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In this study, a really simple and efficient catalytic protocol for the construction of quinazolines from alcohol and diamine has been developed based on CuCoAl layered double hydroxide (CuCoAl-LDH). The developed CuCoAl-LDH catalyst could accelerate the cascade reactions without any additives and tolerate various alcohols with satisfactory yields. Cooperation between the Cu+ and Cu2+ species in CuCoAl-LDH was observed in the cascade reaction, and they are believed to be responsible for the oxidation of alcohol and dehydrogenation of the intermediate, respectively. The promoting effect of the substrate diamine was observed in the oxidation of alcohol, which simplifies the reaction system by eliminating the requirement for a base additive. The catalytic system exhibited highly practical potential for the synthesis of quinazolines, as demonstrated through recyclability investigations and scale-up experiments. A possible catalytic mechanism has been proposed based on a series of control experiments and EPR analysis.
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The concise and highly convergent synthesis of the isodityrosine unit of seongsanamide A-D and its derivatives bearing a diaryl ether moiety is described. In this work, the synthetic strategy features palladium-catalyzed C(sp3)-H functionalization and a Cu/ligand-catalyzed coupling reaction. We report a practical protocol for the palladium-catalyzed mono-arylation of ß-methyl C(sp3)-H of an alanine derivative bearing a 2-thiomethylaniline auxiliary. The reaction is compatible with a variety of functional groups, providing practical access to numerous ß-aryl-α-amino acids; these acids can be converted into various tyrosine and dihydroxyphenylalanine (DOPA) derivatives. Then, a CuI/N,N-dimethylglycine-catalyzed arylation of the already synthesized DOPA derivatives with aryl iodides is described for the synthesis of isodityrosine derivatives.
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Paladio , Tirosina , Paladio/química , Catálisis , DihidroxifenilalaninaRESUMEN
Lung cancer has high morbidity and mortality. This study demonstrated that Bufalin inhibits the proliferation of lung cancer cells in vivo / in vitro by suppressing Hippo-YAP pathway. Here, we found that Bufalin promoted the binding of LATS and YAP to elevate the level of YAP phosphorylation. Phosphorylated YAP could not successfully enter the nucleus to activate the expression of downstream proliferation-related target genes Cyr61 and CTGF, whereas the YAP retained in the cytoplasm further bound to ß-TrCP and underwent ubiquitination and degradation. This study verified the key role of YAP in stimulating the proliferation of lung cancer and revealed the anticancer target of Bufalin. Therefore, this study provides a theoretical basis for the anticancer effect of Bufalin, and suggests that Bufalin can be a potential anticancer drug.
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Neoplasias Pulmonares , Proteínas Serina-Treonina Quinasas , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Transcripción/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Proliferación Celular/genéticaRESUMEN
BACKGROUND: Corosolic acid is a pentacyclic triterpene acid with hypoglycemic, anti-inflammatory, and anti-cancer effects. However, its potential targets in hepatocellular carcinoma (HCC) are unknown, hindering clinical utilization. METHODS: Differentially expressed proteins of the Bel-7404 cell line were identified with tandem mass tag analysis and differentially expressed genes (DEGs) of an HCC TCGA dataset using bioinformatics. Gene functions and pathways were inferred using the DAVID database. Online databases were used to establish P4HA2 expression in HCC (GEPIA2) and its relationship with patient survival (UALCAN and The Human Protein Atlas), the association between P4HA2 expression and immune cell infiltration (TIMER2), and DNA methylation of the P4HA2 gene (MethSurv). Cell proliferation, cell cycle, and cell death were assessed with PI and SYTOX-Green staining, CCK-8, and colony formation assays. Protein expression levels were detected by Western blotting. RESULTS: A total of 44 differentially expressed proteins and 4498 DEGs were identified. Four genes whose proteins were also found in the differential protein profile but with opposing expressions were selected as candidate targets. The candidate gene prolyl 4-hydroxylase subunit alpha 2 (P4HA2) was recognized as the only potential target due to its high expression in public datasets, association with poor patient survival, and relation to immune cell infiltration in HCC tissues. Moreover, the DNA methylation status in 4 CpG islands of the P4HA2 gene correlated with a poor prognosis. Furthermore, corosolic acid treatment inhibited the proliferation of HCC cell lines Bel-7404 and HepG2 in a dose-dependent manner, caused G2/M phase cell cycle arrest, and promoted cell death. In addition, the treatment reduced P4HA2 protein levels. CONCLUSION: Our results indicate that P4HA2 is a potential target of corosolic acid. Thus, they contribute to understanding molecular changes in HCC after corosolic acid treatment and facilitate finding new treatment regimens.