RESUMO
Though Isoimperatorin from Angelicae dahuricae is known to have antiviral, antidiabetic, anti-inflammatory and antitumor effects, its underlying antitumor mechanism remains elusive so far. Hence, the apoptotic mechanism of Isoimperatorin was explored in hepatocellular carcinomas (HCCs). In this study, Isoimperatorin inhibited the viability of Huh7 and Hep3B HCCs and increased the subG1 apoptotic portion and also abrogated the expression of pro-poly-ADP ribose polymerase (pro-PARP) and pro-caspase 3 in Huh7 and Hep3B cells. Also, Isoimperatorin abrogated the expression of cyclin D1, cyclin E1, CDK2, CDK4, CDK6 and increased p21 as G1 phase arrest-related proteins in Huh7 and Hep3B cells. Interestingly, Isoimperatorin reduced the expression and binding of c-Myc and Sirtuin 1 (SIRT1) by Immunoprecipitation (IP), with a binding score of 0.884 in Huh7 cells. Furthermore, Isoimperatorin suppressed the overexpression of c-Myc by the proteasome inhibitor MG132 and also disturbed cycloheximide-treated c-Myc stability in Huh7 cells. Overall, these findings support the novel evidence that the pivotal role of c-Myc and SIRT1 is critically involved in Isoimperatorin-induced apoptosis in HCCs as potent molecular targets in liver cancer therapy.
Assuntos
Apoptose , Carcinoma Hepatocelular , Furocumarinas , Neoplasias Hepáticas , Proteínas Proto-Oncogênicas c-myc , Transdução de Sinais , Sirtuína 1 , Humanos , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/efeitos dos fármacos , Sirtuína 1/metabolismo , Furocumarinas/farmacologiaRESUMO
BACKGROUND: Extensive local invasion of glioblastoma (GBM) cells within the central nervous system (CNS) is one factor that severely limits current treatments. The aim of this study was to uncover genes involved in the invasion process, which could also serve as therapeutic targets. For the isolation of invasive GBM cells from non-invasive cells, we used a three-dimensional organotypic co-culture system where glioma stem cell (GSC) spheres were confronted with brain organoids (BOs). Using ultra-low input RNA sequencing (ui-RNA Seq), an invasive gene signature was obtained that was exploited in a therapeutic context. METHODS: GFP-labeled tumor cells were sorted from invasive and non-invasive regions within co-cultures. Ui-RNA sequencing analysis was performed to find a gene cluster up-regulated in the invasive compartment. This gene cluster was further analyzed using the Connectivity MAP (CMap) database. This led to the identification of SKF83566, an antagonist of the D1 dopamine receptor (DRD1), as a candidate therapeutic molecule. Knockdown and overexpression experiments were performed to find molecular pathways responsible for the therapeutic effects of SKF83566. Finally, the effects of SKF83566 were validated in orthotopic xenograft models in vivo. RESULTS: Ui-RNA seq analysis of three GSC cell models (P3, BG5 and BG7) yielded a set of 27 differentially expressed genes between invasive and non-invasive cells. Using CMap analysis, SKF83566 was identified as a selective inhibitor targeting both DRD1 and DRD5. In vitro studies demonstrated that SKF83566 inhibited tumor cell proliferation, GSC sphere formation, and invasion. RNA sequencing analysis of SKF83566-treated P3, BG5, BG7, and control cell populations yielded a total of 32 differentially expressed genes, that were predicted to be regulated by c-Myc. Of these, the UHRF1 gene emerged as the most downregulated gene following treatment, and ChIP experiments revealed that c-Myc binds to its promoter region. Finally, SKF83566, or stable DRD1 knockdown, inhibited the growth of orthotopic GSC (BG5) derived xenografts in nude mice. CONCLUSIONS: DRD1 contributes to GBM invasion and progression by regulating c-Myc entry into the nucleus that affects the transcription of the UHRF1 gene. SKF83566 inhibits the transmembrane protein DRD1, and as such represents a candidate small therapeutic molecule for GBMs.
