RESUMO
The physiological functions of butyrylcholinesterase (BChE) and its role in malignancy remain unexplained. Our studies in children newly diagnosed with neuroblastoma indicated that BChE expressions is proportional to MYCN amplification suggesting that pathogenesis of high-risk disease may be related to the persistent expression of abnormally high levels of tumor-associated BChE. BChE-deficient neuroblastoma cells (KO [knockout]) were produced from MYCN -amplified BE(2)-C cells (WT [wild-type]) by the CRISPR-Cas9 targeted disruption of the BCHE locus. KO cells have no detectable BChE activity. The compensatory acetylcholinesterase activity was not detected. The average population doubling time of KO cells is 47.0±2.4 hours, >2× longer than WT cells. Reduced proliferation rates of KO cells were accompanied by the loss of N-Myc protein and a significant deactivation of tyrosine kinase receptors associated with the aggressive neuroblastoma phenotype including Ros1, TrkB, and Ltk. Tumorigenicity of WT and KO cells in male mice was essentially identical. In contrast, KO xenografts in female mice were very small (0.37±0.10 g), ~3× smaller compared with WT xenografts (1.11±0.30 g). Unexpectedly, KO xenografts produced changes in plasma BChE similarly to WT tumors but lesser in magnitude. The disruption of BCHE locus in MYCN -amplified neuroblastoma cells decelerates proliferation and produces neuroblastoma cells that are less aggressive in female mice.
Assuntos
Butirilcolinesterase , Neuroblastoma , Acetilcolinesterase/genética , Animais , Butirilcolinesterase/genética , Feminino , Humanos , Masculino , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Tirosina Quinases , Proteínas Proto-OncogênicasRESUMO
BACKGROUND: Neuroblastoma (NB) patients with MYCN amplification or overexpression respond poorly to current therapies and exhibit extremely poor clinical outcomes. PI3K-mTOR signaling-driven deregulation of protein synthesis is very common in NB and various other cancers that promote MYCN stabilization. In addition, both the MYCN and mTOR signaling axes can directly regulate a common translation pathway that leads to increased protein synthesis and cell proliferation. However, a strategy of concurrently targeting MYCN and mTOR signaling in NB remains unexplored. This study aimed to investigate the therapeutic potential of targeting dysregulated protein synthesis pathways by inhibiting the MYCN and mTOR pathways together in NB. METHODS: Using small molecule/pharmacologic approaches, we evaluated the effects of combined inhibition of MYCN transcription and mTOR signaling on NB cell growth/survival and associated molecular mechanism(s) in NB cell lines. We used two well-established BET (bromodomain extra-terminal) protein inhibitors (JQ1, OTX-015), and a clinically relevant mTOR inhibitor, temsirolimus, to target MYCN transcription and mTOR signaling, respectively. The single agent and combined efficacies of these inhibitors on NB cell growth, apoptosis, cell cycle and neurospheres were assessed using MTT, Annexin-V, propidium-iodide staining and sphere assays, respectively. Effects of inhibitors on global protein synthesis were quantified using a fluorescence-based (FamAzide)-based protein synthesis assay. Further, we investigated the specificities of these inhibitors in targeting the associated pathways/molecules using western blot analyses. RESULTS: Co-treatment of JQ1 or OTX-015 with temsirolimus synergistically suppressed NB cell growth/survival by inducing G1 cell cycle arrest and apoptosis with greatest efficacy in MYCN-amplified NB cells. Mechanistically, the co-treatment of JQ1 or OTX-015 with temsirolimus significantly downregulated the expression levels of phosphorylated 4EBP1/p70-S6K/eIF4E (mTOR components) and BRD4 (BET protein)/MYCN proteins. Further, this combination significantly inhibited global protein synthesis, compared to single agents. Our findings also demonstrated that both JQ1 and temsirolimus chemosensitized NB cells when tested in combination with cisplatin chemotherapy. CONCLUSIONS: Together, our findings demonstrate synergistic efficacy of JQ1 or OTX-015 and temsirolimus against MYCN-driven NB, by dual-inhibition of MYCN (targeting transcription) and mTOR (targeting translation). Additional preclinical evaluation is warranted to determine the clinical utility of targeted therapy for high-risk NB patients.
