RESUMO
Glucose-stimulated insulin secretion (GSIS) relies on repetitive, electrical spiking activity of the beta cell membrane. Cyclic activation of voltage-gated potassium channels (K(v) ) generates an outward, 'delayed rectifier' potassium current, which drives the repolarizing phase of each spike and modulates insulin release. Although several K(v) channels are expressed in pancreatic islets, their individual contributions to GSIS remain incompletely understood. We take advantage of a naturally occurring cone-snail peptide toxin, Conkunitzin-S1 (Conk-S1), which selectively blocks K(v) 1.7 channels to provide an intrinsically limited, finely graded control of total beta cell delayed rectifier current and hence of GSIS. Conk-S1 increases GSIS in isolated rat islets, likely by reducing K(v) 1.7-mediated delayed rectifier currents in beta cells, which yields increases in action potential firing and cytoplasmic free calcium. In rats, Conk-S1 increases glucose-dependent insulin secretion without decreasing basal glucose. Thus, we conclude that K(v) 1.7 contributes to the membrane-repolarizing current of beta cells during GSIS and that block of this specific component of beta cell K(v) current offers a potential strategy for enhancing GSIS with minimal risk of hypoglycaemia during metabolic disorders such as Type 2 diabetes.
Assuntos
Glucose/metabolismo , Insulina/metabolismo , Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Animais , Secreções Corporais , Humanos , Secreção de Insulina , Masculino , Venenos de Moluscos/toxicidade , Ratos , Ratos WistarRESUMO
Vaccination using DCs pulsed with tumor lysates or specific tumor-associated peptides has so far yielded limited clinical success for cancer treatment, due mainly to the low immunogenicity of tumor-associated antigens. In this study, we have identified intratumoral virus-induced inflammation as a precondition for effective antitumor DC vaccination in mice. Administration of a tumor-targeted DC vaccine during ongoing virus-induced tumor inflammation, a regimen referred to as oncolysis-assisted DC vaccination (ODC), elicited potent antitumoral CD8+ T cell responses. This potent effect was not replicated by TLR activation outside the context of viral infection. ODC-elicited immune responses mediated marked tumor regression and successful eradication of preestablished lung colonies, an essential prerequisite for potentially treating metastatic cancers. Unexpectedly, depletion of Tregs during ODC did not enhance therapeutic efficacy; rather, it abrogated antitumor cytotoxicity. This phenomenon could be attributed to a compensatory induction of myeloid-derived suppressor cells in Treg-depleted and thus vigorously inflamed tumors, which prevented ODC-mediated immune responses. Consequently, Tregs are not only general suppressors of immune responses, but are essential for the therapeutic success of multimodal and temporally fine-adjusted vaccination strategies. Our results highlight tumor-targeting, replication-competent viruses as attractive tools for eliciting effective antitumor responses upon DC vaccination.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T Reguladores/imunologia , Vírus/imunologia , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos , Neoplasias/virologia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologiaRESUMO
Oncolytic infection elicits antitumoral immunity, but the impact of tumor-selective replication on the balance between antiviral and antitumoral immune responses has not yet been investigated. To address this question, we constructed the highly tumor-selective adenovirus Ad-p53T whose replication in target tumor cells is governed by aberrant telomerase activity and transcriptional p53 dysfunction. Telomerase-dependent or nonselective adenoviruses were constructed as isogenic controls. Following infection of mice with the nonselective adenovirus, viral DNA and mRNA levels correlated with strong stimulation of innate immune response genes and severe liver toxicity, whereas telomerase-/p53-specific replication did not trigger innate immunity and prevented liver damage. Compared to telomerase-dependent or unselective viral replication, telomerase-/p53-specific virotherapy significantly decreased antiviral CD8-specific immune responses and antiviral cytotoxicity in vivo. Consistent with our hypothesis, telomerase-selective replication led to intermediate results in these experiments. Remarkably, all viruses efficiently lysed tumors and induced a therapeutically effective tumor-directed CD8 cytotoxicity. In immunocompetent mice with extended lung metastases burden, treatment of subcutaneous primary tumors with Ad-p53T significantly prolonged survival by inhibition of lung metastases, whereas unselective viral replication resulted in death by liver failure. In summary, the degree of tumor selectivity of viral replication marginally influences antitumoral immune responses, but is a major determinant of antivector immunity and systemic toxicity.
