RESUMO
During in vitro culture, human pluripotent stem cells (hPSCs) often acquire survival advantages characterized by decreased susceptibility to mitochondrial cell death, known as "culture adaptation." This adaptation is associated with genetic and epigenetic abnormalities, including TP53 mutations, copy number variations, trisomy, and methylation changes. Understanding the molecular mechanisms underlying this acquired survival advantage is crucial for safe hPSC-based cell therapies. Through transcriptome and methylome analysis, we discovered that the epigenetic repression of CHCHD2, a mitochondrial protein, is a common occurrence during in vitro culture using enzymatic dissociation. We confirmed this finding through genetic perturbation and reconstitution experiments in normal human embryonic stem cells (hESCs). Loss of CHCHD2 expression conferred resistance to single cell dissociation-induced cell death, a common stress encountered during in vitro culture. Importantly, we found that the downregulation of CHCHD2 significantly attenuates the activity of Rho-associated protein kinase (ROCK), which is responsible for inducing single cell death in hESCs. This suggests that hESCs may survive routine enzyme-based cell dissociation by downregulating CHCHD2 and thereby attenuating ROCK activity. These findings provide insights into the mechanisms by which hPSCs acquire survival advantages and adapt to in vitro culture conditions.
Assuntos
Células-Tronco Embrionárias Humanas , Células-Tronco Pluripotentes , Humanos , Linhagem Celular , Repressão Epigenética , Variações do Número de Cópias de DNA , Células-Tronco Embrionárias Humanas/metabolismo , Diferenciação Celular , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Naïve and primed pluripotent stem cells recapitulate the peri- and post-implantation development, respectively. Thus, investigation of distinct traits between each pluripotent stem cell type would shed light on early embryonic processes. Herein, by screening a fluorescent probe library, we found that intracellular glycogen led to specific reactivity to CDg4, a glycogen fluorescence sensor, in both human and mouse naïve embryonic stem cells (ESCs). The requirement of constant inhibition of Gsk3ß as well as high oxidative phosphorylation (OxPHOS) in naïve compared to primed ESCs was closely associated to high level of intracellular glycogen in naïve ESCs. Both capacity of OxPHOS and stored glycogen, rescued naïve ESCs by transient inhibition of glycolysis, which selectively eliminated primed ESCs. Additionally, naïve ESCs with active OxPHOS were enriched from a mixture with primed ESCs by high reactivity to ATP-Red1, a mitochondrial ATP fluorescence probe. These results indicate the active OxPHOS and high intracellular glycogen as a novel "biomarker" delineating metabolic remodeling during the transition of naïve pluripotency.
Assuntos
Glicogênio , Células-Tronco Pluripotentes , Trifosfato de Adenosina/metabolismo , Animais , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Glucose/metabolismo , Glicogênio/metabolismo , Camundongos , Células-Tronco Pluripotentes/metabolismoRESUMO
BACKGROUND: The existence of differentiated thyroid cells is critical to respond radioactive iodide treatment strategy in thyroid cancer, and loss of the differentiated phenotype is a trademark of iodide-refractive thyroid disease. While high-dose therapy has been beneficial to several cancer patients, many studies have indicated this clinical benefit was limited to patients having BRAF mutation. BRAF-targeted paired box gene-8 (PAX8), a thyroid-specific transcription factor, generally dysregulated in BRAF-mutated thyroid cancer. METHODS: In this study, thyroid iodine-metabolizing gene levels were detected in BRAF-transformed thyroid cells after low and high dose of ionizing radiation. Also, an mRNA-targeted approach was used to figure out the underlying mechanism of low (0.01Gyx10 or 0.1Gy) and high (2Gy) radiation function on thyroid cancer cells after BRAFV600E mutation. RESULTS: Low dose radiation (LDR)-induced PAX8 upregulation restores not only BRAF-suppressive sodium/iodide symporter (NIS) expression, one of the major protein necessary for iodine uptake in healthy thyroid, on plasma membrane but also regulate other thyroid metabolizing genes levels. Importantly, LDR-induced PAX8 results in decreased cellular transformation in BRAF-mutated thyroid cells. CONCLUSION: The present findings provide evidence that LDR-induced PAX8 acts as an important regulator for suppression of thyroid carcinogenesis through novel STAT3/miR-330-5p pathway in thyroid cancers.
