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Combination of cetuximab with chemoradiation, trastuzumab or MAPK inhibitors: mechanisms of sensitisation of cervical cancer cells.

Meira DD, de Almeida VH, Mororó JS, Nóbrega I, Bardella L, Silva RL, Albano RM, Ferreira CG - Br. J. Cancer (2009)

Bottom Line: Cetuximab efficiently decreased MAPK and AKT phosphorylation in A431 cells but slightly less in Caski and C33A cells.In Caski, but not in C33A cells, cetuximab cooperated with the TKI, reducing cell survival and AKT and MAPK phosphorylation.However, cetuximab with trastuzumab or PD98059 reduced survival and MAPK phosphorylation of both cell lines.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Research, Research Coordination, Instituto Nacional de Câncer (INCA), Rio de Janeiro, Cep: 20231-050, RJ, Brazil.

ABSTRACT

Background: Cervical cancer (CC) annually kills 288,000 women worldwide. Unfortunately, responses to chemoradiation are partial and are of short duration. As anti-EGFR monoclonal antibodies sensitise tumours, we investigated cetuximab's toxicity plus chemoradiation on CC cells, which express different EGFR levels.

Methods: EGFR, HER2, AKT and MAPK expression and phosphorylation status were determined by western blotting. Cytotoxicity was assessed by MTT or clonogenic assays (CA) in cell lines treated with cetuximab alone or in combinations.

Results: Cetuximab with cisplatin and radiation achieved maximum cytotoxic effects for A431, Caski and C33A cells (high, intermediate and low EGFR expression, respectively) in CA. Cetuximab efficiently decreased MAPK and AKT phosphorylation in A431 cells but slightly less in Caski and C33A cells. To check whether further EGFR, HER2 or MAPK inhibition would improve cetuximab's cytotoxicity, we combined it with an EGFR tyrosine kinase inhibitor (TKI), trastuzumab or a MEK1/2 inhibitor (PD98059). In Caski, but not in C33A cells, cetuximab cooperated with the TKI, reducing cell survival and AKT and MAPK phosphorylation. However, cetuximab with trastuzumab or PD98059 reduced survival and MAPK phosphorylation of both cell lines.

Conclusion: Our data suggest that cetuximab combined with chemoradiation, trastuzumab or MAPK inhibitors has useful applications for CC treatment, independently of EGFR expression.

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Related in: MedlinePlus

Effects of cetuximab (100 μg ml−1) alone or combined with trastuzumab (10 μg ml−1), an anti-HER2 MAb, or PD153035 (0.1 μM), an EGFR tyrosine kinase inhibitor, or PD98059 (25 μM), a MAPK pathway inhibitor, on Caski and C33A cells. (A) Effects of cetuximab alone or combined to trastuzumab on the survival in CA. (B) Representative pictures of Caski and C33A cells under cetuximab and trastuzumab treatments in CA. (C and D) Western blotting analysis of the inhibition of EGF-induced phosphorylation of AKT and p44/42 (ERK1/2) by cetuximab alone or combined with trastuzumab in Caski and C33A cells, respectively. (E) Effects of cetuximab alone or combined with PD153035 on cell survival in CA. (F) Effects of cetuximab alone or combined with PD98059 on proliferation by MTT assays. (G) Western blotting analysis of the inhibition of EGF-induced phosphorylation of EGFR (Tyr1068), HER-2/neu, AKT and p44/42 (ERK1/2) by cetuximab alone or combined with PD153035. To visualise EGFR in C33A cells, 80 μg of protein were loaded on the SDS–PAGE gels as seen by the higher amount of the endogenous control, HSC-70. (H) Western blotting analysis of the inhibition of phosphorylation of p44/42 (Erk1/2) by cetuximab and PD98059. Student's t-test *P<0.05, when compared to control cells. (I) Proposed model of the effects of the combination of cetuximab with chemoradiation or trastuzumab or MAPK pathway inhibitor on CC cell lines. Ligand binding activates signalling through EGFR and triggers the AKT and MAPK pathways. The binding of cetuximab sensitises CC cells to chemoradiation (1) and to trastuzumab (2), leading to cell death independently of EGFR expression levels, but more dependent on EGFR-HER2 signalling. For cells in which activation of the MAPK pathway occurs also through EGFR independent mechanisms, cetuximab inhibition of EGFR sensitises them to PD98059 (3), a MAPK pathway inhibitor, leading to additive effects on the inhibition of cell proliferation.
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fig4: Effects of cetuximab (100 μg ml−1) alone or combined with trastuzumab (10 μg ml−1), an anti-HER2 MAb, or PD153035 (0.1 μM), an EGFR tyrosine kinase inhibitor, or PD98059 (25 μM), a MAPK pathway inhibitor, on Caski and C33A cells. (A) Effects of cetuximab alone or combined to trastuzumab on the survival in CA. (B) Representative pictures of Caski and C33A cells under cetuximab and trastuzumab treatments in CA. (C and D) Western blotting analysis of the inhibition of EGF-induced phosphorylation of AKT and p44/42 (ERK1/2) by cetuximab alone or combined with trastuzumab in Caski and C33A cells, respectively. (E) Effects of cetuximab alone or combined with PD153035 on cell survival in CA. (F) Effects of cetuximab alone or combined with PD98059 on proliferation by MTT assays. (G) Western blotting analysis of the inhibition of EGF-induced phosphorylation of EGFR (Tyr1068), HER-2/neu, AKT and p44/42 (ERK1/2) by cetuximab alone or combined with PD153035. To visualise EGFR in C33A cells, 80 μg of protein were loaded on the SDS–PAGE gels as seen by the higher amount of the endogenous control, HSC-70. (H) Western blotting analysis of the inhibition of phosphorylation of p44/42 (Erk1/2) by cetuximab and PD98059. Student's t-test *P<0.05, when compared to control cells. (I) Proposed model of the effects of the combination of cetuximab with chemoradiation or trastuzumab or MAPK pathway inhibitor on CC cell lines. Ligand binding activates signalling through EGFR and triggers the AKT and MAPK pathways. The binding of cetuximab sensitises CC cells to chemoradiation (1) and to trastuzumab (2), leading to cell death independently of EGFR expression levels, but more dependent on EGFR-HER2 signalling. For cells in which activation of the MAPK pathway occurs also through EGFR independent mechanisms, cetuximab inhibition of EGFR sensitises them to PD98059 (3), a MAPK pathway inhibitor, leading to additive effects on the inhibition of cell proliferation.

