RESUMO
High performance affinity reagents are essential tools to enable biologists to profile the cellular location and composition of macromolecular complexes undergoing dynamic reorganization. To support further development of such tools, we have assembled a high-throughput phage display pipeline to generate Fab-based affinity reagents that target different dynamic forms of a large macromolecular complex, using the Chromosomal Passenger Complex (CPC), as an example. The CPC is critical for the maintenance of chromosomal and cytoskeleton processes during cell division. The complex contains 4 protein components: Aurora B kinase, survivin, borealin and INCENP. The CPC acts as a node to dynamically organize other partnering subcomplexes to build multiple functional structures during mitotic progression. Using phage display mutagenesis, a cohort of synthetic antibodies (sABs) were generated against different domains of survivin, borealin and INCENP. Immunofluorescence established that a set of these sABs can discriminate between the form of the CPC complex in the midbody versus the spindle. Others localize to targets, which appear to be less organized, in the nucleus or cytoplasm. This differentiation suggests that different CPC epitopes have dynamic accessibility depending upon the mitotic state of the cell. An Immunoprecipitation/Mass Spectrometry analysis was performed using sABs that bound specifically to the CPC in either the midbody or MT spindle macromolecular assemblies. Thus, sABs can be exploited as high performance reagents to profile the accessibility of different components of the CPC within macromolecular assemblies during different stages of mitosis suggesting this high throughput approach will be applicable to other complex macromolecular systems.
Assuntos
Anticorpos , Aurora Quinase B , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Fragmentos Fab das Imunoglobulinas , Complexos Multiproteicos , Survivina , Anticorpos/química , Anticorpos/genética , Aurora Quinase B/análise , Aurora Quinase B/imunologia , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/imunologia , Proteínas Cromossômicas não Histona/análise , Proteínas Cromossômicas não Histona/imunologia , Citoesqueleto/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Mitose , Complexos Multiproteicos/análise , Complexos Multiproteicos/imunologia , Biblioteca de Peptídeos , Fosforilação , Fuso Acromático/metabolismo , Survivina/química , Survivina/metabolismoRESUMO
Targeting specific protein binding sites to interfere with protein-protein interactions (PPIs) is crucial for the rational modulation of biologically relevant processes. Survivin, which is highly overexpressed in most cancer cells and considered to be a key player of carcinogenesis, features two functionally relevant binding sites. Here, we demonstrate selective disruption of the Survivin/Histone H3 or the Survivin/Crm1 interaction using a supramolecular approach. By rational design we identified two structurally related ligands (LNES and LHIS ), capable of selectively inhibiting these PPIs, leading to a reduction in cancer cell proliferation.
Assuntos
Proteínas Inibidoras de Apoptose , Sítios de Ligação , Proliferação de Células , Proteínas Inibidoras de Apoptose/metabolismo , Ligação Proteica , Survivina/química , Survivina/metabolismoRESUMO
Survivin's dual function as apoptosis inhibitor and regulator of cell proliferation is mediated via its interaction with the export receptor CRM1. This protein-protein interaction represents an attractive target in cancer research and therapy. Here, we report a sophisticated strategy addressing Survivin's nuclear export signal (NES), the binding site of CRM1, with advanced supramolecular tweezers for lysine and arginine. These were covalently connected to small peptides resembling the natural, self-complementary dimer interface which largely overlaps with the NES. Several biochemical methods demonstrated sequence-selective NES recognition and interference with the critical receptor interaction. These data were strongly supported by molecular dynamics simulations and multiscale computational studies. Rational design of lysine tweezers equipped with a peptidic recognition element thus allowed to address a previously unapproachable protein surface area. As an experimental proof-of-principle for specific transport signal interference, this concept should be transferable to any protein epitope with a flanking well-accessible lysine.
