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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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Susceptibility of ovarian cancer cells to combined treatment with Cetuximab and TKI. Ovarian cancer cells were treated with Erlotinib and Gefitinib (both 1 nM, 10 nM, 100 nM, 1 μM) and Vandetanib (10 nM, 100 nM, 500 nM and 1 μM). Where indicated in the legend Cetuximab was added in a concentration of 1 μg/mL. After incubation time of 72 h cell viability was determined by the MTT-assay. Means and standard error of three to four independent experiments are shown for IGROV-1 and SKOV-3 in (a). Cell cycle analysis of IGROV-1 and SKOV-3 after combined treatment is shown in (b). Means and standard errors of three independent experiments are indicated. Statistical analysis was performed by unpaired t-test.
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f3-ijms-13-12000: Susceptibility of ovarian cancer cells to combined treatment with Cetuximab and TKI. Ovarian cancer cells were treated with Erlotinib and Gefitinib (both 1 nM, 10 nM, 100 nM, 1 μM) and Vandetanib (10 nM, 100 nM, 500 nM and 1 μM). Where indicated in the legend Cetuximab was added in a concentration of 1 μg/mL. After incubation time of 72 h cell viability was determined by the MTT-assay. Means and standard error of three to four independent experiments are shown for IGROV-1 and SKOV-3 in (a). Cell cycle analysis of IGROV-1 and SKOV-3 after combined treatment is shown in (b). Means and standard errors of three independent experiments are indicated. Statistical analysis was performed by unpaired t-test.

Mentions: The different mode of action of monoclonal antibodies and TKIs might suggest that the combined use of both substances might increase antitumoural activity. Therefore, we investigated whether the combined use of Erlotinib, Gefitinib and Vandetanib with Cetuximab might overcome resistance to the single agents. We assessed the cell viability and changes in cell cycle in IGROV-1, SKOV-3, A2780 and ASC. TKIs were concentrated as described above. Cetuximab was added in a concentration of 1 μg/mL. Cell viability and cell cycle analysis were performed after 72 h incubation time. As depicted in Figure 3a (red lines) in the anti-EGFR-susceptible cell line IGROV-1 cell proliferation was inhibited similar to the levels achieved by the mono-agents (compare Figures 1b and 2a). An additional or synergistic effect of combination therapy was not observed. Reduction of cell viability under high TKI-concentrations could not be further augmented by Cetuximab. The resistant ovarian cancer cells like SKOV-3 and ASC were not affected by combined treatment (Figure 3a for SKOV-3, blue lines). Accordingly, cell cycle studies of both susceptible IGROV-1 cells and resistant SKOV-3 cells revealed no difference between combination treatment versus treatment with mono-agents (Figure 3b). Our data suggest that a combination of TKI and Cetuximab is not superior to single agents alone. This is true for both resistant and susceptible cell lines.


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 combined treatment with Cetuximab and TKI. Ovarian cancer cells were treated with Erlotinib and Gefitinib (both 1 nM, 10 nM, 100 nM, 1 μM) and Vandetanib (10 nM, 100 nM, 500 nM and 1 μM). Where indicated in the legend Cetuximab was added in a concentration of 1 μg/mL. After incubation time of 72 h cell viability was determined by the MTT-assay. Means and standard error of three to four independent experiments are shown for IGROV-1 and SKOV-3 in (a). Cell cycle analysis of IGROV-1 and SKOV-3 after combined treatment is shown in (b). Means and standard errors of three independent experiments are indicated. Statistical analysis was performed by unpaired t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-ijms-13-12000: Susceptibility of ovarian cancer cells to combined treatment with Cetuximab and TKI. Ovarian cancer cells were treated with Erlotinib and Gefitinib (both 1 nM, 10 nM, 100 nM, 1 μM) and Vandetanib (10 nM, 100 nM, 500 nM and 1 μM). Where indicated in the legend Cetuximab was added in a concentration of 1 μg/mL. After incubation time of 72 h cell viability was determined by the MTT-assay. Means and standard error of three to four independent experiments are shown for IGROV-1 and SKOV-3 in (a). Cell cycle analysis of IGROV-1 and SKOV-3 after combined treatment is shown in (b). Means and standard errors of three independent experiments are indicated. Statistical analysis was performed by unpaired t-test.
Mentions: The different mode of action of monoclonal antibodies and TKIs might suggest that the combined use of both substances might increase antitumoural activity. Therefore, we investigated whether the combined use of Erlotinib, Gefitinib and Vandetanib with Cetuximab might overcome resistance to the single agents. We assessed the cell viability and changes in cell cycle in IGROV-1, SKOV-3, A2780 and ASC. TKIs were concentrated as described above. Cetuximab was added in a concentration of 1 μg/mL. Cell viability and cell cycle analysis were performed after 72 h incubation time. As depicted in Figure 3a (red lines) in the anti-EGFR-susceptible cell line IGROV-1 cell proliferation was inhibited similar to the levels achieved by the mono-agents (compare Figures 1b and 2a). An additional or synergistic effect of combination therapy was not observed. Reduction of cell viability under high TKI-concentrations could not be further augmented by Cetuximab. The resistant ovarian cancer cells like SKOV-3 and ASC were not affected by combined treatment (Figure 3a for SKOV-3, blue lines). Accordingly, cell cycle studies of both susceptible IGROV-1 cells and resistant SKOV-3 cells revealed no difference between combination treatment versus treatment with mono-agents (Figure 3b). Our data suggest that a combination of TKI and Cetuximab is not superior to single agents alone. This is true for both resistant and susceptible cell lines.

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