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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Northern Institute for Cancer Research, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom and 2 KuDOS Pharmaceuticals Ltd., Cambridge, United Kingdom
Requests for reprints: Nicola Curtin, Northern Institute for Cancer Research, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, United Kingdom. Phone: 44-191-246-4415; Fax: 44-191-246-4301; E-mail: n.j.curtin{at}ncl.ac.uk.
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H2AX foci and flow cytometry. Tissue distribution of NU7441 and potentiation of etoposide activity were determined in mice bearing SW620 tumors. NU7441 increased the cytotoxicity of ionizing radiation and etoposide in SW620, LoVo, and V3-YAC cells but not in V3 cells, confirming that potentiation was due to DNA-PK inhibition. NU7441 substantially retarded the repair of ionizing radiation–induced and etoposide-induced DSB. NU7441 appreciably increased G2-M accumulation induced by ionizing radiation, etoposide, and doxorubicin in both SW620 and LoVo cells. In mice bearing SW620 xenografts, NU7441 concentrations in the tumor necessary for chemopotentiation in vitro were maintained for at least 4 hours at nontoxic doses. NU7441 increased etoposide-induced tumor growth delay 2-fold without exacerbating etoposide toxicity to unacceptable levels. In conclusion, NU7441 shows sufficient proof of principle through in vitro and in vivo chemosensitization and radiosensitization to justify further development of DNA-PK inhibitors for clinical use. (Cancer Res 2006; 66(10): 5354-62) |
Introduction |
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In mammalian cells, homologous recombination and nonhomologous end-joining (NHEJ) are the major DSB repair pathways (9, 10). The principal factor determining the choice of mechanism is the cell cycle stage (11, 12). Homologous recombination is a complex error-free pathway, which is dependent on the proximity of the sister chromatid and can therefore only take place in late S and the G2 phases of the cell cycle. DSB repair is also achieved by means of NHEJ, which brings the broken ends together and rejoins them without reference to a second template. This pathway is, therefore, predominant during G1 or G0 phases, but it is also operational at other phases of the cell cycle (13).
The core NHEJ machinery consists of the Ku70/80 heterodimer and the catalytic subunit of DNA-dependent protein kinase (DNA-PKCS), which together make the active DNA-PK, XRCC4, ligase IV, and the endonuclease artemis. Ku locates at the break first, recruiting DNA-PKCS to bring about synapsis of the ends. Artemis processes the DNA ends, and the final ligation of the juxtaposed ends is accomplished by the XRCC4/ligase IV complex (14–16). DNA-PK has been shown to phosphorylate several cellular proteins in vivo, including itself and the variant histone H2AX (15). Cells deficient in components of the NHEJ pathway are defective in DSB repair and highly sensitive to ionizing radiation and topoisomerase II poisons (17–24). Moreover, overexpression of DNA-PKCS can accelerate the repair of ionizing radiation–induced, etoposide-induced, and doxorubicin-induced DNA DSBs and confer resistance to these agents (25, 26). DNA-PK may also be responsible for chemoresistance and radioresistance in the clinical setting: studies have shown that DNA-PK protein expression correlates with sensitivity to etoposide in human chronic lymphocytic leukemia samples (27).
Inhibition of NHEJ is therefore an attractive approach to modulating resistance to therapeutically induced DNA DSBs. DNA-PK is a member of the phosphatidylinositol-3 kinase (PI3K)–related protein kinase (PIKK) family of enzymes. Inhibitors of PI3K, such as wortmannin and LY294002, also inhibit DNA-PK in a noncompetitive and competitive manner, respectively (28). Both wortmannin and LY294002 have been reported to retard DSB repair and enhance the cytotoxicity of ionizing radiation and etoposide, which has largely been attributed to inhibition of DNA-PKCS (29–31). Using LY294002 as a lead, a more potent and specific inhibitor of DNA-PKCS, NU7026 (2-(morpholin-4-yl)-benzo[h]chromen-4-one), was developed, which also increased the cytotoxicity of both ionizing radiation and topoisomerase II poisons and retarded DSB repair (24, 32). Further compound elaboration has identified NU7441 (2-N-morpholino-8-dibenzothiophenyl-chromen-4-one) as a yet more potent and specific inhibitor of DNA-PK, with an IC50 of only 14 nmol/L and at least 100-fold selectivity for this enzyme compared with other PI3KK family kinases (33, 34).