Assuntos
Carioferinas/química , Carioferinas/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Survivina/química , Survivina/metabolismo , Sítios de Ligação , Proliferação de Células , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Modelos Moleculares , Sinais de Exportação Nuclear , Ligação Proteica , Conformação Proteica , Proteína Exportina 1RESUMO
Recognizing proteins that lead to a decreased efficiency of treatment in cancer cells constitutes a main goal for biomedical and biotechnological research and applications. Establishing recombinant cells that overexpress a gene of interest stably is important for treatment studies and drug/compound screening. Survivin is an anti-apoptotic protein which can be a potential candidate for regulating cell death and survival. To investigate the association between survivin increment and apoptosis rate, survivin-reconstituted HEK (HEK-S) cell was developed as in vitro model. RT-PCR and Western blot demonstrated that survivin was constitutively overexpressed in HEK-S cells. Both morphological observation and survival assay showed that HEK-S cells were significantly resistant to apoptotic stimuli. Survivin overexpression led to a decrease in caspase 3/7 activity, whereas YM155 led to a corresponding enhance of caspase activity. ROS level was decreased but ATP content increased in HEK-S cells. Also, HEK-S showed less red- fluorescence and reduced cell proliferation compared to HEK after stimulation. Resistance to laser irradiation was clearly visible as compared with control. Moreover, scratching analysis demonstrated the ability of survivin to cause neighboring cells to increase resistance to drug, whereas YM155 enhanced apoptotic rate and declined invasion in HEK-S cells.
Assuntos
Apoptose/genética , Avaliação Pré-Clínica de Medicamentos , Survivina/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Camundongos , Naftoquinonas/farmacologia , Survivina/química , Survivina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The G protein-coupled receptor (GPCR) dimer interface plays an important role in the formation and stabilization of the dimer. Therefore, identifying the potential receptor-receptor interface is an important part of studying GPCRs. Various strategies have been employed to study the GPCR dimer interface and explore its functional significance, but experimental methods lack robustness and calculations are laborious. Herein, we report a combined optimized experimental and calculation approach for identifying and structurally characterizing GPCR dimer interfaces, and constructing atomic resolution models. Using a transmembrane domain (TM) peptide containing a human immunodeficiency virus trans-acting transcriptional activator (HIV-TAT) protein transduction motif, matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MALDITOF-MS), and bioluminescence resonance energy transfer (BRET), we successfully identified Apelin receptor (APJ)/Nociceptin receptor 1 (ORL1) and APJ/Vasopressin receptor 2 (V2R) heterodimer interfaces, and two key sites mediating dimerization. This method can identify dimer interfaces of GPCR homodimers and heterodimers.
Assuntos
Dimerização , Conformação Proteica , Receptores Acoplados a Proteínas G/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Survivina/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Motivos de Aminoácidos/genética , Membrana Celular/química , Proteínas de Ligação ao GTP/química , Células HEK293 , Humanos , Modelos Moleculares , Domínios Proteicos/genética , Multimerização Proteica/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Survivina/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
Survivin is one of the rare proteins that is differentially expressed in normal and cancer cells and is directly or indirectly involved in numerous pathways required for tumor maintenance. It is expressed in almost all cancers and its expression has been detected at early stages of cancer. These traits make survivin an exceptionally attractive target for cancer therapeutics. Even with these promising features to be an oncotherapeutic target, there has been limited success in the clinical trials targeting survivin. Only recently it has emerged that survivin was not being specifically targeted which could have resulted in the negative clinical outcome. Also, focus of research has now shifted from survivin expression in the overall heterogeneous tumor cell populations to survivin expression in cancer stem cells as these cells have proved to be the major drivers of tumors. Therefore, in this review we have analyzed the expression of survivin in normal and cancer cells with a particular focus on its expression in cancer stem cell compartment. We have discussed the major signaling pathways involved in regulation of survivin. We have explored the current development status of various types of interventions for inhibition of survivin. Furthermore, we have discussed the challenges involving the development of potent and specific survivin inhibitors for cancer therapeutics. Finally we have given insights for some of the promising future anticancer treatments.