Assuntos
Antagonistas de Dopamina , Glioblastoma , Glioma , Proteínas Proto-Oncogênicas c-myc , Animais , Humanos , Camundongos , Encéfalo , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Dopamina , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Camundongos Nus , Família Multigênica , Receptores de Dopamina D1/antagonistas & inibidores , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Cisplatin (CPT) is one of the standard treatments for hepatocellular carcinoma (HCC). However, its use is limits as a monotherapy due to drug resistance, and the underlying mechanism remains unclear. To solve this problem, we tried using canagliflozin (CANA), a clinical drug for diabetes, to reduce chemoresistance to CPT, and the result showed that CANA could vigorously inhibit cell proliferation and migration independent of the original target SGLT2. Mechanistically, CANA reduced aerobic glycolysis in HCC by targeting PKM2. The downregulated PKM2 directly bound to the transcription factor c-Myc in the cytoplasm to form a complex, which upregulated the level of phosphorylated c-Myc Thr58 and promoted the ubiquitination and degradation of c-Myc. Decreased c-Myc reduced the expression of GLS1, a key enzyme in glutamine metabolism, leading to impaired glutamine utilization. Finally, intracellular glutamine starvation induced ferroptosis and sensitized HCC to CPT. In conclusion, our study showed that CANA re-sensitized HCC to CPT by inducing ferroptosis through dual effects on glycolysis and glutamine metabolism. This is a novel mechanism to increase chemosensitivity, which may provide compatible chemotherapy drugs for HCC.
Assuntos
Carcinoma Hepatocelular , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas , Humanos , Canagliflozina/farmacologia , Canagliflozina/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Glutamina/metabolismo , Glicólise , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas de Ligação a Hormônio da TireoideRESUMO
As a master transcription factor, c-Myc plays an important role in promoting tumor immune escape. In addition, PPARγ (peroxisome proliferator-activated receptor γ) regulates cell metabolism, inflammation, and tumor progression, while the effect of PPARγ on c-Myc-mediated tumor immune escape is still unclear. Here we found that cells treated with PPARγ agonist pioglitazone (PIOG) reduced c-Myc protein expression in a PPARγ-dependent manner. qPCR analysis showed that PIOG had no significant effect on c-Myc gene levels. Further analysis showed that PIOG decreased c-Myc protein half-life. Moreover, PIOG increased the binding of c-Myc to PPARγ, and induced c-Myc ubiquitination and degradation. Importantly, c-Myc increased PD-L1 and CD47 immune checkpoint protein expression and promoted tumor immune escape, while PIOG inhibited this event. These findings suggest that PPARγ agonist inhibited c-Myc-mediated tumor immune escape by inducing its ubiquitination and degradation.
Assuntos
Neoplasias Colorretais , Pioglitazona , Tiazolidinedionas , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Regulação da Expressão Gênica , Pioglitazona/farmacologia , PPAR gama/agonistas , PPAR gama/metabolismo , Tiazolidinedionas/farmacologia , Evasão Tumoral , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
The use of probiotic and synbiotic is a promising strategy to modulate the intestinal microbiota, and thereby modify the risk of diseases. In this study, the effect of probiotic VSL#3, isolated or associated with a yacon-based product (PBY), on the functional metabolic pathways of the microbiota, in a colorectal carcinogenesis model, was evaluated. For this, mice induced to carcinogenesis were fed with standard diet AIN-93 M (CON), diet AIN-93 M and VSL#3 (PRO) or diet AIN-93 M with yacon and VSL#3 (SYN). The SYN group showed a highly differentiated intestinal community based on the MetaCyc pathways. Of the 351 predicted functional pathways, 222 differed between groups. Most of them were enriched in the SYN group, namely: amino acid biosynthesis pathways, small molecule biosynthesis pathways (cofactors, prosthetic groups, electron carriers and vitamins) carbohydrate degradation pathways and fermentation pathways. In addition, the synbiotic was able to stimulate the anti-inflammatory immune response and reduce the gene expression of PCNA and c-myc. Thus, we conclude that the synbiotic impacted more significantly the metabolic functions of the microbiota compared to the isolated use of probiotic. We believe that the enrichment of these pathways can exert antiproliferative action, reducing colorectal carcinogenesis. The prediction of the functional activity of the microbiota is a promising tool for understanding the influence of the microbiome on tumor development.
