RESUMEN
The impact of etoposide (VP-16) plasma concentrations on the day of allogeneic hematopoietic stem cell transplantation (allo-HSCT) on leukemia-free survival in children with acute lymphoblastic leukemia (ALL) was studied. In addition, the in vitro effects of VP-16 on the lymphocytes proliferation, cytotoxic activity and on Th1/Th2 cytokine responses were assessed. In 31 children undergoing allo-HSCT, VP-16 plasma concentrations were determined up to 120 h after the infusion using the HPLC-UV method. For mentioned in vitro studies, VP-16 plasma concentrations observed on allo-HSCT day were used. In 84 % of children, VP-16 plasma concentrations (0.1-1.5 µg/mL) were quantifiable 72 h after the end of the drug infusion, i.e. when allo-HSCT should be performed. In 20 (65 %) children allo-HSCT was performed 4 days after the end of the drug infusion, and VP-16 was still detectable (0.1-0.9 µg/mL) in plasma of 12 (39 %) of them. Post-transplant ALL relapse occurred in four children, in all of them VP-16 was detectable in plasma (0.1-0.8 µg/mL) on allo-HSCT day, while there was no relapse in children with undetectable VP-16. In in vitro studies, VP-16 demonstrated impact on the proliferation activity of stimulated lymphocytes depending on its concentration and exposition time. The presence of VP-16 in plasma on allo-HSCT day may demonstrate an adverse effect on graft-versus-leukemia (GvL) reaction and increase the risk of post-transplant ALL relapse. Therefore, if 72 h after VP-16 administration its plasma concentration is still above 0.1 µg/mL then the postponement of transplantation for next 24 h should be considered to protect GvL effector cells from transplant material.
Asunto(s)
Antineoplásicos Fitogénicos/farmacocinética , Etopósido/farmacocinética , Efecto Injerto vs Leucemia/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfocitos T Citotóxicos/efectos de los fármacos , Adolescente , Antineoplásicos Fitogénicos/efectos adversos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Niño , Supervivencia sin Enfermedad , Cálculo de Dosificación de Drogas , Etopósido/efectos adversos , Femenino , Humanos , Técnicas In Vitro , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Recurrencia , Riesgo , Linfocitos T Citotóxicos/inmunología , Balance Th1 - Th2/efectos de los fármacos , Trasplante HomólogoRESUMEN
Pancreatic adenocarcinoma is one of the malignancies that is highly resistant to therapy and among the leading causes of cancer-related death. Several factors may influence pancreatic cancer resistance, and expression of ATP-binding cassette transport proteins is one of the major mechanisms of drug resistance. Members of this family's C-branch, also referred to as multidrug resistance-associated proteins (MRPs), might be of particular interest because they are able to efflux nucleoside analogs used in the treatment of pancreatic cancer. Expression of MRP1, MRP3, MRP4, and MRP5 in human pancreas and pancreatic carcinoma has been reported. However, contributions of MRPs to chemoresistance of pancreatic cancer are not fully understood. MRP5 mRNA expression in pancreatic adenocarcinoma cell lines correlated significantly with cellular sensitivity to 5-fluorouracil (5-FU) (r = 0.738, p < 0.05). Long-term treatment with 5-FU increased expression of MRP5 by 2.4-fold and was associated with significant drug resistance [IC(50) values for control and 5-fluorouracil (5-FU)-resistant Patu-T cell lines were 11.3 ± 5.3 and 33.2 ± 6.9 µM, respectively (p < 0.05)]. Consequently, overexpression of MRP5 in Colo-357 cells resulted in significantly reduced accumulation of 5-FU related radioactivity and 5-FU cytotoxicity. Knockdown of MRP5 significantly increased cellular cytotoxicity of 5-FU to Patu-02 cells and enhanced accumulation of radioactivity related to 5-FU and its metabolites. Our results suggest that MRP5 is expressed and functionally active and contributes to variable sensitivities of pancreatic adenocarcinoma cell lines to 5-FU. Further investigations using models that resemble human pancreas tumors are necessary to prove a causative relation between expression and activity of MRP5 and tumor resistance to 5-FU.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Fluorouracilo/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Adenocarcinoma , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Técnicas de Silenciamiento del Gen , Humanos , Páncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , ARN/análisis , Interferencia de ARN , Células Tumorales CultivadasRESUMEN
