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BACKGROUND: Current treatments have a modest impact on survival of pancreatic cancer patients and this study investigates the interaction between sorafenib and gemcitabine and the molecular pharmacodynamics of this combination. METHODS: The pancreatic cancer cells were treated with sorafenib and gemcitabine, alone or in combination. The effects of treatments were evaluated on cell proliferation, cell cycle, apoptosis, phosphorylation of Akt, c-Kit, ERK and VEGFR2, and expression of genes related to drug activity. RESULTS: Gemcitabine and sorafenib synergistically interacted on the inhibition of cell proliferation, and assessment of apoptosis demonstrated that drug associations increased the apoptotic index. Sorafenib reduced c-Kit, ERK and VEGFR2 activation and on the other hand, gemcitabine inhibited Akt phosphorylation. Moreover, quantitative PCR showed that sorafenib modulated the expression of genes related to gemcitabine activity, while gemcitabine induced the expression of RKIP. CONCLUSION: These data demonstrate that gemcitabine and sorafenib combination displays a synergistic effect in pancreatic cancer cells.
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Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Bencenosulfonatos/uso terapéutico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Piridinas/uso terapéutico , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Bencenosulfonatos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Sinergismo Farmacológico , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Terapia Molecular Dirigida , Niacinamida/análogos & derivados , Neoplasias Pancreáticas/patología , Compuestos de Fenilurea , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridinas/farmacología , Transducción de Señal/efectos de los fármacos , Sorafenib , GemcitabinaRESUMEN
PURPOSE: Selecting patients according to key genetic characteristics may help to tailor chemotherapy and optimize the treatment in non-small cell lung cancer (NSCLC). Polymorphisms at the xeroderma pigmentosum group D (XPD), excision repair cross-complementing 1 (ERCC1), and cytidine deaminase (CDA) genes have been associated with alterations in enzymatic activity and may change sensitivity to the widely used cisplatin-gemcitabine regimen. EXPERIMENTAL DESIGN: Analyses of CDA, XPD, and ERCC1 polymorphisms were done on blood samples of 65 chemotherapy-naïve, advanced NSCLC patients treated with cisplatin-gemcitabine. Furthermore, CDA enzymatic activity was evaluated by high-performance liquid chromatography analysis. Association between XPD Asp(312)Asn and Lys(751)Gln, ERCC1 C118T, and CDA Lys(27)Gln polymorphisms and response, clinical benefit, toxicity, time to progression (TTP), and overall survival (OS) was estimated using Pearson's chi(2) tests, the Kaplan-Meier method, the log-rank test, and the Cox proportional hazards model. RESULTS: The CDA Lys(27)Lys polymorphism significantly correlated with better clinical benefit (P = 0.04) and grade > or =3 neutropenia and thrombocytopenia, as well as with longer TTP and OS (P = 0.006 and P = 0.002, respectively), whereas no significant associations were found among ERCC1 and XPD polymorphisms and both response and clinical outcome. Finally, the enzymatic activity assay showed a significant lower mean in subjects harboring the CDA Lys(27)Lys polymorphism. CONCLUSIONS: Our data suggested the role of CDA Lys(27)Lys polymorphism as a possible predictive marker of activity, toxicity, TTP, and OS in advanced NSCLC patients treated with cisplatin and gemcitabine. These results may be explained by the lower enzymatic activity associated with the Lys(27)Lys CDA and offer a potential new tool for treatment optimization.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Citidina Desaminasa/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/mortalidad , Polimorfismo de Nucleótido Simple , Proteína de la Xerodermia Pigmentosa del Grupo D/genética , Adulto , Anciano , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Cisplatino/administración & dosificación , Citidina Desaminasa/metabolismo , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Progresión de la Enfermedad , Femenino , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Análisis de Supervivencia , Resultado del Tratamiento , GemcitabinaRESUMEN
