A selective eradication of human nonhereditary breast cancer cells by phenanthridine-derived polyADP-ribose polymerase inhibitors
© Inbar-Rozensal; licensee BioMed Central Ltd. 2009
Received: 19 February 2009
Published: 9 November 2009
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.
In vitro (cell cultures) and in vivo (xenotransplants) experiments were performed.
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.
These results outline a new therapeutic approach for a selective eradication of abundant nonhereditary human breast cancers.
PolyADP-ribose polymerases (PARPs) catalyze a posttranslational and energy-consuming modification of proteins by polyADP-ribosylation. This enzymatic reaction is initiated by ADP-ribose transferase activity, which proceeds with polymerization of ADP-riboses into long and branched polymers . 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 , 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 . In addition, polyADP-ribosylated PARP-1 highly augmented the activity of phosphorylated ERK, enhancing phosphorylation of ERK-targeted transcription factors, core histone acetylation, and the expression of ERK target genes, some of which are oncogenes [3–6]. 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 [9–12]. 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 [9–12]. 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 ) or in combination with DNA-damaging treatments . 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.
Materials and methods
Human breast cancer cell line MCF-7 and MDA231 and human epithelial cells MCF-10A were supplied by ATCC Co. (American Type Culture Collection, P.O. Box 1549, Manassas, VA 20108, USA. The dealer in Israel is Almog Diagnostic & Medical equipment Ltd.). Mouse embryonic fibroblasts were prepared in the laboratory of Dr. Dantzer (Strasbourg, France).
MCF-7 and MDA231 cells were cultured in six-well multidish plates (Nunc, Thermo Fisher scientific, Reskilde, Denmark). MCF-7 and MDA231 cells were maintained in a medium containing DMEM (cat. 01055-1A), 10% Fetal bovine serum (FBS; cat. 04-124-1A), 1% L-glutamine (cat. 03-020-1B), and 1% Pen-Strep Ampho (cat. 03-033-1B) (Gibco, purchased from Rhenium, Rehovot, Israel).
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%.
Mouse embryonic fibroblasts were cultured in a medium containing DMEM (cat. 01055-1A), 10% FBS (cat. 04-121-1A), 1% L-glutamine (cat. 03-020-1B), and 1% Pen-Strep Ampho (cat. 03-033-1B) (Gibco, purchased from Rhenium, Rehovot, Israel).
Phenanthridine-derived PARP inhibitors
The effect of PJ-34 on the development of MCF-7 and MDA231 xenotransplants
Xenotransplants were developed in female CD-1 nu/nu mice 5 to 6 weeks old (Charles River Labs, Sulzfeld, Germany) purchased in Israel from Harlan Labs, Jerusalem. MCF-7 and MDA231 cells were injected subcutaneously, about 107 MCF-7 or MDA231 cells in 150 μl of PBS and 150 μl of Matrigel Basement Membrane Matrix (Becton Dickinson, Bedford, MA, USA; In Israel, Bactolab Diagnostics). In mice treated with PJ-34, Injection was adjacent to subcutaneous osmotic pumps dripping PJ-34 by a slow release. The mice were maintained under specific pathogen-free conditions with access to mouse chow and water ad libitum. PJ-34 (2 mM dissolved in 100 μl PBS) was inserted in subcutaneously implanted Alzet osmotic pumps, designed to release PJ-34 continuously (at about 0.6 nmol/h) for 14 days. For comparison, in the in vitro experiments, the amount of PJ-34 per dish was approximately 20 nmol. Subcutaneous implantation of these pumps was performed before injection by a veterinarian (Dr. Kastel David). All the experiments with nude mice conform with the Guide for the Care and Use of Laboratory Animals published by the NIH (publication No. 85-23, revised 1996). Approval was granted by the Israeli Ministry of Health ethics review board in the Tel-Aviv University (M08033).
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
Thus, treatment with PJ-34 induced a transient G2/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
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-on-mouse"; Vector Labs/Zotal)).
Tumor-free survival curves  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 .
Findings indicating that polyADP-ribosylation is required for spindle formation  are in accordance with the observed G2/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 G2 arrest while malignant cells die.
G2 arrest could be induced by DNA damage in proliferating cells . 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  nor PTEN (phosphatase and tensin homologue)-deficient cells , in which DNA-repair mechanisms are impaired [15, 23].
Other possible mechanisms underlying G2/M arrest implicate signal-transduction pathways involving p53, p21/WAF1, cdc25c, cdc2, and suppressed cyclin-B gene expression [20, 24–26]. Stabilization of p21 could promote G2 arrest and induce cell death . ERK activation also is implicated in G2/M transition control. Nuclear translocation of cyclin B/Cdc2 complex, which is required for G2/M transition, could be mediated by ERK activation . Other mechanisms involving ERK activation in G2/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 [26–31]. Various impairments in some of these regulatory mechanisms were detected in human cancer cell types.
Numerous primary human tumors and derived cell lines display a constitutive activation of ERK [8, 31–34]. Some of them harbor mutations of the Ras protein that render it and the Raf-MEK-ERK cascade constitutively active [35–37]. Constitutively activated Raf has been found in many human cancers [35–37]. ERK targets are constitutively activated in B-Raf melanomas, colon cancer, PC-3 prostate cancer, and pancreatic cancer cells [37–39]. Growth and proliferation of the human breast cancer MCF-7 and MDA231 cells are also highly dependent on ERK activity [40–42].
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 , 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 .
The recently disclosed augmentation of ERK activity in the nucleus by polyADP-ribosylated PARP-1 , 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 DNA-damaging 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 . 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.
Phenanthridine-derived PARP inhibitors possess a promising potency to arrest proliferation (G2/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).
externally regulated kinase
mitogen-activated protein kinase kinase
phosphatase and tensin homologue
mitogen-activated protein kinase kinase kinase.
This work was supported by The Israeli Science Foundation and the Israeli Ministry of Health, grants of Prof. M. Cohen-Armon.
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