RESUMO
BACKGROUND: Topoisomerase I (Topo I) poisons (e.g., camptothecin (CPT)), used to treat cancer, cause DNA breaks that are most cytotoxic during S phase. PARP-1 promotes DNA repair and PARP inhibitors (PARPi) sensitise cells to Topo I poisons. We aimed to determine whether chemosensitisation is also S phase specific using rucaparib, a potent PARPi in advanced clinical evaluation. METHODS: The impact of rucaparib, on CPT-induced cytotoxicity was measured in human colon cancer (LoVo) and leukaemic (K562) cells in asynchronous and cell cycle phase-separated cultures. Topoisomerase I and PARP levels and activity and the effect of rucaparib on DNA single-strand breaks (SSBs), double-strand breaks (DSBs) and collapsed replication fork induction and repair were determined in cell cycle phase-separated cells. RESULTS: The cytotoxicity of CPT was greatest during S phase, partially attributable to high Topo I activity, and rucaparib preferentially sensitised S-phase cells. Rucaparib increased CPT-induced DNA SSBs in all phases of the cell cycle, and increased DSB and γH2AX foci in S and G2, with γH2AX foci being highest in S-phase cells. Repair of SSBs and DSBs was most rapid during S then G2 phases and was substantially hindered by rucaparib. CONCLUSIONS: Rucaparib preferentially sensitises S-phase cells by increasing the frequency of collapsed replication forks.
Assuntos
Camptotecina/farmacologia , Indóis/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Inibidores da Topoisomerase I/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Células K562 , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
BACKGROUND: Poly(ADP-ribose) polymerase-1 (PARP) inhibitors (PARPi) exploit tumour-specific defects in homologous recombination DNA repair and continuous dosing is most efficacious. Early clinical trial data with rucaparib suggested that it caused sustained PARP inhibition. Here we investigate the mechanism of this durable inhibition and potential exploitation. METHODS: Uptake and retention of rucaparib and persistence of PARP inhibition were determined by radiochemical and immunological assays in human cancer cell lines. The pharmacokinetics and pharmacodynamics of rucaparib were determined in tumour-bearing mice and the efficacy of different schedules of rucaparib was determined in mice bearing homologous recombination DNA repair-defective tumours. RESULTS: Rucaparib accumulation is carrier mediated (Km=8.4±1.2 µM, Vmax=469±22 pmol per 10(6) cells per 10 min), reaching steady-state levels >10 times higher than the extracellular concentration within 30 min. Rucaparib is retained in cells and inhibits PARP ≥50% for ≥72 h days after a 30-min pulse of 400 nM. In Capan-1 tumour-bearing mice rucaparib accumulated and was retained in the tumours, and PARP was inhibited for 7 days following a single dose of 10 mg kg(-1) i.p or 150 mg kg(-1) p.o. by 70% and 90%, respectively. Weekly dosing of 150 mg kg(-1) p.o once a week was as effective as 10 mg kg(-1) i.p daily for five days every week for 6 weeks in delaying Capan-1 tumour growth. CONCLUSIONS: Rucaparib accumulates and is retained in tumour cells and inhibits PARP for long periods such that weekly schedules have equivalent anticancer activity to daily dosing in a pre-clinical model, suggesting that clinical evaluation of alternative schedules of rucaparib should be considered.
Assuntos
Inibidores Enzimáticos/administração & dosagem , Indóis/administração & dosagem , Poli(ADP-Ribose) Polimerases/genética , Animais , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Esquema de Medicação , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/farmacocinética , Recombinação Homóloga/efeitos dos fármacos , Humanos , Indóis/sangue , Indóis/farmacocinética , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Patients with malignant pleural effusions (MPEs) generally have advanced disease with poor survival and few therapeutic options. Cells within MPEs may be used to stratify patients for targeted therapy. Targeted therapy with poly(ADP ribose) polymerase inhibitors (PARPi) depends on identifying homologous recombination DNA repair (HRR)-defective cancer cells. We aimed to determine the feasibility of assaying HRR status in MPE cells. METHODS: A total of 15 MPE samples were collected from consenting patients with non-small-cell lung cancer (NSCLC), mesothelioma and ovarian and breast cancer. Primary cultures were confirmed as epithelial by pancytokeratin, and HRR status was determined by the detection of γH2AX and RAD51 foci following a 24-h exposure to rucaparib, by immunofluorescence microscopy. Massively parallel next-generation sequencing of DNA repair genes was performed on cultured MPE cells. RESULTS: From 15 MPE samples, 13 cultures were successfully established, with HRR function successfully determined in 12 cultures. Four samples - three NSCLC and one mesothelioma - were HRR defective and eight samples - one NSCLC, one mesothelioma, one sarcomatoid, one breast and four ovarian cancers - were HRR functional. No mutations in DNA repair genes were associated with HRR status, but there was probable loss of heterozygosity of FANCG, RPA1 and PARP1. CONCLUSIONS: HRR function can be successfully detected in MPE cells demonstrating the potential to stratify patients for targeted therapy with PARPi.
