Research ArticleBasic Science Investigations

Inhibition of Poly(ADP-Ribose) Polymerase Enhances the Toxicity of 131I-Metaiodobenzylguanidine/Topotecan Combination Therapy to Cells and Xenografts That Express the Noradrenaline Transporter

Anthony G. McCluskey, Robert J. Mairs, Mathias Tesson, Sally L. Pimlott, John W. Babich, Mark N. Gaze, Sue Champion and Marie Boyd
Journal of Nuclear Medicine July 2012, 53 (7) 1146-1154; DOI: https://doi.org/10.2967/jnumed.111.095943

Abstract

Targeted radiotherapy using 131I-metaiodobenzylguanidine (131I-MIBG) has produced remissions in some neuroblastoma patients. We previously reported that combining 131I-MIBG with the topoisomerase I inhibitor topotecan induced long-term DNA damage and supraadditive toxicity to noradrenaline transporter (NAT)–expressing cells and xenografts. This combination treatment is undergoing clinical evaluation. This present study investigated the potential of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP-1) inhibition, in vitro and in vivo, to further enhance 131I-MIBG/topotecan efficacy. Methods: Combinations of topotecan and the PARP-1 inhibitor PJ34 were assessed for synergism in vitro by combination-index analysis in SK-N-BE(2c) (neuroblastoma) and UVW/NAT (NAT-transfected glioma) cells. Three treatment schedules were evaluated: topotecan administered 24 h before, 24 h after, or simultaneously with PJ34. Combinations of PJ34 and 131I-MIBG and of PJ34 and 131I-MIBG/topotecan were also assessed using similar scheduling. In vivo efficacy was measured by growth delay of tumor xenografts. We also assessed DNA damage by γH2A.X assay, cell cycle progression by fluorescence-activated cell sorting analysis, and PARP-1 activity in treated cells. Results: In vitro, only simultaneous administration of topotecan and PJ34 or PJ34 and 131I-MIBG induced supraadditive toxicity in both cell lines. All scheduled combinations of PJ34 and 131I-MIBG/topotecan induced supraadditive toxicity and increased DNA damage in SK-N-BE(2c) cells, but only simultaneous administration induced enhanced efficacy in UVW/NAT cells. The PJ34 and 131I-MIBG/topotecan combination treatment induced G2 arrest in all cell lines, regardless of the schedule of delivery. In vivo, simultaneous administration of PJ34 and 131I-MIBG/topotecan significantly delayed the growth of SK-N-BE(2c) and UVW/NAT xenografts, compared with 131I-MIBG/topotecan therapy. Conclusion: The antitumor efficacy of topotecan, 131I-MIBG, and 131I-MIBG/topotecan combination treatment was increased by PARP-1 inhibition in vitro and in vivo.

Neuroblastoma is a heterogeneous disease, and patients can be assigned to various risk groups using age, stage, and other biologic characteristics (1). There is evidence that, over time, outcomes are gradually improving (2). However, for patients with high-risk disease, outcomes remain poor (3,4), and innovative therapies are required, especially for those whose disease fails to respond well to induction chemotherapy (5).

Approximately 90% of neuroblastoma tumor cells express the noradrenaline transporter (NAT), a 12-spanning integral membrane protein responsible for the active intracellular accumulation of catecholamine neurotransmitters. Metaiodobenzylguanidine (MIBG), a derivative of the adrenergic neurone-blocking drugs bretylium and guanethidine, is a structural analog of noradrenaline and is also selectively concentrated in NAT-expressing tissues and tumors by this process (6,7).

Targeted therapy of neuroblastoma using 131I-MIBG has produced encouraging results (long-term remissions and palliation) in patients with resistant disease (8–10). However, the most effective way to use this drug has yet to be defined, and, increasingly, 131I-MIBG is administered in combination with other treatments (11,12). The optimization of radiation damage induced in target cells can be obtained by the rational combination of 131I-MIBG with radiosensitizing agents, and we have previously reported that pretreatment with the topoisomerase I (Topo-I) poison topotecan increased the intracellular concentration of 131I-MIBG (13). Furthermore, we demonstrated that combinations of 131I-MIBG and topotecan induced disruption of DNA repair in NAT-expressing cells in vitro, increased supraadditive levels of cytotoxicity, and increased efficacy against NAT-expressing xenografts in vivo. Maximal topotecan-induced radiosensitization resulted from the administration of the drug simultaneously with or after 131I-MIBG. Exposure to topotecan before 131I-MIBG was less effective (14,15), suggesting that increased 131I-MIBG uptake due to prior topotecan administration was less important an influence on efficacy than the increased disruption of DNA repair observed in cells treated by combination schedules where topoisomerase I was inhibited concurrently, or after 131I-MIBG administration.

