RESUMEN
Targeted therapies for cutaneous T-cell lymphoma (CTCL) are limited and curative approaches are lacking. Furthermore, relapses and drug induced side effects are major challenges in the therapeutic management of patients with CTCL, creating an urgent need for new and effective therapies. Pathologic constitutive NF-κB activity leads to apoptosis resistance in CTCL cells and, thus, represents a promising therapeutic target in CTCL. In a preclinical study we showed the potential of dimethyl fumarate (DMF) to block NF-κB and, specifically, kill CTCL cells. To translate these findings to applications in a clinical setting, we performed a multicentric phase 2 study evaluating oral DMF therapy in 25 patients with CTCL stages Ib to IV over 24 weeks (EudraCT number 2014-000924-11/NCT number NCT02546440). End points were safety and efficacy. We evaluated skin involvement (using a modified severity weighted assessment tool [mSWAT]), pruritus, quality of life, and blood involvement, if applicable, as well as translational data. Upon skin analysis, 7 of 23 (30.4%) patients showed a response with >50% reduction in the mSWAT score. Patients with high tumor burden in the skin and blood responded best to DMF therapy. Although not generally significant, DMF also improved pruritus in several patients. Response in the blood was mixed, but we confirmed the NF-κB-inhibiting mechanism of DMF in the blood. The overall tolerability of the DMF therapy was very favorable, with mostly mild side effects. In conclusion, our study presents DMF as an effective and excellently tolerable therapeutic option in CTCL to be further evaluated in a phase 3 study or real-life patient care as well as in combination therapies. This trial was registered at www.clinicaltrials.gov as #NCT02546440.
Asunto(s)
Linfoma Cutáneo de Células T , Neoplasias Cutáneas , Humanos , Dimetilfumarato/uso terapéutico , FN-kappa B , Calidad de Vida , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Recurrencia Local de Neoplasia/tratamiento farmacológico , Linfoma Cutáneo de Células T/tratamiento farmacológico , Linfoma Cutáneo de Células T/patología , Prurito/tratamiento farmacológicoRESUMEN
BACKGROUND: Uptake of apoptotic cells induces a tolerogenic phenotype in phagocytes and promotes peripheral tolerance. The highly conserved Annexin core domain, present in all members of the Annexin family, becomes exposed on the apoptotic cell-surface and triggers tolerogenic signalling in phagocytes via the Dectin-1 receptor. Consequently, Annexins exposed on tumour cells upon cell death are expected to induce tolerance towards tumour antigens, inhibiting tumour rejection. METHODS: Expression analysis for all Annexin family members was conducted in cancer cell lines of diverse origins. Presentation of Annexins on the cell surface during apoptosis of cancer cell lines was investigated using surface washes and immunoblotting. Expression data from the GEO database was analysed to compare Annexin levels between malignant and healthy tissue. RESULTS: Six Annexins at least were consistently detected on mRNA and protein level for each investigated cell line. AnxA1, AnxA2 and AnxA5 constituted the major part of total Annexin expression. All expressed Annexins translocated to the cell surface upon apoptosis induction in all cell lines. Human expression data indicate a correlation between immune infiltration and overall Annexin expression in malignant compared to healthy tissue. CONCLUSIONS: This study is the first comprehensive analysis of expression, distribution and presentation of Annexins in cancer.
