RESUMO
BACKGROUND: The search for biomarkers to evaluate ovarian cancer (OC) homologous recombination (HR) function and predict the response to therapy is an urgent clinical need to improve the selection of patients who could benefit from platinum- and olaparib (poly-ADP ribose polymerase inhibitors, PARPi)-based therapies. METHODS: We used a large collection of OC patient-derived xenografts (PDXs) (n = 47) and evaluated their HR status based on BRCA1/2 mutations, BRCA1 promoter methylation and the HRDetect score. RAD51 foci were quantified in formalin-fixed, paraffin-embedded untreated tumour specimens by immunofluorescence and the messenger RNA expression of 21 DNA repair genes by real-time PCR. RESULTS: Tumour HR deficiency predicted both platinum and olaparib responses. The basal level of RAD51 foci evaluated in geminin-positive/replicating cells strongly inversely correlated with olaparib response (p = 0.011); in particular, the lower the foci score, the greater the sensitivity to olaparib, while low RAD51 foci score seems to associate with platinum activity. CONCLUSIONS: The basal RAD51 foci score is a candidate predictive biomarker of olaparib response in OC patients as it can be easily translatable in a clinical setting. Moreover, the findings corroborate the importance of OC-PDXs as a reliable tool to identify and validate biomarkers of response to therapy.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Cisplatino/farmacologia , Recombinação Homóloga , Neoplasias Ovarianas/patologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rad51 Recombinase/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ovarian cancer is the fifth cause of cancer-related deaths in women with high grade serous carcinoma (HGSOC) representing the most common histological subtype. Approximately 50 % of HGSOC are characterized by deficiency in homologous recombination (HR), one of the main cellular pathways to repair DNA double strand breaks and one of the well-described mechanisms is the loss of function of the BRCA1 or BRCA2 genes. Inhibition of the poly-ADP-ribose polymerase (PARP) is synthetic lethal with HR deficiency and the use of PARP inhibitors (PARPi) has significantly improved the outcome of patients with HGSOC with a greater benefit in patients with BRCA1/2 deficient tumors. However, intrinsic or acquired resistance to PARPi inevitably occurs in most HGSOC patients. Distinct heterogeneous mechanisms underlying the resistance to PARPi have been described, including a decrease in intracellular drug levels due to upregulation of multidrug efflux pumps, loss of expression/inactivating mutations in the PARP1 protein, restoration of HR and the protection of the replicative fork. Deciphering the molecular mechanisms of resistance to PARPi is of paramount importance towards the development of new treatment strategies and/or novel pharmacological agents to overcome this chemoresistance and optimize the treatment regimen for individual HGSOC patients. The current review summarizes the mechanisms underlying the resistance to PARPi, the available preclinical and clinical data on new combination treatment strategies (with chemotherapy, anti-angiogenic agents and immune checkpoint inhibitors) as well as agents under investigation which target the DNA damage response.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteína BRCA2/genética , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/fisiologia , Feminino , Humanos , Inibidores de Checkpoint Imunológico/administração & dosagem , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Ensaios Clínicos Controlados Aleatórios como Assunto , Ubiquitina-Proteína Ligases/genética , Regulação para Cima/fisiologiaRESUMO
Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and ß, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Dioxóis/farmacologia , Lipossarcoma Mixoide/genética , Lipossarcoma Mixoide/metabolismo , Tetra-Hidroisoquinolinas/farmacologia , Apoptose , Proteína C-Reativa/genética , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Metilação de DNA , Metilases de Modificação do DNA/deficiência , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/deficiência , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fibronectinas/genética , Humanos , Lipossarcoma Mixoide/tratamento farmacológico , Lipossarcoma Mixoide/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Componente Amiloide P Sérico/genética , Transdução de Sinais , Temozolomida , Trabectedina , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Raios UltravioletaRESUMO
Transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2 can reversibly inhibit the proliferation of hematopoietic progenitor cells in vivo, leading us to hypothesize that such quiescent progenitors might be more resistant to high doses of cell cycle active chemotherapeutic drugs, thereby allowing dose intensification of such agents. Initial studies showed that whereas administration of TGF-beta 1 or TGF-beta 2 did not prevent death in normal mice treated with high doses of 5-fluorouracil (5-FU), those mice that received TGF-beta 2 did exhibit the beginning of a hematologic recovery by day 11 after administration of 5-FU, and were preferentially rescued by a suboptimal number of transplanted bone marrow cells. Subsequently, it was found that the administration of TGF-beta 2 protected recovering progenitor cells from high concentrations of 5-FU in vitro. This protection coincided with the finding that significantly more progenitors for colony-forming unit-culture (CFU-c) and CFU-granulocyte, erythroid, megakaryocyte, macrophage (GEMM) were removed from S-phase by TGF-beta in mice undergoing hematopoietic recovery than in normal mice. Further studies showed that the administration of TGF-beta protected up to 90% of these mice undergoing hematologic recovery from a rechallenge in vivo with high dose 5-FU, while survival in mice not given TGF-beta was < 40%. Pretreatment of mice with TGF-beta 1 or TGF-beta 2 also protected 70-80% of mice from lethal doses of the noncycle active chemotherapeutic drug, doxorubicin hydrochloride (DXR). These results demonstrate that TGF-beta can protect mice from both the lethal hematopoietic toxicity of 5-FU, as well as the nonhematopoietic toxicity of DXR. This report thus shows that a negative regulator of hematopoiesis can be successfully used systemically to mediate chemoprotection in vivo.
Assuntos
Doxorrubicina/toxicidade , Fluoruracila/toxicidade , Fator de Crescimento Transformador beta/farmacologia , Animais , Medula Óssea/efeitos dos fármacos , Transplante de Medula Óssea , Divisão Celular/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/farmacologiaRESUMO
Trabectedin (ET-743, Yondelis) is a natural marine compound with antitumour activity currently undergoing phase II/III clinical trials. The mechanism of the drug's action is still to be defined, even though it has been clearly demonstrated the key role of Nucleotide Excision Repair (NER). To get further insights into the drug's mode of action, we studied the involvement of the DNA-double strand break repair (DNA-DSB) pathways: homologous and non-homologous recombination, both in budding yeasts and in mammalian cells and the possible cross-talk between NER and these repair pathways. Budding yeasts and mammalian cells deficient in the non-homologous end-joining pathway were moderately sensitive to trabectedin, while systems deficient in the homologous recombination pathway were extremely sensitive to the drug, with a 100-fold decrease in the IC50, suggesting that trabectedin-induced lesions are repaired by this pathway. The induction of Rad51 foci and the appearance of gamma-H2AX were chosen as putative markers for DNA-DSBs and were studied at different time points after trabectedin treatment in NER proficient and deficient systems. Both were clearly detected only in the presence of an active NER, suggesting that the DSBs are not directly caused by the drug, but are formed during the processing/repair of the drug- induced lesions.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Dioxóis/farmacologia , Tetra-Hidroisoquinolinas/farmacologia , Animais , Antineoplásicos/farmacologia , Biomarcadores/metabolismo , Células CHO , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cisplatino/farmacologia , Cricetinae , Cricetulus , Reparo do DNA/genética , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Histonas/efeitos dos fármacos , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Metanossulfonato de Metila/farmacologia , Mutação , Fosforilação , Saccharomyces cerevisiae/genética , TrabectedinaRESUMO