On the basis of our findings, clinical investigations of MATIN (MIBG and Topotecan in Neuroblastoma) have commenced (16). To date, more than 70 patients in 5 institutions across Europe have been enrolled in MATIN. Encouraging responses have been observed in a heterogeneous group of patients with relapsed or primary refractory disease. It is our intention to further enhance the effectiveness of MATIN by identifying other clinically relevant drugs that synergize with topotecan, 131I-MIBG, or both agents combined.

Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP-1) is an enzyme involved in DNA repair (17,18), and PARP-1 inhibition has been shown to enhance the efficacy of low-dose radiation (19). Furthermore, PARP-1 binds directly to Topo-I, leading to increased formation of Topo-I–DNA complexes (20). Therefore, we conjectured that PARP-1 inhibition may also influence topotecan-induced toxicity. This study investigated the effects of PARP-1 inhibition on the efficacy of topotecan, 131I-MIBG, and 131I-MIBG/topotecan combination therapy.

MATERIALS AND METHODS

Drugs

Topotecan (topoisomerase I inhibitor) was purchased from Axxora (U.K.) Ltd. and PJ34 (PARP-1 inhibitor) from Merck Chemicals Ltd. For in vitro experiments, no-carrier-added 131I-MIBG was provided by Dr. Sally Pimlott, NHS Greater Glasgow and Clyde. For in vivo experiments, no-carrier-added 131I-MIBG was provided by Molecular Insight Pharmaceuticals Inc.

Cells and Culture Conditions

The following human cell lines were cultured: SK-N-BE(2c), derived from neuroblastoma (21), and UVW/NAT, derived by transfection of the noradrenaline transporter gene into the human glioma cell line UVW (22). UVW/NAT cells were maintained in modified Eagle medium containing 10% fetal calf serum and 2 mM glutamine. SK-N-BE(2c) cells were maintained in Dulbecco modified Eagle medium containing 15% fetal calf serum, nonessential amino acids, and 2 mM glutamine. Cell lines were maintained at 37°C in a 5% CO2 atmosphere. All media and supplements were purchased from Gibco. Cell lines were authenticated routinely using the Applied Biosystems AmpF/STR Identifiler Kit (Applied Biosystems U.K.).

No-Carrier-Added Synthesis of 131I-MIBG via Polymer-Supported Precursor

For in vivo experiments, no-carrier-added 131I-MIBG was prepared using a solid-phase system for which the precursor of 131I-MIBG was attached to an insoluble polymer via the tin-aryl bond (23,24). For in vitro experiments, no-carrier-added 131I-MIBG was prepared using a liquid phase system, with the trimethylsily precursor (ABX) (25). The reaction conditions, high-performance liquid chromatography purification procedure, and radiochemical yield were as described previously (24).

Determination of Cytotoxicity After Combination Therapy

To investigate the effects of PARP-1 inhibition on 131I-MIBG/topotecan therapy, initially combinations of topotecan and PJ34 were evaluated using the following 3 treatment schedules: topotecan administered 24 h before PJ34 (schedule 1), topotecan administered after PJ34 (schedule 2), or topotecan administered simultaneously with PJ34 (schedule 3).

Combinations of PJ34 and 131I-MIBG and triple combinations of PJ34 and 131I-MIBG/topotecan were also evaluated, using similar scheduling. Because we have already reported that the administration of 131I-MIBG and topotecan induced supraadditive responses, in the assessment of PJ34, 131I-MIBG, and topotecan 3-drug combinations, PJ34 was administered 24 h before, after, or simultaneously with 131I-MIBG and topotecan given simultaneously.

Cytotoxicity was measured by clonogenic assay. Monolayers of cells were cultured in 25-cm2 flasks (Nunclon Plastics). UVW/NAT and SK-N-BE(2c) cells were seeded at 2 × 105 and 4 × 105 cells per flask, respectively. After 2 d, when the cultures were 70% confluent, medium was removed and replaced with fresh medium containing the appropriate concentration of test drug. Cells were incubated with 131I-MIBG for 2 h, after which uptake is maximal (26). Cells were incubated with topotecan or PJ34 for 24 h.

