Lenvatinib down-regulates IGF1R/Mek/Erk signaling pathway in the treatment of regorafenib-resistant hepatocellular carcinoma / 中华肿瘤杂志

Objective: To investigate the therapeutic effect and mechanism of lenvatinib on regorafenib-resistant hepatocellular carcinoma cells. Methods: CCK-8 and clone formation assay were used to observe the inhibitory effect of lenvatinib on the growth of hepatocellular carcinoma cells. Flow cytometry was used to detect the apoptosis of regorafenib-resistant hepatocellular carcinoma cells treated with lenvatinib. The expression levels of related proteins were detected by western blot and immunohistochemical staining. The inhibitory effect of lenvatinib on the tumor formation ability of regorafenib-resistant hepatocellular carcinoma cells in vivo was observed by subcutaneous tumor formation experiment in mice. Results: CCK-8 and clone formation assay showed that lenvatinib could inhibit the proliferation of regorafenib-resistant hepatocellular carcinoma cells. The number of clones of HepG2, SMMC7721 and regorafenib-resistant HepG2, SMMC7721 cells in lenvatinib group (120.67±11.06, 53.00±11.14, 55.00±9.54, 78.67±14.64) were all lower than those in control group (478.00±24.52, 566.00±27.87, 333.67±7.02, 210.00±12.77, all P<0.05). Flow cytometry showed that lenvatinib could promote apoptosis of regorafenib-resistant hepatocellular carcinoma cells, the apoptosis rates of HepG2, SMMC7721 and regorafenib-resistant HepG2, SMMC7721 cells in lenvatinib group [(12.30±0.70)%, (9.83±0.38)%, (15.90±1.32)%, (10.60±0.00)%] were all higher than those in control group [(7.50±0.87)%, (5.00±1.21)%, (8.10±1.61)%, (7.05±0.78)%, all P<0.05]. The apoptosis-related protein levels suggested that apoptosis was increased in the treatment of lenvatinib. The animal study showed that lenvatinib can inhibit the growth of regorafenib-resistant cells in vivo. Immunohistochemistry and western blot results showed that lenvatinib could down-regulate the abnormally activated IGF1R/Mek/Erk signaling pathway in regorafenib-resistant cells. Conclusion: Lenvatinib can reverse regorafenib resistance in hepatocellular carcinoma, possibly by down-regulating IGF1R/Mek/Erk signaling pathway.

Isonlosinine induces autophagy of PC9 cells by ERK signaling pathway in lung cancer / 中国临床药理学与治疗学

AIM: To investigate the effects of isonlosinine on proliferation, invasion, migration and autophagy of PC9 cells in non-small cell lung cancer (NSCLC), and to explore its possible molecular mechanism. METHODS: The effect of Isoliensinine on the proliferation of PC9 cells were measured by CCK-8 assay, and the IC50 value of PC9 cells was calculated. Wound healing and transwell experiments were used to study the effect of Isoliensinine on migration and invasion of PC9 cells in vitro, respectively. The formation of autophagosome was observed with acridine orange staining under fluorescence microscope. The expression levels of LC3, pERK and ERK in the PC9 cells were determined by western blot. RESULTS: Isonlosinine significantly inhibited the proliferation of PC9 cells. IC50 of isonlosinine (24 h) for the PC9 cells was 34.11 µmol / L. Isonlosinine significantly inhibited cell migration and invasion of PC9 cells. The results of acridine orange fluorescent staining showed that the number of the intracellular acid dye follicular bright red fluorescence in PC9 cells was significantly increased after isonlosinine treatment, while the autophagic lysosomes were rarely observed in control group. The expression of LC3-II in PC9 cells was significantly enhanced after isonlosinine treatment. Furthermore, molecular mechanism study showed that isonlosinine could activate the expression level of p-ERK. CONCLUSION: Isoliensinine significantly inhibits the proliferation, migration and invasion, and induces autophagy of PC9 cells, which may be correlated with the activation of ERK signaling pathway.