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Triterpenos , Humanos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Línea Celular , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Triterpenos/farmacología , Farmacología en RedRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Pulsatilla chinensis (Bunge) Regel is a traditional Chinese herbal medicine used to treat intestinal amebiasis, malaria, vaginal trichomoniasis, and bacterial infections. Anemoside B4 (AB4), a pentacyclic triterpenoid saponin, is one of the primary bioactive substances in Pulsatilla chinensis (Bunge) Regel, and gavage administration of AB4 to animals has been demonstrated to exhibit anticancer, anti-inflammatory, and antiviral actions. However, AB4 exposure in plasma is very low after oral administration, and the biotransformation of AB4 in vivo after oral administration remains unknown. AIM OF THE STUDY: The reason for conducting this research was to explore at the metabolite profile of AB4 in rats following oral administration. Additionally, we aimed to develop an appropriate extravascular formulation to increase the exposure and duration of AB4 in vivo. MATERIALS AND METHODS: A well-validated HPLC-QQQ-MS/MS method was used for the quantification of AB4 in plasma and was further applied to evaluate and compare the pharmacokinetic properties of AB4 dissolved in a saline solution and AB4 formulations in a rectal suppository or enteric capsule. Reliable UHPLC coupled to Q-Exactive Plus high-resolution MS was used to identify the metabolites in rat plasma, bile, urine, and faeces. RESULTS: AB4 was extensively metabolized, and a total of 29 metabolites were identified. The primary metabolic routes included deglycosylation, oxidation, dehydrogenation, reduction, sulfation, hydration, acetylation, and glucuronidation. The pharmacokinetic comparison showed that both the rectal suppository and enteric capsule increased the exposures of AB4 and one of its active metabolites, 23-hydroxybetulinic acid (23-HA). Notably, rectal suppositories increased systemic AB4 exposure (AUC0-∞) by approximately 49 and 28 times higher than that of the AB4 saline solution and enteric capsules, respectively. The t1/2 of AB4 was extended to approximately 7 h after rectal administration compared to 2 h after oral administration. CONCLUSION: Overall, our study demonstrated that the mismatched exposure-response relationship of AB4 could result from extensive metabolism in the gastrointestinal and circulatory systems. Thus, a rectal suppository could be an alternative formulation of AB4 to obtain both higher and longer exposure.
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Saponinas , Espectrometría de Masas en Tándem , Femenino , Ratas , Animales , Supositorios , Espectrometría de Masas en Tándem/métodos , Solución Salina , Saponinas/farmacología , Administración OralRESUMEN
Aims: Lipid peroxidation occurring in lung adenocarcinoma (LUAD) cells leads to ferroptosis. Lysophosphatidylcholine acyl-transferase 3 (LPCAT3) plays a key role in providing raw materials for lipid peroxidation by promoting esterification of polyunsaturated fatty acids to phospholipids. Whether LPCAT3 determines ferroptosis sensitivity and the mechanism by which its expression is regulated in LUAD has not been reported. Results: LPCAT3 and acyl-coenzyme A (CoA) synthetase long-chain family member (ACSL)4 levels were positively associated with ferroptosis sensitivity in LUAD cell lines. Overexpression of LPCAT3 and ACSL4 sensitized LUAD cells to ferroptosis, while LPCAT3 and ACSL4 knockout showed the opposite effect. Zinc-finger E-box-binding (ZEB) was shown to directly bind the LPCAT3 promoter to stimulate its transcription in a Yes-associated protein (YAP)-dependent manner. An interaction between YAP and ZEB was also observed. E1A-binding protein p300 (EP300) simultaneously bound with YAP and ZEB, and induced H3K27Ac for LPCAT3 transcription. This mechanism was verified in primary LUAD cell and xenograft models. The ACSL4, LPCAT3, and YAP combination can jointly determine LUAD ferroptosis sensitivity. Innovation: The binding site of ZEB exists in the -1600 to -1401 nt region of LPCAT3 promoter, which promotes LPCAT3 transcription after ZEB binding. ZEB and YAP bind, and the ZEB zinc-finger cluster domain and YAP WW domain are crucial for their binding. EP300 may bind with YAP via its Bromo domain and with ZEB via its CBP/p300-HAT domain. In addition, the combination of ACSL4, LPCAT3, and YAP to determine ferroptosis sensitivity of LUAD cells is better than prostaglandin-endoperoxide synthase 2 (PTGS2), transferrin receptor (TFRC), or NADPH oxidase 1 (NOX1). Conclusion: LPCAT3 transcription is regulated by YAP, ZEB, and EP300. LUAD ferroptosis sensitivity can be determined by the combination of ACSL4, LPCAT3, and YAP. Antioxid. Redox Signal. 39, 491-511.