Assuntos
Derrame Pleural Maligno/genética , Reparo de DNA por Recombinação , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos de Viabilidade , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Derrame Pleural Maligno/patologiaRESUMO
Historically, PARP inhibitors (PARPi) were developed to potentiate the cytotoxic effect of certain chemotherapeutic agents and are currently being investigated in combination with chemotherapy in diverse cancer types. These agents are also radiosensitisers and clinical trials of PARPi with concurrent radiation are required. It has long been recognised that defective DNA repair pathways lead to tumour susceptibility. Recent studies indicate that tumour cells with defective homologous recombination (HR) repair pathways, the classic example being BRCA mutations, are exquisitely sensitive to PARPi. Defects in HR are not restricted to BRCA-associated tumours and other cancer types may be enriched for HR defects and hence susceptible to PARP inhibition. The identification of predictive markers for sensitivity to PARP inhibition is a priority area for research.
Assuntos
Reparo do DNA , Poli(ADP-Ribose) Polimerases/metabolismo , Antineoplásicos/uso terapêutico , Ensaios Clínicos como Assunto , Inibidores Enzimáticos/uso terapêutico , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases , Radiossensibilizantes/uso terapêuticoRESUMO
BACKGROUND: The ataxia telangiectasia mutated and Rad3-related kinase (ATR) has a key role in the signalling of stalled replication forks and DNA damage to cell cycle checkpoints and DNA repair. It has long been recognised as an important target for cancer therapy but inhibitors have proved elusive. As NU6027, originally developed as a CDK2 inhibitor, potentiated cisplatin in a CDK2-independent manner we postulated that it may inhibit ATR. METHODS: Cellular ATR kinase activity was determined by CHK1 phosphorylation in human fibroblasts with inducible dominant-negative ATR-kinase dead expression and human breast cancer MCF7 cells. Cell cycle effects and chemo- and radiopotentiation by NU6027 were determined in MCF7 cells and the role of mismatch repair and p53 was determined in isogenically matched ovarian cancer A2780 cells. RESULTS: NU6027 is a potent inhibitor of cellular ATR activity (IC(50)=6.7 µM) and enhanced hydroxyurea and cisplatin cytotoxicity in an ATR-dependent manner. NU6027 attenuated G2/M arrest following DNA damage, inhibited RAD51 focus formation and increased the cytotoxicity of the major classes of DNA-damaging anticancer cytotoxic therapy but not the antimitotic, paclitaxel. In A2780 cells sensitisation to cisplatin was greatest in cells with functional p53 and mismatch repair (MMR) and sensitisation to temozolomide was greatest in p53 mutant cells with functional MMR. Importantly, NU6027 was synthetically lethal when DNA single-strand break repair is impaired either through poly(ADP-ribose) polymerase (PARP) inhibition or defects in XRCC1. CONCLUSION: NU6027 inhibits ATR, impairing G2/M arrest and homologous recombination thus increasing sensitivity to DNA-damaging agents and PARP inhibitors. It provides proof of concept data for clinical development of ATR inhibitors.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Proteínas de Ciclo Celular/antagonistas & inibidores , Compostos Nitrosos/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/uso terapêutico , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Neoplasias da Mama/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Genes p53 , Humanos , Leucemia L1210 , Camundongos , Neoplasias Ovarianas/genética , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
BACKGROUND: Cellular proliferation, driven by cyclin-dependent kinases (CDKs) and their cyclin partners, is deregulated in cancer. Anti-estrogens, such as tamoxifen, antagonise estrogen-induced ERalpha transactivation of cyclin D1, resulting in reduced CDK4/6 activity, p27(Kip1)-mediated inhibition of CDK2 and growth arrest. We hypothesised that direct inhibition of CDK2 and CDK1 may overcome the major clinical problem of anti-estrogen resistance. METHODS: The cellular effects of CDK2/1 siRNA knockdown and purine-based CDK2/1 inhibitors, NU2058 and NU6102, were measured in anti-estrogen-sensitive and resistant breast cancer cell lines. RESULTS: CDK2 knockdown caused G1 accumulation, whereas CDK1 depletion caused G2/M slowing, and dual CDK1/2 depletion resulted in further G2/M accumulation and cell death in both anti-estrogen-sensitive and resistant cells, confirming CDK2 and CDK1 as targets for breast cancer therapy. In contrast to tamoxifen, which only affected hormone-sensitive cells, NU2058 and NU6102 reduced CDK2-mediated phosphorylation of pRb, E2F transcriptional activity and proliferation, ultimately resulting in cell death, in both anti-estrogen-sensitive and resistant cells. Both drugs caused G2/M arrest, reflective of combined CDK2/1 knockdown, with a variable degree of G1 accumulation. CONCLUSION: These studies confirm the therapeutic potential of CDK2 and CDK1 inhibitors for cancer therapy, and support their use as an alternative treatment for endocrine-resistant breast cancer.
