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Anti-epidermal growth factor receptor (EGFR) antibodies overcome resistance of ovarian cancer cells to targeted therapy and natural cytotoxicity.

Gottschalk N, Kimmig R, Lang S, Singh M, Brandau S - Int J Mol Sci (2012)

Bottom Line: Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs.ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1.Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, University of Duisburg-Essen, 45147 Essen, Germany; E-Mails: nina.gottschalk@uk-essen.de (N.G.); stephan.lang@uk-essen.de (S.L.) ; Department of Gynecology and Obstetrics, University of Duisburg-Essen, 45157 Essen, Germany; E-Mail: rainer.kimmig@uk-essen.de.

ABSTRACT
The poor outcome of advanced ovarian cancer under conventional therapy stimulated the exploration of new strategies to improve therapeutic efficacy. In our preclinical in vitro study we investigated a combination of targeted therapy and immunotherapy. Combination treatment with the anti-EGFR-antibody Cetuximab, related tyrosine kinase inhibitors (TKI) and cytolytic NK cells was tested against different ovarian cancer cell lines and primary tumour cells cultured from patient ascites. We found that selected ovarian cancer cells were susceptible to cetuximab and anti-EGFR-TKI-treatment, while the majority of cell lines were resistant to single or combination treatment with both substances. In addition, most ovarian cancer cells displayed low susceptibility to natural cytotoxicity of unstimulated NK cells. Notably, NK cytotoxicity against resistant ovarian cancer cells could be effectively enhanced by addition of Cetuximab mediating antibody-dependent cellular cytotoxicity (ADCC). Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs. ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1. Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions. Future studies should evaluate the combination of targeted therapy and immunotherapeutic approaches in patients with advanced ovarian cancer being resistant to standard treatment.

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Epidermal growth factor receptor (EGFR)-expression of ovarian cancer cells and their susceptibility to Cetuximab. (a) The EGFR-expression of five different ovarian cancer cell lines and one primary ascites culture (ASC) was determined by flow cytometry after staining with PE-coupled anti-EGFR and isotype (Iso); (b) The ovarian cancer cells were seeded in 96-well-plates (10,000 cells/well) and treated with different concentrations of Cetuximab for 72 h at 37 °C. Cell viability was determined by the MTT-assay. Means and standard errors of three to seven independent experiments are shown; (c) Cell cycle was analyzed by flow cytometry after propidium iodide staining. Means and standard error of the percentage of cells in the respective cell cycle phases are shown for three independent experiments performed with IGROV-1 and SKOV-3. Statistical analysis was performed by unpaired t-test, statistical significance (p < 0.05) is indicated.
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f1-ijms-13-12000: Epidermal growth factor receptor (EGFR)-expression of ovarian cancer cells and their susceptibility to Cetuximab. (a) The EGFR-expression of five different ovarian cancer cell lines and one primary ascites culture (ASC) was determined by flow cytometry after staining with PE-coupled anti-EGFR and isotype (Iso); (b) The ovarian cancer cells were seeded in 96-well-plates (10,000 cells/well) and treated with different concentrations of Cetuximab for 72 h at 37 °C. Cell viability was determined by the MTT-assay. Means and standard errors of three to seven independent experiments are shown; (c) Cell cycle was analyzed by flow cytometry after propidium iodide staining. Means and standard error of the percentage of cells in the respective cell cycle phases are shown for three independent experiments performed with IGROV-1 and SKOV-3. Statistical analysis was performed by unpaired t-test, statistical significance (p < 0.05) is indicated.

Mentions: Five ovarian cancer cell lines IGROV-1, SKOV-3, A2780, OVCAR-3, JA-I and one primary ascites culture (ASC) were characterized regarding the expression of EGFR by flow cytometry as shown in Figure 1a. Thereby the strongest EGFR-expression was shown for OVCAR-3, followed by JA-I and SKOV-3. IGROV-1 and ASC showed moderate expression of EGFR, A2780 was EGFR-negative. The cell lines and ASC were coincubated with the anti-EGFR-antibody Cetuximab in concentrations of 1, 3, 10, 50 and 100 μg/mL for 72 h. We evaluated the antiproliferative effect of Cetuximab using the MTT-assay [29]. Changes in cell cycle were analyzed by flow cytometry after propidium iodide staining [30]. The only susceptible cell line was IGROV-1 showing reduced cell growth (Figure 1b). A significant decrease in the cell cycle phase G0/G1 (p ≤ 0.01) of IGROV-1 was observed, while the apoptotic fraction SubG1 was significantly increased (p ≤ 0.05) (Figure 1c). In contrast, SKOV-3 and the other ovarian cancer cells were not affected by Cetuximab-treatment (Figure 1b,c).