PURPOSE: Antitumor potential of angiotensin-converting enzyme inhibitors has been shown in different preclinical settings, which always involved immunocompromised organisms or nonimmunogenic tumor models. In our study, we wanted to evaluate the effect of captopril on growth of immunogenic tumors in immunocompetent animals. EXPERIMENTAL DESIGN: We used different murine tumor models to evaluate the effect of captopril on tumor take and survival of tumor-bearing immunocompetent and immunocompromised mice. We used an orthotopic renal cell cancer model and highly immunogenic tumor model, which were based on kidney subcapsular injection of RenCa cells or s.c. injection of MethA cells, respectively. To show the influence of captopril on antigen-specific immune responses, we have used two model antigens (green fluorescent protein and beta-galactosidase). RESULTS: Captopril decreased survival of RenCa-bearing, immunocompetent mice in a dose-dependent manner and in adjuvant setting. In nephrectomized mice, captopril shortened their survival. Captopril promoted formation of immunogenic MethA sarcoma tumors but had no effect on nonimmunogenic melanoma cells (B78-H1). Treatment of immunocompromised mice bearing MethA tumors or RenCa kidney tumors with captopril did not affect tumor formation nor survival, respectively. Captopril-treated mice immunized with AdLacZ or AdGFP vectors did not generate or generated decreased numbers of antigen-specific CD8+ T cells, respectively. However, they showed B-cell responses represented by infiltration of MethA tumors with activated B cells and dramatically increased serum level of beta-galactosidase-specific antibodies. CONCLUSIONS: Our results show a novel role of captopril in tumor biology and the tumor-promoting properties of captopril seem to be associated with its immunomodulatory potential.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Captopril/farmacología , Sarcoma Experimental/patología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCID , Sarcoma Experimental/inmunología , Trasplante Heterólogo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have constructed and expressed recombinant chimeric soluble TGF-beta type II receptor fused with the Fc portion of human IgG1 (sTbetaRII-Fc) in NS0 mouse myeloma cells and isolated cell lines constitutively secreting very high levels of biologically active protein. The GS-NS0 expression system takes advantage of the strong human cytomegalovirus immediate early promoter expression vector and glutamine synthetase as a selectable marker. The recombinant chimeric receptor could be produced in high amounts and efficiently purified by one step chromatography on a protein A column. Biochemical studies revealed that recombinant sTbetaRII-Fc binds native TGF-beta1 and TGF-beta3 isoforms and neutralizes their activity in vitro.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteínas Recombinantes de Fusión/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Cromatografía de Afinidad , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Unión Proteica , Proteínas Serina-Treonina Quinasas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The ability of various cytokines to hamper tumor growth or to induce anti-tumor immune response has resulted in their study as antitumor agents in gene therapy approaches. In this review we will concentrate on the costimulation of antitumor immune responses using modification of various cell types by cytokine genes. Several strategies have emerged such as (i). modification of tumor cells with cytokine genes ex vivo (whole tumor cell vaccines), (ii). ex vivo modification of other cell types for cytokine gene delivery, (iii). delivery of cytokine genes into tumor microenvironment in vivo, (iv). modification of dendritic cells with cytokine genes ex vivo. Originally single cytokine genes were used. Subsequently, multiple cytokine genes were applied simultaneously, or in combination with other factors such as chemokines, membrane bound co-stimulatory molecules, or tumor associated antigens. In this review we discuss these strategies and their use in cancer treatment as well as the promises and limitations of cytokine based cancer gene therapy. Clinical trials, including our own experience, employing the above strategies are discussed.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Citocinas/genética , Células Dendríticas/inmunología , Técnicas de Transferencia de Gen , Inmunoterapia Adoptiva , Vacunas contra el Cáncer/genética , Citocinas/inmunología , Terapia Genética , HumanosRESUMEN