Assuntos
Neoplasias Colorretais , Microbioma Gastrointestinal , Antígeno Nuclear de Célula em Proliferação , Simbióticos , 1,2-Dimetilidrazina/farmacologia , Animais , Carcinogênese , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/prevenção & controle , Redes e Vias Metabólicas , Camundongos , Antígeno Nuclear de Célula em Proliferação/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
In recent years, multifunctional platinum nanoclusters (Pt-NCs) as new Pt-based anti-cancer drugs exhibit a promising therapeutic efficiency for several cancer diseases, especially for human pulmonary carcinoma. However, the endocytosis behaviors (like uptake pathway, etc.) and induced apoptosis mechanism of Pt-NCs for drug-resistant non-small cell lung cancer (NSCLC), are still inconclusive. In this research, we explored the endocytic pathway of Pt-NCs in both typical NSCLC A549 cells and cisplatin-resistant A549/Cis cells through qualitative confocal laser scanning microscope (CLSM) measurement and quantitative flow cytometry (FCM) and inductive coupled plasma-optical emission spectroscopy (ICP-OES) analysis, by the means of introducing the specific inhibitors which impede the classical ways of endocytosis. It was found that Pt-NCs dominatingly entered A549 cells via caveolin-mediated endocytosis as well as A549/Cis cells through micropinocytosis approach. Pt-NCs possessed an excellent inhibitory effect on the cell proliferation, migration and invasion, which the cell activity of A549 cells reduced to 14% and that of A549/Cis cells went down about four fifths. Moreover, Pt-NCs treatment increased caspase-3 protein levels and downregulated the expression of c-Myc and Bcl-2, proving the Pt-NCs-induced apoptosis of NSCLC cells was related to c-Myc/p53 and Bcl-2/caspase-3 signal pathways. These results demonstrate the explicit uptake pathway and apoptotic signaling pathway of Pt-NCs for NSCLC, which provides an in-depth and reasonable theoretical basis for the development of new Pt-NCs-based chemotherapeutics in future clinical practice.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Compostos de Platina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Células A549 , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Nanoestruturas , Compostos de Platina/administração & dosagem , Ensaio Tumoral de Célula-TroncoRESUMO
Cerebral ischemia-reperfusion injury induces a secondary immune inflammatory reaction that exacerbates brain injury and clinical prognosis. Dendritic cells (DCs) and microglia are both important regulators of neuroinflammation. Studies have confirmed that a large number of cells express the DC surface marker CD11c in the ischemic area, and some of these cells also express microglial markers. However, the specific mechanism of transformation between microglia and DCs and their roles in the process of cerebral ischemia-reperfusion injury are still not clear. In this study, we established a mouse model and flow cytometry was used to detect the expression of mature DC surface molecules in activated microglia. IFN-γ knockout mice were used to determine the regulatory effect of IFN-γ on microglial transformation. We found that CD11c+ cells were derived from microglia after ischemia-reperfusion injury, and this group of cells highly expressed MHC-II molecules and other costimulatory molecules, such as CD80 and CD86, which were regulated by IFN-γ and its downstream signaling molecules ERK/c-myc. In summary, our results showed in cerebral ischemia-reperfusion injury, IFN-γ regulates the transformation of microglia to DC-like cells. Microglial-derived DC-like cells possess the ability to present antigens and activate naïve T cells which is regulated by the ERK/c-myc signaling pathway.