Assuntos
Desenvolvimento de Medicamentos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Células-Tronco/metabolismo , Survivina/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Survivina/química , Survivina/genéticaRESUMO
The Aurora B chromosomal passenger complex (CPC) is a conserved regulator of mitosis. Its functions require localization first to the chromosome arms and then centromeres in mitosis and subsequently the central spindle in anaphase. Here, we analyze the requirements for core CPC subunits, survivin and INCENP, and the mitotic kinesin-like protein 2 (MKLP2) in targeting to these distinct localizations. Centromere recruitment of the CPC requires interaction of survivin with histone H3 phosphorylated at threonine 3, and we provide a complete structure of this assembly. Furthermore, we show that the INCENP RRKKRR-motif is required for both centromeric localization of the CPC in metaphase and MKLP2-dependent transport in anaphase. MKLP2 and DNA bind competitively to this motif, and INCENP T59 phosphorylation acts as a switch preventing MKLP2 binding in metaphase. In anaphase, CPC binding promotes the microtubule-dependent ATPase activity of MKLP2. These results explain how centromere targeting of the CPC in mitosis is coupled to its movement to the central spindle in anaphase.
Assuntos
Anáfase , Aurora Quinase B/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Cinesinas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Aurora Quinase B/química , Aurora Quinase B/genética , Ligação Competitiva , Centrômero/metabolismo , Centrômero/ultraestrutura , Cromatina/ultraestrutura , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , DNA/genética , DNA/metabolismo , Células HeLa , Histonas/química , Histonas/genética , Humanos , Cinesinas/química , Cinesinas/genética , Metáfase , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Survivina/química , Survivina/genética , Survivina/metabolismoRESUMO
The protein Survivin is highly upregulated in most cancers and considered to be a key player in carcinogenesis. We explored a supramolecular approach to address Survivin as a drug target by inhibiting the protein-protein interaction of Survivin and its functionally relevant binding partner Histone H3. Ligand L1 is based on the guanidiniocarbonyl pyrrole cation and serves as a highly specific anion binder in order to target the interaction between Survivin and Histone H3. NMR titration confirmed binding of L1 to Survivin's Histone H3 binding site. The inhibition of the Survivin-Histone H3 interaction and consequently a reduction of cancer cell proliferation were demonstrated by microscopic and cellular assays.
Assuntos
Histonas/metabolismo , Pirróis/química , Survivina/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Histonas/química , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Ligação Proteica , Pirróis/metabolismo , Pirróis/farmacologia , Survivina/químicaRESUMO
Survivin belongs to the inhibitor of apoptosis protein family, which is consistently overexpressed in most cancer cells but rarely expressed in normal adult tissues. Therefore, the detection and inhibition of survivin are regarded as attractive strategies for cancer-specific treatment. In this study, we designed and synthesized 7-19 residues of inner centromere protein (INCENP)-derived small peptides (INC peptides) as novel survivin-targeting agents. The INC peptides showed binding affinity for the human survivin protein (Kd = 91.4-255 nmol L-1 ); INC16-22 , which contains residues 16-22 of INCENP, showed the highest affinity (91.4 nmol L-1 ). Confocal fluorescence imaging showed consistent colocalization of FITC-INC16-22 and survivin in cell lines. Nona-arginine-linked INC16-22 (r9-INC16-22 ) rendered INC16-22 cells penetrable and strongly inhibited cell growth of MIA PaCa-2 cells (52% inhibition at 1.0 µmol L-1 ) and MDA-MB-231 cells (60% inhibition at 10 µmol L-1 ) as determined by MTT assays. The exposure of MIA PaCa-2 cells to 40 µmol L-1 r9-INC16-22 apparently reduced the intracellular protein expression levels of survivin. However, cleaved caspase-3 was significantly increased in cells treated with r9-INC16-22 , even at 10 µmol L-1 , compared to untreated cells. Flow cytometry revealed that r9-INC16-22 strongly induced apoptosis in MIA PaCa-2 cells. These results indicate that the cytotoxic effects of r9-INC16-22 could be mediated mainly through the disruption of survivin-dependent antiapoptotic functions and partly because of the direct degradation of the survivin protein. Our findings suggest that INC peptides can act as useful scaffolds for novel cancer imaging and anticancer agents.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Proteínas Cromossômicas não Histona/genética , Peptídeos/farmacologia , Survivina/isolamento & purificação , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caspases/química , Caspases/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas Cromossômicas não Histona/química , Feminino , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/isolamento & purificação , Imagem Molecular/métodos , Peptídeos/síntese química , Peptídeos/química , Survivina/química , Survivina/genéticaRESUMO