Data presented here demonstrate that vaccine-induced CD8(+) T cells can eliminate their specific tumor-target with a two-staged attack. First, they release interferon-gamma that results in growth arrest of the tumor cells via induction of antiangiogenic mediators. Then, during the latter stages of the immune response, CD8(+) effector T cells eradicate the remaining tumor cells through perforin-mediated lysis. A combination of these two mechanisms is highly effective in the described model, while either pathway alone fails to completely achieve tumor rejection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Interferón gamma/metabolismo , Neoplasias Pulmonares/terapia , Adenoviridae , Animales , Linfocitos T CD8-positivos/fisiología , Vacunas contra el Cáncer/farmacología , División Celular/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Neoplasias Pulmonares/veterinaria , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales , Neovascularización Patológica , Perforina , Proteínas Citotóxicas Formadoras de PorosRESUMEN
This article reviews the evidence for the danger model in the context of immune response to tumors and the insufficiency of the immune system to eliminate tumor growth. Despite their potential immunogenicity tumors do not induce significant immune responses which could destroy malignant cells. According to the danger model, the immune surveillance system fails to detect tumor antigens because transformed cells do not send any danger signals which could activate dendritic cells and initiate an immune response. Instead, tumor cells or antigen presenting cells turn off the responding T cells and induce tolerance. The studies reviewed herein based on model tumor antigens, recombinant viral vectors and detection of tumor specific T cells by MHC/peptide tetramers underscore the critical role of tumor antigen presentation and the context in which it occurs. They indicate that antigen presentation only by activated but not by cancer or resting dendritic cells is necessary for the induction of immune responses to tumor antigens. It becomes apparent that the inability of dendritic cells to become activated provides a biological niche for tumor escape from immune destruction and seems to be a principal mechanism for the failure of tumor immune surveillance.
Asunto(s)
Modelos Biológicos , Neoplasias/inmunología , Neoplasias/patología , Animales , Antígenos de Neoplasias/inmunología , Células Dendríticas/inmunología , Humanos , Tolerancia Inmunológica , Linfocitos T/inmunologíaRESUMEN
Since the invention of the polymerase chain reaction (PCR) several quantitative PCR-based approaches have been described. Recently, the real-time PCR method became a standard in quantitative PCR, although high costs of the necessary equipment and reagents make it unaffordable for many laboratories. In this paper we describe two novel competitive PCR techniques, which were used to determine the frequency of T-cell receptor delta gene (TCRD) rearrangements in peripheral blood leukocytes. In the reference gene competitive PCR (rgc-PCR) the rearranged TCRD gene competes with the reference gene (RAG1) for common reagents (dNTPs and Taq polymerase). The intensity ratio of amplification products, TCRD/RAG1, corresponds to the portion of cells containing a rearrangement. A series of reactions was performed, in which RAG1 primers were added to the PCR after different numbers of cycles. On the basis of the number of cycles needed to obtain equal band intensity, the frequency of cells containing a rearrangement was calculated. In the common primer competitive PCR (cpc-PCR), two gene rearrangements, Vdelta1-Jdelta1 and Vdelta2-Jdelta1, compete for the common Jdelta1 primer. The competing genes are amplified from the same genomic DNA template; therefore unlike in the method using the internal competitor, the results are not affected by the quantity or quality of the analysed sample. We showed that the rgc-PCR and cpc-PCR are reliable and give reproducible results. The methods do not require any expensive equipment or reagents, and can be used to determine the frequency of gene rearrangements.