Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Siembra Neoplásica , Anciano , Femenino , Dosificación de Gen , Humanos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Secuenciación Completa del GenomaRESUMEN
Small-cell lung cancers (SCLCs) initially respond to chemotherapy but are often resistant at recurrence. A potentially new method to overcome resistance is to combine classical chemotherapeutic drugs with apoptosis induction via tumour necrosis factor (TNF) death receptor family members such as Fas. The doxorubicin-resistant human SCLC cell line GLC4-Adr and its parental doxorubicin-sensitive line GLC4 were used to analyse the potential of the Fas-mediated apoptotic pathway and the mitochondrial apoptotic pathway to modulate doxorubicin resistance in SCLC. Western blotting showed that all proteins necessary for death-inducing signalling complex formation and several inhibitors of apoptosis were expressed in both lines. The proapototic proteins Bid and caspase-8, however, were higher expressed in GLC4-Adr. In addition, GLC4-Adr expressed more Fas (3.1x) at the cell membrane. Both lines were resistant to anti-Fas antibody, but plus the protein synthesis inhibitor cycloheximide anti-Fas antibody induced 40% apoptosis in GLC4-Adr. Indomethacin, which targets the mitochondrial apoptotic pathway, induced apoptosis in GLC4-Adr but not in GLC4 cells. Surprisingly, in GLC4-Adr indomethacin induced caspase-8 and caspase-9 activation as well as Bid cleavage, while both caspase-8 and caspase-9 specific inhibitors blocked indomethacin-induced apoptosis. In GLC4-Adr, doxorubicin plus indomethacin resulted in elevated caspase activity and a 2.7-fold enhanced sensitivity to doxorubicin. In contrast, no effect of indomethacin on doxorubicin sensitivity was observed in GLC4. Our findings show that indomethacin increases the cytotoxic activity of doxorubicin in a doxorubicin-resistant SCLC cell line partly via the death receptor apoptosis pathway, independent of Fas.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de la Ciclooxigenasa/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Indometacina/farmacología , Apoptosis/fisiología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Western Blotting , Carcinoma de Células Pequeñas/tratamiento farmacológico , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Caspasas/efectos de los fármacos , Caspasas/metabolismo , Resistencia a Antineoplásicos/fisiología , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Microscopía Confocal , ARN Mensajero/análisis , Receptores del Factor de Necrosis Tumoral/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor fasRESUMEN
Testicular germ cell tumors (TGCTs) are unusually sensitive to cisplatin. In the present study the role of the CD95 death pathway in cisplatin sensitivity of TGCT cells was studied in Tera and its in vitro acquired cisplatin-resistant subclone Tera-CP. Cisplatin induced an increase in CD95 membrane expression, which preceded the onset of apoptosis. Cisplatin-induced apoptosis was efficiently blocked by caspase-8 inhibitor zIETD-fmk in Tera cells, but only partially in Tera-CP cells. In addition, cisplatin induced FADD and caspase-8 recruitment to the CD95 receptor in Tera cells, which was not noticed in Tera-CP cells. Moreover, overexpression of vFLIP reduced apoptosis induction by cisplatin in Tera cells. CD95L-blocking experiments revealed the involvement of CD95/CD95L interactions in cisplatin-induced apoptosis of Tera cells as well as cisplatin-sensitive 833KE TGCT cells. Tera and 833KE cells, treated with low doses of cisplatin, were sensitive for an apoptosis-inducing anti-CD95 antibody. In contrast, CD95L blocking had no effect on cisplatin-induced apoptosis in Tera-CP or Scha, an intrinsic resistant TGCT cell line, nor did anti-CD95 antibody induce additional apoptosis in cisplatin-treated Tera-CP or Scha cells. Taken together, these results show that (1) cisplatin sensitivity of TGCT cells is dependent on the activation of the CD95 death pathway and (2) loss of cisplatin-induced activation of this CD95 signaling pathway may result in resistance to cisplatin.