Survivin is a newly identified tumour-associated antigen and has been demonstrated to be an excellent target for immunotherapy in several cancers, but its role in hepatocellular carcinoma treatment is still unknown. In this study, survivin-derived peptide-specific cytotoxic T lymphocytes (CTLs) from peripheral blood mononuclear cells of healthy donors were induced by multiple stimulations with HLA-A2- restricted survivin peptide-pulsed T2 cells. The induced CTLs exhibited specific lysis of T2 cells pulsed with the peptide and HLA-A2+ hepatocellular carcinoma cells expressing survivin, while HLA-A2+ hepatocellular carcinoma cell lines that did not express survivin were not recognized by the CTLs. These results suggest that the survivin peptide epitope could be a potential target of specific immunotherapy for HLA-A2+ patients with hepatocellular carcinoma.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Antígeno HLA-A2/imunologia , Peptídeos/farmacologia , Survivina/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Células Cultivadas , ELISPOT , Epitopos/química , Epitopos/imunologia , Expressão Gênica , Antígeno HLA-A2/genética , Voluntários Saudáveis , Células Hep G2 , Humanos , Células K562 , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Ativação Linfocitária/efeitos dos fármacos , Peptídeos/química , Peptídeos/imunologia , Survivina/química , Survivina/genética , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Células Th2/citologia , Células Th2/imunologiaRESUMO
Survivin (IAP proteins) is considered as a significant target for anticancer drug research owing to its upregulation in tumor cells to mediate resistance to apoptotic stimulus. The current study aimed to investigate phytochemicals as inhibitors of survivin with caspases to reactivate the functioning of caspases through molecular docking. The compounds namely 2(R), 4(R)-dihydroxypyrrolidine, 4-hydroxy-2-(4-methoxyphenyl)-1,1-dioxo-3,4-dihydrothieno[3,2-e]thiazine-6-sulfonamide, 2,3-Diketo-L-gulonic acid, (3-hydroxy-2-octadeca-9,12-dienoyloxypropyl) octadecanoate, 2-[[4-[[4-[(4-formamido-1-methylimidazole-2-carbonyl)amino]-1-methylimidazole-2-carbonyl]amino]-1-methylimidazole-2-carbonyl]amino]ethyl-dimethylazanium, Picolinic acid and (2-Hydroxy-5-nitrophenyl) dihydrogen phosphate successfully bind inside the pocket of survivin. ADMETsar was used to evaluate the anticancer potential of selected compounds. These compounds can be proposed as effective inhibitors, disrupting the survivin-caspases interaction and reactivating the caspases function of apoptosis. The study might facilitate the development of cost-effective and natural drugs against cancer. However, further validation is essential for confirmation of its drug efficacy and bio-compatibility.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Compostos Fitoquímicos/farmacologia , Survivina/antagonistas & inibidores , Survivina/química , Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/toxicidade , Caspases/metabolismo , Simulação por Computador , Humanos , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Compostos Fitoquímicos/farmacocinética , Compostos Fitoquímicos/toxicidade , Conformação Proteica , Survivina/metabolismo , Distribuição TecidualRESUMO
Herein we report the first small molecule that disrupts the survivin-Smac interaction taking place in mitochondria. The inhibitor, PZ-6-QN, was identified by initially screening a phenothiazine library using a fluorescence anisotropy assay and then conducting a structure-activity relationship study. Mutagenesis and molecular docking studies suggest that PZ-6-QN binds to survivin similarly to the known Smac peptide, AVPI. The results of the effort also show that PZ-6-QN exhibits good anticancer activity against various cancer cells. Moreover, cell-based mechanistic studies provide evidence for the proposal that PZ-6-QN enters mitochondria to inhibit the survivin-Smac interaction and promotes release of Smac and cytochrome c from mitochondria into the cytosol, a process that induces apoptosis in cancer cells. Overall, the present study suggests that PZ-6-QN can serve as a novel chemical probe for study of processes associated with the mitochondrial survivin-Smac interaction and it will aid the discovery of novel anticancer agents.
