RESUMO
INTRODUCTION: The American Academy of Pediatrics Back-to-Sleep Campaign significantly reduced infant mortality from sudden infant death syndrome. As a result of prolonged supine positioning, the incidence of deformational plagiocephaly has also risen 5-fold since its adoption. We aimed to improve the current educational paradigm for new parents with the goal of reducing the incidence of plagiocephaly within the confines of the Back-to-Sleep Campaign. We hypothesized that the early addition of plagiocephaly focused education for parents would reduce cephalic index, the ratio of head width to length, used as an easily measured objective proxy for positional plagiocephaly. METHODS: Children were screened at their newborn visit. Premature newborns and those diagnosed with craniofacial disorders were excluded. For those enrolled, biparietal and anteroposterior measurements of the head were obtained using manual calipers to obtain cephalic index. Subjects randomly assigned to the intervention group were shown a 2-minute video and given an educational pamphlet on methods to prevent plagiocephaly. Unpaired 2-sample t tests comparing mean differences in intervention and control were performed. RESULTS: Thirty-nine subjects were enrolled as of November 2023 with variable lengths of follow-up completed. The average baseline cephalic index for subjects in the control group was 82.7 and 83.8 for intervention group. Unpaired 2-sample t tests were performed at 2-, 4-, and 6-month time points to analyze the difference between groups. At 4 months, average cephalic index for subjects in the control and treatment group, respectively, was 90.6 and 83.4 (P = 0.02). SIGNIFICANCE: Parental education at the newborn visit led to decreases in cephalic index, a proxy for positional plagiocephaly, compared with control patients. This simple intervention has the potential to reduce parental stress and healthcare costs associated with the evaluation and treatment of plagiocephaly.
Assuntos
Plagiocefalia não Sinostótica , Plagiocefalia , Lactente , Humanos , Recém-Nascido , Criança , Plagiocefalia não Sinostótica/prevenção & controle , Plagiocefalia não Sinostótica/diagnóstico , Decúbito Dorsal , Plagiocefalia/prevenção & controle , Plagiocefalia/complicações , Pais , SonoRESUMO
(1) Aim: The aim of this study was to determine the relationship between lower limb bone deformities and body functions, activity, and participation in ambulant children with CP and whether changing bone morphology affects outcomes in these domains. (2) Methods: A systematic literature search (PROSPERO CRD42020208416) of studies reporting correlations between measures of lower limb bone deformities and measures of body function, activity or participation, or post-surgical outcomes in these domains was conducted from 1990 to 2023 in Medline, Scopus, and Cochrane Library. We assessed study quality with the Checklist for Case Series (CCS) and a quality assessment developed by Quebec University Hospital. Meta-analysis was not possible; therefore, descriptive synthesis was performed. (3) Results: A total of 12 of 3373 screened articles were included. No studies evaluated the relationships between bone deformities and activity or participation, or the effect of isolated bone surgery on these domains. Correlations between bone deformities and body functions were poor-to-moderate. Internal hip rotation during gait improved after femoral derotation osteotomy. (4) Conclusions: A shift in paradigm is urgently required for the research and management of bone deformities in children with CP to include the activity and participation domains of the ICF, as well as consider more psychological aspects such as self-image.
RESUMO
NF-κB transcription factors are critical regulators of innate and adaptive immunity and major mediators of inflammatory signaling. The NF-κB signaling is dysregulated in a significant number of cancers and drives malignant transformation through maintenance of constitutive pro-survival signaling and downregulation of apoptosis. Overactive NF-κB signaling results in overexpression of pro-inflammatory cytokines, chemokines and/or growth factors leading to accumulation of proliferative signals together with activation of innate and select adaptive immune cells. This state of chronic inflammation is now thought to be linked to induction of malignant transformation, angiogenesis, metastasis, subversion of adaptive immunity, and therapy resistance. Moreover, accumulating evidence indicates the involvement of NF-κB signaling in induction and maintenance of invasive phenotypes linked to epithelial to mesenchymal transition (EMT) and metastasis. In this review we summarize reported links of NF-κB signaling to sequential steps of transition from epithelial to mesenchymal phenotypes. Understanding the involvement of NF-κB in EMT regulation may contribute to formulating optimized therapeutic strategies in cancer. Video Abstract.