A key focus of research on pancreatic ductal adenocarcinoma (PDAC) is identifying new techniques to tailor gemcitabine and 5-fluorouracil treatments. Availability of tumor tissue is critical for the accurate assessment of gene expression, and laser microdissection (LMD) and primary cell cultures may be useful tools to separate tumor cells from the stromal reaction. The aim of this study was (1) to address the genetic profile relevant to drug activity and (2) to evaluate differences between microdissected and non-microdissected tumors, normal tissues, and primary cell cultures. Quantitative PCR of seven key genes was performed on mRNA from 113 microdissected and 28 non-microdissected tumors, a pool of normal tissues and four established primary cell lines. Protein expression was evaluated by western blot and immunocytochemistry and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. LMD allowed the analysis of 110 samples and revealed significant differences in mRNA levels between microdissected tumors and normal tissues, as well as between non-microdissected and microdissected tumors from the same patients. In contrast, primary cell lines showed similar expression profiles with respect to their respective microdissected tumors. In particular, expression levels of human equilibrative nucleoside transporter-1 and thymydilate synthase were significantly related to gemcitabine and 5-fluorouracil cytotoxicity. We conclude that LMD is a reliable technique for mRNA extraction, and allows detection of significant differences in the expression of specific target genes when compared to non-microdissected specimens and normal tissues. Moreover, expression levels in microdissected tumors are similar to those observed in primary tumor cell cultures, both at mRNA and protein level, and are related to drug chemosensitivity. The use of these ex vivo techniques for molecular analysis of tumors therefore appears to be of some value in implementing the clinical management of PDAC.
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Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/metabolismo , Rayos Láser , Microdisección/métodos , Neoplasias Pancreáticas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/patología , Células Cultivadas , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Femenino , Fluorouracilo/farmacología , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Pancreáticas/patología , ARN Mensajero/biosíntesis , ARN Mensajero/aislamiento & purificación , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , GemcitabinaRESUMEN
Gene expression analysis may help the management of cancer patients, allowing the selection of subjects responding to treatment. The aim of this study was the characterization of expression pattern of genes involved in gemcitabine activity in pancreas tumor specimens and its correlation with treatment outcome. The role of drug transport and metabolism on gemcitabine cytotoxicity was examined with specific inhibitors, whereas transcription analysis of human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-NT), cytidine deaminase (CDA), and ribonucleotide reductase subunits M1 and M2 (RRM1 and RRM2) was done by quantitative reverse transcription-PCR in tumor tissue isolated by laser microdissection from surgical or biopsy samples of 102 patients. Association between clinical outcome and gene expression levels was estimated using Kaplan-Meier method and Cox's proportional hazards model. Transport and metabolism had a key role on gemcitabine sensitivity in vitro; moreover, hENT1, dCK, 5'-NT, CDA, RRM1, and RRM2 were detectable in most tumor specimens. hENT1 expression was significantly correlated with clinical outcome. Patients with high levels of hENT1 had a significantly longer overall survival [median, 25.7; 95% confidence interval (95% CI), 17.6-33.7 months in the higher expression tertile versus median, 8.5; 95% CI, 7.0-9.9 months in the lower expression tertile]. Similar results were obtained with disease-free survival and time to disease progression, and the multivariate analysis confirmed the prognostic significance of hENT1. This study suggests that the expression levels of hENT1 may allow the stratification of patients based on their likelihood of survival, thus offering a potential new tool for treatment optimization.
