Chinese Herbal Medicine Feiyanning Prescription Regulates EMT to Reverse Cisplatin Resistance in Non-small Cell Lung Cancer / 中国实验方剂学杂志

ObjectiveTo observe the effect of Feiyanning prescription （FYN） on cisplatin （DDP） resistance in non-small cell lung cancer （NSCLC） and explore the underlying mechanism. MethodCell counting kit-8 （CCK-8） assay was used to detect the proliferation of A549 and A549/DDP （DDP-resistant） cells treated by DDP （0， 2.0， 4.0， 6.0， 8.0， 10.0 mg⋅L-1） and the proliferation of A549/DDP cells treated by FYN （0， 100， 200， 300， 400， 500， 600 mg⋅L-1）. Based on immunofluorescence staining and Western blot （WB）， the expression of epithelial mesenchymal transition （EMT）-related proteins in A549 and A549/DDP groups was observed. A549/DDP cells were classified into control group， FYN group （200 mg⋅L-1）， DDP group （6.0 mg⋅L-1）， and combination group ［FYN （200 mg⋅L-1） + DDP （6.0 mg⋅L-1）］ and respectively treated with corresponding drugs. Then， invasion ability of each group was examined by transwell assay， and the expression of EMT-related proteins in each group by WB. Moreover， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） and immunofluorescence staining were separately applied to detect the mRNA and protein expression of drug resistance-related factors in each group， respectively. ResultCompared with A549 group， A549/DDP group showed high resistance to DDP （P<0.01）， low expression of E-cadherin， and high protein expression of Vimentin， N-cadherin， and Snail （P<0.05， P<0.01）. As compared with the control group， FYN inhibited the proliferation of A549/DDP cells in a concentration-dependent manner （P<0.01）， and the FYN group， DDP group， and combination group demonstrated low invasion ability （P<0.01）. In addition， the invasion ability in the combination group was particularly lower than that in the DDP group （P<0.01）. The expression of E-cadherin protein was higher and the protein expression of N-cadherin， Vimentin， and Snail was lower in the in FYN group than in the control group （P<0.01）. The protein expression of E-cadherin， N-cadherin， and Vimentin was lower and the expression of Snail was higher in the DDP group than in the control group （P<0.05，P<0.01）. The protein expression of E-cadherin， N-cadherin， Vimentin， and Snail in the combination group decreased as compared with that in the control group （P<0.01）. Compared with the DDP alone， the combination raised the expression of E-cadherin and lowered the protein expression of N-cadherin， Vimentin， and Snail （P<0.01）. The protein and mRNA expression of lung resistance-related protein （LRP） and multidrug resistance 1 （MDR1） was lower and the protein and mRNA expression of topoisomerase Ⅱα （TOPO Ⅱα） was higher in the FYN group than in the control group （P<0.01）. The protein and mRNA expression of LRP， MDR1， and TOPO Ⅱα was higher in the DDP group than in the control group （P<0.01）. The expression of LRP protein and mRNA showed no significant variation， but the protein and mRNA expression of MDR1 and TOPO Ⅱα increased in the combination group compared with those in the control group （P<0.01）. Compared with the DDP group， FYN group and combination group showed low protein and mRNA expression of LRP and MDR1 and high protein and mRNA expression of TOPO Ⅱα （P<0.01）. Compared with FYN， the combination elevated the protein and mRNA expression of LRP， MDR1， and TOPO Ⅱα （P<0.01）. ConclusionFYN prescription can reverse the DDP resistance of NSCLC by modulating EMT.