Assuntos
Acetanilidas/farmacologia , Azepinas/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteína Proto-Oncogênica N-Myc/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Sirolimo/análogos & derivados , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triazóis/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo , Sinergismo Farmacológico , Fator de Iniciação 4E em Eucariotos/efeitos dos fármacos , Fator de Iniciação 4E em Eucariotos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Esferoides Celulares/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: The activity of MP1, a pyrrolomycin, was studied in MYCN amplified neuroblastoma (NB) alone and combined with temsirolimus (TEM). METHODS: Activity of MP1 was tested in MYCN amplified (BE-2c, IMR) and non amplified (SKN-AS) NB cells. The effect of MP1 on MYCN, MCL-1, cleaved PARP, LC3II/LC3I, bcl-2, BAX, and BRD-4 were determined by western blot and RNAseq. The effect of MP1 on metabolism, mitochondrial morphology, and cell cycle was determined. Toxicology and efficacy of MP1 plus TEM were evaluated. RESULTS: The IC50 of MP1 was 0.096 µM in BE-2c cells compared to 0.89 µM in IMR, and >50 µM in SKN-AS. The IC50 of MP1 plus TEM in BE-2c cells was 0.023 µM. MP1 inhibited metabolism leading to quiescence and produced a decline in cell cycle S-phase. Electron microscopy showed cristae loss and rounding up of mitochondria. Gene and protein expression for MYCN and MCL-1 declined while LCII and cleaved PARP increased. Protein expression of BAX, bcl-2, and BRD-4 were not significantly changed after MP1 treatment. The in-vivo concentrations of MP1 in blood and tumor were sufficient to produce the biologic effects seen in-vitro. MP1 plus TEM produced a complete response in 3 out of 5 tumor bearing mice. In a second mouse study, the combination of MP1 and TEM slowed tumor growth compared to control. CONCLUSIONS: MP1 has a potent inhibitory effect on the viability of MYCN amplified NB. Inhibition of metabolism by MP1 induced quiescence and autophagy with a favorable toxicology and drug distribution profile. When combined with TEM anti-tumor activity was potentiated in-vitro and in-vivo.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Amplificação de Genes , Proteína Proto-Oncogênica N-Myc/genética , Pirróis/farmacologia , Sirolimo/análogos & derivados , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Interações Medicamentosas , Humanos , Camundongos , Estrutura Molecular , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/ultraestrutura , Pirróis/química , Sirolimo/química , Sirolimo/farmacologia , Análise Espectral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Medulloblastoma (MB) is a malignant pediatric brain tumor with poor prognosis. Signal transducers and activators of transcription-3 (STAT3) is constitutively activated in MB where it functions as an oncoprotein, mediating cancer progression and metastasis. Here, we have delineated the functional role of activated STAT3 in MB, by using a cell permeable STAT3-NH2 terminal domain inhibitor (S3-NTDi) that specifically perturbs the structure/function of STAT3. We have implemented several biochemical experiments using human MB tumor microarray (TMA) and pediatric MB cell lines, derived from high-risk SHH-TP53-mutated and MYC-amplified Non-WNT/SHH tumors. Treatment of MB cells with S3-NTDi leads to growth inhibition, cell cycle arrest, and apoptosis. S3-NTDi downregulated expression of STAT3 target genes, delayed migration of MB cells, attenuated epithelial-mesenchymal transition (EMT) marker expressions and reduced cancer stem-cell associated protein expressions in MB-spheres. To elucidate mechanisms, we showed that S3-NTDi induce expression of pro-apoptotic gene, C/EBP-homologous protein (CHOP), and decrease association of STAT3 to the proximal promoter of CCND1 and BCL2. Of note, S3-NTDi downregulated microRNA-21, which in turn, de-repressed Protein Inhibitor of Activated STAT3 (PIAS3), a negative regulator of STAT3 signaling pathway. Furthermore, combination therapy with S3-NTDi and cisplatin significantly decreased highly aggressive MYC-amplified MB cell growth and induced apoptosis by downregulating STAT3 regulated proliferation and anti-apoptotic gene expression. Together, our results revealed an important role of STAT3 in regulating MB pathogenesis. Disruption of this pathway with S3-NTDi, therefore, may serves as a promising candidate for targeted MB therapy by enhancing chemosensitivity of MB cells and potentially improving outcomes in high-risk patients.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Chaperonas Moleculares/genética , Peptídeos/farmacologia , Proteínas Inibidoras de STAT Ativados/genética , Fator de Transcrição STAT3/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/farmacologia , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Chaperonas Moleculares/metabolismo , Peptídeos/síntese química , Proteínas Inibidoras de STAT Ativados/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismoRESUMO
The systematics of the molluscan class Bivalvia are explored using a 5-gene Sanger-based approach including the largest taxon sampling to date, encompassing 219 ingroup species spanning 93 (or 82%) of the 113 currently accepted bivalve families. This study was designed to populate the bivalve Tree of Life at the family level and to place many genera into a clear phylogenetic context, but also pointing to several major clades where taxonomic work is sorely needed. Despite not recovering monophyly of Bivalvia or Protobranchia-as in most previous Sanger-based approaches to bivalve phylogeny-our study provides increased resolution in many higher-level clades, and supports the monophyly of Autobranchia, Pteriomorphia, Heteroconchia, Palaeoheterodonta, Heterodonta, Archiheterodonta, Euheterodonta, Anomalodesmata, Imparidentia, and Neoheterodontei, in addition to many other lower clades. However, deep nodes within some of these clades, especially Pteriomorphia and Imparidentia, could not be resolved with confidence. In addition, many families are not supported, and several are supported as non-monophyletic, including Malletiidae, Nuculanidae, Yoldiidae, Malleidae, Pteriidae, Arcidae, Propeamussiidae, Iridinidae, Carditidae, Myochamidae, Lyonsiidae, Pandoridae, Montacutidae, Galeommatidae, Tellinidae, Semelidae, Psammobiidae, Donacidae, Mactridae, and Cyrenidae; Veneridae is paraphyletic with respect to Chamidae, although this result appears to be an artifact. The denser sampling however allowed testing specific placement of species, showing, for example, that the unusual Australian Plebidonax deltoides is not a member of Donacidae and instead nests within Psammobiidae, suggesting that major revision of Tellinoidea may be required. We also showed that Cleidothaerus is sister group to the cementing member of Myochamidae, suggesting that Cleidothaeridae may not be a valid family and that cementation in Cleidothaerus and Myochama may have had a single origin. These results highlight the need for an integrative approach including as many genera as possible, and that the monophyly and relationships of many families require detailed reassessment. NGS approaches may be able to resolve the most recalcitrant nodes in the near future.
Assuntos
Bivalves/classificação , Bivalves/genética , Filogenia , Análise de Sequência de DNA/métodos , Animais , Teorema de Bayes , Funções VerossimilhançaRESUMO
BACKGROUND: Evaluate the anti-tumor activity of ozonide antimalarials using a chemoresistant neuroblastoma cell line, BE (2)-c. METHODS: The activity of 12 ozonides, artemisinin, and two semisynthetic artemisinins were tested for activity against two neuroblastoma cell-lines (BE (2)-c and IMR-32) and the Ewing's Sarcoma cell line A673 in an MTT viability assay. Time course data indicated that peak effect was seen 18 h after the start of treatment thus 18 h pre-treatment was used for all subsequent experiments. The most active ozonide (OZ513) was assessed in a propidium iodide cell cycle flow cytometry analysis which measured cell cycle transit and apoptosis. Metabolic effects of OZ513 in BE (2)-c cells was evaluated. Western blots for the apoptotic proteins cleaved capase-3 and cleaved PARP, the highly amplified oncogene MYCN, and the cell cycle regulator CyclinD1, were performed. These in-vitro experiments were followed by an in-vivo experiment in which NOD-scid gamma immunodeficient mice were injected subcutaneously with 1 × 106 BE (2)-c cells followed by immediate treatment with 50-100 mg/kg/day doses of OZ513 administered IP three times per week out to 23 days after injection of tumor. Incidence of tumor development, time to tumor development, and rate of tumor growth were assessed in DMSO treated controls (N = 6), and OZ513 treated mice (N = 5). RESULTS: It was confirmed that five commonly used chemotherapy drugs had no cytotoxic activity in BE (2)-c cells. Six of 12 ozonides tested were active in-vitro at concentrations achievable in vivo with OZ513 being most active (IC50 = 0.5 mcg/ml). OZ513 activity was confirmed in IMR-32 and A673 cells. The Ao peak on cell-cycle analysis was increased after treatment with OZ513 in a concentration dependent fashion which when coupled with results from western blot analysis which showed an increase in cleaved capase-3 and cleaved PARP supported an increase in apoptosis. There was a concentration dependent decline in the MYCN and a cyclinD1 protein indicative of anti-proliferative activity and cell cycle disruption. OXPHOS metabolism was unaffected by OZ513 treatment while glycolysis was increased. There was a significant delay in time to tumor development in mice treated with OZ513 and a decline in the rate of tumor growth. CONCLUSIONS: The antimalarial ozonide OZ513 has effective in-vitro and in-vivo activity against a pleiotropic drug resistant neuroblastoma cell-line. Treatment with OZ513 increased apoptotic markers and glycolysis with a decline in the MYCN oncogene and the cell cycle regulator cyclinD1. These effects suggest adaptation to cellular stress by mechanism which remain unclear.