Asunto(s)
Anexinas , Neoplasias , Anexina A5 , Anexinas/genética , Anexinas/metabolismo , Antígenos de Neoplasias , Humanos , Neoplasias/genética , ARN MensajeroRESUMEN
Therapeutic options for cutaneous T-cell lymphoma (CTCL) are limited and curative treatment regimens are not available. Thus, new targeted and well-tolerated therapeutic approaches are urgently needed. In this respect, we have recently shown that dimethyl fumerate (DMF) inhibits NF-κB acting as a survival factor in CTCL. Similarly, inhibition of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) has been shown to induce cell death in CTCL especially when combined with histone deacetylase inhibitors. Therefore, we hypothesized that inhibition of Bcl-2 should potentiate NF-κB inhibition in a novel combination treatment of CTCL. We show that, in vitro, the Bcl-2 inhibitors ABT-199 and ABT-263 induced specific cell death in primary CD4+ cells from CTCL patients as well as in the CTCL cell line SeAx, but not in T cells of healthy donors nor in the CTCL cell line HH, which lacks Bcl-2. Combined treatment with ABT-199 and DMF caused synergistic cell death specifically in CTCL cells engaging 2 independent signaling pathways. To verify these findings in vivo, we performed combined ABT-199 and DMF treatment in a xenograft mouse model for CTCL. The combined treatment effectively reduced tumor growth and increased overall survival via synergistic induction of CTCL cell death and suppression of tumor cell proliferation. Essentially, the combination treatment was superior to ABT-199 monotherapy with respect to both efficacy and tolerability. To sum up, our data provide proof of principle for the therapeutic potential of combining Bcl-2 and NF-κB inhibitors in treating CTCL. Next, this potential should be explored further in a clinical study.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Linfoma Cutáneo de Células T/metabolismo , FN-kappa B/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Animales , Apoptosis , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Linfoma Cutáneo de Células T/diagnóstico , Linfoma Cutáneo de Células T/tratamiento farmacológico , Linfoma Cutáneo de Células T/genética , Ratones , FN-kappa B/genética , Estadificación de Neoplasias , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Signal transduction to nuclear factor-kappa B (NF-κB) involves multiple kinases and phosphorylated target proteins, but little is known about signal termination by dephosphorylation. By RNAi screening, we have identified protein phosphatase 4 regulatory subunit 1 (PP4R1) as a negative regulator of NF-κB activity in T lymphocytes. PP4R1 formed part of a distinct PP4 holoenzyme and bridged the inhibitor of NF-κB kinase (IKK) complex and the phosphatase PP4c, thereby directing PP4c activity to dephosphorylate and inactivate the IKK complex. PP4R1 expression was triggered upon activation and proliferation of primary human T lymphocytes and deficiency for PP4R1 caused sustained and increased IKK activity, T cell hyperactivation, and aberrant NF-κB signaling in NF-κB-addicted T cell lymphomas. Collectively, our results unravel PP4R1 as a previously unknown activation-associated negative regulator of IKK activity in lymphocytes whose downregulation promotes oncogenic NF-κB signaling in a subgroup of T cell lymphomas.
Asunto(s)
Fosfoproteínas Fosfatasas/inmunología , Transducción de Señal , Linfocitos T/inmunología , Biocatálisis , Diferenciación Celular , Células Cultivadas , Holoenzimas/inmunología , Humanos , Quinasa I-kappa B/inmunología , Quinasa I-kappa B/metabolismo , Activación de Linfocitos , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosfoproteínas Fosfatasas/genética , Interferencia de ARNRESUMEN
The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.
Asunto(s)
Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Transducción de Señal , Receptor fas/química , Apoptosis , Caspasa 10/metabolismo , Caspasa 8/metabolismo , Dimerización , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Células HeLa , Humanos , Espectrometría de Masas/métodos , Microscopía Fluorescente/métodos , Modelos Biológicos , Modelos Teóricos , Receptor fas/metabolismoRESUMEN
Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4+ T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions.
Asunto(s)
Inmunoprecipitación/métodos , Complejos Multiproteicos/aislamiento & purificación , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Apoptosis , Biotinilación , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Inmunoprecipitación de Cromatina , Técnicas de Silenciamiento del Gen , Humanos , Carioferinas/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Proteína Fosfatasa 2C/metabolismo , Proteómica , Receptores Citoplasmáticos y Nucleares/metabolismo , Reproducibilidad de los Resultados , Receptor fas/metabolismoRESUMEN
Despite intensive efforts in recent years, a curative therapy for cutaneous T-cell lymphoma (CTCL) has not yet been developed. Therefore, the establishment of new therapeutic approaches with higher efficacy rates and milder side effects is strongly desired. A characteristic feature of the malignant T-cell population in CTCL is resistance toward cell death resulting from constitutive NF-κB activation. Therefore, NF-κB-dependent cell death resistance represents an interesting therapeutic target in CTCL because an NF-κB-directed therapy would leave bystander T cells widely unaffected. We investigated the effects of dimethyl fumarate (DMF) on CTCL cells in vitro and in vivo. DMF induced cell death in primary patient-derived CD4(+) cells and CTCL cell lines, but hardly in T cells from healthy donors. DMF-induced cell death was linked specifically to NF-κB inhibition. To study the impact of DMF in vivo, we developed 2 CTCL xenograft mouse models with different cutaneous localizations of the T-cell infiltrate. DMF treatment delayed the growth of CTCL tumors and prevented formation of distant metastases. In addition, DMF induced increased cell death in primary CTCL tumors and in liver metastases. In summary, DMF treatment represents a remarkable therapeutic option in CTCL because it restores CTCL apoptosis in vitro and in preclinical models in vivo and prevents spreading of the disease to distant sites. DMF treatment is of particular promise in CTCL because DMF is already in successful clinical use in the treatment of psoriasis and multiple sclerosis allowing fast translation into clinical studies in CTCL.
