Research progress on function and molecular mechanism of transcription factor ELK1 of Ets / 中国生物制品学杂志

@#Ets transcription factor ELK 1,a member of the ternary complex factor(TCF) subfamily in the Ets family,is directly downstream signal of MAPK/ERK pathway and is activated by phosphorylation to execute the function of ERK signal.ELK1,which is highly expressed and phosphorylated in stem cells and tumor cells,plays a role in promoting proliferation,inhibiting apoptosis and differentiation in stem cells.In tumor cells,ELK1 has shown to promote proliferation,migration,and inhibit apoptosis.In neural cells,ELK1 is involved in long-term memory,learning,addiction and other functions.In this paper,the research progress on the main structure and basic biological characteristics of ELK1,the function and mechanism of ELK 1 in tumor cells,stem cells and nerve cells are reviewed in order to provide new ideas for the follow-up research.

The antitumor mechanism of ginsenoside Rg3 activating MAPK/ERK pathway to promote T cell function / 中国药理学通报

Aim To elucidate the mechanism by which Rg3 regulates the function of CD8

Type III secretory protein SINC of Chlamydia psittaci promotes host cell autophagy by activating the MAPK/ERK signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effects of SINC, a secreted protein of Chlamydia psittaci, on autophagy of host cells and the role of MAPK/ERK signaling pathway in mediating SINC-induced autophagy.@*METHODS@#RAW 264.7 cells treated with recombinant SINC were examined for changes in expression levels of LC3-II, Beclin-1, phosphorylated and total ERK1/2 using Western blotting. The expression level of LC3 in the treated cells was detected using immunofluorescence analysis, and the formation of autophagosomes and autolysosomes was observed with transmission electron microscopy (TEM). The effect of pretreatment with U0126 (a specific ERK inhibitor) on the expression levels of LC3-II and Beclin-1 in RAW 264.7 cells exposed to different concentrations of SINC was examined using Western blotting, and LC3 puncta in the cells was detected with immunofluorescence analysis.@*RESULTS@#The expression levels of LC3-II and Beclin-1 were the highest in RAW 264.7 cells treated with 2 μg/mL SINC for 12h. Immunofluorescence analysis showed exposure to SINC significantly increased the number of cells containing LC3 puncta, where the presence of autophagosomes and autolysosomes was detected. Exposure to 2 μg/mL SINC for 15 min resulted in the most significant increase of the ratios of p-ERK1/2/ERK1/2 in RAW 264.7 cells. Pretreatment of the cells with U0126 prior to SINC exposure significantly decreased the ratio of p-ERK1/2/ERK1/2, lowered the expression levels of LC3-II and Beclin-1, and decreased LC3 aggregation in the cells.@*CONCLUSIONS@#SINC exposure can induce autophagy in RAW 264.7 cells by activating the MAPK/ERK signaling pathway.

Mechanism of Gentisic Acid on Rheumatoid Arthritis Based on miR-19b-3p/RAF1 Axis / 中国结合医学杂志

OBJECTIVE@#To investigate the therapeutic effect of gentisic acid (GA) on rheumatoid arthritis (RA) based on the miR-19b-3p/RAF1 axis.@*METHODS@#The cell counting kit-8 method was used to detect the growth inhibitory effect of different concentrations of GA on MH7A cells, and the drug concentration of GA was determined in the experiment. The quantificational real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-19b-3p and RAF1. RAF1, extracellular regulated protein kinases1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2) were examined by Western blotting. Three methods (dual-luciferase assay, qRT-PCR and Western blot analysis) were used to verify miR-19b-3p targeting RAF1. Flow cytometry was performed to detect MH7A cell apoptosis. Transwell and wound healing assays were used to determine the invasion and migration capacities of MH7A cells.@*RESULTS@#The growth of MH7A cells was gradually inhibited with increasing GA concentration. When the GA concentration exceeded 80 mmol/L, GA was significantly cytotoxic to MH7A cells, so the half maximal inhibitory concentration of GA for MH7A cells was calculated as 67.019 mmol/L. GA upregulated miR-19b-3p expression, downregulated RAF1 expression, inhibited ERK1/2 phosphorylation, induced MH7A cell apoptosis and suppressed MH7A cell invasion and migration (P<0.05 or P<0.01). RAF1 was identified as the target of miR-19b-3p and reversed inhibitory effects on miR-19b-3p expression (P<0.05 or P<0.01). The miR-19b-3p inhibitor upregulated RAF1 expression and ERK1/2 phosphorylation, suppressed MH7A cell apoptosis and induced MH7A cell invasion and migration (P<0.01).@*CONCLUSION@#GA regulated miR-19b-3p/RAF1 axis to mediate ERK pathway and inhibit the development of RA.