We report here the preclinical evaluation of NU7441, showing that (a) potentiation of ionizing radiation and etoposide cytotoxicity by NU7441 is due to DNA-PK inhibition using Chinese hamster ovary cells deficient and complemented with DNA-PKCS; (b) NU7441 increases the persistence of H2AX foci after ionizing radiation–induced or etoposide-induced DNA damage; (c) NU7441 prolongs the G2-M arrest and profoundly increases the cytotoxicity induced by ionizing radiation and etoposide in human colon cancer cell lines differing in their p53 status. Furthermore, NU7441 enhances the antitumor activity of etoposide in a human colon cancer xenograft model.
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Materials and Methods |
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Cell lines and culture. LoVo and SW620 human colon cancer cells were obtained from the American Type Culture Collection (Manassas, VA). V3 (DNA-PKCS deficient Chinese hamster ovary cells) and their derivative V3-YAC, transfected with a yeast artificial chromosome (YAC) carrying the human DNA-PKCS gene, were a kind gift from Dr. Penny Jeggo (University of Sussex, United Kingdom). All cell lines were cultured in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, penicillin (50 units/mL), and streptomycin (50 µg/mL) at 37°C in an atmosphere of 5% CO2 in air. V3-YAC cells were maintained under antibiotic selection to retain the YAC using G-418 sulfate (Invitrogen, Carlsbad, CA) at a final concentration of 500 µg/mL. Cells were free of Mycoplasma contamination. The doubling time of the cells was 
24 hours (LoVo and SW620) and 16 hours (V3 and V3-YAC).
Cytotoxicity and growth inhibition studies. The effect of NU7441 on cellular survival following exposure to etoposide, doxorubicin, and ionizing radiation was measured in SW620, LoVo, V3, and V3-YAC cells by clonogenic assays. Briefly, we exposed exponentially growing cells in six-well plates or 6-cm dishes to etoposide or doxorubicin with or without NU7441 (0.5 or 1.0 µmol/L) for 16 hours. For radiosensitization studies, NU7441 was added to the cells 1 hour before irradiation. V3 and V3-YAC cells were exposed to -irradiation (3.1 Gy/min 137Cesium, Gammacell 1000 Elite, Nordion International Ltd., Ottawa, Ontario, Canada). SW620 and LoVo were exposed to X-irradiation (2.9 Gy/min at 230 kV, 10 mA; Gulmay Medical Ltd., Camberly, United Kingdom) due to the equipment available. After irradiation, the cells were incubated with or without NU7441 for a further 16 hours. Cells were then harvested by trypsinization, counted, and seeded into 10-cm diameter Petri dishes at densities varying from 100 to 100,000 per dish in drug-free medium for colony formation. Colonies were stained with crystal violet after 10 to 14 days and counted with an automated colony counter (ColCount, Oxford Optronics Ltd., Oxford, United Kingdom). The survival reduction factor (SRF) was calculated as the surviving fraction of cells in the absence of NU7441 divided by the surviving fraction of cells in the presence of NU7441 for any given dose or concentration of cytotoxic agent. The dose modification ratio (DMR90) is calculated as the concentration/dose of cytotoxic agent required to kill 90% of the cells in the absence of NU7441 divided by the concentration/dose of cytotoxic agent required to kill 90% of the cells in the presence of NU7441. Cell growth inhibition following 5-day continuous exposure to NU7441 was determined in LoVo and SW620 cells grown in 96-well plates as described previously (36).
