RESUMO
The receptor Triggering Receptor Expressed on Myeloid cells 2 (TREM2) is associated with several neurodegenerative diseases including Alzheimer's Disease and TREM2 stimulation represents a novel therapeutic opportunity. TREM2 can be activated by antibodies targeting the stalk region, most likely through receptor dimerization. Endogenous ligands of TREM2 are suggested to be negatively charged apoptotic bodies, mimicked by phosphatidylserine incorporated in liposomes and other polyanionic molecules likely binding to TREM2 IgV fold. However, there has been much discrepancy in the literature on the nature of phospholipids (PLs) that can activate TREM2 and on the stability of the corresponding liposomes over time. We describe optimized liposomes as robust agonists selective for TREM2 over TREM1 in cellular system. The detailed structure/activity relationship studies of lipid polar heads indicate that negatively charged lipid heads are required for activity and we identified the shortest maximally active PL sidechain. Optimized liposomes are active on both TREM2 common variant and TREM2 R47H mutant. Activity and selectivity were further confirmed in different native TREM2 expressing cell types including on integrated cellular responses such as stimulation of phagocytic activity. Such tool agonists will be useful in further studies of TREM2 biology in cellular systems alongside antibodies, and in the design of small molecule synthetic TREM2 agonists.
Assuntos
Doença de Alzheimer , Lipossomos , Glicoproteínas de Membrana/imunologia , Receptores Imunológicos/imunologia , Doença de Alzheimer/metabolismo , Anticorpos/metabolismo , Encéfalo/metabolismo , Humanos , Ligantes , Microglia/metabolismo , Células Mieloides/metabolismo , Fosfatidilserinas/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismoRESUMO
The Revised International Staging System (R-ISS) stratifies patients affected by Multiple Myeloma (MM) into three distinct risk groups: R-ISS I [ISS Stage I, Standard-Risk cytogenetics and normal Lactase DeHydrogenase (LDH)], R-ISS III (ISS stage III and either high-risk cytogenetics or high LDH) and R-ISS II (any other characteristics). With the aim to verify whether the three R-ISS groups could be divided into subgroups with different prognostic factors based on the detection of Circulating Plasma Cells (CPCs) at diagnosis, in this retrospective analysis, we evaluated 161 patients with MM treated at our centre between 2005 and 2017. In all, 57 patients (33·9%) were staged as R-ISS III, 98 (58·3%) as R-ISS II and six (3·6%) as R-ISS I. CPCs were detected in 125 patients (74·4%), while in 43 patients (25·6%) no CPCs were seen. Our analysis revealed that Overall Survival (OS) and progression-free survival (PFS) rates in R-ISS II patients were higher in the subgroup without CPCs compared to the subgroup with ≥1 CPCs (OS: 44·7% vs. 16·3%, P = 0·0089; PFS: 27·8% vs. 8·1%, P = 0·0118). Our present findings suggest that the detection of CPCs at diagnosis may be used as a further prognostic biomarker to improve the risk stratification of patients with MM staged as R-ISS II.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Mieloma Múltiplo , Células Neoplásicas Circulantes/metabolismo , Plasmócitos/metabolismo , Transplante de Células-Tronco , Adulto , Idoso , Idoso de 80 Anos ou mais , Autoenxertos , Dexametasona/administração & dosagem , Intervalo Livre de Doença , Feminino , Humanos , Fatores Imunológicos/administração & dosagem , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/sangue , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/terapia , Estadiamento de Neoplasias , Inibidores de Proteassoma/administração & dosagem , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Cancer cell stress induced by cytotoxic