BACKGROUND: Epithelial ovarian cancer has a poor prognosis, mostly due to its late diagnosis and the development of drug resistance after a first platinum-based regimen. The presence of a specific population of "cancer stem cells" could be responsible of the relapse of the tumor and the development of resistance to therapy. For this reason, it would be important to specifically target this subpopulation of tumor cells in order to increase the response to therapy. METHOD: We screened a chemical compound library assembled during the COST CM1106 action to search for compound classes active in targeting ovarian stem cells. We here report the results of the high-throughput screening assay in two ovarian cancer stem cells and the differentiated cells derived from them. RESULTS AND CONCLUSION: Interestingly, there were compounds active only on stem cells, only on differentiated cells, and compounds active on both cell populations. Even if these data need to be validated in ad hoc dose response cytotoxic experiments, the ongoing analysis of the compound structures will open up to mechanistic drug studies to select compounds able to improve the prognosis of ovarian cancer patients.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Triagem em Larga Escala , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Antineoplásicos/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Estrutura Molecular , Neoplasias Ovarianas/patologia , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The activity of 4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548), a new alkycycline with high antitumor activity against a broad range of cancer cells, was evaluated in vitro and in vivo in cells selected for resistance to different anticancer agents. Both in vitro and in vivo, PNU-159548 did retain its activity in cells expressing the multidrug resistance (MDR) phenotype, associated to MIDR-1 gene overexpression or with an alteration in the topoisomerase II gene (altered MDR), independently on the drug used for the selection of the resistant cell line. According to these data, the intracellular uptake of PNU-159548 is not influenced by the presence of MDR-1. PNU-159548 was also active, both in vitro and in vivo, against cells showing resistance to various alkylating agents iincluding cisplatin, cyclophosphamide, and melphalan) and topoisomerase I-inhibitors. Cells defective in nucleotide excision repair, which did show hypersensitivity to treatment with UV irradiation and alkylating agents, showed only a marginally increased sensitivity to PNU-159548. Similarly, the activity of the drug was not influenced by the mismatch repair system, as assessed in two different cellular systems deficient in hMLH1 expression and in which hMLH1 activity was restored by chromosome 3 transfer. The results obtained clearly indicate that the new anticancer agent PNU-159548 is able to overcome the classical mechanisms of resistance emerging after treatment with the most clinically used anticancer agents, and it could represent an alternate choice in the treatment of those tumors refractory to conventional therapy.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Daunorrubicina/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Animais , Células CHO , Cricetinae , Daunorrubicina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Concentração Inibidora 50 , Leucemia L1210/tratamento farmacológico , Leucemia P388/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBARESUMO
Resistance of mouse M5076 (M5) ovarian reticular cell sarcoma to cyclophosphamide (CTX) was obtained in vivo by repeated drug treatment followed by transplantation of the regrowing tumor. After 16 passages, we obtained an M5 subline resistant to CTX (M5-CTX-16R). Median survival times were approximately 29 and 39 days for M5 and M5-CTX-16R, respectively. Survival of M5-bearing mice given a single i.p. dose of 200 or 300 mg/kg was 160 and 168% of controls, respectively, whereas in M5-CTX-16R it ws 103 and 123%, respectively. The resistance was not reversible after 14 additional passages with no further CTX treatment. M5 and M5-CTX-16R appear similar in histological features, pattern of metastasis formation, and DNA content, as assessed by flow cytometry (hypotetraploid). Metastases of M5-CTX-16R were also resistant to CTX. Flow cytometry studies 12 and 24 hr after CTX treatment revealed a block in S and G2-M phases in both tumors. After 48 hr and at subsequent times, no cytokinetic pertubation was evident in M5-CTX-16R, whereas in M5 marked accumulation of cells in G2-M was observed at 48, 72, 96, and 120 hr. Cross-resistance was found between CTX, L-phenylalanine mustard, chlorambucil, and hexamethylmelamine. M5-CTX-16R was sensitive, but less so than M5, to cis-platinum, 1,3-bis(2-chloroethyl)-1-nitrosourea, and imidazole-4-carboxamide,5-(3,3-dimethyl-1-triazene). Adriamycin was equally active on M5 and M5-CTX-16R, while 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidine-beta-D-glucopyranoside) was inactive. This model appears to be suitable for studies on the mechanism of resistance to CTX and alkylating agents and for screening new, non-cross-resistant drugs.
