RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Sea buckthorn (Hippophae rhamnoides), a traditional Tibetan medicinal herb, exhibits protective effects against cardiovascular and respiratory diseases. Although Sea buckthorn extract (SBE) has been confirmed to alleviate airway inflammation in mice, its therapeutic effect and underlying mechanism on chronic obstructive pulmonary disease (COPD) requires further clarification. AIM OF THE STUDY: To elucidate the alleviative effect and molecular mechanism of SBE on lipopolysaccharides (LPS)/porcine pancreatic elastase (PPE)-induced COPD by blocking ferroptosis. METHODS: The anti-ferroptotic effects of SBE were evaluated in human BEAS-2B bronchial epithelial cells using CCK8, RT-qPCR, western blotting, and transmission electron microscopy. Transwell was employed to detect chemotaxis of neutrophils. COPD model was induced by intranasally administration of LPS/PPE in mice and measured by alterations of histopathology, inflammation, and ferroptosis. RNA-sequencing, western blotting, antioxidant examination, flow cytometry, DARTS, CETSA, and molecular docking were then used to investigate its anti-ferroptotic mechanisms. RESULTS: In vitro, SBE not only suppressed erastin- or RSL3-induced ferroptosis by suppressing lipid peroxides (LPOs) production and glutathione (GSH) depletion, but also suppressed ferroptosis-induced chemotactic migration of neutrophils via reducing mRNA expression of chemokines. In vivo, SBE ameliorated LPS/PPE-induced COPD phenotypes, and inhibited the generation of LPOs, cytokines, and chemokines. RNA-sequencing showed that p53 pathway and mitogen-activated protein kinases (MAPK) pathway were implicated in SBE-mediated anti-ferroptotic action. SBE repressed erastin- or LPS/PPE-induced overactivation of p53 and MAPK pathway, thereby decreasing expression of diamine acetyltransferase 1 (SAT1) and arachidonate 15-lipoxygenase (ALOX15), and increasing expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11). Mechanistically, erastin-induced elevation of reactive oxygen species (ROS) was reduced by SBE through directly scavenging free radicals, thereby contributing to its inhibition of p53 and MAPK pathways. CETSA, DARTS, and molecular docking further showed that ROS-generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) may be the target of SBE. Overexpression of NOX4 partially impaired the anti-ferroptotic activity of SBE. CONCLUSION: Our results demonstrated that SBE mitigated COPD by suppressing p53 and MAPK pro-ferroptosis pathways via directly scavenging ROS and blocking NOX4. These findings also supported the clinical application of Sea buckthorn in COPD therapy.
Assuntos
Ferroptose , Hippophae , Extratos Vegetais , Doença Pulmonar Obstrutiva Crônica , Espécies Reativas de Oxigênio , Proteína Supressora de Tumor p53 , Ferroptose/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Animais , Humanos , Espécies Reativas de Oxigênio/metabolismo , Hippophae/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Proteína Supressora de Tumor p53/metabolismo , Camundongos , Masculino , Camundongos Endogâmicos C57BL , Linhagem Celular , Lipopolissacarídeos/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Animais de Doenças , Simulação de Acoplamento MolecularRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Tubeimoside-I (TBM) promotes various cancer cell death by increasing the reactive oxygen species (ROS) production. However, the specific molecular mechanisms of TBM and its impact on oxaliplatin-mediated anti-CRC activity are not yet fully understood. AIM OF THE STUDY: To elucidate the therapeutic effect and underlying molecular mechanism of TBM on oxaliplatin-mediated anti-CRC activity. MATERIALS AND METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, wound healing assays and flow cytometry were conducted to investigate the changes in cell phenotypes and ROS generation. Real-time quantitative PCR (qRT-PCR) and western blotting were performed to detect the expressions of related mRNA and proteins. Finally, mouse xenograft models demonstrated that synergistic anti-tumor effects of combined treatment with TBM and oxaliplatin. RESULTS: The synergistic enhancement of the anti-tumor effects of oxaliplatin in colon cancer cells by TBM involved in the regulation of ROS-mediated endoplasmic reticulum (ER) stress, C-jun-amino-terminal kinase (JNK), and p38 MAPK signaling pathways. Mechanistically, TBM increased ROS generation in colon cancer cells by inhibiting heat shock protein 60 (HSPD1) expression. Knocking down HSPD1 increased TBM-induced antitumor activity and ROS generation in colon cancer cells. The mouse xenograft tumor models further validated that the combination therapy exhibited stronger anti-tumor effects than monotherapy alone. CONCLUSIONS: Combined therapy with TBM and oxaliplatin might be an effective therapeutic strategy for some CRC patients.