Mentions: We speculated that cells expressing higher EGFR/HER2 ratios, such as A431 cells, rely more on EGFR signalling for MAPK pathway activation and cell proliferation, whereas cells with a lower EGFR/HER2 ratio, such as C33A cells, depend more on EGFR/HER2 heterodimer signalling. Based on this assumption, the inhibition of the EGFR/HER2 heterodimer by anti-EGFR (cetuximab) and anti-HER2 (trastuzumab) MAbs should interfere with C33A cell proliferation. As expected, this combination markedly reduced C33A cell colony formation leading to a synergistic interaction (R=0.58; Figures 4A and B), with concomitant reduction of MAPK and AKT phosphorylation (Figure 4D). Indeed, an additive effect (R=0.84) was also noted for Caski cells, that express intermediate levels of EGFR and HER2 (Figure 3A), with a decrease of almost 60% in cell survival (Figures 4A and B) and inhibition of downstream signalling pathways (MAPK and AKT; Figure 4C). There were no changes in total AKT and MAPK proteins in Caski and C33A cell lines on treatments (data not shown).


Combination of cetuximab with chemoradiation, trastuzumab or MAPK inhibitors: mechanisms of sensitisation of cervical cancer cells.

Meira DD, de Almeida VH, Mororó JS, Nóbrega I, Bardella L, Silva RL, Albano RM, Ferreira CG - Br. J. Cancer (2009)

Effects of cetuximab (100 μg ml−1) alone or combined with trastuzumab (10 μg ml−1), an anti-HER2 MAb, or PD153035 (0.1 μM), an EGFR tyrosine kinase inhibitor, or PD98059 (25 μM), a MAPK pathway inhibitor, on Caski and C33A cells. (A) Effects of cetuximab alone or combined to trastuzumab on the survival in CA. (B) Representative pictures of Caski and C33A cells under cetuximab and trastuzumab treatments in CA. (C and D) Western blotting analysis of the inhibition of EGF-induced phosphorylation of AKT and p44/42 (ERK1/2) by cetuximab alone or combined with trastuzumab in Caski and C33A cells, respectively. (E) Effects of cetuximab alone or combined with PD153035 on cell survival in CA. (F) Effects of cetuximab alone or combined with PD98059 on proliferation by MTT assays. (G) Western blotting analysis of the inhibition of EGF-induced phosphorylation of EGFR (Tyr1068), HER-2/neu, AKT and p44/42 (ERK1/2) by cetuximab alone or combined with PD153035. To visualise EGFR in C33A cells, 80 μg of protein were loaded on the SDS–PAGE gels as seen by the higher amount of the endogenous control, HSC-70. (H) Western blotting analysis of the inhibition of phosphorylation of p44/42 (Erk1/2) by cetuximab and PD98059. Student's t-test *P<0.05, when compared to control cells. (I) Proposed model of the effects of the combination of cetuximab with chemoradiation or trastuzumab or MAPK pathway inhibitor on CC cell lines. Ligand binding activates signalling through EGFR and triggers the AKT and MAPK pathways. The binding of cetuximab sensitises CC cells to chemoradiation (1) and to trastuzumab (2), leading to cell death independently of EGFR expression levels, but more dependent on EGFR-HER2 signalling. For cells in which activation of the MAPK pathway occurs also through EGFR independent mechanisms, cetuximab inhibition of EGFR sensitises them to PD98059 (3), a MAPK pathway inhibitor, leading to additive effects on the inhibition of cell proliferation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2736849&req=5

