RESUMEN
Chloroethylnitrosoureas (CENUs) are important chemotherapies applied in the treatment of cancer. They exert anticancer activity by inducing DNA interstrand cross-links (ICLs) via the formation of two O6-alkylguanine intermediates, O6-chloroethylguanine (O6-ClEtG) and N1,O6-ethanoguanine (N1,O6-EtG). However, O6-alkylguanine-DNA alkyltransferase (AGT), a DNA-repair enzyme, can restore the O6-alkylguanine damages and thereby obstruct the formation of ICLs (dG-dC cross-link). In this study, the inhibitory mechanism of ICL formation was investigated to elucidate the drug resistance of CENUs mediated by AGT in detail. Based on the structures of the substrate-enzyme complexes obtained from docking and MD simulations, two ONIOM (QM/MM) models with different sizes of the QM region were constructed. The model with a larger QM region, which included the substrate (O6-ClEtG or N1,O6-EtG), a water molecule, and five residues (Tyr114, Cys145, His146, Lys165, and Glu172) in the active pocket of AGT, accurately described the repairing reaction and generated the results coinciding with the experimental outcomes. The repair process consists of two sequential steps: hydrogen transfer to form a thiolate anion on Cys145 and alkyl transfer from the O6 site of guanine (the rate-limiting step). The repair of N1,O6-EtG was more favorable than that of O6-ClEtG from both kinetics and thermodynamics aspects. Moreover, the comparison of the repairing process with the formation of dG-dC cross-link and the inhibition of AGT by O6-benzylguanine (O6-BG) showed that the presence of AGT could effectively interrupt the formation of ICLs leading to drug resistance, and the inhibition of AGT by O6-BG that was energetically more favorable than the repair of O6-ClEtG could not prevent the repair of N1,O6-EtG. Therefore, it is necessary to completely eliminate AGT activity before CENUs medication to enhance the chemotherapeutic effectiveness. This work provides reasonable explanations for the supposed mechanism of AGT-mediated drug resistance of CENUs and will assist in the development of novel CENU chemotherapies and their medication strategies.
Asunto(s)
Reparación del ADN , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , O(6)-Metilguanina-ADN Metiltransferasa , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Humanos , Teoría Cuántica , Resistencia a Antineoplásicos/efectos de los fármacos , Compuestos de Nitrosourea/química , Compuestos de Nitrosourea/farmacología , Compuestos de Nitrosourea/metabolismoRESUMEN
Endometriosis is a common disease. Its pathogenesis still remains uncertain, but it is clear that cell proliferation, apoptosis and chronic inflammation play an important role in its development. This paper aimed to investigate the anti-proliferative and anti-inflammatory effects of a combined therapy with fotemustine and dexamethasone. Endometriosis was induced by intraperitoneal injections of uterine fragments from donor animals to recipient animals. Next, the pathology was allowed to develop for 7 days. On the seventh day, fotemustine was administered once and dexamethasone was administered daily for the next 7 days. On Day 14, the animals were sacrificed, and peritoneal fluids and lesions were explanted. In order to evaluate the gastrointestinal side effects of the drugs, stomachs were harvested as well. The combined therapy of fotemustine and dexamethasone reduced the proinflammatory mediator levels in the peritoneal fluid and reduced the lesions' area and diameter. In particular, fotemustine and dexamethasone administration reduced the heterogeneous development of endometrial stroma and glands (histological analysis of lesions) and hyperproliferation of endometriotic cells (immunohistochemical analysis of Ki67 and Western blot analysis of PCNA) through the mitogen-activated protein kinase (MAPK) signaling pathway. Combined fotemustine and dexamethasone therapy showed anti-inflammatory effects by inducing the synthesis of anti-inflammatory mediators at the transcriptional and post-transcriptional levels (Western blot analysis of NFκB, COX-2 and PGE2 expression). Fotemustine and dexamethasone administration had anti-apoptotic activity, restoring the impaired mechanism (TUNEL assay and Western blot analysis of Bax and Bcl-2). Moreover, no gastric disfunction was detected (histological analysis of stomachs). Thus, our data showed that the combined therapy of fotemustine and dexamethasone reduced endometriosis-induced inflammation, hyperproliferation and apoptosis resistance.