H2AX focus assay. DNA DSBs were quantified by H2AX focus formation in SW620 cells grown to 50% to 70% confluence on round No. 1 coverslips in 6-cm dishes. To investigate the effect of NU7441 on ionizing radiation–induced focus formation and loss, we incubated cells with or without 1 µmol/L NU7441 for 1 hour before irradiation with 2 or 4 Gy X-irradiation (2.9 Gy/min at 230 kV, 10 mA) followed by incubation at 37°C in control medium or that containing 1 µmol/L NU7441 for up to 4 hours. Similarly, to investigate the effect of NU7441 on etoposide-induced focus formation and loss, cells were incubated in the presence or absence of 1 µmol/L NU7441 for 1 hour before adding etoposide (1 µmol/L) for 1 hour followed by extensive washing (thrice in drug-free medium) and incubation at 37°C in medium with or without 1 µmol/L NU7441 for up to 24 hours. Coverslips were washed in PBS and fixed in methanol at –20°C for 5 minutes before washing thrice for 10 minutes each in PBS to rehydrate. Blocking was carried out for 1 to 18 hours at 4°C in KCM buffer [120 mmol/L KCl, 20 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.5), 0.5 mmol/L EDTA, 0.1% (v/v) Triton X-100] containing 10% (w/v) dried milk powder and 2 % (w/v) bovine serum albumin. The coverslips were incubated with the primary mouse monoclonal antibody for -H2AXser139 (Upstate Technology, Buckingham, United Kingdom) diluted 1:200 in KCM for 1 hour at room temperature before washing in KCM and incubating for 1 hour at dark room temperature with FITC-conjugated second antibody anti mouse IgG (Molecular Probes, Eugene, OR) diluted 1:200 in KCM. The coverslips were mounted using Vectorshield mounting medium (Vector Laboratories, Burlingame, CA), which contains 1.5 µg/mL 4',6-diamidino-2-phenylindole (DAPI). Images were obtained using an Olympus BH2-RFCA fluorescence microscope fitted with a xenon lamp and a x40 objective (DplanApo 40UV Olympus, Tokyo, Japan). Separate 16-bit gray-scale images were recorded for DAPI (420 nm) and FITC (590 nm) using a Hamamatu ORCAII BT-1024 cooled CCD camera. Image Pro Plus software (Media Cybernetics, Silver Spring, MD) was used for image capture and quantitative image analysis. Subsequent image handling was carried out in Photoshop (Adobe, San Jose, CA). We counted foci in 30 cells, from three different areas per sample, for each exposure.
Flow cytometric cell cycle analysis. We treated exponentially growing asynchronous populations of SW620 and LoVo cells (seeded 24 hours previously) with control or 1 µmol/L NU7441-containing medium for 1 hour before ionizing radiation or adding etoposide or doxorubicin and incubating for a further 16 hours. Cells were then harvested by trypsinization, washed with ice-cold PBS, fixed in 70% ethanol, and stored at 4°C. Cellular DNA was labeled with propidium iodide (400 µg/mL) in the presence of the RNase (1 mg/mL; Sigma). Flow cytometry was done on a Becton Dickinson FACScan (Heidelberg, Germany) equipped with an argon ion laser (excitation at 488 nm).
NU7441 plasma pharmacokinetics following different routes of administration. All in vivo experiments were reviewed and approved by the relevant institutional animal welfare committees and done according to national law. We determined the plasma pharmacokinetics after administering NU7441 i.v. at 5 mg/kg in 10% DMSO/10% cyclodextrin in saline or i.p. or orally at 10 mg/kg (dissolved at 1 mg/mL in 40% PEG400/saline) to female BALB/c mice. These were the maximum administrable doses by the route used due to the limit of solubility of NU7441. Mice were killed at intervals up to 360 minutes after NU7441 administration; blood was taken and immediately centrifuged, and the plasma fraction was removed and stored at –20°C.
