein and cytokine that signals through two cell adhesion molecules, integrin αvβ3 and CD44, to regulate cancer growth and metastasis. The aim of this study was to investigate the expression of OPN and integrin αv (ITGAV, main receptor of the OPN) in laryngeal and hypopharyngeal squamous cell carcinoma (LHSCC) and correlate the expression quantity with tumor biological behavior.     

Decreased insulin content and secretion in RIN 1046-38 cells overexpressing α2-Adrenergic receptorsreceptors

Several G_i-protein-coupled receptors normally expressed in islet β-cells inhibit insulin secretion on binding of their respective agonists. To study the effect of supraphysiologic expression of such a receptor in insulin-secreting β-cells, we stably transfected cDNA encoding the mouse α_2a-adrenergic receptor into RIN 1046-38 cells. Four different cell lines were selected, each overexpressing the α_2a-adrenergic receptor to varying degrees. Cell lines showing the highest level of receptor expression showed significantly reduced insulin content, and reduced basal and stimulated insulin secretion. Pertussis toxin (PTX) treatment of cells was able to reverse partially the reduced insulin secretory response. Our results suggest that overexpression of a G_i-protein-coupled receptor in β-cells causes tonic inhibition of both insulin synthesis and secretion. Abnormalities in expression or function of such receptors could be a contributory factor in the impaired insulin secretion present in type II diabetes.     

Positive expression of E-cadherin suppresses cell adhesion to fibronectin via reduction of α5β1 integrin in human breast carcinoma cells

E-cadherin mainly mediated the epithelial cell–cell adhesion, and integrin signaling can modulate the signaling pathway of E-cadherin in the different levels. Up to now, however, it is still unclear that whether E-cadherin could interfere with cell–matrix interaction, a typical adhesion through integrins. In this study we investigated the effects of E-cadherin on cell–matrix adhesion and α5β1 integrin expression in human breast carcinoma cells. It was found that either mRNA or protein level of α5 and β1 subunits of integrin decreased in E-cad-231 compared with Mock-231. Furthermore, the promoter activity of α5 gene was inhibited in E-cad-231 compared with Mock-231. Consistently, phosphorylated focal adhesion kinase, a closer key downstream protein kinase of integrin signaling, were also down-regulated in E-cad-231. Furthermore, distribution of β-catenin was observed and data showed β-catenin was accumulated in the nucleus in Mock-231, while disappeared from the nucleus and mainly accumulated near the cell surface membrane in E-cad-231. LiCl, a molecule that can inhibit the GSK-3β activity and down-regulate β-catenin degradation, could inversely stimulate expression of α5 and β1 integrin. Taken together, these results indicated that positive expression of E-cadherin inhibits the cell adhesion to extracellular matrix mediated by α5β1 integrin signaling.