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1.
Anticancer Drugs ; 26(7): 716-27, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25850884

RESUMO

Glioblastoma is a disease characterized by rapid invasive tumour growth. Studies on the proapoptotic CD95/CD95L signalling pathway recently suggested a significant contribution of CD95 signalling towards the high degree of motility in glioma cells. Apogenix has developed APG101, a clinical phase II compound designed to bind and neutralize CD95L, and thus to interfere with CD95/CD95L-based signalling. APG101 has shown clinical efficacy in a controlled randomized phase II trial in patients with recurrent glioma. Because APG101 is not cytotoxic to tumour cells in vitro, we postulated that the anti-invasive function of APG101 is the main mechanism of action for this compound. Using three-dimensional spheroid invasion assays in vitro and in murine brain tissue cultures, we found that knockdown of endogenous CD95L reduced the invasive phenotype in our two glioblastoma model cell lines U87-MG and U251-MG. Invasion was restored in CD95L knockdown cells upon the addition of soluble recombinant CD95L and this effect was inhibited by APG101. We conclude that CD95L from autocrine and paracrine sources contributes towards the invasive phenotype of glioblastoma cells and that APG101 acts as a suppressor of proinvasive signalling by the CD95/CD95L pathway in glioblastoma.


Assuntos
Neoplasias Encefálicas/patologia , Proteína Ligante Fas/metabolismo , Glioblastoma/patologia , Imunoglobulina G/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Esferoides Celulares/efeitos dos fármacos , Receptor fas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Glioblastoma/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Transdução de Sinais , Esferoides Celulares/patologia , Esferoides Celulares/fisiologia , Receptor fas/genética , Receptor fas/farmacologia
2.
Exp Dermatol ; 22(10): 656-9, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24079735

RESUMO

Cutaneous lupus erythematosus (CLE) is a heterogeneous autoimmune disease. Different pathogenetic mechanisms, including the accumulation of apoptotic keratinocytes in CLE, have been reported. Therefore, we investigated whether CLE and other inflammatory skin diseases differ with regard to the epidermal expression of molecules that are crucial for the initiation and regulation of apoptosis. In this study, 241 skin biopsies from patients with CLE, psoriasis (PSO), lichen planus (LP) and healthy controls (HCs) were analysed immunohistochemically using the tissue microarray (TMA) technique. The TUNEL assay and anti-activated caspase-3 antibodies revealed a significant increase of apoptotic keratinocytes in CLE lesions compared with HCs. Furthermore, we detected a significant increase in the epidermal expression of CD95 in CLE specimens compared with PSO, LP and HCs. These data suggest that the accumulation of apoptotic keratinocytes in CLE might be due to the increased epidermal expression of CD95, resulting in increased activity of the extrinsic apoptotic pathway in the disease.


Assuntos
Apoptose , Regulação da Expressão Gênica , Lúpus Eritematoso Cutâneo/imunologia , Lúpus Eritematoso Cutâneo/metabolismo , Pele/metabolismo , Pele/patologia , Adulto , Idoso , Biópsia , Caspase 3/metabolismo , Epiderme/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Inflamação/patologia , Queratinócitos/citologia , Masculino , Pessoa de Meia-Idade , Análise Serial de Tecidos , Receptor fas/metabolismo
3.
BMC Cancer ; 13: 532, 2013 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-24209510

RESUMO

BACKGROUND: An imbalance between proliferation and apoptosis is one of the main features of carcinogenesis. TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis upon binding to the TRAIL death receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2, whereas binding to TRAIL-R3 and TRAIL-R4 might promote cell survival and proliferation. The anti-tumor activity of TRAIL-R1 and TRAIL-R2 agonists is currently investigated in clinical trials. To gain further insight into the regulation of apoptosis in hepatocellular carcinoma (HCC), we investigated the TRAIL pathway and the regulators of apoptosis caspase-8, Bcl-xL and Mcl-1 in patients with HCC regarding patient survival. METHODS: We analyzed 157 hepatocellular carcinoma patients who underwent partial liver resection or orthotopic liver transplantation and healthy control liver tissue using immunohistochemistry on tissue microarrays for the expression of TRAIL-R1 to TRAIL-R4, caspase-8, Bcl-xL and Mcl-1. Immunohistochemical data were evaluated for potential associations with clinico-pathological parameters and survival. RESULTS: Whereas TRAIL-R1 was downregulated in HCC in comparison to normal liver tissue, TRAIL-R2 and -R4 were upregulated in HCC, especially in G2 and G3 tumors. TRAIL-R1 downregulation and upregulation of TRAIL-R2 and TRAIL-R4 correlated with tumor dedifferentiation (G2/G3). TRAIL-R3, Bcl-xL and Mcl-1 showed no differential expression in tumor tissue compared to normal tissue. The expression levels of TRAIL receptors did not correlate with patient survival after partial hepatectomy. Interestingly, in tumor tissue, but not in normal hepatocytes, caspase-8 showed a strong nuclear staining. Low cytosolic and high nuclear staining intensity of caspase-8 significantly correlated with impaired survival after partial hepatectomy, which, for cytosolic caspase-8, was independent from tumor grade. CONCLUSIONS: Assessment of TRAIL-receptor expression patterns may have therapeutic implications for the use of TRAIL receptor agonists in HCC therapy. Tumor-specific nuclear localisation of caspase-8 in HCC suggests an apoptosis-independent function of caspase-8 and correlates with patient survival.


