RESUMO
Resistance to androgen receptor (AR) targeting therapeutics in prostate cancer (PC) is a significant clinical problem. Mechanisms by which this is accomplished include AR amplification and expression of AR splice variants, demonstrating that AR remains a key therapeutic target in advanced disease. For the first time we show that IKBKE drives AR signalling in advanced PC. Significant inhibition of AR regulated gene expression was observed upon siRNA-mediated IKBKE depletion or pharmacological inhibition due to inhibited AR gene expression in multiple cell line models including a LNCaP derivative cell line resistant to the anti-androgen, enzalutamide (LNCaP-EnzR). Phenotypically, this resulted in significant inhibition of proliferation, migration and colony forming ability suggesting that targeting IKBKE could circumvent resistance to AR targeting therapies. Indeed, pharmacological inhibition in the CWR22Rv1 xenograft mouse model reduced tumour size and enhanced survival. Critically, this was validated in patient-derived explants where enzymatic inactivation of IKBKE reduced cell proliferation and AR expression. Mechanistically, we provide evidence that IKBKE regulates AR levels via Hippo pathway inhibition to reduce c-MYC levels at cis-regulatory elements within the AR gene. Thus, IKBKE is a therapeutic target in advanced PC suggesting repurposing of clinically tested IKBKE inhibitors could be beneficial to castrate resistant PC patients.
Assuntos
Quinase I-kappa B/fisiologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Androgênicos/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Humanos , Quinase I-kappa B/antagonistas & inibidores , Masculino , Camundongos Nus , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Sinalização YAPRESUMO
High-risk neuroblastoma, a predominantly TP53 wild-type (wt) tumour, is incurable in >50% patients supporting the use of MDM2 antagonists as novel therapeutics. Idasanutlin (RG7388) shows in vitro synergy with chemotherapies used to treat neuroblastoma. This is the first study to evaluate the in vivo efficacy of the intravenous idasanutlin prodrug, RO6839921 (RG7775), both alone and in combination with temozolomide in TP53 wt orthotopic neuroblastoma models. Detection of active idasanutlin using liquid chromatography-mass spectrometry and p53 pathway activation by ELISA assays and Western analysis showed peak plasma levels 1 h post-treatment with maximal p53 pathway activation 3-6 h post-treatment. RO6839921 and temozolomide, alone or in combination in mice implanted with TP53 wt SHSY5Y-Luc and NB1691-Luc cells showed that combined RO6839921 and temozolomide led to greater tumour growth inhibition and increase in survival compared to vehicle control. Overall, RO6839921 had a favourable pharmacokinetic profile consistent with intermittent dosing and was well tolerated alone and in combination. These preclinical studies support the further development of idasanutlin in combination with temozolomide in neuroblastoma in early phase clinical trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neuroblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Pirrolidinas/farmacologia , Temozolomida/farmacologia , para-Aminobenzoatos/farmacologia , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Camundongos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Pirrolidinas/farmacocinética , Distribuição Aleatória , Temozolomida/administração & dosagem , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , para-Aminobenzoatos/farmacocinéticaRESUMO
Poly(adenosine diphosphate ribose) polymerases (PARPs) are multifunctional proteins which play a role in many cellular processes. Namely, PARP1 and PARP2 have been shown to be involved in DNA repair, and therefore are valid targets in cancer treatment with PARP inhibitors, such as rucaparib, currently in clinical trials. Proton magnetic resonance spectroscopy (1 H-MRS) was used to study the impact of rucaparib in vitro and ex vivo in liver tissue from mice, via quantitative analysis of nicotinamide adenosine diphosphate (NAD+ ) spectra, to assess the potential of MRS as a biomarker of the PARP inhibitor response. SW620 (colorectal) and A2780 (ovarian) cancer cell lines, and PARP1 wild-type (WT) and PARP1 knock-out (KO) mice, were treated with rucaparib, temozolomide (methylating agent) or a combination of both drugs. 1 H-MRS spectra were obtained from perchloric acid extracts of tumour cells and mouse liver. Both cell lines showed an increase in NAD+ levels following PARP inhibitor treatment in comparison with temozolomide treatment. Liver extracts from PARP1 WT mice showed a significant increase in NAD+ levels after rucaparib treatment compared with untreated mouse liver, and a significant decrease in NAD+ levels in the temozolomide-treated group. The combination of rucaparib and temozolomide did not prevent the NAD+ depletion caused by temozolomide treatment. The 1 H-MRS results show that NAD+ levels can be used as a biomarker of PARP inhibitor and methylating agent treatments, and suggest that in vivo measurement of NAD+ would be valuable.
