RESUMO
Treatment-naïve small cell lung cancer (SCLC) is typically susceptible to standard-of-care chemotherapy consisting of cisplatin and etoposide recently combined with PD-L1 inhibitors. Yet, in most cases, SCLC patients develop resistance to first-line therapy and alternative therapies are urgently required to overcome this resistance. In this study, we tested the efficacy of dinaciclib, an FDA-orphan drug and inhibitor of the cyclin-dependent kinase (CDK) 9, among other CDKs, in SCLC. Furthermore, we report on a newly developed, highly specific CDK9 inhibitor, VC-1, with tumour-killing activity in SCLC. CDK9 inhibition displayed high killing potential in a panel of mouse and human SCLC cell lines. Mechanistically, CDK9 inhibition led to a reduction in MCL-1 and cFLIP anti-apoptotic proteins and killed cells, almost exclusively, by intrinsic apoptosis. While CDK9 inhibition did not synergise with chemotherapy, it displayed high efficacy in chemotherapy-resistant cells. In vivo, CDK9 inhibition effectively reduced tumour growth and improved survival in both autochthonous and syngeneic SCLC models. Together, this study shows that CDK9 inhibition is a promising therapeutic agent against SCLC and could be applied to chemo-refractory or resistant SCLC.
Assuntos
Quinase 9 Dependente de Ciclina , Indolizinas , Neoplasias Pulmonares , Compostos de Piridínio , Carcinoma de Pequenas Células do Pulmão , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/metabolismo , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/patologia , Humanos , Animais , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Linhagem Celular Tumoral , Camundongos , Compostos de Piridínio/farmacologia , Compostos de Piridínio/uso terapêutico , Indolizinas/farmacologia , Óxidos N-Cíclicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
BACKGROUND: The risk of an anastomotic leakage (AL) following Ivor-Lewis esophagectomy is increased in patients with calcifications of the aorta or a stenosis of the celiac trunc. Ischemic conditioning (ISCON) of the gastric conduit prior to esophagectomy is supposed to improve gastric vascularization at the anastomotic site. The prospective ISCON trial was conducted to proof the safety and feasibility of this strategy with partial gastric devascularization 14 days before esophagectomy in esophageal cancer patients with a compromised vascular status. This work reports the results from a translational project of the ISCON trial aimed to investigate variables of neo-angiogenesis. METHODS: Twenty esophageal cancer patients scheduled for esophagectomy were included in the ISCON trial. Serum samples (n = 11) were collected for measurement of biomarkers and biopsies (n = 12) of the gastric fundus were taken before and after ISCON of the gastric conduit. Serum samples were analyzed including 62 different cytokines. Vascularization of the gastric mucosa was assessed on paraffin-embedded sections stained against CD34 to detect the degree of microvascular density and vessel size. RESULTS: Between November 2019 and January 2022 patients were included in the ISCON Trial. While serum samples showed no differences regarding cytokine levels before and after ISCON biopsies of the gastric mucosa demonstrated a significant increase in microvascular density after ISCON as compared to the corresponding gastric sample before the intervention. CONCLUSION: The data prove that ISCON of the gastric conduit as esophageal substitute induces significant neo-angiogenesis in the gastric fundus which is considered as surrogate of an improved vascularization at the anastomotic site.