Assuntos
Dendritos/efeitos dos fármacos , Interferon gama/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Microglia/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Traumatismo por Reperfusão/patologia , Animais , Antígenos CD11/metabolismo , Dendritos/patologia , Genes MHC da Classe II , Interferon gama/fisiologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , Cultura Primária de Células , Receptores de Interferon/biossíntese , Linfócitos TRESUMO
Aberrant cellular Myc (c-Myc) is a common feature in the majority of human cancers and has been linked to oncogenic malignancies. Here, we developed a novel c-Myc-targeting compound, N, N-bis (5-ethyl-2-hydroxybenzyl) methylamine (EMD), and present evidence demonstrating its effectiveness in targeting c-Myc for degradation in human lung carcinoma. EMD exhibited strong cytotoxicity toward various human lung cancer cell lines, as well as chemotherapeutic-resistant patient-derived lung cancer cells, through apoptosis induction in comparison with chemotherapeutic drugs. The IC50 of EMD against lung cancer cells was approximately 60 µM. Mechanistically, EMD eliminated c-Myc in the cells and initiated caspase-dependent apoptosis cascade. Cycloheximide chase assay revealed that EMD tended to shorten the half-life of c-Myc by approximately half. The cotreatment of EMD with the proteasome inhibitor MG132 reversed its c-Myc-targeting effect, suggesting the involvement of ubiquitin-mediated proteasomal degradation in the process. We further verified that EMD strongly induced the ubiquitination of c-Myc and promoted protein degradation. c-Myc inhibition and apoptosis induction were additionally shown in hematologic malignant K562 cells, indicating the generality of the observed EMD effects. Altogether, we identified EMD as a novel potent compound targeting oncogenic c-Myc that may offer new opportunities for lung cancer treatment. SIGNIFICANCE STATEMENT: The deregulation of c-Myc is frequently associated with cancer progression. This study examined the effect of a new compound, N, N-bis (5-ethyl-2-hydroxybenzyl) methylamine (EMD), in targeting c-Myc in several lung cancer cell lines and drug-resistant primary lung cancer cells. EMD induced dramatic c-Myc degradation through a ubiquitin-proteasomal mechanism. The promising anticancer and c-Myc-targeted activities of EMD support its use in potential new approaches to treat c-Myc-driven cancer.
Assuntos
Antineoplásicos/síntese química , Neoplasias Pulmonares/metabolismo , Metilaminas/síntese química , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Células K562 , Neoplasias Pulmonares/tratamento farmacológico , Metilaminas/química , Metilaminas/farmacologia , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Ubiquitina/metabolismoRESUMO
6-Bromoindirubin-3'-oxime (6BIO) is a novel small molecule that exerts positive effects on several age-related alterations. However, the anti-aging effects of 6BIO on the aging heart remain unknown. Herein, we aim to investigate the effects of 6BIO on the myocardium and its underlying mechanism in vivo and vitro. Following 6BIO treatment, an increased p53 contents, a reduced p16 and ß-gal levels, and attenuation of cardiac fibrosis were observed, suggesting 6BIO retarded aging of cardiomyocytes. As observed, 6BIO reduced p62 contents, elevated the levels of Beclin-1 and the ratio of LC3II/I, indicating the induction of autophagy, while the reduction of the accumulation of ROS indicated 6BIO alleviated oxidative stress. In addition, 6BIO treatment inhibited both GSK3ß signaling and mTOR signaling. 6BIO might be a promising agent for preventing myocardium from aging.
Assuntos
Envelhecimento/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Coração/efeitos dos fármacos , Indóis/farmacologia , Mioblastos Cardíacos/efeitos dos fármacos , Miocárdio/metabolismo , Oximas/farmacologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Antioxidantes/metabolismo , Proteína Beclina-1/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Linhagem Celular , Inibidor p16 de Quinase Dependente de Ciclina/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Fibrose , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Mioblastos Cardíacos/metabolismo , Miocárdio/patologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Sirolimo/farmacologia , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/efeitos dos fármacos , beta-Galactosidase/metabolismoRESUMO