Asunto(s)
Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Germinoma/tratamiento farmacológico , Germinoma/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Testiculares/tratamiento farmacológico , Neoplasias Testiculares/metabolismo , Receptor fas/efectos de los fármacos , Anticuerpos/farmacología , Antineoplásicos/farmacología , Apoptosis/fisiología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/metabolismo , Caspasa 8 , Inhibidores de Caspasas , Caspasas/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cisplatino/farmacología , Relación Dosis-Respuesta a Droga , Células Madre de Carcinoma Embrionario , Inhibidores Enzimáticos/farmacología , Proteína Ligando Fas , Germinoma/fisiopatología , Humanos , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Células Madre Neoplásicas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Neoplasias Testiculares/fisiopatología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Receptor fas/metabolismoRESUMEN
Chemotherapeutic efficacy is hampered by occurrence of drug resistance. Several mechanisms cause this phenomenon. A final common factor is the reduced capacity of resistant cells to go into apoptosis following treatment with DNA-damaging agents. It is therefore interesting to search for ways to facilitate this apoptotic process following use of chemotherapeutic drugs. The death receptor ligands tumor necrosis factor (TNF), FasL and TNF-related apoptosis-inducing ligand (TRAIL) might be interesting candidates as they are able to induce apoptosis by binding to their cell membrane receptors. Recombinant forms of these ligands potentiate chemotherapeutic drug effects in preclinical models. For the clinical application of TNF, FasL and TRAIL, it is of primary importance that their safety be guaranteed. RhTNF is the only ligand currently used in humans. However, systemic rhTNF has shown low antitumor activity and higher doses induce severe sepsis-like toxicity. Perfusion setting aimed at limb preservation with rhTNF plus melphalan is currently used in sarcoma patients. A number of options have been tested in the preclinical setting that might allow circumvention of TNF toxicity in the clinic. Systemic rhFasL administration in humans is not yet feasible because of observed severe liver toxicity in mice due to Fas-mediated apoptosis of hepatocytes. Measures to circumvent liver toxicity have not yet been exploited. Another option for using FasL in the clinic may be to identify an alternative route of administration. In the animal model, FasL appeared to be less toxic for the liver compared with anti-Fas antibodies when administered intraperitoneally. There are relatively nontoxic modulators of the Fas death pathway, such as interferon and nonsteroidal antiinflammatory drugs (NSAIDs), which might prove interesting in combination with chemotherapy. Finally, it may be possible to produce a modified FasL with a reduced toxicity profile. TRAIL, produced as soluble, zinc-stabilized rhTRAIL seems to be without preclinical toxicity. Agonistic DR4 and DR5 antibodies against their TRAIL death receptor are being studied as another potential clinical option to induce apoptosis. Due to the synergistic effect observed in the preclinical setting between death receptor ligands and other modulators of the death receptor pathways and chemotherapy, it may well be that this approach is especially of value in the clinic when combined with chemotherapy. Ideally, choices for specific (modified) death receptor ligands for the treatment of patients can be rationally made based on tumor characteristics.
Asunto(s)
Antígenos CD/metabolismo , Neoplasias/tratamiento farmacológico , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptor fas/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Ensayos Clínicos como Asunto , Resistencia a Antineoplásicos , Proteína Ligando Fas , Humanos , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/uso terapéutico , Neoplasias/metabolismo , Neoplasias/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores Tipo I de Factores de Necrosis Tumoral , Proteínas Recombinantes/uso terapéutico , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/uso terapéuticoRESUMEN
Immunotherapy of tumours by induction of tumour-specific cytotoxic T-lymphocytes (CTLs) will only be effective for tumours with a functional antigen processing and presentation machinery. However, many tumours are known to down-regulate expression of major histocompatibility complex (MHC) class I molecules and/or to impair antigen processing. It is therefore desirable to evaluate the ability of a given tumour to present antigenic epitopes before developing an immunotherapy protocol. In this study we have used influenza virus as a tool to determine the antigen-presenting capacities of the murine neuroblastoma C1300 cell line NB41A3, a frequently used model for human neuroblastoma. Immunofluorescence analyses revealed low and moderate expression of MHC class I molecules Dd and Kk respectively. Nevertheless, infected NB41 A3 cells were lysed efficiently by influenza-specific CTLs. These results demonstrate that all steps of the antigen-processing pathway function properly in the NB tumour cells, and that the limited MHC class I expression suffices for efficient recognition by CTLs. In addition, lysis of the NB tumour cells shows that the cells are susceptible to CTL-induced apoptosis, a pathway that is often impaired in tumour cells. These characteristics make neuroblastoma a suitable target for immunotherapy. The presented assay allows evaluation of various immunological properties of tumour cells and, thus, represents a valuable tool to assess whether a given tumour will be susceptible to immunotherapy or not.