Assuntos
Neoplasias Esofágicas , Precondicionamento Isquêmico , Laparoscopia , Humanos , Esofagectomia/métodos , Estudos Prospectivos , Precondicionamento Isquêmico/métodos , Estômago/irrigação sanguínea , Isquemia , Neoplasias Esofágicas/cirurgia , Neoplasias Esofágicas/patologiaRESUMO
Authors have only now noticed that in the Figure 3a, the immunohistochemical analysis of IL-4Rα on paraffin-embedded sections from breast is incorrect: IL-4 from breast was duplicated and used for the IL-4Rα staining. The correct Figure 3a has been included in the amendment to this paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand (Apo2L), is a member of the TNF cytokine superfamily. By cross-linking TRAIL-Receptor (TRAIL-R) 1 or TRAIL-R2, also known as death receptors 4 and 5 (DR4 and DR5), TRAIL has the capability to induce apoptosis in a wide variety of tumor cells while sparing vital normal cells. The discovery of this unique property among TNF superfamily members laid the foundation for testing the clinical potential of TRAIL-R-targeting therapies in the cancer clinic. To date, two of these therapeutic strategies have been tested clinically: (i) recombinant human TRAIL and (ii) antibodies directed against TRAIL-R1 or TRAIL-R2. Unfortunately, however, these TRAIL-R agonists have basically failed as most human tumors are resistant to apoptosis induction by them. It recently emerged that this is largely due to the poor agonistic activity of these agents. Consequently, novel TRAIL-R-targeting agents with increased bioactivity are currently being developed with the aim of rendering TRAIL-based therapies more active. This review summarizes these second-generation novel formulations of TRAIL and other TRAIL-R agonists, which exhibit enhanced cytotoxic capacity toward cancer cells, thereby providing the potential of being more effective when applied clinically than first-generation TRAIL-R agonists.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Apoptose/efeitos dos fármacos , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Relação Estrutura-Atividade , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
BACKGROUND: Mastocytosis is characterized by the accumulation of mast cells (MCs) associated with activating mutations of KIT. Tumor necrosis factor-related apoptosis-inducing ligand receptors (TRAIL-Rs) are preferentially expressed on neoplastic cells and induce the extrinsic apoptotic pathway. Recent studies reported on the expression of TRAIL-Rs and TRAIL-induced apoptosis in cultured human MCs, which depend on stem cell factor (SCF)-induced or constitutive KIT activation. MATERIAL AND METHODS: We sought to further define the impact of TRAIL-Rs on MCs in vivo and in vitro. Using Cre/loxP recombination, we generated mice with MC-specific and ubiquitous knockout of TRAIL-R. In these mice, anaphylaxis and numbers of MCs were investigated. We also explored the expression and function of TRAIL-Rs in cultured murine and human MCs upon activation of KIT. By conducting immunofluorescence staining, we analyzed the expression of TRAIL-Rs in MCs infiltrating the bone marrow of patients with mastocytosis. RESULTS: MC-specific deletion of TRAIL-R was associated with a slight, but significant increase in anaphylaxis. Numbers of MCs in MC-specific knockouts of TRAIL-R were comparable to controls. Whereas cultured IL-3-dependent murine MCs from wild-type mice were resistant to TRAIL-induced apoptosis, SCF-stimulated MCs underwent apoptosis in response to TRAIL. Interestingly, activating KIT mutations also promoted sensitivity to TRAIL-mediated apoptosis in human MCs. In line with these findings, MCs infiltrating the bone marrow of patients with mastocytosis expressed TRAIL-R1. CONCLUSIONS: Activation of KIT regulates the function of TRAIL-Rs in MCs. TRAIL-R1 may represent an attractive diagnostic and therapeutic target in diseases associated with KIT mutations, such as mastocytosis.
Assuntos
Mastócitos/imunologia , Mastócitos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Medula Óssea/imunologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Mastocitose/genética , Mastocitose/imunologia , Mastocitose/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Fator de Células-Tronco/metabolismo , Fator de Células-Tronco/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Human papillomavirus (HPV) is causative for a new and increasing form of head and neck squamous cell carcinomas (HNSCCs). Although localised HPV-positive cancers have a favourable response to radio-chemotherapy (RT/CT), the impact of HPV in advanced or metastatic HNSCC remains to be defined and targeted therapeutics need to be tested for cancers resistant to RT/CT. To this end, we investigated the sensitivity of HPV-positive and -negative HNSCC cell lines to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), which induces tumour cell-specific apoptosis in various cancer types. A clear correlation was observed between HPV positivity and resistance to TRAIL compared with HPV-negative head and neck cancer cell lines. All TRAIL-resistant HPV-positive cell lines tested were sensitised to TRAIL-induced cell death by treatment with bortezomib, a clinically approved proteasome inhibitor. Bortezomib-mediated sensitisation to TRAIL was associated with enhanced activation of caspase-8, -9 and -3, elevated membrane expression levels of TRAIL-R2, cytochrome c release and G2/M arrest. Knockdown of caspase-8 significantly blocked cell death induced by the combination therapy, whereas the BH3-only protein Bid was not required for induction of apoptosis. XIAP depletion increased the sensitivity of both HPV-positive and -negative cells to TRAIL alone or in combination with bortezomib. In contrast, restoration of p53 following E6 knockdown in HPV-positive cells had no effect on their sensitivity to either single or combination therapy, suggesting a p53-independent pathway for the observed response. In summary, bortezomib-mediated proteasome inhibition sensitises previously resistant HPV-positive HNSCC cells to TRAIL-induced cell death through a mechanism involving both the extrinsic and intrinsic pathways of apoptosis. The cooperative effect of these two targeted anticancer agents therefore represents a promising treatment strategy for RT/CT-resistant HPV-associated head and neck cancers.
