RESUMO
In the field of drug discovery, understanding how small molecule drugs interact with cellular components is crucial. Our study introduces a novel methodology to uncover primary drug targets using Tandem Affinity Purification for identification of Drug-Binding Proteins (TAP-DBP). Central to our approach is the generation of a FLAG-hemagglutinin (HA)-tagged chimeric protein featuring the FKBP12(F36V) adaptor protein and the TurboID enzyme. Conjugation of drug molecules with the FKBP12(F36V) ligand allows for the coordinated recruitment of drug-binding partners effectively enabling in-cell TurboID-mediated biotinylation. By employing a tandem affinity purification protocol based on FLAG-immunoprecipitation and streptavidin pulldown, alongside mass spectrometry analysis, TAP-DBP allows for the precise identification of drug-primary binding partners. Overall, this study introduces a systematic, unbiased method for identification of drug-protein interactions, contributing a clear understanding of target engagement and drug selectivity to advance the mode of action of a drug in cells.
Assuntos
Proteínas de Transporte , Purificação por Afinidade em Tandem , Purificação por Afinidade em Tandem/métodos , Proteína 1A de Ligação a Tacrolimo/metabolismo , Proteínas/metabolismo , Cromatografia de Afinidade/métodosRESUMO
Two recent studies, by Lin et al. and Liu et al., unveiled the pivotal role of F-box and WD repeat domain containing 10 (FBXW10)-mediated ubiquitination and activation of oncogenic signaling as the primary driver behind the higher prevalence of hepatocellular carcinoma (HCC) in men. These discoveries shed light on underlying mechanisms of sex-biased cancer and provide a promising roadmap for both basic and clinical research.
Assuntos
Carcinoma Hepatocelular , Proteínas F-Box , Neoplasias Hepáticas , Humanos , Masculino , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , UbiquitinaçãoRESUMO
Oncogene Moesin plays critical role in initiation, progression, and metastasis of multiple cancers. It exerts oncogenic activity due to its high-level expression as well as posttranslational modification in cancer. However, factors responsible for its high-level expression remain elusive. In this study, we identified positive as well as negative regulators of Moesin. Our study reveals that Moesin is a cellular target of F-box protein FBXW2. We showed that FBXW2 suppresses breast cancer progression through directing proteasomal degradation of Moesin. In contrast, AKT kinase plays an important role in oncogenic function of Moesin by protecting it from FBXW2-mediated proteasomal degradation. Mechanistically, AKT phosphorylates Moesin at Thr-558 and thereby prevents its degradation by FBXW2 via weakening the association between FBXW2 and Moesin. Further, accumulated Moesin prevents FBXW2-mediated degradation of oncogene SKP2, showing that Moesin functions as an upstream regulator of oncogene SKP2. In turn, SKP2 stabilizes Moesin by directing its non-degradable form of polyubiquitination and therefore AKT-Moesin-SKP2 oncogenic axis plays crucial role in breast cancer progression. Collectively, our study reveals that FBXW2 functions as a tumor suppressor in breast cancer by restricting AKT-Moesin-SKP2 axis. Thus, AKT-Moesin-SKP2 axis may be explored for the development of therapeutics for cancer treatment.