Mechanism of albiflorin in improvement of Alzheimer's disease based on network pharmacology and in vitro experiments / 中国中药杂志

This study aimed to explore the mechanism of albiflorin in the treatment of Alzheimer's disease(AD) based on network pharmacology, molecular docking, and in vitro experiments. Network pharmacology was used to predict the potential targets and pathways of albiflorin against AD, and molecular docking technology was used to verify the binding affinity of albiflorin to key target proteins. Finally, the AD cell model was induced by Aβ_(25-35) in rat pheochromocytoma(PC12) cells and intervened by albiflorin to validate core targets and pathways. The results of network pharmacological analysis showed that albiflorin acted on key targets such as mitogen-activated protein kinase-1(MAPK1 or ERK2), albumin(ALB), epidermal growth factor receptor(EGFR), caspase-3(CASP3), and sodium-dependent serotonin transporter(SLC6A4), and signaling pathways such as MAPK, cAMP, and cGMP-PKG. The results of molecular docking showed that albiflorin had strong binding affinity to MAPK1(ERK2). In vitro experiments showed that compared with the blank group, the model group showed decreased cell viability, decreased expression level of B-cell lymphoma 2(Bcl-2), increased Bcl-2-associated X protein(Bax), and reduced phosphorylation level of extracellular signal-regulated kinase 1/2(ERK1/2) and the relative expression ratio of p-ERK1/2 to ERK1/2. Compared with the model group, the albiflorin group showed potentiated cell viability, up-regulated expression of Bcl-2, down-regulated Bax, and increased phosphorylation level of ERK1/2 and the relative expression ratio of p-ERK1/2 to ERK1/2. These results suggest that the mechanism of albiflorin against AD may be related to its activation of the MAPK/ERK signaling pathway and its inhibition of neuronal apoptosis.

Transgelin inhibits proliferation and invasion of human thyroid cancer cells by blocking the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway / 中华内分泌代谢杂志

Objective:To explore the role of transgelin(TAGLN) in the occurrence and development of papillary thyroid carcinoma (PTC) and its possible signal pathway.Methods:One hundred cases of PTC tissues and corresponding paracancerous normal thyroid tissues were collected. Realtime quantitative PCR (RT-qPCR), Western blotting, and immunohistochemistry were used to analyze the expression of TAGLN in PTC tissues and corresponding paracancerous normal thyroid tissues. PTC cells were transfected with plasmid and shRNA lentivirus vector respectively to up-regulate or down-regulate the expression of TAGLN in order to detect the effects of them on the proliferation, invasion, and migration by cell proliferation assay(cell counting kit-8, CCK-8)and cell invasion and migration assays (Transwell). The effects of TAGLN on mitogen-activated protein kinase (MAPK)/extracellular-signal regulating kinase (ERK) signal pathway was detected with Western blotting.Results:RT-qPCR showed that there was no difference in the expression of TAGLN mRNA between PTC and corresponding paracancerous normal thyroid tissues ( P>0.05); Western blotting demonstrated that the expression of TAGLN protein in PTC tissues was significantly lower than that in corresponding paracancerous normal thyroid tissues ( P<0.01). Immunohistochemical results revealed that the expression of TAGLN in PTC tissues was significantly lower than that in corresponding paracancerous normal thyroid tissues. Overexpression of TAGLN inhibited the proliferation, invasion, and migration of PTC cells ( P<0.01), but knockdown of TAGLN promoted the proliferation, invasion, and migration of PTC cells ( P<0.01). Overexpression of TAGLN decreased the expression of phosphorylated ERK ( P<0.05), whereas silencing TAGLN increased phosphorylated ERK level in PTC cells( P<0.01). Conclusion:The expression of TAGLN in PTC is significantly decreased. It is related to the occurrence and development of PTC, and its mechanism may be related to MAPK/ERK signal pathway.