Assuntos
Indóis/farmacologia , Fígado/metabolismo , NAD/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Espectroscopia de Prótons por Ressonância Magnética/métodos , Animais , Linhagem Celular Tumoral , Humanos , Camundongos Knockout , NAD/químicaRESUMO
BACKGROUND: The phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and mTOR inhibitors have been developed and have now reached clinical trials. Similarly, CDKs have been investigated as cancer drug targets. METHODS: We have synthesised and characterised a series of 6-aminopyrimidines identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2. Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell lines, and in vivo HT29 efficacy studies. RESULTS: 2,6-Diaminopyrimidines with an O(4)-cyclohexylmethyl substituent and a C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were growth inhibitory (GI50<20 µM). Compound 1, but not 2 or 5, potently inhibits CDK2 (IC50=0.1 nM) as well as PI3K, and was cytotoxic at growth inhibitory concentrations. Consistent with kinase inhibition data, compound 1 reduced phospho-Rb and phospho-rS6 at GI50 concentrations. Combination of NU6102 (CDK2 inhibitor) and pictilisib (GDC-0941; pan-PI3K inhibitor) resulted in synergistic growth inhibition, and enhanced cytotoxicity in HT29 cells in vitro and HT29 tumour growth inhibition in vivo. CONCLUSIONS: These studies identified a novel series of mixed CDK2/PI3K inhibitors and demonstrate that dual targeting of CDK2 and PI3K can result in enhanced antitumour activity.
Assuntos
Adenocarcinoma/patologia , Neoplasias Colorretais/patologia , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Adenocarcinoma/enzimologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/enzimologia , Quinase 2 Dependente de Ciclina/fisiologia , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/síntese química , Pirimidinas/farmacologia , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A major drawback of cytotoxic chemotherapy is the lack of selectivity toward noncancerous cells. The targeted delivery of cytotoxic drugs to tumor cells is a longstanding goal in cancer research. We proposed that covalent inhibitors could be adapted to deliver cytotoxic agents, conjugated to the ß-position of the Michael acceptor, via an addition-elimination mechanism promoted by covalent binding. Studies on model systems showed that conjugated 5-fluorouracil (5FU) could be released upon thiol addition in relevant time scales. A series of covalent epidermal growth factor receptor (EGFR) inhibitors were synthesized as their 5FU derivatives. Achieving the desired release of 5FU was demonstrated to depend on the electronics and geometry of the compounds. Mass spectrometry and NMR studies demonstrated an anilinoquinazoline acrylate ester conjugate bound to EGFR with the release of 5FU. This work establishes that acrylates can be used to release conjugated molecules upon covalent binding to proteins and could be used to develop targeted therapeutics.
Assuntos
Citotoxinas , Fluoruracila , Fluoruracila/farmacologia , Receptores ErbB , Ésteres , Espectrometria de MassasRESUMO
There is a wide inter-individual variation in PARP-1 {PAR [poly(ADP-ribose)] polymerase 1} activity, which may have implications for health. We investigated if the variation: (i) is due to polymorphisms in the PARP-1 gene or PARP-1 protein expression; and (ii) affects patients' response to anticancer treatment. We studied 56 HV (healthy volunteers) and 118 CP (cancer patients) with supporting in vivo experiments. PARP activity ranged between 10 and 2600 pmol of PAR/106 cells and expression between 0.02-1.55 ng of PARP-1/µg of protein. PARP-1 expression correlated with activity in HV (R2=0.19, P=0.003) and CP (R2=0.06, P=0.01). A short CA repeat in the promoter was significantly associated with increased cancer risk [OR (odds ratio), 5.22; 95% CI (confidence interval), 1.79-15.24]. PARP activity was higher in men than women (P=0.04) in the HV. Male mice also had higher PARP activity than females or castrated males. Oestrogen supplementation activated PARP in PBMCs (peripheral blood mononuclear cells) from female mice (P=0.003), but inhibited PARP-1 in their livers by 80%. PARP activity and expression were not dependent on the investigated polymorphisms, but there was a modest correlation of PARP activity with expression. Studies in the HV revealed sex differences in PARP activity, which was confirmed in mice and shown to be associated with sex hormones. Toxic response to treatment was not associated with PARP activity and/or expression.