Palmitoylation in Regulating Fyn Kinase Activity Based on FRET Imaging / 医用生物力学

Objective To investigate the molecular mechanism of palmitoylation modification in regulating the activity of non-receptor tyrosine kinase Fyn. Methods The intracellular Fyn activity was detected by applying fluorescence resonance energy transfer (FRET) technology, and the mechanism was investigated by combining with Fyn palmitoylation deficiency and C-terminal Src kinase ( CSK ) plasmid co-expression. ResultsExperimental data showed that single loss of either of ( C3, C6) palmitoylation sites resulted in higher Fyn activity, and C6 seemed more significant. It is known that CSK membrane translocation occurred after activation. FRET assay confirmed that CSK could down-regulate the activity of Fyn in cells, but could not effectively regulate the activity of Fyn(GSS) with the loss of palmitoylation sites. Conclusions The results in this study support the hypothesis on Fyn regulation by spatial localization, namely, non-palmitoylated Fyn (GSS) is less effective in the inhibitory regulation by CSK on cell membrane, thus promoting constitutive high activity expression

Effects of Panaxadiol on Tau Protein Phosphorylation and Fyn/GluN 2B Signaling Pathway in APP-SH-SY 5Y Cells / 中国药房

OBJECTIVE：To study the effects and mechanism of panaxadiol （PD） on Tau protein phosphorylation in the SH-SY5Y cells transfected with APP gene（APP-SH-SY5Y）. METHODS ：The target of PD and non-receptor tyrosine kinases Fyn was verified by molecular docking. SH-SY 5Y cells were cultured in vitro ，and the APP-SH-SY 5Y cell models and green fluorescent （GFP）-SH-SY5Y cell model （control cell ）was constructed. The expression of Aβ1-42 was detected so as to verify the success of APP-SH-SY5Y cell model. Taking GFP-SH-SY 5Y cells as control ，the effects of 5，10，20，30，40 μmol/L PD and 125，250， 500，1 000，2 000 nmol/L PP 2（Fyn inhibitor ，positive control ）on the survival rate of APP-SH-SY 5Y cells were detected by CCK-8 assay after treated for 24 h，so as to confirm the optimal concentration. The concentration of Ca 2 + ，the ratio fophosphorylated Tau protein （p-Tau）/Tau，phosphorylatedn Src（p-Src）/Fyn and phosphorylated glutamate receptor 2B（p-GluN2B）/ GluN2B were detected in APP-SH-SY 5Y cells after trated with the optimal concentration of PD and PP 2 for 24 h. RESULTS ：The results of molecular simulation docking showed that PD could target Fyn protein. Compared with GFP-SH-SY 5Y cells ，the protein expression of Aβ1-42 in APP-SH-SY 5Y cell were increased significantly （P＜0.01）. The optimal concentration of PD and PP 2 were 20 μmol/L and 500 nmol/L. The 20 μmol/L PD and 500 nmol/L PP 2 could increase the survival rate of the cells and reduced the concentration of Ca 2+，the ratio of p-Tau/Tau ，p-Src/Fyn，and p-GluN 2B/GluN2B. CONCLUSIONS：PD can reduce the the phosphorylation of Tau protein through inhibiting Fyn/GluN 2B signaling pathway.

Activated PKB/GSK-3β synergizes with PKC-δ signaling in attenuating myocardial ischemia/reperfusion injury via potentiation of NRF2 activity: Therapeutic efficacy of dihydrotanshinone-I

@#Disrupted redox status primarily contributes to myocardial ischemia/reperfusion injury (MIRI). NRF2, the endogenous antioxidant regulator, might provide therapeutic benefits. Dihydrotanshinone-I (DT) is an active component in Salvia miltiorrhiza with NRF2 induction potency. This study seeks to validate functional links between NRF2 and cardioprotection of DT and to investigate the molecular mechanism particularly emphasizing on NRF2 cytoplasmic/nuclear translocation. DT potently induced NRF2 nuclear accumulation, ameliorating post-reperfusion injuries via redox alterations. Abrogated cardioprotection in NRF2-deficient mice and cardiomyocytes strongly supports NRF2-dependent cardioprotection of DT. Mechanistically, DT phosphorylated NRF2 at Ser40, rendering its nuclear-import by dissociating from KEAP1 and inhibiting degradation. Importantly, we identified PKC-δ-(Thr505) phosphorylation as primary upstream event triggering NRF2-(Ser40) phosphorylation. Knockdown of PKC-δ dramatically retained NRF2 in cytoplasm, convincing its pivotal role in mediating NRF2 nuclear-import. NRF2 activity was further enhanced by activated PKB/GSK-3β signaling via nuclear-export signal blockage independent of PKC-δ activation. By demonstrating independent modulation of PKC-δ and PKB/GSK-3β/Fyn signaling, we highlight the ability of DT to exploit both nuclear import and export regulation of NRF2 in treating reperfusion injury harboring redox homeostasis alterations. Coactivation of PKC and PKB phenocopied cardioprotection of DT in vitro and in vivo, further supporting the potential applicability of this rationale. Graphical abstract

