RESUMO
Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS.
Assuntos
Glândulas Suprarrenais/citologia , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Neurais/citologia , Sistema Nervoso Simpático/citologia , Adaptação Fisiológica , Glândulas Suprarrenais/metabolismo , Animais , Biomarcadores/metabolismo , Antígenos CD57/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Sistema Nervoso Simpático/metabolismoRESUMO
BACKGROUND: Evasion of apoptosis is a hallmark of pancreatic cancer. However, the underlying mechanisms are still only partly understood and may involve antiapoptotic proteins such as c-FLIP. Here, the role of c-FLIP in the regulation of death receptor-mediated apoptosis in pancreatic cancer was investigated. METHODS: Expression of c-FLIP(L) and c-FLIP(S) was analysed in primary pancreatic carcinoma samples, pancreatic carcinoma cell lines and primary tumour cells together with its function as a regulator of death receptor-induced apoptosis by knockdown and overexpression studies and through modulation by chemotherapeutics. RESULTS: c-FLIP is expressed in pancreatic intraepithelial neoplasm (PanIN) lesions and in pancreatic ductal adenocarcinomas, whereas normal pancreatic ducts were consistently negative for c-FLIP. Simultaneous downregulation of c-FLIP(L) and c-FLIP(S) as well as individual knockdown of either isoform by RNA interference significantly enhances TRAIL (tumour necrosis factor-related apoptosis-inducing ligand)- and CD95-induced caspase activation and caspase-dependent apoptosis. Also, pretreatment with chemotherapeutic drugs--that is, 5-fluorouracil (5-FU), cisplatin or gemcitabine--downregulates c-FLIP and renders cells sensitive to death receptor-triggered apoptosis. Similarly, primary cultured pancreatic cancer cells are primed for TRAIL-induced apoptosis by pre-exposure to 5-FU or cisplatin. Mechanistic studies revealed that 5-FU-mediated suppression of c-FLIP results in increased TRAIL-induced recruitment and activation of caspase-8 at the death-inducing signalling complex (DISC), leading to caspase-3 activation and caspase-dependent cell death. Overexpression of c-FLIP(L) rescues cells from 5-FU- or cisplatin-mediated sensitisation for TRAIL-induced apoptosis, indicating that c-FLIP suppression is a key event in this chemotherapy-mediated sensitisation to TRAIL. Further, concomitant neutralisation of c-FLIP and XIAP acts in concert to potentiate TRAIL-induced apoptosis. CONCLUSIONS: Both the long and the short isoform of the antiapoptotic protein c-FLIP are critical regulators of death receptor-induced apoptosis in pancreatic carcinoma cells and are suppressed by chemotherapeutics. Targeting either c-FLIP(L) or c-FLIP(S) is sufficient to promote death receptor-induced apoptosis in pancreatic carcinoma cells. These findings have important implications for the design of TRAIL-based combination protocols in pancreatic cancer.
Assuntos
Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias Pancreáticas/patologia , Receptores de Morte Celular/fisiologia , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Fluoruracila/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Células Tumorais Cultivadas , Receptor fas/fisiologiaRESUMO
Defects in apoptosis contribute to treatment resistance and poor outcome of pancreatic cancer, calling for novel therapeutic strategies. Here, we provide the first evidence that nuclear factor (NF) κB is required for Smac mimetic-mediated sensitization of pancreatic carcinoma cells for gemcitabine-induced apoptosis. The Smac mimetic BV6 cooperates with gemcitabine to reduce cell viability and to induce apoptosis. In addition, BV6 significantly enhances the cytotoxicity of several anticancer drugs against pancreatic carcinoma cells, including doxorubicin, cisplatin, and 5-fluorouracil. Molecular studies reveal that BV6 stimulates NF-κB activation, which is further increased in the presence of gemcitabine. Importantly, inhibition of NF-κB by overexpression of the dominant-negative IκBα superrepressor significantly decreases BV6- and gemcitabine-induced apoptosis, demonstrating that NF-κB exerts a proapoptotic function in this model of apoptosis. In support of this notion, inhibition of tumor necrosis factor α (TNFα) by the TNFα blocking antibody Enbrel reduces BV6- and gemcitabine-induced activation of caspase 8 and 3, loss of mitochondrial membrane potential, and apoptosis. By demonstrating that BV6 and gemcitabine trigger a NF-κB-dependent, TNFα-mediated loop to activate apoptosis signaling pathways and caspase-dependent apoptotic cell death, our findings have important implications for the development of Smac mimetic-based combination protocols in the treatment of pancreatic cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Mitocondriais/química , Mimetismo Molecular , NF-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Antineoplásicos/administração & dosagem , Proteínas Reguladoras de Apoptose , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Humanos , Camundongos , Fator de Necrose Tumoral alfa/metabolismo , GencitabinaRESUMO