Assuntos
Neoplasias Colorretais , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático , Oxaliplatina , Espécies Reativas de Oxigênio , Saponinas , Triterpenos , Animais , Humanos , Masculino , Camundongos , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HCT116 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oxaliplatina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mitogen-activated protein kinases, a family of three stress-related kinases, the Erks and Jnks and p38s, are activated by three-layer transphosphorylation cascades and are important for the activation, differentiation, and effector functions of lymphocytes. Recent studies on the aged immune systems from both humans and mice have uncovered a different mode of MAPK signaling that is independent of canonical activation cascades and instead occurs through simultaneous self-phosphorylation reactions within the sestrin-MAPK activation complex (sMAC), an immune-inhibitory complex not previously observed. In this chapter, we discuss methodologies to study these pathways at the population and single cell level, which allows rejuvenating immune cell differentiation and fate.
Assuntos
Senescência Celular , Linfócitos T , Humanos , Linfócitos T/metabolismo , Linfócitos T/imunologia , Transdução de Sinais , Fosforilação , Sistema de Sinalização das MAP Quinases , Diferenciação Celular , Citometria de Fluxo/métodos , Células CultivadasRESUMO
BACKGROUND: Abnormalities of in utero testis development are strongly associated with reproductive health conditions, including male infertility and testis cancer. In mouse testes, SOX9 and FGF9 support Sertoli cell development, while VEGF signalling is essential for the establishment of vasculature. The mitogen-activated protein kinase (MAPK) pathway is a major signalling cascade, essential for cell proliferation, differentiation and activation of Sry during primary sex-determination, but little is known about its function during fetal testis morphogenesis. We explored potential functions of MAPK signalling immediately after the establishment of testis cords in embryonic day (E)12.5 Oct4-eGFP transgenic mouse testes cultured using a MEK1/2 inhibitor. RESULTS: RNA sequencing in isolated gonadal somatic cells identified 116 and 114 differentially expressed genes after 24 and 72 h of MEK1/2 inhibition, respectively. Ingenuity Pathway Analysis revealed an association of MEK1/2 signalling with biological functions such as angiogenesis, vasculogenesis and cell migration. This included a failure to upregulate the master transcriptional regulators of vascular development, Sox7 and Sox17, VEGF receptor genes, the cell adhesion factor gene Cd31 and a range of other endothelial cell markers such as Cdh5 (encoding VE-cadherin) and gap junction genes Gja4 and Gja5. In contrast, only a small number of Sertoli cell enriched genes were affected. Immunofluorescent analyses of control testes revealed that the MEK1/2 downstream target, ERK1/2 was phosphorylated in endothelial cells and Sertoli cells. Inhibition of MEK1/2 eliminated pERK1/2 in fetal testes, and CD31, VE-cadherin, SOX7 and SOX17 and endothelial cells were lost. Consistent with a role for VEGF in driving endothelial cell development in the testis, inhibition of VEGFR also abrogated pERK1/2 and SOX7 and SOX17 expressing endothelial cells. Moreover, while Sertoli cell proliferation and localisation to the testis cord basement membrane was disrupted by inhibition of MEK1/2, it was unaffected by VEGFR inhibition. Instead, inhibition of FGF signalling compromised Sertoli cell proliferation and localisation to the testis cord basement membrane. CONCLUSIONS: Together, our data highlight an essential role for VEGF-dependent MEK1/2 signalling in promoting vasculature and indicate that FGF signalling through MEK1/2 regulates Sertoli cell organisation in the developing mouse testis.
Assuntos
Camundongos Transgênicos , Fatores de Transcrição SOXF , Testículo , Animais , Masculino , Fatores de Transcrição SOXF/metabolismo , Fatores de Transcrição SOXF/genética , Camundongos , Testículo/metabolismo , Testículo/embriologia , Testículo/irrigação sanguínea , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/metabolismo , MAP Quinase Quinase 2/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Transdução de Sinais , Sistema de Sinalização das MAP Quinases , Neovascularização Fisiológica , MAP Quinase Quinase Quinase 1/metabolismo , MAP Quinase Quinase Quinase 1/genética , Angiogênese , Proteínas HMGBRESUMO
Mutations in the epidermal growth factor receptor (EGFR) gene are common driver oncogenes in non-small cell lung cancer (NSCLC). Studies have shown that afatinib is beneficial for NSCLC patients with rare EGFR mutations. However, the effectiveness of tyrosine kinase inhibitors (TKIs) against the G719X (G719A, G719C and G719S) mutation has not been fully established. Herein, using the CRISPR method, the EGFR G719X mutant cell lines were constructed to assess the sensitivity of the rare mutation G719X in NSCLC. WZ3146, a novel mutation-selective EGFR inhibitor, was conducted transcriptome sequencing and in vitro experiments. The results showed that WZ3146 induced cytotoxic effects, inhibited growth vitality and proliferation via ERK and AKT pathway in the EGFR G719X mutant cells. Our findings suggest that WZ3146 may be a promising treatment option for NSCLC patients with the EGFR exon 18 substitution mutation G719X.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células , Receptores ErbB , Neoplasias Pulmonares , Mutação , Proteínas Proto-Oncogênicas c-akt , Humanos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Antineoplásicos/farmacologiaRESUMO
Despite initial high response rates to first-line EGFR TKI, all non-small-cell lung cancer (NSCLC) with EGFR-activating mutation will ultimately develop resistance to treatment. Identification of resistance mechanisms is critical to adapt treatment and improve patient outcomes. Here, we show that a PPP3CB transcript that encodes full-length catalytic subunit 2B of calcineurin accumulates in EGFR-mutant NSCLC cells with acquired resistance against different EGFR TKIs and in post-progression biopsies of NSCLC patients treated with EGFR TKIs. Neutralization of PPP3CB by siRNA or inactivation of calcineurin by cyclosporin A induces apoptosis in resistant cells treated with EGFR TKIs. Mechanistically, EGFR TKIs increase the cytosolic level of calcium and trigger activation of a calcineurin/MEK/ERK pathway that prevents apoptosis. Combining EGFR, calcineurin, and MEK inhibitors overcomes resistance to EGFR TKI in both in vitro and in vivo models. Our results identify PPP3CB overexpression as a new mechanism of acquired resistance to EGFR TKIs, and provide a promising therapeutic approach for NSCLC patients that progress under TKI treatment.