Improvement of bladder function and repair of axonal injury in rats with traumatic spinal cord injury via MAPK/ERK pathway by nerve growth factor / 西安交通大学学报(医学版)

【Objective】 To explore the effects of nerve growth factor （NGF） on bladder function and axon injury repair in rats with traumatic spinal cord injury （t-SCI） so as to explore its molecular mechanism. 【Methods】 Traumatic spinal cord injury model was constructed in 30 male SD rats by modified Allen’s beating method. The rats were randomly divided into sham-operation group， injury group and NGF group， with 10 rats in each group. We used the BBB score to observe the motor function of the rats’ hind limbs before and after the operation. The BL-420 biometer experimental system detected the urodynamics. Six anterior roots of the left lumbar taken from the distal end of the anastomosis were stained with toluidine blue， and the number of myelinated axons was counted. We used HE to stain rat bladder tissue， TUNEL to stain the rats’ severely injured spinal cord， and observed the spinal cord apoptosis rate. Western blotting was used to detect the protein expressions of Raf-1， p-MEK-2， MEK-2， ERK1/2， and p-ERK1/2 in spinal cord tissue. 【Results】 The BBB score results showed that there was no difference in the scores of the sham-operation group， the injury group and the NGF group before the operation. After the operation， the scores of the injury group and the NGF group were significantly lower than those in the sham-operation group （P0.05）. 【Conclusion】 NGF may hinder the conduction of MAPK/ERK pathway， thereby affecting the repair of axon damage and improving the bladder function of t-SCI rats.

The C-terminus of the amelogenin peptide promotes the proliferation of alc ameloblasts through accelerating cell cycle / 中国组织工程研究

BACKGROUND: C-terminus of the amelogenin peptide (AMG-CP) is a small molecular endogenous peptide that is highly shown that AMG-CP can regulate the proliferation and differentiation of cementoblasts, bone marrow mesenchymal stem cells and periodontal ligament fibroblasts, but the biological function of AMG-CP on ameloblasts has not been elucidated. O conserved among species. It is involved in important physiological processes during tooth development. Some studies have BJECTIVE: To investigate the effects of different concentrations of AMG-CP on the proliferation of ALC ameloblasts and its underlying mechanisms. METHODS: AMG-CP was successfully synthesized and determinated by liquid chromatography and mass spectrometry. The effects of AMG-CP at 0, 0.5, 1, 2 mg/L on the proliferation of ALC ameloblasts were observed by xCELLigence RTCA cell analysis system in real time. The effect of AMG-CP at 0, 1, 2 mg/L on cell cycle of ALC was detected by flow cytometry. Real-time PCR was used to detect the expression of cyclin D1, CDK4, MCM2, MCM5 mRNA in ALC cells treated with AMG-CP at 0, 1, 2 mg/L. Western blot was carried out to evaluate the effect of AGM-CP at 0, 1 mg/L on MAPK-ERK1/2 pathway by detecting the expression of phosphorylated ERK1/2 and total ERK1/2 in ALC cells. Pathway blockade assay was performed by using ERK1/2 blocker U0126 to pretreat ALC cells. Then cell proliferation ability as well as phosphorylated ERK1/2 expression was analyzed by xCELLigence RTCA cell analysis system and western blot. RESULTS AND CONCLUSION: Compared with the control group, AMG-CP promoted the proliferation of ALC cells, and decreased the population doubling time in a dose-depending manner. Flow cytometry detected the acceleration of cell cycle after treatment with AMG-CP. The results of Real-time PCR showed that AMG-CP upregulated cell cycle-related genes (cyclin D1, CDK4, MCM2, MCM5) expression. Western blot results showed that AMG-CP could upregulate the expression of phosphorylated ERK1/2 and activate MAPK-ERK1/2 signaling pathway in ALC cells. After U0126 was used to inhibit the MAPK-ERK1/2 pathway, the ability of AMG-CP promoting ALC proliferation was inhibited. These results suggest that AMG-CP has a potential to activate MAPK-ERK1/2 pathway, accelerate the process of cell cycle, and then promote the proliferation of ALC cells, all of which indicate that AMG-CP has the potential to promote the proliferation of ameloblasts.