Assuntos
Antineoplásicos/farmacologia , Proteína Quinase CDC2/metabolismo , Proliferação de Células/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Neoplasias da Mama , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Moduladores de Receptor Estrogênico/farmacologia , Feminino , HumanosRESUMO
BACKGROUND: Temozolomide shows activity against medulloblastoma, the most common malignant paediatric brain tumour. Poly(ADP-ribose) polymerase (PARP) inhibitors enhance temozolomide activity in extracranial adult and paediatric human malignancies. METHODS: We assessed the effect of AG-014699, a clinically active PARP inhibitor, on temozolomide-induced growth inhibition in human medulloblastoma models. Pharmacokinetic, pharmacodynamic and toxicity assays were performed in tumour-bearing mice. RESULTS: Sensitivity to temozolomide in vitro was consistent with methylguanine methyltransferase (MGMT) and DNA mismatch repair (MMR) status; MGMT(+) MMR(+) D384Med cells (temozolomide GI(50)=220 µM), representative of most primary medulloblastomas, were sensitised fourfold by AG-014699; MGMTâ» MMR(+) D425Med cells were hypersensitive (GI(50)=9 µM) and not sensitised by AG-014699, whereas MGMT(+) MMRâ» temozolomide-resistant D283Med cells (GI50=807 µM) were sensitised 20-fold. In xenograft models, co-administration of AG-014699 produced an increase in temozolomide-induced tumour growth delay in D384Med xenografts. Consistent with the in vitro data, temozolomide caused complete tumour regressions of D425Med xenografts, whereas D283Med xenografts were relatively resistant. AG-014699 was not toxic, accumulated and reduced PARP activity ≥75% in xenograft and brain tissues. CONCLUSION: We show for the first time central nervous system penetration and inhibition of brain PARP activity by AG-014699. Taken together with our in vitro chemosensitisation and toxicity data, these findings support further evaluation of the clinical potential of AG-014699-temozolomide combinations in intra-cranial malignancies.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias do Sistema Nervoso Central/patologia , Dacarbazina/análogos & derivados , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Neoplasias do Sistema Nervoso Central/enzimologia , Criança , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Dacarbazina/uso terapêutico , Humanos , Indóis/uso terapêutico , Meduloblastoma/tratamento farmacológico , Meduloblastoma/enzimologia , Meduloblastoma/patologia , Camundongos , Camundongos Nus , Poli(ADP-Ribose) Polimerase-1 , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Temozolomida , Transplante HeterólogoRESUMO
BACKGROUND: Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA-binding enzyme activated by DNA breaks and involved in DNA repair and other cellular processes. Poly(ADP-ribose) polymerase activity can be higher in cancer than in adjacent normal tissue, but cancer predisposition is reported to be greater in individuals with a single-nucleotide polymorphism (SNP) V762A (T2444C) in the catalytic domain that reduces PARP-1 activity. METHODS: To resolve these divergent observations, we determined PARP-1 polymorphisms, PARP-1 protein expression and activity in a panel of 19 solid and haematological, adult and paediatric human cancer cell lines. RESULTS: There was a wide variation in PARP activity in the cell line panel (coefficient of variation, CV=103%), with the lowest and the highest activity being 2460 pmol PAR/10(6) (HS-5 cells) and 85 750 pmol PAR/10(6) (NGP cells). Lower variation (CV=32%) was observed in PARP-1 protein expression with the lowest expression being 2.0 ng microg(-1) (HS-5 cells) and the highest being 7.1 ng microg(-1) (ML-1 cells). The mean activity in the cancer cells was 45-fold higher than the mean activity in normal human lymphocytes and the PARP-1 protein levels were 23-fold higher. CONCLUSIONS: Surprisingly, there was no significant correlation between PARP activity and PARP-1 protein level or the investigated polymorphisms, T2444C and CA.