agents promotes antitumor immune response. Here, we observed that N6-isopentenyladenosine (iPA), an isoprenoid modified adenosine with a well established anticancer activity, was able to induce a significant upregulation of cell surface expression of natural killer (NK) cell activating receptor NK Group 2 member D (NKG2D) ligands on glioma cells in vitro and xenografted in vivo. Specifically suboptimal doses of iPA (0.1 and 1 µM) control the selective upregulation of UL16-binding protein 2 on p53wt-expressing U343MG and that of MICA/B on p53mut-expressing U251MG cells. This event made the glioblastoma cells a potent target for NK cell-mediated recognition through a NKG2D restricted mechanism. p53 siRNA-mediated knock-down and pharmacological inhibition (pifithrin-α), profoundly prevented the iPA action in restoring the immunogenicity of U343MG cells through a mechanism that is dependent upon p53 status of malignancy. Furthermore, accordingly to the preferential recognition of senescent cells by NK cells, we found that iPA treatment was critical for glioma cells entry in premature senescence through the induction of S and G2/M phase arrest. Collectively, our results indicate that behind the well established cytotoxic and antiangiogenic effects, iPA can also display an immune-mediated antitumor activity. The indirect engagement of the innate immune system and its additional activity in primary derived patient's glioma cell model (GBM17 and GBM37), fully increase its translational relevance and led to the exploitation of the isoprenoid pathway for a valid therapeutic intervention in antiglioma research.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/patologia , Glioma/patologia , Isopenteniladenosina/farmacologia , Células Matadoras Naturais/patologia , Animais , Antineoplásicos/imunologia , Neoplasias Encefálicas/imunologia , Linhagem Celular Tumoral , Glioma/imunologia , Humanos , Isopenteniladenosina/imunologia , Células Matadoras Naturais/imunologia , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The endocannabinoid system, through cannabinoid receptor signaling by endocannabinoids, is involved in a wide range of functions and physiopathological conditions. To date, very little is known concerning the role of the endocannabinoids in the control and regulation of cell proliferation. An anti-proliferative action of CB1 signaling blockade in neurogenesis and angiogenesis argues in favor of proliferation-promoting functions of endocannabinoids through CB1 receptors when pro-growth signals are present. Furthermore, liver regeneration, a useful in vivo model of synchronized cell proliferation, is characterized by a peak of anandamide that elicits through CB1 receptor, the expression of critical mitosis genes. The aim of this study was to focus on the timing of endocannabinoid signaling changes during the different phases of the cell cycle, exploiting the rat liver regeneration model following partial hepatectomy, the most useful to study synchronized cell cycle in vivo. Hepatic regeneration led to increased levels of anandamide and endocannabinoid-like molecules oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in the G1 phase of the cell cycle, with a concomitant increase in CB1 mRNA levels, whose protein expression peaked later during the S phase. Blocking of CB1 receptor with a low dose of the selective antagonist/inverse agonist SR141716 (0.7 mg/kg/dose) affected cell cycle progression reducing the expression of PCNA, and through the inhibition of pERK and pSTAT3 pathways. These results support the notion that the signaling mediated by anandamide through CB1 receptor may be important for the entry and progression of cells into the cell cycle and hence for their proliferation under mitogenic signals.