An Anti-Cancer Drug Candidate CYC116 Suppresses Type I Hypersensitive Immune Responses through the Inhibition of Fyn Kinase in Mast Cells

Mast cells are the most prominent effector cells of Type 1 hypersensitivity immune responses. CYC116 [4-(2-amino-4-methyl-1,3-thiazol-5-yl)-N-[4-(morpholin-4-yl)phenyl] pyrimidin-2-amine] is under development to be used as an anti-cancer drug, but the inhibitory effects of CYC116 on the activation of mast cells and related allergy diseases have not reported as of yet. In this study, we demonstrated, for the first time, that CYC116 inhibited the degranulation of mast cells by antigen stimulation (IC₅₀, ∼1.42 µM). CYC116 also inhibited the secretion of pro-inflammatory cytokines including TNF-α (IC₅₀, ∼1.10 µM), and IL-6 (IC₅₀, ∼1.24 µM). CYC116 inhibited the mast cell-mediated allergic responses, passive cutaneous anaphylaxis (ED50, ∼22.5 mg/kg), and passive systemic anaphylaxis in a dose-dependent manner in laboratory experiments performed on mice. Specifically, CYC116 inhibited the activity of Fyn in mast cells and inhibited the activation of Syk and Syk-dependent signaling proteins including LAT, PLCγ, Akt, and MAP kinases. Our results suggest that CYC116 could be used as an alternative therapeutic medication for mast cell-mediated allergic disorders, such as atopic dermatitis and allergic rhinitis.

Activación de la célula T, alteraciones en el lupus eritematoso sistémico, una revisión narrativa / T-cell activation, alterations in systemic lupus erythematosus: A narrative review

Resumen La activación de los linfocitos T se inicia a través de la presentación de antígenos endógenos o exógenos por células presentadoras de antígenos a través del complejo mayor de histocompatibilidad, el cual se une a un receptor especializado presente en los linfocitos T. Este reconocimiento desencadena una cascada de señalización intracelular que conlleva a un aumento en la expresión de integrinas, modificaciones del citoesqueleto y producción de factores de transcripción involucrados en la liberación de citocinas y mediadores inflamatorios. Uno de los inductores más importantes en la activación celular es el complejo enzimático con acción tirosina cinasa. Las cinasas que pertenecen a la familia SRC (SFK), FYN y LCK están involucradas en un gran número de procesos importantes en la activación, modulación de la respuesta linfocitaria y el desarrollo de enfermedades autoinmunes. La regulación de la señalización de las cinasas, así como de proteínas adaptadoras involucradas en la activación del linfocito T, son fundamentales para mantener el umbral de activación y modulación de la respuesta del linfocito. La fosforilación de sitios de regulación positiva de estas proteínas es importante para permitir una configuración activa de la proteína y de esta forma su máxima capacidad como cinasa. La fosforilación de los sitios de regulación negativa conlleva a una configuración cerrada de la proteína de tal forma que reduce su función de cinasa e inhibe su función. Las alteraciones en la señalización por modificación de algunas proteínas citoplasmáticas se asocian en algunos casos al desarrollo de enfermedades autoinmunes, como el lupus eritematoso sistémico. En condiciones fisiológicas, el complejo receptor de linfocitos T se reagrupa con complejos proteicos que interactúan armónicamente para generar una sen al interna. Los eventos de señalización alterados son en parte los responsables de una expresión anómala de citocinas, entre ellas la interleucina-6 (IL-6), IL-10, IL-2, IFN y CD40 ligando; estas modificaciones alteran la capacidad de los linfocitos T para sobre estimular a los linfocitos B, traduciéndose en un aumento en la producción de autoanticuerpos y en el desencadenamiento de la enfermedad autoinmune.