Assuntos
Ácidos Borônicos/farmacologia , Caspase 8/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/virologia , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/fisiologia , Pirazinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Bortezomib , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Células HEK293 , Humanos , Estabilidade Proteica/efeitos dos fármacos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
BACKGROUND: Current treatment strategies for head and neck cancer are associated with significant morbidity and up to 50% of patients relapse, highlighting the need for more specific and effective therapeutics. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Smac mimetics (SMs) are promising anticancer agents, but their effect on head and neck squamous cell carcinoma (HNSCC) remains unknown. METHODS: We examined the response of a panel of nine HNSCC cell lines to TRAIL and SMs and investigated the mechanism of cell type-specific response by functional analysis. RESULTS: Head and neck cancer cell lines revealed a converse response pattern with three cell lines being highly sensitive to Smac-164 (SM) but resistant to TRAIL, whereas the other six were sensitive to TRAIL but resistant to SM. Distinct protein expression and activation patterns were found to be associated with susceptibility of HNSCC cell lines to TRAIL and SM. Tumour necrosis factor-related apoptosis-inducing ligand sensitivity was associated with high caspase-8 and Bid protein levels, and TRAIL-sensitive cell lines were killed via the type II extrinsic apoptotic pathway. Smac mimetic-sensitive cells expressed low levels of caspase-8 and Bid but had high TNF-α expression. Smac mimetic-induced cell death was associated with caspase-10 activation, suggesting that in the absence of caspase-8, caspase-10 mediates response to SM. Cotreatment with TNF-α sensitised the resistant cells to SM, demonstrating a decisive role for TNF-α-driven feedback loop in SM sensitivity. CONCLUSIONS: Tumour necrosis factor-related apoptosis-inducing ligand and SMs effectively kill HNSCC cell lines and therefore represent potential targeted therapeutics for head and neck cancer. Distinct molecular mechanisms determine the sensitivity to each agent, with levels of TNF-α, caspase-8, Bid and caspase-10 providing important predictive biomarkers of response to these agents.
Assuntos
Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Caspase 10/metabolismo , Caspase 8/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Triazóis/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Biomimética , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Hepatocellular carcinoma (HCC) usually develops in the context of chronic hepatitis triggered by viruses or toxic substances causing hepatocyte death, inflammation and compensatory proliferation of liver cells. Death receptors of the TNFR superfamily regulate cell death and inflammation and are implicated in liver disease and cancer. Liver parenchymal cell-specific ablation of NEMO/IKKγ, a subunit of the IκB kinase (IKK) complex that is essential for the activation of canonical NF-κB signalling, sensitized hepatocytes to apoptosis and caused the spontaneous development of chronic hepatitis and HCC in mice. Here we show that hepatitis and HCC development in NEMO(LPC-KO) mice is triggered by death receptor-independent FADD-mediated hepatocyte apoptosis. TNF deficiency in all cells or conditional LPC-specific ablation of TNFR1, Fas or TRAIL-R did not prevent hepatocyte apoptosis, hepatitis and HCC development in NEMO(LPC-KO) mice. To address potential functional redundancies between death receptors we generated and analysed NEMO(LPC-KO) mice with combined LPC-specific deficiency of TNFR1, Fas and TRAIL-R and found that also simultaneous lack of all three death receptors did not prevent hepatocyte apoptosis, chronic hepatitis and HCC development. However, LPC-specific combined deficiency in TNFR1, Fas and TRAIL-R protected the NEMO-deficient liver from LPS-induced liver failure, showing that different mechanisms trigger spontaneous and LPS-induced hepatocyte apoptosis in NEMO(LPC-KO) mice. In addition, NK cell depletion did not prevent liver damage and hepatitis. Moreover, NEMO(LPC-KO) mice crossed into a RAG-1-deficient genetic background-developed hepatitis and HCC. Collectively, these results show that the spontaneous development of hepatocyte apoptosis, chronic hepatitis and HCC in NEMO(LPC-KO) mice occurs independently of death receptor signalling, NK cells and B and T lymphocytes, arguing against an immunological trigger as the critical stimulus driving hepatocarcinogenesis in this model.