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Adenocarcinoma del Pulmón , Ferroptosis , Neoplasias Pulmonares , Humanos , Ferroptosis/genética , Sitios de Unión , Coenzima A Ligasas/genética , Ciclooxigenasa 2 , Neoplasias Pulmonares/genética , Zinc , Proteína p300 Asociada a E1A , 1-Acilglicerofosfocolina O-AciltransferasaRESUMEN
Non-small cell lung cancer (NSCLC) is the most common pathological type of LC and ranks as the leading cause of cancer deaths. Circulating exosomes have emerged as a valuable biomarker for the diagnosis of NSCLC, while the performance of current electrochemical assays for exosome detection is constrained by unsatisfactory sensitivity and specificity. Here we integrated a ratiometric biosensor with an OR logic gate to form an assay for surface protein profiling of exosomes from clinical serum samples. By using the specific aptamers for recognition of clinically validated biomarkers (EpCAM and CEA), the assay enabled ultrasensitive detection of trace levels of NSCLC-derived exosomes in complex serum samples (15.1 particles µL-1 within a linear range of 102-108 particles µL-1). The assay outperformed the analysis of six serum biomarkers for the accurate diagnosis, staging, and prognosis of NSCLC, displaying a diagnostic sensitivity of 93.3% even at an early stage (Stage I). The assay provides an advanced tool for exosome quantification and facilitates exosome-based liquid biopsies for cancer management in clinics.
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Carcinoma de Pulmón de Células no Pequeñas , Electroquímica , Exoma , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Técnicas Biosensibles , Límite de Detección , Análisis Químico de la Sangre/métodos , Análisis Químico de la Sangre/normas , Humanos , Línea Celular TumoralRESUMEN
N6-Methyladenosine (m6A) RNA modification, methylation at the N6 position of adenosine, plays critical roles in tumorigenesis. m6A readers recognize m6A modifications and thus act as key executors for the biological consequences of RNA methylation. However, knowledge about the regulatory mechanism(s) of m6A readers is extremely limited. In this study, RN7SK was identified as a small nuclear RNA that interacts with m6A readers. m6A readers recognized and facilitated secondary structure formation of m6A-modified RN7SK, which in turn prevented m6A reader mRNA degradation from exonucleases. Thus, a positive feedback circuit between RN7SK and m6A readers is established in tumor cells. From findings on the interaction with RN7SK, new m6A readers, such as EWS RNA binding protein 1 (EWSR1) and KH RNA binding domain containing, signal transduction-associated 1 (KHDRBS1), were identified and shown to boost Wnt/ß-catenin signaling and tumorigenesis by suppressing translation of Cullin1 (CUL1). Moreover, several Food and Drug Administration-approved small molecules were demonstrated to reduce RN7SK expression and inhibit tumorigenesis. Together, these findings reveal a common regulatory mechanism of m6A readers and indicate that targeting RN7SK has strong potential for tumor treatment.