Assuntos
Antimaláricos/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Artemisininas/farmacologia , Biomarcadores , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Metaboloma , Metabolômica/métodos , Camundongos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor prognoses and therapy resistance. However, MYC itself has been one of the most challenging targets for cancer treatment. Here, we identify a novel marinopyrrole natural derivative, MP1, that shows desirable anti-MYC and anti-cancer activities in MB. METHODS: In this study, using MYC-amplified (Group 3) and non-MYC amplified MB cell lines in vitro and in vivo, we evaluated anti-cancer efficacies and molecular mechanism(s) of MP1. RESULTS: MP1 significantly suppressed MB cell growth and sphere counts and induced G2 cell cycle arrest and apoptosis in a MYC-dependent manner. Mechanistically, MP1 strongly downregulated the expression of MYC protein. Our results with RNA-seq revealed that MP1 significantly modulated global gene expression and inhibited MYC-associated transcriptional targets including translation/mTOR targets. In addition, MP1 inhibited MYC-target metabolism, leading to declined energy levels. The combination of MP1 with an FDA-approved mTOR inhibitor temsirolimus synergistically inhibited MB cell growth/survival by downregulating the expression of MYC and mTOR signaling components. Our results further showed that as single agents, both MP1 and temsirolimus, were able to significantly inhibit tumor growth and MYC expression in subcutaneously or orthotopically MYC-amplified MB bearing mice. In combination, there were further anti-MB effects on the tumor growth and MYC expression in mice. CONCLUSION: These preclinical findings highlight the promise of marinopyrrole MP1 as a novel MYC inhibition approach for MYC-amplified MB.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Sirolimo/análogos & derivados , Humanos , Animais , Camundongos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Pontos de Checagem da Fase G2 do Ciclo Celular , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Serina-Treonina Quinases TORRESUMO
A molecular phylogeny of Protobranchia, the subclass of bivalve mollusks sister to the remaining Bivalvia, has long proven elusive, because many constituent lineages are deep-sea endemics, which creates methodological challenges for collecting and preserving genetic material. We obtained 74 representatives of all 12 extant protobranch families and investigated the internal phylogeny of this group using sequence data from five molecular loci (16S rRNA, 18S rRNA, 28S rRNA, cytochrome c oxidase subunit I, and histone H3). Model-based and dynamic homology parsimony approaches to phylogenetic reconstruction unanimously supported four major clades of Protobranchia, irrespective of treatment of hypervariable regions in the nuclear ribosomal genes 18S rRNA and 28S rRNA. These four clades correspond to the superfamilies Nuculoidea (excluding Sareptidae), Nuculanoidea (including Sareptidae), Solemyoidea, and Manzanelloidea. Salient aspects of the phylogeny include (1) support for the placement of the family Sareptidae with Nuculanoidea; (2) the non-monophyly of the order Solemyida (Solemyidae+Nucinellidae); (3) and the non-monophyly of most nuculoid and nuculanoid genera and families. In light of this first family-level phylogeny of Protobranchia, we present a revised classification of the group. Estimation of divergence times in concert with analyses of diversification rates demonstrate the signature of the end-Permian mass extinction in the phylogeny of extant protobranchs.