Assuntos
Calcineurina , Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB , Neoplasias Pulmonares , Sistema de Sinalização das MAP Quinases , Inibidores de Proteínas Quinases , Humanos , Calcineurina/metabolismo , Calcineurina/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Receptores ErbB/metabolismo , Receptores ErbB/genética , Resistencia a Medicamentos Antineoplásicos/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Apoptose/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Mutação , FemininoRESUMO
BACKGROUND: The roles of the transcriptional factor SIX2 have been identified in several tumors. However, its roles in gastric cancer (GC) progression have not yet been revealed. Our objective is to explore the impact and underlying mechanisms of SIX2 on the stemness of GC cells. METHODS: Lentivirus infection was employed to establish stable expression SIX2 or PFN2 in GC cells. Gain- and loss-of-function experiments were conducted to detect changes of stemness markers, flow cytometry profiles, tumor spheroid formation, and tumor-initiating ability. ChIP, RNA-sequencing, tissue microarray, and bioinformatics analysis were performed to reveal the correlation between SIX2 and PFN2. The mechanisms underlying the SIX2/PFN2 loop-mediated effects were elucidated through tissue microarray analysis, RNA stability assay, IP-MS, Co-Immunoprecipitation, and inhibition of the JNK signaling pathway. RESULTS: The stemness of GC cells was enhanced by SIX2. Mechanistically, SIX2 directly bound to PFN2's promoter and promoted PFN2 activity. PFN2, in turn, promoted the mRNA stability of SIX2 by recruiting RNA binding protein YBX-1, subsequently activating the downstream MAPK/JNK pathway. CONCLUSION: This study unveils the roles of SIX2 in governing GC cell stemness, defining a novel SIX2/PFN2 regulatory loop responsible for this regulation. This suggests the potential of targeting the SIX2/PFN2 loop for GC treatment (Graphical Abstracts).
Assuntos
Retroalimentação Fisiológica , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio , Células-Tronco Neoplásicas , Profilinas , Neoplasias Gástricas , Neoplasias Gástricas/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Humanos , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Linhagem Celular Tumoral , Profilinas/metabolismo , Profilinas/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Animais , Regiões Promotoras Genéticas/genética , Estabilidade de RNA/genética , Sistema de Sinalização das MAP Quinases , Ligação ProteicaRESUMO
Gastric cancer is one of the most malignant digestive tract tumors worldwide and its progression is associated with gene expression and metabolic alteration. We revealed that the gastric cancer patients with lower expression level of TOB1 exhibited poorer overall survivals according to the data in Kaplan-Meier Plotter. The unphosphorylated TOB1 protein which is effective expressed lower in gastric cancer cells. The gastric cancer cells with TOB1 gene depletion performed higher abilities of proliferation, migration and invasion and lower ability of apoptosis in vitro. The TOB1 gene depletion also promoted the tumorigenesis of gastric cancer cells in vivo. The gastric cancer cells with TOB1 gene overexpression had the converse behaviors. The transcriptional and metabolic sequencing was performed. The analyzation results showed that genes correlate-expressed with TOB1 gene were enriched in the pathways related to ERK pathway, including focal adhesion pathway, which was verified using real-time quantitative PCR. After inhibiting ERK pathway, the proliferation, colony formation and migration abilities were reduced in gastric cancer cells with low phosphorylated TOB1 protein expression level. Moreover, Pearson correlation analysis was adopted to further analyze the correlation of enriched metabolic products and differentially expressed genes. The expression of Choline, UDP-N-acetylglucosamine, Adenosine and GMP were related to the function of TOB1. This study demonstrates the genes and metabolites related to focal adhesion pathway and ERK pathway are the potential diagnosis and therapeutic targets to gastric cancer with TOB1 depletion.