Assuntos
Ácidos Araquidônicos/metabolismo , Ciclo Celular , Proliferação de Células , Endocanabinoides/metabolismo , Regeneração Hepática , Fígado/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Animais , Antagonistas de Receptores de Canabinoides/farmacologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glicerídeos/metabolismo , Hepatectomia , Fígado/efeitos dos fármacos , Fígado/patologia , Regeneração Hepática/efeitos dos fármacos , Masculino , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
N6-isopentenyladenosine (iPA), an end product of the mevalonate pathway with an isopentenyl chain, is already known to exert a suppressor effect against various tumors. In this work, we investigated whether iPA also directly interferes with the angiogenic process, which is fundamental to tumor growth and progression. To this end, using human umbilical vein endothelial cells (HUVECs) as a suitable in vitro model of angiogenesis, we evaluated their viability, proliferation, migration, invasion, tube formation in response to iPA, and molecular mechanisms involved. Data were corroborated in mice by using a gel plug assay. iPA dose- and time-dependently inhibited all the neoangiogenesis stages, with an IC50 of 0.98 µM. We demonstrated for the first time, by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS), that iPA was monophosphorylated into 5'-iPA-monophosphate (iPAMP) by the adenosine kinase (ADK) inside the cells. iPAMP is the active form that inhibits angiogenesis through the direct activation of AMP-kinase (AMPK). Indeed, all effects were completely reversed by pretreatment with 5-iodotubercidin (5-Itu), an ADK inhibitor. The isoprenoid intermediate isopentenyl pyrophosphate (IPP), which shares the isopentenyl moiety with iPA, was ineffective in the inhibition of angiogenesis, thus showing that the iPA structure is specific for the observed effects. In conclusion, iPA is a novel AMPK activator and could represent a useful tool for the treatment of diseases where excessive neoangiogenesis is the underlying pathology.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Inibidores da Angiogênese/metabolismo , Isopenteniladenosina/metabolismo , Cromatografia Líquida , Ativação Enzimática , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Patológica , Fosforilação , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis. METHODS: The expression and localization of ANXA1 in MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were detected by Western Blotting and Immunofluorescence assay. Expression and activation of Formyl Peptide Receptors (FPRs) were shown through flow cytometry/PCR and FURA assay, respectively. To investigate the role of ANXA1 in PC cell migration and invasion, we performed in vitro wound-healing and matrigel invasion assays. RESULTS: In all the analyzed PC cell lines, a huge expression and a variable localization of ANXA1 in sub-cellular compartments were observed. We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers. Then, we tested MIA PaCa-2 and PANC-1 cell migration and invasiveness rate which was inhibited by specific ANXA1 siRNAs. Both the cell lines expressed FPR-1 and -2. Ac2-26, an ANXA1 mimetic peptide, induced intracellular calcium release, consistent with FPR activation, and significantly increased cell migration/invasion rate. Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis. The importance of the secreted form of ANXA1 in cellular motility was confirmed by the administration of ANXA1 blocking antibody that inhibited migration and invasion rate in MIA PaCa-2 but not in PANC-1 cells that lack the 33 kDa ANXA1 form and show a lower degree of invasiveness. Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells. CONCLUSION: This study provides new insights on the role of ANXA1 protein in PC progression. Our findings suggest that ANXA1 protein could regulate metastasis by favouring cell migration/invasion intracellularly, as cytoskeleton remodelling factor, and extracellularly like FPR ligand.
Assuntos
Anexina A1/metabolismo , Neoplasias Pancreáticas/patologia , Anexina A1/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Citoplasma/metabolismo , Transição Epitelial-Mesenquimal , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica , Neoplasias Pancreáticas/metabolismo , Peptídeos/farmacologia , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Neoplasias PancreáticasRESUMO
Statins are well known competitive inhibitors of hydroxymethylglutaryl-CoA reductase enzyme (HMG-CoA reductase), thus traditionally used as cholesterol-lowering agents. In recent years, more and more effects of statins have been revealed. Nowadays alterations of lipid metabolism have been increasingly recognized as a hallmark of cancer cells. Consequently, much attention has been directed toward the potential of statins as therapeutic agents in the oncological field. Accumulated in vitro and in vivo clinical evidence point out the role of statins in a variety of human malignancies, in regulating tumor cell growth and anti-tumor immune response. Herein, we summarize and discuss, in light of the most recent observations, the anti-tumor effects of statins, underpinning the detailed mode of action and looking for their true significance in cancer prevention and treatment, to determine if and in which case statin repositioning could be really justified for neoplastic diseases.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Ácido Mevalônico/antagonistas & inibidores , Ácido Mevalônico/metabolismo , Neoplasias/metabolismo , Neoplasias/prevenção & controleRESUMO
N6-isopentenyladenosine (iPA) is a modified adenosine with an isopentenyl moiety derived from the mevalonate pathway which displays pleiotropic biological effects, including anti-tumor and anti-angiogenic activity. Previous evidence revealed a biphasic effect of iPA on phytohemagglutinin-stimulated lymphocytes, being pro-proliferative at low doses and anti-proliferative at high doses. Analogously, we have recently shown that low iPA concentrations (<1µM) increased the immune response of natural killer (NK) cells against cancer targets. In the present study, we evaluated the effect of iPA at high concentration (10µM) on IL-2-activated NK cells. iPA, inhibited NK cell proliferation and cytotoxicity against their conventional tumor target, human K562 cells. This inhibition was associated with decreased expression and functionality of NK cell activating receptors NKp44 and NKG2D as well as impaired cyto/chemokines secretion (RANTES, MIP-1α, TNF-α and IFN-γ). ERK/MAPK and STAT5 activation in IL-2-activated NK cells were inhibited by iPA. The results obtained in vitro were validated in vivo in the inflammatory murine model of croton oil-induced ear dermatitis. The topical application of iPA significantly reduced mouse ear oedema, thus suggesting anti-inflammatory properties of this molecule. These results show the ability of iPA to exert anti-inflammatory effects both in vitro and in vivo directly targeting NK cells, providing a novel pharmacological tool in those diseases characterized by a deregulated immune-response, such as cancer or inflammatory conditions.