Asunto(s)
Dexametasona/farmacología , Endometriosis/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Compuestos de Nitrosourea/farmacología , Compuestos Organofosforados/farmacología , Animales , Apoptosis/efectos de los fármacos , Líquido Ascítico/metabolismo , Proliferación Celular/efectos de los fármacos , Endometriosis/complicaciones , Endometriosis/genética , Endometriosis/patología , Endometrio/efectos de los fármacos , Endometrio/patología , Femenino , Humanos , Inflamación/complicaciones , Inflamación/genética , Inflamación/patología , FN-kappa B/genética , Antígeno Nuclear de Célula en Proliferación/genética , Ratas , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/genéticaRESUMEN
One of the systems responsible for maintaining cellular redox homeostasis is the thioredoxin-dependent system. An equally important function of this system is the regulation of the expression of many proteins by the transcription factor NF-κB or the apoptosis regulating kinase (ASK-1). Since it has been shown that the Trx-dependent system can contribute to both the enhancement of tumour angiogenesis and growth as well as apoptosis of neoplastic cells, the search for compounds that inhibit the level/activity of Trx and/or TrxR and thus modulate the course of the neoplastic process is ongoing. It has been shown that many naturally occurring polyphenolic compounds inactivate elements of the thioredoxin system. In addition, the effectiveness of Trx is inhibited by imidazole derivatives, while the activity of TrxR is reduced by transition metal ions complexes, dinitrohalobenzene derivatives, Michael acceptors, nitrosourea and ebselen. In addition, research is ongoing to identify new selective Trx/TrxR inhibitors.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Reductasa de Tiorredoxina-Disulfuro/genética , Tiorredoxinas/genética , Antineoplásicos/síntesis química , Derivados del Benceno/química , Derivados del Benceno/farmacología , Inhibidores Enzimáticos/síntesis química , Regulación Neoplásica de la Expresión Génica , Homeostasis/efectos de los fármacos , Homeostasis/genética , Humanos , Imidazoles/química , Imidazoles/farmacología , Isoindoles/química , Isoindoles/farmacología , MAP Quinasa Quinasa Quinasa 5/genética , MAP Quinasa Quinasa Quinasa 5/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Neovascularización Patológica/enzimología , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Compuestos de Nitrosourea/química , Compuestos de Nitrosourea/farmacología , Compuestos de Organoselenio/química , Compuestos de Organoselenio/farmacología , Oxidación-Reducción , Transducción de Señal , Relación Estructura-Actividad , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/antagonistas & inhibidores , Tiorredoxinas/metabolismoRESUMEN
Melanoma stem cells (MSCs) are characterized by their unique cell surface proteins and aberrant signaling pathways. These stemness properties are either in a causal or consequential relationship to melanoma progression, treatment resistance and recurrence. The functional analysis of CD133+ and CD133- cells in vitro and in vivo revealed that melanoma progression and treatment resistance are the consequences of CD133 signal to PI3K pathway. CD133 signal to PI3K pathway drives two downstream pathways, the PI3K/Akt/MDM2 and the PI3K/Akt/MKP-1 pathways. Activation of PI3K/Akt/MDM2 pathway results in the destabilization of p53 protein, while the activation of PI3K/Akt/MKP-1 pathway results in the inhibition of mitogen-activated protein kinases (MAPKs) JNK and p38. Activation of both pathways leads to the inhibition of fotemustine-induced apoptosis. Thus, the disruption of CD133 signal to PI3K pathway is essential to overcome Melanoma resistance to fotemustine. The pre-clinical verification of in vitro data using xenograft mouse model of MSCs confirmed the clinical relevance of CD133 signal as a therapeutic target for melanoma treatment. In conclusion, our study provides an insight into the mechanisms regulating MSCs growth and chemo-resistance and suggested a clinically relevant approach for melanoma treatment.
