EGF-Induced Connexin43 Negatively Regulates Cell Proliferation in Human Ovarian Cancer.

Connexin43 (Cx43) has been shown to regulate cell proliferation and its downregulation is correlated with poor prognosis and survival in several types of human cancer. Cx43 expression levels are frequently downregulated in human ovarian cancer, suggesting a potential role for Cx43 in regulating the progression of this disease. Epidermal growth factor (EGF) is a well-characterized hormone that stimulates ovarian cancer cell proliferation. Although EGF is able to regulate Cx43 expression in other cell types, it is unclear whether EGF can regulate Cx43 expression in ovarian cancer cells. Additionally, it remains unknown whether Cx43 is involved in EGF-stimulated ovarian cancer cell proliferation. In the present study, we demonstrate that treatment with EGF upregulates Cx43 expression in two ovarian cancer cell lines, SKOV3 and OVCAR4. Although treatment with EGF activates both ERK1/2 and Akt signaling pathways, pharmacological inhibition and siRNA-mediated knockdown suggest that only the activation of Akt1 is required for EGF-induced Cx43 upregulation. Functionally, Cx43 knockdown enhanced basal and EGF-induced cell proliferation, whereas the proliferative effects of EGF were reduced by Cx43 overexpression. Co-treatment with the gap junction inhibitor carbenoxolone did not alter the suppressive effects of Cx43 overexpression on EGF-induced cell proliferation, suggesting a gap junction-independent mechanism. This study reveals an important role for Cx43 as a negative regulator of EGF-induced human ovarian cancer cell proliferation.

EpCAM is up-regulated by EGF via ERK1/2 signaling and suppresses human epithelial ovarian cancer cell migration.

Although epithelial cell adhesion molecule (EpCAM) is overexpressed in human epithelial ovarian cancer (EOC), some contradictory results have been reported regarding the correlation between EpCAM overexpression and patient survival. In addition to this controversy, the function and regulation of EpCAM in EOC remain largely unknown. Here, we show that epidermal growth factor (EGF) up-regulates EpCAM expression by activating ERK1/2 signaling in a human EOC cell line, SKOV3. Additionally, EpCAM overexpression suppresses not only basal but also EGF-stimulated SKOV3 cell migration, whereas EpCAM knockdown increases both basal and EGF-stimulated cell migration in another human EOC cell line, OVCAR4. This study demonstrates the regulation of EpCAM and its role in mediating the effects of EGF on human EOC cell migration.

Transforming growth factor-α induces human ovarian cancer cell invasion by down-regulating E-cadherin in a Snail-independent manner.

Transforming growth factor-α (TGF-α), like epidermal growth factor (EGF) and amphiregulin (AREG) binds exclusively to EGF receptor (EGFR). We have previously demonstrated that EGF, AREG and TGF-α down-regulate E-cadherin and induce ovarian cancer cell invasion, though whether these ligands use the same molecular mediators remains unknown. We now show that, like EGF, TGF-α- and AREG-induced E-cadherin down-regulation involves both EGFR and HER2. However, in contrast to EGF and AREG, the transcription factor Snail is not required for TGF-α-induced E-cadherin down-regulation. This study shows that TGF-α uses common and divergent molecular mediators to regulate E-cadherin expression and cell invasion.

Characterization of a glutamine synthetase gene DvGS1 from Dunaliella viridis and investigation of the impact on expression of DvGS1 in transgenic Arabidopsis thaliana.

A novel glutamine synthetase (GS) gene DvGS1 showing highest amino acid sequence identity of 78 % with the other homologous GS proteins from green algae, was isolated and characterized from Dunaliella viridis. Phylogenetic analysis revealed that DvGS1 occupied an independent phylogenetic position which was different with the GSs from higher plants, animals and microbes. Functional complement in E. coli mutant confirmed that the DvGS1 encoded functional GS enzyme. Real-time PCR analysis of DvGS1 in D. viridis cells under nitrogen starvation revealed that the mRNA level of DvGS1 was positively up-regulated in 12 h. The DvGS1 levels at the points of 12 and 24 h were separately twofold and fourfold of the level before nitrogen starvation. In order to investigate the potential application of DvGS1 in higher plants, the transgenic study of DvGS1 in Arabidopsis thaliana was carried out. Phenotype identification demonstrated that all three transgenic lines of T3 generation showed obviously enhanced root length (26 %), fresh weight (22-46 % at two concentrations of nitrate supplies), stem length (26 %), leaf size (29 %) and silique number (30 %) compared with the wild-type Arabidopsis. Biochemical analysis confirmed that all three transgenic lines had higher total nitrogen content, soluble protein concentration, total amino acid content and the leaf GS activity than the wild type plants. The free NH4 (+) and NO3 (-) concentration in fresh leaves of three transgenic lines were reduced by 17-26 % and 14-15 % separately (at two concentrations of nitrate supplies) compared with those of the wild types. All the results indicated that over-expression of DvGS1 in Arabidopsis significantly results in the improvement of growth phenotype and the host's nitrogen use efficiency.

Cloning and characterization of two novel chloroplastic glycerol-3-phosphate dehydrogenases from Dunaliella viridis.

