A selective eradication of human nonhereditary breast cancer cells by phenanthridine-derived polyADP-ribose polymerase inhibitors

Introduction PARP-1 (polyADP-ribose polymerase-1) is known to be activated in response to DNA damage, and activated PARP-1 promotes DNA repair. However, a recently disclosed alternative mechanism of PARP-1 activation by phosphorylated externally regulated kinase (ERK) implicates PARP-1 in a vast number of signal-transduction networks in the cell. Here, PARP-1 activation was examined for its possible effects on cell proliferation in both normal and malignant cells. Methods In vitro (cell cultures) and in vivo (xenotransplants) experiments were performed. Results Phenanthridine-derived PARP inhibitors interfered with cell proliferation by causing G2/M arrest in both normal (human epithelial cells MCF10A and mouse embryonic fibroblasts) and human breast cancer cells MCF-7 and MDA231. However, whereas the normal cells were only transiently arrested, G2/M arrest in the malignant breast cancer cells was permanent and was accompanied by a massive cell death. In accordance, treatment with a phenanthridine-derived PARP inhibitor prevented the development of MCF-7 and MDA231 xenotransplants in female nude mice. Quiescent cells (neurons and cardiomyocytes) are not impaired by these PARP inhibitors. Conclusions These results outline a new therapeutic approach for a selective eradication of abundant nonhereditary human breast cancers.


Introduction
PolyADP-ribose polymerases (PARPs) catalyze a posttranslational and energy-consuming modification of proteins by poly-ADP-ribosylation. This enzymatic reaction is initiated by ADPribose transferase activity, which proceeds with polymerization of ADP-riboses into long and branched polymers [1]. In the chromatin, polyADP-ribosylation apparently regulates the interaction of PARPs and their substrates with protein partners and DNA. PARP-1 is a highly conserved DNA-binding protein and the most abundant nuclear PARP. The enzyme is known to be activated in response to DNA single-strand breaks [1], and its activation induces chromatin remodeling, rendering the DNA more accessible to transcription factors and repair enzymes [1,2].
Our recent findings in quiescent cells and in cell-free systems disclosed an alternative mechanism, inducing an intense and long-lasting activation of PARP-1 in the absence of DNA damage [3,4]. In this process, PARP-1 interaction with phosphorylated ERK2 (externally regulated kinase) resulted in PARP-1 activation and polyADP-ribosylation in a positive-feedback mechanism that kept PARP-1 polyADP-ribosylated as long as ERK2 was phosphorylated [3]. In addition, polyADP-ribosylated PARP-1 highly augmented the activity of ERK: externally regulated kinase; MEK: mitogen-activated protein kinase kinase; PARP: polyADP-ribose polymerase; PTEN: phosphatase and tensin homologue; Raf: mitogen-activated protein kinase kinase kinase. phosphorylated ERK, enhancing phosphorylation of ERK-targeted transcription factors, core histone acetylation, and the expression of ERK target genes, some of which are oncogenes [3][4][5][6]. Because ERK activity in the nucleus is a key modulator for inducing proliferation versus differentiation in a variety of cancer cells [7,8], these findings suggest that PARP-1 activation might be a possible target for mechanisms inducing cell proliferation.
PARP inhibitors were designed to prevent PARP-1 activation in response to nicked DNA, in an attempt to suppress PARP mediated DNA repair [9][10][11][12]. Several generations of PARP inhibitors were designed to prevent PARP-1 activity by blocking the binding of the nicotinamide moiety of NAD + in the catalytic site of the enzyme. PARP inhibitors differ in their chemical structure, their potency, stability, solubility in water, and apparently even in their therapeutic potential [9][10][11][12]. Several groups of PARP inhibitors (including phenanthridine derivatives) were designed to protect cells under stress conditions from cell death induced by a massive activation of PARP-1 (for example, stroke, inflammation; [10,12]), or to cause cell death in malignant cells by preventing polyADP-ribosylation-dependent DNA repair [9,11]. In accordance with this concept, PARP inhibitors were tested for their therapeutic potential in malignant cells with impaired DNA-repair machinery [13,14] (bearing mutations in the tumor-suppressor genes BRCA1 and BRCA2 that cause an impaired DNA repair [15]) or in combination with DNA-damaging treatments [11]. However, in view of findings indicating that activated PARP-1 highly augments the activity of ERK in the nucleus even in the absence of DNA damage [3,4], a different therapeutic potential of PARP inhibitors is examined in breast cancer cells lacking BRCA mutations. MCF-10A human epithelial cells were cultured in DMEM/F12 (Gibco) with FBS (Gibco) 6%, EGF (100 μg/ml, Cytolab, Rehovot, Israel) 0.02%, hydrocortisone (50 μM, Sigma) 2.8%, insulin (10 mg/ml, Sigma) 0.1%, and Pen/Strep (Gibco) 1%.