Assuntos
Transformação Celular Neoplásica/patologia , Transformação Celular Neoplásica/efeitos da radiação , Proteínas Proto-Oncogênicas B-raf/metabolismo , Glândula Tireoide/patologia , Glândula Tireoide/efeitos da radiação , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Hipotireoidismo/patologia , Iodo/metabolismo , Camundongos Mutantes , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Mutação/genética , Fator de Transcrição PAX8/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Fator de Transcrição STAT3/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/radioterapia , Regulação para Cima/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Since the molecular mechanism of the cell cycle was established, various theoretical models of this process have been developed. A recent study revealed significant variability in cell cycle duration between mother and daughter cells, but this observation has not been incorporated into the theoretical models. METHODS: We used fluorescent ubiquitination-based cell cycle indicator (FUCCI) systems and live-monitored the heterogeneity of cell cycle progression within daughter cells, which accounts for dephasing synchrony. To incorporate the variable cell cycle durations into a model, we modified a two-ordinary differential equation (ODE) model based on reciprocal activation between CDK1 and APC. RESULTS: Our model reproduced the experimental population profile, in which cell cycle synchrony dephased due to variability. Based on this model, we determined parameters for CDK1 and APC in the cell cycle profile after treatment with antimitotic drugs and associated the parameters with the drugs' mode of action as cell cycle inhibitors. CONCLUSION: This suggests that this model is useful for determining the mode of action of unknown small molecules on the cell cycle.
Assuntos
Antimitóticos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Mitose/efeitos dos fármacos , Modelos Biológicos , Neoplasias do Colo do Útero/tratamento farmacológico , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Técnicas Biossensoriais , Proteína Quinase CDC2/metabolismo , Simulação por Computador , Feminino , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Microscopia de Fluorescência , Análise Numérica Assistida por Computador , Processos Estocásticos , Fatores de Tempo , Imagem com Lapso de Tempo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Even when targets responsible for chemoresistance are identified, drug development is often hampered due to the poor druggability of these proteins. We systematically analyzed therapy-resistance with a large-scale cancer cell transcriptome and drug-response datasets and predicted the candidate drugs based on the gene expression profile. Our results implicated the epithelial-mesenchymal transition as a common mechanism underlying resistance to chemotherapeutic drugs. Notably, we identified ITGB3, whose expression was abundant in both drug resistance and mesenchymal status, as a promising target to overcome chemoresistance. We also confirmed that depletion of ITGB3 sensitized cancer cells to conventional chemotherapeutic drugs by modulating the NF-κB signaling pathway. Considering the poor druggability of ITGB3 and the lack of feasible drugs to directly inhibit this protein, we took an in silico screening for drugs mimicking the transcriptome-level changes caused by knockdown of ITGB3. This approach successfully identified atorvastatin as a novel candidate for drug repurposing, paving an alternative path to drug screening that is applicable to undruggable targets.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Integrina beta3/genética , Neoplasias Pulmonares/genética , Células A549 , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Humanos , NF-kappa B/genética , Farmacogenética/métodos , Transdução de Sinais/genéticaRESUMO
Basic fibroblast growth factor (bFGF) supplementation is critical to maintain the pluripotency of human pluripotent stem cells (hPSCs) through activation of PI3K/AKT, rather than MEK/ERK pathway. Thus, elaborate molecular mechanisms that preserve PI3K/AKT signaling upon bFGF stimulation may exist in hPSCs. Protein arginine methyltransferase 8 (PRMT8) was expressed and then its level gradually decreased during spontaneous differentiation of human embryonic stem cells (hESCs). PRMT8 loss- or gain-of-function studies demonstrated that PRMT8 contributed to longer maintenance of hESC pluripotency, even under bFGF-deprived conditions. Direct interaction of membrane-localized PRMT8 with p85, a regulatory subunit of PI3K, was associated with accumulation of phosphoinositol 3-phosphate and consequently high AKT activity. Furthermore, the SOX2 induction, which was controlled by the PRMT8/PI3K/AKT axis, was linked to mesodermal lineage differentiation. Thus, we propose that PRMT8 in hESCs plays an important role not only in maintaining pluripotency but also in controlling mesodermal differentiation through bFGF signaling toward the PI3K/AKT/SOX2 axis. Stem Cells 2017;35:2037-2049.
