RESUMEN
The stress-inducible transcription complex NF-κB induces the transcription of genes that regulate proliferation and apoptosis. Constitutively activated NF-κB is common in breast cancers, and contributes to malignant progression and therapeutic resistance. Ataxia telangiectasia mutated (ATM) is a key regulator of the cellular response to DNA double strand breaks (DSBs), and recent reports have demonstrated that ATM is required for the activation of NF-κB following DNA damage. We investigated the role of ATM in the NF-κB signalling cascade induced by ionising radiation (IR) in breast cancer cell lines using KU55933, a novel and specific inhibitor of ATM. KU55933 suppressed IR-induced IκBα degradation, p50/p65 nuclear translocation and binding to kB consensus sequences. KU55933 also suppressed transcription of an NF-κB dependent reporter gene and inhibited IR-induced DSB repair as assessed by the neutral Comet assay. KU55933 sensitised cells to IR, with a concurrent increase in caspase 3 activity. Importantly, KU55933 sensitised IKKß(+/+) and p65(+/+), but not IKKß(-/-) or p65(-/-), mouse embryonic fibroblasts to IR, despite the equivalent inhibitory effects of KU55933 on DSB repair in both the proficient and the deficient cell lines. P65 siRNA had no effect on DSB repair in either breast cancer cell line. When combined with KU55933, DSB repair was inhibited to the same extent as KU55933 alone in both breast cancer cell lines. P65 siRNA alone sensitised both cell lines to IR. A combination of p65 siRNA and KU55933 resulted in no further sensitisation compared to either one alone. Taken together these data support the hypothesis that KU55933-mediated radio-sensitisation is solely a consequence of its inhibition of NF-κB activation. We conclude that radiotherapy deploying ATM inhibitors may be particularly advantageous in tumours where NF-κB is constitutively activated.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Tolerancia a Radiación/genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Daño del ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Femenino , Humanos , Ratones , Morfolinas/farmacología , FN-kappa B/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Pironas/farmacología , ARN Interferente Pequeño/genética , Tolerancia a Radiación/efectos de los fármacos , Radiación Ionizante , Activación Transcripcional/genética , Activación Transcripcional/fisiología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
Defects in the DNA damage response pathway [e.g. del(17p)] are associated with drug-resistant B-cell chronic lymphocytic leukaemia (CLL). We previously demonstrated that over-expression of DNA-dependent protein kinase (DNA-PK) correlates with chemo-resistance and that inhibition of DNA-PK sensitizes CLL cells to chemotherapeutics. Here, we investigated expression of DNA-PK and other proteins that impact on drug resistance, and evaluated the effects of a DNA-PK inhibitor (NU7441) on mitoxantrone-induced cytotoxicity in CLL cells. NU7441 sensitized cells from 42/49 CLL samples to mitoxantrone, with sensitization ranging from 2- to 200-fold Co-culture of CLL cells in conditioned stromal medium increased chemoresistance but did not reduce sensitization by NU7441. Mitoxantrone treatment induced γH2AX foci and NU7441 increased their longevity (24 h). NU7441 prevented mitoxantrone-induced autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) at Ser 2056, confirming that DNA-PK participates in repair of mitoxantrone-induced DNA damage. del(17p) cases were more resistant to mitoxantrone than del(13q) cases, but were resensitized (7-16 fold) by co-incubation with NU7441. Expression of DNA-PKcs, Ku80, P-glycoprotein and topoisomerase IIß were significantly higher in del(17p) cases. PRKDC mRNA levels correlated with DNA-PKcs protein expression, which predicted shorter survival. These data confirm the potential of DNA-PK as a therapeutic target in poor prognosis CLL.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Muerte Celular/efectos de los fármacos , Cromonas/farmacología , Medios de Cultivo Condicionados , Reparación del ADN/efectos de los fármacos , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Proteína Quinasa Activada por ADN/efectos de los fármacos , Proteína Quinasa Activada por ADN/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos , Humanos , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Mitoxantrona/farmacología , Morfolinas/farmacología , Proteínas de Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