Assuntos
NF-kappa B , Neoplasias , Humanos , NF-kappa B/metabolismo , Transição Epitelial-Mesenquimal/genética , Transdução de Sinais , Neoplasias/metabolismo , Transformação Celular Neoplásica , Fenótipo , Linhagem Celular TumoralRESUMO
With a global increase in chronic kidney disease patients, alternatives to dialysis and organ transplantation are needed. Stem cell-based therapies could be one possibility to treat chronic kidney disease. Here, we used multipotent urine-derived renal progenitor cells (UdRPCs) to study nephrogenesis. UdRPCs treated with the JNK inhibitor-AEG3482 displayed decreased proliferation and downregulated transcription of cell cycle-associated genes as well as the kidney progenitor markers-SIX2, SALL1 and VCAM1. In addition, levels of activated SMAD2/3, which is associated with the maintenance of self-renewal in UdRPCs, were decreased. JNK inhibition resulted in less efficient oxidative phosphorylation and more lipid peroxidation via ferroptosis, an iron-dependent non-apoptotic cell death pathway linked to various forms of kidney disease. Our study is the first to describe the importance of JNK signalling as a link between maintenance of self-renewal and protection against ferroptosis in SIX2-positive renal progenitor cells.
Assuntos
Ferroptose , Sistema de Sinalização das MAP Quinases , Insuficiência Renal Crônica , Humanos , Rim , Diálise Renal , Células-TroncoRESUMO
CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.
Assuntos
Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes , Animais , Camundongos , Quinases Ciclina-Dependentes/metabolismo , Ciclo Celular/fisiologia , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fosforilação , Divisão CelularRESUMO
We recently reported shrinkage of untreatable recurrent glioblastoma (GBM) in an end-stage patient using noninvasive brain stimulation with a spinning oscillating magnetic field (sOMF)-generating device called the Oncomagnetic device. Our in vitro experiments demonstrated selective cancer cell death while sparing normal cells by sOMF-induced increase in intracellular reactive oxygen species (ROS) levels due to magnetic perturbation of mitochondrial electron transport. Here, we describe the results of an in vivo study assessing the toxicity of chronic sOMF stimulation in mice using a newly constructed apparatus comprised of the sOMF-generating active components of the Oncomagnetic device. We chronically stimulated 10 normal 60-day old female C57BL/6 mice in their housing cages for 2 h 3 times a day, as in the patient treatment protocol, over 4 months. We also studied the effects of 2-h acute sOMF stimulation. Our observations and those of blinded independent veterinary staff observers, indicated no significant adverse effects of chronic or acute sOMF stimulation on the health, behavior, electrocardiographic and electroencephalographic activities, hematologic profile, and brain and other tissue and organ morphology of treated mice compared to age-matched untreated control mice. These findings suggest that short- and long-term therapies with the Oncomagnetic device are safe and well tolerated.
Assuntos
Glioblastoma , Campos Magnéticos , Animais , Camundongos , Feminino , Espécies Reativas de Oxigênio , Camundongos Endogâmicos C57BL , EncéfaloRESUMO
Kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), which can progress to end stage renal disease (ESRD), are a worldwide health burden. Organ transplantation or kidney dialysis are the only effective available therapeutic tools. Therefore, in vitro models of kidney diseases and the development of prospective therapeutic options are urgently needed. Within the kidney, the glomeruli are involved in blood filtration and waste excretion and are easily affected by changing cellular conditions. Puromycin aminonucleoside (PAN) is a nephrotoxin, which can be employed to induce acute glomerular damage and to model glomerular disease. For this reason, we generated kidney organoids from three iPSC lines and treated these with PAN in order to induce kidney injury. Morphological observations revealed the disruption of glomerular and tubular structures within the kidney organoids upon PAN treatment, which were confirmed by transcriptome analyses. Subsequent analyses revealed an upregulation of immune response as well as inflammatory and cell-death-related processes. We conclude that the treatment of iPSC-derived kidney organoids with PAN induces kidney injury mediated by an intertwined network of inflammation, cytoskeletal re-arrangement, DNA damage, apoptosis and cell death. Furthermore, urine-stem-cell-derived kidney organoids can be used to model kidney-associated diseases and drug discovery.