MYC-driven lymphomas, especially those with concurrent MYC and BCL2 dysregulation, are currently a challenge in clinical practice due to rapid disease progression, resistance to standard chemotherapy, and high risk of refractory disease. MYC plays a central role by coordinating hyperactive protein synthesis with upregulated transcription in order to support rapid proliferation of tumor cells. Translation initiation inhibitor rocaglates have been identified as the most potent drugs in MYC-driven lymphomas as they efficiently inhibit MYC expression and tumor cell viability. We found that this class of compounds can overcome eIF4A abundance by stabilizing target mRNA-eIF4A interaction that directly prevents translation. Proteome-wide quantification demonstrated selective repression of multiple critical oncoproteins in addition to MYC in B-cell lymphoma including NEK2, MCL1, AURKA, PLK1, and several transcription factors that are generally considered undruggable. Finally, (-)-SDS-1-021, the most promising synthetic rocaglate, was confirmed to be highly potent as a single agent, and displayed significant synergy with the BCL2 inhibitor ABT199 in inhibiting tumor growth and survival in primary lymphoma cells in vitro and in patient-derived xenograft mouse models. Overall, our findings support the strategy of using rocaglates to target oncoprotein synthesis in MYC-driven lymphomas.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Linfoma de Células B , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Aglaia , Animais , Feminino , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patologia , Masculino , Camundongos , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BMVC is the first fluorescent probe designed to detect G-quadruplexes (G4s) in vivo. The MYC oncogene promoter forms a G4 (MycG4) which acts as a transcription silencer. Here, we report the high-affinity and specific binding of BMVC to MycG4 with unusual slow-exchange rates on the NMR timescale. We also show that BMVC represses MYC in cancer cells. We determined the solution structures of the 1:1 and 2:1 BMVC-MycG4 complexes. BMVC first binds the 5'-end of MycG4 to form a 1:1 complex with a well-defined structure. At higher ratio, BMVC also binds the 3'-end to form a second complex. In both complexes, the crescent-shaped BMVC recruits a flanking DNA residue to form a BMVC-base plane stacking over the external G-tetrad. Remarkably, BMVC adjusts its conformation to a contracted form to match the G-tetrad for an optimal stacking interaction. This is the first structural example showing the importance of ligand conformational adjustment in G4 recognition. BMVC binds the more accessible 5'-end with higher affinity, whereas sequence specificity is present at the weaker-binding 3'-site. Our structures provide insights into specific recognition of MycG4 by BMVC and useful information for design of G4-targeted anticancer drugs and fluorescent probes.
Assuntos
Carbazóis/química , Carbazóis/farmacocinética , Quadruplex G/efeitos dos fármacos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Compostos de Piridínio/química , Compostos de Piridínio/farmacocinética , Sítios de Ligação/efeitos dos fármacos , Dicroísmo Circular , DNA/química , DNA/efeitos dos fármacos , DNA/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Especificidade por SubstratoRESUMO
Bromodomain-containing protein 4 (BRD4), an epigenetic reader of acetyl lysine, has emerged as a promising therapeutic target for many diseases including cancer, inflammation and heart failure. Our previous study reported that nitroxoline, an FDA approved antibiotic, showed potential BRD4 inhibitory activity and antiproliferation activity against leukemia cell lines. In this study, we further explored the structure-activity relationship (SAR) around nitroxoline and employed our previously developed machine learning based activity scoring function BRD4LGR for further analysis. To improve the cellular level activity, physico-chemical properties were optimized using computational approaches. Then the candidates were tested for their ADME/T profiles. Finally, based on this rational hit-to-lead optimization strategy, 3 drug-like BRD4 inhibitors were obtained, with different profiles on cell line selectivity for multiple myeloma, leukemia and triple negative breast cancer. Further mechanism study showed these compounds could down-regulate c-Myc to inhibit cancer cell growth. This work illustrates the application of multiple computer-aided drug design techniques in a hit-to-lead optimization scenario, and provides novel potent BRD4 inhibitors with different phenotype propensities for future cancer treatment.