Assuntos
Linhagem da Célula , Células-Tronco Embrionárias Humanas/metabolismo , Proteínas de Membrana/metabolismo , Mesoderma/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Células-Tronco Pluripotentes/citologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Fenótipo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Despite the recent promising results of clinical trials using human pluripotent stem cell (hPSC)-based cell therapies for age-related macular degeneration (AMD), the risk of teratoma formation resulting from residual undifferentiated hPSCs remains a serious and critical hurdle for broader clinical implementation. To mitigate the tumorigenic risk of hPSC-based cell therapy, a variety of approaches have been examined to ablate the undifferentiated hPSCs based on the unique molecular properties of hPSCs. In the present review, we offer a brief overview of recent attempts at selective elimination of undifferentiated hPSCs to decrease the risk of teratoma formation in hPSC-based cell therapy.
Assuntos
Células-Tronco Pluripotentes/citologia , Transplante de Células-Tronco/métodos , Animais , Morte Celular/efeitos dos fármacos , Genes Transgênicos Suicidas , Humanos , MicroRNAs/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
BACKGROUND: Glioma stem cells (GSCs) are a major cause of the frequent relapse observed in glioma, due to their high drug resistance and their differentiation potential. Therefore, understanding the molecular mechanisms governing the 'cancer stemness' of GSCs will be particularly important for improving the prognosis of glioma patients. METHODS: We previously established cancerous neural stem cells (CNSCs) from immortalized human neural stem cells (F3 cells), using the H-Ras oncogene. In this study, we utilized the EGFRviii mutation, which frequently occurs in brain cancers, to establish another CNSC line (F3.EGFRviii), and characterized its stemness under spheroid culture. RESULTS: The F3.EGFRviii cell line was highly tumorigenic in vitro and showed high ERK1/2 activity as well as expression of a variety of genes associated with cancer stemness, such as SOX2 and NANOG, under spheroid culture conditions. Through meta-analysis, PCR super-array, and subsequent biochemical assays, the induction of MEK partner-1 (MP1, encoded by the LAMTOR3 gene) was shown to play an important role in maintaining ERK1/2 activity during the acquisition of cancer stemness under spheroid culture conditions. High expression of this gene was also closely associated with poor prognosis in brain cancer. CONCLUSION: These data suggest that MP1 contributes to cancer stemness in EGFRviii-expressing glioma cells by driving ERK activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores ErbB/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neurais/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , PrognósticoRESUMO
Mice null for wild-type p53-induced phosphatase 1 (WIP1) display defects in testis development and spermatogenesis, resulting in reduced fertility. However, the molecular mechanism underlying these abnormalities in the testis remains uncharacterized. We report that the phosphatase activity of WIP1 increases Wnt activity through Nemo-like kinase (NLK). WIP1 directly interacted with NLK, which is highly homologous to p38 MAPK, a WIP1 substrate, and dephosphorylated its activation site. The WIP1-mediated inhibition of NLK activity markedly decreased the phosphorylation of lymphoid enhancer-binding factor 1 (LEF1), enhancing its interaction with ß-catenin. Additionally, WIP1 depletion impaired germ cell development, as evidenced by the expression of Oct4 and the germ cell-specific markers Ddx4, Nanos3 and Dnd1 during the development of germ cells from Oct4-GFP transgenic (OG2) mouse embryonic stem cells (mESCs). The expression of WIP1, whose level was significantly lower after the differentiation of germ cells from mESCs, occurred in parallel with the expression of germ cell development markers and SRY-box 17 (Sox17), a downstream target of Wnt. These results indicate that WIP1 is essential for germ cell development, which is known to require Wnt activity.