Asunto(s)
Antígeno AC133/metabolismo , Melanoma/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Células Madre/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Fosfatasa 1 de Especificidad Dual/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Compuestos de Nitrosourea/farmacología , Compuestos Organofosforados/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transducción de Señal , Células Madre/efectos de los fármacos , Células Madre/patologíaRESUMEN
Glioblastoma multiforme (GBM) is a severe brain tumor whose ability to mutate and adapt to therapies is at the base for the extremely poor survival rate of patients. Despite multiple efforts to develop alternative forms of treatment, advances have been disappointing and GBM remains an arduous tumor to treat. One of the leading causes for its strong resistance is the innate upregulation of DNA repair mechanisms. Since standard therapy consists of a combinatory use of ionizing radiation and alkylating drugs, which both damage DNA, targeting the DNA damage response (DDR) is proving to be a beneficial strategy to sensitize tumor cells to treatment. In this review, we will discuss how recent progress in the availability of the DDR kinase inhibitors will be key for future therapy development. Further, we will examine the principal existing DDR inhibitors, with special focus on those currently in use for GBM clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Reparación del ADN , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glioblastoma/tratamiento farmacológico , Terapia Molecular Dirigida , Antineoplásicos/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Ensayos Clínicos Fase I como Asunto , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Cadena Simple/efectos de los fármacos , Daño del ADN , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/genética , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Proteína Quinasa Activada por ADN/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/uso terapéutico , Glioblastoma/genética , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Compuestos de Nitrosourea/farmacología , Compuestos de Nitrosourea/uso terapéutico , Transducción de Señal/efectos de los fármacos , Temozolomida/farmacología , Temozolomida/uso terapéuticoRESUMEN
Alkylating agents have been used since the 60ties in brain cancer chemotherapy. Their target is the DNA and, although the DNA of normal and cancer cells is damaged unselectively, they exert tumor-specific killing effects because of downregulation of some DNA repair activities in cancer cells. Agents exhibiting methylating properties (temozolomide, procarbazine, dacarbazine, streptozotocine) induce at least 12 different DNA lesions. These are repaired by damage reversal mechanisms involving the alkyltransferase MGMT and the alkB homologous protein ALKBH2, and through base excision repair (BER). There is a strong correlation between the MGMT expression level and therapeutic response in high-grade malignant glioma, supporting the notion that O6-methylguanine and, for nitrosoureas, O6-chloroethylguanine are the most relevant toxic damages at therapeutically relevant doses. Since MGMT has a significant impact on the outcome of anti-cancer therapy, it is a predictive marker of the effectiveness of methylating anticancer drugs, and clinical trials are underway aimed at assessing the influence of MGMT inhibition on the therapeutic success. Other DNA repair factors involved in methylating drug resistance are mismatch repair, DNA double-strand break (DSB) repair by homologous recombination (HR) and DSB signaling. Base excision repair and ALKBH2 might also contribute to alkylating drug resistance and their downregulation may have an impact on drug sensitivity notably in cells expressing a high amount of MGMT and at high doses of temozolomide, but the importance in a therapeutic setting remains to be shown. MGMT is frequently downregulated in cancer cells (up to 40% in glioblastomas), which is due to CpG promoter methylation. Astrocytoma (grade III) are frequently mutated in isocitrate dehydrogenase (IDH1). These tumors show a surprisingly good therapeutic response. IDH1 mutation has an impact on ALKBH2 activity thus influencing DNA repair. A master switch between survival and death is p53, which often retains transactivation activity (wildtype) in malignant glioma. The role of p53 in regulating survival via DNA repair and the routes of death are discussed and conclusions as to cancer therapeutic options were drawn.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Reparación del ADN/efectos de los fármacos , Compuestos de Nitrosourea/farmacología , Medicina de Precisión/métodos , Temozolomida/farmacología , Neoplasias Encefálicas/patología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Humanos , Compuestos de Nitrosourea/uso terapéutico , Temozolomida/uso terapéuticoRESUMEN
BACKGROUND: Glioblastoma multiforme (GBM) is a rare and deadly disease, with a reported average incidence rate of 3.19 cases per 100,000 inhabitants. Fotemustine, a third-generation nitrosourea with an alanine phosphor carrier that facilitates cellular penetration, has been extensively investigated in the setting of recurrent/progressive disease after initial treatment. Fotemustine is usually administered following a schedule consisting of 3 doses every week, followed by maintenance doses administered every 3 weeks. METHODS: In this phase I/II trial, we aimed to assess whether the use of a biweekly regimen allowed administration of higher dose than the standard 100âmg/m dose approved per label indication in a population of patients with recurrent GBM. In this phase I/II trial, fotemustine was administered intravenously over 1âhour every 2 weeks at either 120 or 140âmg/m doses for up to 1 year, until disease progression, unacceptable toxicity, or patient's request to withdraw from the study. The phase I part of the trial was conducted following the classic 3+3 study design. The phase II part of the trial was a single-arm study. The primary efficacy endpoint was the percentage of patients who had not progressed after 24 weeks (PFS-24). RESULTS: Thirty-seven patients were enrolled in this phase I/II trial from August 2006 to November 2011. Treatment was well tolerated in the overall population. Main severe toxicity was grades 3 and 4 thrombocytopenia, which occurred in 4 of 6 patients treated at the 140âmg/m dose level and in 3 of 31 patients treated at 120âmg/m. Median PFS and overall survival were 12.1 (1-40.2) weeks and 19.7 (1-102) weeks, respectively. CONCLUSION: We conclude that fotemustine can be safely administered at 120âmg/m biweekly. The efficacy of such modified schedule and doses should be compared to the biweekly schedule at 80âmg and the standard weekly schedule at 80 to 100âmg/m.