Assuntos
Apoptose/fisiologia , Carcinoma Hepatocelular/etiologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , Hepatite/etiologia , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Hepáticas/etiologia , Receptores de Morte Celular/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Hepatite/imunologia , Hepatite/metabolismo , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismoRESUMO
Unlike other members of the TNF superfamily, the TNF-related apoptosis-inducing ligand (TRAIL, also known as Apo2L) possesses the unique capacity to induce apoptosis selectively in cancer cells in vitro and in vivo. This exciting discovery provided the basis for the development of TRAIL-receptor agonists (TRAs), which have demonstrated robust anticancer activity in a number of preclinical studies. Subsequently initiated clinical trials testing TRAs demonstrated, on the one hand, broad tolerability but revealed, on the other, that therapeutic benefit was rather limited. Several factors that are likely to account for TRAs' sobering clinical performance have since been identified. First, because of initial concerns over potential hepatotoxicity, TRAs with relatively weak agonistic activity were selected to enter clinical trials. Second, although TRAIL can induce apoptosis in several cancer cell lines, it has now emerged that many others, and importantly, most primary cancer cells are resistant to TRAIL monotherapy. Third, so far patients enrolled in TRA-employing clinical trials were not selected for likelihood of benefitting from a TRA-comprising therapy on the basis of a valid(ated) biomarker. This review summarizes and discusses the results achieved so far in TRA-employing clinical trials in the light of these three shortcomings. By integrating recent insight on apoptotic and non-apoptotic TRAIL signaling in cancer cells, we propose approaches to introduce novel, revised TRAIL-based therapeutic concepts into the cancer clinic. These include (i) the use of recently developed highly active TRAs, (ii) the addition of efficient, but cancer-cell-selective TRAIL-sensitizing agents to overcome TRAIL resistance and (iii) employing proteomic profiling to uncover resistance mechanisms. We envisage that this shall enable the design of effective TRA-comprising therapeutic concepts for individual cancer patients in the future.
Assuntos
Neoplasias/terapia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Humanos , Neoplasias/metabolismoRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many cancer cells without causing toxicity in vivo. However, to date, TRAIL-receptor agonists have only shown limited therapeutic benefit in clinical trials. This can, most likely, be attributed to the fact that 50% of all cancer cell lines and most primary human cancers are TRAIL resistant. Consequently, future TRAIL-based therapies will require the addition of sensitizing agents that remove crucial blocks in the TRAIL apoptosis pathway. Here, we identify PIK-75, a small molecule inhibitor of the p110α isoform of phosphoinositide-3 kinase (PI3K), as an exceptionally potent TRAIL apoptosis sensitizer. Surprisingly, PI3K inhibition was not responsible for this activity. A kinome-wide in vitro screen revealed that PIK-75 strongly inhibits a panel of 27 kinases in addition to p110α. Within this panel, we identified cyclin-dependent kinase 9 (CDK9) as responsible for TRAIL resistance of cancer cells. Combination of CDK9 inhibition with TRAIL effectively induced apoptosis even in highly TRAIL-resistant cancer cells. Mechanistically, CDK9 inhibition resulted in downregulation of cellular FLICE-like inhibitory protein (cFlip) and Mcl-1 at both the mRNA and protein levels. Concomitant cFlip and Mcl-1 downregulation was required and sufficient for TRAIL sensitization by CDK9 inhibition. When evaluating cancer selectivity of TRAIL combined with SNS-032, the most selective and clinically used inhibitor of CDK9, we found that a panel of mostly TRAIL-resistant non-small cell lung cancer cell lines was readily killed, even at low concentrations of TRAIL. Primary human hepatocytes did not succumb to the same treatment regime, defining a therapeutic window. Importantly, TRAIL in combination with SNS-032 eradicated established, orthotopic lung cancer xenografts in vivo. Based on the high potency of CDK9 inhibition as a cancer cell-selective TRAIL-sensitizing strategy, we envisage the development of new, highly effective cancer therapies.