The Inhibitory Effects of HYDAMTIQ, a Novel PARP Inhibitor, on Growth in Human Tumor Cell Lines With Defective DNA Damage Response Pathways

Abstract

The poly(ADP-ribose) polymerase (PARP) enzymes play a key role in the regulation of cellular processes (e.g., DNA damage repair, genomic stability). It has been shown that PARP inhibitors (PARPIs) are selectively cytotoxic against cells having dysfunctions in genes involved in DNA repair mechanisms (synthetic lethality). Drug-induced PARP inhibition potentiates the activity of anticancer drugs such as 5-fluorouracil in enhancing DNA damage, whose repair involves PARP-1 activity. The aim of this study was to evaluate the inhibitory effects of a novel PARPI, HYDAMTIQ, on growth in human tumor cell lines characterized by different features with regard to DNA damage response pathways (BRCA mutational status, microsatellite status, and ATM expression level) and degree of sensitivity/resistance to 5-fluorouracil. HYDAMTIQ showed a more potent inhibitory effect on cell growth in a BRCA2 mutant cell line (CAPAN-1) compared with wild-type cells (C2-6, C2-12, and C2-14 CAPAN-1 clones, and MCF-7). No statistically significant difference was observed after HYDAMTIQ exposure between cells having a different MS status or a different MRE11 mutational status. HYDAMTIQ induced greater antiproliferative effects in SW620 cells expressing a low level of ATM than in H630 cells expressing a high level of ATM. Finally, the combination of HYDAMTIQ and 5-fluorouracil exerted a synergistic effect on the inhibition of SW620 cell growth and an antagonistic effect on that of H630 cell growth. Our results show that the novel PARP inhibitor HYDAMTIQ potently inhibits the growth of human tumor cells with defective DNA damage response pathways and exerts synergistic cytotoxicity in combination with 5-fluorouracil. These data provide relevant examples of synthetic lethality and evidence for further development of this novel PARPI.

Figure 1

Inhibition of human colon carcinoma cell growth: dose-dependent effects of HYDAMTIQ (A) and olaparib (B). Black curves indicate microsatellite stable (MSS) cell lines [MRE11 wild type (wt)]; blue curves indicate microsatellite instable (MSI) cell lines with heterozygous status of MRE11; and red curves indicate MSI cell lines with homozygous mutational status of MRE11. Each curve indicates the concentration–cell growth inhibition curve of a single cell line following 240 h of exposure.

Figure 2

Baseline levels of ATM (ataxia-telangiectasia mutated) protein expression in colon carcinoma cell lines. (A) Immunofluorescence staining of cell lines labeled with anti-ATM polyclonal antibody (green) and nuclei (blue) counterstained with DAPI (4,6-diamidino-2 phenylindole) (40×). Images show the baseline levels of ATM in cell lines. ATM plays a critical role in signaling DNA damage to cell cycle checkpoints and DNA repair pathways. (B) Baseline ATM protein level optical density according to the different cell lines tested. *au, arbitrary units, mean ± standard error.

Figure 3

Correlations between baseline ATM expression and HYDAMTIQ IC₅₀ values in colon carcinoma cell lines. Scatter plots indicate relationships between baseline ATM expression and IC₅₀ of HYDAMTIQ at 144 (A) and 240 (B) h or IC₅₀ of olaparib at 144 (C) and 240 (D) h in cell lines. *au, arbitrary units, mean ± standard error. Black circles indicate the intersection of IC₅₀/ATM expression values of a single cell line.

Figure 4

Dose-dependent effects of HYDAMTIQ and olaparib in SW620 and H630 cell lines and changes of ATM levels following 2 × IC₅₀ HYDAMTIQ treatment. Inhibition of cell growth: dose-dependent effects of HYDAMTIQ (A) or olaparib (B) in cell lines with the highest (H630) or the lowest (SW620) level of ATM protein. Inhibitory cell growth effects were evaluated after 240 h of exposure. ATM protein expression levels in H630 (C) and SW620 (D) cells exposed to HYDAMTIQ 2 × IC₅₀ for 240 h. *au, arbitrary units, mean ± standard error.

Figure 5

Pharmacodynamic interactions between HYDAMTIQ or olaparib and 5-fluorouracil in SW620 and H630 cell lines. Synergistic inhibitory effects of HYDAMTIQ 20 μM (∼IC₅₀) (white bar), 5-FU 3 μM (∼IC₂₅) (black bar), HYDAMTIQ 20 μM plus 5-FU 3 μM (gray bar) (A) or olaparib 5 μM (∼IC₅₀) (white bar), 5-FU 3 μM (∼IC₂₅) (black bar), and olaparib 5 μM plus 5-FU 3 μM (gray bar) (B) on the growth of SW620 cells characterized by low levels of ATM after 144 h of exposure. Antagonistic inhibitory effects of HYDAMTIQ 45 μM (∼IC₅₀) (white bar), 5-FU 1 μM (∼IC₂₅) (black bar), HYDAMTIQ 45 μM plus 5-FU 1 μM (gray bar) (C) or olaparib 30 μM (∼IC₅₀) (white bar), 5-FU 1 μM (∼IC₂₅) (black bar), and olaparib 30 μM plus 5-FU 1 μM (gray bar) (D) on the growth of H630 cells characterized by high levels of ATM after 144 h of exposure. Data from a representative experiment. The dotted line indicates expected growth. CI, observed/expected growth ratio.