Abstract P5-06-09: Cell Cycle Effects of Iniparib, a PARP Inhibitor, in Combination with Gemcitabine and Carboplatin in the MDA-MB-468(-) Triple-Negative Breast Cancer (TNBC) Cell Line
Authors: Ossovskaya V , Lim C-u , Schools G , Kalurupalle S , Roninson IB , Broude E. 
Published: April 2011

BACKGROUND: TNBC is an aggressive breast cancer subtype defined by lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2), and for which no approved targeted therapies have shown benefit to date. Inhibition of PARP1, a key regulator of DNA repair, has been associated with antitumor activity in both clinical and preclinical settings. Iniparib, a PARP inhibitor, has demonstrated promising efficacy and safety for treatment of patients with metastatic TNBC when combined with gemcitabine (G) and carboplatin (C), two standard chemotherapeutic agents that synergize to induce DNA damage (O'Shaughnessy et al. SABCS 2009). We hypothesized that by inhibiting DNA repair, iniparib potentiates antiproliferative effects and cell cycle arrest induced by G+C. METHODS: Triple-negative MDA-MB-468(-) cells were selected by fluorescence-activated cell sorting (FACS) of HER2-negative cells. Cells confirmed negative for ER, PR, and HER2 were treated for 72 hours with iniparib (100 uM), G (1 or 2 nM), and/or C (5 or 10 μM), with vehicle as a negative control. Cell proliferation was evaluated with the Hoechst 33342 cell proliferation assay and FACS-based cell cycle analysis, including the propidium iodide (PI) exclusion assay and bromodeoxyuridine (BrdU) incorporation. Apoptotic cells were quantified by TUNEL staining. Cell cycle arrest and DNA damage were evaluated by immunofluorescencestaining of treated cells with γH2AX (marker of DNA double strand breaks) and GF7 (marker of mitotic arrest).

RESULTS: FACS analysis showed that while G+C alone reduced cell viability, addition of iniparib further reduced the percentage of viable cells from 55% to 35% at low-dose G+C (1 nM G; 5 μM C) and 40% to 28% at high-dose G+C (2 nM G; 10 μM C). Addition of single dose of iniparib to G (1 nM), C (5 μM), or G+C (1 nM G; 5 μM C) resulted in an increase in TUNEL-positive apoptotic cells from 3.2% to 5.7%, 2.3% to 5.7%, and 4.7% to 8.8%, respectively, compared with 1.3% in the vehicle-treated control cells. Although iniparib alone had only modest effects on the cell cycle, addition of iniparib to C and G+C potentiated S-phase and G2/M cell cycle arrest at 72 hours after treatment, compared with the vehicle-treated control. Immunofluorescent staining of cells treated with iniparib in combination with G, C, or G+C demonstrated an increase in γH2AX foci, concurrent with increased GF7 staining, compared with G, C, or G+C alone.

CONCLUSIONS: Iniparib potentiated the effects of G and/or C in triple-negative MDA-MB-468 cells, demonstrating an increase in induction of apoptosis, S-phase, and G2/M cell cycle arrest, and DNA damage coinciding with mitotic arrest. These results support the clinical rationale of combining iniparib with G+C in treatment of patients with TNBC.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-09.