Anti-epidermal growth factor receptor (EGFR) antibodies overcome resistance of ovarian cancer cells to targeted therapy and natural cytotoxicity.

Gottschalk N, Kimmig R, Lang S, Singh M, Brandau S - Int J Mol Sci (2012)

Epidermal growth factor receptor (EGFR)-expression of ovarian cancer cells and their susceptibility to Cetuximab. (a) The EGFR-expression of five different ovarian cancer cell lines and one primary ascites culture (ASC) was determined by flow cytometry after staining with PE-coupled anti-EGFR and isotype (Iso); (b) The ovarian cancer cells were seeded in 96-well-plates (10,000 cells/well) and treated with different concentrations of Cetuximab for 72 h at 37 °C. Cell viability was determined by the MTT-assay. Means and standard errors of three to seven independent experiments are shown; (c) Cell cycle was analyzed by flow cytometry after propidium iodide staining. Means and standard error of the percentage of cells in the respective cell cycle phases are shown for three independent experiments performed with IGROV-1 and SKOV-3. Statistical analysis was performed by unpaired t-test, statistical significance (p < 0.05) is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472788&req=5

f1-ijms-13-12000: Epidermal growth factor receptor (EGFR)-expression of ovarian cancer cells and their susceptibility to Cetuximab. (a) The EGFR-expression of five different ovarian cancer cell lines and one primary ascites culture (ASC) was determined by flow cytometry after staining with PE-coupled anti-EGFR and isotype (Iso); (b) The ovarian cancer cells were seeded in 96-well-plates (10,000 cells/well) and treated with different concentrations of Cetuximab for 72 h at 37 °C. Cell viability was determined by the MTT-assay. Means and standard errors of three to seven independent experiments are shown; (c) Cell cycle was analyzed by flow cytometry after propidium iodide staining. Means and standard error of the percentage of cells in the respective cell cycle phases are shown for three independent experiments performed with IGROV-1 and SKOV-3. Statistical analysis was performed by unpaired t-test, statistical significance (p < 0.05) is indicated.
Mentions: Five ovarian cancer cell lines IGROV-1, SKOV-3, A2780, OVCAR-3, JA-I and one primary ascites culture (ASC) were characterized regarding the expression of EGFR by flow cytometry as shown in Figure 1a. Thereby the strongest EGFR-expression was shown for OVCAR-3, followed by JA-I and SKOV-3. IGROV-1 and ASC showed moderate expression of EGFR, A2780 was EGFR-negative. The cell lines and ASC were coincubated with the anti-EGFR-antibody Cetuximab in concentrations of 1, 3, 10, 50 and 100 μg/mL for 72 h. We evaluated the antiproliferative effect of Cetuximab using the MTT-assay [29]. Changes in cell cycle were analyzed by flow cytometry after propidium iodide staining [30]. The only susceptible cell line was IGROV-1 showing reduced cell growth (Figure 1b). A significant decrease in the cell cycle phase G0/G1 (p ≤ 0.01) of IGROV-1 was observed, while the apoptotic fraction SubG1 was significantly increased (p ≤ 0.05) (Figure 1c). In contrast, SKOV-3 and the other ovarian cancer cells were not affected by Cetuximab-treatment (Figure 1b,c).

Bottom Line: Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs.ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1.Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, University of Duisburg-Essen, 45147 Essen, Germany; E-Mails: nina.gottschalk@uk-essen.de (N.G.); stephan.lang@uk-essen.de (S.L.) ; Department of Gynecology and Obstetrics, University of Duisburg-Essen, 45157 Essen, Germany; E-Mail: rainer.kimmig@uk-essen.de.

ABSTRACT
The poor outcome of advanced ovarian cancer under conventional therapy stimulated the exploration of new strategies to improve therapeutic efficacy. In our preclinical in vitro study we investigated a combination of targeted therapy and immunotherapy. Combination treatment with the anti-EGFR-antibody Cetuximab, related tyrosine kinase inhibitors (TKI) and cytolytic NK cells was tested against different ovarian cancer cell lines and primary tumour cells cultured from patient ascites. We found that selected ovarian cancer cells were susceptible to cetuximab and anti-EGFR-TKI-treatment, while the majority of cell lines were resistant to single or combination treatment with both substances. In addition, most ovarian cancer cells displayed low susceptibility to natural cytotoxicity of unstimulated NK cells. Notably, NK cytotoxicity against resistant ovarian cancer cells could be effectively enhanced by addition of Cetuximab mediating antibody-dependent cellular cytotoxicity (ADCC). Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs. ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1. Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions. Future studies should evaluate the combination of targeted therapy and immunotherapeutic approaches in patients with advanced ovarian cancer being resistant to standard treatment.

Show MeSH
Related in: MedlinePlus