Cell survival in cells treated with PJ-34
MCF-7 or MDA231 cells (seeded about 500,000 cells/3-mm well in six-well plates) were treated with PJ-34 applied only once, 24 hours after seeding. In some experiments, cells were reseeded in PJ-34-free medium in 10-cm plates for colony formation. In these experiments, after 2 weeks of incubation without application of PJ-34, cells were fixed (methanol/acetic acid, 3:1), stained with crystal violet, and counted to determine cell survival.
Flow cytometry was used to monitor changes in the ploidy level of malignant and normal cells labeled with propidium Iodide (PI) staining. Counting the cells with flow cytometry in selected time periods for several hours, indicated changes in their cell cycle caused by treatment with PJ-34. Flow cytometry was performed by using a Becton Dickenson machine and the FlowJo software (Tree Star, Ashland, OR, USA). Untreated cells were used as controls for each cell type.

Phenanthridine-derived PARP inhibitors efficiently eradicated MCF-7 and MDA231 breast cancer cells without impairing human epithelial MCF-10A or mouse embryonic fibroblasts
We examined the effect of PARP inhibitors on MCF-7 and MDA231 human breast cancer cells. Cells were treated with the potent PARP inhibitors, PJ-34, Tiq-A, and Phen ( Figure 1 However, unlike the malignant cells, MCF10A cells were only temporarily arrested (no arrest observed after 18 hours of incubation with PJ-34), and this transient arrest was not accompanied by cell death (Figure 4a and 4b). MCF-10A cells overcame the cell-cycle arrest, and continued to proliferate as normal cells, even when incubated with the same concentrations of PJ-34 and for the same durations used to eradicate MDA231 cells (compare Figures 2d and 4a). Also, proliferation of MCF-10A cells was not significantly reduced, even after a long incubation of 14 days with 10 μM PJ-34 (Figure 4a). G 2 /M cell-cycle arrest also was detected in mouse embryonic fibroblasts (Figure 5a) after 6 hours of incubation with PJ-34 (10 μM) (Figure 5b). These cells also overcame the cell-cycle arrest, and the arrest in cell cycle was not accompanied by cell death (Figure 5b).
Thus, treatment with PJ-34 induced a transient G 2 /M arrest in these normal proliferating cells, which was not accompanied by cell death (Figures 4 and 5), whereas the cell cycle of malignant cells MCF-7 and MDA231 was permanently arrested, and these cells were eradicated by incubation with PJ-34 applied only once 24 hours after seeding (Figures 2 and 3). An efficient eradication of MCF-7 cells was observed after 48 hours of incubation with 10 μM PJ-34, whereas MDA231 cells were massively eradicated only after 72 hours of incubation with PJ-34, 20-30 μM. Quiescent cells, brain cortical neurons, and cardiomyocytes were not impaired by incubation with the examined phenanthridine-derived PARP inhibitors (10 to 20 μM PJ-34, 100 μM Tiq-A, and 50 μM Phen [3,10,16,17]).

PJ-34 prevented the development of MCF-7 and MDA231 xenotransplants in nude female mice
In vivo experiments were carried out in nude female mice (nu/ nu) injected subcutaneously with MCF-7 or MDA231 cells ( Figure 6 and Additional data file 1). To test the effect of PJ-34 on the development of xenotransplants in the injected mice, PJ-34 (2 mM dissolved in 100 μl PBS) was inserted into subcutaneously implanted osmotic pumps (Alzet) that enable its constant slow release for 14 days (Methods). In the control nude mice, pumps contained only PBS, or pumps were not implanted. On the next day, 24 hours after pump implantation, each mouse was injected subcutaneously with approximately 10 7 MCF-7 or MDA231 cells dispersed in Matrigel (Methods). Tumors developed within 6 to 7 weeks in the control mice injected with MCF-7 cells and within 10 days in the control mice injected with MDA231 cells. One mouse died 3 weeks after being injected with MDA231 cells. In contrast, no visible tumors developed in the PJ-34-treated mice during 4 months after injection of MCF-7 cells and during the 10 weeks after injection with MDA231 cells (Figure 6a and 6b and Additional data file 1). Importantly, the 14-day treatment with a slow release of PJ-34 did not affect the vitality, growth, development, or any other behavior of the treated mice during the follow-up periods.     Tumors were not detected during 4 months after injection with the MCF-7 cells. Each female CD-1 nu/nu mouse was injected with about 10 7 MCF-7 cells collected from 80% to 90% confluent cell cultures. Cells were immersed in Matrigel/PBS (Methods). Cells were injected near the pump, one hour after pump implantation. (b) Each CD-1 nu/nu female mouse was injected with about 10 7 MDA231 cells collected from 80% to 90% confluent cell cultures and immersed in Matrigel/PBS (Methods). Cells were injected near the pump, 24 hours after pump implantation. Xenotransplants of MDA231 developed within 10 days in five female mice that were not treated with PJ-34 (untreated mice). Tumors did not develop in five female mice treated for 14 days with a slow release from a subcutaneously implanted osmotic pump (Alzet pump). Tumors were not detected in these mice during 10 weeks after injection with MDA231 cells and 8 weeks after the treatment with PJ-34. A detailed presentation of this experiment is included in Additional data file 1. (c) Kaplan-Meier survival analysis is used for presenting tumor-free survival curves of mice injected with MCF-7 cells and mice injected with MDA231 cells, without or after treatment with PJ-34, as described earlier. The significance (log-rank significance test) was P = 0.0253 for mice injected with MCF-7 cells, and P = 0.0023 for mice injected with MDA231 cells.
After 10 weeks, we detected tumors in two of the five mice that were injected with MDA231 cells and treated with PJ-34. These tumors were of human origin, as indicated by histochemistry (labeling with mouse anti-human mitochondria antibody (Millipore/Biotest) applied after blocking ("mouse-onmouse"; Vector Labs/Zotal)).
Tumor-free survival curves [18] for mice injected with MCF-7 cells and for mice injected with MDA231 cells are presented in Figure 6c. The effect of treatment with PJ-34 on tumor-free survival is indicated, and significance was calculated with the log-rank significance test [18].