NU7441 tissue distribution study. Female athymic nude mice (CD1 nu/nu, Charles River, Wilmington, MA) were maintained and handled in isolators under specific pathogen-free conditions for tissue distribution and efficacy studies. We implanted SW620 colorectal tumor cells (1 x 107 in 50 µL culture medium per animal) s.c. into the flanks of the mice and did tissue distribution studies when tumors had reached a size of 650 mm3 (10-14 days after implantation). We administered NU7441 (10 mg/kg i.p.) to tumor-bearing female CD-1 nude mice, which were then killed 10, 30, 60, 90, or 240 minutes later, and the blood and tissues were taken for analysis. Tissues were excised rapidly, wrapped in foil, and snap frozen in liquid nitrogen before storage at –80°C; plasma was obtained as above. Before assay, tissue samples were homogenized in PBS [1:3 (w/v)], using a stirrer mascercarator homogenizer (IKA, Werke GmbH and Co., KG., Staufen, Germany), in 10 second bursts, on ice to prevent warming.
High-performance liquid chromatography analysis of NU7441 in plasma and tissue homogenates. NU7441 was extracted from plasma and tissue homogenates (50 µL) using protein precipitation with acetonitrile [1:4 (v/v)] followed by centrifugation (5,000 x g at room temperature), evaporation of the supernatant to dryness under nitrogen at 30°C, and reconstitution in high-performance liquid chromatography mobile phase before analysis. NU7441 was separated on a Waters Alliance 2780 separation module (Waters, Watford, United Kingdom) using a Genesis 4-µm C18 column (4.6 x 100 mm; Jones Chromatography Ltd., Mid Glamorgan, United Kingdom) and a mobile phase of sodium acetate buffer [0.02 mol/L (pH 5)] and acetonitrile [75: 25 (v/v)] at a flow rate of 1 mL/min. Detection was by UV absorption (329 nm; PDA 996 detector, Waters). Plasma samples were quantified using a standard curve, for NU7441, prepared in plasma, which was linear over the range of 0.05 to 10 µg/mL (r2 = 0.9) with duplicate QA standards (at 0.1, 1, and 10 µg/mL). Tissue concentrations were calculated using the method of addition (37) to account for the efficiency of recovery and compensate for intersample variation.
Antitumor efficacy study. CD-1 nude mice bearing palpable SW620 colorectal cancer xenografts s.c. (5 mm x 5 mm, 8-10 days after implantation, n = 5 per group) were treated with normal saline (control animals), single agent NU7441 (dissolved in 40% PEG 400 in saline), or etoposide phosphate (11.35 mg/kg in saline) i.p. daily for 5 days. For combinations, NU7441 was given immediately before etoposide phosphate. Etoposide phosphate was given as a water-soluble equivalent to etoposide, the dosage being equivalent to free etoposide at 10 mg/kg.
Tumor volume was calculated from two-dimensional electronic caliper (Mitutoyo, Andover, United Kingdom) measurements using the equation a2 x b/2, where a is the smallest measurement, and b is the largest measurement. Data are presented as median relative tumor volume (RTV), where the tumor volume on the initial day of treatment (day 0) is assigned an RTV of 1.
Statistical analysis. We analyzed data using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). For the in vitro studies, the significant differences were determined by Student's t test (parametric), and for in vivo studies, Mann-Whitney nonparametric test was used.
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Results |
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2-fold more sensitive than V3-YAC cells to ionizing radiation (LD90 = 2.4 and 4.5 Gy in V3 and V3-YAC cells, respectively) and 3-fold more sensitive to etoposide (LD90 = 0.6 and 1.8 µmol/L in V3 and V3-YAC cells, respectively). NU7441 alone had no significant effect on the survival of either V3 or V3-YAC cells (94 ± 17% and 97 ± 16% survival compared with DMSO controls, respectively). In V3-YAC cells, NU7441 increased the cytotoxicity of ionizing radiation 1.6-fold (LD90 in the presence of NU7441 = 2.8 Gy) and etoposide 2.3-fold (LD90 in the presence of NU7441 = 0.8 µmol/L) but had little effect in V3 cells (Fig. 1A and B
). In contrast, the ATM inhibitor KU-55933 increased the cytotoxicity of ionizing radiation 2-fold in the V3-YAC and 3-fold in V3 cells and etoposide by 6-fold in both cell lines (Fig. 1C and D).