Effect of oxidative stress in SH-SY5Y cell injury induced by deguelin / 中国药理学通报

Aim To study the correlation between oxidative stress and neurotoxicity induced by deguelin, providing the mechanism basis for the structural modification and drug combination about deguelin. Methods SH-SY5Y cells were exposed to deguelin ranging from 1. 56-100 jimol • L"1 for 24 to 72 h, whose survival rate was determined by CCK-8 assay. The content of LDH, MDA levels, GPx and antioidant enzyme activities of SOD were determined using the assay kits ac-cording the manufacturer's protocol. And the content of ROS was determined by flow cytometry. The expressions of MEK, EGF, ERK and CREB were determined by immunoblotting. Results Deguelin obviously inhibited the growth of SH-SY5Y cells in a concentra-tion-and time-dependent manner ( P < 0. 05 ). After the cells were injured by deguelin, the content of ROS, LDH and MDA in SH-SY5Y cells increased obviously (P <0. 05), while the vitality of GPx and SOD decreased obviously ( P < 0. 05) in a dose-dependent manner. Furthermore, the protein expression levels of MEK, EGF, RAS and CREB in MAPK/ERK signaling pathway decreased obviously when treated with deguelin in a dose-dependent manner. (P < 0. 05). Conclusions The trauma of SH-SY5Y cells induced by deguelin is closely related to oxidative stress, in which MAPK/ERK signaling pathway may play a mediating role.

MicroRNA-206 Reduces Osteosarcoma Cell Malignancy In Vitro by Targeting the PAX3-MET Axis

PURPOSE: This study was undertaken to explore how miR-206 represses osteosarcoma (OS) development. MATERIALS AND METHODS: Expression levels of miR-206, PAX3, and MET mRNA were explored in paired OS and adjacent tissue specimens. A patient-derived OS cell line was established. miR-206 overexpression and knockdown were achieved by lentiviral transduction. PAX3 and MET overexpression were achieved by plasmid transfection. Treatment with hepatocyte growth factor (HGF) was utilized to activate c-Met receptor. Associations between miR-206 and PAX3 or MET mRNA in OS cells were verified by AGO2-RNA immunoprecipitation assay and miRNA pulldown assay. OS cell malignancy was evaluated in vitro by cell proliferation, metastasis, and apoptosis assays. PAX3 and MET gene expression in OS cells was assayed by RT-qPCR and Western blot. Activation of PI3K-AKT and MAPK-ERK in OS cells were assayed by evaluating Akt1 Ser473 phosphorylation and total threonine phosphorylation of Erk1/2, respectively. RESULTS: Expression levels of miR-206 were significantly decreased in OS tissue specimens, compared to adjacent counterparts, and were inversely correlated with expression of PAX3 and MET mRNA. miR-206 directly interacted with PAX3 and MET mRNA in OS cells. miR-206 overexpression significantly reduced PAX3 and MET gene expression in OS cells in vitro, resulting in significant decreases in Akt1 and Erk1/2 activation, cell proliferation, and metastasis, as well as increases in cell apoptosis, while miR-206 knockdown showed the opposite effects. The effects of miR-206 overexpression on OS cells were reversed by PAX3 or MET overexpression, but only partially attenuated by HGF treatment. CONCLUSION: miR-206 reduces OS cell malignancy in vitro by targeting PAX3 and MET gene expression.