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Regulação para Baixo , Feminino , Células HCT116 , Células HeLa , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Distribuição Aleatória , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Smac mimetics (SMs) comprise a class of small molecules that target members of the inhibitor of apoptosis family of pro-survival proteins, whose expression in cancer cells hinders the action of conventional chemotherapeutics. Herein, we describe the activity of SM83, a newly synthesised dimeric SM, in two cancer ascites models: athymic nude mice injected intraperitoneally with IGROV-1 human ovarian carcinoma cells and immunocompetent BALB/c mice injected with murine Meth A sarcoma cells. SM83 rapidly killed ascitic IGROV-1 and Meth A cells in vivo (prolonging mouse survival), but was ineffective against the same cells in vitro. IGROV-1 cells in nude mice were killed within the ascites by a non-apoptotic, tumour necrosis factor (TNF)-dependent mechanism. SM83 administration triggered a rapid inflammatory event characterised by host secretion of TNF, interleukin-1ß and interferon-γ. This inflammatory response was associated with the reversion of the phenotype of tumour-associated macrophages from a pro-tumoural M2- to a pro-inflammatory M1-like state. SM83 treatment was also associated with a massive recruitment of neutrophils that, however, was not essential for the antitumoural activity of this compound. In BALB/c mice bearing Meth A ascites, SM83 treatment was in some cases curative, and these mice became resistant to a second injection of cancer cells, suggesting that they had developed an adaptive immune response. Altogether, these results indicate that, in vivo, SM83 modulates the immune system within the tumour microenvironment and, through its pro-inflammatory action, leads cancer cells to die by necrosis with the release of high-mobility group box-1. In conclusion, our work provides evidence that SMs could be more therapeutically active than expected by stimulating the immune system.
Assuntos
Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Necrose/induzido quimicamente , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Materiais Biomiméticos/uso terapêutico , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Células HCT116 , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação/induzido quimicamente , Proteínas Inibidoras de Apoptose , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transplantation is invariably associated with ischemia-reperfusion injury (IRI), inflammation and rejection. Resultant cell death has morphological features of necrosis but programmed cell death has been synonymous with apoptosis until pathways of regulated necrosis (RN) have been described. The best-studied RN pathway, necroptosis, is triggered by perturbation of caspase-8-mediated apoptosis and depends on receptor-interacting protein kinases 1 and 3 (RIPK1/RIPK3) as well as mixed linage kinase domain like to form the necroptosome. The release of cytosolic content and cell death-associated molecular patterns (CDAMPs) can trigger innate and promote adaptive immune responses. Thus, the form of cell death can substantially influence alloimmunity and graft survival. Necroptosis is a key element of IRI, and RIPK1 interference by RN-specific inhibitors such as necrostatin-1 protects from IRI in kidney, heart and brain. Necroptosis may be a general mechanism in response to other forms of inflammatory organ injury, and will likely emerge as a promising target in solid organ transplantation. As second-generation RIPK1 and RIPK3 inhibitors become available, clinical trials for the prevention of delayed graft function and attenuation of allograft rejection-mediated injury will emerge. These efforts will accelerate upon further identification of critical necroptosis-triggering receptor(s).