Assuntos
Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Neoplasias Gástricas , Proteínas Supressoras de Tumor , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Movimento Celular/genética , Linhagem Celular Tumoral , Animais , Camundongos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adesões Focais/genética , Adesões Focais/metabolismo , Apoptose/genéticaRESUMO
BACKGROUND: Pulmonary fibrosis (PF) is an aging-related progressive lung disorder. The aged lung undergoes functional and structural changes termed immunosenescence and inflammaging, which facilitate the occurrence of fibrosis. Interleukin-10 (IL-10) is a potent anti-inflammatory and immunoregulatory cytokine, yet it remains unclear how IL-10 deficiency-induced immunosenescence participates in the development of PF. METHODS: Firstly we evaluated the susceptibility to fibrosis and IL-10 expression in aged mice. Then 13-month-old wild-type (WT) and IL-10 knockout (KO) mice were subjected to bleomycin(BLM) and analyzed senescence-related markers by PCR, western blot and immunohistochemistry staining of p16, p21, p53, as well as DHE and SA-ß-gal staining. We further compared 18-month-old WT mice with 13-month-old IL-10KO mice to assess aging-associated cell senescence and inflamation infiltration in both lung and BALF. Moreover, proliferation and apoptosis of alveolar type 2 cells(AT2) were evaluated by FCM, immunofluorescence, TUNEL staining, and TEM analysis. Recombinant IL-10 (rIL-10) was also administered intratracheally to evaluate its therapeutic potential and related mechanism. For the in vitro experiments, 10-week-old naïve pramily lung fibroblasts(PLFs) were treated with the culture medium of 13-month PLFs derived from WT, IL-10KO, or IL-10KO + rIL-10 respectively, and examined the secretion of senescence-associated secretory phenotype (SASP) factors and related pathways. RESULTS: The aged mice displayed increased susceptibility to fibrosis and decreased IL-10 expression. The 13-month-old IL-10KO mice exhibited significant exacerbation of cell senescence compared to their contemporary WT mice, and even more severe epithelial-mesenchymal transition (EMT) than that of 18 month WT mice. These IL-10 deficient mice showed heightened inflammatory responses and accelerated PF progression. Intratracheal administration of rIL-10 reduced lung CD45 + cell infiltration by 15%, including a 6% reduction in granulocytes and a 10% reduction in macrophages, and increased the proportion of AT2 cells by approximately 8%. Additionally, rIL-10 significantly decreased α-SMA and collagen deposition, and reduced the expression of senescence proteins p16 and p21 by 50% in these mice. In vitro analysis revealed that conditioned media from IL-10 deficient mice promoted SASP secretion and upregulated senescence genes in naïve lung fibroblasts, which was mitigated by rIL-10 treatment. Mechanistically, rIL-10 inhibited TGF-ß-Smad2/3 and PTEN/PI3K/AKT/ERK pathways, thereby suppressing senescence and fibrosis-related proteins. CONCLUSIONS: IL-10 deficiency in aged mice leads to accelerated cell senescence and exacerbated fibrosis, with IL-10KO-PLFs displaying increased SASP secretion. Recombinant IL-10 treatment effectively mitigates these effects, suggesting its potential as a therapeutic target for PF.
Assuntos
Bleomicina , Senescência Celular , Interleucina-10 , Camundongos Knockout , PTEN Fosfo-Hidrolase , Proteínas Proto-Oncogênicas c-akt , Fibrose Pulmonar , Animais , Interleucina-10/metabolismo , Interleucina-10/genética , Camundongos , Fibrose Pulmonar/patologia , Fibrose Pulmonar/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Camundongos Endogâmicos C57BL , Sistema de Sinalização das MAP Quinases , Apoptose , Pulmão/patologia , Pulmão/metabolismo , Masculino , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Modelos Animais de Doenças , Proliferação de Células , Envelhecimento , Transdução de SinaisRESUMO
Aneuploidy is a hallmark of human cancer, yet the molecular mechanisms to cope with aneuploidy-induced cellular stresses remain largely unknown. Here, we induce chromosome mis-segregation in non-transformed RPE1-hTERT cells and derive multiple stable clones with various degrees of aneuploidy. We perform a systematic genomic, transcriptomic and proteomic profiling of 6 isogenic clones, using whole-exome DNA, mRNA and miRNA sequencing, as well as proteomics. Concomitantly, we functionally interrogate their cellular vulnerabilities, using genome-wide CRISPR/Cas9 and large-scale drug screens. Aneuploid clones activate the DNA damage response and are more resistant to further DNA damage induction. Aneuploid cells also exhibit elevated RAF/MEK/ERK pathway activity and are more sensitive to clinically-relevant drugs targeting this pathway, and in particular to CRAF inhibition. Importantly, CRAF and MEK inhibition sensitize aneuploid cells to DNA damage-inducing chemotherapies and to PARP inhibitors. We validate these results in human cancer cell lines. Moreover, resistance of cancer patients to olaparib is associated with high levels of RAF/MEK/ERK signaling, specifically in highly-aneuploid tumors. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, and reveals a therapeutically-relevant cellular dependency of aneuploid cells.