Assuntos
Injúria Renal Aguda , Células-Tronco Pluripotentes Induzidas , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Humanos , Rim , Organoides/metabolismo , Puromicina Aminonucleosídeo/metabolismo , Puromicina Aminonucleosídeo/farmacologiaRESUMO
BACKGROUND AND OBJECTIVE: This study aimed to identify the degree of concordance between fluorescein angiograms (FA) and fundus photographs (FP) in assessing the severity and potential need for treatment in infants 45 weeks or older postmenstrual age (PMA) with type 2 or less retinopathy of prematurity (ROP). PATIENTS AND METHODS: An observational retrospective case series performed at Associated Retinal Consultants, William Beaumont Hospital in Royal Oak, Michigan. All infants born between 2006 and 2016 with stage 1 to 3 ROP that did not meet type 1 ROP criteria (type 2 or less) who received ablative laser therapy during or after age 45 weeks PMA. Pretreatment FP and FA images were randomized and sent to nine expert retina specialist graders to assess severity and inter-grader variability. RESULTS: A total of 10 babies (19 eyes) were enrolled in this study, and 53 FAs and 27 FPs of these 19 eyes were selected to be interpreted by the nine graders. The number of eyes deemed to be abnormal and warranted for treatment was higher with FA, whereas more eyes were deemed "normal" with FP. CONCLUSION: Although still controversial, knowledge of these findings may encourage retina specialists to closely examine infants with mild ROP older than age 45 weeks PMA and consider ablative laser therapy under certain conditions (even if not meeting type 1 Early Treatment for ROP criteria). [Ophthalmic Surg Lasers Imaging Retina. 2021;52:636-641.].
Assuntos
Retinopatia da Prematuridade , Pré-Escolar , Angiofluoresceinografia , Idade Gestacional , Humanos , Lactente , Recém-Nascido , Retina , Retinopatia da Prematuridade/diagnóstico , Retinopatia da Prematuridade/cirurgia , Estudos RetrospectivosRESUMO
The CDK4/6 inhibitor, palbociclib (PAL), significantly improves progression-free survival in HR+/HER2- breast cancer when combined with anti-hormonals. We sought to discover PAL resistance mechanisms in preclinical models and through analysis of clinical transcriptome specimens, which coalesced on induction of MYC oncogene and Cyclin E/CDK2 activity. We propose that targeting the G1 kinases CDK2, CDK4, and CDK6 with a small-molecule overcomes resistance to CDK4/6 inhibition. We describe the pharmacodynamics and efficacy of PF-06873600 (PF3600), a pyridopyrimidine with potent inhibition of CDK2/4/6 activity and efficacy in multiple in vivo tumor models. Together with the clinical analysis, MYC activity predicts (PF3600) efficacy across multiple cell lineages. Finally, we find that CDK2/4/6 inhibition does not compromise tumor-specific immune checkpoint blockade responses in syngeneic models. We anticipate that (PF3600), currently in phase 1 clinical trials, offers a therapeutic option to cancer patients in whom CDK4/6 inhibition is insufficient to alter disease progression.
Assuntos
Ciclo Celular/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Feminino , Humanos , Masculino , Neoplasias/imunologiaRESUMO
Alternating electric field therapy has been approved for glioblastoma (GBM). We have preclinical evidence for anticancer effects in GBM cell cultures and mouse xenografts with an oscillating magnetic field (OMF) generating device. Here we report OMF treatment of end-stage recurrent glioblastoma in a 53-year-old man who had undergone radical surgical excision and chemoradiotherapy, and experimental gene therapy for a left frontal tumor. He experienced tumor recurrence and progressive enlargement with leptomeningeal involvement. OMF for 5 weeks was well tolerated, with 31% reduction of contrast-enhanced tumor volume and reduction in abnormal T2-weighted Fluid-Attenuated Inversion Recovery volume. Tumor shrinkage appeared to correlate with treatment dose. These findings suggest a powerful new noninvasive therapy for glioblastoma.