Assuntos
Imunidade Celular , Necrose , Traumatismo por Reperfusão/patologia , Animais , HumanosRESUMO
Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality. Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling. Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity. Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance. To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications. By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention. By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models. Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination. Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Linhagem Celular Tumoral/efeitos dos fármacos , Cisplatino/farmacologia , Técnicas de Cocultura , Sinergismo Farmacológico , Humanos , Melanoma/secundário , Neoplasias Cutâneas/patologia , Pele Artificial , Esferoides Celulares/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Interleukin-24 (IL-24), a member of the IL-10 cytokine family whose physiological function remains largely unknown, has been shown to induce apoptosis when expressed in an adenoviral background. It is yet little understood, why IL-24 alone induced apoptosis only in a limited number of tumor cell lines. Analyzing an influenza A virus vector expressing IL-24 for its oncolytic potential revealed enhanced pro-apoptotic activity of the chimeric virus compared with virus or IL-24 alone. Interestingly, IL-24-mediated enhancement of influenza-A-induced apoptosis did not require viral replication but critically depended on toll-like receptor 3 (TLR3) and caspase-8. Immunoprecipitation of TLR3 showed that infection by influenza A virus induced formation of a TLR3-associated signaling complex containing TRIF, RIP1, FADD, cFLIP and pro-caspase-8. Co-administration of IL-24 decreased the presence of cFLIP in the TLR3-associated complex, converting it into an atypical, TLR3-associated death-inducing signaling complex (TLR3 DISC) that induced apoptosis by enabling caspase-8 activation at this complex. The sensitizing effect of IL-24 on TLR3-induced apoptosis, mediated by influenza A virus or the TLR3-specific agonist poly(I:C), was also evident on tumor spheroids. In conclusion, rather than acting as an apoptosis inducer itself, IL-24 sensitizes cancer cells to TLR-mediated apoptosis by enabling the formation of an atypical DISC which, in the case of influenza A virus or poly(I:C), is associated with TLR3.
Assuntos
Apoptose/fisiologia , Interleucinas/biossíntese , Interleucinas/farmacologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Receptor 3 Toll-Like/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Citocinas/biossíntese , Ativação Enzimática , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Interleucinas/genética , Masculino , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Melanoma/terapia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Esferoides Celulares , Transgenes , Regulação para CimaRESUMO
BACKGROUND: XIAP (X-linked inhibitor of apoptosis protein) is an anti-apoptotic protein exerting its activity by binding and suppressing caspases. As XIAP is overexpressed in several tumours, in which it apparently contributes to chemoresistance, and because its activity in vivo is antagonised by second mitochondria-derived activator of caspase (SMAC)/direct inhibitor of apoptosis-binding protein with low pI, small molecules mimicking SMAC (so called SMAC-mimetics) can potentially overcome tumour resistance by promoting apoptosis. METHODS: Three homodimeric compounds were synthesised tethering a monomeric SMAC-mimetic with different linkers and their affinity binding for the baculoviral inhibitor repeats domains of XIAP measured by fluorescent polarisation assay. The apoptotic activity of these molecules, alone or in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and/or Bortezomib, was tested in melanoma cell lines by MTT viability assays and western blot analysis of activated caspases. RESULTS: We show that in melanoma cell lines, which are typically resistant to chemotherapeutic agents, XIAP knock-down sensitises cells to TRAIL treatment in vitro, also favouring the accumulation of cleaved caspase-8. We also describe a new series of 4-substituted azabicyclo[5.3.0]alkane monomeric and dimeric SMAC-mimetics that target various members of the IAP family and powerfully synergise at submicromolar concentrations with TRAIL in inducing cell death. Finally, we show that the simultaneous administration of newly developed SMAC-mimetics with Bortezomib potently triggers apoptosis in a melanoma cell line resistant to the combined effect of SMAC-mimetics and TRAIL. CONCLUSION: Hence, the newly developed SMAC-mimetics effectively synergise with TRAIL and Bortezomib in inducing cell death. These findings warrant further preclinical studies in vivo to verify the anticancer effectiveness of the combination of these agents.