Assuntos
Neoplasias da Mama , Proteínas F-Box , Proteínas Proto-Oncogênicas c-akt , Humanos , Transformação Celular Neoplásica , Proteínas F-Box/genética , Proteínas dos Microfilamentos , Oncogenes , Neoplasias da Mama/genética , Neoplasias da Mama/patologiaRESUMO
Loss of the tumor suppressive activity of the protein phosphatase 2A (PP2A) is associated with cancer, but the underlying molecular mechanisms are unclear. PP2A holoenzyme comprises a heterodimeric core, a scaffolding A subunit and a catalytic C subunit, and one of over 20 distinct substrate-directing regulatory B subunits. Methylation of the C subunit regulates PP2A heterotrimerization, affecting B subunit binding and substrate specificity. Here, we report that the leucine carboxy methyltransferase (LCMT1), which methylates the L309 residue of the C subunit, acts as a suppressor of androgen receptor (AR) addicted prostate cancer (PCa). Decreased methyl-PP2A-C levels in prostate tumors is associated with biochemical recurrence and metastasis. Silencing LCMT1 increases AR activity and promotes castration-resistant prostate cancer growth. LCMT1-dependent methyl-sensitive AB56αCme heterotrimers target AR and its critical coactivator MED1 for dephosphorylation, resulting in the eviction of the AR-MED1 complex from chromatin and loss of target gene expression. Mechanistically, LCMT1 is regulated by S6K1-mediated phosphorylation-induced degradation requiring the ß-TRCP, leading to acquired resistance to anti-androgens. Finally, feedforward stabilization of LCMT1 by small molecule activator of phosphatase (SMAP) results in attenuation of AR-signaling and tumor growth inhibition in anti-androgen refractory PCa. These findings highlight methyl-PP2A-C as a prognostic marker and that the loss of LCMT1 is a major determinant in AR-addicted PCa, suggesting therapeutic potential for AR degraders or PP2A modulators in prostate cancer treatment.
Assuntos
Neoplasias da Próstata , Proteína Fosfatase 2 , Humanos , Masculino , Antagonistas de Androgênios , Leucina , Metiltransferases , Próstata , Neoplasias da Próstata/genética , Proteína Fosfatase 2/genéticaRESUMO
F-box proteins form SCF (Cullin1, SKP1 and F-box-protein) ubiquitin ligase complexes to ubiquitinate cellular proteins. They play key role in several biological processes, including cell cycle progression, cellular signaling, stress response and cell death pathways. Therefore, deregulation of F-box proteins is closely associated with cancer progression. However, the role of most of the F-box proteins, including FBXO41, in cancer progression remains elusive. Here, we unravel the role of FBXO41 in cancer progression. We show that FBXO41 suppresses cancer cell proliferation and tumor growth by inducing autophagic cell death through an alternative pathway. Results revealed that FBXO41-mediated autophagic cell death induction is dependent on accumulation of cell cycle checkpoint protein p21. We found that FBXO41 increases the expression levels of p21 at the post-translational level by promoting the proteasomal degradation of SKP2, an oncogenic F-box protein. Mechanistically, FBXO41 along with p21 disrupts the inhibitory BCL2 (anti-apoptotic protein)-Beclin1 (autophagy initiating factor) complex of autophagy induction to release Beclin1, thereby inducing autophagy. Overall, the present study establishes a new FBXO41-SKP2-p21 axis for induction of autophagic cell death to prevent cancer growth, which could be explored to develop promising cancer therapeutics.
Assuntos
Morte Celular Autofágica , Fenômenos Biológicos , Neoplasias da Mama , Proteínas F-Box/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Proteína Beclina-1/metabolismo , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Proteínas Culina/genética , Proteínas F-Box/genética , Feminino , Humanos , Oncogenes , Proteínas Quinases Associadas a Fase S/genética , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismoRESUMO
FBXO31, a member of F-box protein family, has been shown to play an important role in preventing tumorigenesis by preserving genomic stability during cell proliferation as well as upon genotoxic stress. Inactivation of FBXO31 due to loss of heterozygosity is associated with various cancers, including ovarian cancer, one of the deadliest forms of gynecological cancers. However, the role and regulation of FBXO31 in ovarian cancer remained elusive. Here, using biochemical and molecular biology techniques, we show that c-Myc suppresses the mRNA levels of FBXO31 in ovarian cancer. Chromatin immunoprecipitation experiment showed that c-Myc is recruited to the promoter region of FBXO31 and prevents FBXO31 mRNA synthesis. In contrast, FBXO31 maintains the c-Myc expression at an optimum through proteasome pathway. FBXO31 interacts with and facilitates the polyubiquitination of c-Myc through the SCF complex and thereby inhibits ovarian cancer growth both in vitro and in vivo. Moreover, FBXO31-mediated proteasomal degradation of c-Myc is unique. Unlike other negative regulators, FBXO31 recognizes c-Myc in phosphorylation independent manner to direct its degradation. Further, expression levels analysis revealed that c-Myc and FBXO31 share a converse correlation of expression in ovarian cancer cell lines and patient samples. We observed an increase in the expression levels of c-Myc with a concomitant decrease in the levels of FBXO31 in higher grades of ovarian cancer patient samples. In conclusion, our study demonstrated that oncogene c-Myc impairs the tumor-suppressive functions of FBXO31 to promote ovarian cancer progression, and therefore c-Myc-FBXO31 axis can be explored to develop better cancer therapy.