PURPOSE: We previously reported that small molecule X-linked inhibitor of apoptosis (XIAP) inhibitors synergize with soluble TRAIL to trigger apoptosis in pancreatic carcinoma cells. Because cancers may preferentially signal via 1 of the 2 agonistic TRAIL receptors, we investigated these receptors as a therapeutic target in pancreatic cancer in the present study. EXPERIMENTAL DESIGN: We examined TRAIL receptor expression and cytotoxicity of specific monoclonal antibodies to TRAIL-R1 (HGS-ETR1, mapatumumab) or TRAIL-R2 (HGS-ETR2, lexatumumab) and of TRAIL receptor selective mutants alone and in combination with small molecule XIAP inhibitors in pancreatic cancer cell lines, in primary specimens, and in a xenotransplant model in vivo. RESULTS: The majority of primary pancreatic carcinoma samples and all cell lines express one or both agonistic TRAIL receptors. Nine of 13 cell lines are more sensitive to mapatumumab-induced apoptosis, whereas lexatumumab requires cross-linking for maximal activity. Similarly, TRAIL-R1 selective mutants display higher cytotoxicity than TRAIL-R2 selective mutants. Small molecule XIAP inhibitors preferentially act in concert with mapatumumab to trigger caspase activation, caspase-dependent apoptosis, and suppress clonogenic survival. Also, primary cultured pancreatic carcinoma cells are more susceptible to mapatumumab than lexatumumab, which is significantly enhanced by a XIAP inhibitor. Importantly, combined treatment with mapatumumab and a XIAP inhibitor cooperates to suppress tumor growth in vivo. CONCLUSIONS: Mapatumumab exerts antitumor activity, especially in combination with XIAP inhibitors against most pancreatic carcinoma cell lines, whereas lexatumumab requires cross-linking for optimal cytotoxicity. These findings have important implications for the design of TRAIL-based protocols for pancreatic cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma/patologia , Neoplasias Pancreáticas/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Idoso , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/administração & dosagem , Carcinoma/metabolismo , Linhagem Celular Tumoral , Embrião de Galinha , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Feminino , Humanos , Neoplasias Pancreáticas/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagemRESUMO
Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Caspase 3/metabolismo , Sinergismo Farmacológico , Ativação Enzimática , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Resistance to apoptosis is a hallmark of pancreatic cancer, a leading cause of cancer deaths. Therefore, novel strategies are required to target apoptosis resistance. Here, we report that the combination of X-linked inhibitor of apoptosis (XIAP) inhibition and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an effective approach to trigger apoptosis despite Bcl-2 overexpression and to suppress pancreatic cancer growth in vitro and in vivo. Knockdown of XIAP by RNA interference cooperates with TRAIL to induce caspase activation, loss of mitochondrial membrane potential, cytochrome c release, and apoptosis in pancreatic carcinoma cells. Loss of mitochondrial membrane potential and cytochrome c release are extensively inhibited by a broad range or caspase-3 selective caspase inhibitor and by RNAi-mediated silencing of caspase-3, indicating that XIAP inhibition enhances TRAIL-induced mitochondrial damage in a caspase-3-dependent manner. XIAP inhibition combined with TRAIL even breaks Bcl-2-imposed resistance by converting type II cells that depend on the mitochondrial contribution to the death receptor pathway to type I cells in which TRAIL-induced activation of caspase-3 and caspase-9 and apoptosis proceeds irrespective of high Bcl-2 levels. Most importantly, XIAP inhibition potentiates TRAIL-induced antitumor activity in two preclinical models of pancreatic cancer in vivo. In the chicken chorioallantoic membrane model, XIAP inhibition significantly enhances TRAIL-mediated apoptosis and suppression of tumor growth. In a tumor regression model in xenograft-bearing mice, XIAP inhibition acts in concert with TRAIL to cause even regression of established pancreatic carcinoma. Thus, this combination of XIAP inhibition plus TRAIL is a promising strategy to overcome apoptosis resistance of pancreatic cancer that warrants further investigation.