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H2AX phosphorylation. To examine the effect of NU7441 on the repair of ionizing radiation–induced and etoposide-induced DNA DSBs, we counted H2AX phosphorylation foci (Fig. 3
). There was a very low level of background focus formation of 0.13 ± 0.06 foci per cell in DMSO-treated control cells, which was not increased by NU7441 alone (0.13 ± 0.12 foci per cell). Exposure to 2 Gy alone induced 20 ± 1.9 foci per cell 15 minutes after irradiation, which declined rapidly, such that by 4 hours, only 13% of foci remained (2.6 ± 0.7 foci per cell). NU7441 did not affect the level of ionizing radiation–induced focus formation (20 ± 1.0 foci per cell) but did significantly retard the loss of foci, such that at 4 hours, 74% (15 ± 2.3 foci per cell P = 0.001 compared with cells irradiated in the absence of NU7441) remained (Fig. 3A and C, left). Similar results were seen after exposure to 4 Gy ionizing radiation (data not shown). After exposure to etoposide, foci accumulated gradually, reaching a peak of 22 ± 3.2 foci per cell 30 minutes after etoposide removal (Fig. 3B and C, right). The rate of loss of foci was slower compared with ionizing radiation–induced foci, and at 4 hours, 39% (8.8 ± 1.5 foci per cell) remained. Continuing the incubation for 16 hours after etoposide removal resulted in almost complete loss of foci, and only 5.3% of the peak (1.3 ± 3.6 foci per cell) remained. As with ionizing radiation–induced foci, NU7441 did not affect etoposide-mediated induction of H2AX foci (peak level at 30 minutes was 22 ± 3.6 foci per cell) but did significantly inhibit loss of foci, such that at 4 and 16 hours after etoposide removal, 69% (15 ± 1.4 foci per cell P = 0.005 compared with cells treated with etoposide alone) and 41% (9.1 ± 0.21 foci per cell P < 0.001 compared with cells treated with etoposide alone) remained, respectively.
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Plasma pharmacokinetics of NU7441 following different routes of administration. The plasma pharmacokinetic variables obtained after administration of NU7441 to BALB/c mice by various routes are given in Table 1 . We administered NU7441 at the maximum administrable dose by all routes, which was 5 mg/kg i.v. and 10 mg/kg i.p. and orally. Following i.p. administration, the half-life (T1/2) and clearance were similar to those after i.v. injection, and the area under the concentration/time curve after 10 mg/kg i.p. was approximately double that of 5 mg/kg given i.v. The calculated bioavailability for i.p. administration was 100%. Similar pharmacokinetic analysis of NU7441 after oral administration revealed substantial differences compared with i.v. administration, and the calculated bioavailability was only 33%, indicating less than complete absorption by this route. For this reason, we used i.p. administration for all subsequent studies with NU7441.
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Antitumor activity of etoposide and NU7441 in SW620 tumor-bearing mice. The initial proposed clinical development of DNA-PK inhibitors is chemosensitization, we therefore investigated whether the marked potentiation of etoposide cytotoxicity in human colon cancer cells by NU7441 we observed in vitro could be reproduced in vivo. We treated mice bearing SW620 tumor xenografts with etoposide phosphate alone and in combination with NU7441 (Fig. 5B). Tumors in control mice reached four times their starting volume (RTV4) at a median time of 5.6 days (i.e., time to RTV4 = 5.6 days). Treatment with etoposide phosphate alone caused a tumor growth delay of 2.7 days (time to RTV4 = 8.3 days), which was extended to 5.4 days (time to RTV4 = 11 days, P = 0.0159 compared with etoposide alone) by coadministration of NU7441. Thus, NU7441 enhanced etoposide phosphate efficacy by 100%.
In this study, neither NU7441 nor etoposide phosphate alone caused any significant toxicity (maximum body weight loss = 6%), and the combination of drugs did not cause unacceptable toxicity (maximum body weight loss = 12%; Fig. 5C).