Exploration on mechanism of anti-coronavirus of Sophora flavescens through multi-pathway / 中草药

The coronavirus disease 2019 (COVID-19), originated in Wuhan city, Hubei Province, China in December 2019, and then quickly spread to most provinces and regions in China and even spread to many countries abroad. COVID-19 is characterized by wide epidemic, strong infectivity, rapid onset and critical condition. In the face of this epidemic, all parts of the country quickly set off a peak in the fight against COVID-19, but no effective drug for COVID-19 has been developed in the short term. Recently, many hospitals have combined traditional Chinese medicine with western medicine in treatment, and the clinical effect is remarkable, which proves the antiviral effect of traditional Chinese medicine. A large number of pharmacological and clinical studies have proved that the Chinese materia medica S. flavescens has significant antiviral effect. In this paper, the mechanism of anti-coronavirus effect of S. flavescens is expounded from multiple pathways, such as type I interferon, NF-κB signal pathway, ERK signal pathway, PI3K/Akt signal pathway and matrine alkaloids, etc. It is intended to provide reference for clinical treatment of coronavirus infection pneumonia and research and development of related drugs of S. flavescens.

Effects of Src on cervical cancer cells proliferation and apoptosis through ERK signal transduction pathway / 中华流行病学杂志

Objective To explore the effect of Src on cervical cancer cells through ERK signal transduction pathway.Methods Experimental study was carried out in vitro.Cervical cancer cell lines Hela (HPV-positive) and C33A (HPV-negative) were treated with Src kinase inhibitor PP2.Then,the cell cycle and apoptosis of each group were evaluated by using flow cytometry (FCM).Western blotting and Real-time PCR were used to detect the levels of the expression of ERK 1/2,c-Fos and c-Jun mRNA and protein respectively.The database was established and analyzed with SPSS statistical software (version 20.0).Results After down-regulating Src,the cell proliferation was inhibited and cell apoptosis was induced.The proportions of G0/G 1 stage of Hela and C33A cell in cell cycle increased while G2/M and S stages decreased.Meanwhile,the mRNA levels of ERK 1,ERK 2,c-Fos and c-Jun increased.And the expression levels of ERK 1/2,phosphorylated ERK 1/2 (p-ERK 1/2)and phosphorylated c-Fos (p-c-Fos) protein decreased,while c-Jun and phosphorylated c-Jun (p-c-Jun)protein expression increased.In addtion,the change level of Hela cell,p-ERK 1/2 and c-Fos protein were lower than that of C33A cell before and after the Src inhibition.Conclusions Src,involved in regulating the expression of key factors of the ERK signal transduction pathway including p-ERK 1/2 and p-c-Fos,might be capable of promoting the proliferation of cervical cancer cells and inhibiting their apoptosis.The infection with HPV might have adjustable effect on this process.

Effects of Src on cervical cancer cells proliferation and apoptosis through ERK signal transduction pathway / 中华流行病学杂志

Objective To explore the effect of Src on cervical cancer cells through ERK signal transduction pathway.Methods Experimental study was carried out in vitro.Cervical cancer cell lines Hela (HPV-positive) and C33A (HPV-negative) were treated with Src kinase inhibitor PP2.Then,the cell cycle and apoptosis of each group were evaluated by using flow cytometry (FCM).Western blotting and Real-time PCR were used to detect the levels of the expression of ERK 1/2,c-Fos and c-Jun mRNA and protein respectively.The database was established and analyzed with SPSS statistical software (version 20.0).Results After down-regulating Src,the cell proliferation was inhibited and cell apoptosis was induced.The proportions of G0/G 1 stage of Hela and C33A cell in cell cycle increased while G2/M and S stages decreased.Meanwhile,the mRNA levels of ERK 1,ERK 2,c-Fos and c-Jun increased.And the expression levels of ERK 1/2,phosphorylated ERK 1/2 (p-ERK 1/2)and phosphorylated c-Fos (p-c-Fos) protein decreased,while c-Jun and phosphorylated c-Jun (p-c-Jun)protein expression increased.In addtion,the change level of Hela cell,p-ERK 1/2 and c-Fos protein were lower than that of C33A cell before and after the Src inhibition.Conclusions Src,involved in regulating the expression of key factors of the ERK signal transduction pathway including p-ERK 1/2 and p-c-Fos,might be capable of promoting the proliferation of cervical cancer cells and inhibiting their apoptosis.The infection with HPV might have adjustable effect on this process.