Assuntos
Anti-Inflamatórios/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Interleucina-2/farmacologia , Isopenteniladenosina/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Administração Tópica , Animais , Anti-Inflamatórios/administração & dosagem , Células Cultivadas , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Edema/induzido quimicamente , Edema/tratamento farmacológico , Humanos , Isopenteniladenosina/administração & dosagem , Células K562 , Células Matadoras Naturais/metabolismo , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptor 2 Desencadeador da Citotoxicidade Natural/metabolismo , Fator de Transcrição STAT5/metabolismoRESUMO
The mutational status of the IGHV gene is routinely assessed in patients with chronic lymphocytic leukaemia (CLL), since it is both prognostic of clinical outcome and predictive of response to treatment. This study evaluates the IGHV mutational status, assessed in newly diagnosed CLL patients, as a stand-alone predictor of time to first treatment (TTFT). We analysed the data of 236 CLL patients, diagnosed at our centre between January 2004 and September 2020, with a minimum follow-up period of 3.0 years, Binet A-B and Rai 0-II stages. IGHV was unmutated in 38.1â¯% and mutated in 61.9â¯% of cases. The univariate analysis showed a statistically significant difference (p < 0.001) in TTFT based on unmutated (85.2â¯% at 14 years, 95â¯% CI = 63.3-94.5â¯%) or mutated (41.3â¯% at 14 years, 95â¯% CI = 29.5-51.8â¯%) and the need for treatment at 1, 3 and 5 years was of 20.0â¯% vs 4.1â¯% (p < 0.001), 42.7â¯% vs 11.4â¯% (p < 0.001) and 55.8â¯% vs 20.0â¯% (p < 0.001) in unmutated and mutated IGHV patients, respectively. Multivariate analysis confirmed that unmutated IGHV status negatively affects TTFT (p < 0.001), in addition to high-risk genomic aberration (p = 0.025), Rai stage I (p = 0.007) and II (p-valueâ¯<â¯0.001). The difference in TTFT based on unmutated or mutated IGHV status remains statistically significant also when considering the subgroups by the genomic aberrations and Rai stages. Our findings suggest that, with the single analysis of the IGHV mutational status at CLL diagnosis, along with clinical and laboratory data, and without karyotype and TP53 data, clinicians will have prognostic and predictive indications for the first clinical treatment and appropriate follow-up of patients.