Assuntos
Proteínas F-Box , Neoplasias Ovarianas , Proteínas Supressoras de Tumor , Carcinogênese/genética , Linhagem Celular Tumoral , Proteínas F-Box/genética , Retroalimentação , Feminino , Humanos , Neoplasias Ovarianas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro , Proteínas Supressoras de Tumor/genéticaRESUMO
F-box protein FBXW8 is known to interact with scaffolding protein Cullin1 and Cullin7 to form SCF (SKP1, Cullin and F-box protein) complex. However, detail understanding about the importance of both Cullins for SCF-FBXW8 complex formation as well as its ubiquitin ligase activity remains elusive. Here, we show that, through in vitro and in vivo studies, Cullin1 and Cullin7 increase each other's binding to FBXW8 synergistically. Interestingly, absence of either Cullin results in abrogation of binding of other Cullin to FBXW8. Binding of SKP1 to FBXW8 also increases in the presence of both the Cullins. Thus, SKP1, Cullin1 and Cullin7 are essential to form Cullin1-SKP1-FBXW8-Cullin7 functional ubiquitin ligase complex. Further, using computational, mutational and biochemical analysis, we found that Cullin1 binds to N-terminus of FBXW8 through SKP1 while Cullin7 associates with C-terminus of FBXW8 to form Cullin1-SKP1-FBXW8-Cullin7 functional complex in a cooperative manner. Results showed that Cullin1-SKP1-FBXW8-Cullin7 complex plays a key role in maintaining the basal level expression of ß-TrCP1. Moreover, Cullin1-SKP1-FBXW8-Cullin7 complex promotes cell migration by activating ß-catenin via directing proteasomal degradation of ß-TrCP1. Overall, our study reveals the intriguing molecular mechanism of assembly of SKP1, Cullin1, Cullin7 and FBXW8 to form Cullin1-SKP1-FBXW8-Cullin7 functional complex that control the function of ß-TrCP1.
Assuntos
Proteínas Culina/metabolismo , Proteínas F-Box/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Movimento Celular , Proteínas Culina/química , Proteínas F-Box/química , Humanos , Células MCF-7 , Ligação Proteica , Estabilidade Proteica , Proteólise , Proteínas Quinases Associadas a Fase S/química , Especificidade por Substrato , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/químicaRESUMO
Sequential alteration in the expression levels of cell cycle regulatory proteins is crucial for faithful cell cycle progression to maintain the cellular homeostasis. F-box protein ß-TrCP1 is known to control the expression levels of several important cell cycle regulatory proteins. However, how the function of ß-TrCP1 is regulated in spatiotemporal manner during cell cycle progression remains elusive. Here, we show that expression levels of ß-TrCP1 oscillate during cell cycle progression with a minimum level at the G1 and S phases of cell cycle. Using biochemical, flow cytometry, and immunofluorescence techniques, we found that oscillation of ß-TrCP1 expression is controlled by another F-box protein FBXW8. FBXW8 directs the proteasomal degradation of ß-TrCP1 in MAPK pathway-dependent manner. Interestingly, we found that the attenuation of ß-TrCP1 by FBXW8 is important for Cdc25A-mediated cell cycle transition from G1 phase to S phase as well as DNA damage-free progression of S phase. Overall, our study reveals the intriguing molecular mechanism and significance of maintenance of ß-TrCP1 levels during cell cycle progression by FBXW8-mediated proteasomal degradation.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , Proteínas Contendo Repetições de beta-Transducina/genética , Fosfatases cdc25/genética , Divisão Celular/genética , Dano ao DNA/genética , Citometria de Fluxo , Fase G1/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células HEK293 , Humanos , Células MCF-7 , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , Fase S/genética , Ubiquitina/genéticaRESUMO
