RESUMEN
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.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Cisplatino/farmacología , Recombinación Homóloga , Neoplasias Ováricas/patología , Ftalazinas/farmacología , Piperazinas/farmacología , Recombinasa Rad51/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Neoplasias Ováricas/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteína BRCA2/genética , Daño del ADN/efectos de los fármacos , Reparación del ADN/fisiología , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Ensayos Clínicos Controlados Aleatorios como Asunto , Ubiquitina-Proteína Ligasas/genética , Regulación hacia Arriba/fisiologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Ensayos Analíticos de Alto Rendimiento , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Antineoplásicos/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Estructura Molecular , Neoplasias Ováricas/patología , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Daño del ADN/genética , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/terapia , Neoplasias Ováricas/genética , Neoplasias Ováricas/terapia , Polimorfismo de Nucleótido Simple , Proteínas de la Ataxia Telangiectasia Mutada/genética , Biomarcadores de Tumor/genética , Carcinoma Epitelial de Ovario , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa de Punto de Control 2/genética , Estudios de Cohortes , Quinasas Ciclina-Dependientes/genética , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Análisis Multivariante , Modelos de Riesgos Proporcionales , ARN Mensajero/genética , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Línea Celular Tumoral , Dioxoles/farmacología , Liposarcoma Mixoide/genética , Liposarcoma Mixoide/metabolismo , Tetrahidroisoquinolinas/farmacología , Apoptosis , Proteína C-Reactiva/genética , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/genética , Metilación de ADN , Metilasas de Modificación del ADN/deficiencia , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Reparación del ADN , Enzimas Reparadoras del ADN/deficiencia , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Resistencia a Antineoplásicos , Fibronectinas/genética , Humanos , Liposarcoma Mixoide/tratamiento farmacológico , Liposarcoma Mixoide/patología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Regiones Promotoras Genéticas , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Componente Amiloide P Sérico/genética , Transducción de Señal , Temozolomida , Trabectedina , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Rayos UltravioletaRESUMEN
The purpose of this study was to investigate retrospectively the mRNA expression of genes involved in different DNA repair pathways implicated in processing platinum-induced damage in 171 chemotherapy-naïve ovarian tumours and correlate the expression of the different genes with clinical parameters. The expression of genes involved in DNA repair pathways (PARP1, ERCC1, XPA, XPF, XPG, BRCA1, FANCA, FANCC, FANCD2, FANCF and PolEta), and in DNA damage transduction (Chk1 and Claspin) was measured by RT-PCR in 13 stage I borderline and 77 stage I and 88 III ovarian carcinomas. ERCC1, XPA, XPF and XPG genes were significantly less expressed in stage III than in stage I carcinoma; BRCA1, FANCA, FANCC, FANCD2 gene expressions were low in borderline tumours, higher in stage I carcinomas and lower in stage III samples. High levels of ERCC1, XPA, FANCC, XPG and PolEta correlated with an increase in Overall Survival (OS) and Progression Free Survival (PFS), whilst high BRCA1 levels were associated with PFS on univariate analysis. With multivariate analyses no genes retained an association when adjusted by stage, grade and residual tumour. A tendency towards a better PFS was observed in patients with the highest level of ERCC1 and BRCA1 after platinum-based therapy than those given both platinum and taxol. The expression of DNA repair genes differed in borderline stage I, stage I and stage III ovarian carcinomas. The role of DNA repair genes in predicting the response in ovarian cancer patients seems far from being established.
Asunto(s)
Reparación del ADN , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/genética , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/farmacología , Proteínas de Unión al ADN/genética , Supervivencia sin Enfermedad , Endonucleasas/genética , Femenino , Genes BRCA1 , Humanos , Persona de Mediana Edad , Análisis Multivariante , Paclitaxel/farmacología , ARN Mensajero/metabolismo , Resultado del TratamientoRESUMEN
One of the main reasons why most patients with advanced cancer are not curable with the therapies available is the broad heterogeneity of cancer cells, inherently related to their genomic instability that reflects defects of cell cycle checkpoints and DNA mismatch repair (MMR). The present paper reviews Today's knowledge of MMR. Microsatellite (DNA repetitive sequences) instability (MSI) used as a surrogate marker of MMR defects was associated with a predisposition to somatic mutations of several genes including those involved in the neoplastic transformation and tumor progression. Lynch syndrome is an autosomal dominant cancer predisposition syndrome caused by germ line mutation in genes involved in MMR such as hMLH1 or hMLH2, or less frequently hMLH6 or hPMS2; it is associated with a high risk of intestinal cancer (CRC) and other tumors including endometrial, stomach, kidney and brain. There is ample preclinical evidence that cells deficient in MMR are resistant to methylating agents and to some antimetabolites, including 5FU, which is the drug used most for the CRC, whereas they are equally sensitive to oxaliplatin and possibly more sensitive to irinotecan. More studies are needed on the importance of MMR for sensitivity to different anticancer regimens and drugs, so this knowledge can guide rational therapy according to the tumor MMR status.