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Carcinogénesis , ARN Nuclear Pequeño , Humanos , ARN Nuclear Pequeño/metabolismo , Retroalimentación , Carcinogénesis/genética , Metilación , Transformación Celular Neoplásica , Vía de Señalización Wnt , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
YAP is a transcriptional co-activator with critical roles in tumorigenesis. However, its upstream regulatory mechanism, especially how its mRNA stability is regulated, remains to be further studied. Here, we validated that YAP expression was higher in lung adenocarcinoma (LUAD) tissues compared to adjacent normal tissues, and found that YAP m5C modification occurred in its 328-331 3' UTR region under the promotion NSUN2 and ALYREF, and increased the stability of YAP mRNA. This m5C modification also inhibited miR-582-3p binding and m6A modification in the nearby region. In addition, YAP m5C modification enhanced the exosome secretion effect, which was caused by two YAP-dependent transcription factors, Mycn and SOX10, and then stimulating the transcription of seven downstream exosome-promoting genes. Furthermore, we found that YAP m5C modification and its exosome-secretion-promoting function contributed to the malignant phenotype and AZD9291 (a third-generation EGFR-TKI) resistance of LUAD cells. Collectively, YAP is promoted by its m5C modification, and blocking YAP m5C modification will be helpful for future LUAD treatment.
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Adenocarcinoma del Pulmón , Exosomas , Neoplasias Pulmonares , MicroARNs , Humanos , 5-Metilcitosina/metabolismo , Exosomas/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Neoplasias Pulmonares/patología , Estabilidad del ARN , MicroARNs/genéticaRESUMEN
A lack of promising targets leads to poor prognosis in patients with lung adenocarcinoma (LUAD). Therefore, it is urgent to identify novel therapeutic targets. The importance of the N6-methyladenosine (m6A) RNA modification has been demonstrated in various types of tumors; however, knowledge of m6A-related proteins in LUAD is still limited. Here, we found that insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), an m6A reader protein, is highly expressed in LUAD and associated with poor prognosis. IGF2BP3 desensitizes ferroptosis (a new form of regulated cell death) in a manner dependent on its m6A reading domain and binding capacity to m6A-methylated mRNAs encoding anti-ferroptotic factors, including but not limited to glutathione peroxidase 4 (GPX4), solute carrier family 3 member 2 (SLC3A2), acyl-CoA synthetase long chain family member 3 (ACSL3), and ferritin heavy chain 1 (FTH1). After IGF2BP3 overexpression, expression levels and mRNA stabilities of these anti-ferroptotic factors were successfully sustained. Notably, significant correlations between SLC3A2, ACSL3, and IGF2BP3 were revealed in clinical LUAD specimens, further establishing the essential role of IGF2BP3 in desensitizing ferroptosis. Inducing ferroptosis has been gradually accepted as an alternative strategy to treat tumors. Thus, IGF2BP3 could be a potential target for the future development of new biomaterial-associated therapeutic anti-tumor drugs.
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Abnormal nuclear structure caused by dysregulation of skeletal proteins is a common phenomenon in tumour cells. However, how skeletal proteins promote tumorigenesis remains uncovered. Here, we revealed the mechanism by which skeletal protein Emerin (EMD) promoted glucose metabolism to induce lung adenocarcinoma (LUAD). Firstly, we identified that EMD was highly expressed and promoted the malignant phenotypes in LUAD. The high expression of EMD might be due to its low level of ubiquitination. Additionally, the ISGylation at lysine 37 of EMD inhibited lysine 36 ubiquitination and upregulated EMD stability. We further explored that EMD could inhibit aerobic oxidation and stimulate glycolysis. Mechanistically, via its ß-catenin interaction domain, EMD bound with PDHA, stimulated serine 293 and 300 phosphorylation and inhibited PDHA expression, facilitated glycolysis of glucose that should enter the aerobic oxidation pathway, and EMD ISGylation was essential for EMD-PDHA interaction. In clinical LUAD specimens, EMD was negatively associated with PDHA, while positively associated with EMD ISGylation, tumour stage and diameter. In LUAD with higher glucose level, EMD expression and ISGylation were higher. Collectively, EMD was a stimulator for LUAD by inhibiting aerobic oxidation via interacting with PDHA. Restricting cancer-promoting role of EMD might be helpful for LUAD treatment.
