Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.     

The Influence of Perceived Stress on Cortical Reactivity: A Proof-Of-Principle Study

The aim of this study was to investigate how perceived stress may affect electroencephalographical (EEG) activity in a stress paradigm in a sample of 76 healthy participants. EEG activity was analyzed using multilevel modeling, allowing estimation of nested effects (EEG time segments within subjects). The stress paradigm consisted of a 3-minute pre-stimulus stress period and a 2-minute post-stimulus phase. At t=3 minutes, a single electrical stimulus was delivered. Participants were unaware of the precise moment of stimulus delivery and its intensity level. In the EEG time course of alpha activity, a stronger increase was observed during the post-stimulus period as compared to the pre-stimulus period. An opposite time course effect was apparent for gamma activity. Both effects were in line with a priori expectations and support the validity of this experimental EEG-stress paradigm. Secondly, we investigated whether interaction effects of stress and coping, as measured with the Perceived Stress Scale-10 questionnaire (PSS-10), could be demonstrated. A higher perceived stress score was accompanied by a greater increase in delta- and theta-activity during the post-stimulus phase, compared to low scores. In contrast, low coping capacity was associated with a stronger decrease in slow beta, fast beta and gamma activity during the post-stimulus phase. The results of the present article may be interpreted as proof-of-principle that EEG stress-related activity depends on the level of subjectively reported perceived stress. The inclusion of psychosocial variables measuring coping styles as well as stress-related personality aspects permits further examination of the interconnection between mind and body and may inform on the process of transformation from acute to chronic stress.     

Overexpression of homologous phytochrome genes in tomato: exploring the limits in photoperception

Transgenic tomato [Lycopersicon esculentum (=Solanum lycopersicum)] lines overexpressing tomato PHYA, PHYB1, or PHYB2, under control of the constitutive double-35S promoter from cauliflower mosaic virus (CaMV) have been generated to test the level of saturation in individual phytochrome-signalling pathways in tomato. Western blot analysis confirmed the elevated phytochrome protein levels in dark-grown seedlings of the respective PHY overexpressing (PHYOE) lines. Exposure to 4 h of red light resulted in a decrease in phytochrome A protein level in the PHYAOE lines, indicating that the chromophore availability is not limiting for assembly into holoprotein and that the excess of phytochrome A protein is also targeted for light-regulated destruction. The elongation and anthocyanin accumulation responses of plants grown under white light, red light, far-red light, and end-of-day far-red light were used for characterization of selected PHYOE lines. In addition, the anthocyanin accumulation response to different fluence rates of red light of 4-d-old dark-grown seedlings was studied. The elevated levels of phyA in the PHYAOE lines had little effect on seedling and adult plant phenotype. Both PHYAOE in the phyA mutant background and PHYB2OE in the double-mutant background rescued the mutant phenotype, proving that expression of the transgene results in biologically active phytochrome. The PHYB1OE lines showed mild effects on the inhibition of stem elongation and anthocyanin accumulation and little or no effect on the red light high irradiance response. By contrast, the PHYB2OE lines showed a strong inhibition of elongation, enhancement of anthocyanin accumulation, and a strong amplification of the red light high irradiance response.     

Cloning and expression of an adhesin antigen of Streptococcus sanguis G9B in Escherichia coli

A genomic library of Streptococcus sanguis, strain G9B, was constructed and expressed in Escherichia coli using a lambda gt11 expression vector. The amplified library was probed with polyclonal anti-G9B IgG and 13 antigen-positive clones were isolated. A lysate of one clone, designated PP39, absorbed the adhesion-inhibitory activity of anti-G9B IgG. This clone contained an insert of approximately 2000 bp and expressed unique 200 and 53 kDa proteins that reacted with monospecific anti-adhesin antibody. The 200 kDa protein also reacted with anti-beta-galactosidase IgG, indicating that it is a fusion protein of which 84 kDa represents the streptococcal adhesin. The 84 and 53 kDa proteins are similar in size to the major polypeptides in a streptococcal antigen complex which is associated with the adhesion of G9B to saliva-coated hydroxyapatite. The 53 kDa fragment may result from post-translational cleavage of the recombinant polypeptide.     

