HEY Cell Lines Research Articles

Imatinib mesylate inhibits C-kit and PDGF mediated cell growth in vitro in ovarian cancer

3130 Background: Enhanced and altered signaling at tyrosinkinase receptors (TKR) as abl, c-kit and PDGF-R (receptors) influences tumor growth in a number of solid malignancies. The drug Imatinib is able to reduce cell proliferation by blocking these TKR. Expression patterns of TKR and their impact on growth control in ovarian cancer are investigated. Also the potential therapeutic role of Imatinib for ovarian cancer is examined in vitro. Methods:The ovarian cancer cell lines BG-1, HEY, OVCAR 3, OVCAR 8 and SK-OV 3 are screened for abl, c-kit, PDGF-R-α and -β expression by immunohistochemistry and PCR. Cell lines are incubated 3, 5 and 7 days with rising concentrations of single Imatinib, single Paclitaxel and single Paraplatin. Also combinations of Imatinib with Paclitaxel and Imatinib with Paraplatin are used. Finally ovarian cancer cells are incubated with triple combination of Imatinib, Paclitaxel and Paraplatin. After incubation drug sensibility assays (proliferation and apoptosis) are performed. Results:Abl and PDGF-R-β are expressed in all cell lines. PDGF-R-α is deteceted in BG-1, Skov 3, OVCAR 3 and weakly in OVCAR 8. C-kit expression is only detected in Skov-3 and OVCAR 3. High dose Imatinib incubation (>15 μM for 72 h) leads to receptor independent decrease of cell numbers (IC 50) and pro-apoptotic effects in all cell lines. 72 h combinations of low dose Imatinib (≤ 2 μM) with Paraplatin (IC50), as well as triple combination of Imatinib (≤ 2 μM) with Paclitaxel (IC25) and Paraplatin (IC25) show synergistic effects. Imatinib (≤ 2 μM) in combination with Paclitaxel (IC50) has an additive effect. The chemo-sensitizing effect of low dose Imatinib appears to be mediated by multiple factors, but is enhanced in c-kit positive cell lines (OVCAR 3 and Skov-3). Conclusions: High dose Imatinib leads to unspecific growth inhibition in ovarian cancer cell lines. Additive and even synergistic anti-cancer effects of Imatinib are seen in combination with cytostatic drugs as Paraplatin and Paclitaxel. Further interactions of Imatinib with PDGF-R and c-kit need to be investigated. Additional pre-clinical and clinical studies have to be performed to confirm results in vivo. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Pharmaceutical, Germany Novartis Novartis

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.

Endothelin-1 promotes proteolytic activity of ovarian carcinoma.

Endothelin-1 (ET-1) is a potent mitogenic and angiogenic factor for ovarian carcinoma cell lines, which acts selectively through the ET(A) receptor (ET(A)R). A previous study demonstrated that ET-1 is present at high concentrations in ovarian cancer ascites, indicating a direct role in the progression and metastasis of ovarian carcinoma. In this study, we investigated whether ET-1 could induce production and activation of tumour-associated proteinases in ovarian carcinoma cells. As demonstrated by ELISA, we found that the secretion of matrix metalloproteinase (MMP)-2 and MMP-9, urokinase-type plasminogen activator and plasminogen activator inhibitor type-1 and -2 was upregulated by ET-1 in a dose-dependent manner in the HEY cell line. In addition, the MMPs in ET-1-treated cells are consistently active, as shown by MMP gelatinase activity assay. Finally, we demonstrated that BQ-123, an antagonist of ET(A)R, inhibited the ET-1-induced tumour protease secretion and activity, suggesting that ET-1/ET(A)R may play an important role in the progression and metastasis of ovarian carcinoma, activating multiple proteinase cascades.

RhG-CSF affects genes involved in mitogen signalling and early gene expression in the ovarian cancer cell line HEY.

The ovarian adenocarcinoma cell line HEY was used as an in vitro model to study the influence of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on epithelial tumours such as ovarian cancer. Serum-starved cells were treated with rhG-CSF in a time- and dose-dependent manner. Cell proliferation, measured as cell division and DNA synthesis, was stimulated about 40% by rhG-CSF. After harvesting, cells were examined for the presence of G-CSF receptor (FACS analysis and RT-PCR), as well as for expression of genes involved in mitogen signalling (ERKs, JNKs) and early gene expression (c-jun). rhG-CSF affected mitogen-activated pathways and was receptor-mediated if the G-CSF receptor was present. After rhG-CSF induction, Janus N-terminal kinases (JNK 1 and 2) were simultaneously increased in the cytosol, up to 30-fold as measured by Western blotting), whereas ERK 1 and 2 accumulated maximally by 2.5-fold 1 hr after rhG-CSF induction. c-Jun was up-regulated strongly by this cytokine at the translational level. Our data suggest that rhG-CSF affects genes involved in mitogen signalling and early gene expression in solid tumours. We also noted the presence of G-CSF receptor on ovarian cancer cell lines.

Estrogen receptor alpha (ER-alpha) and beta (ER-beta) mRNAs in normal ovary, ovarian serous cystadenocarcinoma and ovarian cancer cell lines: down-regulation of ER-beta in neoplastic tissues.