Neuroprotective effect of melatonin in cerebellums in Alzheimer's disease animal models via MAPK/ERK signaling pathway / 中国药理学通报

Aim: To investigate the effect of melatonin on neuroprotection in cerebellums of rats with Alzheimer' s disease via MAPK/ERK signaling pathway. Methods: Thirty-two male Sprague-Dawley rats were randomly divided into four groups: control group, Aβ1-42 lateral ventricle injection group (AD), and melatonin intraperitoneal injection group (MT), and Aβ1-42 lateral ventricle injection combined with melatonin intraperitoneal injection group (AD + MT). The pathological changes of rat cerebellar cortex were detected by HE staining; the expression of NeuN (neuronal marker), Calbindin(Purkinje cell marker) and p-ERK protein in each group was detected by immunofluorescence; the expression of ERK and p-ERK in each group was determined by Western blot. Results: The HE staining showed that the expression of neurons decreased, followed with the disordered arrangement and morphological alteration of cells in AD. Melatonin could significantly alleviate the pathological damage in cerebellum. Immunofluorescence results showed that compared with AD group, the expression of NeuN (neuronal marker) increased, the number of Purkinje cells marked by Calbindin significantly was up-regulated(P < 0. 01), and the expression of p-ERK was down-regulated in AD + MT group. Western blot showed that the expression of p-ERK was down-regulated by melatonin. Conclusions: Melatonin may exert the neuroprotective effect and relieve the pathological changes by inhibiting the activation of MAPK/ERK signaling pathway.

Role of M A P K / E R K signaling pathway in apoptosis of nasopharyngeal carcinoma cells by Yiqi Jiedu formula / 中国药理学通报

;Aim To study the effect of Yiqi Jiedu formula extract on the apoptosis of nasopharyngeal carcinoma cells, and explore the mechanism of its induction of apoptosis from MAPK/ERK signaling pathway. Methods The effect of Yiqi Jiedu Formula extract on the proliferation of CNE1 and CNE2 cells was detected by CCK-8. The apoptosis of CNE1 and CNE2 cells was detected by Hoechst 33342 staining, JC-10 staining and fluorescence double dye flow cytometer staining. The protein expression of CNE1 and CNE2 cells was detected by Western blot. Results Yiqi Jiedu formula extract inhibited the proliferation (P < 0. 05) and induced apoptosis (P <0. 05) of CNE1 and CNE2 cells. After 48 h of drug treatment, the expression of Survivin, XIAP and Bcl-2 decreased, Bax increased, and p-c-Raf, p-MEK and p-ERKl/2 of MAPK/ERK signaling pathway significantly decreased (P < 0. 05). On this basis, after adding activator of MAPK/ERK signaling pathway ISO, the expression of p-c-Raf, p-MEK and p-ERKl/2,Survival, XIAP and Bcl-2 increased, while the expression of Bax decreased compared with the extract of YQ group. Drug-induced apoptosis effects were also reduced. Conclusions Yiqi Jiedu formula extract can induce the apoptosis of nasopharyngeal carcinoma cells, which inhibits the expression of key proteins p-c-Raf, p-MEK and p-ERKl/2 in MAPK/ERK signaling pathway, and then down-regulates the expression of Survivin, XIAP, Bcl-2 and up-regulates the expression of Bax.

Label-free quantitative proteomic analysis of acquired herceptin resistance in gastric cancer cells / 药学学报