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Discussion |
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There is evidence of competition between NHEJ and homologous recombination, and cells lacking components of the NHEJ pathway have been reported to have higher levels of homologous recombination, which may partially compensate for the defect (39–41). However, rather than stimulating homologous recombination activity, DNA-PK inhibitors have been reported to block homologous recombination in a dominant-negative fashion by preventing DNA-PK dissociation from DNA, thereby blocking the access of homologous recombination proteins (42). Similar studies have also suggested that DNA-PK inhibition may also block the access of poly(ADP-ribose) polymerase-1 (PARP-1) to DSB and thus inhibit PARP-1-mediated repair as well (24, 43). The knock-on effect of DNA-PK inhibitors on other DNA repair pathways, as well as NHEJ, may therefore contribute to the pronounced chemosensitization and radiosensitization by NU7441 that we observed in our models of human colon cancer. We found that low, noncytotoxic concentrations of NU7441 (1 µmol/L) caused a 3- to 4-fold reduction in the ionizing radiation dose needed to kill 90% of cells, with a substantial 20- to 30-fold reduction in survival at 2 Gy in both cell lines studied. Chemopotentiation studies also showed that these low concentrations of NU7441 were capable of causing a sizeable increase in etoposide-induced cytotoxicity of between 2- and 10-fold. Potentiation of ionizing radiation and topoisomerase II poisons was not apparently dependent on the p53 status of the cells. Hitherto, higher concentrations of DNA-PK inhibitor have been required for similar levels of radiopotentiation and chemopotentiation. For example, 20 µmol/L wortmannin and 50 µmol/L LY294002 were required to cause a radiosensitization DMR90 of 2.8 and 1.9, respectively, in SW480 cells (31). In another human colon cancer cell line (HCT116), the novel DNA-PK inhibitor, IC86621 (100 µmol/L) enhanced the cytotoxicity of ionizing radiation 4-fold and etoposide 15-fold (44). Hence, NU7441 is 20 to 100x more potent at the cellular level than previously described molecules.
We verified that the radiosensitization and chemosensitization by NU7441 was due to the inhibition of DNA DSB repair by measuring H2AX foci. H2AX foci are generated rapidly at DSBs induced by ionizing radiation and disappear as repair proceeds. For topoisomerase II poisons, H2AX foci are thought to appear as topoisomerase cleavable complexes are processed to frank DSBs and at stalled replication forks. Although H2AX phosphorylation is both ATM and DNA-PK dependent, NU7441 did not affect focus induction by ionizing radiation or etoposide, in agreement with the observed redundancy of ATM and DNA-PK for this process (45). The profound suppression by NU7441 of the rate of focus loss shows its ability to inhibit repair of both ionizing radiation–induced and etoposide–induced DSB. Previous studies show that radiosensitivity and chemosensitivity correlates with persistence of H2AX phosphorylation after exposure to DSB-inducing drugs and ionizing radiation (46, 47). Our data reinforce these observations by showing, for the first time, DNA-PK inhibitor–mediated increase in persistence of H2AX foci is related to an increase in cytotoxicity.
We found that chemosensitization and radiosensitization was accompanied by an increase in the ionizing radiation–induced G2 arrest, as has been reported with wortmannin (31), and topoisomerase II poison–induced G2 arrest, as has been reported with NU7026 (32), suggesting that cytotoxin-induced DSBs, persisting in the presence of DNA-PK inhibitor, signal cell cycle arrest at the G2-M checkpoint, possibly via ATM. Interestingly, in the p53 wt LoVo cells, NU7441 alone caused G1 arrest, resulting in cell growth inhibition, but NU7441 did not cause such a marked effect in the p53 mutant SW620 cells. It is tempting to speculate that DNA-PK inhibition can trigger a p53-dependent G1-S checkpoint, although further studies with isogenically matched cell lines would be needed to verify this supposition.