After experimental therapy, cells were washed twice in phosphate-buffered saline (PBS), detached by treatment with 0.05% (v/v) trypsin-ethylenediaminetetraacetic acid (Gibco), and counted and seeded, in triplicate, in 60 × 15 mm plastic dishes (Nunclon Plastics) at 2.5 × 102 cells per dish, for every test concentration. Cultures were incubated at 37°C in 5% CO2 for 14 d. Colonies were fixed in 100% methanol and visualized by staining with a solution of 1% (v/v) Giemsa (BDH Laboratory Supplies) and counted.

Synergy Analysis

The efficacy of the various scheduled combinations was examined according to the method of Chou and Talalay, which is based on the median-effect principle (27). Briefly, from the results of clonogenic assays, dose–effect curves were plotted using the equation log[fa/fu] = mlogD − mlogIC50, where D is the drug dose; fa and fu are, respectively, the fraction of cells affected by drug dose D and the unaffected fraction, and IC50 is the dose that inhibited 50% of colony formation. From these survival plots, the x-intercept (log [IC50]) and slope m were generated for each treatment. These parameters were used to calculate D, the doses of component agents (and combinations) required to produce various levels of toxicity.

The effectiveness of combination therapy was then assessed by combination-index (CI) analysis (27). The toxicities induced by single drugs and scheduled combinations were investigated using the equation CI = (D)1/(Dx)1 + (D)2/(Dx)2, where (D)1 and (D)2 are the doses of each agent that inhibit x% of cell growth when used in combination, and (Dx)1 and (Dx)2 are the doses of each drug, administered as single agents, that inhibit x% of colonies. The resultant numeric values, the combination indices, were plotted against toxicity level.

Assessment of Cell Cycle Progression by Fluorescence-Activated Cell Sorting Analysis

Cells were plated in 25-cm2 flasks and exposed to single drug and multidrug combinations as described above. To directly compare the effects on cell cycle progression with the results of clonogenic assay, the same drug concentrations and incubation times were used. Cultures were then trypsinized, counted, washed twice with PBS, and resuspended in PBS at a concentration of 1 × 106 cells/mL. Cells were fixed by treatment with 75% (v/v) ethanol for 1 h at 4°C. Fixed cells were washed twice with PBS and resuspended in 1 mL of PBS containing 50 mg of propidium iodide per milliliter (Sigma Chemicals) and 5 μg of RNase A per milliliter (Qiagen Ltd.). Cells were stained for 3 h at 4°C before flow cytometry, using a FACScan analyzer (Becton Dickinson Systems). Data were analyzed using CellQuest Pro software (version 5.1.1; BD Biosciences).

Assessment of Double-Strand (ds) DNA Breaks by H2A.X Phosphorylation

Phosphorylation of histone H2A.X at serine 139 (γH2A.X) was assessed using the H2A.X phosphorylation assay kit (Millipore). Briefly, cells were seeded and drug-treated, as described above. Immediately after treatment, when DNA damage was expected to be maximal, cells were trypsinized, counted, washed twice with PBS, and fixed by addition of formaldehyde and methanol for 20 min at 4°C. Cells were then washed 3 times with PBS and resuspended, at a concentration of 2 × 106 cells/mL, in permeabilization solution (5% saponin; 100 mM N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid], pH 7.4; 1.4 M NaCl; and 25 mM CaCl2) containing antiphospho-histone H2A.X (Ser139)-fluorescein isothiocyanate conjugate for 20 min at 4°C. Fluorescence was measured using a FACScan analyzer, and data were analyzed using CellQuest Pro software, version 5.1.1.

PARP-1 Assay

Cellular PARP-1 activity was assessed using the commercially available HT Universal colorimetric assay kit (AMS Biotechnology Ltd.), according to the manufacturer’s instructions. Briefly, following the drug treatment as described in the “Determination of Cytotoxicity After Combination Therapy” section, PARP-1 activity was assayed by incorporation of biotinylated poly(ADP-ribose) onto histone proteins, followed by incubation with streptavidin-horseradish peroxidase and TACS (Trevigen Apoptotic Cell System; Trevigen) sapphire colorimetric substrate. Absorbance at 450 nm was measured using a Sunrise plate reader (Tecan U.K. Ltd.), with Magellan CE software (version 5.04).

Experimental Animals

Six-week-old female, congenitally athymic nude mice of strain MF1 nu/nu were obtained from Charles River PLC. Experiments were performed in accordance with the U.K. Coordinating Committee on Cancer Research guidelines (28).