Assuntos
Bivalves/classificação , Complexo IV da Cadeia de Transporte de Elétrons/classificação , Especiação Genética , Histonas/classificação , Filogenia , RNA Ribossômico/classificação , Algoritmos , Animais , Bivalves/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Extinção Biológica , Variação Genética , Histonas/genética , Funções Verossimilhança , Modelos Genéticos , Oceanos e Mares , RNA Ribossômico/genética , Análise de Sequência de DNARESUMO
Left ventricular assist devices (LVADs) comprise a primary treatment choice for advanced heart failure patients. Treatment with LVAD is commonly associated with complications like stroke and gastro-intestinal (GI) bleeding, which adversely impacts treatment outcomes, and causes fatalities. The etiology and mechanisms of these complications can be linked to the fact that LVAD outflow jet leads to an altered state of hemodynamics in the aorta as compared to baseline flow driven by aortic jet during ventricular systole. Here, we present a framework for quantitative assessment of aortic hemodynamics in LVAD flows realistic human vasculature, with a focus on quantifying the differences between flow driven by LVAD jet and the physiological aortic jet when no LVAD is present. We model hemodynamics in the aortic arch proximal to the LVAD outflow graft, as well as in the abdominal aorta away from the LVAD region. We characterize hemodynamics using quantitative descriptors of flow velocity, stasis, helicity, vorticity and mixing, and wall shear stress. These are used on a set of 27 LVAD scenarios obtained by parametrically varying LVAD outflow graft anastomosis angles, and LVAD flow pulse modulation. Computed descriptors for each of these scenarios are compared against the baseline flow, and a detailed quantitative characterization of the altered state of hemodynamics due to LVAD operation (when compared to baseline aortic flow) is compiled. These are interpreted using a conceptual model for LVAD flow that distinguishes between flow originating from the LVAD outflow jet (and its impingement on the aorta wall), and flow originating from aortic jet during aortic valve opening in normal physiological state.
Assuntos
Insuficiência Cardíaca , Coração Auxiliar , Humanos , Coração Auxiliar/efeitos adversos , Modelos Cardiovasculares , Aorta , Aorta Torácica/fisiologia , HemodinâmicaRESUMO
Left ventricular assist device (LVAD) provides mechanical circulatory support for patients with advanced heart failure. Treatment using LVAD is commonly associated with complications such as stroke and gastro-intestinal bleeding. These complications are intimately related to the state of hemodynamics in the aorta, driven by a jet flow from the LVAD outflow graft that impinges into the aorta wall. Here we conduct a systematic analyses of hemodynamics driven by an LVAD with a specific focus on viscous energy transport and dissipation. We conduct a complementary set of analysis using idealized cylindrical tubes with diameter equivalent to common carotid artery and aorta, and a patient-specific model of 27 different LVAD configurations. Results from our analysis demonstrate how energy dissipation is governed by key parameters such as frequency and pulsation, wall elasticity, and LVAD outflow graft surgical anastomosis. We find that frequency, pulsation, and surgical angles have a dominant effect, while wall elasticity has a weaker effect, in determining the state of energy dissipation. For the patient-specific scenario, we also find that energy dissipation is higher in the aortic arch and lower in the abdominal aorta, when compared to the baseline flow without an LVAD. This further illustrates the key hemodynamic role played by the LVAD outflow jet impingement, and subsequent aortic hemodynamics during LVAD operation.
Assuntos
Insuficiência Cardíaca , Coração Auxiliar , Humanos , Aorta Torácica/cirurgia , Hemodinâmica , Insuficiência Cardíaca/cirurgia , Aorta AbdominalRESUMO
Stroke remains a leading cause of complications and mortality in heart failure patients treated with LVAD circulatory support. Hemodynamics plays a central role in affecting risk and etiology of stroke during LVAD support. Yet, detailed quantitative assessment of hemodynamic variables and their relation to stroke outcomes in patients with an implanted LVAD remains a challenge. We present an in silico hemodynamics analysis in a set of 12 patients on LVAD support; 6 with reported stroke outcomes and 6 without. We conducted patient-specific hemodynamics simulations for models with the LVAD outflow graft reconstructed from cardiac-gated CT images. A pre-implantation baseline flow model was virtually generated for each case by removing the LVAD outflow graft and driving flow from the aortic root. Hemodynamics was characterized using quantitative descriptors for helical flow, vortex generation, and wall shear stress. Our analysis showed higher average values for descriptors of positive helical flow, vortex generation, and wall shear stress, across the 6 cases with stroke outcomes on LVAD support, when compared with cases without stroke. When the descriptors for LVAD-driven flow were compared against estimated baseline flow pre-implantation, extent of positive helicity was higher, and vorticity and wall shear were lower in cases with stroke compared to those without. The study suggests that quantitative analysis of hemodynamics after LVAD implantation; and hemodynamic alterations from a pre-implant flow scenario, can potentially reveal hidden information linked to stroke outcomes during LVAD support. This has broad implications on understanding stroke etiology, LVAD treatment planning, surgical optimization, and efficacy assessment.