Asunto(s)
Apoptosis/efectos de los fármacos , Dimetilfumarato/uso terapéutico , Inmunosupresores/uso terapéutico , Linfoma Cutáneo de Células T/tratamiento farmacológico , FN-kappa B/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Piel/efectos de los fármacos , Animales , Humanos , Linfoma Cutáneo de Células T/inmunología , Linfoma Cutáneo de Células T/patología , Ratones , FN-kappa B/inmunología , Metástasis de la Neoplasia/inmunología , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/prevención & control , Transducción de Señal/efectos de los fármacos , Piel/inmunología , Piel/patología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
Chronic lymphocytic leukemia is a malignancy of mature B cells that strongly depend on microenvironmental factors, and their deprivation has been identified as a promising treatment approach for this incurable disease. Cytokine array screening of 247 chronic lymphocytic leukemia serum samples revealed elevated levels of tumor necrosis factor (TNF) receptor-1 which were associated with poor clinical outcome. We detected a microenvironment-induced expression of TNF receptor-1 in chronic lymphocytic leukemia cells in vitro, and an aberrantly high expression of this receptor in the proliferation centers of patients' lymph nodes. Stimulation of TNF receptor-1 with TNF-α enhanced nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activity and viability of chronic lymphocytic leukemia cells, which was inhibited by wogonin. The therapeutic effects of wogonin were analyzed in mice after adoptive transfer of Eµ-T-cell leukemia 1 (TCL1) leukemic cells. Wogonin treatment prevented leukemia development when given early after transplantation. The treatment of full-blown leukemia resulted in the loss of the TNF receptor-1 on chronic lymphocytic leukemia cells and their mobilization to blood. Targeting TNF receptor-1 signaling is therefore proposed for the treatment of chronic lymphocytic leukemia.
Asunto(s)
Flavanonas/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Traslado Adoptivo , Animales , Técnicas de Cocultivo , Humanos , Leucemia/patología , Leucemia/prevención & control , Leucemia Linfocítica Crónica de Células B/metabolismo , Ganglios Linfáticos/metabolismo , Ratones , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Células Tumorales Cultivadas , Microambiente Tumoral/efectos de los fármacosRESUMEN
During the course of an immune response, antigen-reactive T cells clonally expand and then are removed by apoptosis to maintain immune homeostasis. Life and death of T cells is determined by multiple factors, such as T-cell receptor triggering, co-stimulation or cytokine signalling, and by molecules, such as caspase-8 (FLICE)-like inhibitory protein (FLIP) and haematopoietic progenitor kinase 1 (HPK1), which regulate the nuclear factor-kappaB (NF-kappaB) pathway. Here, we discuss the concepts of activation-induced cell death (AICD) and activated cell-autonomous death (ACAD) in the regulation of life and death in T cells.