Study on the Mechanism of Autophagy of HeLa Cells in Human Cervical Cancer Induced by Fucoxanthin / 中国药房

OBJECTIVE:To study the mechanism of autophagy of HeLa cell in human cervical cancer induced by fucoxanthin. METHODS:MTT was adopted to determine the cell activity and calculate the inhibition rate after HeLa cells were cultured by 1, 10,20,40 and 80 μmol/L of fucoxanthin for 48 h. Flow cytometry was used to determine the cell cycle and apoptosis rate after HeLa cells were cultured by 0(blank control),10,20 and 40 μmol/L of fucoxanthin for 48 h;acridine orange staining,LysoTrack-er Red staining,HeLa-GFP-LC3 method and fluorescence microscope were used to observe the autophagy state;Western blot was used to determine the expressions of proteins related to autophagy. RESULTS:The cells had obvious inhibition effect on the cell growth after being cultured by 0,10,20,40 and 80 μmol/L of fucoxanthin. The cell was blocked in G0/G1 stage after being cul-tured by 10,20 and 40μmol/L of fucoxanthin,and had no obvious effect on the apoptosis rate;autophagy degree was increased af-ter the cells were cultured by 40 μmol/L fucoxanthin for 48 h. Compared with blank control,40 μmol/L fucoxanthin could promote LC3Ⅰ transferring into LC3Ⅱ and the expressions of Beclin-1,PTEN,p21;and inhibit the phosphorylation of p-Akt,p-p70S6K and p-mTOR. The pre-treatment by autophagy inhibitor 3-methyladenine(5 mmol/L)could reverse the autophagy of HeLa cells in-duced by fucoxanthin;U0126 could partly reverse the autophagy of HeLa cells induced by fucoxanthin. CONCLUSIONS:Fucoxan-thin can induce the authphagy of HeLa cells by inhibiting Akt signaling pathway and activating MEK/ERK signaling pathway.

The study of the mechanism in the endochondral ossification of bone mesenchymal stem cells in mice by continued enhanced function of fibroblast growth factor type Ⅱ receptor mutation / 重庆医学

Objective To study the role of ERK signal pathway in the endochondral ossification of bone mesenchymal stem cells ,and to explore the mechanism of ERK signal pathway in persistent enhanced FGFR2 function on development of mice BMSCs by a knock‐in mouse model with the FGFR2S252W/+ .Methods Mice with neo‐FGFR2 gain‐of‐function mutation were mated with EII‐Cre mice .The genotype of generation mice were identified by PCR and divided into wild type group and mutant type group ac‐cording to their genotype .6 week‐old mice were sacrificed to receive bone mesenchymal stem cells .The western blot was used to compare the level of P‐ERK and ERK and the RT‐PCR was applied to detect the genes of Col2 ,Col10 ,OC ,OP in chondrogenic dif‐ferentiation medium of BMSCs .Then ,treatment of cultured BMSCs with PD98059 ,compare the changes of genes and utilize the in vitro culture of long bones detect the role of ERK signal pathway in the endochondral ossification by FGFR2 mutant .Results We successfully derive BMSCs from FGFR2S252W/+ mutant mice and found the activity of ERK signal pathway of FGFR2S252W/+ was en‐hanced .After been cultured in chondrogenic differentiation medium ,the expressions of the BMSCs mRNA of Col2 ,Col10 from mu‐tant group were decreased ,while the expressions of OC ,OP were increased .Those OC ,OP genes levels showed an increased treated by PD98059 .Using in vitro culture of long bones ,we found the retardation of total length growth of long bones has been rescued by PD98059 treatment ,suggesting that ERK signal pathways was responsible for the retarded long bone development in FGFRS252W/+mice .Conclusion The results indicate these effects are mediated by the ERK signal pathway .Furthermore ,the retardation of long bones has been recued by PD98059 treatment ,suggesting that ERK signal pathway is responsible for the retarded long bone devel‐opment in FGFR2S252W/+ mice .