Assuntos
Ácidos Borônicos/farmacologia , Morte Celular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Melanoma/tratamento farmacológico , Proteínas Mitocondriais/farmacologia , Pirazinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas Reguladoras de Apoptose , Bortezomib , Caspase 8/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Interações Medicamentosas , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/administração & dosagem , Proteínas Mitocondriais/administração & dosagem , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagemRESUMO
Although nuclear factor-kappaB (NF-kappaB) usually exerts anti-apoptotic activity, upon activation by interleukin-1 (IL-1) it enhances ultraviolet-B radiation (UVB)-induced apoptosis. This paradoxical effect is associated with NF-kappaB-dependent pronounced secretion of tumour necrosis factor-alpha (TNF) which activates TNF-R1 in an autocrine fashion to enhance UVB-induced apoptosis. We demonstrate that sustained TNF transcription in UVB+IL-1-treated cells involves complete abrogation of the negative feedback loop of NF-kappaB preventing IkappaBalpha resynthesis, hence allowing uncontrolled NF-kappaB activity. We show that IkappaBalpha is not transcriptionally inhibited but resynthesized protein is immediately marked for degradation due to persistent inhibitor of kappaB kinasebeta (IKKbeta) activity. Continuous IKKbeta phosphorylation and activation is caused by UVB-mediated inhibition of the phosphatase PP2A. This study demonstrates that the cellular response to different NF-kappaB activators may be converted to the opposite reaction when both stimuli act in concert. Our data shed new light on the significance of negative feedback regulation of NF-kappaB and identifies PP2A as the key regulator of this process.
Assuntos
Apoptose/efeitos da radiação , Retroalimentação Fisiológica/efeitos da radiação , NF-kappa B/metabolismo , Proteína Fosfatase 2/metabolismo , Raios Ultravioleta , Apoptose/efeitos dos fármacos , Comunicação Autócrina/efeitos dos fármacos , Comunicação Autócrina/efeitos da radiação , Linhagem Celular Tumoral , Retroalimentação Fisiológica/efeitos dos fármacos , Humanos , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/antagonistas & inibidores , Interleucina-1/farmacologia , Modelos Biológicos , Inibidor de NF-kappaB alfa , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos da radiação , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiação , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We investigated the mechanisms involved in the resistance to cell death observed in epithelial cancers. Here, we identify that primary epithelial cancer cells from colon, breast and lung carcinomas express high levels of the antiapoptotic proteins PED, cFLIP, Bcl-xL and Bcl-2. These cancer cells produced interleukin-4 (IL-4), which amplified the expression levels of these antiapoptotic proteins and prevented cell death induced upon exposure to TRAIL or other drug agents. IL-4 blockade resulted in a significant decrease in the growth rate of epithelial cancer cells and sensitized them, both in vitro and in vivo, to apoptosis induction by TRAIL and chemotherapy via downregulation of the antiapoptotic factors PED, cFLIP, Bcl-xL and Bcl-2. Furthermore, we provide evidence that exogenous IL-4 was able to upregulate the expression levels of these antiapoptotic proteins and potently stabilized the growth of normal epithelial cells rendering them apoptosis resistant. In conclusion, IL-4 acts as an autocrine survival factor in epithelial cells. Our results indicate that inhibition of IL-4/IL-4R signaling may serve as a novel treatment for epithelial cancers.
Assuntos
Apoptose , Comunicação Autócrina , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Interleucina-4/metabolismo , Neoplasias Pulmonares/metabolismo , Adulto , Idoso , Anticorpos Monoclonais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Morte Celular , Proliferação de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Interleucina-4/imunologia , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Regulação para Cima , Proteína bcl-X/metabolismoRESUMO
In many tumor cell types, ionizing radiation (IR) or DNA-damaging anticancer drugs enhance sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, which is of great clinical interest. We have investigated the molecular mechanism underlying the response to combined modality treatment in p53-mutant Jurkat T leukemic cells overexpressing Bcl-2. These cells are largely resistant to individual treatment with TRAIL or IR, but sensitive to combined treatment, in vitro as well as in vivo. We demonstrate that IR and DNA-damaging anticancer drugs enable TRAIL receptor-2 and CD95/Fas to bypass the mitochondrial pathway for effector caspase activation. This was validated by RNA interference for Bax and Bak and by overexpression of dominant-negative Caspase-9. Improved effector caspase activation was neither caused by altered expression of proapoptotic components nor by impaired activity of inhibitor of apoptosis proteins or nuclear factor-kappaB signaling. Rather, we found that pretreatment of cells with IR caused quantitative and qualitative changes in death receptor signaling. It strongly improved the capacity of ligand-bound receptors to recruit FADD and activate Caspase-8 and -10 in the death-inducing signaling complex, while c-FLIP(L) levels were unaffected.