F-box proteins ß-TrCP1 and ß-TrCP2 are paralogs present in the human genome. They control several cellular processes including cell cycle and DNA damage signaling. Moreover, it is reported that they facilitate DNA damage-induced accumulation of p53 by directing proteasomal degradation of MDM2, a protein that promotes p53 degradation. However, the individual roles of ß-TrCP1 and ß-TrCP2 in the genotoxic stress-induced activation of cell cycle checkpoints and DNA damage repair remain largely unknown. Here, using biochemical, molecular biology, flow cytometric, and immunofluorescence techniques, we show that ß-TrCP1 and ß-TrCP2 communicate during genotoxic stress. We found that expression levels of ß-TrCP1 are significantly increased while levels of ß-TrCP2 are markedly decreased upon induction of genotoxic stress. Further, our results revealed that DNA damage-induced activation of ATM kinase plays an important role in maintaining the reciprocal expression levels of ß-TrCP1 and ß-TrCP2 via the phosphorylation of ß-TrCP1 at Ser158. Phosphorylated ß-TrCP1 potently promotes the proteasomal degradation of ß-TrCP2 and MDM2, resulting in the activation of p53. Additionally, ß-TrCP1 impedes MDM2 accumulation via abrogation of its lysine 63-linked polyubiquitination by ß-TrCP2. Thus, ß-TrCP1 helps to arrest cells at the G2/M phase of the cell cycle and promotes DNA repair upon DNA damage through attenuation of ß-TrCP2. Collectively, our findings elucidate an intriguing posttranslational regulatory mechanism of these two paralogs under genotoxic stress and revealed ß-TrCP1 as a key player in maintaining the genome integrity through the attenuation of ß-TrCP2 levels in response to genotoxic stress.
Assuntos
Pontos de Checagem do Ciclo Celular , Dano ao DNA , Reparo do DNA , Proteólise , Ubiquitinação , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Sobrevivência Celular , Humanos , Fosforilação , Transdução de Sinais , Proteínas Contendo Repetições de beta-Transducina/genéticaRESUMO
Multiple myeloma (MM) is a plasma cellassociated cancer and accounts for 13% of all hematological malignancies, worldwide. MM still remains an incurable plasma cell malignancy with a poor prognosis due to a lack of suitable markers. Therefore, discovering novel markers and targets for diagnosis and therapeutics of MM is essential. The present study aims to identify markers associated with MM malignancy using patientderived MM mononuclear cells (MNCs). Labelfree quantitative proteomics analysis revealed a total of 192 differentially regulated proteins, in which 79 proteins were upregulated and 113 proteins were found to be downregulated in MM MNCs as compared to nonhematological malignant samples. The identified differentially expressed candidate proteins were analyzed using various bioinformatics tools, including Ingenuity Pathway Analysis (IPA), Protein Analysis THrough Evolutionary Relationships (PANTHER), Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Database for Annotation, Visualization and Integrated Discovery (DAVID) to determine their biological context. Among the 192 candidate proteins, marginal zone B and B1 cell specific protein (MZB1) was investigated in detail using the RPMI-8226 cell line model of MM. The functional studies revealed that higher expression of MZB1 is associated with promoting the progression of MM pathogenesis and could be established as a potential target for MM in the future.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Mieloma Múltiplo/patologia , Proteínas Adaptadoras de Transdução de Sinal/análise , Idoso , Biomarcadores Tumorais/análise , Biópsia , Medula Óssea/patologia , Linhagem Celular Tumoral , Biologia Computacional , Progressão da Doença , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Proteômica , Regulação para CimaRESUMO
In view of the potent anticancer activity of the d-arabino-configured cytosine nucleoside (ara-C), apioarabinofuranosyl pyrimidine nucleosides were designed and synthesized from d-ribose as starting material. The synthetic strategy signifies that tosylation followed by in situ cyclization, one-pot controlled oxidative cleavage and acetylation by Pb(OAc)4 , stereoselective nucleobase condensation, inversion of hydroxyl group and uracil group converted to cytosine as the key steps. Synthesized apioarabinofuranosyl pyrimidine nucleosides were tested using breast, colon, and ovarian cancer cell lines. However, only compound 19a, 19b, and 22b have a moderate growth-suppressive effect against the luminal A breast cancer cell line MCF7.