Assuntos
Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Caspase 8/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Núcleo Celular/metabolismo , Citosol/metabolismo , Feminino , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Transporte Proteico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
4.
Front Immunol ; 14: 1160116, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37304285

RESUMO

Introduction: The ability to modulate and enhance the anti-tumor immune responses is critical in developing novel therapies in cancer. The Tumor Necrosis Factor (TNF) Receptor Super Family (TNFRSF) are potentially excellent targets for modulation which result in specific anti-tumor immune responses. CD40 is a member of the TNFRSF and several clinical therapies are under development. CD40 signaling plays a pivotal role in regulating the immune system from B cell responses to myeloid cell driven activation of T cells. The CD40 signaling axis is well characterized and here we compare next generation HERA-Ligands to conventional monoclonal antibody based immune modulation for the treatment of cancer. Methods & results: HERA-CD40L is a novel molecule that targets CD40 mediated signal transduction and demonstrates a clear mode of action in generating an activated receptor complex via recruitment of TRAFs, cIAP1, and HOIP, leading to TRAF2 phosphorylation and ultimately resulting in the enhanced activation of key inflammatory/survival pathway and transcription factors such asNFkB, AKT, p38, ERK1/2, JNK, and STAT1 in dendritic cells. Furthermore, HERA-CD40L demonstrated a strong modulation of the tumor microenvironment (TME) via the increase in intratumoral CD8+ T cells and the functional switch from pro-tumor macrophages (TAMs) to anti-tumor macrophages that together results in a significant reduction of tumor growth in a CT26 mouse model. Furthermore, radiotherapy which may have an immunosuppressive modulation of the TME, was shown to have an immunostimulatory effect in combination with HERA-CD40L. Radiotherapy in combination with HERA-CD40L treatment resulted in an increase in detected intratumoral CD4+/8+ T cells compared to RT alone and, additionally, the repolarization of TAMs was also observed, resulting in an inhibition of tumor growth in a TRAMP-C1 mouse model. Discussion: Taken together, HERA-CD40L resulted in activating signal transduction mechanisms in dendritic cells, resulting in an increase in intratumoral T cells and manipulation of the TME to be pro-inflammatory, repolarizing M2 macrophages to M1, enhancing tumor control.


Assuntos
Ligante de CD40 , Neoplasias , Animais , Camundongos , Antígenos CD40 , Células Apresentadoras de Antígenos , Macrófagos , Neoplasias/radioterapia , Modelos Animais de Doenças , Microambiente Tumoral
5.
Exp Dermatol ; 20(7): 600-2, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21692859

RESUMO

Recently, it was discovered that the receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL) is part of an important signal transduction pathway for tissue homoeostasis. Therefore, we were interested in investigating RANKL expression in the epidermis of skin lesions from patients with different subtypes of cutaneous lupus erythematosus (CLE) and psoriasis as well as normal healthy donors. Using the tissue microarray technique, skin biopsy specimens were evaluated by immunohistochemistry. RANKL showed a significantly increased expression in the epidermis of skin biopsy specimens from patients with psoriasis (median: 4, range: 0-5) compared to patients with CLE (median: 0, range: 0-4) (P<0.001). No significant differences in epidermal RANKL expression between the CLE subtypes were detected. These data show a different expression of RANKL in the epidermis of skin lesions from patients with CLE compared to those with psoriasis suggesting that RANKL might play an important role in the pathogenesis of the disease.