Characteristics of a protease of Streptococcus sanguis G9B which degrades the major salivary adhesin

An endogenous enzyme present in cell surface extracts of Streptococcus sanguis strain G9B degraded the major salivary adhesin of the organism. The enzyme showed optimal activity between 50 and 65 degrees C and was inactivated at higher temperatures. The activity at these unusually high temperatures seemed to be a consequence of release from the cell surface since intact whole G9B cells showed greater activity at 37 degrees C. The enzyme was not found in culture supernatants of G9B cells. The pH range for the enzyme was between 5 and 9. It was inhibited by iodoacetic acid, Hg2+, Cu2+, EDTA, SDS, and PMSF, but not by TLCK, TPCK, soybean trypsin inhibitor, cysteine, dithiothreitol, leupeptin, Ca2+, Mg2+ or saliva. The enzyme did not show any activity against human or rabbit IgG or human IgA. Enzyme activity was also found in S. sanguis strains Adh- (a spontaneously occurring non-adherent mutant of G9B), and M-5.     

ZD4054, a potent endothelin receptor A antagonist, inhibits ovarian carcinoma cell proliferation

Endothelin-1 (ET-1) is present at high concentrations in ovarian cancer ascites and is overexpressed in primary and metastatic ovarian carcinomas. In these tumors, the presence of ET-1 correlates with tumor grade, enhanced neovascularization, and with vascular endothelial growth factor (VEGF) expression. ET-1 acts as an autocrine factor selectively through ET(A) receptor (ET(A)R), predominantly expressed in ovarian carcinoma cells resulting in increased VEGF production and VEGF-mediated angiogenic effects. Previous results demonstrated that in ovarian carcinoma cells, activation of the ET-1/ET(A)R axis promotes cell proliferation, neovascularization, and invasion, which are the principal hallmarks of tumor progression. The present study was designed to investigate the in vitro effects of trans, trans-2(4-methoxydhenyl)-4-(1-3-benzodiazol-5-yl)-1-(dibutylaminocarbonylmethyl)-pyrrolidine-3-carboxylic acid (ZD4054), an orally active specific ET(A)R antagonist, on the ET-1-induced mitogenic effect in OVCA 433 and HEY ovarian carcinoma cell lines secreting ET-1 and expressing ET(A)R and ET(B)R mRNA. We show that ET(A)R blockade by ZD4054 inhibits ET-1-induced mitogenic effects, while the ET(B)R antagonist, BQ 788, is ineffective. In conclusion, our data demonstrate that ZD4054 is capable in inhibiting the proliferative activity of ET-1, indicating that this specific ET(A)R antagonist may be a potential candidate in developing novel treatment of ovarian carcinoma.     

Endothelin-1 is required during epithelial to mesenchymal transition in ovarian cancer progression

In a range of human cancers, tumorigenesis is promoted by activation of the endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis. ET-1 and ET(A)R are overexpressed in primary and metastatic ovarian carcinomas, and high levels of ET-1 are detectable in patient ascites, suggesting that ET-1 may promote tumor dissemination. Moreover, in these tumors, engagement of ET(A) receptor by ET-1 triggers tumor growth, survival, angiogenesis, and invasiveness. Thus, ET-1 enhances the secretion of matrix metalloproteinases, disrupts intercellular communications, and stimulates cell migration and invasion. Therefore, we investigated the role of the ET-1/ET(A)R autocrine axis in promoting epithelial to mesenchymal transition (EMT) in ovarian tumor cells, a key event in cancer metastasis, in which epithelial cells depolarize, disassemble cell-cell contacts, and adopt an invasive phenotype. Here, we examine the potential role of ET-1 in regulating cell morphology and behavior and epithelial and mesenchymal proteins employing an in vitro 3-D culture system. We found that in 3-D serum-free collagen I gel cultures, HEY and OVCA 433 ovarian carcinoma cells undergo fibroblast-like morphologic changes between 3 and 5 days of ET-1 treatment. In these cells, ET-1 induces loss of adherens and tight-junction protein expression, E-cadherin, beta-catenin, and zonula occludens-1, and gain of N-cadherin and vimentin expression. These results confirm the ability of ET-1 to promote EMT, a metastable process involving sustained loss of epithelial markers and gain of mesenchymal markers. Collectively, these findings provide evidence of a critical role for the ET-1/ET(A)R axis during distinct steps of ovarian carcinoma progression, thus underlining this axis as a potential target in the treatment of ovarian cancer.