A Combination of Two Receptor Tyrosine Kinase Inhibitors, Canertinib and PHA665752 Compromises Ovarian Cancer Cell Growth in 3D Cell Models

Abstract

Introduction: Advanced ovarian cancer is often a fatal disease as chemotherapeutic drugs have limited effectiveness. Better targeted therapy is needed to improve the survival and quality of life for these women. Receptor tyrosine kinases including EGFR, Her-2 and c-Met are associated with a poor prognosis in ovarian cancer. Therefore, the co-activation of these receptors may be crucial for growth promoting activity. In this study, we explored the effect of combining two small molecule inhibitors that target the EGFR/Her-2 and c-Met receptor tyrosine kinases in two ovarian cancer cell lines. The aim of this study was to investigate the combined inhibition activity of a dual EGFR/Her-2 inhibitor (canertinib) and a c-Met inhibitor (PHA665752) in ovarian cancer cell lines in 3D cell aggregates.

Methods: OVCAR-5 and SKOV-3 ovarian cancer cell lines were cultured on a non-adherent surface to produce 3D cell clusters and aggregates. Cells were exposed to canertinib and PHA665752, both individually and in combination, for 48 h. The effect on growth, metabolism and the expression/phosphorylation of selective signaling proteins associated with EGFR, Her-2 and c-Met were investigated.

Results: The single drug treatments significantly decreased cell growth and altered the expression of signaling proteins in OVCAR-5 and SKOV-3 cell lines. The combination treatment showed greater reduction of cell numbers for both cell lines. Total expression and phosphorylation of signaling proteins were further reduced in the combination drug treatments, compared to the single inhibitor treatments.

Conclusion: Our findings suggest that the concurrent targeting of more than one receptor tyrosine kinase may be useful in developing more effective targeted drug regimens for patients, who have EGFR, Her-2 and c-Met positive ovarian cancer cells.

Keywords: Canertinib; Cell clusters; EGFR; Ovarian cancer; PHA665752; Tyrosine kinase inhibitors; c-MET.

Fig. 1

Morphology of cell clusters and the expression of EGFR, Her-2 and c-Met. Microscopic images of OVCAR-5 ovarian cancer cell clusters (a) and SKOV-3 ovarian cancer cell aggregates (b). The expression of epidermal growth factor receptor (EGFR) in OVCAR-5 (c) and SKOV-3 (d). Total expression of human epidermal growth factor receptor-2 (Her-2) in OVCAR-5 (e) and SKOV-3 (f). Total expression of mesenchymal epithelial transition factor (c-Met) receptors in OVCAR-5 (g) and SKOV-3 (h)

Fig. 2

Combined growth factors induce cell growth and canertinib inhibits cell growth of ovarian cancer cells. Growth stimulation of OVCAR-5 (a), SKOV-3 (b) cells incubated with 0.2 and 20 ng/ml epidermal growth factor, EGF, or hepatocyte growth factor, HGF. Canertinib inhibited cell growth of OVCAR-5 in a dose-dependent manner in the presence of 0.2 ng/ml (c) and 20 ng/ml (d) combined growth factors. Cell growth of SKOV-3 aggregates was markedly reduced by canertinib in the presence of combined growth factors (e, f). All experiments were independently performed at least three times with triplicates. Data analyzed logarithmically using one-way ANOVA. Data considered statistically significant are indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) when compared to the control or the other doses

Fig. 3

Inhibition of c-Met inhibitor, PHA665752 on cell growth of ovarian cancer cells. PHA665752 inhibited cell growth of OVCAR-5 clusters in a dose-dependent manner in the presence of 0.2 ng/ml (a) and 20 ng/ml (b) combined growth factors. Cell growth of SKOV-3 aggregates was marginally reduced by PHA665752 in the presence of combined growth factors (c, d). All experiments were independently performed at least three times with triplicates. Data analyzed logarithmically using one-way ANOVA. Data considered statistically significant are indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) when compared to the control or the other doses

Fig. 4

The effect of the combination of canertinib and PHA665752 on the growth of ovarian cancer cells. The expression of proliferative cell nuclear antigen (PCNA) and a reference protein (GAPDH) in ovarian cancer cells treated with single inhibitors and combination in OVCAR-5 (a) and SKOV-3 (b). Cell numbers of OVCAR-5 cells (c) and SKOV-3 (d) were reduced with single and combination of both inhibitors compared to untreated cells in presence of 0.2 ng/ml combined growth factors. All experiments were independently performed at least three times with triplicates. Data analyzed using one-way ANOVA. Data considered statistically significant are indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) when compared to the control or the other doses

Fig. 5

The effect of the combination of canertinib and PHA665752 on the growth of ovarian cancer cell in the presence of 20 ng/ml combined growth factors. Expression of proliferative cell nuclear antigen (PCNA) and a reference protein (GAPDH) in ovarian cancer cell clusters treated with single inhibitors and combination in OVCAR-5 (a) and SKOV-3 (b). Cell numbers of OVCAR-5 cells (c) and SKOV-3 (d) were reduced with single and combination of both inhibitors compared to untreated cells in presence of 20 ng/ml combined growth factors. All experiments were independently performed at least three times with triplicates. Data analyzed using one-way ANOVA. Data considered statistically significant are indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) when compared to the control or the other doses

Fig. 6

Inhibition of selective protein molecules in the presence of canertinib and PHA665752 in OVCAR-5 cell clusters with 0.2 ng/ml combined growth factors. Total protein expression and phosphorylation of EGFR, Her-2, c-Met, Akt and Erk in single and combination of inhibitors. All experiments were independently performed at least three times with triplicates. Data analyzed logarithmically using one-way ANOVA. Data considered statistically significant are indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) when compared to the control or other doses

Fig. 7

Inhibition of selective protein molecules in the presence of canertinib and PHA665752 in OVCAR-5 cell clusters with 20 ng/ml combined growth factors. Total protein expression and phosphorylation of EGFR, Her-2, c-Met, Akt and Erk in single and combination of inhibitors. All experiments were independently performed at least three times with triplicates. Data analyzed logarithmically using one-way ANOVA. Data considered statistically significant are indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) when compared to the control or the other doses

Fig. 8

Inhibition of selective protein molecules in the presence of canertinib and PHA665752 in SKOV-3 cell clusters with 0.2 ng/ml combined growth factors. Total protein expression and phosphorylation of EGFR, Her-2, c-Met, Akt and Erk in single and combination of inhibitors (a). All experiments were independently performed at least three times with triplicates. Data analyzed logarithmically using one-way ANOVA. Data considered statistically significant are indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) when compared to the control or the other doses

Fig. 9

Inhibition of selective protein molecules in the presence of canertinib and PHA665752 in SKOV-3 cell aggregates with 20 ng/ml combined growth factors. Total protein expression and phosphorylation of EGFR, Her-2, c-Met, Akt and Erk in single and combination of inhibitors. All experiments were independently performed at least three times with triplicates. Data analyzed logarithmically using one-way ANOVA. Data considered statistically significant are indicated as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) when compared to the control or the other doses