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Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Adenocarcinoma del Pulmón/genética , Glucosa , Humanos , Neoplasias Pulmonares/patología , Lisina , Proteínas de la Membrana , Proteínas Nucleares , Piruvato Deshidrogenasa (Lipoamida) , Serina , beta CateninaRESUMEN
Circulating microRNAs (miRNAs) can be used as noninvasive biomarkers and are also found circulating in body fluids such as blood. Dysregulated miRNA expression is associated with many diseases, including non-small cell lung cancer (NSCLC), and the miRNA assay is helpful in cancer diagnosis, prognosis, and monitoring. In this work, a versatile electrochemical biosensing system is developed for miRNA detection by DNAzyme-cleavage cycling amplification and hybridization chain reaction (HCR) amplification. With cleavage by Mn2+ targeted DNAzyme, DNA-walker can move along the predesigned DNA tracks and contribute to the transduction and enhancement of signals. For the electrochemical process, the formation of multiple G-quadruplex-incorporated long double-stranded DNA (dsDNA/G-quadruplex) structures is triggered through HCR amplification. The introduction of G-quadruplex allows sensitive measurement of miRNA down to 5.68 fM with good specificity. Furthermore, by profiling miRNA in the NSCLC cohort, this designed strategy shows high efficiency (area under the curve (AUC) of 0.879 using receiver operating characteristic (ROC) analysis) with the sensitivity of 80.0% for NSCLC early diagnosis (stage I). For the discrimination of NSCLC and benign disease, the assay displays an AUC of 0.907, superior to six clinically-acceptable protein tumor markers. Therefore, this platform holds promise in clinical application toward NSCLC diagnosis and prognosis.
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Técnicas Biosensibles , Carcinoma de Pulmón de Células no Pequeñas , MicroARN Circulante , ADN Catalítico , Neoplasias Pulmonares , MicroARNs , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , ADN/química , ADN Catalítico/metabolismo , Técnicas Electroquímicas , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , MicroARNs/genéticaRESUMEN
Yes-associated protein (YAP) activation is crucial for tumor formation and development, and its stability is regulated by ubiquitination. ISGylation is a type of ubiquitination like post-translational modification, whereas whether YAP is ISGylated and how ISGylation influences YAP ubiquitination-related function remains uncovered. In addition, YAP can activate glucose metabolism by activating the hexosamine biosynthesis pathway (HBP) and glycolysis, and generate a large number of intermediates to promote tumor proliferation. However, whether YAP stimulates the pentose phosphate pathway (PPP), another tumor-promoting glucose metabolism pathway, and the relationship between this stimulation and ISGylation needs further investigation. Here, we found that YAP was ISGylated and this ISGylation inhibited YAP ubiquitination, proteasome degradation, interaction with-beta-transducin repeat containing E3 ubiquitin-protein ligase (ßTrCP) to promote YAP stability. However, ISGylation-induced pro-YAP effects were abolished by YAP K497R (K, lysine; R, arginine) mutation, suggesting K497 could be the major YAP ISGylation site. In addition, YAP ISGylation promoted cell viability, cell-derived xenograft (CDX) and patient-derived xenograft (PDX) tumor formation. YAP ISGylation also increased downstream genes transcription, including one of the key enzymes of PPP, 6-phosphogluconolactonase (6PGL). Mechanistically, YAP promoted 6PGL transcription by simultaneously recruiting SMAD family member 2 (SMAD2) and TEA domain transcription factor 4 (TEAD4) binding to the 6PGL promoter to activate PPP. In clinical lung adenocarcinoma (LUAD) specimens, we found that YAP ISGylation degree was positively associated with 6PGL mRNA level, especially in high glucose LUAD tissues compared to low glucose LUAD tissues. Collectively, this study suggested that YAP ISGylation is critical for maintaining its stability and further activation of PPP. Targeting ISGylated YAP might be a new choice for hyperglycemia cancer treatment.