Asunto(s)
Antineoplásicos/farmacología , Reparación de la Incompatibilidad de ADN , Neoplasias/genética , Progresión de la Enfermedad , Resistencia a Antineoplásicos , Predisposición Genética a la Enfermedad , Inestabilidad Genómica , Humanos , Repeticiones de Microsatélite , Neoplasias/tratamiento farmacológico , Neoplasias/patologíaRESUMEN
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.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Dioxoles/farmacología , Tetrahidroisoquinolinas/farmacología , Animales , Antineoplásicos/farmacología , Biomarcadores/metabolismo , Células CHO , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cisplatino/farmacología , Cricetinae , Cricetulus , Reparación del ADN/genética , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Histonas/efectos de los fármacos , Histonas/metabolismo , Humanos , Inmunohistoquímica , Metilmetanosulfonato/farmacología , Mutación , Fosforilación , Saccharomyces cerevisiae/genética , TrabectedinaRESUMEN
It was previously suggested that the peculiar mechanism of action of the novel anticancer drug Yondelis (ET-743, trabectedin) was due to part of the molecule, units A and B, binding to DNA in the minor groove, causing an alkylation at the N2 of guanine, while unit C protrudes out of DNA, possibly interacting with transcription factors or other DNA binding proteins. To test this hypothesis, we have compared the biological activity and the mode of action of Yondelis with its analogue ET-637, which has the same chemical structure except for the lack of the C ring. Yondelis and ET-637 showed similar cytotoxic potency and cell cycle perturbations. As already reported for Yondelis, the UV-96 cell line, deficient in ERCC-1, was less sensitive to ET-637 than the parental cell line. The binding of Yondelis or ET-637 to DNA-oligonucleotides was demonstrated by gel shift assay and SDS did not reverse the binding. Both compounds blocked the temperature-induced activation of the HSP40 promoter in the range of 1-10 nM. This study indicates that ET-637 acts similarly to Yondelis and demonstrates that the C ring of Yondelis may not be required for its biological activity.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Dioxoles/farmacología , Isoquinolinas/farmacología , Antineoplásicos Alquilantes/química , Ciclo Celular/efectos de los fármacos , ADN/metabolismo , Reparación del ADN , Dioxoles/química , Femenino , Humanos , Isoquinolinas/química , Neoplasias Ováricas/patología , Regiones Promotoras Genéticas , Temperatura , Tetrahidroisoquinolinas , Trabectedina , Células Tumorales CultivadasRESUMEN
This paper illustrates some biochemical and pharmacological properties of two natural marine products such as trabectedin (ET-743, Yondelis) and aplidine. Trabectedin has shown clinical antitumor activity in refractory soft tissue sarcoma and ovarian cancer. The lack of cross resistance of trabectedin with other chemotherapeutic drugs is presumably related to its peculiar mode of action, that seems to be related to a promoter-dependent transcription modulation. Aplidine is a potent pro-apoptotic inducer in human leukemia and has antiangiogenic properties. These examples support the view that more research should be carried out to investigate new natural marine products since there are compounds among them with antitumor properties. Some of them appear to act by mechanisms different from those of conventional chemotherapeutic drugs and thus may be effective against tumors for which no active drugs are available.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Apoptosis/efectos de los fármacos , Depsipéptidos/farmacología , Dioxoles/farmacología , Isoquinolinas/farmacología , Preparaciones Farmacéuticas/química , Animales , Antineoplásicos Alquilantes/química , Depsipéptidos/química , Dioxoles/química , Humanos , Isoquinolinas/química , Biología Marina , Péptidos Cíclicos , Sensibilidad y Especificidad , Relación Estructura-Actividad , Tetrahidroisoquinolinas , Trabectedina , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Antineoplásicos/farmacología , Cisplatino/farmacología , Proteínas de Unión al ADN , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Nucleares , Paclitaxel/farmacología , Células Tumorales Cultivadas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Androstadienos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Neoplasias del Colon/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Proteína Quinasa Activada por ADN , Femenino , Humanos , Neoplasias Ováricas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Transcripción Genética , Células Tumorales Cultivadas/efectos de los fármacos , WortmaninaRESUMEN