Assuntos
Lúpus Eritematoso Cutâneo/metabolismo , Psoríase/metabolismo , Ligante RANK/metabolismo , Pele/metabolismo , Análise Serial de Tecidos , Adulto , Idoso , Biópsia , Epiderme/metabolismo , Feminino , Humanos , Queratinócitos/metabolismo , Lúpus Eritematoso Cutâneo/diagnóstico , Lúpus Eritematoso Discoide/metabolismo , Masculino , Pessoa de Meia-Idade
6.
Biochemistry ; 49(7): 1418-24, 2010 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-20082520

RESUMO

Histone deacetylases have proven to be attractive novel targets for the treatment of cancer. The first inhibitor of histone deacetylases was approved for the treatment of cutaneous T-cell lymphoma in 2006. The identification of new lead structures with improved effectiveness and fewer side effects is necessary. This report investigates the mechanism of inhibition of a histone deacetylase-like amidohydrolase by stopped-flow and equilibrium titration techniques. The interaction between the inhibitor (E)-3-(furan-2-yl)-N-hydroxyacrylamide and the enzyme generates a fluorescence resonance energy transfer from the intrinsic tryptophan residues of the enzyme to the chromophore of the inhibitor. The apparent equilibrium binding constant was determined to be 1.9 muM. Several independent experimental results provide evidence of the existence of solely one HDAH conformer. The association kinetics showed two phases representing two unimolecular processes. Kinetic arguments and accurate investigation of the very fast time range suggest a fast pre-equilibrium, in which the inhibitor binds to the surface of the enzyme. In the next step, the first complex undergoes a conformational change that allows the inhibitor to translocate into the active site. Finally, the intermediate complex is stabilized by another conformational rearrangement. All kinetic data are in agreement with a reversible three-step mechanism and analyzed using a global fit, yielding the association constant of the pre-equilibrium (K(1) = 0.28 x 10(6) M(-1)) and the forward and reverse rate constants of the consecutive conformational changes (k(2) = 6.6 s(-1), k(-2) = 1.5 s(-1), k(3) = 0.8 s(-1), and k(-3) = 0.3 s(-1)).


Assuntos
Benzofuranos/química , Benzofuranos/metabolismo , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/metabolismo , Alcaligenes/enzimologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Bordetella/enzimologia , Cristalização , Transferência Ressonante de Energia de Fluorescência , Cinética
7.
J Neurooncol ; 97(2): 171-85, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19768634

RESUMO

TNF-related apoptosis-inducing ligand (TRAIL), a promising novel anti-cancer cytokine of the TNF superfamily, and Bortezomib, the first-in-class clinically used proteasome inhibitor, alone or in combination have been shown to efficiently kill numerous tumor cell lines. However, data concerning primary human tumor cells are very rare. Using primary esthesioneuroblastoma cells we analyzed the anti-tumor potential and the mechanism employed by Bortezomib in combination with TRAIL for the treatment of this rare but aggressive tumor. Expression of components of the TRAIL pathway was analyzed in tumor specimens and isolated primary tumor cells at the protein level. Cells were treated with TRAIL, Bortezomib, and a combination thereof, and apoptosis induction was quantified. Clonogenicity assays were performed to elucidate the long-term effect of this treatment. Despite expressing all components of the TRAIL pathway, freshly isolated primary esthesioneuroblastoma cells were completely resistant to TRAIL-induced apoptosis. They could, however, be very efficiently sensitized by subtoxic doses of Bortezomib. The influence of Bortezomib on the TRAIL pathway was analyzed and showed upregulation of TRAIL death receptor expression, enhancement of the TRAIL death-inducing signaling complex (DISC), and downregulation of anti-apoptotic proteins of the TRAIL pathway. Of clinical relevance, TRAIL-resistant primary tumor cells could be repeatedly sensitized by Bortezomib, providing the basis for repeated clinical application schedules. This is the first report on the highly synergistic induction of apoptosis in primary esthesioneuroblastoma cells by Bortezomib and TRAIL. This combination, therefore, represents a promising novel therapeutic option for esthesioneuroblastoma.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Estesioneuroblastoma Olfatório/metabolismo , Neoplasias Nasais/metabolismo , Pirazinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Adulto , Apoptose/fisiologia , Western Blotting , Bortezomib , Separação Celular , Células Cultivadas , Sinergismo Farmacológico , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Masculino , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
8.
Clin Cancer Res ; 15(2): 650-9, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19147771