Assuntos
Mitocôndrias/metabolismo , Oligopeptídeos/metabolismo , Fenotiazinas/química , Survivina/metabolismo , Apoptose/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Citocromos c/metabolismo , Humanos , Simulação de Acoplamento Molecular , Oligopeptídeos/química , Fenotiazinas/metabolismo , Fenotiazinas/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Relação Estrutura-Atividade , Survivina/químicaRESUMO
An ultimate goal of synthetic DNA motor studies is to mimic natural protein motors in biological systems. Here, we rationally designed a highly integrated and biostable DNA motor system with high potential for living body operation, through simple assembly of a Mn2+-dependent DNAzyme-powered DNA motor with a degradable MnO2 nanosheet. The motor system shows outstanding high integration and improved biostability. High integration confers the motor system with the ability to deliver all the core components to the target sites as a whole, thus, enabling precise control of the spatiotemporal distribution of these components and achieving high local concentrations. At the target sites, reduction of the MnO2 nanosheet by intracellular glutathione (GSH) not only releases the DNA motor, which can then be initiated by the intracellular target, but also produces Mn2+ in situ to power the autonomous and progressive operation of the DNA motor. Interestingly, the resultant consumption of GSH in turn protects the DNA motor from destruction by physiological GSH, thus, conferring our motor system with improved biostability, reduced false-positive outputs, and consequently, an increased potential to be applied in a living body. As a proof of concept, the highly integrated DNA motor system was demonstrated to work well for amplified imaging detection of survivin mRNA (mRNA), an important tumor biomarker, in both living cancer cells and living tumor-bearing mice. This work reveals concepts and strategies promoting synthetic DNA motor applications in biological systems.
Assuntos
DNA de Neoplasias/química , Animais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , DNA Catalítico/química , DNA Catalítico/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Glutationa/química , Células HeLa , Humanos , Compostos de Manganês/química , Compostos de Manganês/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanoestruturas/química , Neoplasias Experimentais/diagnóstico por imagem , Imagem Óptica , Óxidos/química , Óxidos/metabolismo , Tamanho da Partícula , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Propriedades de Superfície , Survivina/química , Survivina/genética , Survivina/metabolismoRESUMO
INTRODUCTION: The regulation of apoptosis via compounds originated from marine organisms signifies a new wave in the field of drug discovery. Marine organisms produce potent compounds as they hold the phenomenal diversity in chemical structures. The main focus of drug development is anticancer therapy. METHODS: Expertise on manifold activities of compounds helps in the discovery of their derivatives for preclinical and clinical experiment that promotes improved activity of compounds for cancer patients. RESULTS: These marine derived compounds stimulate apoptosis in cancer cells by targeting Bcl-2 and Survivin, highlighting the fact that instantaneous targeting of these proteins by novel derivatives results in efficacious and selective killing of cancer cells. CONCLUSION: Our study reports the identification of Aplysin and Haterumaimide J as Bcl-2 inhibitors and Cortistatin A as an inhibitor of survivin protein, from a sequential virtual screening approach.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Organismos Aquáticos/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Survivina/química , Survivina/farmacologia , Animais , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Survivina/síntese químicaRESUMO
With steroid as a carrier nucleus and introducing a pyridine heterocycle as a pharmacophore on the D ring, a series of steroidal pyridine derivatives were designed and studied for their antitumor activity by molecular docking software. The compounds were synthesized as small molecule inhibitors and studied as anticancer agents. The synthesis of the analogs was performed in a one-pot multi-component reaction and the corresponding compounds were screened in vitro for their antitumor activity. Four adherently growing cancer cell lines were used and arranged before dosing. Among all compounds screened for their antitumor activity, compounds 2f and 2p were found to be the most active. Here, the most obvious changes in the morphology of the treated cells could be observed.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Desenho de Fármacos , Simulação de Acoplamento Molecular , Piridinas/química , Esteroides/química , Esteroides/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Técnicas de Química Sintética , Humanos , Conformação Proteica , Esteroides/síntese química , Esteroides/metabolismo , Relação Estrutura-Atividade , Survivina/química , Survivina/metabolismoRESUMO