PURPOSE: del(17p), del(11q), and associated p53 dysfunction predict for short survival and chemoresistance in B-cell chronic lymphocytic leukemia (CLL). DNA-dependent protein kinase (DNA-PK) is activated by DNA damage and mediates DNA double-strand break repair. We hypothesized that inhibiting DNA-PK would sensitize CLL cells to drug-induced DNA damage and that this approach could increase the therapeutic index of agents used to treat CLL. EXPERIMENTAL DESIGN: Fifty-four CLL cases were characterized for poor prognosis markers [del(17p), del(11q), CD38, and ZAP-70]. In selected cases, DNA-PK catalytic subunit (DNA-PKcs) expression and activity and p53 function were also measured. Ex vivo viability assays established sensitivity to fludarabine and chlorambucil and also tested the ability of a novel DNA-PK inhibitor (NU7441) to sensitize CLL cells to these drugs. The effects of NU7441 on fludarabine-induced DNA damage repair were also assessed (Comet assays and detection of gammaH2AX). RESULTS: DNA-PKcs levels correlated with DNA-PK activity and varied 50-fold between cases but were consistently higher in del(17p) (P = 0.01) and del(11q) cases. NU7441 sensitized CLL cells to chlorambucil and fludarabine, including cases with del(17p), del(11q), p53 dysfunction, or high levels of DNA-PKcs. NU7441 increased fludarabine-induced double-strand breaks and abrogated drug-induced autophosphorylation of DNA-PKcs at Ser2056. High DNA-PK levels predicted for reduced treatment-free interval. CONCLUSIONS: These data validate the concept of targeting DNA-PKcs in poor risk CLL, and demonstrate a mechanistic rationale for use of a DNA-PK inhibitor. The novel observation that DNA-PKcs is overexpressed in del(17p) and del(11q) cases indicates that DNA-PK may contribute to disease progression in CLL.
Asunto(s)
Cromonas/uso terapéutico , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Proteína Quinasa Activada por ADN/fisiología , Sistemas de Liberación de Medicamentos , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Morfolinas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/fisiología , Proteína Quinasa Activada por ADN/metabolismo , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Células Tumorales CultivadasRESUMEN
BACKGROUND: The histone variant histone H2A.X comprises up to 25% of the H2A complement in mammalian cells. It is rapidly phosphorylated following exposure of cells to double-strand break (DSB) inducing agents such as ionising radiation. Within minutes of DSB generation, H2AX molecules are phosphorylated in large chromatin domains flanking DNA double-strand breaks (DSBs); these domains can be observed by immunofluorescence microscopy and are termed gammaH2AX foci. H2AX phosphorylation is believed to have a role mounting an efficient cellular response to DNA damage. Theoretical considerations suggest an essentially random chromosomal distribution of X-ray induced DSBs, and experimental evidence does not consistently indicate otherwise. However, we observed an apparently uneven distribution of gammaH2AX foci following X-irradiation with regions of the nucleus devoid of foci. METHODOLOGY/PRINCIPLE FINDINGS: Using immunofluorescence microscopy, we show that focal phosphorylation of histone H2AX occurs preferentially in euchromatic regions of the genome following X-irradiation. H2AX phosphorylation has also been demonstrated previously to occur at stalled replication forks induced by UV radiation or exposure to agents such as hydroxyurea. In this study, treatment of S-phase cells with hydroxyurea lead to efficient H2AX phosphorylation in both euchromatin and heterochromatin at times when these chromatin compartments were undergoing replication. This suggests a block to H2AX phosphorylation in heterochromatin that is at least partially relieved by ongoing DNA replication. CONCLUSIONS/SIGNIFICANCE: We discuss a number of possible mechanisms that could account for the observed pattern of H2AX phosphorylation. Since gammaH2AX is regarded as forming a platform for the recruitment or retention of other DNA repair and signaling molecules, these findings imply that the processing of DSBs in heterochromatin differs from that in euchromatic regions. The differential responses of heterochromatic and euchromatic compartments of the genome to DSBs will have implications for understanding the processes of DNA repair in relation to nuclear and chromatin organization.
Asunto(s)
Eucromatina/metabolismo , Histonas/metabolismo , Radiación Ionizante , Línea Celular Tumoral , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/química , Daño del ADN , Replicación del ADN , Heterocromatina/química , Humanos , Microscopía Fluorescente , Modelos Biológicos , Fosforilación , Estructura Terciaria de ProteínaRESUMEN
DNA-PK and ATM are members of the phosphatidylinositol 3'-kinase like kinase (PIKK) family of serine/threonine protein kinases and have critical roles in the cellular response to DNA double-strand breaks. Genetic loss of either activity leads to pronounced sensitivity to ionizing radiation (IR). Hence, these enzymes are potential targets to confer enhanced radiosensitivity on tumour cells. We show that novel inhibitors of either DNA-PK or ATM sensitize breast carcinoma cells to IR. Radiosensitization was accompanied by an apparent DNA repair deficit as measured by the persistence of IR-induced foci of phosphorylated histone H2AX (gammaH2AX foci). These specific inhibitors also allowed us to probe the biochemistry and kinetics of histone H2AX phosphorylation following gamma-irradiation in breast cancer cells with the aim of validating H2AX as a biomarker for DNA-PK or ATM inhibition in vivo. ATM inhibition reduced the initial average intensity of gammaH2AX foci while inhibition of DNA-PK had only a small effect on the initial phosphorylation of H2AX. However, simultaneous treatment with both compounds dramatically reduced gammaH2AX focus intensity, consistent with the reported role of ATM and DNA-PK in IR induced phosphorylation of H2AX.