Assuntos
Aneuploidia , Dano ao DNA , Sistema de Sinalização das MAP Quinases , Ftalazinas , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ftalazinas/farmacologia , Linhagem Celular Tumoral , Piperazinas/farmacologia , Quinases raf/metabolismo , Quinases raf/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Resistencia a Medicamentos Antineoplásicos/genéticaRESUMO
Myocardial ischemia-reperfusion (I/R) injury exacerbates cellular damage upon restoring blood flow to ischemic cardiac tissue, causing oxidative stress, inflammation, and apoptosis. This study investigates Nicotinamide Riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD+), for its cardioprotective effects. Administering NR to mice before I/R injury and evaluating heart function via echocardiography showed that NR significantly improved heart function, increased left ventricular ejection fraction (LVEF) and fractional shortening (FS), and reduced left ventricular end-diastolic (LVDd) and end-systolic diameters (LVSd). NR also restored E/A and E/e' ratios. It reduced cardiomyocyte apoptosis both in vivo and in vitro, inhibiting elevated caspase-3 activity and returning Bax protein levels to normal. In vitro, NR reduced the apoptotic rate in hydrogen peroxide (H2O2)-treated HL-1 cells from 30% to 10%. Mechanistically, NR modulated the SIRT3/mtROS/JNK pathway, reversing H2O2-induced SIRT3 downregulation, reducing mitochondrial reactive oxygen species (mtROS), and inhibiting JNK activation. Using SIRT3-knockout (SIRT3-KO) mice, we confirmed that NR's cardioprotective effects depend on SIRT3. Echocardiography showed that NR's benefits were abrogated in SIRT3-KO mice. In conclusion, NR provides significant cardioprotection against myocardial I/R injury by enhancing NAD+ levels and modulating the SIRT3/mtROS/JNK pathway, suggesting its potential as a novel therapeutic agent for ischemic heart diseases, meriting further clinical research.
Assuntos
Apoptose , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica , Niacinamida , Compostos de Piridínio , Espécies Reativas de Oxigênio , Sirtuína 3 , Animais , Sirtuína 3/metabolismo , Sirtuína 3/genética , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Camundongos , Compostos de Piridínio/farmacologia , Compostos de Piridínio/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Humanos , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Modelos Animais de Doenças , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacosRESUMO
The KRAS oncogene drives many common and highly fatal malignancies. These include pancreatic, lung, and colorectal cancer, where various activating KRAS mutations have made the development of KRAS inhibitors difficult. Here we identify the scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a RAS interactor that binds active KRAS, including mutant forms, competes with RAF and limits oncogenic KRAS downstream signalling, maintaining mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity at an optimal level. SHANK3 depletion breaches this threshold, triggering MAPK/ERK signalling hyperactivation and MAPK/ERK-dependent cell death in KRAS-mutant cancers. Targeting this vulnerability through RNA interference or nanobody-mediated disruption of the SHANK3-KRAS interaction constrains tumour growth in vivo in female mice. Thus, inhibition of SHANK3-KRAS interaction represents an alternative strategy for selective killing of KRAS-mutant cancer cells through excessive signalling.
Assuntos
Sistema de Sinalização das MAP Quinases , Mutação , Proteínas do Tecido Nervoso , Proteínas Proto-Oncogênicas p21(ras) , Animais , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Humanos , Camundongos , Linhagem Celular Tumoral , Feminino , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Sistema de Sinalização das MAP Quinases/genética , Morte Celular/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos Nus , Proteínas dos MicrofilamentosRESUMO
An increasing body of evidence suggests that acylphosphatase-2 (ACYP2) polymorphisms are correlated with an increased susceptibility to a range of malignancies. Nevertheless, its potential functions, molecular mechanisms in hepatocellular carcinoma (HCC) and whether it can be act as a therapeutic target remain uninvestigated. Herein, ACYP2 was found to be lowly expressed in HCC and was negatively correlated with tumor size, tumor differentiation, microvascular invasion and the prognosis of HCC patients. Functional investigations revealed that overexpression of ACYP2 inhibited the proliferation and metastasis of HCC cells while promoting apoptosis; knockdown of ACYP2 had the exact opposite effect. Additionally, it was observed that ACYP2 was distributed in both the cytoplasm and nucleus of HCC cells. According to the mechanistic studies, the expression of potassium calcium-activated channel subfamily N member 4 (KCNN4) was negatively regulated by cytoplasmic ACYP2, resulting in the inhibition of K+ outflow and subsequent inactivation of the ERK pathway, which impeded the growth and metastasis of HCC. Furthermore, the activity of telomerase reverse transcriptase (TERT) was inhibited by nuclear ACYP2, leading to the reduction in length of telomeres and consequent reversal of HCC cell immortalization. Additionally, a novel targeted nanotherapy strategy was developed wherein the pcDNA-ACYP2 vector was encapsulated within polyetherimide nanoparticles (PEI/NPs), which were subsequently coated with HCC cell membranes (namely pcDNA/PEI/NPs@M). Safety and targeting characteristics abound for these nanocomposites, in both subcutaneous graft tumor models and orthotopic mouse models, they inhibited the progression of HCC by impeding TERT activity and the KCNN4/ERK pathway. In conclusion, our research identifies novel molecular mechanisms involving cytoplasmic and nuclear ACYP2 that inhibit the progression of HCC. Moreover, pcDNA/PEI/NPs@M represents a targeted therapeutic strategy for HCC that holds great promising.