Asunto(s)
Virus de la Influenza A/inmunología , Neuroblastoma/inmunología , Neuroblastoma/terapia , Linfocitos T Citotóxicos/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Femenino , Genes MHC Clase I , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Interferón gamma/farmacología , Células L , Ratones , Ratones Endogámicos A , Proteínas Recombinantes , Linfocitos T Citotóxicos/virología , Células Tumorales CultivadasRESUMEN
The mutant rat thyroid cell line FRTL-5/TA, isolated from a non-functional tumour which originated spontaneously from wild-type FRTL-5 cells, shows autonomous TSH-independent growth and loss of the thyroid-specific phenotype, lacking thyroid-specific expression of thyroglobulin (Tg) and thyroid peroxidase (TPO) genes. To investigate the role of the transcription factors Pax-8 and thyroid transcription factor-1 (TTF-1) in rat thyroid tumorigenesis, RNA expression of these two thyroid-specific nuclear factors was measured in FRTL-5/TA tumour cells and compared with the expression in wild-type FRTL-5 cells. TTF-1 gene expression was similar to that in wild-type FRTL-5, and showed a similar down-regulation after stimulation with TSH. The finding suggested normal TTF-1 mRNA and protein expression in both cell lines. By contrast, Pax-8 mRNA transcript signal was markedly reduced in FRTL-5/TA cells, reaching levels as low as 8% of the normal, basal level in FRTL-5 cells. These data indicated that the loss of thyroid-specific expression of Tg and TPO genes in FRTL-5/TA cells was not related to changes in TTF-1 gene expression but rather to reduced Pax-8 gene expression. It was concluded that a disruption of the co-ordinated expression of TTF-1 and Pax-8 is implicated in the loss of thyroid phenotype of FRTL-5/TA cells in terms of reduced Tg and TPO expression.
Asunto(s)
Proteínas de Homeodominio/fisiología , Neoplasias de la Tiroides/metabolismo , Factores de Transcripción/fisiología , Animales , Northern Blotting , Línea Celular , Proteínas de Unión al ADN/genética , Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Yoduro Peroxidasa/genética , Proteínas Nucleares/genética , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box , ARN Mensajero/análisis , Ratas , Tiroglobulina/genética , Glándula Tiroides/metabolismo , Factor Nuclear Tiroideo 1 , Tirotropina/farmacología , Transactivadores/genética , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
Migration patterns of leukemic cells in bone marrow are largely regulated by cell contacts between leukemic cells and stromal cells or extra-cellular matrix. The mechanism of this interaction with bone-marrow stromal cells was studied in a human in vitro model. Migration behavior of erythroleukemia cell line K562, derived from a patient with chronic myeloid leukemia, was compared with that of the erythroleukemia cell line HEL92.1.7 and the promyelocytic leukemia cell line HL60 from acute leukemias. Interaction varied between low binding affinity (K562) to intensive cell interaction (HEL92.1.7) followed by invasion into the stromal cell monolayer. Some of the HL60 cells adhered to stromal cells, while the remainder migrated into the stromal cell monolayer. The role of adhesion molecules in these cell interactions was determined. Distinct expression of beta1-integrins ICAM-1, CD44 and VCAM-1 was detected on the different cell lines. Inhibition studies pointed to a dominant role of VLA-4- and VLA-5-mediated interactions. K562 lacked VLA-4 and a low binding affinity of the VLA-5 on these cells resulted in an absence of binding to the bone-marrow stroma. These results indicate the VLA-5/fibronectin, VLA-4/fibronectin and the VLA-4/VCAM-1 interaction pathways between leukemic cells and bone-marrow stroma.
Asunto(s)
Médula Ósea/patología , Integrina beta1/fisiología , Leucemia/patología , Adhesión Celular , Moléculas de Adhesión Celular/análisis , Movimiento Celular , Humanos , Receptores de Fibronectina/fisiología , Células del Estroma/patología , Células Tumorales CultivadasRESUMEN
Adhesion of tumour cells to cultured bone marrow stromal cells has been studied in an in vitro model system. Stromal cells were isolated from bone marrow aspirates. Immunohistochemical and electron microscopical analysis revealed a uniform cell monolayer of myofibroblastic cells, expressing fibroblast antigens and smooth muscle actin. Cell interactions with tumour cells lines showed different patterns. The K562 cells bound in low numbers to stromal cells. HEL-DR- and HL60 cells adhered to stromal cells showing an enlarged cell contact area (spreading) attenuated by distinct contact sites and they invaded the monolayer. Adhesion molecules, important for cell contacts, were detected on tumor cells. Different VLA antigens were detected on tumour cells, but on stromal cells only VLA-5 and CD29 were found. In vitro inhibition studies with mAbs against adhesion molecules indicated two major pathways for binding of tumour cells to stromal cells: VCAM-1/VLA-4 and fibronectin/VLA-5. Variation in inhibition of mAbs to VLA-4 and VCAM-1 indicated the existence of critical epitopes in the adhesion of tumour cells.