In Vivo Investigations

SK-N-BE(2c) and UVW/NAT xenografts were established and analyzed as previously described (14,15). Briefly, SK-N-BE(2c) xenografts were established by intrasplenic injection of 3 × 106 exponentially growing cells. After the growth of tumors in the spleen and liver, animals were euthanized and tumor fragments 2–3 mm in diameter were then implanted subcutaneously in the subcostal flanks of other nude mice. Experimental therapy commenced 17 d after tumor implantation when tumors had reached approximately 10 mm in diameter (500 mm3). UVW/NAT xenografts were established by subcutaneous injection of 2 × 106 cells. Experimental therapy was initiated 9 d later, when tumor volume was approximately 60 mm3.

Groups of 6 mice with SK-N-BE(2c) or UVW/NAT tumors were randomized into 6 treatment groups that received, by intraperitoneal injection, saline (control), PJ34 (20 mg/kg) alone, 18 MBq of 131I-MIBG plus 1.75 mg of topotecan per kilogram (SK-N-BE(2c)), 5 MBq of 131I-MIBG plus 0.825 mg of topotecan per kilogram (UVW/NAT), or PJ34 given simultaneously with 131I-MIBG/topotecan.

Experimental xenografts were measured with calipers immediately before treatment and every 2–3 d thereafter. Measurements were converted to an approximate volume on the assumption of ellipsoidal geometry as previously described (14,15).

For each treatment group, the mean time taken for a 2-fold (T2) (SK-N-BE(2c)) or 10-fold (T10) (UVW/NAT) increase in tumors was calculated. Tumor cure was defined as the failure of experimental xenografts to grow over the experimental time course.

Statistical Analysis

Unless otherwise stated, experimental results are expressed as means and SDs of 3 separate experiments, performed in triplicate. Statistical analyses were performed using Prism software (version 4.03; GraphPad Software Inc.). One-way ANOVA was used to compare, between treatments, formation of dsDNA damage and PARP-1 activity. Post hoc testing used Bonferroni correction for multiple comparisons. Differences in tumor growth between experimental therapy groups were assessed by the Kruskal–Wallis test, followed by post hoc testing by the Mann–Whitney U test. With Bonferroni correction, a P value of less than 0.01667 was considered significant.

RESULTS

Cytotoxicity of 2-Drug Combination Therapy

For both cell lines, the efficacy of topotecan and PJ34 as single agents was assessed, and results were plotted graphically, according to the median-effect principle (Supplemental Fig. 1A; supplemental materials are available online only at http://jnm.snmjournals.org). On the basis of single-drug toxicity, a fixed ratio of topotecan-to-PJ34 was used in subsequent analyses of alternative combination schedules. The topotecan (nM)–to–PJ34 (μM) ratios were 8.8:31.97 for SK-N-BE(2c) cells and 10:29.1 for UVW/NAT cells. The median-effect plots for alternative topotecan and PJ34 combination schedules are shown in Supplemental Figure 1B.

Combination-index analysis of topotecan and PJ34 treatments in SK-N-BE(2c) and UVW/NAT cells are shown in Figure 1. Both cell lines were resistant to doses of combinations of topotecan and PJ34, which induced low levels of toxicity. Schedule 3 (topotecan and PJ34 simultaneously) was the most effective treatment, inducing supraadditive responses in both lines. In SK-N-BE(2c) cells, supraadditive responses were also observed after the administration of schedule 2 treatment (topotecan after PJ34, but not schedule 1, topotecan before PJ34). All 3 schedules induced supraadditivity in UVW/NAT cells at high levels of toxicity.

FIGURE 1.
FIGURE 1.

Combination-index analysis of PJ34/topotecan treatment in SK-N-BE(2c) (A) and UVW/NAT (B) cells. On the basis of results shown in Supplemental Figure 1, SK-N-BE(2c) cells were treated with topotecan and PJ34 in a ratio of 8.8:31.97. UVW/NAT cells were treated with PJ34 and topotecan in a ratio of 10:29.1. Data are means and SDs of triplicate determinations from 3 experiments. CI < 1, CI = 1, and CI > 1 indicate synergism, additivity, and antagonism, respectively.

Dose responses for PJ34 and 131I-MIBG as single agents are shown in Supplemental Figure 2A. On the basis of these results, the ratios of PJ34 (μM)–to–131I-MIBG (MBq/mL) used in subsequent combination studies were 31.97:1.29 for SK-N-BE(2c) cells and 29.1:2.76 for UVW/NAT cells. The effects of scheduled combinations are shown in Supplemental Figure 2B. The resultant combination-index analyses of SK-N-BE(2c) and UVW/NAT cells are shown in Figure 2.