Assuntos
Neoplasias/enzimologia , Neoplasias/genética , Poli(ADP-Ribose) Polimerases/genética , Linhagem Celular Tumoral , Humanos , Linfócitos/enzimologia , Repetições de Microssatélites , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/biossíntese , Poli(ADP-Ribose) Polimerases/metabolismo , Polimorfismo de Nucleotídeo Único , Regiões Promotoras GenéticasRESUMO
The thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid (CB3717) inhibits the growth of human lung carcinoma A549 cells. The cytotoxicity of CB3717 is potentiated by the nucleoside transport inhibitor dipyridamole (DP), which not only inhibits the uptake and therefore salvage of thymidine but also inhibits the efflux of deoxyuridine, thereby enhancing the intracellular accumulation of deoxyuridine nucleotides. Measurement of intracellular deoxyuridine triphosphate (dUTP) pools, by sensitive radioimmunoassay, demonstrated a large increase in response to CB3717, in a dose- and time-related manner, and this accumulation was enhanced by coincubation with DP. In untreated cells and those treated with DP alone, dUTP was close to or below the limit of detection of the assay. In cells treated for 24 h with 3 microM CB3717 (concentration producing 50% growth inhibition) the intracellular dUTP was 46.1 +/- 9.6 (SEM) pmol/10(6) cells and after 24 h exposure to 30 microM CB3717, 337.5 +/- 37.9 pmol dUTP/10(6) cells was detected. There was significant enhancement by DP of the accumulation of dUTP in cells treated with CB3717; coincubation of cells with 1 microM DP + 3 microM CB3717 for 24 h resulted in intracellular dUTP levels of 174.7 +/- 57.7 pmol/10(6) cells. Accumulation of DNA strand breaks, measured by alkaline elution, also increased in response to CB3717 concentration and exposure period. Newly synthesized (nascent) DNA was more sensitive to damage by CB3717 than was mature DNA. As with the accumulation of dUTP, coincubation with DP also enhanced the accumulation of strand breaks, whereas DP alone had little or no effect on DNA fragmentation. When data for cells treated with CB3717 alone and CB3717 in combination with DP were combined, there was a significant correlation of intracellular dUTP levels with the level of DNA strand breaks. This strongly suggests that growth inhibition following thymidylate synthase inhibition is mediated through an increase in intracellular dUTP, leading to uracil misincorporation into DNA, its subsequent excision, and resultant strand breakage.
Assuntos
Divisão Celular/efeitos dos fármacos , Sobrevivência Celular , Dano ao DNA , Nucleotídeos de Desoxiuracil/metabolismo , Dipiridamol/farmacologia , Ácido Fólico/análogos & derivados , Quinazolinas/farmacologia , Timidilato Sintase/antagonistas & inibidores , Sinergismo Farmacológico , Ácido Fólico/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Timidina/biossíntese , Fatores de Tempo , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
The impact of p53 status on cellular sensitivity to antifolate drugs has been examined in seven human cell lines (A549, MCF7, T-47D, CCRF-CEM, COR-L23, A2780, and HCT-116) and p53 nonfunctional counterparts of two of the cell lines (HCT-116/N7 and A2780/CP70). p53 status was determined by sequencing and functional assays. The sensitivities of the cell lines to growth inhibition (sulphorhodamine B assay) produced by four antifolate drugs (Alimta, methotrexate, raltitrexed, and lometrexol) were studied. There was no clear relationship between functional p53 status and sensitivity to methotrexate or lometrexol, whereas a functional p53 status was possibly associated with resistance to Alimta- and raltitrexed-induced growth inhibition. In contrast, in the two pairs of related human tumor cell lines (HCT-116 and HCT-116/N7 and A2780 and A2780/CP70) cells with functional p53 were more sensitive to Alimta- and raltitrexed-induced growth inhibition (P = 0.002). Detailed studies were performed with the A2780 cell lines, and in the parental cells sensitivity to Alimta- and raltitrexed-induced cytotoxicity (clonogenic assay) was similar to the sensitivity determined in the sulphorhodamine B assay. However, in A2780/CP70 cells, 1 microM of drug resulted in only 40-60% growth inhibition yet > or = 85% cytotoxicity. After Alimta and raltitrexed exposure for < or = 72 h, there were no differences between the A2780 and A278/CP70 cell lines in cell cycle phase distribution, absolute cell number, or the induction of apoptosis. However, the cellular protein content of the A2780/CP70 cells was 3-6-fold higher than in A2780 cells after Alimta and raltitrexed treatment, suggesting that cells without functional p53 can maintain protein synthesis in the absence of cell division (unbalanced cell growth). In conclusion, the apparent impact of functional p53 status on sensitivity to antifolate drugs may depend upon the phenotypic/genotypic background as well as the assay used to measure cellular sensitivity.