Abstract The activation of T cells is initiated by the presentation of exogenous or endogenous antigens, by antigen presenting cells through the major histocompatibility complex, which binds to a special receptor on T cells. This acknowledgement triggers a cascade of intracellular signalling that leads to an increase in integrin expression, cytoskeletal modifications, and transcription factors production involved in the liberation of cytokines and inflammatory mediators. One of the most important inducers in cell activation is the enzymatic complex with tyrosine kinase action. The kinases which belong to the SRC (SFK) LCK and FYN family have been involved in a large number of important processes in the activation and modulation of the T cells response, as well as in the development of autoimmune diseases. Regulating the kinases signalling, as well as the adapter proteins involved in T cell activation, is essential for maintaining an activation threshold, as well as the modulation of cell response. The phosphorylation of the positive regulation sites of these proteins is important to allow an active configuration of the protein and thereby its maximum capacity as kinase. The phosphorylation of negative regulation sites leads to a closed configuration of the protein that reduces its kinase function, and thereby inhibits its own function. The alteration in signalling by the modification of certain cytoplasmic proteins in some cases is associated with the development of autoimmune diseases, such as systemic lupus erythematosus. Under physiological conditions the T cell receptor complex regroups with protein complexes that interact harmonically to generate an internal signal. The altered signalling events are partly responsible for an anomalous expression of cytokines, including the interleukin-6 (IL-6), IL-10, IL-2, IFN, and CD40 linking, these modifications affects the cells ability to over-stimulate T and B cells, resulting in an increased production of autoantibodies and the triggering of the autoimmune disease.

The study of the protective effect of panoxadiol on Alzheimer’s disease cells based on network pharmacology / 中国药理学通报

Aim To explore the therapeutic effects of main active compounds of panaxadiol ( PD ) in on Alzheimer’s disease ( AD) via network pharmacologi-cal analysis and Mmolecular docking. Methods A to-tal of 107 prescriptions for AD treatment were screened by using network pharmacology, screening for the high-est frequency of ginseng and its target for AD. Use mo-lecular docking technology was used to find components with the highest score for non-receptor tyrosine kinase ( FYN) docking. Then we successfully estimatedestab-lished AD cell model with overexpressinged APP pro-teins in vitro. Next,the cell viability was detected by MTT assay,the cell damage was detected by LDH as-say,the apoptosis and intracellular Ca2+concentration were detected by flow cytometry, and phosphorylated FYN protein expression was detected by Western blot detection of . phosphorylated FYN protein expression. Results Eighteen active components of Gginseng and 29 AD-related targets were screened by the method of network pharmacology. The results of molecular doc-king showed that PD had strong binding effects with FYN. The results showed that PD could increase the survival rate of cells,reduce the release of LDH,reduce apoptosis,and improve AD cells’ intracellular Ca2+o-verload and reduce the expression of FYN-Y416 pro-tein. Conclusion The experimental results of network pharmacology were are verified and the protective effect of PD on AD may be related to inhibition of FYN signa-ling pathway.

Role of tyrosine kinase of mast cell in allergy and its drug target / 中国药理学通报

The degranulation of mast cells represents a pivotal e-vent in the allergic disorders.The Src family kinases(SFKs)are as a starting signal in the activation of mast cell.Lyn,Fyn and Syk play important regulatory role in the degranulation of mast cells.Regulating SFKs can reduce the degranlation process and inhibit the allergic disorders.Therefore,SFKs inhibitors can be potential drugs in the allergy.It is necessary to study the targeted medicine of SFKs,which will be a new direction of drug develop-ment.