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BACKGROUND: Resistance to ferroptosis, a regulated cell death caused by iron-dependent excessive accumulation of lipid peroxides, has recently been linked to lung adenocarcinoma (LUAD). Intracellular antioxidant systems are required for protection against ferroptosis. The purpose of the present study was to investigate whether and how extracellular system desensitizes LUAD cells to ferroptosis. METHODS: Established human lung fibroblasts MRC-5, WI38, and human LUAD H1650, PC9, H1975, H358, A549, and H1299 cell lines, tumor and matched normal adjacent tissues of LUAD, and plasma from healthy individuals and LUAD patients were used in this study. Immunohistochemistry and immunoblotting were used to analyze protein expression, and quantitative reverse transcription-PCR was used to analyze mRNA expression. Cell viability, cell death, and the lipid reactive oxygen species generation were measured to evaluate the responses to ferroptosis. Exosomes were observed using transmission electron microscope. The localization of arachidonic acid (AA) was detected using click chemistry labeling followed by confocal microscopy. Interactions between RNAs and proteins were detected using RNA pull-down, RNA immunoprecipitation and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation methods. Proteomic analysis was used to investigate RNA-regulated proteins, and metabolomic analysis was performed to analyze metabolites. Cell-derived xenograft, patient-derived xenograft, cell-implanted intrapulmonary LUAD mouse models and plasma/tissue specimens from LUAD patients were used to validate the molecular mechanism. RESULTS: Plasma exosome from LUAD patients specifically reduced lipid peroxidation and desensitized LUAD cells to ferroptosis. A potential explanation is that exosomal circRNA_101093 (cir93) maintained an elevation in intracellular cir93 in LUAD to modulate AA, a poly-unsaturated fatty acid critical for ferroptosis-associated increased peroxidation in the plasma membrane. Mechanistically, cir93 interacted with and increased fatty acid-binding protein 3 (FABP3), which transported AA and facilitated its reaction with taurine. Thus, global AA was reduced, whereas N-arachidonoyl taurine (NAT, the product of AA and taurine) was induced. Notably, the role of NAT in suppressing AA incorporation into the plasma membrane was also revealed. In pre-clinical in vivo models, reducing exosome improved ferroptosis-based treatment. CONCLUSION: Exosome and cir93 are essential for desensitizing LUAD cells to ferroptosis, and blocking exosome may be helpful for future LUAD treatment.
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Adenocarcinoma del Pulmón , Exosomas , Ferroptosis , Neoplasias Pulmonares , Adenocarcinoma del Pulmón/genética , Animales , Exosomas/genética , Exosomas/metabolismo , Exosomas/patología , Humanos , Neoplasias Pulmonares/patología , Ratones , Proteómica , ARN Circular/genética , TaurinaRESUMEN
BACKGROUND: Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer. Patient prognosis is poor, and the existing therapeutic strategies for LUAD are far from satisfactory. Recently, targeting N6-methyladenosine (m6A) modification of RNA has been suggested as a potential strategy to impede tumor progression. However, the roles of m6A modification in LUAD tumorigenesis is unknown. METHODS: Global m6A levels and expressions of m6A writers, erasers and readers were evaluated by RNA methylation assay, dot blot, immunoblotting, immunohistochemistry and ELISA in human LUAD, mouse models and cell lines. Cell viability, 3D-spheroid generation, in vivo LUAD formation, experiments in cell- and patient-derived xenograft mice and survival analysis were conducted to explore the impact of m6A on LUAD. The RNA-protein interactions, translation, putative m6A sites and glycolysis were explored in the investigation of the mechanism underlying how m6A stimulates tumorigenesis. RESULTS: The elevation of global m6A level in most human LUAD specimens resulted from the combined upregulation of m6A writer methyltransferase 3 (METTL3) and downregulation of eraser alkB homolog 5 (ALKBH5). Elevated global m6A level was associated with a poor overall survival in LUAD patients. Reducing m6A levels by knocking out METTL3 and overexpressing ALKBH5 suppressed 3D-spheroid generation in LUAD cells and intra-pulmonary tumor formation in mice. Mechanistically, m6A-dependent stimulation of glycolysis and tumorigenesis occurred via enolase 1 (ENO1). ENO1 mRNA was m6A methylated at 359 A, which facilitated it's binding with the m6A reader YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and resulted in enhanced translation of ENO1. ENO1 positively correlated with METTL3 and global m6A levels, and negatively correlated with ALKBH5 in human LUAD. In addition, m6A-dependent elevation of ENO1 was associated with LUAD progression. In preclinical models, tumors with a higher global m6A level showed a more sensitive response to the inhibition of pan-methylation, glycolysis and ENO activity in LUAD. CONCLUSIONS: The m6A-dependent stimulation of glycolysis and tumorigenesis in LUAD is at least partially orchestrated by the upregulation of METTL3, downregulation of ALKBH5, and stimulation of YTHDF1-mediated ENO1 translation. Blocking this mechanism may represent a potential treatment strategy for m6A-dependent LUAD.