The prognosis in ovarian carcinoma, the most lethal of the gynecologic neoplasms, is poor and has changed little in the last three decades. Only a small number respond to antiestrogen therapy, although the classic estrogen receptor, ER-alpha, has been identified in ovarian surface epithelium, from which approximately 90% of ovarian cancers originate. We have previously shown that ER-beta mRNA is most abundant in human fetal ovaries, suggesting that it might play an important role in ovarian development. Therefore, we investigated the mRNA levels of both ERs in normal ovaries, ovarian serous cystadenocarcinomas, granulosa cells from patients undergoing in vitro fertilization (IVF), the ovarian surface epithelium cell line IOSE-Van, and the ovarian cancer cell lines SKOV3, HEY and OCC1. Northern blots of normal and neoplastic ovaries were hybridized with an ER-beta riboprobe that spans the A/B domain. We detected two major hybridizing bands at approximately 8 and 10 kb. An RNase protection assay using the same probe revealed a single band of the expected size. Hybridizing the same blot with an ER-alpha riboprobe showed a strong hybridizing band at approximately 6.5 kb. In ovarian cancer samples, ER-beta mRNA level was decreased when compared to normal ovaries. Using 25 cycles of RT-PCR followed by Southern blotting, we found equal amounts of ER-alpha and -beta mRNAs in normal ovaries in all age groups from 33 to 75 years; however, in ovarian cancer tissue, the level of ER-alpha mRNA was similar or slightly higher, comparable to 10(3) to 10(4) copies of plasmid DNA, but ER-beta mRNA levels were markedly decreased. Granulosa cells from IVF patients expressed high levels of ER-beta mRNA. The OSE cell line expressed a low level of ER-alpha, detectable after 40 cycles of RT-PCR and no ER-beta mRNA. SKOV3 showed a low level of ER-alpha and -beta mRNAs, whereas OCC1 showed a low level of ER-beta and a relatively high level of ER-alpha. HEY did not contain detectable amounts of either ER after 40 cycles of RT-PCR. We found no evidence of differential splicing or major deletions in almost the entire coding region of ER-beta in either normal ovaries or tumor samples.

C‐jun expression and growth stimulation in human ovarian carcinoma cell lines following exposure to cytokines

We evaluated the effects of recombinant human G-CSF, rhGM-CSF, rhM-CSF and rhIL-1 alpha on proliferation and regulation of c-jun gene expression in 4 human ovarian-carcinoma (HOC) cell lines, NIH:OVCAR-3, SK-OV-3, HEY and BG-1, and in one primary ovarian tumor in vitro. The cytokines were administered in concentrations of 0.1 U/ml to 1000 U/ml. Cell growth was measured by crystal-violet- and thiazolyl-blue(MTT)-based cell counts. c-jun transcripts were measured by the solution hybridization/RNAse protection assay. RhM-CSF and rhGM-CSF showed no growth stimulation of any of the 5 cell lines tested. Results from exposure to rhG-CSF were different. The cell lines NIH:OVCAR-3, SK-OV-3, BG-1 and the primary ovarian tumor showed no proliferative response. A 2- to 3-fold increase in proliferation was observed in the HEY HOC cell line. rhIL-1 alpha led to growth stimulation in the BG-1 cell line, but showed an inhibitory effect in the NIH:OVCAR-3 cells. No effects of rhIL-1 alpha were observed in the remaining 2 cell lines nor in the primary ovarian tumor. Growth stimulation was accompanied by an increase in c-jun expression in the HEY cell line, and the BG-1 cell line. No alterations in c-jun expression were observed in the remaining 3 cell lines. Our results indicate that rhG-CSF or rhIL-1 alpha influence cell proliferation in 2 out of 5 human ovarian-tumor cell lines, accompanied by an increase in c-jun expression.

Targeting of [ 111In]biocytin to cultured ovarian adenocarcinoma cells using covalent monoclonal antibody—Streptavidin conjugates

Three monoclonal antibodies (mAb) directed against the human ovarian adenocarcinoma cell line HEY, were substituted with maleimide and covalently bonded to thiolated streptavidin. The conjugates were separated from unreacted reagents by successive affinity chromatography on protein A—Sepharose and iminobiotin columns. Purified conjugates consisted of an immunoglobulin (Ig) monomer bound to a streptavidin tetramer through a covalent bond between the Ig molecule and one of the streptavidin subunits. The conjugates were able to specifically target [ 111In]biocytin to HEY cells in vitro in the presence of human serum and ascitic fluid from ovarian cancer patients.

Characterization of binding of four monoclonal antibodies to the human ovarian adenocarcinoma cell line HEY.

Four mouse monoclonal antibodies (mAb) (10B, IgG1; 8C, IgG2a; M2A, IgG2a; M2D, IgG2b) were characterized with respect to their binding to the ovarian adenocarcinoma cell line HEY, using displacement assays and Scatchard plot analyses. The four mAb reacted with different antigens on the surface of HEY cells, with affinity constants ranging from 1 X 10(9) to 3 X 10(9) M-1. The number of binding sites per cell for each antibody was approximately 2 X 10(4). mAb 8C and M2D remained associated with the cell surface following binding to their respective antigens, while mAb 10B was rapidly internalized, with 50% of the bound mAb being lost from the cell surface during 4 h of incubation at 37 degrees C. These different binding characteristics of the mAb may influence their ability to target radioactivity and cytotoxic drugs to HEY cells.