Assuntos
Células Germinativas/citologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Proteína Fosfatase 2C/metabolismo , Proteínas Wnt/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Células HEK293 , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Fosforilação , Proteína Fosfatase 2C/genética , Proteínas Serina-Treonina Quinases , Testículo/citologia , Testículo/metabolismo , beta Catenina/metabolismoRESUMO
Tumorigenesis is a relatively rare event in the human body considering the enormous number of cells composing our body and the frequent occurrence of genetic mutations in each cell. Nevertheless, the cells that happen to meet the minimum requirements can be transformed when stressed by a variety of oncogenic stimulations, then progress to form tumors. The vigorous competition between oncogenic signaling and tumor-suppressor defense is a critical determinant of cellular fate, which can be either tumorigenic transformation or cellular senescence/apoptosis depending on "who wins the battle." Recently, a number of cancers have been reported to originate from stem cells, whose self-renewing properties are normally reduced by innate tumor suppressors. Therefore, exploring the innate mechanism by which stem cells modulate tumor suppressors to maintain their "stemness" may provide valuable clues to characterize the distinctive oncogenic susceptibility of stem cells. This review is focused on the recent advances in the field of tumorigenesis of stem cells and on the associated molecular mechanisms.
Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Estresse Fisiológico , Animais , HumanosRESUMO
The future of safe cell-based therapy rests on overcoming teratoma/tumor formation, in particular when using human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation, complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile, we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e., survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors, leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular, a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g., quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast, differentiated cell types (e.g., dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together, these results provide the "proof of concept" that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy.
Assuntos
Células-Tronco Pluripotentes/citologia , Bibliotecas de Moléculas Pequenas , Teratoma/patologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Apoptose , Proteína 10 de Linfoma CCL de Células B , Diferenciação Celular , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Mitocôndrias/metabolismo , Naftoquinonas/farmacologia , Células-Tronco Pluripotentes/metabolismo , Transplante de Células-Tronco , Survivina , Teratoma/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Wip1 belongs to the protein phosphatase C (PP2C) family, of which expression is up-regulated by a number of external stresses, and serves as a stress modulator in normal physiological conditions. When overexpressed, premature dephosphorylation of stress-mediators by Wip1 results in abrogation of tumor surveillance, thus Wip1 acts as an oncogene. Previously, the functional regulation of Wip1 in cell-cycle progression by counteracting cellular G1 and G2/M checkpoint activity in response to DNA damage was reported. However, other than in stress conditions, the function and regulatory mechanism of Wip1 has not been fully determined. Herein, we demonstrated that protein regulation of Wip1 occurs in a cell cycle-dependent manner, which is directly governed by APC/C(Cdh1) at the end of mitosis. In particular, we also showed evidence that Wip1 phosphatase activity is closely associated with its own protein stability, suggesting that reduced phosphatase activity of Wip1 during mitosis could trigger its degradation. Furthermore, to verify the physiological role of its phosphatase activity during mitosis, we established doxycycline-inducible cell models, including a Wip1 wild type (WT) and phosphatase dead mutant (Wip1 DA). When ectopically expressing Wip1 WT, we observed a delay in the transition from metaphase to anaphase. In conclusion, these studies show that mitotic degradation of Wip1 by APC/C(Cdh1) is important for normal mitotic progression.