Assuntos
Antineoplásicos/farmacologia , Citarabina/farmacologia , Pirimidinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Citarabina/análogos & derivados , Citarabina/química , Feminino , Humanos , Células MCF-7 , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
In this study, one step hydrothermal synthetic strategy was adopted for preparing carbon dots (C. dots) from jeera (Cumin: Cuminum cyminum), a naturally abundant and cost effective carbon source. The physical, optical and surface functional properties of C. dots were extensively studied by different techniques such as Transmission electron microscopy (TEM), Scanning electron microscopy (SEM), spectrophotometry, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The obtained C. dots were highly water dispersible and photostable with a quantum yield of 5.33%. The antioxidant property of C. dots was investigated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. The C. dots were then capped with cystamine using 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) coupling chemistry to design a selective sensing system for chromium (VI) (Cr (VI)). The minimum detection limit of Cr (VI) was found to be 1.57 µM. Biocompatibility and low toxicity of C. dots obtained from jeera made it a potential tool for bioimaging application. The internalisation of C. dots by MCF-7 breast cancer cells and Multi Drug Resistant (MDR) pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa were proved by the bioimaging of respective cells.
Assuntos
Antibacterianos/química , Materiais Biocompatíveis/química , Cromo/análise , Poluentes Químicos da Água/análise , Antibacterianos/síntese química , Antibacterianos/farmacologia , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Carbono/química , Carbono/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cuminum/química , Cistamina/química , Cistamina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Humanos , Células MCF-7 , Testes de Sensibilidade Microbiana , Imagem Óptica , Tamanho da Partícula , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pontos Quânticos/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Propriedades de SuperfícieRESUMO
F-box protein 31 (FBXO31) is a reported putative tumor suppressor, and its inactivation due to loss of heterozygosity is associated with cancers of different origins. An emerging body of literature has documented FBXO31's role in preserving genome integrity following DNA damage and in the cell cycle. However, knowledge regarding the role of FBXO31 during normal cell-cycle progression is restricted to its functions during the G2/M phase. Interestingly, FBXO31 levels remain high even during the early G1 phase, a crucial stage for preparing the cells for DNA replication. Therefore, we sought to investigate the functions of FBXO31 during the G1 phase of the cell cycle. Here, using flow cytometric, biochemical, and immunofluorescence techniques, we show that FBXO31 is essential for maintaining optimum expression of the cell-cycle protein cyclin A for efficient cell-cycle progression. Stable FBXO31 knockdown led to atypical accumulation of cyclin A during the G1 phase, driving premature DNA replication and compromised loading of the minichromosome maintenance complex, resulting in replication from fewer origins and DNA double-strand breaks. Because of these inherent defects in replication, FBXO31-knockdown cells were hypersensitive to replication stress-inducing agents and displayed pronounced genomic instability. Upon entering mitosis, the cells defective in DNA replication exhibited a delay in the prometaphase-to-metaphase transition and anaphase defects such as lagging and bridging chromosomes. In conclusion, our findings establish that FBXO31 plays a pivotal role in preserving genomic integrity by maintaining low cyclin A levels during the G1 phase for faithful genome duplication and segregation.