Mouse Fyn induces pseudopodium formation in Chinese hamster ovary cells

Molecular mechanisms underlying the effects of Fyn on cell morphology, pseudopodium movement, and cell migration were investigated. The Fyn gene was subcloned into pEGFP-N1 to produce pEGFP-N1-Fyn. Chinese hamster ovary (CHO) cells were transfected with pEGFP-N1-Fyn. The expression of Fyn mRNA and proteins was monitored by reverse transcription-PCR and Western blotting. Additionally, transfected cells were stained with 4',6-diamidino-2-phenylindole and a series of time-lapse images was taken. Sequences of the recombinant plasmids pMD18-T-Fyn and pEGFP-N1-Fyn were confirmed by sequence identification using National Center for Biotechnology Information in USA, and Fyn expression was detected by RT-PCR and Western blotting. The morphology of CHO cells transfected with the recombinant vector was significantly altered. Fyn expression induced filopodia and lamellipodia formation. Based on these results, we concluded that overexpression of mouse Fyn induces the formation of filopodia and lamellipodia in CHO cells, and promotes cell movement.

The Serotonin-6 Receptor as a Novel Therapeutic Target

Serotonin (5-hydroxytryptamine, 5-HT) is an important neurotransmitter that is found in both the central and peripheral nervous systems. 5-HT mediates its diverse physiological responses through 7 different 5-HT receptor families: 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7 receptors. Among them, the 5-HT6 receptor (5-HT6R) is the most recently cloned serotonin receptor and plays important roles in the central nervous system (CNS) and in the etiology of neurological diseases. Compared to other 5-HT receptors, the 5-HT6R has been considered as an attractive CNS therapeutic target because it is expressed exclusively in the CNS and has no known isoforms. This review evaluates in detail the role of the 5-HT6R in the physiology and pathophysiology of the CNS and the potential usefulness of 5-HT6R ligands in the development of therapeutic strategies for the treatment of CNS disorders. Preclinical studies provide support for the use of 5-HT6R ligands as promising medications to treat the cognitive dysfunction associated with Alzheimer's disease, obesity, depression, and anxiety.

Assessment of Relationship between Fyn-related Kinase Gene Polymorphisms and Overweight/Obesity in Korean Population

The fyn-related kinase (FRK) belongs to the tyrosine kinase family of protein kinases. Recent studies have shown that Frk affects pancreatic beta cell number during embryogenesis and promotes beta cell cytotoxic signals in response to streptozotocin. To investigate the genetic association between FRK polymorphisms and the risk of obesity in Korean population, single nucleotide polymorphisms (SNPs) in the FRK gene region were selected and analyzed. The body mass index (BMI) was calculated, and biochemical data (systolic blood pressure, diastolic blood pressure, hemoglobin A1C, triglyceride, total cholesterol, high density lipoprotein, and low density lipoprotein) of blood sample from each subject were also measured. One hundred fifty five healthy control and 204 overweight/obesity subjects were recruited. Genotype frequencies of six SNPs [rs6568920 (+8391G>A), rs3756772 (+56780A>G), rs3798234 (+75687C>T), rs9384970 (+68506G>A), rs1933739 (+72978G>A), and rs9400883 (+75809A>G)] in the FRK gene were determined by Affymetrix Targeted Genotyping Chip data. According to the classification of Korean Society for the Study of Obesity, control (BMI 18 to or =23) subjects were recruited. For the analysis of genetic data, EM algorithm, SNPStats, Haploview, HapAnalyzer, SNPAnalyzer, and Helixtree programs were used. Multiple logistic regression analysis (codominant, dominant, and recessive models) was performed. Age and gender as covariates were adjusted. For biochemical data, Student's t test was used. The mean value of BMI in the control and overweigh/obesity groups was 21.1+/-1.2 (mean+/-SD) and 25.6+/-2.0, respectively. All biochemical data of the overweight/obesity group were statistically significance, compared with the control group. Among six SNPs, two linkage disequilibrium (LD) blocks were discovered. One block consisted of rs1933739 and rs9400883, and the other comprised rs3756772 and rs3798234. One SNP (rs9384970, +68506G>A) showed an association with overweight/obesity in the codominant model (p=0.03). Interestingly, the AA genotype distribution in the overweight/obesity group (n=7, 3.5%) was higher than those in the control group (n=1, 0.6%), which is not found in either Japanese or Chinese subjects. Therefore, the AA genotype of rs9384970 may be a risk factor for development of obesity in Korean population. The results suggest that FRK may be associated with overweight/obesity in Korean population.