Asunto(s)
Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fármacos Fotosensibilizantes/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Radiación Ionizante , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Cromonas/farmacología , Técnica del Anticuerpo Fluorescente , Humanos , Morfolinas/farmacología , Pironas/farmacologíaRESUMEN
DNA-dependent protein kinase (DNA-PK) and poly (ADP-ribose) polymerase-1 (PARP-1) participate in nonhomologous end joining and base excision repair, respectively, and are key determinants of radio- and chemo-resistance. Both PARP-1 and DNA-PK have been identified as therapeutic targets for anticancer drug development. Here we investigate the effects of specific inhibitors on enzyme activities and DNA double-strand break (DSB) repair. The enzyme activities were investigated using purified enzymes and in permeabilized cells. Inhibition, or loss of activity, was compared using potent inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361), and cell lines proficient or deficient for DNA-PK or PARP-1. Inactive DNA-PK suppressed the activity of PARP-1 and vice versa. This was not the consequence of simple substrate competition, since DNA ends were provided in excess. The inhibitory effect of DNA-PK on PARP activity was confirmed in permeabilized cells. Both inhibitors prevented ionizing radiation-induced DSB repair, but only AG14361 prevented single-strand break repair. An increase in DSB levels caused by inhibition of PARP-1 was shown to be caused by a decrease in DSB repair, and not by the formation of additional DSBs. These data point to combined inhibition of PARP-1 and DNA-PK as a powerful strategy for tumor radiosensitization.
Asunto(s)
Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Azulenos , Benzodiazepinas/farmacología , Unión Competitiva , Línea Celular , Línea Celular Tumoral , Cromonas/síntesis química , Cromonas/farmacología , Proteína Quinasa Activada por ADN , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Morfolinas/síntesis química , Morfolinas/farmacología , Proteínas Nucleares , Fosforilación , Poli(ADP-Ribosa) PolimerasasRESUMEN
We report for the first time the use of a selective small-molecule inhibitor of DNA repair to potentiate topoisomerase II (topo II) poisons, identifying DNA-dependent protein kinase (DNA-PK) as a potential target for leukemia therapy. Topo II poisons form cleavable complexes that are processed to DNA double-strand breaks (DSBs). DNA-PK mediates nonhomologous end joining (NHEJ). Inhibition of this DSB repair pathway may sensitize cells to topo II poisons. We investigated the effects of a novel DNA-PK inhibitor, NU7026 (2-(morpholin-4-yl)-benzo[h]chomen-4-one), on the response to topo II poisons using K562 leukemia cells. NU7026 (10 microM) potentiated the growth inhibition of idarubicin, daunorubicin, doxorubicin, etoposide, amsacrine (mAMSA), and mitroxantrone with potentiation factors at 50% growth inhibition ranging from approximately 19 for mAMSA to approximately 2 for idarubicin (potentiation of etoposide was confirmed by clonogenic assay). In contrast, NU7026 did not potentiate camptothecin or cytosine arabinoside (araC). NU7026 did not affect the levels of etoposide-induced topo IIalpha or beta cleavable complexes. NU7026 alone had no effect on cell cycle distribution, but etoposide-induced accumulation in G2/M was increased by NU7026. A concentration-dependent increase in etoposide-induced DSB levels was increased by NU7026. The mechanism of NU7026 potentiation of topo II poisons involves inhibition of NHEJ and a G2/M checkpoint arrest.