The Effects ofβ-Sheet Breaker Peptide H102 on ERK Signal Transduction Pathway in Brain of PAP Double Transgenic Mice / 天津医药

Objective To investigate the activation of β-sheet breaker peptide H102 on ERK signal transduction pathway in brain of PAP double transgenic mice. Methods PAP double transgenic mice were randomly divided into model group and H102 treatment group (n=10 for each group). A group of C57BL/6J mice with the same genetic background was served as controls. H102 (5.8 mg/kg) 5 μL was infused by intranasal administration to mice in H102 treatment group, and equal volume of blank solution of H102 (chitosan, BSA) was given to mice in control group and model group. The ability of spatial reference memory was tested by Morris water maze after 30 days of treatment. Then immunohistochemistry tests and Western blot technique were used to detect the content of RAS, P-MEK and P-ERK proteins in mouse brain. Results (1) The ability of learning and memory was significantly lower in model group than that of control group. The ability of learning and memory was significantly improved in treatment group than that in model group (P<0.05). (2) The contents of RAS, P-MEK and P-ERK in mouse brain were significantly lower in model group than those of control group, and these protein ex-pressions were significantly increased in treatment group than those in model group (P<0.01). Conclusion β-sheet break-er peptide H102 can activate ERK signal transduction pathway in brain of PAP double transgenic mice, increase PAS, P-MEK and P-ERK levels in nerve cells, and improve the ability of learning and memory in PAP mice.

Effect of 2,3,5,4′-tetrahydroxy Stilbene-2-O-β-D-glucoside on Activation of ERK in APP Transgenic Model of Alzheimer's Disease / 中国康复理论与实践

@#Objective To investigate the deficit of extracellular-signal regulated kinase (ERK)1/2 activation in the different age of Alzheimer's disease (AD)-like animal model and the protective effect of 2,3,5,4′-tetrahydroxy stilbene-2-O-β-D-glucoside(TSG), which is the main component of Polygonum multiflorum, on ERK activation. Methods A generally accepted animal model of AD - PDAPPV717I transgenic (Tg) mouse was observed from 4 to 16 months old. Tg mice were randomly divided into 3 model groups(4, 10 and 16 months old mice)and TSG treated (at doses 120 and 240 μmol/kg/d) groups. TSG was administered to some Tg mice with an age range 4-10 months. In untreated 10 months old Tg mice, the TSG was administrated to those falling in the age range 10-16 months. For the control group we adopted the same age and background C57BL/6J mice. The ERK1/2 expression and phosphorylation were detected by Western blotting.Results In the 4-month-old PDAPPV717I Tg mice, phosphorylation of ERK1/2 decreased significantly in hippocampus and cortex compared with age matched control. In the 10-month-old Tg mice, decrease of ERK1/2 activation was aggravated in cortex but was less in hippocampus. The treatment of TSG at the doses of 120 and 240 μmol/kg for 6 months (from the age of 4 to 10 months) significantly up-regulated ERK1/2 activation in Tg mice. In the 16-month-old Tg mice, over-activation of ERK1/2 occurred in both hippocampus and cortex. The transgenic mice treated by TSG for 6 months (from the age of 10 months to 16 months) showed significant inhibition of over-activation of ERK1/2. Expression of total ERK1/2 showed no difference among control, Tg model and TSG treated groups.Conclusion PDAPPV717I transgenic mice with an age range from 4 to 16 months revealed the time-dependent deficit of ERK1/2 activation. TSG can bring the down or over activation of ERK1/2 into normal. Because ERK1/2 activation plays the crucial role in cellular signal transduction and learning-memory ability, TSG may have beneficial potential to the prevention and treatment of neurodegenerative diseases like AD.

Regulation of proliferation and apoptosis of K562 cells by changes of Ras-ERK pathway induced by simvastatin / 国际肿瘤学杂志

Objective To investigate the changes in molecule levels of Ras-ERK signal pathway of K562 cells treated with simvastatin in vitro,and to illustrate that simvastatin inducing the changes in molecule levels of Ras- ERK signal pathway is involved in regulation of proliferation and apoptosis of K562 cells. Methods K562 cells,the chronic myelocytic leukemia(CML) cell lines,were cultured and treated with simvastatin in vitro and proliferation activity of K562 cells was detected by MTT. The changes of apoptosis rate and cell cycle of K562 cells were measured by flow cytometry(FCM). The molecular changes of Ras-ERK signal pathway were analyzed by RT- PCR in transcriptional level. Results The proliferation of K562 cells was inhibited by simvastatin,and G_0-G_1 arrested in K562 cells and significant apoptosis rate was observed with FCM. Most molecules of Ras- ERK signal pathway expressed differentially at transctiptional level. Conclusion Simvastatin probablely inhibit proliferation and induces apoptosis of K562 cells,depending on Ras-ERK signal pathway which is involved in cell proliferation and apoptosis.