Assuntos
Desenho Assistido por Computador , Imidazóis/química , Nitroquinolinas/química , Proteínas Nucleares/antagonistas & inibidores , Quinolinas/química , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Humanos , Leucemia/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Relação Estrutura-Atividade , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
c-MYC proto-oncogene harbours a transcription-inhibitory quadruplex-forming scaffold (Pu27) upstream P1 promoter providing anti-neoplastic therapeutic target. Previous reports showed the binding profile of human Cathelicidin peptide (LL37) and telomeric G-quadruplex. Here, we truncated the quadruplex-binding domain of LL37 to prepare a small library of peptides through site-specific amino acid substitution. We investigated the intracellular selectivity of peptides for Pu27 over other oncogenic quadruplexes and their role in c-MYC promoter repression by dual-luciferase assays. We analysed their thermodynamics of binding reactions with c-MYC quadruplex isomers (Pu27, Myc22, Pu19) by Isothermal Titration Calorimetry. We discussed how amino acid substitutions and peptide helicity enhanced/weakened their affinities for c-MYC quadruplexes and characterized specific non-covalent inter-residual interactions determining their selectivity. Solution NMR structure indicated that KR12C, the best peptide candidate, selectively stabilized the 5'-propeller loop of c-MYC quadruplex by arginine-driven electrostatic-interactions at the sugar-phosphate backbone while KR12A peptide destabilized the quadruplex inducing a single-stranded hairpin-like conformation. Chromatin immunoprecipitations envisaged that KR12C and KR12A depleted and enriched Sp1 and NM23-H2 (Nucleoside diphosphate kinase) occupancy at Pu27 respectively supporting their regulation in stabilizing and unfolding c-MYC quadruplex in MCF-7 cells. We deciphered that selective arresting of c-MYC transcription by KR12C triggered apoptotic-signalling pathway via VEGF-A-BCL-2 axis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Apoptose/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Neoplasias/patologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Genes myc/efeitos dos fármacos , Humanos , Células MCF-7 , Mutagênese Sítio-Dirigida , Peptídeos/química , Peptídeos/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , CatelicidinasRESUMO
Adipose tissue-derived adipokines (i.e., leptin/adiponectin/resistin) play important roles in the regulation of several pathophysiologic processes through the activation of specific receptors. However, although adipokines and their receptors are widely distributed in many tissues and exhibit a clear modulation according to particular metabolic conditions (e.g., obesity and/or fasting), their expression, regulation, and putative action on normal prostate glands (PGs; a hormone-dependent organ tightly regulated by the endocrine-metabolic milieu) are still to be defined. Different in vivo/in vitro models were used to comprehensively characterize the expression pattern and actions of different adipokine systems (i.e., leptin/adiponectin/resistin/receptors) in mouse PGs. Adiponectin, resistin, and adiponectin receptors (1 and 2) and leptin receptor are coexpressed at different levels in PG cells, wherein they are finely regulated under fasting and/or obesity conditions. Furthermore, treatment with different adipokines exerted both homologous and heterologous regulation of specific adipokines/receptor-synthesis and altered the expression of key proliferation and oncogenesis markers (i.e., Ki67/c-Myc/p53) in mouse PG cell cultures, wherein some of these actions might be elicited through extracellular signal-regulated kinase (ERK) activation. Moreover, treatment with leptin, adiponectin, and resistin differentially regulated key functional parameters [i.e., proliferation and migration capacity and/or prostate-specific antigen (PSA) secretion] in human normal and/or tumoral prostate cell lines. Altogether, our data show that various adipokine and receptor systems are differentially expressed in normal PG cells; that their expression is under a complex ligand- and receptor-selective regulation under extreme metabolic conditions; and that they mediate distinctive and common direct actions in normal and tumoral PG cells (i.e., homologous and heterologous regulation of ligand and receptor synthesis, ERK signaling activation, modulation of proliferation markers, proliferation and migration capacity, and PSA secretion), suggesting a relevant role of these systems in the regulation of PG pathophysiology.
Assuntos
Adipocinas/metabolismo , Jejum/metabolismo , Obesidade/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Receptores de Adiponectina/metabolismo , Receptores para Leptina/metabolismo , Adipocinas/farmacologia , Adiponectina/metabolismo , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Antígeno Ki-67/efeitos dos fármacos , Antígeno Ki-67/metabolismo , Leptina/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Antígeno Prostático Específico/efeitos dos fármacos , Antígeno Prostático Específico/metabolismo , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Resistina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
In parallel with the continuous use of conventional insecticides, introduction of more environmentally friendly substances continues to grow in modern agriculture. In the present study, we evaluated chlorpyrifos, and imidacloprid and α-cypermethrin as two representatives of green insecticides for their genotoxic activity. We conducted a 14-day treatment in extended human lymphocytes cultures using real life exposure relevant concentrations. An alkaline comet assay was used to detect primary DNA damage. Simultaneously, the effect on the specific action towards the TP 53 and c-Myc genes in terms of fragmentation and copy number were determined. Both genes are responsible for cell cycle regulation; thus playing an active role in carcinogenesis. Contrary to what was expected, imidacloprid showed the highest genotoxicity potential, irrespective of the fact that none of the insecticides induced a significant level of primary DNA damage at all tested concentrations. Similar, no significant effect towards the TP 53 and c-Myc gene was recorded. The present study indicates that low level use of chlorpyrifos as a conventional insecticide and imidacloprid and α-cypermethrin as green insecticides does not pose a risk to DNA in general, nor to the TP 53 and c-Myc gene structural integrity.