Asunto(s)
Apoptosis/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Animales , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo , Humanos , Transducción de Señal/inmunologíaRESUMEN
Immunological tolerance is constantly being maintained in the periphery by dendritic cells processing material from apoptotic cells (ACs) in the steady-state. Although research has focused on the uptake of ACs by phagocytes, tolerogenic signals exposed by the ACs are much less well defined. In this article, we show that the annexin (Anx) family members AnxA5 and AnxA13 translocate to the surface of ACs to function as redundant tolerogenic signals in vitro and in vivo. Exposure of bone marrow-derived dendritic cells to AnxA5 or AnxA13 in vitro resulted in the inhibition of both proinflammatory cytokine secretion and the upregulation of costimulatory molecules upon TLR stimulation. The highly conserved Anx core domain was sufficient to mediate these effects, whereas recognition by N-formyl peptide receptor family members was dispensable. In vivo, coinjection of OVA-expressing and Anx-expressing ACs prevented induction of Ag-specific CD8(+) T cells. Moreover, mice immunized with Anx-expressing ACs became refractory to an antigenic challenge. These results suggest that several Anxs contribute to AC-induced suppression of dendritic cell activation. Therefore, manipulating Anx-mediated immunosuppression may prove beneficial for patients with cancer or autoimmune diseases and chronic inflammatory disorders.
Asunto(s)
Anexina A5/inmunología , Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Animales , Anexina A1/genética , Anexina A5/farmacología , Enfermedades Autoinmunes/inmunología , Células de la Médula Ósea/inmunología , Citocinas/metabolismo , Femenino , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/inmunología , Fagocitos/inmunología , Estructura Terciaria de Proteína , Receptores de Formil Péptido/inmunología , Receptores Toll-Like/inmunologíaRESUMEN
Monoclonal antibodies directed to the B-cell-specific CD20-antigen are successfully used for the treatment of lymphomas and autoimmune diseases. Here, we compare the anti-B-cell activity of three different antibodies directed to CD20: (i) a chimeric, monospecific antibody, (ii) an Fc-optimized variant thereof, and (iii) a bispecific CD20×CD95-antibody in a newly developed recombinant format, termed Fabsc. The bispecific antibody specifically triggers the CD95 death receptor on malignant, as well as activated, normal B-cells. We found that the capability of this antibody to suppress the growth of malignant B-cells in vitro and in vivo and to specifically deplete normal, activated B-cells from peripheral blood mononuclear cell (PBMC) cultures was superior to that of the Fc-optimized monospecific antibody. This antibody in turn was more effective than its nonoptimized variant. Moreover, the bispecific antibody was the only reagent capable of significantly suppressing antibody production in vitro. Our findings imply that the bispecific CD20×CD95-antibody might become a new, prototypical reagent for the treatment of B-cell-mediated autoimmune disease.
Asunto(s)
Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Antígenos CD20/inmunología , Linfocitos B/efectos de los fármacos , Linfoma de Células B/tratamiento farmacológico , Receptor fas/inmunología , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/farmacología , Linfocitos B/inmunología , Linfocitos B/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Linfoma de Células B/inmunología , RatonesRESUMEN
Over-expression of Bcl-2, Bcl-xL and Bcl-w is frequently associated with cancer resistance to chemotherapy. Navitoclax (ABT-263), an orally bio-available small-molecule mimetic of the Bcl-2 homology domain 3, specifically inhibits Bcl-2, Bcl-xL and Bcl-w. Despite promising results obtained from the clinical trials, the use of Navitoclax in patients is dose-limited due to induction of death of platelets via inhibition of Bcl-xL and subsequent thrombocytopenia. This side effect limits the use of Navitoclax in low doses and to very sensitive tumors. In this study, we show that HTLV-1-associated adult T-cell leukemia/lymphoma (ATL) cells, which over-express Bcl-2, Bcl-xL and Bcl-w, show a 10- to 20-fold higher sensitivity (EC50 = â¼ 25-50 nM) to Navitoclax compared to non-HTLV-1-associated leukemic cells (EC50 = â¼ 1 µM). Investigation of the molecular mechanisms revealed that the HTLV-1 oncogenic protein Tax up-regulates expression of the pro-apoptotic protein Bax which enhances the therapeutic efficacy of Navitoclax. In addition, we show that agents that inhibit the transcription elongation or translation initiation such as Wogonin and Roc-A can further decrease the effective dose of Navitoclax. Our study suggests that HTLV-1 ATL may be a good candidate disease for low dose Navitoclax therapy and probably with less risk of thrombocytopenia.