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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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Related in: MedlinePlus

Susceptibility of ovarian cancer cells to anti-EGFR-tyrosine-kinase-inhibitors (TKI). Ovarian cancer cells were incubated with Erlotinib, Gefitinib and Vandetanib in the indicated concentrations for 72 h at 37 °C. Cell viability was determined by the MTT-assay. Means and standard errors of three to four independent experiments are shown for IGROV-1 (a) and SKOV-3 (b). Cell cycle analysis was performed. Means and standard errors of three independent experiments are shown in (c) and (d). Representative experiments of flow cytometric cell cycle analysis are depicted for IGROV-1 (e) and SKOV-3 (f) showing percentage of positive cells for each cell cycle phase. Statistical analysis was performed by unpaired t-test, statistical significance (p < 0.05) is indicated.
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f2-ijms-13-12000: Susceptibility of ovarian cancer cells to anti-EGFR-tyrosine-kinase-inhibitors (TKI). Ovarian cancer cells were incubated with Erlotinib, Gefitinib and Vandetanib in the indicated concentrations for 72 h at 37 °C. Cell viability was determined by the MTT-assay. Means and standard errors of three to four independent experiments are shown for IGROV-1 (a) and SKOV-3 (b). Cell cycle analysis was performed. Means and standard errors of three independent experiments are shown in (c) and (d). Representative experiments of flow cytometric cell cycle analysis are depicted for IGROV-1 (e) and SKOV-3 (f) showing percentage of positive cells for each cell cycle phase. Statistical analysis was performed by unpaired t-test, statistical significance (p < 0.05) is indicated.

Mentions: Next we investigated the response of ASC and the ovarian cancer cell lines described above to various anti-EGFR-tyrosine kinase inhibitors (TKI’s). We used Erlotinib and Gefitinib in the concentrations 1 μM, 100 nM, 10 nM and 1 nM and Vandetanib in a dose range of 1 μM down to 10 nM. Cell growth inhibition and effects on cell cycle phases were determined by MTT-assay and flow cytometry, respectively. IGROV-1 showed a strongly dose-dependent decrease of cell proliferation in the presence of all three TKI’s (Figure 2a). Major changes in cell cycle phases with increase of the apoptotic fraction SubG1 (p ≤ 0.01 for each TKI) and decrease of the G0/G1-phase (p ≤ 0.02 per TKI) and G2/M-phase (p ≤ 0.05 for each TKI) in response to all three TKIs (Figure 2c,e) were observed. SKOV-3 and the other cell lines including ASC remained unaffected by TKI-treatment as depicted for SKOV-3 regarding cell proliferation (Figure 2b) and cell cycle changes (Figure 2d,f). Our data show that resistance to anti-EGFR-TKI’s was paralleled by the resistance to cetuximab. In turn, susceptibility to Cetuximab predicted sensitivity to anti-EGFR-TKI-treatment.


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)

Susceptibility of ovarian cancer cells to anti-EGFR-tyrosine-kinase-inhibitors (TKI). Ovarian cancer cells were incubated with Erlotinib, Gefitinib and Vandetanib in the indicated concentrations for 72 h at 37 °C. Cell viability was determined by the MTT-assay. Means and standard errors of three to four independent experiments are shown for IGROV-1 (a) and SKOV-3 (b). Cell cycle analysis was performed. Means and standard errors of three independent experiments are shown in (c) and (d). Representative experiments of flow cytometric cell cycle analysis are depicted for IGROV-1 (e) and SKOV-3 (f) showing percentage of positive cells for each cell cycle phase. 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

f2-ijms-13-12000: Susceptibility of ovarian cancer cells to anti-EGFR-tyrosine-kinase-inhibitors (TKI). Ovarian cancer cells were incubated with Erlotinib, Gefitinib and Vandetanib in the indicated concentrations for 72 h at 37 °C. Cell viability was determined by the MTT-assay. Means and standard errors of three to four independent experiments are shown for IGROV-1 (a) and SKOV-3 (b). Cell cycle analysis was performed. Means and standard errors of three independent experiments are shown in (c) and (d). Representative experiments of flow cytometric cell cycle analysis are depicted for IGROV-1 (e) and SKOV-3 (f) showing percentage of positive cells for each cell cycle phase. Statistical analysis was performed by unpaired t-test, statistical significance (p < 0.05) is indicated.
Mentions: Next we investigated the response of ASC and the ovarian cancer cell lines described above to various anti-EGFR-tyrosine kinase inhibitors (TKI’s). We used Erlotinib and Gefitinib in the concentrations 1 μM, 100 nM, 10 nM and 1 nM and Vandetanib in a dose range of 1 μM down to 10 nM. Cell growth inhibition and effects on cell cycle phases were determined by MTT-assay and flow cytometry, respectively. IGROV-1 showed a strongly dose-dependent decrease of cell proliferation in the presence of all three TKI’s (Figure 2a). Major changes in cell cycle phases with increase of the apoptotic fraction SubG1 (p ≤ 0.01 for each TKI) and decrease of the G0/G1-phase (p ≤ 0.02 per TKI) and G2/M-phase (p ≤ 0.05 for each TKI) in response to all three TKIs (Figure 2c,e) were observed. SKOV-3 and the other cell lines including ASC remained unaffected by TKI-treatment as depicted for SKOV-3 regarding cell proliferation (Figure 2b) and cell cycle changes (Figure 2d,f). Our data show that resistance to anti-EGFR-TKI’s was paralleled by the resistance to cetuximab. In turn, susceptibility to Cetuximab predicted sensitivity to anti-EGFR-TKI-treatment.

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