Effect of Oxaliplatin, Olaparib and LY294002 in Combination on Triple-Negative Breast Cancer Cells

Abstract

Triple-negative breast cancer (TNBC) has a poor prognosis as the therapy has several limitations, most importantly, treatment resistance. In this study we examined the different responses of triple-negative breast cancer line MDA-MB-231 and hormone receptor-positive breast cancer line MCF7 to a combined treatment including olaparib, a poly-(ADP ribose) polymerase (PARP) inhibitor, oxaliplatin, a third-generation platinum compound and LY294002, an Akt pathway inhibitor. We applied the drugs in a single, therapeutically relevant concentration individually and in all possible combinations, and we assessed the viability, type of cell death, reactive oxygen species production, cell-cycle phases, colony formation and invasive growth. In agreement with the literature, the MDA-MB-231 cells were more treatment resistant than the MCF7 cells. However, and in contrast with the findings of others, we detected no synergistic effect between olaparib and oxaliplatin, and we found that the Akt pathway inhibitor augmented the cytostatic properties of the platinum compound and/or prevented the cytoprotective effects of PARP inhibition. Our results suggest that, at therapeutically relevant concentrations, the cytotoxicity of the platinum compound dominated over that of the PARP inhibitor and the PI3K inhibitor, even though a regression-based model could have indicated an overall synergy at lower and/or higher concentrations.

Keywords: Akt pathway inhibitor; MCF7; TNBC; olaparib; oxaliplatin.

Figure 1

The effects of Ola, Oxa and LY on the viability of TNBC MDA-MB-231 (A,B) and non-TNBC MCF7 (C,D) cells were evaluated by MTT assay. Cells were treated with 2 µM olaparib, 25 µM oxaliplatin, 1 µM LY294002 alone and in combination for 72 h. Data are shown as mean ± SEM of at least three separate experiments. * p < 0.05 compared with the untreated cells. Ola—olaparib; Oxa—oxaliplatin; LY—LY294002; TNBC—triple-negative breast cancer; MTT-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 2

The effects of Ola, Oxa and LY on MDA-MB-231 (A,B) and MCF7 (C,D) cell apoptosis were detected by MUSE Cell Analyzer using MUSE Annexin V and Dead Cell Kit after 72 h treatment with 2 µM olaparib, 25 µM oxaliplatin, 1 µM LY294002 alone and in combination. Results are shown as mean ±SEM of at least three separate experiments. * p < 0.05 compared with the untreated cells. Ola—olaparib; Oxa—oxaliplatin; LY—LY294002.

Figure 3

Effects of Ola, Oxa and LY on ROS production by MDA-MB-231 (A,B) and MCF7 (C,D) cells treated with 2 µM olaparib, 25 µM oxaliplatin and 1 µM LY294002. Results are shown as mean ±SEM of at least three separate experiments. * p < 0.05 compared with the untreated cells. Ola—olaparib; Oxa—oxaliplatin; LY—LY294002.

Figure 4

Flow cytometry analysis of MDA-MB-231 (A–D) and MCF7 (E–H) cell-cycle distribution. Cells were cultured and treated for 72 h with 2 µM olaparib, 25 µM oxaliplatin and 1 µM LY294002 alone and in combination. The cell-cycle distribution was determined with propidium iodide staining. The histograms show cell-cycle phases of control cells (A,E) and cells treated with combination of olaparib, oxaliplatin and LY294002 (B,F). The bar charts represent effect of single and combined treatment on cell cycle phase distribution (C,D,G,H). Results are expressed as mean ±SEM of at least three separate experiments. * p < 0.05 compared with the untreated cells.

Figure 5

Effects of Ola, Oxa and LY on colony formation by MDA-MB-231 (A,B) and MCF7 (C,D) cells. Cells were seeded in six-well plates and after one day treated with 2 µM olaparib, 25 µM oxaliplatin, 1 µM LY294002 alone and in combination. Cells were treated for 14 days before being stained with Coomassie Blue. Results are shown as mean ± SEM of at least three separate experiments. * p < 0.05. Ola—olaparib; Oxa—oxaliplatin; LY—LY294002.

Figure 6

Effect of olaparib and oxaliplatin on invasive growth of MDA-MB-231 (A) and MCF7 (B) cells treated with 2 µM olaparib and 25 µM oxaliplatin. Line A—control, Line B—2 µM olaparib treatment, Line C—25 µM oxaliplatin treatment, Line D—combination treatment.

Figure 7

Effects of olaparib, oxaliplatin and LY294002 on invasive growth of MDA-MB-231 (A) and MCF7 (B) cells treated with 2 µM olaparib, 25 µM oxaliplatin and 1 µM LY294002. Line A—control, Line B—1 µM LY294002 treatment, Line C—2 µM olaparib and 25 µM oxaliplatin, Line D—combination treatment of 1 µM LY294002, 2 µM olaparib and 25 µM oxaliplatin.