FIGURE 2.
FIGURE 2.

Combination-index analysis of PJ34/131I-MIBG treatment in SK-N-BE(2c) (A) and UVW/NAT (B) cells. On the basis of results shown in Supplemental Figure 2, SK-N-BE(2c) cells were treated with PJ34 and 131I-MIBG in a ratio of 31.97:1.29. UVW/NAT cells were treated with PJ34 and 131I-MIBG in a ratio of 29.1:2.76. Data are means and SDs of triplicate determinations from 3 experiments. CI < 1, CI = 1, and CI > 1 indicate synergism, additivity, and antagonism, respectively.

Both cell lines were resistant to doses of combinations of PJ34 and 131I-MIBG, which induced low levels of toxicity. Schedule 3 (PJ34 and 131I-MIBG simultaneously) induced supraadditive responses in both cell lines. In SK-N-BE(2c) cells, schedule 1 (PJ34 before 131I-MIBG) induced an additive response, whereas schedule 2 (PJ34 after 131I-MIBG) induced infraadditive toxicity. In UVW/NAT cells, supraadditive responses were also observed after administration of schedule 2, whereas schedule 1 induced infraadditive toxicity.

Cytotoxicity of 3-Drug Combination Therapy

Dose responses for PJ34 as a single agent and 131I-MIBG/topotecan 2-drug combinations are shown in Supplemental Figure 3A. On the basis of these results, the ratios of PJ34 (μM) to 131I-MIBG (MBq/mL) to topotecan (nM) used in subsequent 3-drug combination studies were 31.97:1.29:8.8 for SK-N-BE(2c) cells and 29.1:2.76:10 for UVW/NAT cells. Therefore, 1 arbitrary dose unit (au) contained 0.76 μM PJ34, 0.03 MBq of 131I-MIBG per milliliter, and 0.21 nM topotecan for SK-N-BE(2c) cells, or 0.69 μM PJ34, 0.07 MBq of 131I-MIBG per milliliter, and 0.24 nM topotecan for UVW/NAT cells.

The effects of scheduled triple combinations are shown in Supplemental Figure 3B. The resultant combination-index analyses of SK-N-BE(2c) and UVW/NAT cells are shown in Figure 3.

Supraadditive toxicity was observed in SK-N-BE(2c) cells following all 3 schedules at every level of toxicity assessed. Only schedule 3 (PJ34 and 131I-MIBG/topotecan simultaneously) induced enhanced efficacy in UVW/NAT cells. Schedule 2 (PJ34 after 131I-MIBG/topotecan) induced an additive response in UVW/NAT cells, whereas schedule 1 (PJ34 before 131I-MIBG/topotecan) was antagonistic.

FIGURE 3.
FIGURE 3.

Combination index analysis of PJ34/131I-MIBG/topotecan in SK-N-BE(2c) (A) and UVW/NAT (B) cells. On the basis of results shown in Supplemental Figure 3, SK-N-BE(2c) cells were treated with PJ34, 131I-MIBG, and topotecan in a ratio of 31.97:1.29:8.8. UVW/NAT cells were treated with PJ34, 131I-MIBG, and topotecan in a ratio of 29.1:2.76:10. Data are means and SDs of triplicate determinations from 3 experiments. CI < 1, CI = 1, and CI > 1 indicate synergism, additivity, and antagonism, respectively.

Cell Cycle Redistribution

Cell cycle redistribution induced by PJ34 in SK-N-BE(2c) and UVW/NAT cells is shown in Table 1. The effects of triple-combination treatments on cell cycle phases are shown in Tables 2 (SK-N-BE(2c)) and 3 (UVW/NAT). PJ34 as a single agent induced G2/M arrest. Likewise, all 3 scheduled combinations of PJ34 and 131I-MIBG/topotecan caused an increase in the number of cells in G2/M.

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TABLE 1

Effects of PJ34 on Cell Cycle of SK-N-BE(2c) and UVW/NAT Cells

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TABLE 2

Effects of PJ34 and 131I-MIBG/Topotecan on Cell Cycle of SK-N-BE(2c) Cells

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TABLE 3

Effects of PJ34 and 131I-MIBG/Topotecan on Cell Cycle of UVW/NAT Cells

γH2A.X Analysis of dsDNA Damage

The effects of 3-drug administration on formation of dsDNA breaks are shown in Figure 4A. Cells treated with PJ34 and 131I-MIBG/topotecan combinations displayed increased phosphorylation of H.2AX foci compared with untreated controls. In SK-N-BE(2c) cells, all treatment schedules induced a 28- to 45-fold increase in dsDNA damage. ANOVA demonstrated no significant difference between the potency of alternative combination schedules. However, in UVW/NAT cells, administration of alternative schedules gave rise to various levels of DNA damage. Schedule 1 induced a 2-fold increase in γH2A.X foci, whereas schedule 3 induced a 60-fold increase. Schedule 2 induced a 20-fold increase in H2A.X phosphorylation. ANOVA demonstrated significant variation between the responses induced by alternative schedules of delivery and in post hoc testing. Schedule 3 induced significantly higher levels of γH2A.X phosphorylation than either of the other treatment schedules (P < 0.01667).