RESUMO

PURPOSE: The death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R) are involved in immune surveillance and tumor development. Here, we studied a possible association between the expression of TRAIL/TRAIL-Rs and the prognosis in patients with renal cell carcinomas (RCC). EXPERIMENTAL DESIGN: A tissue microarray containing RCC tumor tissue samples and corresponding normal tissue samples from 838 patients was generated. Expression of TRAIL and TRAIL-Rs was examined by immunohistochemistry and the effect of TRAIL and TRAIL-R expression on disease-specific survival was assessed. RESULTS: High TRAIL-R2 expression levels were associated with high-grade RCCs (P < 0.001) and correlated negatively with disease-specific survival (P = 0.01). Similarly, high TRAIL expression was associated with a shorter disease-specific survival (P = 0.01). In contrast, low TRAIL-R4 expression was associated with high-stage RCCs (P < 0.001) as well as with the incidence of distant metastasis (P = 0.03) and correlated negatively with disease-specific survival (P = 0.02). In patients without distant metastasis, multivariate Cox regression analyses revealed that TRAIL-R2 and TRAIL are independent prognostic factors for cancer-specific survival (in addition to tumor extent, regional lymph node metastasis, grade of malignancy, and type of surgery). CONCLUSION: High TRAIL-R2, high TRAIL, and low TRAIL-R4 expression levels are associated with a worse disease-specific survival in patients with RCCs. Therefore, the assessment of TRAIL/TRAIL-R expression offers valuable prognostic information that could be used to select patients for adjuvant therapy studies. Moreover, our findings are of relevance for a potential experimental therapeutic administration of TRAIL-R agonists in patients with RCCs.


Assuntos
Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/diagnóstico , Neoplasias Renais/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Idoso , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Modelos de Riscos Proporcionais , Resultado do Tratamento
10.
J Immunother Cancer ; 7(1): 191, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324216

RESUMO

BACKGROUND: Glucocorticoid-induced TNFR-related protein (TNFRSF18, GITR, CD357), expressed by T cells, and its ligand (TNFSF18, GITRL), expressed by myeloid populations, provide co-stimulatory signals that boost T cell activity. Due to the important role that GITR plays in regulating immune functions, agonistic stimulation of GITR is a promising therapeutic concept. Multiple strategies to induce GITR signaling have been investigated. The limited clinical efficacy of antibody-based GITR agonists results from structural and functional characteristics of antibodies that are unsuitable for stimulating the well-defined trimeric members of the TNFRSF. METHODS: To overcome limitations of antibody-based TNFRSF agonists, we have developed HERA-GITRL, a fully human hexavalent TNF receptor agonist (HERA) targeting GITR and mimicking the natural signaling concept. HERA-GITRL is composed of a trivalent but single-chain GITRL-receptor-binding-domain (scGITRL-RBD) unit fused to an IgG1 derived silenced Fc-domain serving as dimerization scaffold. A specific mouse surrogate, mmHERA-GITRL, was also generated to examine in vivo activity in respective mouse tumor models. RESULTS: For functional characterization of HERA-GITRL in vitro, human immune cells were isolated from healthy-donor blood and stimulated with anti-CD3 antibody in the presence of HERA-GITRL. Consistently, HERA-GITRL increased the activity of T cells, including proliferation and differentiation, even in the presence of regulatory T cells. In line with these findings, mmHERA-GITRL enhanced antigen-specific clonal expansion of both CD4+ (OT-II) and CD8+ (OT-I) T cells in vivo while having no effect on non-specific T cells. In addition, mmHERA-GITRL showed single-agent anti-tumor activity in two subcutaneous syngeneic colon cancer models (CT26wt and MC38-CEA). Importantly, this activity is independent of its FcγR-binding functionality, as both mmHERA-GITRL with a functional Fc- and a silenced Fc-domain showed similar tumor growth inhibition. Finally, in a direct in vitro comparison to a bivalent clinical benchmark anti-GITR antibody and a trivalent GITRL, only the hexavalent HERA-GITRL showed full biological activity independent of additional crosslinking. CONCLUSION: In this manuscript, we describe the development of HERA-GITRL, a true GITR agonist with a clearly defined mechanism of action. By clustering six receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional FcγR-mediated crosslinking.


Assuntos
Receptores do Fator de Necrose Tumoral/agonistas , Anticorpos de Cadeia Única/administração & dosagem , Linfócitos T Reguladores/imunologia , Fatores de Necrose Tumoral/química , Animais , Linhagem Celular Tumoral , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Ativação Linfocitária , Macaca fascicularis , Camundongos , Proteínas Recombinantes de Fusão/imunologia , Transdução de Sinais , Anticorpos de Cadeia Única/imunologia , Fatores de Necrose Tumoral/metabolismo
11.
Eur J Pharmacol ; 579(1-3): 98-103, 2008 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-18054910

RESUMO

The antihypertensive drug doxazosin has been associated with an increased risk for congestive heart failure and cardiomyocyte apoptosis. Human ether-a-go-go-related gene (hERG) K(+) channels, previously shown to be blocked by doxazosin at therapeutically relevant concentrations, represent plasma membrane receptors for the antihypertensive drug. To elucidate the molecular basis for doxazosin-associated pro-apoptotic effects, cell death was studied in human embryonic kidney cells using three independent apoptosis assays. Doxazosin specifically induced apoptosis in hERG-expressing HEK cells, while untransfected control groups were insensitive to treatment with the antihypertensive agent. An unexpected biological mechanism has emerged: binding of doxazosin to its novel membrane receptor, hERG, triggers apoptosis, possibly representing a broader pathophysiological mechanism in drug-induced heart failure.