Assuntos
Cadeias Pesadas de Imunoglobulinas , Leucemia Linfocítica Crônica de Células B , Mutação , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/terapia , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Seguimentos , Prognóstico , Adulto , Idoso de 80 Anos ou mais , Cadeias Pesadas de Imunoglobulinas/genética , Tempo para o Tratamento , Região Variável de Imunoglobulina/genética , Estadiamento de Neoplasias , Estudos RetrospectivosRESUMO
In this study we investigated the role of CB1 receptor signaling in angiogenesis and the therapeutic exploitation of CB1 inactivation as an antiangiogenic strategy. We started from the observation that CB1 receptor expression is induced during angiogenesis and that the endocannabinoid anandamide stimulated bFGF-induced angiogenesis in the nanomolar physiologic range. To define the functional involvement of CB1 receptor signaling during angiogenesis, 2 different strategies have been carried out: siRNA-mediated knockdown and pharmacologic antagonism of CB1 receptors. CB1 receptors inactivation resulted in the inhibition of bFGF-induced endothelial proliferation, migration, and capillary-like tube formation, through prosurvival and migratory pathways involving ERK, Akt, FAK, JNK, Rho, and MMP-2. To corroborate the potential therapeutic exploitation of CB1 blockade as an antiangiogenic strategy, we performed in vivo assays founding that CB1 blockade was able to inhibit bFGF-induced neovascular growth in the rabbit cornea assay. A relevant finding was the ability to reduce ocular pathologic neo-vascularization in mouse oxygen-induced retinopathy. These results demonstrate that CB1 signaling participates to the proliferative response elicited by proangiogenic growth factors in angiogenesis and that for this reason CB1 receptor could represent a novel target for the treatment of diseases where excessive neoangiogenesis is the underlying pathology.
Assuntos
Neovascularização Fisiológica , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/deficiência , Animais , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Córnea/irrigação sanguínea , Córnea/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Recém-Nascido , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , RNA Interferente Pequeno/genética , Coelhos , Receptor CB1 de Canabinoide/genética , Retinopatia da Prematuridade/tratamento farmacológico , Transdução de Sinais , Quinases Associadas a rho/metabolismoRESUMO
CD33/Sialic acid-binding Ig-like lectin 3 (SIGLEC3) is an innate immune receptor expressed on myeloid cells and mediates inhibitory signaling via tyrosine phosphatases. Variants of CD33 are associated with Alzheimer's disease (AD) suggesting that modulation of CD33 signaling might be beneficial in AD. Hence, there is an urgent need for reliable cellular CD33 reporter systems. Therefore, we generated a CD33 reporter cell line expressing a fusion protein consisting of the extracellular domain of either human full-length CD33 (CD33M) or the AD-protective variant CD33ΔE2 (D2-CD33/CD33m) linked to TYRO protein tyrosine kinase binding protein (TYROBP/DAP12) to investigate possible ligands and antibodies for modulation of CD33 signaling. Application of the CD33-specific antibodies P67.6 and 1c7/1 to the CD33M-DAP12 reporter cells resulted in increased phosphorylation of the kinase SYK, which is downstream of DAP12. CD33M-DAP12 but not CD33ΔE2-DAP12 expressing reporter cells showed increased intracellular calcium levels upon treatment with CD33 antibody P67.6 and partially for 1c7/1. Furthermore, stimulation of human induced pluripotent stem cell-derived microglia with the CD33 antibodies P67.6 or 1c7/1 directly counteracted the triggering receptor expressed on myeloid cells 2 (TREM2)-induced phosphorylation of SYK and decreased the phagocytic uptake of bacterial particles. Thus, the developed reporter system confirmed CD33 pathway activation by CD33 antibody clones P67.6 and 1c7/1. In addition, data showed that phosphorylation of SYK by TREM2 activation and phagocytosis of bacterial particles can be directly antagonized by CD33 signaling.