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Desoxicitidina/análogos & derivados , Tranportador Equilibrativo 1 de Nucleósido/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , 5'-Nucleotidasa/biosíntesis , 5'-Nucleotidasa/genética , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Citidina Desaminasa/biosíntesis , Citidina Desaminasa/genética , Desoxicitidina/uso terapéutico , Desoxicitidina Quinasa/biosíntesis , Desoxicitidina Quinasa/genética , Tranportador Equilibrativo 1 de Nucleósido/biosíntesis , Femenino , Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/metabolismo , Valor Predictivo de las Pruebas , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleósido Difosfato Reductasa/biosíntesis , Ribonucleósido Difosfato Reductasa/genética , Activación Transcripcional , Resultado del Tratamiento , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/genética , GemcitabinaRESUMEN
INTRODUCTION: Gemcitabine (2',2'-difluorodeoxycytidine) (GEM) has been demonstrated to be active in the salvage setting of ovarian cancer (OC) patients. CASE REPORT: A 57-year-old woman, with a diagnosis of FIGO Stage IIIC clear cell OC, judged inoperable to optimal residual tumor, was administered neo-adjuvant chemotherapy with carboplatin/paclitaxel/topotecan, and cytoreduction. After 5 months the patient progressed, and pegylated liposomal doxorubicin was started with rapid progression. GEM (1,000 mg/m2, d1,8,15, q28) was then started, and a complete clinical response was documented after seven cycles of treatment, and was maintained for 9 months; at progression fourth line carboplatin was attempted but 1 month after the last course of carboplatin, progression occurred, and the patient was re-challenged with GEM obtaining a partial response, of 6 months duration. Currently, the patient is still under treatment, without any complaints relative to her quality of life/specific symptoms. CONCLUSION: We described the case of a drug resistant clear cell ovarian cancer showing a selective susceptibility only to GEM at first administration and at re-challenge. Moreover, this case expressed a molecular profile very likely to support high tumour cell sensitivity to GEM.
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Antimetabolitos Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Neoplasias Ováricas/tratamiento farmacológico , Antimetabolitos Antineoplásicos/administración & dosificación , Desoxicitidina/administración & dosificación , Desoxicitidina/uso terapéutico , Esquema de Medicación , Resistencia a Antineoplásicos , Femenino , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/patología , Resultado del Tratamiento , GemcitabinaRESUMEN
PURPOSE: Standard treatments have modest effect against pancreatic cancer, and current research focuses on agents targeting molecular pathways involved in tumor growth and angiogenesis. This study investigated the interactions between ZD6474, an inhibitor of tyrosine kinase activities of vascular endothelial growth factor receptor-2 and epidermal growth factor receptor (EGFR), gemcitabine, and ionizing radiation in human pancreatic cancer cells and analyzed the molecular mechanisms underlying this combination. EXPERIMENTAL DESIGN: ZD6474, ionizing radiation, and gemcitabine, alone or in combination, were given in vitro to MIA PaCa-2, PANC-1, and Capan-1 cells and in vivo to MIA PaCa-2 tumor xenografts. The effects of treatments were studied by the evaluation of cytotoxicity, apoptosis, cell cycle, EGFR and Akt phosphorylation, modulation of gene expression of enzymes related to gemcitabine activity (deoxycytidine kinase and ribonucleotide reductase), as well as vascular endothelial growth factor immunohistochemistry and microvessel count. RESULTS: In vitro, ZD6474 dose dependently inhibited cell growth, induced apoptosis, and synergistically enhanced the cytotoxic activity of gemcitabine and ionizing radiation. Moreover, ZD6474 inhibited phosphorylation of EGFR and Akt and triggered cell apoptosis. PCR analysis showed that ZD6474 increased the ratio between gene expression of deoxycytidine kinase and ribonucleotide reductase. In vivo, ZD6474 showed significant antitumor activity alone and in combination with radiotherapy and gemcitabine, and the combination of all three modalities enhanced MIA PaCA-2 tumor growth inhibition compared with gemcitabine alone. CONCLUSIONS: ZD6474 decreases EGFR and Akt phosphorylation, enhances apoptosis, favorably modulates gene expression in cancer cells, and acts synergistically with gemcitabine and radiotherapy to inhibit tumor growth. These findings support the investigation of this combination in the clinical setting.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Desoxicitidina/análogos & derivados , Receptores ErbB/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/radioterapia , Piperidinas/farmacología , Quinazolinas/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Terapia Combinada , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Desoxicitidina Quinasa/genética , Desoxicitidina Quinasa/efectos de la radiación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta en la Radiación , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Neoplasias Pancreáticas/diagnóstico , Fosforilación , Piperidinas/uso terapéutico , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Quinazolinas/uso terapéutico , Radiación Ionizante , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal/efectos de los fármacos , Trasplante Heterólogo , GemcitabinaRESUMEN