Assuntos
Infecções por Adenoviridae/imunologia , Adenoviridae/fisiologia , Hepatopatias/imunologia , Neoplasias Pulmonares/imunologia , Vírus Oncolíticos/fisiologia , Replicação Viral/fisiologia , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/virologia , Animais , Western Blotting , Células Cultivadas , Feminino , Fibroblastos/imunologia , Fibroblastos/virologia , Humanos , Técnicas Imunoenzimáticas , Hepatopatias/terapia , Hepatopatias/virologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Nus , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/genética , Telomerase/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND AIMS: Viral infection of a dying cell dictates the immune response against intracellular antigens, suggesting that virotherapy may be an effective tool to induce immunogenic cell death during systemic cancer treatment. Since viruses and proteasome inhibitors both induce accumulation of misfolded proteins, endoplasmic reticulum (ER) stress and immune responses during treatment of hepatocellular carcinoma (HCC) with bortezomib and the tumour-specifically replicating virus hTert-Ad (human telomerase reverse transcriptase promoter-regulated adenovirus) were investigated. METHODS: Unfolded protein response (UPR) pathways and ER stress-mediated apoptosis were investigated by western blots, caspase-3 assays, 4',6-diamidino-2-phenylindole (DAPI) and Annexin V staining in HCC cells following hTert-Ad/bortezomib treatment. Oncolysis was assessed in subcutaneous HCC mouse models. Antiviral/antitumoural immune responses were characterised in immunocompetent HCC mouse models by ELISA, ELISpot assays and pentamer staining. Systemic efficacy of antitumoural immunity was investigated by determination of lung metastases burden. RESULTS: Bortezomib and hTert-Ad trigger complementary UPR pathways but negatively interfere with important recovery checkpoints, resulting in enhanced apoptosis of HCC cells in vitro and improved oncolysis in vivo. In immunocompetent mice, bortezomib inhibited antiviral immune responses, whereas ER stress-induced apoptosis of infected HCC resulted in caspase-dependent triggering of antitumoural immunity. In therapeutic settings in immunocompetent, but not in immunodeficient or CD8-depleted mice, virotherapy-induced antitumoural immunity efficiently inhibited outgrowth of non-infected lung metastases. Immunotherapeutic efficacy could be significantly improved by bortezomib in experiments with low viral doses. CONCLUSION: Proteasome inhibition during virotherapy disrupts the UPR, leading to enhanced ER stress-induced apoptosis, improved local oncolysis and antitumoural immunity. The results suggest that combining intratumoural virotherapy with adjuvant systemic therapies, which specifically support the function of the virotherapy as an antitumoural vaccine, is a promising immunotherapeutic strategy against HCC.
Assuntos
Carcinoma Hepatocelular/secundário , Neoplasias Hepáticas/terapia , Neoplasias Pulmonares/secundário , Terapia Viral Oncolítica/métodos , Adenoviridae , Animais , Antineoplásicos/uso terapêutico , Apoptose , Ácidos Borônicos/uso terapêutico , Bortezomib , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Terapia Combinada , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/fisiologia , Feminino , Imunidade Celular , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Inibidores de Proteassoma , Pirazinas/uso terapêutico , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/fisiologia , Células Tumorais Cultivadas , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
A hallmark of human tumors is the loss of p53 or its transcriptional functions. In this study, we describe the generation of the conditionally replicating adenovirus Adp53sensor for the treatment of p53-dysfunctional tumors. p53-selective attenuation of viral replication was achieved by using p53-dependent expression of the transcriptional repressor Gal4-KRAB that was directed against the adenoviral E1A locus. Adp53sensor shows efficient replication in p53-dysfunctional, but not in p53-active cells. In p53-dysfunctional cells, p53-analogous transcriptional activity by other p53 family members was not sufficient to compromise replication of Adp53sensor. In comparison with a genetically similar, but p53-insensitive virus, Adp53sensor replication was inhibited after systemic infection of p53-wt-mice, but not in p53-ko-mice thus confirming the correct function of the chosen approach. Adp53sensor showed efficient lytic and replicative properties in all investigated cells with p53-dysfunction and successfully inhibited the growth of subcutaneous xenotransplants in vivo. We further demonstrated that intravenous injection of Adp53sensor lead to significantly reduced liver damage compared to the control virus. Together, our data show that Adp53sensor is an oncolytic, p53-selective adenovirus for efficient treatment of p53-dysfunctional tumors with a favorable toxicity profile. Moreover, Adp53sensor provides a strategy that should be applicable to other transcriptionally regulated DNA viruses.
Assuntos
Adenoviridae/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Replicação Viral/fisiologia , Adenoviridae/genética , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Interferência de RNA , Proteína Supressora de Tumor p53/genética , Replicação Viral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ion channels are membrane-spanning proteins that allow ions to permeate at high rates. The kinetic characteristics of the channels present in a cell determine the cell signaling profile and therefore cell function in many different physiological processes. We found that Kv1.7 channels from mouse heart muscle have two putative translation initiation start sites that generate two channel isoforms with different functional characteristics, mKv1.7L (489 aa) and a shorter mKv1.7S (457 aa). The electrophysiological analysis of mKv1.7L and mKv1.7S channels revealed that the two channel isoforms have different inactivation kinetics. The channel resulting from the longer protein (L) inactivates faster than the shorter channels (S). Our data supports the hypothesis that mKv1.7L channels inactivate predominantly due to an N-type related mechanism, which is impaired in the mKv1.7S form. Furthermore, only the longer version mKv1.7L is regulated by the cell redox state, whereas the shorter form mKv1.7S is not. Thus, expression starting at each translation initiation site results in significant functional divergence. Our data suggest that the redox modulation of mKv1.7L may occur through a site in the cytoplasmic N-terminal domain that seems to encompass a metal coordination motif resembling those found in many redox-sensitive proteins. The mRNA expression profile and redox modulation of mKv1.7 kinetics identify these channels as molecular entities of potential importance in cellular redox-stress states such as hypoxia.