Assuntos
Radiação Ionizante , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Antineoplásicos/farmacologia , Apoptose , Caspases/metabolismo , Dano ao DNA , Ativação Enzimática , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Células Jurkat , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Interferência de RNA , Transdução de Sinais , Receptor fas/imunologiaRESUMO
PEA-15 (phosphoprotein enriched in astrocytes 15 kDa) is a death effector domain-containing protein, which is involved in the regulation of apoptotic cell death. Since PEA-15 is highly expressed in cells of glial origin, we studied the role of PEA-15 in human malignant brain tumors. Immunohistochemical analysis of PEA-15 expression shows strong immunoreactivity in astrocytomas and glioblastomas. Phosphorylation of PEA-15 at Ser(116) is found in vivo in perinecrotic areas in glioblastomas and in vitro after glucose deprivation of glioblastoma cells. Overexpression of PEA-15 induces a marked resistance against glucose deprivation-induced apoptosis, whereas small interfering RNA (siRNA)-mediated downregulation of endogenous PEA-15 results in the sensitization to glucose withdrawal-mediated cell death. This antiapoptotic activity of PEA-15 under low glucose conditions depends on its phosphorylation at Ser(116). Moreover, siRNA-mediated knockdown of PEA-15 abolishes the tumorigenicity of U87MG glioblastoma cells in vivo. PEA-15 regulates the level of phosphorylated extracellular-regulated kinase (ERK)1/2 in glioblastoma cells and the PEA-15-dependent protection from glucose deprivation-induced cell death requires ERK1/2 signaling. PEA-15 transcriptionally upregulates the Glucose Transporter 3, which is abrogated by the inhibition of ERK1/2 phosphorylation. Taken together, our findings suggest that Ser(116)-phosphorylated PEA-15 renders glioma cells resistant to glucose deprivation-mediated cell death as encountered in poor microenvironments, for example in perinecrotic areas of glioblastomas.
Assuntos
Apoptose/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Glioblastoma/enzimologia , Glucose/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Fosfoproteínas/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Glioblastoma/metabolismo , Glioblastoma/patologia , Glucose/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Nus , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , FosforilaçãoRESUMO
Death ligands such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and certain forms of CD95L are attractive therapeutic options for metastatic melanoma. Since knowledge about the regulation of death receptor sensitivity in melanoma is sparse, we have analysed these signaling pathways in detail. The loss of CD95 or TRAIL-R1, but not of TRAIL-R2, surface expression correlated with apoptosis sensitivity in a panel of melanoma cell lines. In contrast, the expression of proteins of the apical apoptosis signaling cascade (FADD, initiator caspases-8 and cFLIP) did not predict apoptosis sensitivity. Since both TRAIL-R1 and -R2 transmit apoptotic signals, we asked whether cFLIP, highly expressed in several of the cell lines tested, is sufficient to maintain resistance to TRAIL-R2-mediated apoptosis. Downregulation of cFLIP in TRAIL-R2-positive, TRAIL-resistant IGR cells dramatically increased TRAIL sensitivity. Conversely ectopic expression of cFLIP in TRAIL-sensitive, TRAIL-R2-expressing RPM-EP melanoma cells inhibited TRAIL- and CD95L-mediated cell death. Thus, modulation of cFLIP is sufficient to sensitize TRAIL-R2-expressing cells for TRAIL. Taken together, albeit expressing all proteins necessary for death receptor-mediated apoptosis, TRAIL-R1 negative melanoma cells cannot undergo TRAIL- or CD95L-induced apoptosis due to expression of cFLIP. Hence, cFLIP represents an attractive therapeutic target for melanoma treatment, especially in combination with TRAIL receptor agonists.