RESUMO
Control of the cell cycle through selective pharmacological inhibition of CDK4/6 has proven beneficial in the treatment of breast cancer. Extending this level of control to additional cell cycle CDK isoforms represents an opportunity to expand to additional tumor types and potentially provide benefits to patients that develop tumors resistant to selective CDK4/6 inhibitors. However, broad-spectrum CDK inhibitors have a long history of failure due to safety concerns. In this approach, we describe the use of structure-based drug design and Free-Wilson analysis to optimize a series of CDK2/4/6 inhibitors. Further, we detail the use of molecular dynamics simulations to provide insights into the basis for selectivity against CDK9. Based on overall potency, selectivity, and ADME profile, PF-06873600 (22) was identified as a candidate for the treatment of cancer and advanced to phase 1 clinical trials.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Cães , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Injeções Intravenosas , Camundongos , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Metabolism is a key regulator of hematopoietic stem cell (HSC) functions. There is a lack of real-time, non-invasive approaches to evaluate metabolism in single HSCs. Using fluorescence lifetime imaging microscopy, we developed a set of metabolic optical biomarkers (MOBs) from the auto-fluorescent properties of metabolic coenzymes NAD(P)H and FAD. The MOBs revealed the enhanced glycolysis, low oxidative metabolism, and distinct mitochondrial localization of HSCs. Importantly, the fluorescence lifetime of enzyme-bound NAD(P)H (τbound) can non-invasively monitor the glycolytic/lactate dehydrogenase activity in single HSCs. As a proof of concept for metabolism-based cell sorting, we further identified HSCs within the Lineage-cKit+Sca1+ (KLS) hematopoietic stem/progenitor population using MOBs and a machine-learning algorithm. Moreover, we revealed the dynamic changes of MOBs, and the association of longer τbound with enhanced glycolysis under HSC stemness-maintaining conditions during HSC culture. Our work thus provides a new paradigm to identify and track the metabolism of single HSCs non-invasively and in real time.
RESUMO
Human urine is a non-invasive source of renal stem cells with regeneration potential. Urine-derived renal progenitor cells were isolated from 10 individuals of both genders and distinct ages. These renal progenitors express pluripotency-associated proteins- TRA-1-60, TRA-1-81, SSEA4, C-KIT and CD133, as well as the renal stem cell markers -SIX2, CITED1, WT1, CD24 and CD106. The transcriptomes of all SIX2+ renal progenitors clustered together, and distinct from the human kidney biopsy-derived epithelial proximal cells (hREPCs). Stimulation of the urine-derived renal progenitor cells (UdRPCs) with the GSK3ß-inhibitor (CHIR99021) induced differentiation. Transcriptome and KEGG pathway analysis revealed upregulation of WNT-associated genes- AXIN2, JUN and NKD1. Protein interaction network identified JUN- a downstream target of the WNT pathway in association with STAT3, ATF2 and MAPK1 as a putative negative regulator of self-renewal. Furthermore, like pluripotent stem cells, self-renewal is maintained by FGF2-driven TGFß-SMAD2/3 pathway. The urine-derived renal progenitor cells and the data presented should lay the foundation for studying nephrogenesis in human.
Assuntos
Autorrenovação Celular/genética , Autorrenovação Celular/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Rim/citologia , Células-Tronco Pluripotentes/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Urina/citologia , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , Diferenciação Celular/genética , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Humanos , Masculino , Células-Tronco Pluripotentes/metabolismo , Transcriptoma , Fator de Crescimento Transformador beta/metabolismoRESUMO
Single cell analysis methods are increasingly being utilized to investigate how individual cells process information and respond to diverse stimuli. Soluble proteins play a critical role in controlling cell populations and tissues, but directly monitoring secretion is technically challenging. Microfabricated well arrays have been developed to assess secretion at the single cell level, but these systems are limited by low detection sensitivity. Semiconductor quantum dots (QD) exhibit remarkably bright and photostable luminescence signal, but to date they have not been evaluated in single cell secretion studies using microfabricated well arrays. Here, we used QDs in a sandwich immunoassay to detect secretion of the soluble cytokine tumor necrosis factor-α (TNF-α) from single cells. To enhance detection sensitivity, we employed two different strategies. First, we used a unique single QD imaging approach, which provided a detection threshold (180 attomolar) that was >100-fold lower than previously reported results using QDs. We also amplified QD binding to each captured TNF-α molecule using the bioorthogonal cycloaddition reaction between trans-cyclooctene and tetrazine, which further lowered detection threshold to 60 attomolar. This is 6 orders of magnitude more sensitive than organic fluorophores that have been used for single cell secretion studies, and far surpasses single molecule resolution within sub-picoliter microwells that are used to assess single cell secretion. Finally, single cell secretion studies were performed using phorbol 12-myristate 13-acetate (PMA) differentiated and lipopolysaccharide (LPS) activated U-937 cells. TNF-α secretion was detected from 3-fold more single cells using the QD-based method in comparison to rhodamine, which was accomplished by extending sensitivity into the range of â¼2 to 10 000 molecules captured per microwell. In future work, we will apply this technique to assess immune cell secretion dynamics under diverse stimuli and disease settings. We will also incorporate multiplexing capabilities to evaluate the secretome at the resolution of single molecules.