Assuntos
Antagonistas do Ácido Fólico/farmacologia , Guanina/análogos & derivados , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ciclinas/metabolismo , Análise Mutacional de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Genótipo , Glutamatos/farmacologia , Guanina/farmacologia , Humanos , Metotrexato/farmacologia , Mutação , Pemetrexede , Quinazolinas/farmacologia , Sensibilidade e Especificidade , Tetra-Hidrofolatos/farmacologia , Tiofenos/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
The antifolate LY309887 is a specific glycinamide ribonucleotide formyltransferase inhibitor that blocks de novo purine synthesis and produces a depletion of purine nucleotides. The activity of LY309887 in six human tumor cell lines has been examined by growth inhibition and clonogenic assay after continuous exposure for three cell doubling times and by ATP depletion at 24 h. Three cell lines (CCRF-CEM, MCF7, and GC3) were sensitive to LY309887-induced growth inhibition (IC50: 5.6-8.1 nM), whereas the other cell lines (COR-L23, T-47D, and A549) were comparatively resistant (IC50: 36-55 nM). Sensitivity to LY309887 cytotoxicity was consistent with sensitivity to growth inhibition in four of five cell lines tested (MCF7/GC3: 0.01% survival and COR-L23/T-47D: 1-5% survival at 100 nM LY309887). LY309887-induced ATP depletion was measured by luciferase-based ATP assay and confirmed by high performance liquid chromatography measurements. There was a linear relationship between ATP depletion and growth inhibition when data were analyzed for all six cell lines (r2 = 0.93; P < 0.0001). Depletion of 24-h cellular ATP concentrations to < 1 mM was associated with both cell growth inhibition and cytotoxicity in all cell lines studied. In conclusion, cellular ATP depletion induced by LY309887 can be used to predict growth inhibition and cytotoxicity in human tumor cells.
Assuntos
Trifosfato de Adenosina/metabolismo , Antineoplásicos/toxicidade , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Tetra-Hidrofolatos/toxicidade , Neoplasias da Mama , Tamanho Celular/efeitos dos fármacos , Feminino , Humanos , Hidroximetil e Formil Transferases/antagonistas & inibidores , Neoplasias Pulmonares , Fosforribosilglicinamido Formiltransferase , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
The activity of antimetabolite inhibitors of de novo deoxyribonucleotide biosynthesis can be compromised by the salvage of extracellular preformed nucleosides and nucleobases. Dipyridamole (DP) is a nucleoside transport inhibitor that has been used clinically in an attempt to increase antimetabolite activity; however, DP binds tightly to the serum protein alpha1-acid glycoprotein (AGP) thereby rendering this therapeutic strategy largely ineffective. Four novel DP analogues (NU3076, NU3084, NU3108, and NU3121) have been developed with substitutions at the 2,6- and 4,8-positions of the pyrimidopyrimidine ring. The novel DP analogues inhibit thymidine (dThd) uptake into L1210 cells in vitro (NU3076 IC(50), 0.25 microM; NU3084 IC(50), 0.27 microM; NU3108 IC(50), 0.31 microM; NU3121 IC(50), 0.26 microM; and DP IC(50), 0.37 microM), but, unlike DP, their activity remains largely unaffected in the presence of 5 mg/ml AGP. The four DP analogues inhibit dThd and hypoxanthine rescue from Alimta (multitargeted antifolate)-induced growth inhibition in A549 and COR L23 human lung carcinoma cell lines in the presence of 2.5 mg/ml AGP, whereas the activity of DP is completely abolished. i.p. administration of 10 mg/kg NU3108, NU3121, and DP produced peak plasma concentrations of 4.4, 2.1, and 6.7 microM, respectively, and levels were sustained above 1 microM for approximately 45 min (DP) and 120 min (NU3108 and NU3121). [3H]thymidine incorporation into COR L23 xenografts grown in CD1 nude mice was reduced by 64% (NU3108), 44% (NU3121), and 65% (DP) 2 h after administration of the nucleoside transport inhibitors. In conclusion, two novel DP analogues (NU3108 and NU3121) have been identified that do not bind to AGP and that display superior pharmacokinetic profiles in comparison to DP and inhibit [3H]thymidine incorporation into human tumor xenografts in vivo.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Dipiridamol/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Guanina/análogos & derivados , Proteínas de Membrana/antagonistas & inibidores , Animais , Proteínas de Transporte/metabolismo , Divisão Celular/efeitos dos fármacos , Dipiridamol/química , Dipiridamol/farmacocinética , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Glutamatos/farmacologia , Guanina/farmacologia , Humanos , Hipoxantina/farmacologia , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Transporte de Nucleosídeos , Orosomucoide/farmacologia , Pemetrexede , Tetra-Hidrofolatos/farmacologia , Timidina/metabolismo , Fatores de Tempo , Trítio , Células Tumorais CultivadasRESUMO