Assuntos
Carcinoma Hepatocelular , Proliferação de Células , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Neoplasias Hepáticas , Sistema de Sinalização das MAP Quinases , Telomerase , Humanos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Telomerase/metabolismo , Telomerase/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Camundongos , Masculino , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Nus , Apoptose/efeitos dos fármacos , Feminino , Progressão da Doença , Camundongos Endogâmicos BALB C , Nanopartículas/química , Pessoa de Meia-IdadeRESUMO
This study explored the mechanism of curcumin (CUR) suppressing osteoclastogenesis and evaluated its effects on osteoarthritis (OA) mouse. Bone marrow-derived macrophages were isolated as osteoclast precursors. In the presence or absence of CUR, cell proliferation was detected by CCK-8, osteoclastogenesis was detected by tartrate-resistant acid phosphatase (TRAP) staining, F-actin rings formation was detected by immunofluorescence, bone resorption was detected by bone slices, IκBα, nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were detected using western blot, osteoclastogenesis-related gens were measured using quantitative polymerase chain reaction. A knee OA mouse model was designed by destabilizing the medial meniscus (DMM). Thirty-six male mice were divided into sham+vehicle, OA+vehicle, and OA+CUR groups. Mice were administered with or without CUR at 25 mg/kg/d from the first post-operative day until sacrifice. After 4 and 8 weeks of OA induction, micro-computed tomography was performed to analyze microstructure changes in subchondral bone, hematoxylin and eosin staining was performed to calculate the thickness of the calcified and hyaline cartilage layers, toluidine blue O staining was performed to assess the degenerated cartilage, TRAP-stained osteoclasts were counted, and NF-κB, phosphorylated Jun N-terminal Kinases (p-JNK), and receptor activator of nuclear factor κB ligand (RANKL) were detected using immunohistochemistry. CUR suppressed osteoclastogenesis and bone resorption without cytotoxicity. CUR restrained RANKL-induced activation of NF-κB, p-JNK and up-regulation of osteoclastogenesis-related genes. CUR delayed cartilage degeneration by suppressing osteoclastogenesis and bone resorption in early OA. The mechanism of CUR inhibiting osteoclastogenesis might be associated with NF-κB/JNK signaling pathway, indicating a novel strategy for OA treatment.
Assuntos
Curcumina , Sistema de Sinalização das MAP Quinases , NF-kappa B , Osteoclastos , Osteogênese , Animais , Camundongos , Masculino , NF-kappa B/metabolismo , Curcumina/farmacologia , Osteogênese/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Osteoartrite/patologia , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologiaRESUMO
Pulmonary fibrosis (PF) is an interstitial lung disease characterized by inflammation and fibrotic changes, with an unknown cause. In the early stages of PF, severe inflammation leads to the destruction of lung tissue, followed by upregulation of fibrotic factors like Transforming growth factor-ß (TGF-ß) and connective tissue growth factor (CTGF), which disrupt normal tissue repair. Geniposide, a natural iridoid glycoside primarily derived from the fruits of Gardenia jasminoides Ellis, possesses various pharmacological activities, including liver protection, choleretic effects, and anti-inflammatory properties. In this study, we investigated the effects of Geniposide on chronic inflammation and fibrosis induced by bleomycin (BLM) in mice with pulmonary fibrosis (PF). PF was induced by intratracheal instillation of bleomycin, and Geniposide(100/50/25mgâ¢kg-1) was orally administered to the mice once a day until euthanasia(14 day/28 day). The Raw264.7 cell inflammation induced by LPS was used to evaluate the effect of Geniposide on the activation of macrophage. Our results demonstrated that Geniposide reduced lung coefficients, decreased the content of Hydroxyproline, and improved pathological changes in lung tissue. It also reduced the number of inflammatory cells and levels of pro-inflammatory cytokines in bronchoalveolar lavage fluid (BALF) of bleomycin-induced PF mice. At the molecular level, Geniposide significantly down-regulated the expression of TGF-ß1, Smad2/3, p38, and CTGF in lung tissues of PF mice induced by bleomycin. Molecular docking results revealed that Geniposide exhibited good binding activity with TGF-ß1, Smad2, Smad3, and p38. In vitro study showed Geniposide directly inhibited the activation of macrophage induced by LPS. In conclusion, our findings suggest that Geniposide can ameliorate bleomycin-induced pulmonary fibrosis in mice by inhibiting the TGF-ß/Smad and p38MAPK signaling pathways.