Survivin is a small protein that belongs to the inhibitor of apoptosis protein family. It is abundantly expressed in tumors compared with adult differentiated tissues, being associated with poor prognosis in many human neoplasms. This apoptotic inhibitor has a relevant role in both the promotion of cancer cell survival and in the inhibition of cell death. Consequently, aberrant survivin expression stimulates tumor progression and confers resistance to several therapeutic strategies in a variety of tumors. In fact, efficient survivin downregulation or inhibition results in spontaneous apoptosis or sensitization to chemotherapy and radiotherapy. Therefore, all these features make survivin an attractive therapeutic target to treat cancer. Currently, there are several survivin inhibitors under clinical evaluation, although more specific and efficient survivin inhibitors are being developed. Moreover, novel combination regimens targeting survivin together with other therapeutic approaches are currently being designed and assessed. In this review, recent progress in the therapeutic options targeting survivin for cancer treatment is analyzed. Direct survivin inhibitors and their current development status are explored. Besides, the major signaling pathways implicated in survivin regulation are described and different therapeutic approaches involving survivin indirect inhibition are evaluated. Finally, promising novel inhibitors under preclinical or clinical evaluation as well as challenges of developing survivin inhibitors as a new therapy for cancer treatment are discussed.
Assuntos
Terapia de Alvo Molecular , Neoplasias/terapia , Survivina/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacos , Survivina/química , Survivina/metabolismoRESUMO
INTRODUCTION: Survivin is a nodal protein involved in several cellular pathways. It is a member of the IAP family and an integral component of the chromosomal passenger complex, where it binds to borealin and INCENP through its dimerization interface. By targeting survivin with a small molecule at its dimerization interface, inhibition of the proliferation of cancer cells has been suggested. With Abbott 8, a small-molecule dimerization inhibitor has been recently reported. The structure-activity relationship of this series of inhibitors implied that the middle pyridin-2(1H)-one ring did not tolerate modifications of any kind. METHODS: Based on the synthetic strategy of Abbott 8 using multicomponent reactions, we synthesized a series of small molecules bearing a novel rigidized core scaffold. This rigidization strategy was accomplished by integrating the pyridin-2(1H)-one and its 6-phenyl substituent into a tricyclic structure, linking position 5 of pyridin-2(1H)-one to the phenyl substituent by rings of different sizes. The new scaffolds were designed based on in silico molecular dynamics of survivin. RESULTS: Binding of these rigidized scaffolds to the recombinant L54M mutant of survivin was evaluated, revealing affinities in the low micromolar range. CONCLUSION: This easily accessible, new class of survivin-dimerization modulators is an interesting starting point for further lead optimization.
Assuntos
Simulação de Dinâmica Molecular , Multimerização Proteica , Piridonas/química , Bibliotecas de Moléculas Pequenas/química , Survivina/química , Humanos , Estrutura Molecular , Mutação , Piridonas/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Bibliotecas de Moléculas Pequenas/síntese química , Survivina/genética , Survivina/isolamento & purificaçãoRESUMO
Accurate tumor margin demarcation in situ remains a paramount challenge. Herein, a NanoFlare (also known as spherical-nucleic-acid technology) based strategy is reported for in situ tumor margin delineation by transforming and amplifying the pathophysiological redox signals of tumor microenvironment. The NanoFlare designed (named AuNS-ASON) is based on gold nanostar (AuNS) coated with a dense shell of disulfide bridge-inserted and cyanine dyes-labeled antisense oligonucleotides (ASON) targeting survivin mRNA. The unique anisotropic ASON-spike nanostructure endows the AuNS-ASON with universal cellular internalization of tumor cells, while the disulfide bridge inserted confers response specificity toward redox activation. In vitro experiments demonstrate that the AuNS-ASON can discriminate tumor cells rapidly with activated fluorescence signals (>100-fold) in 2 h, and further achieve synergistic gene/photothermal tumor cells ablation upon near-infrared laser irradiation. Remarkably, in situ tumor margin delineation with high accuracy and outstanding spatial resolution (<100 µm) in mice bearing different tumors is obtained based on the AuNS-ASON, providing intraoperative guidance for tumor resection. Moreover, the AuNS-ASON can enable efficient neoadjuvant gene/photothermal therapy before surgery to reduce tumor extent and increase resectability. The concept of NanoFlare-based microenvironment signal transformation and amplification could be used as a general strategy to guide the design of activatable nanoprobes for cancer theranostics.
