J Breast Cancer.  2015 Dec;18(4):329-338. 10.4048/jbc.2015.18.4.329.

Synergistic Effect of Trabectedin and Olaparib Combination Regimen in Breast Cancer Cell Lines

• Avila-Arroyo S
• Nunez GS
• Garcia-Fernandez LF
• Galmarini

• KMID: 2176281
• DOI: http://doi.org/10.4048/jbc.2015.18.4.329

Abstract

PURPOSE
Trabectedin induces synthetic lethality in tumor cells carrying defects in homologous recombinant DNA repair. We evaluated the effect of concomitant inhibition of nucleotide-excision repair and poly (ADP-ribose) polymerase (PARP) activity with trabectedin and PARP inhibitors, respectively, and whether the synthetic lethality effect had the potential for a synergistic effect in breast cancer cell lines. Additionally, we investigated if this approach remained effective in BRCA1-positive breast tumor cells.
METHODS
We have evaluated the in vitro synergistic effect of combinations of trabectedin and three different PARP inhibitors (veliparib, olaparib, and iniparib) in four breast cancer cell lines, each presenting a different BRCA1 genetic background. Antiproliferative activity, DNA damage, cell cycle perturbations and poly(ADP-ribosyl)ation were assessed by MTT assay, comet assay, flow cytometry and western blot, respectively.
RESULTS
The combination of trabectedin and olaparib was synergistic in all the breast cancer cell lines tested. Our data indicated that the synergy persisted regardless of the BRCA1 status of the tumor cells. Combination treatment was associated with a strong accumulation of double-stranded DNA breaks, G2/M arrest, and apoptotic cell death. Synergistic effects were not observed when trabectedin was combined with veliparib or iniparib.
CONCLUSION
Collectively, our results indicate that the combination of trabectedin and olaparib induces an artificial synthetic lethality effect that can be used to kill breast cancer cells, independent of BRCA1 status.

Keyword

BRCA1 protein; Breast neoplasms; Drug combinations; Olaparib; Trabectedin

MeSH Terms

Blotting, Western
BRCA1 Protein
Breast Neoplasms*
Breast*
Cell Cycle
Cell Death
Cell Line*
Comet Assay
DNA Breaks, Double-Stranded
DNA Damage
DNA, Recombinant
Drug Combinations
Flow Cytometry
BRCA1 Protein
DNA, Recombinant
Drug Combinations

Figure

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  Figure 1 Expression of proteins involved in different DNA repair systems. MCF7, MDA-MB-231, MDA-MB-436, and HCC-1937 were lysed and protein expression analyzed by western blot. α-Tubulin was used as loading control.

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  Figure 2 Flow cytometric cell cycle perturbations. Quantification of sub-G1, G0/1 (), S-phase (), and G/2M () cell cycle phases in untreated and drug-treated MCF7 (A), MDA-MB-236 (B), MDA-MB-436 (C), and HCC-1937 (D) breast cancer cells. All cell lines were analyzed after 24 hours exposure to olaparib, trabectedin, and the combination of trabectedin/olaparib as described in Methods section. For (C), only the diploid population is depicted. NT=non-treated.

• 

  Figure 3 Synergistic effects of the combination of trabectedin and olaparib on poly(ADP-ribosyl)ation and DNA damage in four different breast cancer cell lines. MCF7, MDA-MB-231, MDA-MB-436, and HCC-1937 cells were treated with the indicated concentrations of trabectedin, olaparib or the combination of both drugs for 24 hours. Then, each cell line was lysed and analyzed by western blot with anti-PAR and γ-H2AX antibodies. α-Tubulin was used as loading control. NT=non-treated cells; C1=combination of 0.05 nM trabectedin and 5 µM olaparib; C2=combination of 0.1 nM trabectedin and 5 µM olaparib; C3=combination of 0.5 nM trabectedin and 5 µM olaparib.

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  Figure 4 Synergistic effects of the combination of trabectedin and olaparib on DNA damage as detected by COMET assay in four different breast cancer cell lines. Representative images and quantification of damaged DNA in the comet assay in untreated and drug-treated MCF7 (A), MDA-MB-236 (B), MDA-MB-436 (C), and HCC-1937 (D) breast cancer cells. All cell lines were analyzed through comet assay followed by exposure to olaparib, trabectedin, and the combination of trabectedin/olaparib as described in Methods section. NT=non-treated; Olap =olaparib; Trab=trabectedin; Combo=combination.

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