Potent poly(ADP-ribose) polymerase (PARP) inhibitors have been developed that potentiate the cytotoxicity of ionizing radiation and anticancer drugs. The biological effects of two novel PARP inhibitors, NU1025 (8-hydroxy-2-methylquinazolin-4-[3H]one, Ki = 48 nM) and NU1085 [2-(4-hydroxyphenyl)benzamidazole-4-carboxamide, Ki = 6 nM], in combination with temozolomide (TM) or topotecan (TP) have been studied in 12 human tumor cell lines (lung, colon, ovary, and breast cancer). Cells were treated with increasing concentrations of TM or TP +/- NU1025 (50, 200 microM) or NU1085 (10 microM) for 72 h. The potentiation of growth inhibition by NU1025 and NU1085 varied between the cell lines from 1.5- to 4-fold for TM and 1- to 5-fold for TP and was unaffected by p53 status. Clonogenic assays undertaken in two of the cell lines confirmed that the potentiation of growth inhibition reflected the potentiation of cytotoxicity. NU1025 (50 microM) was about as effective as 10 microM NU1085 at potentiating growth inhibition and cytotoxicity, consistent with the relative potencies of the two molecules as PARP inhibitors. Potentiation of cytotoxicity was obtained at concentrations of NU1025 and NU1085 that were not toxic per se; however, NU1085 alone was 3-fold more cytotoxic (LC50 values ranged from 83 to 94 microM) than NU1025 alone (LC50 > 900 microM). These data demonstrate that PARP inhibitors are effective resistance-modifying agents in human tumor cell lines and have provided a comprehensive assessment protocol for the selection of optimum combinations of anticancer drugs, PARP inhibitors, and cell lines for in vivo studies.
Assuntos
Antineoplásicos/toxicidade , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dacarbazina/análogos & derivados , Inibidores Enzimáticos/toxicidade , Inibidores de Poli(ADP-Ribose) Polimerases , Quinazolinas/toxicidade , Topotecan/toxicidade , Neoplasias da Mama , Neoplasias do Colo , Dacarbazina/toxicidade , Sinergismo Farmacológico , Feminino , Humanos , Neoplasias Pulmonares , Neoplasias Ovarianas , Temozolomida , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
The cytotoxicity of the antifolate inhibitors of de novo purine biosynthesis, lometrexol (LTX) and LY309887, can be abolished by hypoxanthine (HPX) salvage. The nucleoside transport inhibitor, dipyridamole (DP) can prevent HPX rescue from LTX growth inhibition in a cell line-specific manner. The studies described here have shown that, excluding colon and hematological malignancies, DP prevents HPX rescue from LTX growth inhibition in approximately one-third of cell lines with otherwise limited tissue specificity. The clinical dose-limiting toxicities of antipurine antifolates are to the bone marrow and gastrointestinal tract. In vitro models of these normal tissues were established, and the effect of DP on HPX rescue from LY309887 treatment was studied. Growth inhibition assays are not feasible in these primary cultures; therefore, an alternative assay, cellular ATP depletion, was validated in four tumor cell lines as a marker of de novo and salvage purine synthesis. In LY309887-treated cells, DP prevented HPX-mediated maintenance of ATP levels only in cell lines in which DP inhibited HPX rescue from antifolate cytotoxicity. Hence, ATP depletion is a reliable indicator of sensitivity of HPX transport to DP when direct cell growth measurement is impractical. In primary cultures of human hematopoetic progenitor cells and mouse small intestine, coincubation with HPX prevented LY309887-mediated ATP depletion, which was not blocked by DP. These data suggest that DP would not prevent HPX rescue from antipurine antifolate growth inhibition in sensitive normal tissues, whereas activity against certain solid human tumors would be maintained.