Chemotherapy has produced unsatisfactory results in pancreas cancer and novel approaches, including treatment tailoring by pharmacogenetic analysis and new molecular-targeted drugs, are required. The scarcity of effective therapies may reflect the lack of knowledge about the influence of tumor-related molecular abnormalities on responsiveness to drugs. Advances in the understanding of pancreas cancer biology have been made over the past decade, including the discovery of critical mutations in oncogenes (i.e., K-Ras) as well as the loss of tumor suppressor genes, such as TP53 and p16(INK4). Other studies showed the dysregulation of the expression of proteins involved in the control of cell cycle, proliferation, apoptosis, and invasiveness, such as Bcl-2, Akt, mdm2, and epidermal growth factor receptor. These characteristics might contribute to the aggressive behavior of pancreatic cancer and influence response to treatment. Indeed, the inactivation of p53 may explain the relative resistance to 5-fluorouracil, whereas Bcl-2 overexpression is associated with reduced sensitivity to gemcitabine. However, the future challenge of pancreas cancer chemotherapy relies on the identification of molecular markers that help in the selection of drugs best suited to the individual patient. Recent pharmacogenetic studies focused on genes encoding proteins directly involved in drug activity, showing the role of thymidylate synthase and human equilibrative nucleoside transporter-1 as prognostic factor in 5-fluorouracil- and gemcitabine-treated patients, respectively. Finally, inhibitors of signal transduction and angiogenesis are under extensive investigation, and several prospective trials have been devoted to this area. Pharmacogenetics is likely to play a central role in the personalization of treatment, to stratify patients based on their likelihood of response to both standard agents (i.e., gemcitabine/nucleoside transporters) and targeted treatments (i.e., epidermal growth factor receptor gene mutations and/or amplification and tyrosine kinase inhibitors), Thus, molecular analysis should be implemented in the optimal management of the patient affected by pancreatic adenocarcinoma.
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Antineoplásicos/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Diseño de Fármacos , Humanos , FarmacogenéticaRESUMEN
PURPOSE: Gemcitabine is an inhibitor of ribonucleotide reductase (RR) and DNA synthesis and is an effective agent in the treatment of pancreas cancer. The present study investigates whether the multitargeted antifolate pemetrexed would be synergistic with gemcitabine against MIA PaCa-2, PANC-1, and Capan-1 pancreatic cancer cell lines. EXPERIMENTAL DESIGN: Cells were treated with gemcitabine and pemetrexed, and the type of drug interaction was assessed using the combination index. Cytotoxicity of gemcitabine was examined with inhibitors of (a) deoxycytidine kinase (dCK), which activates gemcitabine by phosphorylation, and (b) 5'-nucleotidase (drug dephosphorylation) and cytidine deaminase (drug deamination), the main inactivating enzymes. The effects of gemcitabine and pemetrexed on cell cycle were analyzed by flow cytometry, and apoptosis was examined by fluorescence microscopy. Finally, quantitative, real-time PCR was used to study the pharmacogenetics of the drug combination. RESULTS: Synergistic cytotoxicity and enhancement of apoptosis was demonstrated, mostly with the sequence pemetrexed-->gemcitabine. Pemetrexed increased cells in S phase, the most sensitive to gemcitabine, and a positive correlation was found between the expression ratio of dCK:RR and gemcitabine sensitivity. Indeed, pemetrexed significantly enhanced dCK gene expression (+227.9, +86.0, and +135.5% in MIA PaCa-2, PANC-1, and Capan-1 cells, respectively), and the crucial role of this enzyme was confirmed by impairment of gemcitabine cytotoxicity after dCK saturation with 2'-deoxycytidine. CONCLUSIONS: These data demonstrate that the gemcitabine and pemetrexed combination displays schedule-dependent synergistic cytotoxic activity, favorably modulates cell cycle, induces apoptosis, and enhances dCK expression in pancreatic cancer cells.