Assuntos
Imunoensaio/métodos , Pontos Quânticos , Análise de Célula Única/métodos , Fator de Necrose Tumoral alfa/análise , Humanos , Limite de Detecção , Células U937RESUMO
In most organ systems, regeneration is a coordinated effort that involves many stem cells, but little is known about whether and how individual stem cells compensate for the differentiation deficiencies of other stem cells. Functional compensation is critically important during disease progression and treatment. Here, we show how individual hematopoietic stem cell (HSC) clones heterogeneously compensate for the lymphopoietic deficiencies of other HSCs in a mouse. This compensation rescues the overall blood supply and influences blood cell types outside of the deficient lineages in distinct patterns. We find that highly differentiating HSC clones expand their cell numbers at specific differentiation stages to compensate for the deficiencies of other HSCs. Some of these clones continue to expand after transplantation into secondary recipients. In addition, lymphopoietic compensation involves gene expression changes in HSCs that are characterized by increased lymphoid priming, decreased myeloid priming, and HSC self-renewal. Our data illustrate how HSC clones coordinate to maintain the overall blood supply. Exploiting the innate compensation capacity of stem cell networks may improve the prognosis and treatment of many diseases.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Contagem de Células , Diferenciação Celular , Proliferação de Células , Células Clonais , Regulação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Linfopoese , Camundongos Endogâmicos C57BL , Mutação/genéticaRESUMO
A new series of lactam-derived EZH2 inhibitors was designed via ligand-based and physicochemical-property-based strategies to address metabolic stability and thermodynamic solubility issues associated with previous lead compound 1. The new inhibitors incorporated an sp3 hybridized carbon atom at the 7-position of the lactam moiety present in lead compound 1 as a replacement for a dimethylisoxazole group. This transformation enabled optimization of the physicochemical properties and potency compared to compound 1. Analysis of relationships between calculated log D (clogD) values and in vitro metabolic stability and permeability parameters identified a clogD range that afforded an increased probability of achieving favorable ADME data in a single molecule. Compound 23a exhibited the best overlap of potency and pharmaceutical properties as well as robust tumor growth inhibition in vivo and was therefore advanced as a development candidate (PF-06821497). A crystal structure of 23a in complex with the three-protein PRC2 complex enabled understanding of the key structural features required for optimal binding.
Assuntos
Desenho de Fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Isoquinolinas/farmacologia , Isoquinolinas/farmacocinética , Administração Oral , Disponibilidade Biológica , Linhagem Celular Tumoral , Humanos , Isoquinolinas/administração & dosagem , Isoquinolinas/química , Modelos Moleculares , Conformação MolecularRESUMO
A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound 18. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound 31. Compound 31 displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound 18. Inhibitor 31 also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Desenho de Fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Piridonas/química , Piridonas/farmacologia , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ciclização , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Humanos , Isoquinolinas/química , Isoquinolinas/farmacologia , Isoquinolinas/uso terapêutico , Lactamas/química , Lactamas/farmacologia , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Piridonas/uso terapêuticoRESUMO
The cinobufagin (CB) has a broad spectrum of cytotoxicity to inhibit cell proliferation of various human cancer cell lines, but the molecular mechanisms still remain elusive. Here we observed that CB inhibited the cell proliferation and tumor growth, but induced cell cycle arrest and apoptosis in a dose-dependent manner in non-small cell lung cancer (NSCLC) cells. Treatment with CB significantly increased the reactive oxygen species but decreased the mitochondrial membrane potential in NSCLC cells. These effects were markedly blocked when the cells were pretreated with N-acetylcysteine, a specific reactive oxygen species inhibitor. Furthermore, treatment with CB induced the expression of BAX but reduced that of BCL-2, BCL-XL and MCL-1, leading to an activation of caspase-3, chromatin condensation and DNA degradation in order to induce programmed cell death in NSCLC cells. In addition, treatment with CB reduced the expressions of p-AKTT308 and p-AKTS473 and inhibited the AKT/mTOR signaling pathway in NSCLC cells in a time-dependent manner. Our results suggest that CB inhibits tumor growth by inducing intrinsic apoptosis through the AKT signaling pathway in NSCLC cells.