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Compuestos de Nitrosourea/farmacología , Compuestos Organofosforados/farmacología , Trombocitopenia/inducido químicamente , Administración Intravenosa , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/epidemiología , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Dacarbazina/análogos & derivados , Dacarbazina/uso terapéutico , Supervivencia sin Enfermedad , Esquema de Medicación , Femenino , Glioblastoma/epidemiología , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/mortalidad , Compuestos de Nitrosourea/administración & dosificación , Compuestos de Nitrosourea/efectos adversos , Compuestos Organofosforados/administración & dosificación , Compuestos Organofosforados/efectos adversos , Temozolomida , Trombocitopenia/clasificaciónRESUMEN
OBJECTIVES: Bevacizumab is an anti-vascular endothelial growth factor antibody used in the treatment of recurrent glioblastoma (GBM). Despite the large number of studies carried out in patients with recurrent GBM, little is known about the administration of this angiogenesis inhibitor after the failure of the second-line chemotherapy. MATERIALS AND METHODS: In this retrospective multicenter study, on behalf of the Italian Association of Neuro-Oncology, we reported the results obtained in 51 patients with recurrent GBM treated with single-agent bevacizumab after the failure of second-line chemotherapy with fotemustine. RESULTS: In March 2016, at the time of data analysis, 3 patients (14.4%) were still alive with stable disease, whereas 48 died due to disease progression. Kaplan-Meier estimated median survival from the diagnosis of GBM was 28 months (95% confidence interval [CI], 22.1-33.9 mo). Median survival measured from the beginning of fotemustine and bevacizumab therapy were 11.3 (95% CI, 8.4-13.6 mo) and 6 months (95% CI, 3.8-8.1 mo), respectively. The 6- and 12-month progression free survival rates from the beginning of bevacizumab treatment were 18% and 13%, respectively. CONCLUSIONS: On the basis of our data, in patients with recurrent GBM, the failure of a second-line chemotherapy with cytotoxic agents might not exclude the administration of bevacizumab as third-line chemotherapy.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Bevacizumab/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Compuestos de Nitrosourea/farmacología , Compuestos Organofosforados/farmacología , Adolescente , Adulto , Anciano , Antineoplásicos/farmacología , Neoplasias Encefálicas/patología , Femenino , Estudios de Seguimiento , Glioblastoma/patología , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Pronóstico , Estudios Retrospectivos , Terapia Recuperativa , Tasa de Supervivencia , Adulto JovenRESUMEN
The toxic effects of combined cisplatin/docetaxel therapy cycles on erythroid and granulocytic hematopoietic lineages as well as their intercycle recovery were examined in patients with stage III-IV non-small-cell lung carcinoma. Responsiveness of the blood system to this therapy remained at a high level. Combined therapy pronouncedly activated the key elements of the erythroid and granulocytic hematopoietic lineages leading to accumulation of immature and mature myelokaryocytes in the bone marrow, enlargement of the medullary pool of mature neutrophils, and increase in the count of medullary erythroid and granulocytic precursor cells under conditions of their accelerated maturation.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Granulocitos/metabolismo , Neoplasias Pulmonares/metabolismo , Antraciclinas/farmacología , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Cisplatino/farmacología , Disacáridos/farmacología , Docetaxel , Doxorrubicina/farmacología , Eritropoyesis/efectos de los fármacos , Granulocitos/citología , Granulocitos/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Macrólidos/farmacología , Compuestos de Nitrosourea/farmacología , Compuestos Organoplatinos/farmacología , Taxoides/farmacologíaRESUMEN
Chloroethylating nitrosoureas (CNU), such as lomustine, nimustine, semustine, carmustine and fotemustine are used for the treatment of malignant gliomas, brain metastases of different origin, melanomas and Hodgkin disease. They alkylate the DNA bases and give rise to the formation of monoadducts and subsequently interstrand crosslinks (ICL). ICL are critical cytotoxic DNA lesions that link the DNA strands covalently and block DNA replication and transcription. As a result, S phase progression is inhibited and cells are triggered to undergo apoptosis and necrosis, which both contribute to the effectiveness of CNU-based cancer therapy. However, tumor cells resist chemotherapy through the repair of CNU-induced DNA damage. The suicide enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes the precursor DNA lesion O6-chloroethylguanine prior to its conversion into ICL. In cells lacking MGMT, the formed ICL evoke complex enzymatic networks to accomplish their removal. Here we discuss the mechanism of ICL repair as a survival strategy of healthy and cancer cells and DNA damage signaling as a mechanism contributing to CNU-induced cell death. We also discuss therapeutic implications and strategies based on sequential and simultaneous treatment with CNU and the methylating drug temozolomide.