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Antimetabolitos Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Glutamatos/farmacología , Guanina/análogos & derivados , Guanina/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Desoxicitidina/metabolismo , Desoxicitidina Quinasa/genética , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Pemetrexed , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/genética , GemcitabinaRESUMEN
BACKGROUND: Radiotherapy (RT) with or without chemotherapy (CT) plays an important role as exclusive treatment in patients with head and neck squamous cell cancer (HNSCC). Unfortunately, in some cases, benefit for patients is not recorded and only treatment-related complications are registered. MATERIALS AND METHODS: Data relating to Akt1 single nucleotide polymorphism (SNP) and response to treatment of 46 patients treated with exclusive RT or RT-CT for HNSCC were evaluated. RESULTS: For heterozygous patients median overall survival was 28.5 months, while for the wild-type group median overall survival was 10.9 (p=0.019). Three-year survival was 85% for mutated Akt1 homozygosis and 40% for patients with a heterozygous status (p=0.019, hazard ratio (HR)=7.960). CONCLUSION: SNP of rs2498804 can recognize patients resistant to RT-CT. Further studies are needed to confirm our data and to investigate the role of Akt SNPs in HNSCC patients.
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Carcinoma de Células Escamosas/genética , Neoplasias de Cabeza y Cuello/genética , Proteínas Proto-Oncogénicas c-akt/genética , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/radioterapia , Femenino , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Estimación de Kaplan-Meier , Masculino , Polimorfismo de Nucleótido Simple , Modelos de Riesgos Proporcionales , Tolerancia a Radiación/genética , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
This study was aimed at investigating the antitumor activity of novel 2-oxindole derivatives against a well-characterized human nonsmall cell lung cancer (NSCLC) cell line. Test compounds produced an antiproliferative activity in the low micromolar/submicromolar range of concentrations and significantly induced typical apoptotic morphology with cell shrinkage, nuclear condensation and fragmentation, and rupture of cells into debris in a relatively low percentage of A549 cells. Cell cycle arrest occurred at the G1/S phase (1a and 2), and Akt phosphorylation was significantly inhibited at Thr308 and Ser473. The most active compound (1a) has an IC50 6-fold lower than the Akt inhibitor, perifosine. These data suggest that the new compounds may be cytostatic and may have maximum clinical effects in NSCLC patients who do not respond to EGFR inhibitors. These findings prompt us to further explore the oxindole structure as leading scaffold to design new molecules with potent antitumor activity against NSCLC.
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INTRODUCTION: Lung cancer is one of the most lethal tumors and, although standard chemotherapy produces clinical response, there has been little improvement in prognosis. Therefore, research effort has focused on target-specific agents, such as sorafenib, which blocks both the RAF/MEK/ERK signalling pathways and receptors involved in neovascularization and tumor progression, including VEGFR-2 and c-Kit. We investigated whether sorafenib would be synergistic with gemcitabine against NSCLC cell lines. MATERIALS AND METHODS: Human lung cancer cells A549, CALU-1, CALU-6, H23 and HCC 827 were treated with sorafenib and gemcitabine, alone or in combination, and the cytotoxicity was assessed with CellTiter 96 Non-radioactive cell proliferation kit. Cell cycle and apoptosis were investigated with flow cytometry and fluorescence microscopy, respectively. Moreover, the effects of drugs on Akt (S473), c-Kit (Y823) and ERK (pTpY185/187) phosphorylation were studied with ELISA. Finally, quantitative PCR analysis was performed to assess whether sorafenib and gemcitabine modulated the expression of genes related to drug activity. RESULTS: Gemcitabine and sorafenib synergistically interacted on the inhibition of cell proliferation, and assessment of apoptosis demonstrated that drug associations increased the apoptotic index. Sorafenib reduced c-Kit and ERK activation and gemcitabine inhibited Akt phosphorylation. Moreover quantitative PCR showed that sorafenib modulated the expression of targets related to gemcitabine activity, while gemcitabine induced the expression of RKIP. CONCLUSIONS: These data demonstrate that sorafenib and gemcitabine synergistically interact against NSCLC cells, through suppression of Akt, c-Kit and ERK phosphorylation, induction of apoptosis and modulation of dCK, RRM1, RRM2 and RKIP gene expression. The association between traditional cytotoxic agents with new target-specific agents, such as sorafenib, is a challenge for both clinical and preclinical future investigations in lung cancer treatments.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas , Apoptosis/efectos de los fármacos , Bencenosulfonatos/administración & dosificación , Bencenosulfonatos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/fisiopatología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/fisiopatología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Neovascularización Patológica , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Piridinas/administración & dosificación , Piridinas/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Sorafenib , Quinasas raf/antagonistas & inhibidores , GemcitabinaRESUMEN