Assuntos
Ciclina A/metabolismo , Replicação do DNA/genética , Proteínas F-Box/metabolismo , Genoma Humano/genética , Proteínas Supressoras de Tumor/metabolismo , Ciclo Celular/genética , Cromatina/genética , Proteínas F-Box/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Cinética , Células MCF-7 , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Ubiquitinação/genéticaRESUMO
Aberrant activation of ß-catenin has been implicated in a variety of human diseases, including cancer. In spite of significant progress, the regulation of active Wnt/ß-catenin-signaling pathways is still poorly understood. In this study, we show that F-box protein 16 (FBXO16) is a putative tumor suppressor. It is a component of the SCF (SKP1-Cullin1-F-box protein) complex, which targets the nuclear ß-catenin protein to facilitate proteasomal degradation through the 26S proteasome. FBXO16 interacts physically with the C-terminal domain of ß-catenin and promotes its lysine 48-linked polyubiquitination. In addition, it inhibits epithelial-to-mesenchymal transition (EMT) by attenuating the level of ß-catenin. Therefore, depletion of FBXO16 leads to increased levels of ß-catenin, which then promotes cell invasion, tumor growth, and EMT of cancer cells. Furthermore, FBXO16 and ß-catenin share an inverse correlation of cellular expression in clinical breast cancer patient samples. In summary, we propose that FBXO16 functions as a putative tumor suppressor by forming an SCFFBXO16 complex that targets nuclear ß-catenin in a unique manner for ubiquitination and subsequent proteasomal degradation to prevent malignancy. This work suggests a novel therapeutic strategy against human cancers related to aberrant ß-catenin activation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Proteínas F-Box/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Transição Epitelial-Mesenquimal/genética , Genes Supressores de Tumor/fisiologia , Humanos , Proteínas Nucleares/metabolismo , Via de Sinalização Wnt/fisiologiaRESUMO
BACKGROUND: Modified nucleosides established a prime role as therapeutic drugs. OBJECTIVE: Design and synthesis of novel truncated carbocyclic nucleoside with modified nucleobases and evaluation of their anticancer properties. METHODS: Novel truncated carbocyclic nucleoside analogues were synthesized from a versatile starting material D-ribose. The synthetic route includes stereoselective Grignard reaction, Wittig olefination, ring closing metathesis, double bond hydrogenation and Mitsunobu nucleobase condensation as the key steps. Cytotoxicity was measured using MTT assay in breast cancer cell lines (MCF7 and MDA-MB-231), ovarian cancer cell lines (IGROV1 and OVCAR8). RESULT & CONCLUSION: The synthesized compounds were characterized using spectroscopy techniques. The synthesized compounds induced cytotoxicity in breast cancer cell lines (MCF7 and MDA-MB-231), ovarian cancer cell lines (IGROV1 and OVCAR8) while minimal effect on primary cell line. Among the eight analogues, 1b and 1d showed the highest cytotoxicity effect and induced autophagy mode of cell death. These compounds induced autophagy by inactivating AKT and mTOR pathway. In addition, PARP1 was found to be stabilized upon treatment with compound 1b and 1d and is one of the known markers associated with induction of autophagy through the AMPK/mTOR pathway after DNA damage. Taken together, these results suggest that compounds 1b and 1d inhibit cancer cell proliferation through mTOR inactivation-mediated induction of autophagy.