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Infecciones por HTLV-I/patología , Leucemia-Linfoma de Células T del Adulto/patología , Sulfonamidas/farmacología , Proteína X Asociada a bcl-2/biosíntesis , Adulto , Western Blotting , Línea Celular Tumoral , Infecciones por HTLV-I/metabolismo , Virus Linfotrópico T Tipo 1 Humano , Humanos , Leucemia-Linfoma de Células T del Adulto/metabolismo , Leucemia-Linfoma de Células T del Adulto/virología , TransfecciónRESUMEN
Combination therapy of cancer is based on the cumulative effects mediated by several drugs. Although molecular mechanisms of action of each particular drug are partially elucidated, understanding of the dynamic cross-talk between different cell death pathways at the quantitative level induced by combination therapy is still missing. Here, we exemplified this question for the death receptor (DR) networks in pancreatic cancer cells. We demonstrate that the combined action of CD95L and gemcitabine in pancreatic cancer cells leads to the simultaneous induction of caspase-dependent and caspase-independent cell death. The pro-apoptotic effects are mediated through down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, while caspase-independent cell death was blocked by inhibition of the kinase activity of RIP1. Furthermore, gemcitabine co-treatment strongly increased the amount of cells undergoing CD95-induced RIP1-regulated necrosis. Imaging flow cytometry has enabled us to get the quantitative insights into the apoptosis-necroptosis network and reveal that the majority of the cells upon the CD95L/gemcitabine co-treatment undergoes necroptosis. Our data underlie the importance of the quantitative understanding of the interplay between different cell death modalities, which is essential for the development of anti-cancer therapies. Taken together, our results are important for combination therapy of pancreatic cancer comprising chemotherapeutics and DR-agonists and offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-type-mediated death.
Asunto(s)
Apoptosis/efectos de los fármacos , Desoxicitidina/análogos & derivados , Proteína Ligando Fas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Neoplasias Pancreáticas/patología , Proteínas de Unión al ARN/metabolismo , Adenosina Trifosfato/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Western Blotting , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Terapia Combinada , Desoxicitidina/farmacología , Humanos , Necrosis , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Receptor fas/metabolismo , GemcitabinaRESUMEN
NF-κB is an important transcription factor in the immune system, and aberrant NF-κB activity contributes to malignant diseases and autoimmunity. In T cells, NF-κB is activated upon TCR stimulation, and signal transduction to NF-κB activation is triggered by a cascade of phosphorylation events. However, fine-tuning and termination of TCR signaling are only partially understood. Phosphatases oppose the role of kinases by removing phosphate moieties. The catalytic activity of the protein phosphatase PP2A has been implicated in the regulation of NF-κB. PP2A acts in trimeric complexes in which the catalytic subunit is promiscuous and the regulatory subunit confers substrate specificity. To understand and eventually target NF-κB-specific PP2A functions it is essential to define the regulatory PP2A subunit involved. So far, the regulatory PP2A subunit that mediates NF-κB suppression in T cells remained undefined. By performing a siRNA screen in Jurkat T cells harboring a NF-κB-responsive luciferase reporter, we identified the PP2A regulatory subunit B56γ as negative regulator of NF-κB in TCR signaling. B56γ was strongly up-regulated upon primary human T cell activation, and B56γ silencing induced increased IκB kinase (IKK) and IκBα phosphorylation upon TCR stimulation. B56γ silencing enhanced NF-κB activity, resulting in increased NF-κB target gene expression including the T cell cytokine IL-2. In addition, T cell proliferation was increased upon B56γ silencing. These data help to understand the physiology of PP2A function in T cells and the pathophysiology of diseases involving PP2A and NF-κB.