PURPOSE: Oxaliplatin effect in the treatment of colorectal cancer is improved upon combination with thymidylate synthase (TS) inhibitors. Pemetrexed is polyglutamated by the folylpolyglutamate synthase (FPGS) and blocks folate metabolism and DNA synthesis by inhibiting TS, dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study evaluates the pharmacological interaction between oxaliplatin and pemetrexed in colorectal cancer cells. METHODS: Human HT29, WiDr, SW620 and LS174T cells were treated with oxaliplatin and pemetrexed. Drug interaction was studied using the combination index method, while cell cycle was investigated with flow cytometry. The effects of drugs on Akt phosphorylation and apoptosis were studied with ELISA and fluorescence microscopy, respectively. RT-PCR analysis was performed to assess whether drugs modulated the expression of pemetrexed targets and of genes involved in DNA repair (ERCC1 and ERCC2). Finally, platinum-DNA adduct levels were detected by ultra-sensitive multi-collector inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: A dose-dependent inhibition of cell growth was observed after drug exposure, while a synergistic interaction was detected preferentially with sequential combinations. Oxaliplatin enhanced cellular population in the S-phase. Drug combinations increased apoptotic indices with respect to single agents, and both drugs inhibited Akt phosphorylation. RT-PCR analysis showed a correlation between the FPGS/(TS x DHFR x GARFT) ratio and pemetrexed sensitivity, as well as a downregulation of ERCC1, ERCC2, TS, DHFR and GARFT after drug exposure. In addition, pretreatment with pemetrexed resulted in an increase of oxaliplatin-DNA adducts. CONCLUSION: These data demonstrate that oxaliplatin and pemetrexed synergistically interact against colon cancer cells, through modulation of cell cycle, inhibition of Akt phosphorylation, induction of apoptosis and modulation of gene expression.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Glutamatos/administración & dosificación , Guanina/administración & dosificación , Guanina/análogos & derivados , Humanos , Microscopía Fluorescente , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Pemetrexed , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: Gemcitabine (2',2'-difluorodeoxycytidine) (GEM) is one of the most actively investigated drugs in ovarian cancer. Many molecular mechanisms have been proposed to be involved in GEM sensitivity/resistance including the equilibrative nucleoside transporter-1 (hENT1), the concentrative nucleoside transporter-1 (hCNT1), and deoxycytidine kinase (dCK). The expression of the ribonucleotide reductase regulatory subunits M1 (RRM1) and M2 (RRM2), and the catabolic enzymes 5'nucleotidase (5'NT), and cytidine deaminase (CDA) play also an important role. The aim of the study is to investigate the potential clinical role of hENT1, hCNT1, dCK, RRM1, RRM2, CDA, and 5'NT in a single institutional series of 25 primary ovarian carcinomas. METHODS: The expression levels of the target genes were assayed by means of real-time quantitative PCR. The clinical role of the expression levels of each parameter was analyzed by Kaplan and Meier method and the Cox's proportional hazards model. RESULTS: hENT1, hCNT1, dCK, CDA, 5'NT, RRM1, and RRM2 gene expression was documented in all samples; undifferentiated and clear cell carcinoma exhibited higher levels of hENT1, dCK, 5'NT, and RRM1 compared to serous ovarian tumors. As of May 2009, the median follow-up was 32 months (range 10-80), and death of disease were observed in 17 cases (68.0%). A statistically significant direct association of RRM2 expression levels and the relative risk of death was observed (Chi (2) = 8.18, P = 0.0043). Moreover, cases with high RRM2 expression had a shorter OS (median OS = 19 months) than cases with low RRM2 levels (median OS = 36 months) (P = 0.0506). CONCLUSIONS: We first reported that the most relevant genes involved in gemcitabine metabolism are expressed in ovarian carcinoma, and might be associated with more aggressive histotypes. The assessment of the expression levels of RRM2 as marker of clinical outcome deserves further investigation in a larger series of ovarian cancer patients.