Assuntos
Anti-Hipertensivos/farmacologia , Apoptose/efeitos dos fármacos , Doxazossina/farmacologia , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Antagonistas Adrenérgicos alfa/farmacologia , Linhagem Celular , Eletrofisiologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Coloração e Rotulagem
12.
Clin Cancer Res ; 13(11): 3403-12, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17545549

RESUMO

PURPOSE: Malignant gliomas are the most aggressive human brain tumors without any curative treatment. The antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in gliomas has thus far only been thoroughly established in tumor cell lines. In the present study, we investigated the therapeutic potential of TRAIL in primary human glioma cells. EXPERIMENTAL DESIGN: We isolated primary tumor cells from 13 astrocytoma and oligoastrocytoma patients of all four WHO grades of malignancy and compared the levels of TRAIL-induced apoptosis induction, long-term tumor cell survival, caspase, and caspase target cleavage. RESULTS: We established a stable culture model for isolated primary human glioma cells. In contrast to cell lines, isolated primary tumor cells from all investigated glioma patients were highly TRAIL resistant. Regardless of the tumor heterogeneity, cotreatment with the proteasome inhibitor bortezomib efficiently sensitized all primary glioma samples for TRAIL-induced apoptosis and tremendously reduced their clonogenic survival. Due to the pleiotropic effect of bortezomib-enhanced TRAIL DISC formation upon TRAIL triggering, down-regulation of cFLIP(L) and activation of the intrinsic apoptosis pathway seem to cooperatively contribute to the antitumor effect of bortezomib/TRAIL cotreatment. CONCLUSION: TRAIL sensitivity of tumor cell lines is not a reliable predictor for the behavior of primary tumor cells. The widespread TRAIL resistance in primary glioma cells described here questions the therapeutic clinical benefit of TRAIL as a monotherapeutic agent. Overcoming TRAIL resistance by bortezomib cotreatment might, however, provide a powerful therapeutic option for glioma patients.


Assuntos
Apoptose , Astrocitoma/tratamento farmacológico , Ácidos Borônicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Imunoterapia/métodos , Pirazinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Adolescente , Adulto , Idoso , Astrocitoma/metabolismo , Bortezomib , Neoplasias Encefálicas/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
13.
Front Oncol ; 8: 387, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30298117

RESUMO

Tumor necrosis factor receptor superfamily member 7 (TNFRSF7, CD27), expressed primarily by T cells, and its ligand CD27L (TNFSF7, CD70) provide co-stimulatory signals that boost T cell activation, differentiation, and survival. Agonistic stimulation of CD27 is therefore a promising therapeutic concept in immuno-oncology intended to boost and sustain T cell driven anti-tumor responses. Endogenous TNFSF/TNFRSF-based signal transmission is a structurally well-defined event that takes place during cell-to-cell-based contacts. It is well-established that the trimeric-trivalent TNFSF-receptor binding domain (TNFSF-RBD) exposed by the conducting cell and the resulting multi-trimer-based receptor clustering on the receiving cell are essential for agonistic signaling. Therefore, we have developed HERA-CD27L, a novel hexavalent TNF receptor agonist (HERA) targeting CD27 and mimicking the natural signaling concept. HERA-CD27L is composed of a trivalent but single-chain CD27L-receptor-binding-domain (scCD27L-RBD) fused to an IgG1 derived silenced Fc-domain serving as dimerization scaffold. The hexavalent agonist significantly boosted antigen-specific T cell responses while having no effect on non-specific T cells and was superior over stabilized recombinant trivalent CD27L. In addition, HERA-CD27L demonstrated potent single-agent anti-tumor efficacy in two different syngeneic tumor models, MC38-CEA and CT26wt. Furthermore, the combination of HERA-CD27L and an anti-PD-1 antibody showed additive anti-tumor effects highlighting the importance of both T cell activation and checkpoint inhibition in anti-tumor immunity. In this manuscript, we describe the development of HERA-CD27L, a true CD27 agonist with a clearly defined forward-signaling mechanism of action.