Assuntos
Antineoplásicos/uso terapêutico , Ciclofosfamida/uso terapêutico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Animais , Carmustina/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Dacarbazina/uso terapêutico , Resistência a Medicamentos , Feminino , Linfoma Difuso de Grandes Células B/fisiopatologia , Melfalan/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Ovarianas/fisiopatologiaRESUMO
Previous studies have demonstrated that interleukin 1 (IL-1) can protect most mice from the acute lethal toxicity mediated by high doses of radiation and/or some chemotherapeutic drugs. The results presented herein demonstrate that the pretreatment of mice with recombinant human interleukin 1 alpha (rhIL-1 alpha) protects mice from the lethal effects of several myelotoxic chemotherapeutic drugs, including 5-fluorouracil (5FUra), cyclophosphamide, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II), and 1,3-bis-(2-chloroethyl)-1-nitrosourea. However, pretreatment with rhIL-1 alpha was not effective against the acute lethal toxicity generated by doxorubicin and cisplatin. The chemoprotective effects appear to be at least partially due to myeloprotection/restoration, since the recovery of myeloid colony-forming units and the total cellularity in the bone marrow and spleen were accelerated in the rhIL-1 alpha-pretreated mice. However, the chemoprotective effects of rhIL-1 alpha are apparently not limited to myeloprotection, since pretreatment with rhIL-1 alpha protects mice against the lethal toxicity of both 5FUra and cyclophosphamide, yet bone marrow transplants rescue mice treated with 5FUra but not those treated with cyclophosphamide. The chemoprotective effects of rhIL-1 alpha may be at least partially indirect, since the efficacy of chemoprotection by rhIL-1 alpha is reduced in athymic mice, and interleukin 6, but not tumor necrosis factor alpha, can substitute for rhIL-1 alpha in chemoprotection from 5FUra.
Assuntos
Antineoplásicos/toxicidade , Medula Óssea/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Interleucina-1/uso terapêutico , Baço/efeitos dos fármacos , Animais , Medula Óssea/patologia , Carboplatina/toxicidade , Carmustina/toxicidade , Cisplatino/toxicidade , Ciclofosfamida/toxicidade , Morte , Doxorrubicina/toxicidade , Fluoruracila/toxicidade , Células-Tronco Hematopoéticas/patologia , Humanos , Interleucina-1/farmacologia , Interleucina-6/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Baço/patologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Late stage epithelial ovarian cancer has a dismal prognosis. Identification of pharmacogenomic markers (i.e. polymorphisms) to stratify patients to optimize individual therapy is of paramount importance. We here report the retrospective analysis of polymorphisms in 5 genes (ATM, ATR, Chk1, Chk2 and CDK12) involved in the cellular response to platinum in a cohort of 240 cancer patients with late stage ovarian cancer. The aim of the present study was to evaluate associations between the above mentioned SNPs and patients' clinical outcomes: overall survival (OS) and progression free survival (PFS). None of the ATM, ATR, Chk1 and Chk2 polymorphisms was found to significantly affect OS nor PFS in this cohort of patients. Genotype G/G of CDK12 polymorphism (rs1054488) predicted worse OS and PFS than the genotype A/A-A/G in univariate analysis. The predictive value was lost in the multivariate analysis. The positive correlation observed between this polymorphism and age, grade and residual tumor may explain why the CDK12 variant was not confirmed as an independent prognostic factor in multivariate analysis.The importance of CDK12 polymorphism as possible prognostic biomarker need to be confirmed in larger ovarian cancer cohorts, and possibly in other cancer population responsive to platinum agents.