Asunto(s)
Muerte Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Compuestos de Nitrosourea/farmacología , Compuestos de Nitrosourea/uso terapéutico , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Replicación del ADN/efectos de los fármacos , HumanosRESUMEN
BACKGROUND: The combination of stem cell-based gene therapy with chemotherapy comprises an advantageous strategy that results in a reduction of system toxicity effects and an improvement in the general efficacy of treatment. In the present study, we estimated the efficacy of adipose tissue-derived mesenchymal stem cells (AT-MSCs) expressing cytosine deaminase (CDA) combined with lysomustine chemotherapy in mice bearing late stage Lewis lung carcinoma (LLC). METHODS: Adipose tissue-derived mesenchymal stem cells were transfected with non-insert plasmid construct transiently expressing fused cytosine deaminase-uracil phosphoribosyltransferase protein (CDA/UPRT) or the same construct fused with Herpes Simplex Virus Type1 tegument protein VP22 (CDA/UPRT/VP22). Systemic administration of 5-fluorocytosine (5FC) and lysomustine was implemented after a single intratumoral injection of transfected AT-MSCs. RESULTS: We demonstrated that direct intratumoral transplantation of AT-MSCs expressing CDA/UPRT or CDA/UPRT/VP22 followed by systemic administration of 5FC resulted in a significant tumor growth inhibition. There was a 56% reduction in tumor volume in mice treated by AT-MSCs-CDA/UPRT + 5FC or with AT-MSCs-CDA/UPRT/VP22 + 5FC compared to control animals grafted with lung carcinoma alone. Transplantation of AT-MSCs-CDA/UPRT + 5FC and AT-MSCs-CDA/UPRT/VP22 + 5FC prolonged the life span of mice bearing LLC by 27% and 31%, respectively. Co-administration of lysomustine and AT-MSCs-CDA/UPRT + 5FC led to tumor growth inhibition (by 86%) and life span extension (by 60%) compared to the control group. CONCLUSIONS: Our data indicate that a combination CDA/UPRT-expressing AT-MSCs with lysomustine has a superior antitumor effect in the murine lung carcinoma model compared to monotherapies with transfected AT-MSCs or lysomustine alone, possibly because of a synergistic effect of the combination therapy. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Carcinoma Pulmonar de Lewis/terapia , Citosina Desaminasa/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Compuestos de Nitrosourea/farmacología , Tejido Adiposo/citología , Animales , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patología , Línea Celular Tumoral , Terapia Combinada , Citosina Desaminasa/genética , Femenino , Flucitosina/administración & dosificación , Flucitosina/farmacología , Terapia Genética/métodos , Masculino , Ratones Endogámicos C57BL , Compuestos de Nitrosourea/administración & dosificación , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Análisis de Supervivencia , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genéticaRESUMEN
The drug resistance of CENUs induced by O(6)-alkylguanine-DNA alkyltransferase (AGT), which repairs the O(6)-alkylated guanine and subsequently inhibits the formation of dG-dC cross-links, hinders the application of CENU chemotherapies. Therefore, the discovery of CENU analogs with AGT inhibiting activity is a promising approach leading to novel CENU chemotherapies with high therapeutic index. In this study, a new combi-nitrosourea prodrug 3-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-1-(2-chloroethyl)-1-nitrosourea (6), designed to release a DNA cross-linking agent and an inhibitor of AGT, was synthesized and evaluated for its antitumor activity and ability to induce DNA interstrand cross-links (ICLs). The results indicated that 6 exhibited higher cytotoxicity against mer(+) glioma cells compared with ACNU, BCNU, and their respective combinations with O(6)-benzylguanine (O(6)-BG). Quantifications of dG-dC cross-links induced by 6 were performed using HPLC-ESI-MS/MS. Higher levels of dG-dC cross-link were observed in 6-treated human glioma SF763 cells (mer(+)), whereas lower levels of dG-dC cross-link were observed in 6-treated calf thymus DNA, when compared with the groups treated with BCNU and ACNU. The results suggested that the superiority of 6 might result from the AGT inhibitory moiety, which specifically functions in cells with AGT activity. Molecular docking studies indicated that five hydrogen bonds were formed between the O(6)-BG analogs released from 6 and the five residues in the active pocket of AGT, which provided a reasonable explanation for the higher AGT-inhibitory activity of 6 than O(6)-BG.