FIGURE 4.
FIGURE 4.

(A) γH2A.X analysis of dsDNA damage in SK-N-BE(2c) (i) and UVW/NAT (ii) cells. (B) PARP-1 activity in SK-N-BE(2c) and UVW/NAT cells after PJ34 (i) and 131I-MIBG/topotecan (ii) treatment. (C) PARP-1 activity in SK-N-BE(2c) (i) and UVW/NAT (ii) cells after PJ34 and 131I-MIBG/topotecan combination treatment. Ratios of PJ34 and 131I-MIBG/topotecan used in 3-drug combinations were 31.97:1.29:8.8 and 29.1:2.76:10 for SK-N-BE(2c) and UVW/NAT cells, respectively. Ratios of 131I-MIBG and topotecan used in 2-drug combinations were 1.29:8.8 and 2.76:10 for SK-N-BE(2c) and UVW/NAT cells, respectively. Data are means and SD of triplicate determinations from 3 experiments.

PARP-1 Activity

The effects of PJ34 treatment and 131I-MIBG/topotecan 2-drug therapy on PARP-1 activity in SK-N-BE(2c) and UVW/NAT cells are shown in Figure 4B. PJ34 induced a dose-dependent reduction in PARP-1 activity, compared with untreated controls. SK-N-BE(2c) cells were more sensitive to PJ34 than UVW/NAT cells. The dose that reduced PARP-1 activity by 50% (EC50) was 8.8 and 14.6 μM in SK-N-BE(2c) cells and UVW/NAT cells, respectively.

In SK-N-BE(2c) cells, compared with untreated controls, administration of 131I-MIBG/topotecan induced a reduction in PARP-1 activity at concentrations less than or equal to 5.05 au. 131I-MIBG/topotecan also reduced PARP-1 activity in UVW/NAT cells at concentrations less than or equal to 6.38 au. As with PJ34, SK-N-BE(2c) cells were more sensitive to 131I-MIBG/topotecan than UVW/NAT cells (EC20 values were 1.65 and 4.4 au for SK-N-BE(2c) and UVW/NAT cells, respectively). However, in both cell lines, after administration of the highest administered dose of 131I-MIBG/topotecan (7.57 au for SK-N-BE(2c) cells and 9.57 au for UVW/NAT cells), 100% recovery of PARP-1 activity was observed, suggesting an adaptive response to 131I-MIBG/topotecan–induced disruption of PARP-1 function.

The effects of PJ34 and 131I-MIBG/topotecan combination therapy on PARP-1 activity in SK-N-BE(2c) and UVW/NAT cells are shown in Figure 4C. Three-drug therapy reduced PARP-1 activity in SK-N-BE(2c) cells, compared with untreated controls. Schedule 3 (simultaneous administration) was the most effective schedule. However, unlike 131I-MIBG/topotecan 2-drug therapy, there was no recovery in PARP-1 activity at higher doses in SK-N-BE(2c) cells. In UVW/NAT cells, the administration of schedules 2 and 3 (PJ34 simultaneously with or after 131I-MIBG/topotecan) also induced a reduction in PARP-1 activity. Again, there was no evidence of a recovery of PARP-1 function at higher doses. However, schedule 1 (PJ34 before 131I-MIBG/topotecan) had no effect on PARP-1 activity, suggesting that inhibition of PARP-1 function by PJ34 was not only reversed after removal of the drug but also provoked resistance to subsequent 131I-MIBG/topotecan–induced inhibition of PARP-1 function in this cell line.

In Vivo Investigations

None of the animals in this study showed signs of distress. Figure 5 shows the effect on the growth of SK-N-BE(2c) and UVW/NAT tumor xenografts of the administration of PJ34 or 131I-MIBG/topotecan either alone or in combination. Tumor growth times and cure rates for SK-N-BE(2c) and UVW/NAT xenografts are presented in Table 4.