14.
J Immunother ; 41(9): 385-398, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30273198

RESUMO

CD40 ligand (TNFSF5/CD154/CD40L), a member of the tumor necrosis factor (TNF) superfamily is a key regulator of the immune system. The cognate receptor CD40 (TNFRSF5) is expressed broadly on antigen-presenting cells and many tumor types, and has emerged as an attractive target for immunologic cancer treatment. Most of the CD40 targeting drugs in clinical development are antibodies which display some disadvantages: their activity typically depends on Fcγ receptor-mediated crosslinking, and depletion of CD40-expressing immune cells by antibody-dependent cellular cytotoxicity compromises an efficient antitumor response. To overcome the inadequacies of antibodies, we have developed the hexavalent receptor agonist (HERA) Technology. HERA compounds are fusion proteins composed of 3 receptor binding domains in a single chain arrangement, linked to an Fc-silenced human IgG1 thereby generating a hexavalent molecule. HERA-CD40L provides efficient receptor agonism on CD40-expressing cells and, importantly, does not require FcγR-mediated crosslinking. Strong activation of NFκB signaling was observed upon treatment of B cells with HERA-CD40L. Monocyte treatment with HERA-CD40L promoted differentiation towards the M1 spectrum and repolarization of M2 spectrum macrophages towards the M1 spectrum phenotype. Treatment of in vitro co-cultures of T and B cells with HERA-CD40L-triggered robust antitumor activation of T cells, which depended upon direct interaction with B cells. In contrast, bivalent anti-CD40 antibodies and trivalent soluble CD40L displayed weak activity which critically depended on crosslinking. In vivo, a murine surrogate of HERA-CD40L-stimulated clonal expansion of OT-I-specific murine CD8 T cells and showed single agent antitumor activity in the CD40 syngeneic MC38-CEA mouse model of colorectal cancer, suggesting an involvement of the immune system in controlling tumor growth. We conclude that HERA-CD40L is able to establish robust antitumor immune responses both in vitro and in vivo.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Antígenos CD40/antagonistas & inibidores , Ligante de CD40/farmacologia , Imunoglobulina G/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia
15.
Mol Cancer Res ; 16(5): 767-776, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29453321

RESUMO

CD95 (Fas/APO-1), a death receptor family member, activity has been linked to tumorigenicity in multiple cancers, including glioblastoma multiforme (GBM). A phase II clinical trial on relapsed glioblastoma patients demonstrated that targeted inhibition of CD95 signaling via the CD95 ligand (CD95L) binding and neutralizing Fc-fusion protein APG101 (asunercept) prolonged patient survival. Although CD95 signaling may be relevant for multiple aspects of tumor growth, the mechanism of action of APG101 in glioblastoma is not clear. APG101 action was examined by in vitro proliferation, apoptosis, and invasion assays with human and murine glioma and human microglial cells, as well as in vivo therapy studies with orthotopic gliomas and clinical data. APG101 inhibits CD95L-mediated invasion of glioma cells. APG101 treatment was effective in glioma-bearing mice, independently of the presence or absence of CD4 and CD8 T lymphocytes, which should be sensitive to CD95L. Combined with radiotherapy, APG101 demonstrated a reduction of tumor growth, fewer tumor satellites, reduced activity of matrix metalloproteinases (MMP) as well as prolonged survival of tumor-bearing mice compared with radiotherapy alone. Inhibiting rather than inducing CD95 activity is a break-of-paradigm therapeutic approach for malignant gliomas. Evidence, both in vitro and in vivo, is provided that CD95L-binding fusion protein treatment enhanced the efficacy of radiotherapy and reduced unwanted proinfiltrative effects by reducing metalloproteinase activity by directly affecting the tumor cells.Implications: APG101 (asunercept) successfully used in a controlled phase II glioblastoma trial (NCT01071837) acts anti-invasively by inhibiting matrix metalloproteinase signaling, resulting in additive effects together with radiotherapy and helping to further develop a treatment for this devastating disease. Mol Cancer Res; 16(5); 767-76. ©2018 AACR.


Assuntos
Proteína Ligante Fas/antagonistas & inibidores , Glioblastoma/radioterapia , Imunoglobulina G/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Receptor fas/uso terapêutico , Animais , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Imunoglobulina G/farmacologia , Camundongos , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais
16.
Clin Cancer Res ; 12(8): 2640-6, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16638878