Assuntos
Antineoplásicos/farmacologia , Dipiridamol/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Hipoxantina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antimetabólitos Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores do Crescimento/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Tetra-Hidrofolatos/farmacologia , Células Tumorais CultivadasRESUMO
Expression of the multidrug resistance-associated protein (MRP) is widespread in human malignancies, high levels are associated with poor prognosis and may be responsible for intrinsic and radiotherapy-induced chemoresistance. In this study, the nucleoside transport inhibitor, dipyridamole (DP), was investigated as a chemosensitiser of MRP. In growth inhibition assays MRP-over-expressing COR L23/R cells were 20 times more resistant to VP16 and doxorubicin compared with the parental COR L23/R human lung carcinoma cells. DP caused an approximately 8-fold sensitisation of the resistant cells and a 2-fold sensitisation of the parental cells. DP enhanced the accumulation of VP16 1.5 to 2-fold in the parental cells, but had only a modest effect on VP16 accumulation in the resistant cells. VP16 efflux was rapid in both cell lines. DP caused a modest and transient inhibition of the initial efflux in the resistant cells but not the parental cells. Incubation with DP caused a progressive decrease in GSH levels which was more rapid and profound in COR L23/R cells than in COR L23/P cells. Thus, chemosensitisation to VP16 by DP in MRP-overexpressing COR L23/R cells appears to be caused by depletion of cellular GSH rather than a direct effect of DP on MRP-mediated drug accumulation and efflux.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Dipiridamol/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Fosfodiesterase/uso terapêutico , Antineoplásicos/uso terapêutico , Divisão Celular , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/uso terapêutico , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Células Tumorais CultivadasRESUMO
Poly(ADP-ribose) polymerase (PARP) plays an important role in a number of cellular processes including DNA repair. Since poly(ADP-ribosyl)ation occurs in response to radiation- or drug-induced DNA damage, inhibitors of the enzyme may enhance the antitumour activity of radiotherapy or cytotoxic drug treatment. In this review the development of PARP inhibitors is discussed, and structure-activity relationships amongst inhibitors of the enzyme are presented. Studies to date regarding the in vitro and in vivo activity of PARP inhibitors, as resistance modifying agents in cancer therapy, are also overviewed.
Assuntos
Reparo do DNA/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Neoplasias/terapia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Estrutura Molecular , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Relação Estrutura-AtividadeRESUMO
The novel multitargeted antifolate, MTA (N-[4[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-ethy l]-benzoyl]-L-glutamic acid; LY23 1514) inhibits thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase. The resultant inhibition of the de novo thymidylate and purine biosynthesis can be circumvented by salvage of extracellular thymidine and hypoxanthine. The first step in the salvage pathway is the transport of nucleosides and bases across the cell membrane. Dipyridamole inhibits nucleoside transport and in vitro studies have demonstrated that dipyridamole can prevent thymidine salvage rescue from antifolate thymidylate synthase inhibitors. More recently, dipyridamole also has been shown to prevent hypoxanthine rescue from antipurine antifolates in some cell lines but not others. The effects of dipyridamole on MTA growth inhibition and end product reversal by thymidine and hypoxanthine was investigated in two lung cancer cell lines with (A549) and without (COR L23) dipyridamole-sensitive hypoxanthine rescue. The IC50 values for MTA-induced growth inhibition were 28 and 640 nmol/L for COR L23 and A549 cells, respectively. End product reversal studies show that thymidine can completely reverse growth inhibition by IC50 concentration of MTA but only partially rescue cells from 10 times the IC50 concentration of MTA. The combination of thymidine and hypoxanthine was required for complete reversal from MTA at 10 times the IC50 concentration. Dipyridamole blocked the partial rescue from MTA-induced growth inhibition by thymidine alone as well as the complete rescue by thymidine plus hypoxanthine not only in A549 cells, which have dipyridamole-sensitive hypoxanthine transport, but also in COR L23 cells, in which hypoxanthine uptake is insensitive to dipyridamole. These studies demonstrate that nucleoside and base salvage can compromise the activity of MTA in human tumor cell lines, but that dipyridamole can readily prevent salvage and restore growth inhibition.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Dipiridamol/farmacologia , Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Glutamatos/farmacologia , Guanina/análogos & derivados , Hipoxantina/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Fosfodiesterase/farmacologia , Timidina/metabolismo , Timidilato Sintase/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Divisão Celular/efeitos dos fármacos , Glutamatos/uso terapêutico , Guanina/farmacologia , Guanina/uso terapêutico , Humanos , Neoplasias Pulmonares/metabolismo , Pemetrexede , Células Tumorais CultivadasRESUMO
Clinical studies concerning the role of poly(ADP-ribose) polymerase (PARP) in the repair of drug- and radiation-induced DNA damage have been impeded by the poor solubility, lack of potency, and limited specificity of currently available inhibitors. A series of 2-alkyl- and 2-aryl-substituted 8-hydroxy-, 8-methoxy-, and 8-methylquinazolin-4(3H)-ones has been synthesized and evaluated for PARP inhibitory activity in permeabilized L1210 murine leukemia cells. 8-Methoxy- and 8-methylquinazolinones (14-34) were readily prepared by acylation of 3-substituted anthranilamides with the appropriate acid chloride, followed by base-catalyzed cyclization. The requisite 8-hydroxyquinazolinones (6, 35-39) were synthesized by demethylation of the corresponding 8-methoxyquinazolinones with BBr3. N-Methylation of 8-methoxy-2-methylquinazolinone (15) with MeI, followed by O-demethylation by BBr3, afforded the control N3-methylquinazolinones 42 and 43, respectively. In general, an 8-hydroxy or 8-methyl substituent enhanced inhibitory activity in comparison with an 8-methoxy group. 2-Phenylquinazolinones were marginally less potent than the corresponding 2-methylquinazolinones, but the introduction of an electron-withdrawing or electron-donating 4'-substituent on the 2-aryl ring invariably increased potency. This was particularly evident in the 8-methylquinazolinone series (IC50 values 0.13-0.27 microM), which are among the most potent PARP inhibitors reported to date. N3-Methylquinazolinones 42 and 43 were essentially devoid of activity (IC50 values > 100 microM). In studies with L1210 cells in vitro, a concentration of 200 microM 8-hydroxy-2-methylquinazolinone (6, NU1025) (IC50 value 0.40 microM) potentiated the cytotoxicity of the monomethylating agent 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide and gamma-radiation 3.5- and 1.4-fold, respectively, at the 10% survival level.