Asunto(s)
Antineoplásicos/toxicidad , Cromonas/farmacología , Proteínas de Unión al ADN , Morfolinas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Inhibidores de Topoisomerasa II , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/genética , Proteína Quinasa Activada por ADN , Humanos , Células K562 , Proteínas Nucleares , Fármacos Sensibilizantes a Radiaciones/farmacologíaRESUMEN
PURPOSE: Mismatch repair (MMR) deficiency confers resistance to temozolomide, a clinically active DNA-methylating agent. The purpose of the current study was to investigate the reversal mechanism of temozolomide resistance by the potent novel poly(ADP-ribose) polymerase (PARP)-1 inhibitor, AG14361, in MMR-proficient and -deficient cells. EXPERIMENTAL DESIGN: The effects of AG14361, in comparison with the methylguanine DNA methyltransferase inhibitor, benzylguanine, on temozolomide-induced growth inhibition were investigated in matched pairs of MMR-proficient (HCT-Ch3, A2780, and CP70-ch3) and -deficient (HCT116, CP70, and CP70-ch2) cells. RESULTS: AG14361 enhanced temozolomide activity in all MMR-proficient cells (1.5-3.3-fold) but was more effective in MMR-deficient cells (3.7-5.2-fold potentiation), overcoming temozolomide resistance. In contrast, benzylguanine only increased the efficacy of temozolomide in MMR-proficient cells but was ineffective in MMR-deficient cells. The differential effect of AG14361 in MMR-deficient cells was not attributable to differences in PARP-1 activity or differences in its inhibition by AG14361, nor was it attributable to differences in DNA strand breaks induced by temozolomide plus AG14361. MMR-deficient cells are resistant to cisplatin, but AG14361 did not sensitize any cells to cisplatin. PARP-1 inhibitors potentiate topotecan-induced growth inhibition, but AG14361 did not potentiate topotecan in MMR-deficient cells more than in MMR-proficient cells. CONCLUSIONS: MMR defects are relatively common in sporadic tumors and cancer syndromes. PARP-1 inhibition represents a novel way of selectively targeting such tumors. The underlying mechanism is probably a shift of the cytotoxic locus of temozolomide to N(7)-methylguanine and N(3)-methyladenine, which are repaired by the base excision repair pathway in which PARP-1 actively participates.
Asunto(s)
Disparidad de Par Base , Benzodiazepinas/farmacología , Reparación del ADN , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Adenina/farmacología , Antineoplásicos/farmacología , Antineoplásicos Alquilantes/farmacología , Azulenos , División Celular , Línea Celular Tumoral , Cisplatino/farmacología , Daño del ADN , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Genotipo , Guanina/análogos & derivados , Guanina/farmacología , Humanos , Modelos Químicos , NAD , TemozolomidaRESUMEN
BACKGROUND: Poly(ADP-ribose) polymerase-1 (PARP-1) facilitates the repair of DNA strand breaks. Inhibiting PARP-1 increases the cytotoxicity of DNA-damaging chemotherapy and radiation therapy in vitro. Because classical PARP-1 inhibitors have limited clinical utility, we investigated whether AG14361, a novel potent PARP-1 inhibitor (inhibition constant <5 nM), enhances the effects of chemotherapy and radiation therapy in human cancer cell cultures and xenografts. METHODS: The effect of AG14361 on the antitumor activity of the DNA alkylating agent temozolomide, topoisomerase I poisons topotecan or irinotecan, or x-irradiation or gamma-radiation was investigated in human cancer cell lines A549, LoVo, and SW620 by proliferation and survival assays and in xenografts in mice by tumor volume determination. The specificity of AG14361 for PARP-1 was investigated by microarray analysis and by antiproliferation and acute toxicity assays in PARP-1-/- and PARP-1+/+ cells and mice. After intraperitoneal administration, the concentration of AG14361 was determined in mouse plasma and tissues, and its effect on PARP-1 activity was determined in tumor homogenates. All statistical tests were two-sided. RESULTS: AG14361 at 0.4 micro M did not affect cancer cell gene expression or growth, but it did increase the antiproliferative activity of temozolomide (e.g., in LoVo cells by 5.5-fold, 95% confidence interval [CI] = 4.9-fold to 5.9-fold; P =.004) and topotecan (e.g., in LoVo cells by 1.6-fold, 95% CI = 1.3-fold to 1.9-fold; P =.002) and inhibited recovery from potentially lethal gamma-radiation damage in LoVo cells by 73% (95% CI = 48% to 98%). In vivo, nontoxic doses of AG14361 increased the delay of LoVo xenograft growth induced by irinotecan, x-irradiation, or temozolomide by two- to threefold. The combination of AG14361 and temozolomide caused complete regression of SW620 xenograft tumors. AG14361 was retained in xenografts in which PARP-1 activity was inhibited by more than 75% for at least 4 hours. CONCLUSION: AG14361 is, to our knowledge, the first high-potency PARP-1 inhibitor with the specificity and in vivo activity to enhance chemotherapy and radiation therapy of human cancer.