Asunto(s)
FN-kappa B/inmunología , Proteína Fosfatasa 2/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Western Blotting , Células Cultivadas , Expresión Génica/genética , Expresión Génica/inmunología , Humanos , Quinasa I-kappa B/inmunología , Quinasa I-kappa B/metabolismo , Proteínas I-kappa B/inmunología , Proteínas I-kappa B/metabolismo , Interleucina-2/genética , Interleucina-2/inmunología , Interleucina-2/metabolismo , Isoenzimas/inmunología , Isoenzimas/metabolismo , Células Jurkat , Activación de Linfocitos/inmunología , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Fosforilación/inmunología , Proteína Quinasa C/inmunología , Proteína Quinasa C/metabolismo , Proteína Quinasa C-theta , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Interferencia de ARN , Receptores de Antígenos de Linfocitos T/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Natural compounds are an important source for drug development. With an increasing cancer rate worldwide there is an urgent quest for new anti-cancer drugs. In this study, we show that a group of dolabrane-type of diterpenes, collectively named tagalsins, isolated from the Chinese mangrove genus Ceriops has potent cytotoxicity on a panel of hematologic cancer cells. Investigation of the molecular mechanisms by which tagalsins kill malignant cells revealed that it induces a ROS-mediated damage of DNA. This event leads to apoptosis induction and blockage of cell cycle progression at S-G2 phase via activation of the ATM/ATR-Chk1/Chk2 check point pathway. We further show that tagalsins suppress growth of human T-cell leukemia xenografts in vivo. Tagalsins show only minor toxicity on healthy cells and are well tolerated by mice. Our study shows a therapeutic potential of tagalsins for the treatment of hematologic malignancies and a new source of anticancer drugs.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Quinasa de Punto de Control 2/metabolismo , Diterpenos/farmacología , Proteínas Quinasas/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN/efectos de los fármacos , Fase G2/efectos de los fármacos , Humanos , Células Jurkat , Leucemia de Células T/tratamiento farmacológico , Leucemia de Células T/metabolismo , Ratones , Especies Reactivas de Oxígeno/metabolismo , Rhizophoraceae/química , Fase S/efectos de los fármacos , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Tumor initiation, progression and resistance to therapies are tightly associated with over-expression of anti-apoptotic proteins Bcl-2, Bcl-x(L), Bcl-w and Mcl-1. ABT-263 (Navitoclax), an orally bio-available small-molecule mimetic of the Bcl-2 homology domain 3, inhibits Bcl-2, Bcl-x(L), and Bcl-w and has shown anti-cancer effects mainly on lymphomas and lymphocytic leukemia. Despite promising results obtained from the clinical trials, the use of ABT-263 in patients is dose-limited due to causing thrombocytopenia via inhibition of Bcl-x(L) in platelets. ABT-199 specifically inhibits Bcl-2; however, its use is limited to tumors over-expressing only Bcl-2. Besides, many tumors resist treatment due to high levels of Mcl-1 expression or develop resistance via up-regulation of Mcl-1 during long-term exposure. These obstacles highlight the demand to improve the ABT-263-based therapy. In this study, we show that anti-cancer flavones, e.g., wogonin, baicalein, apigenin, chrysin and luteolin enhance ABT-263-induced apoptosis in different cancer cell lines and in primary AML and ALL cells by down-regulation of Mcl-1 expression. Importantly, wogonin does not enhance the toxicity of ABT-263 to proliferating normal T cells and thrombocytes. Wogonin also potentiates the lethality of ABT-263 in cancer cells which have acquired resistance to ABT-263. Furthermore, we show that combination of wogonin with ABT-263 promotes in vivo tumor regression in a human T-cell leukemia xenograft mouse model. Our study demonstrates that wogonin (and related flavones) reduce the effective dose of ABT-263 thereby possibly decreasing the risk of adverse side effects.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Flavanonas/farmacología , Flavonas/farmacología , Sulfonamidas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The endogenous metabolite of estradiol, 2-Methoxyestradiol (2ME2), is an antimitotic and antiangiogenic cancer drug candidate that also exhibits disease-modifying activity in animal models of rheumatoid arthritis (RA). We found that 2ME2 dramatically suppresses development of mouse experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). 2ME2 inhibits in vitro lymphocyte activation, cytokine production, and proliferation in a dose-dependent fashion. 2ME2 treatment of lymphocytes specifically reduced the nuclear translocation and transcriptional activity of nuclear factor of activated T-cells (NFAT) c1, whereas NF-κB and activator protein 1 (AP-1) activation were not adversely affected. We therefore propose that 2ME2 attenuates EAE through disruption of the NFAT pathway and subsequent lymphocyte activation. By extension, our findings provide a molecular rationale for the use of 2ME2 as a tolerable oral immunomodulatory agent for the treatment of autoimmune disorders such as MS in humans.