Whole animal studies show that concentrations of NU7441 required for chemosensitization and radiosensitization in vitro were achievable and maintained within the tumor tissue for at least 4 hours after administration of nontoxic doses of NU7441. However, the poor aqueous solubility of this compound precluded the investigation of higher doses that might have led to these levels of the drug being retained within the tumor for longer periods. Nevertheless, based on the promising pharmacokinetics, efficacy studies in combination with etoposide were conducted, and these did indeed show that NU7441 doubled the etoposide-induced tumor growth delay without increasing the toxicity to an unacceptable degree. Similar results have been reported using the HCT116 human colon cancer xenograft system, where IC86621 caused a 4-fold enhancement of ionizing radiation–induced tumor growth delay and increase in survival. However, this compound required administration at a dose of 1,600 mg/kg to maintain levels above 50 µmol/L (concentration required for potentiation in vitro) for 4 hours (44) due its pharmacokinetic properties, limiting its further use.
In conclusion, NU7441 is a potent and specific DNA-PK inhibitor in in vitro enzyme inhibition assays; its cellular radiosensitization and chemosensitization are due to DNA-PK inhibition and are accompanied by increased persistence of DSBs and G2-M arrest. NU7441 potentiates the activity of radiation, doxorubicin, and etoposide in human tumor cell lines in vitro and etoposide in a human tumor xenograft model in vivo. Concentrations (in vitro) and doses (in vivo) of NU7441 required for potentiation are lower than those reported necessary for similar effects using previously reported DNA-PK inhibitors and are not toxic per se, with daily dosing in vivo being sufficient to produce potentiation. The limited aqueous solubility and oral bioavailability of NU7441 restrict further development of this compound. However, NU7441 has provided excellent proof of principle in vitro and in vivo chemosensitization and radiosensitization data to warrant further development of this class of compound for clinical use.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Justin Leahy, Marc Hummersone, and Laurent Rigoreau for the kind gifts of NU7441 and KU55933.
Received 11/30/05. Revised 1/23/06. Accepted 3/ 6/06.
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B. E. Lally, G. A. Geiger, S. Kridel, A. E. Arcury-Quandt, M. E. Robbins, N. D. Kock, K. Wheeler, P. Peddi, A. Georgakilas, G. D. Kao, et al. Identification and Biological Evaluation of a Novel and Potent Small Molecule Radiation Sensitizer via an Unbiased Screen of a Chemical Library Cancer Res., September 15, 2007; 67(18): 8791 - 8799. [Abstract] [Full Text] [PDF]
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X. Hong, L. Lei, B. Kunert, R. Naredla, S. E. Applequist, A. Grandien, and R. Glas Tripeptidyl-peptidase II Controls DNA Damage Responses and In vivo {gamma}-Irradiation Resistance of Tumors Cancer Res., August 1, 2007; 67(15): 7165 - 7174. [Abstract] [Full Text] [PDF]
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P. Douglas, X. Cui, W. D. Block, Y. Yu, S. Gupta, Q. Ding, R. Ye, N. Morrice, S. P. Lees-Miller, and K. Meek The DNA-Dependent Protein Kinase Catalytic Subunit Is Phosphorylated In Vivo on Threonine 3950, a Highly Conserved Amino Acid in the Protein Kinase Domain Mol. Cell. Biol., March 1, 2007; 27(5): 1581 - 1591. [Abstract] [Full Text] [PDF]
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) or presence (
) of 1 µmol/L NU7441 with further incubation in presence or absence of NU7441 for 16 hours before seeding for colony formation in drug-free medium. Data points, mean of three replicates in a single representative experiment; bars, SD.
), NU7441 alone (10 mg/kg daily x 5, 
), etoposide phosphate alone 11.5 mg/kg (equivalent to etoposide-free drug at 10 mg/kg daily x 5, 
), or NU7441 and etoposide phosphate (
). Shaded bar, dosing period. C, toxicity evaluation of etoposide and NU7441 in SW620 tumor-bearing nude mice. Points, mean body weight of mice treated as described in (B): vehicle control (