Dunaliella, a unicellular green alga, has the unusual ability to survive dramatic osmotic stress by accumulating high concentrations of intracellular glycerol as a compatible solute. The chloroplastic glycerol-3-phosphate dehydrogenase (GPDH) has been considered to be the key enzyme that produces glycerol for osmoregulation in Dunaliella. In this study, we cloned the two most prominent GPDH cDNAs (DvGPDH1 and DvGPDH2) from Dunaliella viridis, which encode two polypeptides of 695 and 701 amino acids, respectively. Unlike higher plant GPDHs, both proteins contained extra phosphoserine phosphatase (SerB) domains at their N-termini in addition to C-terminal GPDH domains. Such bi-domain GPDHs represent a novel type of GPDH and are found exclusively in the chlorophyte lineage. Transient expression of EGFP fusion proteins in tobacco leaf cells demonstrated that both DvGPDH1 and DvGPDH2 are localized in the chloroplast. Overexpression of DvGPDH1 or DvGPDH2 could complement a yeast GPDH mutant (gpd1Delta), but not a yeast SerB mutant (ser2Delta). In vitro assays with purified DvGPDH1 and DvGPDH2 also showed apparent GPDH activity for both, but no SerB activity was detected. Surprisingly, unlike chloroplastic GPDHs from plants, DvGPDH1 and DvGPDH2 could utilize both NADH and NADPH as coenzymes and exhibited significantly higher GPDH activities when NADH was used as the coenzyme. Q-PCR analysis revealed that both genes exhibited transient transcriptional induction of gene expression upon hypersalinity shock, followed by a negative feedback of gene expression. These results shed light on the regulation of glycerol synthesis during salt stress in Dunaliella.

Sphingosine-1-phosphate promotes ovarian cancer cell proliferation by disrupting Hippo signaling.

Epithelial ovarian carcinomas account for more than 90% of human ovarian cancers and have become the primary cause of death for gynecological malignancies. Unlimited cell proliferation and resistance to cell apoptosis contribute to the development of ovarian cancers. However, the underlying mechanisms involved in these processes in epithelial ovarian carcinomas are yet poorly understood. In the present study, we examined the Hippo signaling gene expression and investigated the effects of Sphingosine 1-phosphate (S1P) on cell proliferation and the underlying mechanisms in human ovarian cancer cell lines, OVCAR3 and SKOV3. Our results demonstrate that S1P disrupts Hippo signaling by reducing YAP phosphorylation and increasing the expression of CCN1 and CCN2 in both ovarian cancer cells. Furthermore, the increase in CCN1/CCN2 expression contributes to the S1P-induced increase in cancer cell proliferation.

Loss of Sprouty2 in human high-grade serous ovarian carcinomas promotes EGF-induced E-cadherin down-regulation and cell invasion.

Sprouty (SPRY) proteins are well-characterized factors that inhibit receptor tyrosine kinase signaling. Our Human Exonic Evidence-Based Oligonucleotide (HEEBO) microarray results showed that the mRNA levels of SPRY2, but not of SPRY1 or SPRY4, are down-regulated in high-grade serous ovarian carcinoma (HGSC) tissues and epithelial ovarian cancer (EOC) cell lines. Molecular inversion probe (MIP) copy number analysis showed the deletion of the SPRY2 locus in HGSC. Overexpression of SPRY2 reduced EGF-induced cell invasion by attenuating EGF-induced E-cadherin down-regulation. Moreover, a positive correlation between SPRY2 and E-cadherin protein levels was observed in HGSC tissues. This study reveals the loss of SPRY2 in HGSC and indicates an important tumor-suppressive role for SPRY2 in mediating the stimulatory effect of EGF on human EOC progression.

Characterization of a glutamine synthetase gene DvGS2 from Dunaliella viridis and biochemical identification of DvGS2-transgenic Arabidopsis thaliana.

The salt-tolerant green alga Dunaliella has remarkable capability to survive in some extreme environments such as nitrogen starvation, which makes Dunaliella be a proper model for mining novel genes on nitrogen uptake or assimilation. In this study, a glutamine synthetase (GS) gene DvGS2 with amino acid identity of 72% to other homologous GS proteins, was isolated and characterized from Dunaliella viridis. Phylogenetic comparison with other GSs revealed that DvGS2 occupied an independent phylogenetic position. Expressional analysis in D. viridis cells under nitrogen starvation confirmed that DvGS2 increased its mRNA level in 12h. Subcellular localization study and functional analysis in a GS-deficient Escherichia coli mutant proved that DvGS2 was a chloroplastic and functional GS enzyme. In order to investigate the potential application of DvGS2 in higher plants, the transgenic studies of DvGS2 in Arabidopsis thaliana were carried out. Results showed that the transgenic lines expressed the DvGS2 gene and demonstrated obviously enhanced root length (29%), fresh weight (40%-48% at two concentrations of nitrate supplies), stem length (21%), leaf size (39%) and silique number (44%) in contrast with the wild-type Arabidopsis. Furthermore, the transgenic lines had higher total nitrogen content (35%-43%), total GS activity (39%-45%) and soluble protein concentration (23%-24%) than the wild type. These results indicated that the overexpression of DvGS2 in A. thaliana resulted in higher biomass and the improvement of the host's nitrogen use efficiency.

Amphiregulin induces human ovarian cancer cell invasion by down-regulating E-cadherin expression.

Aberrant epidermal growth factor receptor (EGFR) activation is associated with ovarian cancer progression. In this study, we report that the EGFR ligand amphiregulin (AREG) stimulates cell invasion and down-regulates E-cadherin expression in two human ovarian cancer cell lines, SKOV3 and OVCAR5. In addition, AREG increases the expression of transcriptional repressors of E-cadherin including SNAIL, SLUG and ZEB1. siRNA targeting SNAIL or SLUG abolishes AREG-induced cell invasion. Moreover, ERK1/2 and AKT pathways are involved in AREG-induced E-cadherin down-regulation and cell invasion. Finally, we show that three EGFR ligands, AREG, epidermal growth factor (EGF) and transforming growth factor-α (TGF-α), exhibit comparable effects in down-regulating E-cadherin and promoting cell invasion. This study demonstrates that AREG induces ovarian cancer cell invasion by down-regulating E-cadherin expression.