Assuntos
Antineoplásicos/síntese química , Reparo do DNA , Inibidores Enzimáticos/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases , Quinazolinas/síntese química , Alquilantes/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Raios gama , Leucemia L1210/patologia , Camundongos , Quinazolinas/química , Quinazolinas/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The nuclear enzyme poly(ADP-ribose) polymerase (PARP) facilitates the repair of DNA strand breaks and is implicated in the resistance of cancer cells to certain DNA-damaging agents. Inhibitors of PARP have clinical potential as resistance-modifying agents capable of potentiating radiotherapy and the cytotoxicity of some forms of cancer chemotherapy. The preclinical development of 2-aryl-1H-benzimidazole-4-carboxamides as resistance-modifying agents in cancer chemotherapy is described. 1H-Benzimidazole-4-carboxamides, particularly 2-aryl derivatives, are identified as a class of potent PARP inhibitors. Derivatives of 2-phenyl-1H-benzimidazole-4-carboxamide (23, K(i) = 15 nM), in which the phenyl ring contains substituents, have been synthesized. Many of these derivatives exhibit K(i) values for PARP inhibition < 10 nM, with 2-(4-hydroxymethylphenyl)-1H-benzimidazole-4-carboxamide (78, K(i) = 1.6 nM) being one of the most potent. Insight into structure-activity relationships (SAR) for 2-aryl-1H-benzimidazole-4-carboxamides has been enhanced by studying the complex formed between 2-(3-methoxyphenyl)-1H-benzimidazole-4-carboxamide (44, K(i) = 6 nM) and the catalytic domain of chicken PARP. Important hydrogen-bonding and hydrophobic interactions with the protein have been identified for this inhibitor. 2-(4-Hydroxyphenyl)-1H-benzimidazole-4-carboxamide (45, K(i) = 6 nM) potentiates the cytotoxicity of both temozolomide and topotecan against A2780 cells in vitro (by 2.8- and 2.9-fold, respectively).
Assuntos
Benzimidazóis/química , Benzimidazóis/síntese química , Dacarbazina/análogos & derivados , Inibidores Enzimáticos/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases , Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Cristalografia por Raios X , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Relação Estrutura-Atividade , Temozolomida , Topotecan/farmacologia , Células Tumorais CultivadasRESUMO
A series of O(6)-allyl- and O(6)-(2-oxoalkyl)guanines were synthesized and evaluated, in comparison with the corresponding O(6)-alkylguanines, as potential inhibitors of the DNA-repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT). Simple O(6)-alkyl- and O(6)-cycloalkylguanines were weak AGT inactivators compared with O(6)-allylguanine (IC(50) = 8.5 +/- 0.6 microM) with IC(50) values ranging from 100 to 1000 microM. The introduction of substituents at C-2 of the allyl group of O(6)-allylguanine reduced activity compared with the parent compound, while analogous compounds in the O(6)-(2-oxoalkyl)guanine series exhibited very poor activity (150-1000 microM). O(6)-Cycloalkenylguanines proved to be excellent AGT inactivators, with 1-cyclobutenylmethylguanine (IC(50) = 0.55 +/- 0.02 microM) and 1-cyclopentenylmethylguanine (IC(50) = 0.39 +/- 0.04 microM) exhibiting potency approaching that of the benchmark AGT inhibitor O(6)-benzylguanine (IC(50) = 0.18 +/- 0.02 microM). 1-Cyclopentenylmethylguanine also inactivated AGT in intact HT29 human colorectal carcinoma cells (IC(50) = 0.20 +/- 0.07 microM) and potentiated the cytotoxicity of the monomethylating antitumor agent Temozolomide by approximately 3- and 10-fold, respectively, in the HT29 and Colo205 tumor cell lines. The observation that four mutant AGT enzymes resistant to O(6)-benzylguanine also proved strongly cross-resistant to 1-cyclopentenylmethylguanine indicates that the O(6)-substituent of each compound makes similar binding interactions within the active site of AGT.