Aurora kinase inhibitor VE 465 synergistically enhances cytotoxicity of carboplatin in ovarian cancer cells through induction of apoptosis and downregulation of histone 3

Abstract

Aurora kinases are essential for regulation of chromosome segregation and cytokinesis during mitosis and play a role in growth and progression of human tumors, including ovarian cancer. Aurora A and Aurora B are frequently overexpressed in high-grade and low-grade ovarian cancers. Targeting Aurora kinases has great potential for improving the efficacy of chemotherapies of ovarian cancer. In this study, we investigated whether the Aurora kinase inhibitor, VE 465, can enhance the anti-tumor activity of carboplatin in human ovarian cancer cells. The antitumor activity of VE 465 was tested by MTT proliferative assay in multiple established human epithelial ovarian cancer cell lines of varying p53 status. VE 465 and carboplatin had a synergistic effect on cell viability in both platinum-sensitive and -resistant ovarian cancers. The growth-inhibitory effect was accompanied by reduction in expression of histone 3 and an increase in apoptosis. We conclude that VE 465 enhances the efficacy of carboplatin agents in ovarian carcinoma.

Figure 1. Growth-inhibitory effect of VE 465 on ovarian cancer cell lines as detected by MTT proliferative assay. (A) VE 465 on cell lines 2774, A2780, 2008/C13, Hey, NMP1, OVCAR3, and SKOV3. (B) IC₅₀ of VE 465 in different ovarian cancer cell lines after treatment for 96 h. The experiments were repeated three times. (C) Western blot analysis of the expression of Aurora kinases A and B, p53, and ER-α in ovarian cancer cell lines.

Figure 2. The effect of VE 465 on the cell cycle of 2008/C13 cells showing arrest in G₂/M phase. (A) Flow cytometry analysis of the effect of VE 465 on 2008/C13 cells. (B) Flow cytometry analysis of cells arrested in G₂/M phase (left panel) and sub-G₁ cells (right panel) after exposure to VE 465. (C) Western blot analysis of expression of phosphorylated H3, Aurora kinase B (left panel), and cleaved PARP (right panel) in 2008/C13 ovarian cancer cells after treatment for 48 h.

Figure 3. Growth-inhibitory effects of VE 465 or carboplatin or a combination of the two for 72 h on ovarian cancer cell lines 2008/C13 (A) and A2780 (B) as detected by MTT proliferative assay.

Figure 4. Flow cytometry analysis of the effects of VE 465 plus carboplatin on the cell cycle of 2008/C13 ovarian cancer cells. (A) The effects of VE 465 plus carboplatin on 2008/C13 cells. (B) Percentage of cells arrested in G₂/M phase after exposure to VE 465 and carboplatin.

Figure 5. Western blot analysis of expression of phosphorylated H3 and Aurora kinase B (A) and ER-α (B) in 2008/C13 ovarian cancer cells after treatment with VE 465 and carboplatin for 48 h. V6 and V7: VE 465 at concentration of 10⁻⁶ and 10⁻⁷ M; C16 and C32: Carboplatin at concentration of 16 and 32 μg/mL.

Figure 6. Effects of treatment with VE 465 plus carboplatin for 48 h on apoptosis in ovarian cancer cell line 2008/C13 as detected by TUNEL (A), flow cytometry (B), and western blot analysis of cleaved PARP expression.