Assuntos
Dano ao DNA/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/terapia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Polimorfismo de Nucleotídeo Único , Proteínas Mutadas de Ataxia Telangiectasia/genética , Biomarcadores Tumorais/genética , Carcinoma Epitelial do Ovário , Quinase 1 do Ponto de Checagem/genética , Quinase do Ponto de Checagem 2/genética , Estudos de Coortes , Quinases Ciclina-Dependentes/genética , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Análise Multivariada , Modelos de Riscos Proporcionais , RNA Mensageiro/genética , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP) and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20), only DDP treatment induced p53 phosphorylation at serine 15 (Ser15). Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM) cell lines, the role of ATM in drug-induced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Proteínas de Ligação a DNA , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Nucleares , Paclitaxel/farmacologia , Células Tumorais Cultivadas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Androstadienos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Neoplasias do Colo/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Proteína Quinase Ativada por DNA , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Transcrição Gênica , Células Tumorais Cultivadas/efeitos dos fármacos , WortmaninaRESUMO
DNA repair has been proposed to be an important determinant of cancer cell sensitivity to alkylating agents and cisplatin (DDP). Nucleotide excision repair (NER), which represents one of the most important cellular DNA repair processes able to remove a broad spectrum of DNA lesions, is involved in the recognition and repair of the crosslinks caused by DDP and melphalan (L-PAM). In this study, the mRNA levels of the different genes involved in NER (ERCC1, XPA, XPB, XPC, XPD, XPF) were examined in a panel of eight different human cancer cell lines, together with the overall DNA repair capacity using a host cell reactivation assay of a damaged plasmid. A statistically significant correlation was observed between the relative expression of XPA/XPC (P < 0.05) and ERCC1/XPC (P < 0.05) mRNAs. No correlation was found between the DDP and L-PAM IC50S and the relative mRNA expression of the tested NER genes. When the overall cellular DNA repair capacity was studied, carcinomas seemed to have a higher repair activity than leukaemias; but this repair DNA activity correlated neither with the mRNA expression of the different NER genes nor with DDP and L-PAM IC50S. These data seem to suggest that even if the NER pathway is an important determinant for the cytotoxicity of alkylating agents, as demonstrated by the extremely high sensitivity to alkylating agents in cells lacking this repair system, other factors have to play a role in regulating the cellular sensitivity/resistance to these antitumour drugs.
Assuntos
Antineoplásicos/uso terapêutico , Reparo do DNA , Neoplasias/tratamento farmacológico , Antineoplásicos Alquilantes/uso terapêutico , Northern Blotting , Cisplatino/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Feminino , Expressão Gênica , Humanos , Leucemia/tratamento farmacológico , Leucemia/genética , Melfalan/administração & dosagem , Neoplasias/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The mode of action of Ecteinascidin-743 (ET-743), a marine tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, which has shown very potent antitumour activity in preclinical systems and encouraging results in Phase I clinical trials was investigated at a cellular level. Both SW620 and LoVo human intestinal carcinoma cell lines exposed for 1 h to ET-743 progress through S phase more slowly than control cells and then accumulate in the G2M phase. The sensitivity to ET-743 of G1 synchronised cells was much higher than that of cells synchronised in S phase and even higher than that of cells synchronised in G2M. ET-743 concentrations up to four times higher than the IC(50) value caused no detectable DNA breaks or DNA-protein cross-links as assessed by alkaline elution techniques. ET-743 induced a significant increase in p53 levels in cell lines expressing wild-type (wt) (p53). However, the p53 status does not appear to be related to the ET-743 cytotoxic activity as demonstrated by comparing the drug sensitivity in p53 (-/-) or (+/+) mouse embryo fibroblasts and in A2780 ovarian cancer cells or the A2780/CX3 sub-line transfected with a dominant-negative mutant TP53. The cytotoxic potency of ET-743 was comparatively evaluated in CHO cell lines proficient or deficient in nucleotide excision repair (NER), and it was found that ET-743 was approximately 7-8 times less active in ERCC3/XPB and ERCC1-deficient cells than control cells. The findings that G1 phase cells are hypersensitive and that NER-deficient cells are resistant to ET-743 indicate that the mode of action of ET-743 is unique and different from that of other DNA-interacting drugs.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Dioxóis/uso terapêutico , Isoquinolinas/uso terapêutico , Antineoplásicos Alquilantes/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular , Neoplasias do Colo/patologia , Ciclinas/metabolismo , Dano ao DNA , DNA de Neoplasias/análise , Dioxóis/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isoquinolinas/farmacologia , Tetra-Hidroisoquinolinas , Trabectedina , Células Tumorais Cultivadas/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