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TABLE 4

Delayed Tumor Growth Resulting from Administration of PJ34 or 131I-MIBG/Topotecan Alone or in Combination

FIGURE 5.
FIGURE 5.

Effects of PJ34 and 131I-MIBG/TPT on SK-N-BE(2c) (A) and UVW/NAT (B) xenografts. Each treatment group consisted of 6 animals. Mice bearing SK-N-BE(2c) xenografts were treated with either 20 mg of PJ34 per kilogram, 18 MBq of 131I-MIBG plus 1.75 mg of topotecan per kilogram, or combinations of PJ34 and 131I-MIBG/topotecan. UVW/NAT-bearing animals were treated with either 20 mg of PJ34 per kilogram, 5 MBq of 131I-MIBG plus 0.875 mg of topotecan per kilogram, or 3-drug combinations.

For both xenograft models, overall differences in the effectiveness of the different treatments were highly significant (Kruskal–Wallis test, P < 0.005). Single treatment with PJ34 did not significantly affect tumor growth. Treatment with 131I-MIBG/topotecan or PJ34 in combination with 131I-MIBG/topotecan significantly delayed SK-N-BE(2c) and UVW/NAT tumor growth, compared with PBS-treated controls (P < 0.01667). Furthermore, 3-drug therapy also significantly increased the delay in tumor growth, compared with 131I-MIBG/topotecan double combinations in both in vivo models (P < 0.01667). No SK-N-BE(2c) tumors were cured during the course of these experiments. In contrast, 60% of UVW/NAT tumors were cured by 131I-MIBG/topotecan treatment, whereas 3-drug treatment cured all UVW/NAT tumors.

DISCUSSION

Previously, we reported that topotecan (topoisomerase I inhibitor) synergized with 131I-MIBG. The present study indicated that PJ34 enhanced the efficacy of topotecan and 131I-MIBG in vitro and 131I-MIBG/topotecan combination therapy in vitro and in vivo. Enhanced 131I-MIBG/topotecan efficacy was associated with disruption of PARP-1 activity, increased formation of dsDNA breaks, and G2/M cell cycle arrest.

Enhanced efficacy was most likely caused by disruption of DNA damage repair pathways. PARP-1 is involved in the repair of single-strand DNA breaks through the base excision repair pathway (18) and may also be involved in repair of double-strand breaks, through the homologous recombination (HR) pathway (29). Furthermore, we have previously demonstrated that topotecan and 131I-MIBG, either alone or in combination, induced G2-phase cell cycle arrest (15). In this study, treatment with PJ34 either as a single agent or in combination with 131I-MIBG/topotecan also caused G2 arrest. Cells in G2- and M-phase are more radiosensitive than cells in other phases of the cell cycle (30). Thus, cell cycle redistribution induced by treatment with PJ34 and 131I-MIBG/topotecan combinations probably contributed to the enhanced efficacy of 131I-MIBG.

After topotecan treatment, Topo-I becomes strongly associated with DNA via stabilization of Topo-I–DNA complexes, leading to stalled DNA replication. PARP-1–mediated ADP-ribosylation of Topo-I reprograms the trapped enzyme to remove itself from cleaved DNA (31). PARP-1 also collaborates with Mre11, a core subunit of the Mre11/Rad50/Nbs1 damage recognition complex, to promote replication fork restart after release from replication blocks (29). Thus, by counteracting topoisomerase I–induced DNA damage, PARP-1 activity acts as a positive regulator of genomic stability in eukaryotic cells.

It has previously been demonstrated that after the induction of DNA damage by x-irradiation, ultraviolet light, and γ-irradiation, binding of PARP-1 to Topo-I induces a rapid sequestration of Topo-I onto the sites of the DNA lesions (20,32,33). Furthermore, Topo-I–mediated unwinding of supercoiled DNA is reduced after irradiation, possibly by abrogation of Topo-I catalytic activity (34,35) or reduced longevity of Topo-I–DNA complexes (36). This effect appears to be due to PARP-1–induced ADP-ribosylation of Topo-I and is prevented by the addition of PARP-1 inhibitors (34,35),

Therefore, in cells treated with combinations of 131I-MIBG and topotecan, inhibition of PARP-1 activity by PJ34 leads to the simultaneous generation of multiple effects. Deregulation of Topo-I function via ADP-ribosylation and prevention of removal of topotecan-mediated aberrant Topo-I-DNA adducts will enhance the efficacy of the Topo-I poison, whereas disruption of base excision repair and, potentially, HR mechanisms of repair would increase the effects of 131I-MIBG–induced DNA damage.