Asunto(s)
Antineoplásicos/farmacología , Reactivos de Enlaces Cruzados/química , Inhibidores Enzimáticos/farmacología , Compuestos de Nitrosourea/farmacología , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Profármacos/farmacología , HumanosRESUMEN
PURPOSE: Uveal melanoma (UM) is associated with a high risk of metastases and lack of efficient therapies. Reduced capacity for apoptosis induction by chemotherapies is one obstacle to efficient treatments. Human UM is characterized by high expression of the anti-apoptotic protein Bcl-2. Consequently, regulators of apoptosis such as Bcl-2 family inhibitors may constitute an attractive approach to UM therapeutics. In this aim, we have investigated the efficacy of the Bcl-2/Bcl-XL inhibitor S44563 on 4 UM Patient-Derived Xenografts (PDXs) and derived-cell lines. EXPERIMENTAL DESIGN: Four well characterized UM PDXs were used for in vivo experiments. S44563 was administered alone or combined with fotemustine either concomitantly or after the alkylating agent. Bcl-2, Bcl-XL, and Mcl-1 expressions after S44563 administration were evaluated by immunohistochemistry (IHC). RESULTS: S44563 administered alone by at 50 and 100 mg/kg i.p. induced a significant tumour growth inhibition in only one xenograft model with a clear dose effect. However, when S44563 was concomitantly administered with fotemustine, we observed a synergistic activity in 3 out of the 4 tested models. In addition, S44563 administered after fotemustine induced a tumour growth delay in 2 out of 3 tested xenografts. Finally, IHC analyses showed that Bcl-2, Bcl-XL, and Mcl-1 expression were not modified after S44563 administration. CONCLUSION: The novel anti-apoptotic experimental compound S44563, despite a relative low efficacy when administered alone, increased the efficacy of fotemustine in either concomitant or sequential combinations or indeed subsequent to fotemustine. These data support further exploration of potential therapeutic effect of Bcl-2/Bcl-xl inhibition in human UM.
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Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Compuestos Heterocíclicos con 3 Anillos/farmacología , Compuestos Heterocíclicos con 3 Anillos/uso terapéutico , Melanoma/tratamiento farmacológico , Terapia Molecular Dirigida , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Neoplasias de la Úvea/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Polarización de Fluorescencia , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Humanos , Inmunohistoquímica , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones SCID , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Compuestos de Nitrosourea/farmacología , Compuestos de Nitrosourea/uso terapéutico , Compuestos Organofosforados/farmacología , Compuestos Organofosforados/uso terapéutico , Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Sulfonamidas/administración & dosificación , Análisis de Supervivencia , Neoplasias de la Úvea/metabolismo , Neoplasias de la Úvea/patología , Proteína bcl-X/metabolismoRESUMEN
In the treatment of metastatic melanoma, a highly therapy-refractory cancer, alkylating agents are used and, for the subgroup of BRAFV600E cancers, the B-Raf inhibitor vemurafenib. Although vemurafenib is initially beneficial, development of drug resistance occurs leading to tumor relapse, which necessitates the requirement for combined or sequential therapy with other drugs, including genotoxic alkylating agents. This leads to the question whether vemurafenib and alkylating agents act synergistically and whether chronic vemurafenib treatment alters the melanoma cell response to alkylating agents. Here we show that a) BRAFV600E melanoma cells are killed by vemurafenib, driving apoptosis, b) BRAFV600E melanoma cells are neither more resistant nor sensitive to temozolomide/fotemustine than non-mutant cells, c) combined treatment with vemurafenib plus temozolomide or fotemustine has an additive effect on cell kill, d) acquired vemurafenib resistance of BRAFV600E melanoma cells does not affect MGMT, MSH2, MSH6, PMS2 and MLH1, nor does it affect the resistance to temozolomide and fotemustine, e) metastatic melanoma biopsies obtained from patients prior to and after vemurafenib treatment did not show a change in the MGMT promoter methylation status and MGMT expression level. The data suggest that consecutive treatment with vemurafenib and alkylating drugs is a reasonable strategy for metastatic melanoma treatment.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Melanoma/tratamiento farmacológico , Línea Celular Tumoral , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Sinergismo Farmacológico , Humanos , Indoles/administración & dosificación , Indoles/farmacología , Melanoma/patología , Recurrencia Local de Neoplasia/tratamiento farmacológico , Compuestos de Nitrosourea/administración & dosificación , Compuestos de Nitrosourea/farmacología , Compuestos Organofosforados/administración & dosificación , Compuestos Organofosforados/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Sulfonamidas/administración & dosificación , Sulfonamidas/farmacología , Temozolomida , VemurafenibRESUMEN
Selection for targeted therapies in melanoma is currently based on the search for mutations in selected genes. We aimed at evaluating the interest of signalling and chemosensitivity studies in addition to genotyping for assessing the best suitable treatment in an individual patient. We extracted genomic DNA and melanoma cells from tumor tissue of a skin metastasis of a 17-year-old woman with stage IV melanoma progressing despite three successive lines of treatment. Despite the absence of mutation in BRAF, NRAS cKIT, the MAPK pathway was activated and a significant response to sorafenib, a mitogen-activated protein kinase (MAPK)/RAF inhibitor, was found in signalling and chemosensitivity assays. A treatment combining sorafenib and dacarbazine produced a partial response for 9 months, with marked necrosis in some lesions. Chemosensitivity assays and signalling pathway studies could be of great value in addition to genotyping for assessing the most appropriate treatment in melanoma.
