Differential anti-proliferative activities of poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer cells - PubMed (original) (raw)

Differential anti-proliferative activities of poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer cells

Hsiao-Ching Chuang et al. Breast Cancer Res Treat. 2012 Jul.

Abstract

Despite recent advances in the clinical evaluation of various poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer (TNBC) patients, data defining potential anti-tumor mechanisms beyond PARP inhibition for these agents are lacking. To address this issue, we investigated the effects of four different PARP inhibitors (AG-014699, AZD-2281, ABT-888, and BSI-201) in three genetically distinct TNBC cell lines (MDA-MB-468, MDA-MB-231, and Cal-51). Assays of cell viability and colony formation and flow cytometric analysis were used to determine effects on cell growth and cell cycle progression. PARP-dependent and -independent signaling mechanisms of each PARP inhibitor were investigated by western blotting and shRNA approaches. Potential synergistic interactions between PARP inhibitors and cisplatin in suppressing TNBC cell viability were assessed. These PARP inhibitors exhibited differential anti-tumor activities, with the relative potencies of AG-014699 > AZD-2281 > ABT-888 > BSI-201. The higher potencies of AG-014699 and AZD-2281 were associated with their effects on G(2)/M arrest and DNA damage as manifested by γ-H2AX formation and, for AG-014699, its unique ability to suppress Stat3 phosphorylation. Abilities of individual PARP inhibitors to sensitize TNBC cells to cisplatin varied to a great extent in a cell context- and cell line-specific manner. Differential activation of signaling pathways suggests that the PARP inhibitors currently in clinical trials have different anti-tumor mechanisms beyond PARP inhibition and these PARP-independent mechanisms warrant further investigation.

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Conflict of interest statement

Conflict of interest The authors declare no conflict of interest.

Figures

Fig. 1

Fig. 1

Differential effects of PARP inhibitors on the viability and survival of TNBC cells. a Structures and reported IC50 values for PARP inhibition of AG-014699 [15], AZD-2281 [16], ABT-888 [17], and BSI-201 [18]. b MTT assays of the dose-dependent suppressive effects of AG-014699, AZD-2281, ABT-888, and BSI-201 on the viability of MDA-MB-468, MDA-MB-231, and Cal-51 cells in 5 % FBS-supplemented DMEM medium after 72 h of treatment. Mean ± SD (n = 6). c Clonogenic assays of the dose-dependent suppressive effects of AG-014699, AZD-2281, ABT-888, and BSI-201 on the survival of MDA-MB-468, MDA-MB-231, and Cal-51 cells in 5 % FBS-supplemented DMEM medium after 14–21 days of treatment. Mean ± SD (n = 3)

Fig. 2

Fig. 2

Differential effects of PARP inhibitors on PARP inhibition, DNA damage, and apoptosis in TNBC cells. Effects of AG-014699, AZD-2281, ABT-888, or BSI-201 on the levels of PAR, γ-H2AX, and PARP cleavage in MDA-MB-468, MDA-MB-231, and Cal-51 cells. Cells were treated with the indicated concentrations of PARP inhibitors in the presence of 5 % FBS-supplemented DMEM medium for 72 h

Fig. 3

Fig. 3

Differential effects of PARP inhibitors on the activation status of signaling effectors pertinent to cell proliferation and survival in TNBC cells, suggesting the heterogeneity in their modes of action, a Effects of AG-014699, AZD-2281, ABT-888, or BSI-201 on the phosphorylation status of Stat3, Akt, ERK, and p38 in MDA-MB-468, MDA-MB-231, and Cal-51 cells after 72 h of treatment, bLeft, verification by western blotting of ectopic expression of Stat3 in MDA-MB-468 cells transfected with an empty pcDNA vector or plasmid encoding Flag-tagged constitutively active Stat3 (CA-Stat3). Right, effect of the ectopic expression of CA-Stat3 versus the pcDNA vector on AG-014699-mediated suppression of clonogenic survival of MDA-MB-468 cells. Mean ± SD (n = 6). * P < 0.05

Fig. 4

Fig. 4

AG-014699- and AZD-2281-mediated activation of Akt and ERK might be attributable to downregulation of PHLPP expression in MDA-MB-468 cells, a Dose-dependent effects of AG-014699 and AZD-2281 versus ABT-888 on PHLPP expression after 72 h of treatment, b Effect of LY-294002 (10 µM) on AG-014699- and AZD-2281-mediated phosphorylation of Ser473-Akt and ERKs

Fig. 5

Fig. 5

Effect of shRNA-mediated silencing of p53 on the sensitivity of Cal-51 cells to the anti-proliferative effects of PARP inhibitors, a Verification by western blotting of shRNA-mediated knockdown of p53 expression in the stably transfected Cal-51 subclone. b Effects of p53 knockdown on the potencies of AG-014699, AZD-2281, ABT-888, and BSI-201 in suppressing the viability of Cal-51 cells after 72 h of treatment. Mean ± SD (n = 6). * P< 0.05. c Effects of p53 knockdown on the potencies of AG-014699, AZD-2281, ABT-888, and BSI-201 in suppressing the clonogenic survival of Cal-51 cells. Mean ± SD (n = 3). * P<0.05. d Effects of p53 knockdown on the DNA damage response, as indicated by γ-H2AX formation, to AG-014699 and AZD-2281

Fig. 6

Fig. 6

Sensitization of TNBC cells to cisplatin-mediated cell death and DNA damage by PARP inhibitors, a Differential susceptibility of MDA-MB-468 (468), MDA-MB-231 (231), and Cal-51 (51) cells to cisplatin-mediated suppression of cell viability after 72 h of treatment. Mean ± SD (n = 6). b Effects of AZD-2281, AG-014699, ABT-888, and BSI-201 on the sensitivity of MDA-MB-468 cells to cisplatin. Mean ± SD (n = 6). § CI > 1; ≠ CI = 1; * 1 > CI > 0.5; ** CI < 0.5. c Western blot analysis of the effects of AZD-2281, AG-014699, ABT-888, and BSI-201 on the sensitivity of MDA-MB-468 cells to cisplatin-induced DNA damage, as indicated by γ-H2AX formation, d Effects of AZD-2281, AG-014699, ABT-888, and BSI-201 on the sensitivity of Cal-51 and MDA-MB-231 cells to cisplatin. Mean ± SD (n = 6). e Western blot analysis of the effects of AZD-2281, AG-014699, ABT-888, and BSI-201 on the sensitivity of Cal-51 and MDA-MB-231 cells to cisplatin-induced DNA damage, as indicated by γ-H2AX formation

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