RESUMO

PURPOSE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) exhibits potent antitumor activity on systemic administration in nonhuman primates without deleterious side effects for normal tissue. However, there is a controversy about the potential toxicity of TRAIL on human hepatocytes. The use of different recombinant TRAIL forms only partially explains the contradicting reports on TRAIL sensitivity in primary human hepatocytes (PHH). EXPERIMENTAL DESIGN: To clarify this issue, we comprehensively tested four different recombinant forms of TRAIL for their apoptosis-inducing capacity on PHH obtained from a total of 55 human livers between day 1 and day 8 of in vitro culture. RESULTS: One day after single-cell isolation, all but one recombinant form of TRAIL [i.e., an untagged form of TRAIL (TRAIL.0)] induced apoptosis in PHH. Apoptosis induction by TRAIL in these cells could only be fully inhibited by concomitant blockade of TRAIL receptor 1 and TRAIL receptor 2. At day 4 of in vitro culture, when surrogate markers indicated optimal hepatocyte in vitro function, only high doses of cross-linked FLAG-TRAIL killed PHH whereas the other three recombinant TRAIL forms did not. Strikingly, cotreatment of day 4 PHH with cisplatin sensitized for TRAIL-induced apoptosis whereas 5-fluorouracil, etoposide, gemcitabine, irinotecan, or oxaliplatin, which are commonly used in the treatment of gastrointestinal cancers, did not. CONCLUSION: Our data show that whereas TRAIL alone or together with selected chemotherapeutic drugs seems to be safe, the combination of TRAIL with cisplatin is toxic to PHH.


Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/farmacologia , Apoptose/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Glicoproteínas de Membrana/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Western Blotting , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cisplatino/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Etoposídeo/farmacologia , Fluoruracila/farmacologia , Proteínas Ligadas por GPI , Expressão Gênica/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Imuno-Histoquímica , Irinotecano , Glicoproteínas de Membrana/genética , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Membro 10c de Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ligante Indutor de Apoptose Relacionado a TNF , Fatores de Tempo , Receptores Chamariz do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/genética , Gencitabina
17.
Cell Death Discov ; 2: 16081, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28028438

RESUMO

In recent years, new treatment options for malignant melanoma patients have enhanced the overall survival for selected patients. Despite new hope, most melanoma patients still relapse with drug-resistant tumors or experience intrinsic resistance to the therapy. Therefore, novel treatment modalities beneficial for subgroups of patients are needed. TRAIL receptor agonists have been suggested as promising candidates for use in cancer treatment as they preferentially induce apoptosis in cancer cells. Unfortunately, the first generation of TRAIL receptor agonists showed poor clinical efficacy. hvTRA is a second-generation TRAIL receptor agonist with improved composition giving increased potency, and in the present study, we showed hvTRA-induced activation of apoptosis leading to an efficient and sustained reduction in melanoma cell growth in cell lines and xenograft models. Furthermore, the potential of hvTRA in a clinical setting was demonstrated by showing efficacy on tumor cells harvested from melanoma patients with lymph node metastasis in an ex vivo drug sensitivity assay. Inhibition of mutated BRAF has been shown to regulate proteins in the intrinsic apoptotic pathway, making the cells more susceptible for apoptosis induction. In an attempt to increase the efficacy of hvTRA, combination treatment with the mutated BRAF inhibitor vemurafenib was investigated. A synergistic effect by the combination was observed for several cell lines in vitro, and an initial cytotoxic effect was observed in vivo. Unfortunately, the initial increased reduction in tumor growth compared with hvTRA mono treatment was not sustained, and this was related to downregulation of the DR5 level by vemurafenib. Altogether, the presented data imply that hvTRA efficiently induce apoptosis and growth delay in melanoma models and patient material, and the potential of this TRAIL receptor agonist should be further evaluated for treatment of subgroups of melanoma patients.

18.
Cancer Lett ; 194(1): 125-31, 2003 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-12706866

RESUMO

Microscopic detection of structural alterations is the most reliable method to identify apoptotic cells, which however, does not allow any correlation with cell cycle phases. Discrimination of individual cells within solid human tumors undergoing apoptotic death is possible by flow cytometry where apoptotic cells appear in a hypodiploid sub G0/1-peak as a consequence of partial DNA loss. To refer induction of apoptosis to cell cycle phases we adopted the terminal deoxynucleotidyl transferase nick-end-labelling (TUNEL) technique to flow cytometry which enables the detection of cellular DNA content and DNA fragmentation by multiparametric analysis. One thousand seven hundred human breast carcinomas were screened. In 40 cases (2.3%) of 1700 carcinomas we detected a hypodiploid sub -G0/1 apoptotic peak. The spontaneous apoptotic fractions within individual tumors ranged between 1.5 and 25%. A correlation (r(2)=0.78) was found between apoptotic cells in sub-G0/1-peak measured by DNA-cytometry and TUNEL positive cells measured by multiparametric cytometry, because TUNEL reaction signed also cells with strand breaks. High proliferation indices correspond well (r(2)=0.807) with the increased amount of TUNEL positive cells. Multiparametric flow cytometry for the combined determination of DNA-content and DNA-fragmentation by TUNEL offers not only the advantage of a higher apoptosis sensitivity but also enables the quantification of DNA fragmentation related to any cell cycle phase.