fig4: Effects of cetuximab (100 μg ml−1) alone or combined with trastuzumab (10 μg ml−1), an anti-HER2 MAb, or PD153035 (0.1 μM), an EGFR tyrosine kinase inhibitor, or PD98059 (25 μM), a MAPK pathway inhibitor, on Caski and C33A cells. (A) Effects of cetuximab alone or combined to trastuzumab on the survival in CA. (B) Representative pictures of Caski and C33A cells under cetuximab and trastuzumab treatments in CA. (C and D) Western blotting analysis of the inhibition of EGF-induced phosphorylation of AKT and p44/42 (ERK1/2) by cetuximab alone or combined with trastuzumab in Caski and C33A cells, respectively. (E) Effects of cetuximab alone or combined with PD153035 on cell survival in CA. (F) Effects of cetuximab alone or combined with PD98059 on proliferation by MTT assays. (G) Western blotting analysis of the inhibition of EGF-induced phosphorylation of EGFR (Tyr1068), HER-2/neu, AKT and p44/42 (ERK1/2) by cetuximab alone or combined with PD153035. To visualise EGFR in C33A cells, 80 μg of protein were loaded on the SDS–PAGE gels as seen by the higher amount of the endogenous control, HSC-70. (H) Western blotting analysis of the inhibition of phosphorylation of p44/42 (Erk1/2) by cetuximab and PD98059. Student's t-test *P<0.05, when compared to control cells. (I) Proposed model of the effects of the combination of cetuximab with chemoradiation or trastuzumab or MAPK pathway inhibitor on CC cell lines. Ligand binding activates signalling through EGFR and triggers the AKT and MAPK pathways. The binding of cetuximab sensitises CC cells to chemoradiation (1) and to trastuzumab (2), leading to cell death independently of EGFR expression levels, but more dependent on EGFR-HER2 signalling. For cells in which activation of the MAPK pathway occurs also through EGFR independent mechanisms, cetuximab inhibition of EGFR sensitises them to PD98059 (3), a MAPK pathway inhibitor, leading to additive effects on the inhibition of cell proliferation.
Mentions: We speculated that cells expressing higher EGFR/HER2 ratios, such as A431 cells, rely more on EGFR signalling for MAPK pathway activation and cell proliferation, whereas cells with a lower EGFR/HER2 ratio, such as C33A cells, depend more on EGFR/HER2 heterodimer signalling. Based on this assumption, the inhibition of the EGFR/HER2 heterodimer by anti-EGFR (cetuximab) and anti-HER2 (trastuzumab) MAbs should interfere with C33A cell proliferation. As expected, this combination markedly reduced C33A cell colony formation leading to a synergistic interaction (R=0.58; Figures 4A and B), with concomitant reduction of MAPK and AKT phosphorylation (Figure 4D). Indeed, an additive effect (R=0.84) was also noted for Caski cells, that express intermediate levels of EGFR and HER2 (Figure 3A), with a decrease of almost 60% in cell survival (Figures 4A and B) and inhibition of downstream signalling pathways (MAPK and AKT; Figure 4C). There were no changes in total AKT and MAPK proteins in Caski and C33A cell lines on treatments (data not shown).

Bottom Line: Cetuximab efficiently decreased MAPK and AKT phosphorylation in A431 cells but slightly less in Caski and C33A cells.In Caski, but not in C33A cells, cetuximab cooperated with the TKI, reducing cell survival and AKT and MAPK phosphorylation.However, cetuximab with trastuzumab or PD98059 reduced survival and MAPK phosphorylation of both cell lines.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Research, Research Coordination, Instituto Nacional de Câncer (INCA), Rio de Janeiro, Cep: 20231-050, RJ, Brazil.

ABSTRACT

Background: Cervical cancer (CC) annually kills 288,000 women worldwide. Unfortunately, responses to chemoradiation are partial and are of short duration. As anti-EGFR monoclonal antibodies sensitise tumours, we investigated cetuximab's toxicity plus chemoradiation on CC cells, which express different EGFR levels.

Methods: EGFR, HER2, AKT and MAPK expression and phosphorylation status were determined by western blotting. Cytotoxicity was assessed by MTT or clonogenic assays (CA) in cell lines treated with cetuximab alone or in combinations.

Results: Cetuximab with cisplatin and radiation achieved maximum cytotoxic effects for A431, Caski and C33A cells (high, intermediate and low EGFR expression, respectively) in CA. Cetuximab efficiently decreased MAPK and AKT phosphorylation in A431 cells but slightly less in Caski and C33A cells. To check whether further EGFR, HER2 or MAPK inhibition would improve cetuximab's cytotoxicity, we combined it with an EGFR tyrosine kinase inhibitor (TKI), trastuzumab or a MEK1/2 inhibitor (PD98059). In Caski, but not in C33A cells, cetuximab cooperated with the TKI, reducing cell survival and AKT and MAPK phosphorylation. However, cetuximab with trastuzumab or PD98059 reduced survival and MAPK phosphorylation of both cell lines.

Conclusion: Our data suggest that cetuximab combined with chemoradiation, trastuzumab or MAPK inhibitors has useful applications for CC treatment, independently of EGFR expression.

Show MeSH
Related in: MedlinePlus