Assuntos
Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Ativação Enzimática , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Humanos , Células K562 , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Neoplasias/genética , Farmacogenética , Poli(ADP-Ribose) Polimerase-1 , Polimorfismo Genético , Fatores SexuaisRESUMO
Poly(ADP-ribose) polymerase (PARP1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of damage. Nevertheless, PARP1-/- mice are viable, fertile and do not develop early onset tumours. Here, we show that PARP inhibitors trigger gamma-H2AX and RAD51 foci formation. We propose that, in the absence of PARP1, spontaneous single-strand breaks collapse replication forks and trigger homologous recombination for repair. Furthermore, we show that BRCA2-deficient cells, as a result of their deficiency in homologous recombination, are acutely sensitive to PARP inhibitors, presumably because resultant collapsed replication forks are no longer repaired. Thus, PARP1 activity is essential in homologous recombination-deficient BRCA2 mutant cells. We exploit this requirement in order to kill BRCA2-deficient tumours by PARP inhibition alone. Treatment with PARP inhibitors is likely to be highly tumour specific, because only the tumours (which are BRCA2-/-) in BRCA2+/- patients are defective in homologous recombination. The use of an inhibitor of a DNA repair enzyme alone to selectively kill a tumour, in the absence of an exogenous DNA-damaging agent, represents a new concept in cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína BRCA2/deficiência , Genes BRCA2 , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Azulenos , Proteína BRCA2/genética , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Replicação do DNA , Camundongos , Camundongos Nus , Neoplasias/enzimologia , Neoplasias/patologia , Poli(ADP-Ribose) Polimerases/deficiência , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.
Assuntos
Alcinos/farmacologia , Cisteína/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Alcinos/síntese química , Alcinos/química , Cisteína/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade , Quinase Induzida por NF-kappaBRESUMO
PURPOSE: High-risk neuroblastoma is characterized by poor survival rates, and the development of improved therapeutic approaches is a priority. Temozolomide and topotecan show promising clinical activity against neuroblastoma. Poly(ADP-ribose) polymerase-1 (PARP-1) promotes DNA repair and cell survival following genotoxic insult; we postulated that its inhibition may enhance the efficacy of these DNA-damaging drugs in pediatric cancers. EXPERIMENTAL DESIGN: We evaluated the chemosensitizing properties of the PARP inhibitor AG014699 (Pfizer, Inc.) in combination with temozolomide and topotecan, against human neuroblastoma cells and xenografts, alongside associated pharmacologic and toxicologic indices. RESULTS: Addition of PARP-inhibitory concentrations of AG014699 significantly potentiated growth inhibition by both topotecan (1.5- to 2.3-fold) and temozolomide (3- to 10-fold) in vitro, with equivalent effects confirmed in clonogenic assays. In two independent in vivo models (NB1691 and SHSY5Y xenografts), temozolomide caused a xenograft growth delay, which was enhanced by co-administration of AG014699, and resulted in complete and sustained tumor regression in the majority (6 of 10; 60%) of cases. Evidence of enhanced growth delay by topotecan/AG014699 co-administration was observed in NB1691 xenografts. AG014699 metabolites distributed rapidly into the plasma (Cmax, 1.2-1.9 nmol/L at 30 min) and accumulated in xenograft tissues (Cmax, 1-2 micromol/L at 120 min), associated with a sustained suppression of PARP-1 enzyme activity. Doses of AG014699 required for potentiation were not toxic per se. CONCLUSIONS: These data show enhancement of temozolomide and topotecan efficacy by PARP inhibition in neuroblastoma. Coupled with the acceptable pharmacokinetic, pharmacodynamic, and toxicity profiles of AG014699, our findings provide strong rationale for investigation of PARP inhibitors in pediatric early clinical studies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Dacarbazina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Neuroblastoma/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases , Topotecan/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dacarbazina/farmacologia , Sinergismo Farmacológico , Humanos , Camundongos , Neuroblastoma/patologia , Poli(ADP-Ribose) Polimerase-1 , Temozolomida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.