Discussion
Findings indicating that polyADP-ribosylation is required for spindle formation [19] are in accordance with the observed G 2 /M arrest in both normal and malignant dividing cells treated with the potent PARP inhibitor PJ-34 (Figures 3, 4b, 5b). It is puzzling how normal cells overcome the G 2 arrest while malignant cells die. G 2 arrest could be induced by DNA damage in proliferating cells [20]. Because of their genomic instability and fast proliferation, malignant cells might be more susceptible to inhibition of PARP mediated DNA repair than are normal proliferating cells [13,14]. However, eradication of MCF-7 and MDA231 cells was not shared by other non-phenanthridine-derived PARP inhibitors, and the examined cell lines MCF-7 and MDA231 were neither BRCA-deficient cells [21] nor PTEN (phosphatase and tensin homologue)-deficient cells [22], in which DNA-repair mechanisms are impaired [15,23].
Other possible mechanisms underlying G 2 /M arrest implicate signal-transduction pathways involving p53, p21/WAF1, cdc25c, cdc2, and suppressed cyclin-B gene expression [20,[24][25][26]. Stabilization of p21 could promote G 2 arrest and induce cell death [25]. ERK activation also is implicated in G 2 / M transition control. Nuclear translocation of cyclin B/Cdc2 complex, which is required for G 2 /M transition, could be mediated by ERK activation [20]. Other mechanisms involving ERK activation in G 2 /M transition control are not fully resolved. They include autostabilization of the p21cip/cyclin D1 complex leading to cell-cycle arrest, and mechanisms promoting degradation of cyclin-dependent inhibitors such as p27kip1 [26][27][28][29][30][31]. Various impairments in some of these regulatory mechanisms were detected in human cancer cell types.
Blocking the activity of ERK by blocking the Ras/Raf/MEK/ ERK pathway is one of the main targets for human cancer treatment. However, previous clinical studies showed an insufficient antitumor activity of MEK inhibitors [39], suggesting that the MEK/ERK phosphorylation pathway is resilient to diminution of the activity of MEK and can adapt rapidly to maintain nearly normal ERK activation [39].
The recently disclosed augmentation of ERK activity in the nucleus by polyADP-ribosylated PARP-1 [3], and the fact that PARP-1 silencing with targeted siRNA downregulated ERK phosphorylation in nuclei of both MCF-7 and MDA231 cells (not shown), urged us to examine PARP inhibitors for their possible effect on cell proliferation in the absence of DNAdamaging factors. However, whereas phenanthridine-derived PARP inhibitors efficiently arrested the cell proliferation of MCF-7 and MDA231 cancer cells, other potent PARP inhibitors did not cause a similar effect. PARP inhibitor, ABT-888 (benzimidazole-4-carboxamide) hardly affected MCF-7 and MDA231 cells (not shown), and potent PARP inhibitors efficiently eradicating BRCA mutated breast cancer cells did not similarly eradicate MCF-7 cells [13,14] and cancer cells not carrying BRCA mutations [43]. Hence, additional features of the phenanthridine-derived PARP inhibitors apparently underlie their very promising potency to arrest proliferation and cause cell death in cancer cells lacking mutations that impair DNA repair, without impairing normal proliferating or quiescent cells.

Conclusions
Phenanthridine-derived PARP inhibitors possess a promising potency to arrest proliferation (G 2 /M arrest) and cause cell death in MCF-7 and MDA231 human breast cancer cells, without impairing normal proliferating human epithelial cells, fibroblasts, or quiescent cells (neurons, cardiomyocytes).