In order to simulate drug resistance observed in the clinic, two cisplatin-resistant cell lines were produced from a murine ovarian reticulosarcoma, M5076 (M5), by pulse (M5/CDDP) and continuous (M5/CDDPc) treatment with cis-diamminedichloroplatinum(II)(CDDP). These cell lines showed a similar stable low level of resistance (approximately 3-fold) to CDDP and cross-resistance to carboplatin, iproplatin and the new alkylating agent tallimustine, but not to L-PAM (L-phenylalanine mustard) and BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea). Collateral sensitivity to two inhibitors of topoisomerase II, VP16 (etoposide) and doxorubicin (Dox), but cross-resistance to the topoisomerase I inhibitor, camptothecin, were observed. The two cell lines were also sensitive to 5-fluorouracil. No increase in the level of glutathione or activity of glutathione S-transferase could be observed in resistant cells compared with the parental M5 cells. Total DNA platination immediately after treatment was similar in the parental and resistant cell lines. Repair of total DNA platination, measured after 24 h of recovery, was undetectable in M5 and M5/CDDP cells, but was 33% in M5/ CDDPc cells. Initial DNA-interstrand cross-links (DNA-ISC) were six times higher in M5 than in M5/CDDP cells, but 24 h after treatment, both lines had completely repaired this damage. M5/ CDDPc cells did not show formation of DNA-ISC at any time after treatment. The two resistant cell lines were tumorigenic when implanted in mice and resistant to CDDP treatment in vivo. The CDDP resistant tumours were not cross-resistant in vivo to L-PAM, BCNU and Dox, which had been active in vitro, nor to tallimustine, which had been cross-resistant in vitro. Mechanisms of resistance in M5/CDDP and M5-CDDPc seem to be based on a lower formation of DNA-ISC combined, for the latter cell line, with a higher repair capacity for total DNA platination.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Linfoma não Hodgkin/tratamento farmacológico , Animais , Adutos de DNA/efeitos dos fármacos , Esquema de Medicação , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Linfoma não Hodgkin/patologia , Camundongos , Camundongos Endogâmicos C57BL , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Altretamine (hexamethylmelamine) is an antitumour drug with demonstrated antitumour activity in refractory ovarian cancer. Due to its poor water solubility, the drug has been given by the oral route. In all animal species, including humans, altretamine undergoes oxidative N-demethylation with the formation of hydroxymethyl derivatives as intermediates. Hydroxymethylmelamines are believed to be responsible for the cytotoxic and antitumour activity of the drug. The inter-hand intrapatient variability of the bioavailability of altretamine after oral administration represents an important drawback for effective clinical use of this drug. The variability appears to be mostly related to the first-pass effect and therefore may be overcome by intravenous administration of the drug. Although attempts to administer the drug intravenously have not been successful in the past, some investigations on the use of the new parental formulation of altretamine are in progress.
Assuntos
Altretamine/farmacocinética , Administração Oral , Altretamine/administração & dosagem , Altretamine/química , Animais , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Humanos , Infusões Parenterais , Injeções Intravenosas , Estrutura MolecularRESUMO
We investigated the cytotoxic activity and some aspects of the mode of action of 5-aza-2'-deoxycytidine (Aza-dC) in 21 primary cultures of leukemic cells freshly obtained from patients with chronic myeloid leukemia (CML) in blast crisis. The cytotoxic potency of Aza-dC was comparable or even greater than that of 1-beta-D-arabinofuranosylcytosine (Ara-C) in most cases, suggesting that this drug has potential in the therapy of blast crisis of CML. Drug incorporation into DNA was evaluated by exposing leukemic cells simultaneously to 3H-Aza-dC at the concentration of 0.1 micrograms/ml and 14C-thymidine (TdR) used as internal standard. Incorporation of Aza-dC into DNA was detectable in all cases. In 17 samples we evaluated the DNA integrity of leukemic cells exposed to Aza-dC using alkaline elution techniques. The drug caused a detectable amount of DNA alkali labile sites (ALS). DNA-ALS increased in cells exposed to Aza-dC concentrations from 0.1 to 1 microgram/ml but did not further increase at 10 micrograms/ml. A plateau in the levels of DNA-ALS was also seen in human K562 cells exposed to increasing concentrations of Aza-dC from 5 to 10 micrograms/ml, whereas in these cells Aza-dC incorporation into DNA increased with increasing Aza-dC concentrations. Therefore, DNA-ALS caused by Aza-dC are not simply the result of the chemical decomposition of azacytosine molecules incorporated into DNA, but are presumably the result of a saturable DNA repair mechanism (e.g., glycosylases) leading to formation of the apyrimidinic sites.