RESUMO
MB is a common childhood malignancy of the central nervous system, with significant morbidity and mortality. Among the four molecular subgroups, MYC-amplified Group 3 MB is the most aggressive type and has the worst prognosis due to therapy resistance. The present study aimed to investigate the role of activated STAT3 in promoting MB pathogenesis and chemoresistance via inducing the cancer hallmark MYC oncogene. Targeting STAT3 function either by inducible genetic knockdown (KD) or with a clinically relevant small molecule inhibitor reduced tumorigenic attributes in MB cells, including survival, proliferation, anti-apoptosis, migration, stemness and expression of MYC and its targets. STAT3 inhibition attenuates MYC expression by affecting recruitment of histone acetyltransferase p300, thereby reducing enrichment of H3K27 acetylation in the MYC promoter. Concomitantly, it also decreases the occupancy of the bromodomain containing protein-4 (BRD4) and phosphoSer2-RNA Pol II (pSer2-RNAPol II) on MYC, resulting in reduced transcription. Importantly, inhibition of STAT3 signaling significantly attenuated MB tumor growth in subcutaneous and intracranial orthotopic xenografts, increased the sensitivity of MB tumors to cisplatin, and improved the survival of mice bearing high-risk MYC-amplified tumors. Together, the results of our study demonstrate that targeting STAT3 may be a promising adjuvant therapy and chemo-sensitizer to augment treatment efficacy, reduce therapy-related toxicity and improve quality of life in high-risk pediatric patients.
RESUMO
BACKGROUND: Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor clinical outcomes and respond poorly to current therapies. Epigenetic deregulation is very common in MYC-driven MB. The bromodomain extra-terminal (BET) proteins and histone deacetylases (HDACs) are epigenetic regulators of MYC transcription and its associated tumorigenic programs. This study aimed to investigate the therapeutic potential of inhibiting the BET proteins and HDACs together in MB. METHODS: Using clinically relevant BET inhibitors (JQ1 or OTX015) and a pan-HDAC inhibitor (panobinostat), we evaluated the effects of combined inhibition on cell growth/survival in MYC-amplified MB cell lines and xenografts and examined underlying molecular mechanism(s). RESULTS: Co-treatment of JQ1 or OTX015 with panobinostat synergistically suppressed growth/survival of MYC-amplified MB cells by inducing G2 cell cycle arrest and apoptosis. Mechanistic investigation using RNA-seq revealed that co-treatment of JQ1 with panobinostat synergistically modulated global gene expression including MYC/HDAC targets. SYK and MSI1 oncogenes were among the top 50 genes synergistically downregulated by JQ1 and panobinostat. RT-PCR and western blot analyses confirmed that JQ1 and panobinostat synergistically inhibited the mRNA and protein expression of MSI1/SYK along with MYC expression. Reduced SYK/MSI expression after BET (specifically, BRD4) gene-knockdown further confirmed the epigenetic regulation of SYK and MSI1 genes. In addition, the combination of OTX015 and panobinostat significantly inhibited tumor growth in MYC-amplified MB xenografted mice by downregulating expression of MYC, compared to single-agent therapy. CONCLUSIONS: Together, our findings demonstrated that dual-inhibition of BET and HDAC proteins of the epigenetic pathway can be a novel therapeutic approach against MYC-driven MB.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Humanos , Camundongos , Animais , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Histona Desacetilases/metabolismo , Proteínas Nucleares/metabolismo , Panobinostat/farmacologia , Panobinostat/uso terapêutico , Azepinas/farmacologia , Epigênese Genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Apoptose , Proliferação de Células , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
The MYC oncogene is frequently amplified in patients with medulloblastoma, particularly in group 3 patients, who have the worst prognosis. mTOR signaling-driven deregulated protein synthesis is very common in various cancers, including medulloblastoma, that can promote MYC stabilization. As a transcription factor, MYC itself is further known to regulate transcription of several components of protein synthesis machinery, leading to an enhanced protein synthesis rate and proliferation. Thus, inhibiting enhanced protein synthesis by targeting the MYC and mTOR pathways together may represent a highly relevant strategy for the treatment of MYC-driven medulloblastoma. Here, using siRNA and small-molecule inhibitor approaches, we evaluated the effects of combined