Treatment with 131I-MIBG/topotecan induced a reduction in PARP-1 activity, reaching a nadir at a combination dose equivalent to 5.05 and 6.36 au in SK-N-BE(2c) and UVW/NAT cells, respectively. It has long been recognized that MIBG is an inhibitor of mono-ADP-ribosylation (37). Furthermore, it has recently been suggested that, in the absence of DNA damage, PARP-1 function is regulated by the mono-ADP-ribosyl polymerase activity of the related enzyme PARP-3 (38). Therefore, one possible explanation for these findings is that reduced PARP-1 function after administration of low doses of 131I-MIBG/topotecan is due to MIBG-induced inhibition of PARP-3 regulation of PARP-1. However, after administration of doses of 131I-MIBG/topotecan greater than 5.05 au (SK-N-BE(2c)) or 6.36 au (UVW/NAT), PARP-1 function recovered in both cell lines. Increased DNA damage was observed with increasing dose of 3-drug therapy. Taken together, these results suggest that if, as speculated, MIBG inhibits PARP-3, increasing DNA damage may induce activation of PARP-1 via a PARP-3–independent pathway, leading to the observed recovery of PARP-1 function after high-dose 131I-MIBG/topotecan treatment.

UVW/NAT cells treated with PJ34 before 131I-MIBG/topotecan displayed no disruption of PARP-1 function. PJ34 induced a reduction of PARP-1 activity, which was restored by removal of the drug. The subsequent addition of 131I-MIBG/topotecan had no effect on PARP-1 function, suggesting that UVW/NAT cells were primed to resist 131I-MIBG/topotecan–induced disruption of PARP-1 activity by PJ34 pretreatment. It is possible that the recovery of PARP-1 function in PJ34 pretreated cells may be induced by a PARP-3–independent pathway, which would be unaffected by PARP-3 inhibition by MIBG. This result suggests that the efficacy of PJ34/131I-MIBG/topotecan treatment may be affected by alternative PARP-1 activation pathways and warrants further study.

Although the involvement of PARP-1 in repair of dsDNA damage is as yet unclear, this present study does suggest that PARP-1 activity is involved in this process in some way. SK-N-BE(2c) cells, which exhibited reduced PARP-1 function in this phase, also displayed increased generation of dsDNA damage after 3-drug treatment, leading to supraadditive cytotoxicity. Conversely, UVW/NAT cells treated with PJ34 before 131I-MIBG/topotecan exhibited normal PARP-1 function and DNA damage was negligible, suggesting less inhibition of the repair of dsDNA breaks, leading to infraadditive toxicity in cells treated by this schedule. Combination therapy also induced G2/M arrest, in which the predominant dsDNA damage repair pathway is HR. Therefore, it is possible that PARP-1 may play a role in HR; however, involvement with nonhomologous end joining cannot be discounted.

PJ34 alone induced cytotoxicity and G2/M arrest. Therefore, nontarget effects may influence overall response. These nontarget effects will be addressed by further mechanistic studies, using PJ34 and also second- and third-generation inhibitors with greater PARP-1 specificity.

Previously, we demonstrated that 131I-MIBG/topotecan combination therapy significantly inhibited SK-N-BE(2c) and UVW/NAT tumor growth in vivo (14,15). In this study, whereas PJ34 treatment alone had no effect on tumor growth, administration of PJ34 concurrently with 131I-MIBG/topotecan significantly delayed the growth of SK-N-BE(2c) and UVW/NAT xenografts, compared with 131I-MIBG/topotecan.

CONCLUSION

This study indicates that inhibition of PARP-1 has the potential to increase the efficacy of 131I-MIBG/topotecan combination therapy by increasing radiosensitivity and disrupting DNA repair. Taking into account the responses observed both in vitro and in vivo, this study suggests that enhancement of 131I-MIBG/topotecan efficacy may be best achieved by the simultaneous inhibition of PARP-1 function. Elucidation of the basis for resistance after pretreatment with PJ34 may allow further refinements to this combination and are worthy of investigation.

DISCLOSURE STATEMENT

The costs of publication of this article were defrayed in part by the payment of page charges. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

This work was supported by grants from Cancer Research U.K., the Neuroblastoma Society, and Sparks. No other potential conflict of interest relevant to this article was reported.

Footnotes

  • ↵* Contributed equally to this work.

  • Published online Jun. 11, 2012.

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  • Received for publication July 27, 2011.
  • Accepted for publication March 5, 2012.
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