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Adenocarcinoma del Pulmón/genética , Glucólisis/genética , Neoplasias Pulmonares/genética , Fosfopiruvato Hidratasa/metabolismo , Proteómica/métodos , ARN Mensajero/genética , Adenocarcinoma del Pulmón/patología , Animales , Carcinogénesis , Modelos Animales de Enfermedad , Humanos , Neoplasias Pulmonares/patología , Ratones , Pronóstico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ferroptosis is an iron- and lipid peroxidation-dependent form of regulated cell death. The release of labile iron is one of the important factors affecting sensitivity to ferroptosis. Yes-associated protein (YAP) controls intracellular iron levels by affecting the transcription of ferritin heavy chain (FTH) and transferrin receptor (TFRC). However, whether YAP regulates iron metabolism through other target genes remains unknown. Here, we observed that the system Xc- inhibitor erastin inhibited the binding of the WW domain and PSY motif between YAP and transcription factor CP2 (TFCP2), and then suppressed the transcription of ferritin light chain (FTL) simultaneously mediated by YAP, TFCP2 and forkhead box A1 (FOXA1). Furthermore, inhibition of FTL expression abrogated ferroptosis-resistance in cells with sustained YAP expression. Unlike FTH, which exhibited first an increase and then a decrease in transcription, FTL transcription continued to decline after the addition of erastin, and a decrease in lysine acetyltransferase 5 (KAT5)-dependent acetylation of FTL was also observed. In lung adenocarcinoma (LUAD) tissues, lipid peroxidation and labile iron decreased, while YAP, TFCP2 and FTL increased compared to their adjacent normal tissues, and the lipid peroxidation marker 4-hydroxynonenal (4-HNE) was negatively correlated with the level of FTL or the degree of LUAD malignancy, but LUAD tissues with lower levels of 4-HNE showed a higher sensitivity to ferroptosis. In conclusion, the findings from this study indicated that the suppression of FTL transcription through the inhibition of the YAP-TFCP2-KAT5 complex could be another mechanism for elevating ferroptosis sensitivity and inducing cell death, and ferroptotic therapy is more likely to achieve better results in LUAD patients with a lower degree of lipid peroxidation.
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Advanced liver fibrosis can lead to cirrhosis, resulting in an accelerated risk of hepatocellular carcinoma and liver failure. Fuzheng Huayu formula (FZHY) is a traditional Chinese medicine formula treated liver fibrosis in China approved by a Chinese State Food and Drug Administration (NO: Z20050546), composed of Salvia Miltiorrhiza bge., Prunus davidiana (Carr.) Franch., cultured Cordyceps sinensis (BerK.) Sacc. Mycelia, Schisandra chinensis (Turcz.) Baill., Pinus massoniana Lamb., and Gynostemma pentaphyllum (Thunb.) Makino. However, the main active substances and mechanism of FZHY are unclear. The aim of this study is to identify a novel anti-fibrotic compound, which consists of the main active ingredients of FZHY, and investigate its mechanism of pharmacological action. The main active ingredients of FZHY were investigated by quantitative analysis of FZHY extracts and FZHY-treated plasma and liver in rats. The anti-fibrotic composition of the main active ingredients was studied through uniform design in vivo, and its mechanism was evaluated in carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced liver fibrosis models in rats and mice, and transforming growth factor beta 1-induced LX-2 cell activation model in vitro. A novel Chinese medicine, namely JY5 formula, consisting of salvianolic acid B, schisantherin A, and amygdalin, the main active ingredients of FZHY, significantly alleviated hepatic hydroxyproline content and collagen deposition in CCl4-and BDL-induced fibrotic liver in rats and mice. In addition, JY5 inhibited the activation of hepatic stellate cells (HSCs) by inactivating Notch signaling in vitro and in vivo. In this study, we found a novel JY5 formula, which exerted anti-hepatic fibrotic effects by inhibiting the Notch signaling pathway, consequently suppressing HSCs activation. These results provide an adequate scientific basis for clinical research and application of the JY5 formula, which may be a potential novel therapeutic candidate for liver fibrosis.