Asunto(s)
Antineoplásicos/farmacología , Benzodiazepinas/farmacología , Dacarbazina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Azulenos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/radioterapia , Dacarbazina/farmacología , Rayos gamma/uso terapéutico , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/radioterapia , Ratones , Poli(ADP-Ribosa) Polimerasas/metabolismo , Temozolomida , Trasplante HeterólogoRESUMEN
The DNA repair enzymes, DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase-1 (PARP-1), are key determinants of radio- and chemo-resistance. We have developed and evaluated novel specific inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361) for use in anticancer therapy. PARP-1- and DNA-PK-deficient cell lines were 4-fold more sensitive to ionizing radiation (IR) alone, and showed reduced potentially lethal damage recovery (PLDR) in G(0) cells, compared with their proficient counterparts. NU7026 (10 micro M) potentiated IR cytotoxicity [potentiation factor at 90% cell kill (PF(90)) = 1.51 +/- 0.04] in exponentially growing DNA-PK proficient but not deficient cells. Similarly, AG14361 (0.4 micro M) potentiated IR in PARP-1(+/+) (PF(90) = 1.37 +/- 0.03) but not PARP-1(-/-) cells. When NU7026 and AG14361 were used in combination, their potentiating effects were additive (e.g., PF(90) = 2.81 +/- 0.19 in PARP-1(+/+) cells). Both inhibitors alone reduced PLDR approximately 3-fold in the proficient cell lines. Furthermore, the inhibitor combination completely abolished PLDR. IR-induced DNA double strand break (DNA DSB) repair was inhibited by both NU7026 and AG14361, and use of the inhibitor combination prevented 90% of DNA DSB rejoining, even 24-h postirradiation. Thus, there was a correlation between the ability of the inhibitors to prevent IR-induced DNA DSB repair and their ability to potentiate cytotoxicity. Thus, individually, or in combination, the DNA-PK and PARP-1 inhibitors act as potent radiosensitizers and show potential as tools for anticancer therapeutic intervention.
Asunto(s)
Benzodiazepinas/farmacología , Cromonas/farmacología , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN , Inhibidores Enzimáticos/farmacología , Morfolinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Fármacos Sensibilizantes a Radiaciones/farmacología , Azulenos , Daño del ADN , Proteína Quinasa Activada por ADN , Células HeLa , Humanos , Proteínas Nucleares , Tolerancia a Radiación/efectos de los fármacosRESUMEN
The nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) facilitates DNA repair, and is, therefore, an attractive target for anticancer chemo- and radio-potentiation. Novel benzimidazole-4-carboxamides (BZ1-6) and tricyclic lactam indoles (TI1-5) with PARP-1 K(i) values of <10 nM have been identified. Whole cell PARP-1 inhibition, intrinsic cell growth inhibition, and chemopotentiation of the cytotoxic agents temozolomide (TM) and topotecan (TP) were evaluated in LoVo human colon carcinoma cells. The acute toxicity of the inhibitors was investigated in PARP-1 null and wild-type mice. Tissue distribution and in vivo chemopotentiation activity was determined in nude mice bearing LoVo xenografts. At a nontoxic concentration (0.4 micro M) the PARP-1 inhibitors potentiated TM-induced growth inhibition 1.0-5.3-fold and TP-induced inhibition from 1.0-2.1-fold. Concentrations of the PARP-1 inhibitors that alone inhibited cell growth by 50% ranged from 8 to 94 micro M. Maximum potentiation of TM activity was achieved at nongrowth inhibitory concentrations (=1 micro M) of potent PARP-1 inhibitors BZ5 and TI4. Whole cell PARP-1 inhibition by BZ3, BZ5, BZ6, TI1, and TI4 was confirmed by attenuation of DNA damage-induced NAD(+) depletion. Selected inhibitors (TI1, TI3, and TI4), in contrast to the benchmark compound PD128763, caused only mild hypothermia in both PARP-1 null and wild-type mice. Excellent distribution of BZ5, TI1, and TI3 into tumor tissue was observed, and TI3 enhanced TM antitumor activity in vivo. These studies have identified potent nontoxic PARP-1 inhibitors with structural modifications that promote aqueous solubility, tolerability, and tissue distribution. These compounds are important leads in the development of clinically viable PARP-1 inhibitors.