Assuntos
Bleomicina , Iridoides , Fibrose Pulmonar , Fator de Crescimento Transformador beta , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Bleomicina/efeitos adversos , Bleomicina/toxicidade , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Iridoides/farmacologia , Camundongos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Masculino , Células RAW 264.7 , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
Humans with monogenic inborn errors responsible for extreme disease phenotypes can reveal essential physiological pathways. We investigated germline mutations in GNAI2, which encodes Gαi2, a key component in heterotrimeric G protein signal transduction usually thought to regulate adenylyl cyclase-mediated cyclic adenosine monophosphate (cAMP) production. Patients with activating Gαi2 mutations had clinical presentations that included impaired immunity. Mutant Gαi2 impaired cell migration and augmented responses to T cell receptor (TCR) stimulation. We found that mutant Gαi2 influenced TCR signaling by sequestering the guanosine triphosphatase (GTPase)-activating protein RASA2, thereby promoting RAS activation and increasing downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT S6 signaling to drive cellular growth and proliferation.
Assuntos
Subunidade alfa Gi2 de Proteína de Ligação ao GTP , Mutação em Linhagem Germinativa , Receptores de Antígenos de Linfócitos T , Linfócitos T , Proteínas Ativadoras de ras GTPase , Humanos , Movimento Celular/genética , Proliferação de Células , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Imunidade/genética , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Proteínas ras/metabolismo , Proteínas ras/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , LinhagemRESUMO
Vitamin B12 is an essential nutritional co-factor for the folate and methionine cycles, which together constitute one-carbon metabolism. Here, we show that dietary uptake of vitamin B12 modulates cell fate decisions controlled by the conserved RAS/MAPK signaling pathway in C. elegans. A bacterial diet rich in vitamin B12 increases vulval induction, germ cell apoptosis and oocyte differentiation. These effects are mediated by different one-carbon metabolites in a tissue-specific manner. Vitamin B12 enhances via the choline/phosphatidylcholine metabolism vulval induction by down-regulating fat biosynthesis genes and increasing H3K4 tri-methylation, which results in increased expression of RAS/MAPK target genes. Furthermore, the nucleoside metabolism and H3K4 tri-methylation positively regulate germ cell apoptosis and oocyte production. Using mammalian cells carrying different activated KRAS and BRAF alleles, we show that the effects of methionine on RAS/MAPK-regulated phenotype are conserved in mammals. Our findings suggest that the vitamin B12-dependent one-carbon metabolism is a limiting factor for diverse RAS/MAPK-induced cellular responses.
Assuntos
Apoptose , Caenorhabditis elegans , Diferenciação Celular , Metionina , Vitamina B 12 , Animais , Vitamina B 12/metabolismo , Vitamina B 12/farmacologia , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Feminino , Metionina/metabolismo , Apoptose/efeitos dos fármacos , Oócitos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas ras/metabolismo , Carbono/metabolismo , Vulva/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células Germinativas/metabolismo , Colina/metabolismo , Fosfatidilcolinas/metabolismo , Camundongos , Humanos , Histonas/metabolismo , Transdução de SinaisRESUMO
Sepsis, a frequently fatal condition, emerges from an exaggerated inflammatory response to infection, resulting in multi-organ dysfunction and alarmingly high mortality rates. Despite the urgent need for effective treatments, current therapeutic options remain limited to antibiotics, with no other efficacious alternatives available. Echinatin (Ecn), a potent bioactive compound extracted from the roots and rhizomes of licorice, has gained significant attention for its broad pharmacological properties, particularly its ability to combat oxidative stress. Recent research highlights the crucial role that oxidative stress plays in the onset and progression of sepsis further emphasizing the potential therapeutic value of Ecn in this context. In this study, we explored the protective effects of Ecn in a murine model of sepsis induced by cecal ligation and puncture (CLP). Ecn demonstrated a significant reduction in the levels of inflammatory cytokines and reactive oxygen species (ROS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Network pharmacology analysis identified 41 targets and top 15 pathways involved in the Ecn-mediated signaling network, revealing that Ecn might exert its effects through key targets including the NF-κB and MAPK signaling pathways. Molecular docking studies suggested a strong affinity between Ecn and MEK, with kinetic simulations and binding energy calculations confirming a stable interaction. Mechanistically, Ecn treatment inhibited NF-κB and the MEK/ERK signaling pathway, as evidenced by decreased phosphorylation of IκBα and nuclear p65, along with reduced phosphorylation of MEK and ERK in both LPS-stimulated RAW 264.7 macrophages and septic mice. Furthermore, the administration of MEK signaling agonists reversed the anti-inflammatory effects of Ecn, indicating the involvement of this signaling pathway in Ecn's protective mechanism. Notably, our investigation revealed that Ecn did not affect bacterial proliferation either in vivo or in vitro, underscoring its specific immunomodulatory effects rather than direct antimicrobial activity. In summation, our findings underscored the potential of Ecn as an innovative therapeutic remedy for sepsis-induced injury, particularly through the regulation of the NF-κB and MEK/ERK signaling pathway. This exploration unveiled a promising therapeutic approach for treating sepsis, supplementing existing interventions and addressing their constraints.