Assuntos
Dano ao DNA/efeitos dos fármacos , Inseticidas/toxicidade , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Animais , Linhagem Celular , Clorpirifos/toxicidade , Ensaio Cometa , Humanos , Imidazóis/toxicidade , Hibridização in Situ Fluorescente , Linfócitos/efeitos dos fármacos , Neonicotinoides , Nitrocompostos/toxicidade , Piretrinas/toxicidadeRESUMO
The ribozyme-sensitive element NHE-III1 in the P1 promoter region of the important proto-oncogene c-myc contains many guanine (G)-rich sequences. Induction and stabilization of the G-quadruplex formed by NHE-III1 can downregulate c-myc expression. In the present study, we found that QPB-15e, a quinazoline derivative designed and synthesized by our laboratory, binds to and stabilizes the c-myc G-quadruplex in vitro, thereby inhibiting double-stranded DNA replication, downregulating c-myc gene expression and arresting cancer cell proliferation. PCR termination experiments showed that QPB-15e blocked double-stranded DNA replication by inducing or stabilizing the c-myc G-quadruplex. FRET-melting further confirmed that QPB-15e improved the stability of the G-quadruplex, and CD spectroscopy indicated that the compound interacted directly with the G-rich sequence. In competitive dialysis experiments, QPB-15e bound preferentially to quadruplex DNA in various structures, especially the G-quadruplex within the c-myc promoter region. Moreover, QPB-15e reduced the weights and volumes of tumors transplanted into nude mice. These findings strongly suggest that QPB-15e is a c-myc G-quadruplex ligand with anti-tumor properties, and may be efficacious for treating cancer in humans.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Quadruplex G/efeitos dos fármacos , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Quinazolinas/farmacologia , Animais , Antineoplásicos/síntese química , Benzamidas/síntese química , Linhagem Celular Tumoral , Genes myc/efeitos dos fármacos , Genes myc/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Regiões Promotoras Genéticas/efeitos dos fármacos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/genética , Quinazolinas/síntese química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Various carcinogens induce EGFR/RAS/MAPK signaling, which is critical in the development of lung cancer. In particular, constitutive activation of extracellular signal-regulated kinase 2 (ERK2) is observed in many lung cancer patients, and therefore developing compounds capable of targeting ERK2 in lung carcinogenesis could be beneficial. We examined the therapeutic effect of catechol in lung cancer treatment. Catechol suppressed anchorage-independent growth of murine KP2 and human H460 lung cancer cell lines in a dose-dependent manner. Catechol inhibited ERK2 kinase activity in vitro, and its direct binding to the ERK2 active site was confirmed by X-ray crystallography. Phosphorylation of c-Myc, a substrate of ERK2, was decreased in catechol-treated lung cancer cells and resulted in reduced protein stability and subsequent down-regulation of total c-Myc. Treatment with catechol induced G1 phase arrest in lung cancer cells and decreased protein expression related to G1-S progression. In addition, we showed that catechol inhibited the growth of both allograft and xenograft lung cancer tumors in vivo. In summary, catechol exerted inhibitory effects on the ERK2/c-Myc signaling axis to reduce lung cancer tumor growth in vitro and in vivo, including a preclinical patient-derived xenograft (PDX) model. These findings suggest that catechol, a natural small molecule, possesses potential as a novel therapeutic agent against lung carcinogenesis in future clinical approaches.