Assuntos
Caderinas/metabolismo , Mitose , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Antígenos CD , Doxiciclina/farmacologia , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Células MCF-7 , Mutação , Proteína Fosfatase 2C , ProteóliseRESUMO
Although loss of Sirt1 leads to chromosome aneuploidy, which accounts for higher tumor susceptibility, the molecular mechanisms remain unclear. Herein, we demonstrate that Sirt1 directly regulates Plk1, of which activity is critical for mitotic progression and spindle dynamics. Depletion or inhibition of Sirt1 significantly perturbs the formation of the mitotic spindle, leading to defective chromosome segregation. Elevated depolymerization of the mitotic spindle following loss of Sirt1 was associated with the deregulation of Plk1 activity. Thus, we conclude that Sirt1 may contribute to a mitotic regulator that controls spindle dynamics through Plk1 activity, resulting in fine-tuning of Plk1 dependent microtubule dynamics.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sirtuína 1/metabolismo , Fuso Acromático/metabolismo , Segregação de Cromossomos , Células HEK293 , Células HeLa , Humanos , Fosforilação , Sirtuína 1/genética , Quinase 1 Polo-LikeRESUMO
Genetic instability is intimately associated with tumour development. In particular, liver cancers associated with hepatitis B virus (HBV) exhibit high genetic instability; however, our understanding of the underlying molecular mechanisms remains limited. In this study, we found that γ-H2AX, a marker of DNA double-strand breaks (DSBs), and the levels of phospho-Chk2 (p-Chk2, the activated form) were significantly elevated in HBV-associated hepatocellular carcinomas and neighbouring regenerating nodules. Likewise, introduction of the pHBV or pMyc-HBx plasmids into cells induced accumulation of γ-H2AX foci and increased the p-Chk2 level. In these cells, inhibitory phosphorylation of Cdc25C phosphatase (Ser(216)) and CDK1 (Tyr(15)) was elevated; consequently, cell-cycle progression was delayed at G2/M phase, suggesting that activation of the ATM-Chk2 pathway by the HBV X protein (HBx) induces cell-cycle delay. Accordingly, inhibition of ataxia telangiectasia mutated (ATM) by caffeine or siRNA abolished the increase in the p-Chk2 level and restored the delayed CDK1 kinase activity in ChangX cells. We also found that cytoplasmic HBx, but not nuclear HBx, induced reactive oxygen species (ROS) production and led to the accumulation of γ-H2AX foci and the increased p-Chk2 level. Together, these data indicate that HBx-induced ROS accumulation induces DNA damage that activates the ATM-Chk2 pathway. Our findings provide insight into the mechanisms of HBV pathogenesis.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ciclo Celular , Quinase do Ponto de Checagem 2/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B/enzimologia , Transativadores/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2/genética , Dano ao DNA , Feminino , Hepatite B/genética , Hepatite B/fisiopatologia , Hepatite B/virologia , Vírus da Hepatite B/genética , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Transativadores/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
We aimed to identify a novel flavonoid from the in-house natural products to suppress matrix metalloproteases (MMPs), which is responsible for degradation of collagen and other extracellular matrix proteins. Total eight natural products were screened for identification of a novel MMP-9 suppressor using MMP-9 reporter system, where the prompt initial screening with multiple samples is readily examined. Among the extracts used in the present study, one extract (Citrus unshiu) was found active in this assay system. Furthermore, three representative flavonoids in this active extract of Citrus unshiu peel were tested in MMP-9 reporter system. Nobiletin (NB) of the tested flavonoids suppressed MMP-9 expression without cytotoxicity, which was validated by both real-time polymerase chain reaction (PCR) and zymography analyses. Sustained p38 mitogen activated protein kinase (MAPK) activity, closely associated with induction of MMP-9 under stress condition, was markedly reduced by NB treatment, which implies that modulation of p38MAPK by nobiletin is responsible for reduction of MMP9 expression. Hence, nobiletin, identified from MMP-9 reporter system based screening, may be further applied for the purpose of delaying collagen degradation in skin fibroblasts.
Assuntos
Citrus/química , Fibroblastos/efeitos dos fármacos , Flavonas/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Extratos Vegetais/farmacologia , Pele/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Fibroblastos/metabolismo , Humanos , Fosforilação , Pele/citologia , Pele/metabolismoRESUMO
The sequential change from totipotency to multipotency occurs during early mammalian embryo development. However, due to the lack of cellular models to recapitulate the distinct potency of stem cells at each stage, their molecular and cellular characteristics remain ambiguous. The establishment of isogenic naïve and primed pluripotent stem cells to represent the pluripotency in the inner cell mass of the pre-implantation blastocyst and in the epiblast from the post-implantation embryo allows the understanding of the distinctive characteristics of two different states of pluripotent stem cells. This review discusses the prominent disparities between naïve and primed pluripotency, including signaling pathways, metabolism, and epigenetic status, ultimately facilitating a comprehensive understanding of their significance during early mammalian embryonic development.