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Antineoplásicos/uso terapéutico , Melanoma/tratamiento farmacológico , Terapia Molecular Dirigida/métodos , Mutación/genética , Medicina de Precisión/métodos , Transducción de Señal/efectos de los fármacos , Adolescente , Antineoplásicos/farmacología , Bencenosulfonatos/farmacología , Bencenosulfonatos/uso terapéutico , Butadienos/farmacología , Supervivencia Celular/efectos de los fármacos , Dacarbazina/farmacología , Dacarbazina/uso terapéutico , Quimioterapia Combinada/métodos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Resultado Fatal , Femenino , Genes ras/genética , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Niacinamida/análogos & derivados , Nitrilos/farmacología , Compuestos de Nitrosourea/farmacología , Compuestos Organofosforados/farmacología , Compuestos de Fenilurea , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-kit/genética , Piridinas/farmacología , Piridinas/uso terapéutico , Sorafenib , Resultado del TratamientoRESUMEN
The paper covers investigation of cytogenetic activity of chiral mutagens and their specific effects on the plant cells chromosomes of soft winter wheat (Triticum aestivum L.). Comparative analysis of cytogenetic activity of chiral NEU: S(+)1-N-nitroso- 1-N-methyl-3-N-sec-buthylureas (S(+)NMsBU) and R(-)1-N-nitroso- 1N-methyl-3-Nsec-buthylureas (R(-)NMsBU) on winter wheat was performed. As it was shown by the frequency of chromosomal aberrations the S(+) stereoisomer was twice more active than R(-). In addition to typical anaphase aberrations (fragments, bridges, lagging chromosomes) the numerous mitosis pathologies were revealed - K-mitoses, hyperspiralization and despiralization of chromosomes, unequal allocation of chromosomes between the daughter nuclei, mass fragmentation, nondisjunction and chromosome adhesion, three-pole mitoses, etc. Neither of the mentioned pathologies was observed under the action of NEU and gamma-rays.
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Núcleo Celular/efectos de los fármacos , Aberraciones Cromosómicas/efectos de los fármacos , Cromosomas de las Plantas/efectos de los fármacos , Mitosis/efectos de los fármacos , Mutágenos/farmacología , Compuestos de Nitrosourea/farmacología , Triticum , Núcleo Celular/genética , Núcleo Celular/efectos de la radiación , Núcleo Celular/ultraestructura , Aberraciones Cromosómicas/efectos de la radiación , Cromosomas de las Plantas/efectos de la radiación , Cromosomas de las Plantas/ultraestructura , Rayos gamma , Mitosis/efectos de la radiación , Mutágenos/síntesis química , Compuestos de Nitrosourea/síntesis química , Estereoisomerismo , Triticum/citología , Triticum/efectos de los fármacos , Triticum/genética , Triticum/efectos de la radiaciónAsunto(s)
Antineoplásicos/farmacología , Neoplasias de la Conjuntiva/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Línea Celular Tumoral , Neoplasias de la Conjuntiva/patología , Humanos , Melanoma/patología , Mitomicina/farmacología , Compuestos de Nitrosourea/farmacología , Compuestos Organofosforados/farmacología , Tretinoina/farmacologíaRESUMEN
Nephronophthisis (NPHP) is an autosomal recessive kidney disease that is often associated with vision and/or brain defects. To date, 11 genes are known to cause NPHP. The gene products, while structurally unrelated, all localize to cilia or centrosomes. Although mouse models of NPHP are available for 9 of the 11 genes, none has been described for nephronophthisis 4 (Nphp4). Here we report a novel, chemically induced mutant, nmf192, that bears a nonsense mutation in exon 4 of Nphp4. Homozygous mutant Nphp4(nmf192/nmf192) mice do not exhibit renal defects, phenotypes observed in human patients bearing mutations in NPHP4, but they do develop severe photoreceptor degeneration and extinguished rod and cone ERG responses by 9 weeks of age. Photoreceptor outer segments (OS) fail to develop properly, and some OS markers mislocalize to the inner segments and outer nuclear layer in the Nphp4(nmf192/nmf192) mutant retina. Despite NPHP4 localization to the transition zone in the connecting cilia (CC), the CC appear to be normal in structure and ciliary transport function is partially retained. Likewise, synaptic ribbons develop normally but then rapidly degenerate by P14. Finally, Nphp4(nmf192/nmf192) male mutants are sterile and show reduced sperm motility and epididymal sperm counts. Although Nphp4(nmf192/nmf192) mice fail to recapitulate the kidney phenotype of NPHP, they will provide a valuable tool to further elucidate how NPHP4 functions in the retina and male reproductive organs.