Assuntos
Antineoplásicos/farmacologia , Bufanolídeos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: In prostate cancer cells, there is CD24-dependent inactivation of mutant p53, but the mechanism and its significance remain largely unknown. Here, we validated this observation and explored the therapeutic potential of targeting CD24 in TP53 mutant prostate cancer cells. EXPERIMENTAL DESIGN: Overall, 553 prostate cancers (522 formalin-fixed paraffin-embedded and 31 frozen tissues) were assessed for protein or mRNA expression of CD24 and TP53 The effects of CD24 on p53-dependent transcriptional regulation, cancer cell growth, the cell cycle, apoptosis, and mutant p53 restoration were also determined. RESULTS: As determined with three sample cohorts, CD24 and p53 were not expressed in prostate epithelial cells but in prostate cancer cells in 48% of cases for CD24 and 16% of cases for p53 (mutant form). Expressions of CD24 and mutant p53 were more frequently observed in late-stage and metastatic prostate tumors. Mutant p53 accompanied with CD24 was expressed in most cases (91.6%, 76/83). Silencing of CD24 increased the transcriptional activity of p53 target genes, such as CDKNA1, VDR, and TP53INP1, leading to suppression of p53-dependent cell growth, cell-cycle arrest, and apoptosis in most TP53-mutant prostate cancer cells. Silencing of CD24 enhanced restoration of PRIMA-1-induced mutant p53 in endogenous TP53(P223L/V274F) DU145 cells and in PC3 cells transfected with TP53(R273H) CONCLUSIONS: In human prostate cancers, there is CD24-dependent inactivation of mutant p53. The coexpression of CD24 and p53 may help identify aggressive cancers. Targeting CD24 provides a strategy to enhance mutant p53-restoring therapies, especially in patients with TP53(R273H) prostate cancer. Clin Cancer Res; 22(10); 2545-54. ©2015 AACR.
Assuntos
Antígeno CD24/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Próstata/metabolismo , Neoplasias da Próstata/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD) is thought to be a genetic disease caused by de novo mutations, though causative mutations have yet to be identified. We searched for de novo coding mutations among a carefully-diagnosed and clinically homogeneous cohort of 35 ROHHAD patients. METHODS: We sequenced the exomes of seven ROHHAD trios, plus tumours from four of these patients and the unaffected monozygotic (MZ) twin of one (discovery cohort), to identify constitutional and somatic de novo sequence variants. We further analyzed this exome data to search for candidate genes under autosomal dominant and recessive models, and to identify structural variations. Candidate genes were tested by exome or Sanger sequencing in a replication cohort of 28 ROHHAD singletons. RESULTS: The analysis of the trio-based exomes found 13 de novo variants. However, no two patients had de novo variants in the same gene, and additional patient exomes and mutation analysis in the replication cohort did not provide strong genetic evidence to implicate any of these sequence variants in ROHHAD. Somatic comparisons revealed no coding differences between any blood and tumour samples, or between the two discordant MZ twins. Neither autosomal dominant nor recessive analysis yielded candidate genes for ROHHAD, and we did not identify any potentially causative structural variations. CONCLUSIONS: Clinical exome sequencing is highly unlikely to be a useful diagnostic test in patients with true ROHHAD. As ROHHAD has a high risk for fatality if not properly managed, it remains imperative to expand the search for non-exomic genetic risk factors, as well as to investigate other possible mechanisms of disease. In so doing, we will be able to confirm objectively the ROHHAD diagnosis and to contribute to our understanding of obesity, respiratory control, hypothalamic function, and autonomic regulation.