Assuntos
Doença de Alzheimer/imunologia , Anticorpos/imunologia , Células-Tronco Pluripotentes Induzidas/imunologia , Microglia/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Doença de Alzheimer/genética , Linhagem Celular , Humanos , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genéticaRESUMO
Voltage-gated ion channels produce rapid transmembrane currents responsible for action potential generation and propagation at the neuronal, muscular, and cardiac levels. They represent attractive clinical targets because their altered firing frequency is often the hallmark of pathological signaling leading to several neuromuscular disorders. Therefore, a method to study their functioning upon repeated triggers at different frequencies is desired to develop new drug molecules selectively targeting pathological phenotype. Optogenetics provides powerful tools for millisecond switch of cellular excitability in contactless, physiological, and low-cost settings. Nevertheless, its application to large-scale drug-screening operations is still limited by long processing time (due to sequential well read), rigid flashing pattern, lack of online compound addition, or high consumable costs of existing methods. Here, we developed a method that enables simultaneous analysis of 384-well plates with optical pacing, fluorescence recording, and liquid injection. We used our method to deliver programmable millisecond-switched depolarization through light-activated opsin in concomitance with continuous optical recording by a fluorescent indicator. We obtained 384-well pacing of recombinant voltage-activated sodium or calcium channels, as well as induced pluripotent stem cell (iPSC)-derived cardiomyocytes, in all-optical parallel settings. Furthermore, we demonstrated the use-dependent behavior of known ion channel blockers by optogenetic pacing at normal or pathological firing frequencies, obtaining very good signal reproducibility and accordance with electrophysiology data. Our method provides a novel physiological approach to study frequency-dependent drug behavior using reversible programmable triggers. The all-optical parallel settings combined with contained operational costs make our method particularly suited for large-scale drug-screening campaigns as well as cardiac liability studies.
Assuntos
Bioensaio , Bloqueadores dos Canais de Cálcio/farmacologia , Optogenética/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Proteínas de Algas/antagonistas & inibidores , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Chlamydomonas reinhardtii , Corantes Fluorescentes/química , Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Imagem Óptica/métodos , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Rodopsina/antagonistas & inibidores , Rodopsina/genética , Rodopsina/metabolismoAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Feminino , Seguimentos , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Pentostatina/administração & dosagem , Pentostatina/efeitos adversosRESUMO
OBJECTIVES: The therapy of advanced, relapsed or refractory primary cutaneous lymphomas is often unsatisfactory. Recent data indicate a favourable pharmacokynetic, pharmacodynamic and toxicity profile of pegylated liposomal doxorubicin (Peg-Doxo) in primary cutaneous T-cell lymphomas, while in primary cutaneous B-cell lymphomas (PCBCLs), the drug efficacy has never been assessed so far. METHODS: We performed a prospective phase II pilot clinical trial of Peg-Doxo monotherapy (20 mg/m(2)) in PCBCLs. One patient had a marginal zone B-cell lymphoma and four were affected by diffuse large B-cell lymphoma-leg type, all with widespread nodular lesions. RESULTS: All the patients achieved a complete response (CR = 100%) in a short period of time (median 3 months), even when pretreated with radio-chemotherapy. Two experienced a relapse. At follow-up, one patient died for progressive disease; four are in CR after 5, 52, 63 and 69 months. As concerning the toxicity profile, the treatment was well-tolerated, no one decreased or delayed the dose. The haematological toxicity was mild with only one case of grade III neutropenia; a patient showed a grade I neurotoxicity. Dermatological toxicity, in particular the palmar-plantar erythrodysesthesia, did not occurred, probably because of both the low dosages of Peg-Doxo monotherapy and the oral prophylaxis with pyridoxine. CONCLUSIONS: In spite of the small number of patients, it emerges that monochemotherapy with Peg-Doxo has a significantly high clinical activity and a good safety profile in PCBCLs, even in aggressive forms, compared with other therapeutic regimens, which are completely reviewed. It suggests the need of further investigations in this field.