Assuntos
Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Crise Blástica , Citarabina/farmacologia , DNA de Neoplasias/metabolismo , Decitabina , Humanos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Single lung transplantation (SLT) is now successfully used in patients with severe emphysema. Mechanical imbalance between the native emphysematous and the healthy transplanted lung can be easily managed, unless severe graft failure occurs, leading to acute respiratory failure. Emergency retransplantation has been used in this setting, since the conventional approach to adult respiratory distress syndrome (ARDS) (mechanical ventilation and positive end-expiratory pressure [PEEP]) fails, due to the mechanical discrepancy between the two lungs. We describe two cases of severe graft failure following SLT in emphysema patients that were successfully treated with prolonged independent respiratory treatment. Mechanical ventilation and PEEP were applied to the failing transplanted lung while the native emphysematous lung was maintained on spontaneous breathing to avoid hyperexpansion and barotrauma. The independent lung respiratory treatment lasted 35 and 25 days, respectively: to our knowledge, these are among the longest-lasting independent respiratory treatments reported. The management was simplified by the early use of a double-lumen tracheostomy cannula as an alternative to orotracheal double lumen tube.
Assuntos
Transplante de Pulmão/fisiologia , Pulmão/fisiopatologia , Enfisema Pulmonar/fisiopatologia , Enfisema Pulmonar/cirurgia , Respiração Artificial/métodos , Rejeição de Enxerto , Humanos , Ventilação com Pressão Positiva Intermitente , Intubação Intratraqueal/instrumentação , Intubação Intratraqueal/métodos , Complacência Pulmonar/fisiologia , Masculino , Pessoa de Meia-Idade , Respiração com Pressão Positiva , Troca Gasosa Pulmonar/fisiologia , Ventilação Pulmonar/fisiologia , TraqueostomiaRESUMO
A new model lung with the capacity for simulated spontaneous breathing is described. It consists of a modified commercial mechanical ventilator (Kontron ABT 4100), connected in parallel to a compliant system, a cylindric acrylic box with a latex thin membrane substituting for the top. Volume and compliance of the model are 2500 ml and 50 ml cmH2O-1, respectively. The modified ventilator simulates physiologic inspiratory flow at a rate of 10 to 30 min-1 and tidal volume up to 1000 ml, with an inspiratory to expiratory time ratio continuously variable between 1:4 and 4:1. The model has been tested under different respiratory assist techniques, connected either to continuous positive airway pressure proved to be reliable, versatile and bearing satisfactory resemblance to human ventilatory physiology.
Assuntos
Pulmão/fisiologia , Modelos Biológicos , Respiração , Humanos , Ventiladores MecânicosRESUMO
Six lambs were anesthetized and connected venovenous mode to a Membrane Lung for Extracorporeal CO2 removal. The animals underwent several hours periods of continuous positive pressure ventilation (CPPV), at 5 cmH2O positive end expiratory pressure (PEEP), alternated with several hours periods of low frequency positive pressure ventilation (5 cmH2O PEEP, 2 b.p.m.) with extracorporeal CO2 removal (LFPPV-ECCO2R). During LFPPV-ECCO2R compared with CPPV, cardiac output increased by 26%, pulmonary vascular resistances and systemic vascular resistances decreased by 28% and 22% respectively. The renal function improved significantly during LFPPV-ECCO2R compared with CPPV, i.e. urinary flow, creatinine clearance and osmolar clearance increased by 50%, 37% and 52% respectively. In these experiments LFPPV-ECCO2R, a form of completely artificial ventilation, seems to prevent hemodynamic and renal complications of CPPV.