Assuntos
Carcinoma Hepatocelular , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Neoplasias Hepáticas Experimentais , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/patologia , Proteína Quinase Ativada por DNA/metabolismo , Doxorrubicina/farmacologia , Humanos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/patologia , Células MCF-7 , Camundongos , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (â¼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82⯵M for ERK5; IC50â¯>â¯120⯵M for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.
Assuntos
Antineoplásicos/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Disponibilidade Biológica , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by ionizing radiation and topoisomerase II poisons, such as etoposide and doxorubicin. A major pathway for the repair of DSB is nonhomologous end joining, which requires DNA-dependent protein kinase (DNA-PK) activity. We investigated the therapeutic use of a potent, specific DNA-PK inhibitor (NU7441) in models of human cancer. We measured chemosensitization by NU7441 of topoisomerase II poisons and radiosensitization in cells deficient and proficient in DNA-PK(CS) (V3 and V3-YAC) and p53 wild type (LoVo) and p53 mutant (SW620) human colon cancer cell lines by clonogenic survival assay. Effects of NU7441 on DSB repair and cell cycle arrest were measured by gammaH2AX 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 G(2)-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.
Assuntos
Cromonas/farmacologia , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Células CHO , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromonas/administração & dosagem , Cromonas/farmacocinética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Cricetinae , Dano ao DNA , Sinergismo Farmacológico , Etoposídeo/administração & dosagem , Etoposídeo/farmacologia , Feminino , Histonas/metabolismo , Humanos , Camundongos , Camundongos Nus , Morfolinas/administração & dosagem , Morfolinas/farmacocinética , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Radiossensibilizantes/administração & dosagem , Radiossensibilizantes/farmacocinética , Radiossensibilizantes/farmacologia , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Poly(ADP-ribose) polymerase (PARP)-1 (EC 2.4.2.30) is a nuclear enzyme that promotes the base excision repair of DNA breaks. Inhibition of PARP-1 enhances the efficacy of DNA alkylating agents, topoisomerase I poisons, and ionizing radiation. Our aim was to identify a PARP inhibitor for clinical trial from a panel of 42 potent PARP inhibitors (K(i), 1.4-15.1 nmol/L) based on the quinazolinone, benzimidazole, tricyclic benzimidazole, tricyclic indole, and tricyclic indole-1-one core structures. We evaluated chemosensitization of temozolomide and topotecan using LoVo and SW620 human colorectal cells; in vitro radiosensitization was measured using LoVo cells, and the enhancement of antitumor activity of temozolomide was evaluated in mice bearing SW620 xenografts. Excellent chemopotentiation and radiopotentiation were observed in vitro, with 17 of the compounds causing a greater temozolomide and topotecan sensitization than the benchmark inhibitor AG14361 and 10 compounds were more potent radiosensitizers than AG14361. In tumor-bearing mice, none of the compounds were toxic when given alone, and the antitumor activity of the PARP inhibitor-temozolomide combinations was unrelated to toxicity. Compounds that were more potent chemosensitizers in vivo than AG14361 were also more potent in vitro, validating in vitro assays as a prescreen. These studies have identified a compound, AG14447, as a PARP inhibitor with outstanding in vivo chemosensitization potency at tolerable doses, which is at least 10 times more potent than the initial lead, AG14361. The phosphate salt of AG14447 (AG014699), which has improved aqueous solubility, has been selected for clinical trial.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos da radiação , Neoplasias Colorretais/radioterapia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Raios gama , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Dose Máxima Tolerável , Camundongos , Camundongos Nus , Poli(ADP-Ribose) Polimerases/metabolismo , Relação Estrutura-Atividade , Temozolomida , Inibidores da Topoisomerase I , Topotecan/farmacologiaRESUMO