Assuntos
Antineoplásicos/farmacologia , Nucleosídeos/farmacologia , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células MCF-7 , Conformação Molecular , Nucleosídeos/análogos & derivados , Nucleosídeos/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Cyclin F protein, also known as FBXO1, is the largest among all cyclins and oscillates in the cell cycle like other cyclins. Apart from being a G2/M cyclin, cyclin F functions as the substrate-binding subunit of SCFcyclin F E3 ubiquitin ligase. In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4+ T cells, we observed down-regulation of the cyclin F gene (CCNF). Later, using gene overexpression and knockdown studies, we identified cyclin F as negatively influencing HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that cyclin F negatively regulates the expression of viral protein Vif (viral infectivity factor) at the protein level. We also identified a novel host-pathogen interaction between cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a cyclin F-specific amino acid motif in the C-terminal region of Vif indicated rescue of the protein from cyclin F-mediated down-regulation. Subsequently, we showed that Vif is a novel substrate of the SCFcyclin F E3 ligase, where cyclin F mediates the ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we showed that cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate that cyclin F is a novel F-box protein that functions as an intrinsic cellular regulator of HIV-1 Vif and has a negative regulatory effect on the maintenance of viral infectivity by restoring APOBEC3G expression.
Assuntos
Ciclinas/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Vírion/patogenicidade , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Desaminase APOBEC-3G/metabolismo , Linfócitos T CD4-Positivos , Células Cultivadas , Ciclinas/genética , Ciclinas/metabolismo , Proteínas F-Box , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Although the cure of amyloid related neurodegenerative diseases, non-neuropathic amyloidogenic diseases and non-neuropathic systemic amyloidosis are appealing energetic research attempts, beneficial medication is still to be discovered. There is a need to explore intensely stable therapeutic compounds, potent enough to restrict, disrupt or wipe out such toxic aggregates. We had performed a comprehensive biophysical, computational and cell based assay, that shows Nordihydroguaiaretic acid (NA) not only significantly inhibits heat induced hen egg white lysozyme (HEWL) fibrillation but also disaggregates preformed HEWL fibrils and reduces the cytoxicity of amyloid fibrils as well as disaggregated fibrillar species. The inhibitory potency of NA was determined by an IC50 of 26.3 µM. NA was also found to effectively inhibit human lysozyme (HL) fibrillation. NA interferes in the amyloid fibrillogenesis process by interacting hydrophobically with the amino acid residues found in highly prone amyloid fibril forming region of HEWL as explicated by molecular docking results. The results recommend NA as a probable neuroprotective and promising inhibitor for the therapeutic advancement prospective against amyloid related diseases.
Assuntos
Amiloidose/metabolismo , Masoprocol/química , Amiloide/química , Amiloidose/tratamento farmacológico , Benzotiazóis , Linhagem Celular Tumoral , Temperatura Alta , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Amiloidose de Cadeia Leve de Imunoglobulina , Concentração Inibidora 50 , Cinética , Luz , Microscopia de Fluorescência , Simulação de Acoplamento Molecular , Muramidase/química , Nefelometria e Turbidimetria , Agregados Proteicos , Ligação Proteica , Conformação Proteica , Espalhamento de Radiação , Espectrometria de Fluorescência , Tiazóis/químicaRESUMO
Glycation plays an important role in various oxidative stress related diseases. Superoxide dismutase (SOD) constitutes an essential defense against oxidative stress. The damage caused by oxidative stress is exacerbated if the antioxidant enzymes themselves are inactivated by glycation. Thymoquinone (TQ) has been reported to have various pharmacological activities. Therefore, the glycation of SOD by glucose or methylglyoxal (MG) and its protection by TQ has been investigated. Incubation of SOD with glucose, MG or both at 37 °C resulted in a progressive decrease in the activity of the enzyme, and a parallel decrease in the amount of protein on SDS-PAGE gels for glucose incubated SOD and formation of high molecular weight aggregates for MG or both glucose and MG incubated enzyme. TQ offered protection against glucose or MG induced loss in SOD activity and fragmentation/cross-linking. The antiglycating activity of TQ appears to be better for mild glycating agents. It is also effective in protecting against strong glycating agents, more when the exposure time to the glycating agent is short. TQ has also earlier been reported to have anti-diabetic effects, and this along with the observed antiglycating effect makes it an effective compound against diabetes and its complications.