The cytotoxic activity of ecteinascidin 743 (ET-743), a natural product derived from the marine tunicate Ecteinascidia turbinata that exhibits potent anti-tumor activity in pre-clinical systems and promising activity in phase I and II clinical trials, was investigated in a number of cell systems with well-defined deficiencies in DNA-repair mechanisms. ET-743 binds to N2 of guanine in the minor groove, but its activity does not appear to be related to DNA-topoisomerase I poisoning as the drug is equally active in wild-type yeast and in yeast with a deletion in the DNA-topoisomerase I gene. Defects in the mismatch repair pathway, usually associated with increased resistance to methylating agents and cisplatin, did not affect the cytotoxic activity of ET-743. However, ET-743 did show decreased activity (from 2- to 8-fold) in nucleotide excision repair (NER)-deficient cell lines compared to NER-proficient cell lines, from either hamsters or humans. Restoration of NER function sensitized cells to ET-743 treatment. The DNA double-strand-break repair pathway was also investigated using human glioblastoma cell lines MO59K and MO59J, respectively, proficient and deficient in DNA-dependent protein kinase (DNA-PK). ET-743 was more effective in cells lacking DNA-PK; moreover, pre-treatment of HCT-116 colon carcinoma cells with wortmannin, a potent inhibitor of DNA-PK, sensitized cells to ET-743. An increase in ET-743 sensitivity was also observed in ataxia telangiectasia-mutated cells. Our data strongly suggest that ET-743 has a unique mechanism of interaction with DNA.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Reparación del ADN , Proteínas de Unión al ADN , Dioxoles/farmacología , Isoquinolinas/farmacología , Androstadienos/farmacología , Animales , Western Blotting , Células CHO , Camptotecina/farmacología , Supervivencia Celular , Cricetinae , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Proteína Quinasa Activada por ADN , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/metabolismo , Tetrahidroisoquinolinas , Trabectedina , Células Tumorales Cultivadas , WortmaninaRESUMEN
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.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Daunorrubicina/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Animales , Células CHO , Cricetinae , Daunorrubicina/análogos & derivados , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Concentración 50 Inhibidora , Leucemia L1210/tratamiento farmacológico , Leucemia P388/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBARESUMEN
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.
Asunto(s)
Antineoplásicos Alquilantes/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Dioxoles/uso terapéutico , Isoquinolinas/uso terapéutico , Antineoplásicos Alquilantes/farmacología , Ciclo Celular/efectos de los fármacos , División Celular , Neoplasias del Colon/patología , Ciclinas/metabolismo , Daño del ADN , ADN de Neoplasias/análisis , Dioxoles/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Isoquinolinas/farmacología , Tetrahidroisoquinolinas , Trabectedina , Células Tumorales Cultivadas/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The levels of the human checkpoint gene hCHK1 were measured in human cancer cells growing in vitro after treatment with the DNA damaging agent cis-dichlorodiammine platinum(II) (DDP). Treatment of human cancer cell lines with DDP induced a decrease in the hCHK1 protein levels starting 6 h after treatment, with a further decline at 24 and 48 h. A similar decrease in the levels of hCHK1 was found at the mRNA level by using Northern blot analysis. By using isogenic cell systems in which p53 was disrupted either by transfection with HPV-E6 or by targeted homologous recombination, we found that the DNA damage-induced down-regulation of hCHK1 was only observable in wild type p53-expressing cells, with only a minor decline in the hCHK1 levels observable 48 h after treatment in cells with disrupted p53. Similarly, treatment of mutant p53-expressing human cancer cell lines with DDP did not result in changes in the levels of hCHK1. The p53-dependent down-regulation of hCHK1 is likely to be at transcriptional levels, as suggested by the lack of down-regulation of the hCHK1 when transfected under the control of a heterologous viral promoter. In addition, p53 is able to down-regulate the luciferase activity under the control of the 5' flanking region of the hCHK1 gene. The data suggest a strict link between p53 and hCHK1 governing the activation and repression of the G(2) checkpoint in which both proteins participate.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Daño del ADN/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes cdc , Genes p53 , Regulación hacia Abajo , Humanos , Células Tumorales CultivadasRESUMEN
Alkylating agents are the most widely used anticancer drugs whose main target is the DNA, although how exactly the DNA lesions cause cell death is still not clear. The emergence of resistance to this class of drugs as well as to other antitumor agents is one of the major causes of failure of cancer treatment. This paper reviews some of the best characterized mechanisms of resistance to alkylating agents. Pre- and post-target mechanisms are recognized, the former able to limit the formation of lethal DNA adducts, and the latter enabling the cell to repair or tolerate the damage. The role in the pre-target mechanisms of reduced drug accumulation and the increased detoxification or activation systems (such as DT-diaphorase, metallothionein, GST/GSH system, etc...) are discussed. In the post-target mechanisms the different DNA repair pathways, tolerance to alkylation damage and the 'downstream' effects (cell cycle arrest and/or apoptosis) are examined.
RESUMEN
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.