The antitumour effect of thymidylate synthase inhibitors such as raltitrexed (RTX) may be reversed by salvage of thymidine (Thd). Since thymidine phosphorylase (TP) depletes Thd, the potential for tumour-selective depletion of Thd using antibody-mediated delivery of TP to tumours was investigated. In vitro studies demonstrated that 25 x 10(-3) units/ml TP depleted extracellular Thd (3 microM) and restored sensitivity to the growth inhibitory effects of RTX in Lovo and HT29 cell lines. Thymidine concentrations in xenograft tumours were inversely proportional to the activity of TP in the tumour, and the presence of a subcutaneous Lovo xenograft reduced plasma Thd concentrations from 0.92 +/- 0.07 to 0.37 +/- 0.04 microM. Intravenous administration of native TP enzyme depleted plasma Thd to 5 nM, but following rapid elimination of TP, plasma Thd returned to pretreatment values. There was no effect on tumour TP or Thd. Conjugation of TP to the A5B7 F(ab)2 antibody fragment, which targets carcinoembryonic antigen (CEA) expressed on colorectal cell-lines such as Lovo, did result in selective accumulation of TP in the tumour. However, there was no tumour-selective depletion of Thd and there did not appear to be any potential benefit of combining antibody-targeted TP with RTX.
Assuntos
Quinazolinas/uso terapêutico , Tiofenos/uso terapêutico , Timidina Fosforilase/metabolismo , Timidina/metabolismo , Timidilato Sintase/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Antígeno Carcinoembrionário/imunologia , Antígeno Carcinoembrionário/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Células HT29 , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Injeções Intravenosas , Camundongos , Camundongos Nus , Quinazolinas/farmacologia , Reprodutibilidade dos Testes , Tiofenos/farmacologia , Timidina Fosforilase/administração & dosagem , Timidina Fosforilase/imunologia , Timidilato Sintase/metabolismoRESUMO
BACKGROUND: Cell lines provide a powerful model to study cancer and here we describe a new spontaneously immortalised epithelial ovarian cancer cell line (NUOC-1) derived from the ascites collected at a time of primary debulking surgery for a mixed endometrioid / clear cell / High Grade Serous (HGS) histology. RESULTS: This spontaneously immortalised cell line was found to maintain morphology and epithelial markers throughout long-term culture. NUOC-1 cells grow as an adherent monolayer with a doubling time of 58 hours. The cells are TP53 wildtype, positive for PTEN, HER2 and HER3 expression but negative for oestrogen, progesterone and androgen receptor expression. NUOC-1 cells are competent in homologous recombination and non-homologous end joining, but base excision repair defective. Karyotype analysis demonstrated a complex tetraploid karyotype. SNP array analysis of parent and derived subpopulations (NUOC-1-A1 and NUOC-1-A2) cells demonstrated heterogeneous cell populations with numerous copy number alterations and a pro-amplification phenotype. The characteristics of this new cell line lends it to be an excellent model for investigation of a number of the identified targets. MATERIALS AND METHODS: The cell line has been characterised for growth, drug sensitivity, expression of common ovarian markers and mutations, clonogenic potential and ability to form xenografts in SCID mice. Copy number changes and clonal evolution were assessed by SNP arrays.
Assuntos
Linhagem Celular Tumoral , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Animais , Bandeamento Cromossômico , Evolução Clonal/genética , Variações do Número de Cópias de DNA , Reparo do DNA , Modelos Animais de Doenças , Feminino , Amplificação de Genes , Genes myc , Xenoenxertos , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Células-Tronco Neoplásicas/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
PURPOSE: Two children with soft tissue sarcomas receiving etoposide as part of their standard clinical treatment had external biliary drainage due to obstruction of the bile duct. These unusual cases provided an opportunity to investigate the biliary clearance of etoposide by determining etoposide concentrations in bile and plasma samples obtained during chemotherapy. PATIENTS AND METHODS: Etoposide was administered to patient 1 at a dose of 150 mg/m(2), as a 4 h infusion, on each of three days of treatment. Patient 2 received a daily etoposide dose of 800 mg/m(2) as a 24 h continuous infusion, also over a 3-day treatment period. Bile and plasma samples were obtained at regular intervals from both patients and etoposide levels quantified by LC/MS analysis. RESULTS AND DISCUSSION: Biliary etoposide clearance was approximately equal to the flow of bile, with an average clearance of 0.32 ml/min determined in patient 1. Less than 2% of the etoposide dose administered was excreted in the bile in either patient studied, indicating that biliary clearance of etoposide is relatively minor. These results suggest that etoposide dose adjustment is unnecessary in patients with biliary obstruction.
Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Bile/metabolismo , Etoposídeo/farmacocinética , Sarcoma/tratamento farmacológico , Adolescente , Antineoplásicos Fitogênicos/sangue , Antineoplásicos Fitogênicos/uso terapêutico , Pré-Escolar , Drenagem , Etoposídeo/sangue , Etoposídeo/uso terapêutico , Feminino , Humanos , Masculino , Taxa de Depuração Metabólica , Sarcoma/metabolismoRESUMO
PURPOSE: Tumours frequently have defects in multiple oncogenic pathways, e.g. MAPK and PI3K signalling pathways, and combinations of targeted therapies may be required for optimal activity. This study evaluated the novel MEK inhibitor WX-554 and the novel PI3K inhibitor WX-037, as single agents and in combination, in colorectal carcinoma cell lines and tumour xenograft-bearing mice. METHODS: In vitro growth inhibition, survival and signal transduction were measured using the Sulforhodamine B, clonogenic and Western blotting assays, respectively, in HCT116 and HT29 cell lines. In vivo anti-tumour efficacy and pharmacokinetic properties were assessed in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice. RESULTS: The combination of WX-554 and WX-037 exhibited marked synergistic growth inhibition in vitro, which was associated with increased cytotoxicity and enhanced inhibition of ERK and S6 phosphorylation, compared to either agent alone. Pharmacokinetic analyses indicated that there was no PK interaction between the two drugs at low doses, but that at higher doses, WX-037 may delay the tumour uptake of WX-554. In vivo efficacy studies revealed that the combination of WX-037 and WX-554 was non-toxic and exhibited marked tumour growth inhibition greater than observed with either agent alone. CONCLUSION: These studies show for the first time that combination treatment with the novel MEK inhibitor WX-554 and the novel PI3K inhibitor WX-037 can induce synergistic growth inhibition in vitro, which translates into enhanced anti-tumour efficacy in vivo.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/administração & dosagem , Animais , Sinergismo Farmacológico , Células HCT116 , Células HT29 , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Studies to identify predictive biomarkers can be carried out in isogenic cancer cell lines, which enable interrogation of the effect of a specific mutation. We assessed the effects of four drugs, the PI3K-mammalian target of rapamycin inhibitor dactolisib, the PI3K inhibitor pictrelisib, and the MEK (MAPK/ERK Kinase) inhibitors PD 0325901 and selumetinib, in isogenic DLD1 parental, KRAS(+/-), KRAS(G13D/-), PIK3CA(+/-) and PIK3CA(E545K/-) colorectal carcinoma cell lines. Importantly, we found substantial differences in the growth of these cells and in their drug sensitivity depending on whether they were studied under 2D (standard tissue culture on plastic) or 3D (in vitro soft agar and in vivo xenograft) conditions. DLD1 KRAS(+/-) and DLD1 PIK3CA(+/-) cells were more sensitive to MEK inhibitors than parental, DLD1 KRAS(G13D/-) and DLD1 PIK3CA(E545K/-) cells under 2D conditions, whereas DLD1 KRAS(G13D/-) and DLD1 PIK3CA(E545K/-) xenografts were sensitive to 10 mg/kg daily ×14 PD 0325901 in vivo (p ≤ 0.02) but tumours derived from parental DLD1 cells were not. These findings indicate that KRAS and PIK3CA mutations can influence the response of DLD1 colorectal cancer cell lines to MEK and PI3K inhibitors, but that the effect is dependent on the experimental model used to assess drug sensitivity.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais , Neoplasias Colorretais/tratamento farmacológico , MAP Quinase Quinase Quinases/antagonistas & inibidores , Mutação , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Feminino , Humanos , MAP Quinase Quinase Quinases/metabolismo , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.