Fyn Tyrosine Kinase-mediated Tyrosine Phosphorylation of Roundabout (Robo), the Slit Receptor / 대한해부학회지

In this study, the molecular mechanism of tyrosine phosphorylation of Roundabout (Robo), the transmembrane receptor for slits, was investigated. The tyrosine phosphorylation of intracellular portion of Robo was increased by the treatment of tyrosine phosphatase inhibitors in human embryonic kidney cells transfected with Robo. The Robo tyrosine phosphorylation was inhibited by the treatment of Src family kinase inhibitor, PP2. The co-transfection of constitutively active form of Fyn, not the dominant negative form of Fyn, and Robo dramatically enhanced the tyrosine phosphorylation of Robo. Furthermore, the SH2 domain of Fyn, which binds to phosphorylated tyrosine residues, interact with Robo, and the interaction was increased by the inhibition of tyrosine phosphatases. These findings indicate that the tyrosine phosphorylation of Robo is regulated by Fyn.

Screening of the protein interacting with inhibitor of differentiation 1′ using yeast two hybrid method / 第三军医大学学报

Objective To obtain the protein interacting with inhibitor of differentiation1′(Id1′). Methods The recombinant bait plasmid pHybLex/Zeo Id1′ was constructed and transformed into yeast strain EGY48/ pSH18 34 to test pHybLex/Zeo Id1′ for non specific activation. Adult human lung cDNA libraries were screened to obtain true positive library plasmid. The true positive library clone was obtained by sequencing and basic local alignment sequence tool (BLAST). Results The recombinant bait vector, named as pHybLex/Zeo Id1′, was confirmed by sequencing. pHybLex/Zeo Id1′ was transformed into yeast strain EGY48/pSH18 34 and the transformants had no autonomously activated reporter genes. One true positive clone, obtained by screening of the adult human lung cDNA libraries, was confirmed to be Fyn by sequencing and BLAST. Conclusion Id1′ can interact with Fyn.

Mice Lacking Protein Tyrosine Kinase Fyn Develop a T Helper-Type 1 Response and Resist \it{Leishmania major} Infection

Fyn is a Src family protein tyrosine kinase associated with TCR/CD3 complex. Fyn appears to play a role in the activation of T cells based on its enzymatic activation and tyrosine phosphorylation following the ligation of TCR/CD3, and it also plays a critical role in the calcium flux and interleukin-2 (IL-2) production. The protective response against murine Leishmania major infection is associated with the T helper-type 1 (Th1) responses and the ability to modulate Th1 cytokines such as IL-2 and interferon-γ, respectively. The role of Fyn tyrosine kinase in vivo was directly examined by the response to infection with L. major in C57BL/6 fyn-deficient mice. Despite the absence of Fyn, the mice remained resistant to this infection with only mild lesion development, and, they demonstrated Th1 responses as assessed by the delayed-type hypersensitivity response and cytokine milieu. The findings in the fyn-deficient mice failed to support a relationship between the anticipated functions of Fyn in vitro and the immune response to L. major infection in vivo. As a result, in leishmanial disease, Fyn probably plays a minor role in the protective immune response and is, therefore, not a key factor in such a response.