Assuntos
Apoptose , Neoplasias da Mama/patologia , Marcação In Situ das Extremidades Cortadas , Divisão Celular , Núcleo Celular/metabolismo , DNA/metabolismo , Fragmentação do DNA , Citometria de Fluxo/métodos , Humanos , Linfócitos/metabolismo
19.
Pathol Res Pract ; 210(2): 83-91, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24268735

RESUMO

BACKGROUND AND AIMS: Treatment with pegylated interferon-alpha (PEG-IFN) and ribavirin is the backbone of standard therapy of HCV by mechanisms that are not completely understood. Besides a direct antiviral effect, different immunomodulatory and apoptotic effects have been discussed. Tumor necrosis factor-related apoptosis inducing-ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family with immunomodulatory as well as pro- and antiapoptotic effects and is putatively involved in control of HCV infection. Thus, we analyzed the expression of the TRAIL/TRAIL-receptor system, caspase-8 and cFLIP and examined their prognostic and predictive value for HCV infection and antiviral therapy, respectively. METHODS: We immunohistochemically analyzed liver biopsies of 116 therapy-naive HCV patients before treatment with PEG-IFNα and ribavirin in comparison to healthy liver tissue. Expression levels of TRAIL, TRAIL-R1 to TRAIL-R4, caspase-8 and cFLIP were correlated with sustained virologic response (SVR), genotype and staging of chronic hepatitis. RESULTS: Caspase-8, cFLIP, TRAIL-R2 and TRAIL-R4 were strongly upregulated in HCV patients, whereas TRAIL-R3 was downregulated. SVR correlated with high expression of TRAIL and pro-apoptotic TRAIL-R2 on HCV infected hepatocytes. CONCLUSIONS: Our results suggest a pathophysiological role of TRAIL in both, HCV infection and therapy. Further studies need to elaborate possible TRAIL-related targets for clinical applications.


Assuntos
Antivirais/uso terapêutico , Caspase 8/metabolismo , Regulação Neoplásica da Expressão Gênica , Hepatite C Crônica/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Adulto , Apoptose , Quimioterapia Combinada , Feminino , Proteínas Ligadas por GPI/metabolismo , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/patologia , Hepatócitos/metabolismo , Humanos , Interferon alfa-2 , Interferon-alfa/uso terapêutico , Fígado/metabolismo , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Polietilenoglicóis/uso terapêutico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Membro 10c de Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes/uso terapêutico , Ribavirina/uso terapêutico
20.
Mol Cancer Ther ; 12(12): 2735-47, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24101228

RESUMO

Cancer cells can be specifically driven into apoptosis by activating Death-receptor-4 (DR4; TRAIL-R1) and/or Death-receptor-5 (DR5; TRAIL-R2). Albeit showing promising preclinical efficacy, first-generation protein therapeutics addressing this pathway, especially agonistic anti-DR4/DR5-monoclonal antibodies, have not been clinically successful to date. Due to their bivalent binding mode, effective apoptosis induction by agonistic TRAIL-R antibodies is achieved only upon additional events leading to antibody-multimer formation. The binding of these multimers to their target subsequently leads to effective receptor-clustering on cancer cells. The research results presented here report on a new class of TRAIL-receptor agonists overcoming this intrinsic limitation observed for antibodies in general. The main feature of these agonists is a TRAIL-mimic consisting of three TRAIL-protomer subsequences combined in one polypeptide chain, termed the single-chain TRAIL-receptor-binding domain (scTRAIL-RBD). In the active compounds, two scTRAIL-RBDs with three receptor binding sites each are brought molecularly in close proximity resulting in a fusion protein with a hexavalent binding mode. In the case of APG350-the prototype of this engineering concept-this is achieved by fusing the Fc-part of a human immunoglobulin G1 (IgG1)-mutein C-terminally to the scTRAIL-RBD polypeptide, thereby creating six receptor binding sites per drug molecule. In vitro, APG350 is a potent inducer of apoptosis on human tumor cell lines and primary tumor cells. In vivo, treatment of mice bearing Colo205-xenograft tumors with APG350 showed a dose-dependent antitumor efficacy. By dedicated muteins, we confirmed that the observed in vivo efficacy of the hexavalent scTRAIL-RBD fusion proteins is-in contrast to agonistic antibodies-independent of FcγR-based cross-linking events.


Assuntos
Antineoplásicos/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores de IgG/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Modelos Biológicos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/química , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Ensaios Antitumorais Modelo de Xenoenxerto
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