Assuntos
Antineoplásicos/uso terapêutico , Doxorrubicina/análogos & derivados , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Adulto , Idoso , Antineoplásicos/efeitos adversos , Doxorrubicina/efeitos adversos , Doxorrubicina/uso terapêutico , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Projetos PilotoRESUMO
The lack of miniaturized and cost-effective methods to control cellular excitability with dosable and temporally precise electrical perturbations represents a long-lasting and unsolved bottleneck for ion channel drug discovery pipelines. Here we developed a high-throughput-compatible fluorescent-based cellular assay that combines optogenetics and co-culture approaches to obtain spatial, temporal, and quantitative control of ion channel activity. The modularity and increased flexibility of control of this light-tandem assay, combined with contained costs and compatibility with conventional drug-screening platforms, make this system suitable for temporally precise screening of ion channel function in controlled conformations and can also be used to recapitulate other complexly regulated biological processes.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Canais Iônicos/química , Optogenética/métodos , Canais de Cálcio Tipo L/química , Linhagem Celular , Técnicas de Cocultura , Humanos , Canais Iônicos/agonistas , Canais Iônicos/antagonistas & inibidores , Cinética , LigantesRESUMO
Targeting the autophagic process is considered a promising therapeutic strategy in cancer since a great number of tumors, including melanoma, show high basal levels of protective autophagy that contributes to tumor progression and chemoresistance. Here, exploiting both in vitro and in vivo approaches, we identified N6-isopentenyladenosine (iPA), an end product of the mevalonate pathway, as a novel autophagy inhibitor with an interesting anti-melanoma activity. iPA, after being phosphorylated by adenosine kinase into 5'-iPA-monophosphate, induces autophagosome accumulation through AMPK activation, measured by increased fluorescent GFP-LC3 puncta and enhanced conversion into the lipidated autophagosome-associated LC3-II. However, at a later stage iPA blocks the autophagic flux monitored by p62 accumulation, Luciferase reporter-based assay for LC3 turnover in living cells and fluorescence of a tandem RFP-GFP-LC3 construct. Impaired autophagic flux is due to the block of autophagosome-lysosome fusion through the defective localization and function of Rab7, whose prenylation is inhibited by iPA, resulting in a net inhibition of autophagy completion that finally leads to melanoma apoptotic cell death. AMPK silencing prevents apoptosis upon iPA treatment, whereas basal autophagosome turnover is still inhibited due to unprenylated Rab7. These results strongly support the advantage of targeting autophagy for therapeutic gain in melanoma and provide the preclinical rational to further investigate the antitumor action of iPA, able to coordinately induce autophagosome accumulation and inhibit the autophagic flux, independently targeting AMPK and Rab7 prenylation. This property may be particularly useful for the selective killing of tumors, like melanoma, that frequently develop chemotherapy resistance due to protective autophagy activation.
Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Isopenteniladenosina/farmacologia , Melanoma/tratamento farmacológico , Prenilação/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Isopenteniladenosina/química , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Células Tumorais Cultivadas , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
INTRODUCTION: First and second generation tyrosine kinase inhibitors, represent a new, fully biologic and targeted approach to chronic lymphocytic leukemia and allowed to obtain high responses and acceptable tolerability even in elderly and high risk patients. On the other hand, prolonged experience with these agents has raised some questions on unexpected toxicities, response quality, treatment duration and onset of resistances. AREAS COVERED: This review analyzes the main published studies with the aim to discuss whether, in future, new agents could become a part of standard treatments or even replace the chemo-immunotherapy in chronic lymphocytic leukemia. Expert commentary: Despite different trials has been already published and many are still ongoing, follow up times are, at present, too short. A chemo-free approach surely represents a desirable goal for chronic lymphocytic leukemia, nevertheless longer observation is warranted to better define the correct use of targeted therapies.
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Antineoplásicos/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores , Ensaios Clínicos como Assunto , Descoberta de Drogas , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Terapia de Alvo Molecular , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
The management of patients with chronic lymphocytic leukemia (CLL) has radically improved over the last few years with the addition of anti-CD20 monoclonal antibodies (MoAbs) to chemotherapy. Chlorambucil has been considered for decades as a suitable therapeutic option for frail patients. Taking into account the advantage offered by the addition of MoAbs to chemotherapy, different studies up to now have explored the feasibility of chlorambucil-based chemoimmunotherapies in treatment-naïve CLL. COMPLEMENT1 is a prospective, randomized, open-label trial evaluating the efficacy and safety of ofatumumab added to chlorambucil, compared with chlorambucil in monotherapy, in the setting of untreated patients with CLL considered unsuitable for a fludarabine-based approach. Progression-free survival was significantly longer in the chemoimmunotherapy arm when compared with the single-agent chlorambucil (22.4 months versus 13.1 months). Response rate and quality were also improved in the combination arm. Furthermore, the addition of ofatumumab did not lead to an unmanageable toxicity. While the employment of anti-CD20 antibodies represents an advantage in the treatment of the CLL symptomatic population, at present different patient selection and treatment schedules do not allow a reliable comparison between chlorambucil-based regimens. The addition of ofatumumab to chlorambucil represents a further therapeutic gain in CLL. Longer follow up and direct comparison with other MoAbs are warranted to establish the preferred first-line treatment in elderly and unfit patients.