Asunto(s)
5'-Nucleotidasa/genética , Citidina Desaminasa/genética , Desoxicitidina Quinasa/genética , Proteínas de Transporte de Nucleósidos/genética , Neoplasias Ováricas/genética , Ribonucleósido Difosfato Reductasa/genética , 5'-Nucleotidasa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antimetabolitos Antineoplásicos/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citidina Desaminasa/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/metabolismo , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Tranportador Equilibrativo 1 de Nucleósido/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Proteínas de Transporte de Membrana/genética , Persona de Mediana Edad , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Proteínas Supresoras de Tumor/genética , GemcitabinaRESUMEN
Irinotecan is a topoisomerase-I (Top-I) inhibitor used for the treatment of colorectal cancer. DNA demethylating agents, including 5-azacytidine (5-aza), display synergistic antitumor activity with several chemotherapy drugs. 5-Aza may enhance irinotecan cytotoxicity by at least one of the following mechanisms: (a) Top-I promoter demethylation, (b) activation of genes involved in Top-I transcriptional regulation (p16 or Sp1), and (c) modulation of the cell cycle and apoptosis after DNA damage. The growth-inhibitory effects of SN38, the active metabolite of irinotecan, 5-aza, and their combinations, were studied in four colorectal cancer cell lines. The effects of treatments on cell cycle were analyzed by flow cytometry, and apoptosis was measured by fluorescence microscopy. Top-I, Sp1, and p53 expression modulated by 5-aza were measured by real-time PCR. Methylation of Top-I, p16, 14-3-3sigma, and hMLH1 promoters before and after 5-aza treatment were measured by MethyLight PCR and DNA bisulfite sequencing. Low-dose 5-aza significantly enhanced the apoptotic effect of irinotecan in all colorectal cancer cells, whereas a synergistic cytotoxic effect was observed only in p53-mutated cells (HT29, SW620, and WiDr). This synergistic effect was significantly correlated with Top-I up-regulation by 5-aza, and coupled to p16 demethylation and Sp1 up-regulation. p16 demethylation was also associated with enhanced cell cycle arrest after irinotecan treatment. In contrast, 5-aza down-regulated Top-I expression in the p53 wild-type LS174T cells in a p53-dependent manner, thereby reducing SN38 cytotoxicity. In conclusion, 5-aza modulates Top-I expression by several mechanisms involving Sp1, p16, and p53. If confirmed in other models, these results suggest that p16 and p53 status affects the 5-aza-irinotecan interaction.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Camptotecina/análogos & derivados , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Metilación de ADN , Epigénesis Genética , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Apoptosis , Azacitidina/farmacología , Camptotecina/farmacología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Daño del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Quimioterapia Combinada , Humanos , Irinotecán , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Gemcitabine is an inhibitor of ribonucleotide reductase (RR) and DNA polymerization with promising activity in hematologic malignancies. Gemcitabine enters the cell mostly via the human equilibrative nucleoside transporter-1 (hENT1), while drug metabolism occurs by phosphorylation by deoxycytidine kinase (dCK), 5'-nucleotidase (cN-II) and cytidine deaminase (CDA) are the main inactivating enzymes. The aim of this study was to investigate the role of these determinants in gemcitabine cytotoxicity and analyze their expression in lymphoid cells. Cytotoxicity was assessed by MTT, and modulated by simultaneous addition of 2'-deoxycytidine (dCK natural substrate), tetrahydrouridine (CDA competitive inhibitor) and