RESUMO
Epidermal growth factor receptor inhibitors (EGFRi) have exhibited promising clinical outcomes in the treatment of various cancers. However, their widespread application has been limited by low patient eligibility and the emergence of resistance. Leveraging a multi-omics approach (>1000 cancer cell lines), we explored molecular signatures linked to EGFRi responsiveness and found that expression signatures involved in the estrogen response could recapitulate cancer cell dependency on EGFR, a phenomenon not solely attributable to EGFR-activating mutations. By correlating genome-wide function screening data with EGFRi responses, we identified chemokine receptor 6 (CCR6) as a potential druggable target to mitigate EGFRi resistance. In isogenic cell models, pharmacological inhibition of CCR6 effectively reversed acquired EGFRi resistance, disrupting mitochondrial oxidative phosphorylation, a cellular process commonly associated with therapy resistance. Our data-driven strategy unveils drug-response biomarkers and therapeutic targets for resistance, thus potentially expanding EGFRi applicability and efficacy.
RESUMO
Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of p53DD, a dominant negative p53, and three UNG inhibitor, engineered to specifically diminish base excision repair, improves cytosine base editor efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional prime editor system also significantly enhances prime editor efficiency in hPSCs. Thus, combined inhibition of the distinct cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.
Assuntos
Sistemas CRISPR-Cas , Dano ao DNA , Reparo do DNA , Edição de Genes , Proteína Supressora de Tumor p53 , Edição de Genes/métodos , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Linhagem Celular , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Citosina/metabolismoRESUMO
Sorafenib (BAY 43-9006) was developed as a multi-kinase inhibitor to treat advanced renal cell, hepatocellular, and thyroid cancers. The cytotoxic effect of sorafenib on cancer cells results from not only inhibiting the MEK/ERK signaling pathway (the on-target effect) but also inducing oxidative damage (the off-target effect). The inhibitory effect of sorafenib on system Xc- (xCT), a cystine/glutamate antiporter, promotes ferroptosis induction and accounts for oxidative damage. While emerging studies on ferroptosis in cancers have garnered increasing attention, the lack of consideration for ferroptosis inducers (FINs) with favorable pharmacokinetics could be problematic. Herein, we remodeled the chemical structure of sorafenib, of which pharmacokinetics and safety are already assured, to customize the off-target effect (i.e., ferroptosis induction) to on-target by disrupting the adenine-binding motif. JB3, a sorafenib derivative (i.e., JB compounds), with a tenfold higher IC50 toward RAF1 because of chemical remodeling, induced strong cytotoxicity in the elastin-sensitive lung cancer cells, while it was markedly reduced by ferrostatin-1. The 24% oral bioavailability of JB3 in rats accounted for a significant anti-tumor effect of orally administrated JB3 in xenograft models. These results indicate that JB3 could be further developed as an orally bioavailable FIN in novel anti-cancer therapeutics.
Assuntos
Antineoplásicos , Ferroptose , Neoplasias Pulmonares , Sorafenibe , Sorafenibe/farmacologia , Sorafenibe/administração & dosagem , Ferroptose/efeitos dos fármacos , Humanos , Animais , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Administração Oral , Antineoplásicos/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Linhagem Celular Tumoral , Camundongos , Ratos , Ensaios Antitumorais Modelo de Xenoenxerto , Masculino , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Camundongos NusRESUMO
Genomic instability has been considered to be one of the prominent factors for carcinogenesis and the development of a number of degenerative disorders, predominantly related to the aging. The cellular machineries involved in the maintenance of genomic integrity such as DNA repair and DNA damage responses are extensively characterized by a large number of studies. The failure of proper actions of such cellular machineries may lead to the devastating effects mostly inducing cancer or premature aging, even with no acute exogenous DNA damage stimuli. In this review, we especially focus on the pathophysiological aspects of the defective DNA damage responses in carcinogenesis and premature aging. Clear understanding the causes of carcinogenesis and age-related degenerative diseases will provide novel and efficient approaches for prevention and rational treatment of cancer and premature aging.