RESUMO
Some compounds of a series of novel pyrrolo-1,5-benzoxa(thia)zepine, a well-known group of tubulin targeting agents, display anti-tumor effects mainly inducing cell cycle arrest and apoptosis in several human cancer models. A member of this family, pyrrolo-1,5-benzoxazepine-15 (PBOX-15), has previously shown potent pro-apoptotic activity in a variety of human tumor cell types, with minimal toxicity toward normal blood and bone marrow cells. In this study, we evaluated the PBOX-15-mediated effects in human colorectal cancer cell (CRC) lines, DLD-1 and HT-29. The compound, used at concentrations equal to or greater than 1 µM, inhibited the proliferation of human CRC cells, inducing a significant cell cycle arrest in the G2/M phase. In DLD-1 cells, treatments prolonged over 48 h triggered a strong activation of the intrinsic apoptotic pathway as indicated by activation of caspase-9, caspase-3 and PARP cleavage. Moreover, nanomolar concentrations of PBOX-15, significantly improved the oxaliplatin and 5-fluouracil-induced anti-proliferative effects in DLD1 cell line. The observed synergistic interaction of both PBOX-15/Oxaliplatin and PBOX-15/5FU may involve activation of p38 MAPK and JNK pathway, which in turn significantly increased caspase-3 cleavage in DLD-1 cells, treated with PBOX-5/Oxaliplatin but not with PBOX-15/5FU. Moreover, PBOX-15/5FU-treated cells showed an increase in expression of the pro-apoptotic protein Bax. Taken together, these results show that PBOX-15 could represent a promising compound for the treatment of human CRC and a strong candidate for novel therapeutic options.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/farmacologia , Compostos Organoplatínicos/farmacologia , Oxazepinas/farmacologia , Pirróis/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Sinergismo Farmacológico , Fluoruracila/administração & dosagem , Células HT29 , Humanos , Compostos Organoplatínicos/administração & dosagem , Oxaliplatina , Oxazepinas/administração & dosagem , Pirróis/administração & dosagemRESUMO
Treatment aim for chronic lymphocytic leukemia has been radically changed over the past years from providing only a palliative approach to reaching disease eradication and improving survival. Ofatumumab is a monoclonal humanized antibody with peculiar in vitro and in vivo properties, at present approved for double fludarabine and alemtuzumab refractory chronic lymphocytic leukemia. Its efficacy in this subset of patients, who typically have an unfavorable prognosis, facilitated its use in different Phase II and III trials. Ofatumumab as single agent or combined with chemotherapeutic or biologic agents, led to sundry results in the setting of both previously treated or untreated patients. Its role in maintenance therapy is also under investigation. Further advances concerning ofatumumab administration as first line therapy in combination with chlorambucil, came recently from the COMPLEMENT 1 study. Results from this trial will open the door to new perspectives of its use in treatment-naïve patients. Ofatumumab was well tolerated in almost all the studies, with the main adverse events relating mostly to infusion reaction. Hematologic toxicity, especially neutropenia, was also common. A significant improvement in patients' quality of life was reported following ofatumumab treatment and this was mainly due to its effect on constitutional symptoms. Nevertheless, some concerns remain regarding the long-term efficacy of the drug in terms of response duration and survival. The real strength of this drug needs to be confirmed by further studies and direct comparative trials.