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Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Estradiol/análogos & derivados , 2-Metoxiestradiol , Animales , Autoinmunidad , Linfocitos T CD4-Positivos/citología , Citocinas/biosíntesis , Estradiol/farmacología , Humanos , Activación de Linfocitos , Linfocitos/citología , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Moduladores de Tubulina/farmacologíaRESUMEN
Interleukin-4 (IL-4) is crucial for the differentiation of naive T helper (T(H)) cells into the T(H)2 effector cells that promote humoral (antibody) immunity and provide protection against intestinal helminths. IL-4 also has a central role in the pathogenesis of allergic inflammation. Many transcription factors are involved in the regulation of expression of the gene encoding IL-4. Initiation of transcription of the gene encoding IL-4 in naive T(H) cells is regulated by the T(H)2-specific transcription factor GATA3, whereas acute expression of the gene encoding IL-4 in T(H)2 cells is mediated by inducible, ubiquitous transcription factors after antigen encounter. This review focuses on acute activation of the gene encoding IL-4 in T cells and discusses therapeutic perspectives at the transcriptional level.
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Interleucina-4/genética , Proteínas Nucleares , Linfocitos T/inmunología , Animales , Proteínas de Unión al ADN/inmunología , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hipersensibilidad/etiología , Inmunosupresores/farmacología , Interleucina-4/biosíntesis , Activación de Linfocitos , Factores de Transcripción NFATC , Regiones Promotoras Genéticas , Factores de Transcripción/inmunologíaRESUMEN
The role of Wilms' tumor suppressor 1 (WT1) in leukemogenesis has been investigated mostly in acute (AML) and chronic (CML) myeloid leukemias. So far, its oncogenic role has been controversially discussed because both overexpression and inactivating mutations are found. A recent study on primary samples from patients with acute T-cell leukemia (T-ALL) revealed that most of them do not express WT1 proteins although they express WT1 mRNA. In our study, we investigated WT-1 expression in ten T-ALL cell lines established from leukemia/lymphoma patients. We show that consistent with the finding in primary T-ALL cells, most of the leukemic T-cell lines tested do not overexpress WT1 proteins. We found that leukemic T-cells overexpressing WT1 protein produce higher levels of CD95L and show elevated CD95L-mediated activation-induced cell death (AICD) compared to cells lacking or expressing low levels of WT1. Ectopic expression of WT1 in the WT1-nonexpressing leukemic T-cell line increases CD95L expression and elevates activation-induced apoptosis, whereas silencing WT1 expression in the WT1-overexpressing leukemic T-cell line by siRNA confers reduced CD95L expression and reduction in AICD. Chromatin immunoprecipitation and luciferase-promoter reporter analysis demonstrate that WT1 binds to and enhances CD95L promoter activity through the Egr-binding sites. Our study provides a new role of WT1 in regulation of CD95L-mediated cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteína Ligando Fas/metabolismo , Leucemia de Células T/patología , Factores de Transcripción/metabolismo , Proteínas WT1/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Proteína Ligando Fas/genética , Humanos , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Activación de Linfocitos , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteínas WT1/antagonistas & inhibidores , Proteínas WT1/genéticaRESUMEN
Targeting the cancer cell cycle machinery is an important strategy for cancer treatment. Cdc25A is an essential regulator of cycle progression and checkpoint response. Over-expression of Cdc25A occurs often in human cancers. In this study, we show that Rocaglamide-A (Roc-A), a natural anticancer compound isolated from the medicinal plant Aglaia, induces a rapid phosphorylation of Cdc25A and its subsequent degradation and, thereby, blocks cell cycle progression of tumor cells at the G1-S phase. Roc-A has previously been shown to inhibit tumor proliferation by blocking protein synthesis. In this study, we demonstrate that besides the translation inhibition Roc-A can induce a rapid degradation of Cdc25A by activation of the ATM/ATR-Chk1/Chk2 checkpoint pathway. However, Roc-A has no influence on cell cycle progression in proliferating normal T lymphocytes. Investigation of the molecular basis of tumor selectivity of Roc-A by a time-resolved microarray analysis of leukemic vs. proliferating normal T lymphocytes revealed that Roc-A activates different sets of genes in tumor cells compared with normal cells. In particular, Roc-A selectively stimulates a set of genes responsive to DNA replication stress in leukemic but not in normal T lymphocytes. These findings further support the development of Rocaglamide for antitumor therapy.