Assuntos
Sistema de Sinalização das MAP Quinases , NF-kappa B , Sepse , Animais , Sepse/tratamento farmacológico , Sepse/metabolismo , NF-kappa B/metabolismo , Camundongos , Células RAW 264.7 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Espécies Reativas de Oxigênio/metabolismo , Simulação de Acoplamento Molecular , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Anti-Inflamatórios/farmacologia , Transdução de Sinais/efeitos dos fármacos , Modelos Animais de Doenças , ChalconasRESUMO
Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide. Serotonin (5-HT) is a biogenic monoamine that acts as a neurotransmitter in the central nervous system and as a paracrine, exocrine, or endocrine messenger in peripheral tissues. In this study, we hypothesized that inhibition of serotonin signaling using 5-HT receptor 2B (HTR2B) inhibitors could potentially impede the progression of CRC. We treated CT26 and COLO-205 cells with SB204741, an inhibitor of HTR2B, and evaluated CRC cell proliferation and migration. We then evaluated the effects of HTR2B inhibition in a xenograft mouse model of human colorectal cancer. We also evaluated the role of a novel inhibitor, GM-60186, using both in vitro and in vivo models. RNA sequencing analysis was performed to elucidate the underlying mechanism of the anti-tumor effects of pharmacological inhibition of HTR2B on CRC. In both CRC cell lines and xenograft mouse models, we show that pharmacological inhibition of HTR2B with SB204741 and GM-60186 significantly inhibits CRC cell proliferation and migration. HTR2B inhibition leads to the suppression of extracellular signal-regulated kinase (ERK) signaling, a critical pathway in CRC pathogenesis. Notably, transcriptomic analysis reveals distinct gene expression changes associated with HTR2B inhibition, providing insight into its therapeutic potential. In this study, we found that pharmacological inhibition of HTR2B suppressed CRC proliferation via ERK signaling. In addition, we proposed a novel HTR2B inhibitor for the treatment of CRC. This study highlights the potential role of HTR2B signaling in CRC. These inhibitors may contribute to new therapeutics for CRC treatment.
Assuntos
Movimento Celular , Proliferação de Células , Neoplasias Colorretais , Sistema de Sinalização das MAP Quinases , Receptor 5-HT2B de Serotonina , Serotonina , Animais , Neoplasias Colorretais/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Humanos , Proliferação de Células/efeitos dos fármacos , Receptor 5-HT2B de Serotonina/metabolismo , Linhagem Celular Tumoral , Serotonina/metabolismo , Serotonina/farmacologia , Movimento Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Camundongos Endogâmicos BALB C , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
This study aimed to investigate the effect and potential mechanism of evodiamine (EVO) on proliferation and apoptosis of nasopharyngeal carcinoma (NPC) cells. EVO inhibited proliferation, blocked cell cycle progression, and induced apoptosis of NPC cells. There are 27 known anti-NPC targets of EVO, of which eight are core targets, namely SRC, ERBB2, STAT3, MAPK8, NOS3, CXCL8, APP, and HDAC1. Molecular docking analysis showed that the binding of EVO with its key targets (SRC, ERBB2) was good. EVO also reduced the expression of SRC and ERBB2, the key proteins p-MEK and p-ERK1/2 of the MAPK/ERK signaling pathway, and the downstream proteins PCNA and XIAP. EVO inhibited the growth of NPC xenografts in nude mice and reduced the expression levels of SRC, ERBB2, ERK1/2, p-ERK1/2, PCNA and XIAP in NPC tissue. When the MAPK/ERK signaling pathway was activated by epidermal growth factor (EGF), the expression levels of PCNA and XIAP increased, the cell proliferation index increased, and the apoptosis rate decreased in the EGF + EVO treatment group compared to treatment with EVO alone. These changes indicated that the inhibitory effect of EVO on proliferation and apoptosis of NPC cells was related to the down-regulation of SRC and ERBB2 expression, and further inhibition of the MAPK/ERK signaling pathway.