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Chronic kidney disease is an increasingly serious public health problem worldwide. Our recent studies have shown that Huangjinsan has a renal protective effect on chronic kidney disease, but the specific mechanism by which this effect occurs is not clear. To study the therapeutic effect of Huangjinsan on chronic kidney disease and to explore its possible mechanism of action through nontargeted metabolomics methods, a chronic kidney disease rat model was induced by adenine, and the Huangjinsan extract was given by oral gavage. Body weight, the kidney index, pathological sections, and a series of biochemical indicators were measured. High-performance liquid chromatography quadrupole time-of-flight mass spectrometry was used to analyze the changes in the plasma metabolome. Huangjinsan significantly reduced indicators of kidney damage, including total protein, albumin, the total protein to creatinine ratio, and the albumin to creatinine ratio in urine, as well as IL-2, MCP-1α, and blood urea levels in plasma. Based on nontargeted metabolomics, 13 metabolites related to chronic kidney disease were discovered. These metabolites are closely related to glycerophospholipid metabolism, arginine and proline metabolism, and sphingolipid metabolism. We found that Huangjinsan can restore the renal function of adenine-induced chronic kidney disease by regulating the metabolic profile.
Asunto(s)
Adenina/toxicidad , Metabolómica/métodos , Insuficiencia Renal Crónica/prevención & control , Animales , Cromatografía Líquida de Alta Presión/métodos , Masculino , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal Crónica/inducido químicamente , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Pulmonary hypertension (PH) is an extremely serious cardiopulmonary disease, finally leading to progressive right ventricular failure and death. Our previous studies have nominated HLQ2g, a pyrazolo[3,4-b] pyridine derivative stimulating soluble guanylate cyclase (sGC), as a new candidate for the treatment of PH, but the specific mechanism is still not clear. The PH model induced by hypoxia was established in rats. Right ventricular systolic pressure (RVSP) was assessed by jugular vein catheterization. RV weight was the index to evaluate RV hypertrophy. The protein levels of cGMP-dependent protein kinase type I (cGKI), bone morphogenetic protein receptor 2 (BMPR2), phosphorylated Smad1/5/8 (p-Smad1/5/8), and inhibitor of differention 1 (Id1) in pulmonary artery and human pulmonary artery smooth muscle cells (HPASMCs) were determined by western blotting. Cell proliferation and migration were evaluated. In the whole experiment, the first clinically available sGC stimulator Riociguat was used as the reference. In hypoxic PH rat model, elevated RVSP and RV hypertrophy were significantly reduced by HLQ2g treatment. Both Riociguat and HLQ2g attenuated vascular remodeling accompanied with up-regulated cGKI expression and BMP signaling pathway, which was characterized by elevated expression of BMPR2, p-Smad1/5/8, and Id1 in HPH rats. In addition, HLQ2g inhibited proliferation and migration of HPASMCs induced by hypoxia and platelet-derived growth factor (PDGF), restored BMPR2 signaling, which was recalled by Rp-8-Br-PET-cGMPS, the inhibitor of cGKI. In summary, the novel pyrazolo[3,4-b] pyridine derivative HLQ2g can alleviate HPH progression by up-regulating cGKI protein and BMP signaling pathway.
RESUMEN
A convenient catalytic protocol for efficiently constructing indoline-fused tetrahydroisoquinolines based on CuCoFe layered double hydroxide (LDH) has been described. Preliminary mechanistic studies show that indoline-fused tetrahydroisoquinolines are produced via domino coupling/cyclization reactions between tetrahydroisoquinolines and active methylene compounds, including malononitrile, malonates, and analogues. CuCoFe-LDH can accelerate the Csp3-Csp3 and Csp3-Csp2 formation reactions in a single step. The research thus presents a unique opportunity to develop a synthetic methodology for N-containing polycyclic compounds.