Assuntos
Antineoplásicos/farmacologia , Catecóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Camundongos SCID , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Deciding appropriate therapy for multiple myeloma (MM) is challenging because of the occurrence of multiple chromosomal changes and the fatal nature of the disease. In the current study, gamabufotalin (GBT) was isolated from toad venom, and its tumor-specific cytotoxicity was investigated in human MM cells. We found GBT inhibited cell growth and induced apoptosis with the IC50 values <50 nM. Mechanistic studies using functional approaches identified GBT as an inhibitor of c-Myc. Further analysis showed that GBT especially evoked the ubiquitination and degradation of c-Myc protein, thereby globally repressing the expression of c-Myc target genes. GBT treatment inhibited ERK and AKT signals, while stimulating the activation of JNK cascade. An E3 ubiquitin-protein ligase, WWP2, was upregulated following JNK activation and played an important role in c-Myc ubiquitination and degradation through direct protein-protein interaction. The antitumor effect of GBT was validated in a xenograft mouse model and the suppression of MM-induced osteolysis was verified in a SCID-hu model in vivo. Taken together, our study identified the potential of GBT as a promising therapeutic agent in the treatment of MM.
Assuntos
Inibidores da Angiogênese/farmacologia , Bufanolídeos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos SCID , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The bromodomain (BRD) and extraterminal (BET) proteins including BRD2, BRD3 and BRD4 have been identified as key targets for leukemia maintenance. A novel oral inhibitor of BRD2/3/4, the thienotriazolodiazepine compound OTX015, suitable for human use, is available. Here we report its biological effects in AML and ALL cell lines and leukemic samples. Exposure to OTX015 lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profiles in sensitive cell lines, including a c-MYC decrease and an HEXIM1 increase. OTX015 exposure also induced a strong decrease of BRD2, BRD4 and c-MYC and increase of HEXIM1 proteins, while BRD3 expression was unchanged. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels did not correlate however with viability following exposure to OTX015. Sequential combinations of OTX015 with other epigenetic modifying drugs, panobinostat and azacitidine have a synergic effect on growth of the KASUMI cell line. Our results indicate that OTX015 and JQ1 have similar biological effects in leukemic cells, supporting OTX015 evaluation in a Phase Ib trial in relapsed/refractory leukemia patients.
Assuntos
Acetanilidas/farmacologia , Antineoplásicos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Leucemia/patologia , Proteínas Nucleares/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , Fatores de Transcrição/biossíntese , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Feminino , Imunofluorescência , Humanos , Immunoblotting , Masculino , Proteínas Nucleares/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/efeitos dos fármacos , Proteínas de Ligação a RNA/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/efeitos dos fármacos , TranscriptomaRESUMO
Tetramethylpyrazine (TMP) has been proven to be an anticancer agent in many studies. However, its effectiveness in acute lymphoblastic leukemia (ALL) and its molecular mechanisms are still unclear. The present study aimed to evaluate the effect of TMP against Jurkat and SUP-B15 ALL cell lines and to investigate the possible detailed mechanism of action of TMP. A Cell Counting Kit-8 (CCK-8) assay was employed to examine the proliferation of Jurkat and SUP-B15 cells. Flow cytometric analysis was conducted to detect the cell cycle distribution and apoptotic rate. The expression of total glycogen synthase kinase-3ß (GSK-3ß), cox-2, survivin, bcl-2 and p27 RNA and protein levels was detected by quantitative real-time PCR and western blot assay, respectively. Additionally, western blot analysis was used to determine the whole-cell and nuclear protein levels of GSK-3ß downstream transcription factors, NF-κB (p65) and c-myc. TMP inhibited the proliferation of Jurkat and SUP-B15 cells in a dose- and time-dependent manner, with IC50 values of 120 and 200 µg/ml, respectively at 48 h. TMP induced the apoptosis of Jurkat and SUP-B15 cells and synergistically blocked cell cycle progression at the G0/G1 phase. Cells treated with TMP exhibited significantly attenuated GSK-3ß, NF-κB (p65) and c-myc expression, followed by downregulation of bcl-2, cox-2 and survivin and an upregulation of p27. The results showed that TMP induced apoptosis and caused cell cycle arrest in Jurkat and SUP-B15 cells through the downregulation of GSK-3ß, which may have further prevented the induced translocation of NF-κB and c-myc from the cytoplasm to the nucleus.