IFN-alpha Downregulates ERK Phosphorylation of CD4+ T Lymphocytes in Systemic Lupus Erythematosus / 대한류마티스학회지

OBJECTIVE: CD4+ T cells from patients with systemic lupus erythematosus (SLE) display aberrant TCR signaling and IFN-alpha plays critical roles in the pathogenesis of SLE; however, the effects of IFN-alpha on disease-associated TCR signaling defects remain unknown. This study investigated the ERK phosphorylation during TCR triggering and the effects of IFN-alpha on ERK signaling in CD4+ T cells. METHODS: CD4+ T lymphocytes were sorted from PBMC using magnetic beads in patients with SLE who met the 1982 revised ACR criteria for SLE and age-matched healthy controls. The phosphorylation of ERK 1/2 was analyzed by flow cytometry and mean fluorescent intensity was measured to define the degree of phosphorylation of ERK. In some experiments, anti-CD3 stimulation was performed after preincubation with patient or control serum, diluted in tissue culture media, with or without addition of an anti-IFN-alpha antibody. The serum level of IFN-alpha was measured by ELISA. RESULTS: ERK-1/2 phosphorylation was decreased in CD4+ T cells of lupus patients than healthy controls and associated with disease activity. Pre-incubation of control CD4+ T cells with allogeneic lupus plasma decreased ERK-1/2 phosphorylation more than allogeneic control and RA plasma and this was reversed by anti-IFN-alpha Ab. Accordingly, ERK-1/2 phosphorylation was decreased in control CD4+ T cells pre-incubation with lupus plasma with high IFN-alpha levels more than lupus plasma with non-detectable IFN-alpha levels. Recombinant IFN-alpha inhibited TCR-mediated ERK-1/2 phosphorylation dose-dependently. CONCLUSION: These results suggest that IFN-alpha stimulation in vivo may underlie the aberrant TCR-mediated MAPK signaling in lupus CD4+ T cells and associated with disease pathogenesis.

Preliminary study of aspisol inhibition on proliferation of breast cancer cells / 中国癌症杂志

Background and purpose:Many studies indicate that nonsteroidal anti-inflammatory drugs(NSAIDs) may inhibit cancer growth.However,the molecular mechanisms may involve different pathway and still remain unclear.The aim of this experiment was to investigate the influence of aspisol against breast cancer lines,including MDA-MB-231(estrogen receptor-negative),MCF-7(estrogen receptor-positive),and reveal the potential signaling pathway mechanism of aspisol effect on breast cancer lines.Methods:MDA-MB-231 and MCF-7 human breast cancer cell lines were treated with aspisol in vitro.Cell proliferation was evaluated by MTT assay and rate of apoptosis were determined by flow cytometry.Extracellular signal regulated kinase(ERK),phosphor-ERK(P-ERK) protein expressed in breast cancer cell lines were analyzed by Western blot.Results:①The results of MTT assay demonstrated that the growth of MDA-MB-231,MCF-7 cells were inhibited by aspisol in a time-and dose-dependent fashion(P0.05).Conclusions:aspisol inhibits proliferation and induces apoptosis not only in ER-positive but also in ER-negative breast cancer cells.The mechanism may relate to ERK signal pathway.

Study of MEK2/ERK signal transduction pathway in the colorectal cancer / 中国普通外科杂志

Objective To study the role of MEK2/ERK signal transduction pathway in the development of colorectal cancer. Methods (1)Western blot analysis was performed on cancerous tissues and adjacent colonic tissues in 45 patients with colorectal cancers.(2)Human colorectal cancer cell line SW480 was treated with MEK inhibitor,and then MTT assay was used to measure the SW480 cells proliferation;and the expression of MEK2, p-ERK and C-myc in SW480 cells were measured by western blot. Results MEK2 protein level was increased in colorectal cancer compared with adjacent mucosa (P