inhibition of MYC transcription and mTOR signaling on medulloblastoma cell growth/survival and associated molecular mechanism(s) in MYC-amplified (group 3) medulloblastoma cell lines and xenografts. Combined inhibition of MYC and mTOR synergistically suppressed medulloblastoma cell growth and induced G1 cell-cycle arrest and apoptosis. Mechanistically, the combined inhibition significantly downregulated the expression levels of key target proteins of MYC and mTOR signaling. Our results with RNA-sequencing revealed that combined inhibition synergistically modulated global gene expression including MYC/mTOR components. In addition, the combination treatment significantly delayed tumor growth and prolonged survival of MYC-amplified medulloblastoma xenografted mice by downregulating expression of MYC and the key downstream components of mTOR signaling, compared with single-agent therapy. Together, our findings demonstrated that dual inhibition of MYC (transcription) and mTOR (translation) of the protein synthesis pathway can be a novel therapeutic approach against MYC-driven medulloblastoma.
Assuntos
Azepinas/farmacologia , Neoplasias Cerebelares/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Meduloblastoma/tratamento farmacológico , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Quinolinas/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose , Ciclo Celular , Proliferação de Células , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Feminino , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Proto-Oncogênicas c-myc/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aberrant activation/expression of pathways/molecules including NF-kB, mTOR, hedgehog and polo-like-kinase-1 (PLK1) are correlated with poor-prognosis neuroblastoma. Therefore, to identify a most efficacious treatment for neuroblastoma, we investigated the efficacy of NF-kB/mTOR dual-inhibitor 13-197, hedgehog inhibitor vismodegib and PLK1 inhibitor BI2536 alone or combined with topotecan against high-risk neuroblastoma. The in vitro efficacy of the inhibitors alone or combined with topotecan on cell growth/apoptosis and molecular mechanism(s) were investigated. Results showed that as single agents 13-197, BI2536 and vismodegib significantly decreased neuroblastoma cell growth and induced apoptosis by targeting associated pathways/molecules. In combination with topotecan, 13-197 did not show significant additive/synergistic effects against neuroblastoma. However, BI2536 or vismodegib further significantly decreased neuroblastoma cell growth/survival. These results clearly showed that vismodegib combination with topotecan was synergistic and more efficacious compared with BI2536 in combination. Together, in vitro data demonstrated that vismodegib was most efficacious in potentiating topotecan-induced antineuroblastoma effects. Therefore, we tested the combined efficacy of vismodegib and topotecan against neuroblastoma in vivo using NSG mice. This resulted in significantly (p<0.001) reduced tumor growth and increased survival of mice. Together, the combination of vismodegib and topotecan showed a significant enhanced antineuroblastoma efficacy by targeting associated pathways/molecules which warrants further preclinical evaluation for translation to the clinic.
Assuntos
Anilidas/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Piridinas/farmacologia , Topotecan/farmacologia , Animais , Quimioterapia Combinada , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neuroblastoma/patologia , Inibidores da Topoisomerase I/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Early diagnosis of Parkinson's disease (PD) is required to improve therapeutic responses. Indeed, a clinical diagnosis of resting tremor, rigidity, movement and postural deficiencies usually reflect >50% loss of the nigrostriatal system in disease. In a step to address this, quantitative diffusion tensor magnetic resonance imaging (DTI) was used to assess nigrostriatal degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication model of dopaminergic nigral degeneration. We now demonstrate increased average diffusion (p<0.005) and decreased fractional anisotropy (p<0.03) in the substantia nigra (SN) of 5- to 7-day MPTP-treated animals when compared to saline controls. Transverse diffusivity demonstrated the most significant differences (p < or = 0.002) and correlated with the numbers of SN dopaminergic neurons (r=-0.75, p=0.012). No differences were found in the striatum, corpus callosum, cerebral cortex, or ventricles. These results demonstrate that DTI may be used as a surrogate biomarker of nigral dopaminergic neuronal degeneration.