Assuntos
Ouro/química , Terapia Neoadjuvante/métodos , Oligonucleotídeos Antissenso/química , Fototerapia/métodos , Nanocompostos/química , Oxirredução , RNA Mensageiro/química , Survivina/química , Microambiente Tumoral/efeitos dos fármacosRESUMO
BACKGROUND: The success of gene therapy is mostly dependent on the development of gene carrier. Graphene oxide (GO) possesses excellent aqueous solubility and biocompatibility, which is important for its biochemical and medical applications. Our previous work proved that GO can deliver siRNA into cells efficiently and downregulate the expression of desired protein. METHODS: In this study, a novel delivery carrier, GO-R8/anti-HER2 (GRH), was developed by conjugating octaarginine (R8) and anti-HER2 antibody with GO as a tumor active-targeting vector for survivin-siRNA delivery. RESULTS: GRH/survivin-siRNA formed nanoglobes of 195±10 nm in diameter. Real-time polymerase chain reaction analysis revealed that survivin messenger RNA expression showed a 42.4%±2.69% knockdown. The expression of survivin protein was downregulated to 50.86%±2.94% in enzyme-linked immunosorbent assay. In MTT tests, GRH exhibited no testable cytotoxicity. In vivo, GRH/survivin-siRNA showed gene silencing and inhibition of tumor growth. CONCLUSION: The in vitro and in vivo results consistently demonstrated that GRH/survivin-siRNA has potential to be an efficient gene silencing carrier for siRNA delivery in cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Sistemas de Liberação de Medicamentos , RNA Interferente Pequeno/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Survivina/farmacologia , Antineoplásicos/química , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Feminino , Grafite/química , Grafite/farmacologia , Humanos , Células MCF-7 , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Óxidos/química , Óxidos/farmacologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Receptor ErbB-2/metabolismo , Relação Estrutura-Atividade , Survivina/química , Survivina/genética , Células Tumorais CultivadasRESUMO
Survivin is overexpressed in various types of human cancer, but rarely expressed in terminally differentiated adult tissues. Thus, survivin is a potential target antigen for a cancer vaccine. However, self-tumor-associated antigens are not highly immunogenic. Bacteria-derived lipoproteins can activate antigen-presenting cells through their toll-like receptors to enhance immune responses. In this context, lipidated survivin is an attractive candidate for cancer immunotherapy. In the present study, recombinant lipidated human survivin (LSur) was prepared from an Escherichia coli-based system. We investigated whether LSur is efficiently captured by antigen-presenting cells then facilitating effective induction of survivin cross-presentation and generation of immunity against cancer cells. Our results demonstrate that LSur, but not its non-lipidated counterpart, can activate mouse bone-marrow-derived-dendritic cells (BMDCs) to enhance cytokine (IL-6, TNF-α, and IL-12) secretion and costimulatory molecules (CD40, CD80, CD86, and MHC II) expression. However, the pathways involved in the capture of the recombinant lipidated antigen by antigen-presenting cells have not yet been elucidated. To this end, we employ various endocytosis inhibitors to study the effect on LSur internalization. We show that the internalization of LSur is suppressed by the inhibition of various routes of endocytosis. These results suggest that endocytosis of LSur by BMDCs can be mediated by multiple mechanisms. Furthermore, LSur is trafficked to the early endosome after internalization by BMDCs. These features of LSur are advantageous for cross-presentation and the induction of antitumor immunity. We demonstrate that immunization of C57BL/6 mice with LSur under treatment with exogenous adjuvant-free formulation induce survivin-specific CD8+ T-cell responses and suppress tumor growth. The antitumor responses are mediated by CD8+ cells. Our findings indicate that LSur is a potential candidate for stimulating protective antitumor immunity. This study suggests that lipidated tumor antigens may be a promising approach for raising a robust antitumor response in cancer immunotherapy.