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Células Fotorreceptoras de Vertebrados/fisiología , Células Fotorreceptoras de Vertebrados/ultraestructura , Proteínas/genética , Proteínas/fisiología , Degeneración Retiniana/genética , Maduración del Esperma/genética , Sinapsis/fisiología , Sinapsis/ultraestructura , Animales , Western Blotting , Mapeo Cromosómico , Cilios/metabolismo , Codón sin Sentido , Electrorretinografía , Infertilidad Masculina/genética , Enfermedades Renales Quísticas/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Mutación , Compuestos de Nitrosourea/farmacología , Fenotipo , Retina/anomalías , Análisis de Secuencia de ADN , Motilidad EspermáticaRESUMEN
Molecular chaperones and co-chaperones are crucial for cellular development and maintenance as they assist in protein folding and stabilization of unfolded or misfolded proteins. Prefoldin (PFDN), a ubiquitously expressed heterohexameric co-chaperone, is necessary for proper folding of nascent proteins, in particular, tubulin and actin. Here we show that a genetic disruption in the murine Pfdn5 gene, a subunit of prefoldin, causes a syndrome characterized by photoreceptor degeneration, central nervous system abnormalities, and male infertility. Our data indicate that a missense mutation in Pfdn5, may cause these phenotypes through a reduction in formation of microtubules and microfilaments, which are necessary for the development of cilia and cytoskeletal structures, respectively. The diversity of phenotypes demonstrated by models carrying mutations in different PFDN subunits suggests that each PFDN subunit must confer a distinct substrate specificity to the prefoldin holocomplex.
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Modelos Animales , Chaperonas Moleculares/metabolismo , Células Receptoras Sensoriales/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animales , Secuencia de Bases , Proliferación Celular , Supervivencia Celular/genética , Sistema Nervioso Central/anomalías , Sistema Nervioso Central/metabolismo , Femenino , Humanos , Hipogonadismo/genética , Masculino , Ratones , Microtúbulos/genética , Microtúbulos/metabolismo , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutagénesis , Mutación Missense/efectos de los fármacos , Compuestos de Nitrosourea/farmacología , Mutación Puntual/efectos de los fármacos , Conformación Proteica , Segmento Interno de las Células Fotorreceptoras Retinianas/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Saccharomyces cerevisiae/citología , Células Receptoras Sensoriales/citologíaRESUMEN
N epsilon-Nitroso-N epsilon- [N'-(2-chloroethyl)carbamoyl]-L-lysine (I) and N epsilon- [N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-L-lysine (II), the isomers being the constituents of antitumor agent Lysomustine, were obtained by RFHPLC. The study of cytotoxicity of the above compounds against K562 cells showed that the lesions induced by isomer (II) produce a significant cytotoxic effect but can be efficiently repaired by the action of MGMT (O6-methylaguanine DNA methyltransferase). Under similar conditions, the lesions induced by isomer (I) produce substantially smaller effect but are weakly if at all repairable by MGMT. The effects of a clinically approved agent Lysomustine, which is the mixture of isomers (I) and (II), are similar to those of isomer (II). The results obtained point to a different chemical nature of DNA lesions induced by two Lysomustine isomers. Our data indicate that Lysomustine and its isomer (II) can be used for in vitro selection of cells expressing MGMT.