diethylpyrocarbonate (cN-II non-competitive inhibitor), while the expression of hENT1, dCK, cN-II, CDA and RR in WIL2-S, Jurkat and CCRF-CEM cells as well as in lymphoid cells from 25 chronic lymphocytic B-leukemia (B-CLL) patients was studied with quantitative-PCR. Cell cycle modulation and induction of apoptosis were analyzed by cytofluorimetry and bisbenzimide staining. Gemcitabine was highly cytotoxic, increased the cells in S-phase and significantly enhanced apoptosis. The crucial role of metabolism in gemcitabine activity was confirmed by the significant modulation of cytotoxicity by inhibitors of dCK, CDA and cN-II. Furthermore, PCR demonstrated a correlation between gemcitabine sensitivity and expression of its determinants, and that their values were within those observed in patients. These data indicate that gemcitabine is cytotoxic against lymphoid cells, affecting cell cycle and apoptosis. Furthermore, chemosensitivity may be predicted on the basis of gene expression profile of critical determinants involved in gemcitabine mechanism of action, suggesting the use of pharmacogenetic profiling for treatment optimization.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Anciano , Antígenos CD/análisis , Antimetabolitos Antineoplásicos/metabolismo , Antimetabolitos Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/metabolismo , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Desoxicitidina Quinasa/genética , Desoxicitidina Quinasa/metabolismo , Relación Dosis-Respuesta a Droga , Tranportador Equilibrativo 1 de Nucleósido/genética , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Inmunofenotipificación , Células Jurkat , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Persona de Mediana Edad , Selección de Paciente , Valor Predictivo de las Pruebas , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleósido Difosfato Reductasa/genética , Ribonucleósido Difosfato Reductasa/metabolismo , Resultado del Tratamiento , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , GemcitabinaRESUMEN
Gemcitabine and pemetrexed are effective agents in the treatment of non-small-cell lung cancer (NSCLC), and the present study investigates cellular and genetic aspects of their interaction against A549, Calu-1, and Calu-6 cells. Cells were treated with pemetrexed and gemcitabine, and their interaction was assessed using the combination index. The role of drug metabolism in gemcitabine cytotoxicity was examined with inhibitors of deoxycytidine kinase (dCK), 5'-nucleotidase, and cytidine deaminase, whereas the role of pemetrexed targets, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) in drug chemosensitivity was analyzed in cytotoxicity rescue studies. The effect of gemcitabine and pemetrexed on Akt phosphorylation was investigated with enzyme-linked immunosorbent assay, whereas quantitative polymerase chain reaction (PCR) was used to study target gene-expression profiles and its modulation by each drug. Synergistic cytotoxicity was demonstrated, and pemetrexed significantly decreased the amount of phosphorylated Akt, enhanced apoptosis, and increased the expression of dCK in A549 and Calu-6 cells, as well as the expression of the human nucleoside equilibrative transporter 1 (hENT1) in all cell lines. PCR demonstrated a correlation between dCK expression and gemcitabine sensitivity, whereas expression of TS, DHFR, and GARFT was predictive of pemetrexed chemosensitivity. These data demonstrated that 1) gemcitabine and pemetrexed synergistically interact against NSCLC cells through the suppression of Akt phosphorylation and induction of apoptosis; 2) the gene expression profile of critical genes may predict for drug chemosensitivity; and 3) pemetrexed enhances dCK and hENT1 expression, thus suggesting the role of gene-expression modulation for rational development of chemotherapy combinations.