This study was designed to explore proteins differentially expressed in HER2 positive gastric cancer N87 cells and N87/R cells with an acquired resistance to herceptin based on label-free quantitative proteomics. The extracted proteins were reduced and alkylated, then digested using filter aided sample preparation (FASP); peptides were separated via small manual reversed phase column, analyzed by LC-MS/MS, and identified with protein database 2.1 search engine. Proteins were quantified by intensity based quantification (IBQ) to search for differential proteins by comparison with relatively quantified proteins. The enrichment and network construction in gene ontology (GO) terms, genes-disease and Wikipathway of differential proteins were established through Web Gestalt. A total of 8 509 proteins were detected, among them, 7 163 proteins were further analyzed by bioinformatics, of which 110 proteins were up-regulated and 70 were down-regulated in N87/R cells. The differential proteins showed a significant difference in cellular component, biological process and molecular function in GO terms, respectively. Genes-disease network analysis indicated the association of these differential proteins with neoplasm metastasis, neoplasm invasiveness and inflammation, etc. Wikipathway enrichment analysis revealed the relevance of several signaling pathways to herceptin resistance, which included IL-2, MAPK/ERK, mTOR, aurora A, Ret, NF-κB, immune-regulatory and metabolic pathway. Western blot showed a significant increase of ERK1/2 activities in N87/R cells compared with N87 cells. Correspondingly, SCH772984, a MAPK/ERK inhibitor, preferentially reduced the viability of N87/R cells. Taken together, our data suggested that the MAPK/ERK signaling pathway is one of the key pathways that mediate herceptin resistance. This study provides the basic information for exploring the mechanisms of acquired resistance to herceptin in gastric cancer cells.

Panaxadiol saponins induce activation of MAPK/ERK signaling pathway in bone marrow cells of aplastic anemia mice / 中国病理生理杂志

AIM:To observe the effects of panaxadiol saponins(PDS)on up-regulation of MAPK/ERK signal pathway in bone marrow cells and increase in regulatory T(Treg)cells in spleen tissue of aplastic anemia(AA)mice,and to explore the mechanisms.METHODS:For preparation of immune-mediated AA model,BALB/c mice were exposed to sublethal dose(5.0 Gy)of [60Co]-γradiation, followed by transplantation of lymphocytes from DBA /2 donor mice. BALB/c mice(n=60)were randomly divided into 6 groups,including normal mouse group,AA model group,PDS treat-ment groups at low,medium and high doses,and cyclosporine group as positive control.PDS and cyclosporine were given by gavage for 14 d.The peripheral blood cell counts and bone marrow pathological examination were tested.The protein levels of MEK1/2,p-MEK1/2,ERK1/2 and p-ERK1/2 in the bone marrow cells were analyzed by Western blot and im-munohistochemistry experiment.Flow cytometry was used to detect the proportion of Treg cells in spleen tissue of each group.RESULTS:The peripheral blood cell counts were significantly decreased in AA mouse group as compared with nor -mal mouse group(P<0.05).The bone marrow sections showed markedly inhibition status of hematopoiesis and the de -crease in cellularity.In response to PDS treatment,the peripheral blood cell counts and Treg cells in the spleen tissues of AA mouse treated with PDS were significantly increased in a dose-dependent manner(P<0.05).Treatment with PDS at medium and high doses up-regulated the protein levels of MEK1/2,p-MEK1/2,ERK1/2 and p-ERK1/2 in the bone mar-row of AA mice(P<0.05).CONCLUSION:PDS is effective to enhance recovery of hematopoietic function in AA mice. This effect may be related to up-regulating multiple protein kinases of MAPK/ERK signal pathway in the bone marrow cells of AA mice.In addition,PDS has an impact on immune function of AA mice.

Effects of marrow stromal cell-mediated microenvironment on human lung adenocarcinoma A549 cells / 中国病理生理杂志

AIM: To investigate the effects of marrow stromal cell line HS-5 on human lung adenocarcinoma A549 cells in the tumor microenvironment.METHODS:The effects of HS-5 cell-conditioned medium(HS-5-CM)on the viability and migration ability of A549 cells were detected by MTT assay and wound-healing assay.After treatment with HS-5-CM,the expression of CX3C chemokine receptor 1(CX3CR1)at mRNA level in the A549 cells was examined by qPCR. The protein levels of p-ERK and ERK in the A549 cells treated with MAPK/ERK pathway inhibitor U0126 were observed by Western blot,the migration ability of the A549 cells was measured by wound-healing assay,and the protein expression of CX3CR1 was determined by Western blot.RESULTS: HS-5-CM promoted the viability and migration ability of the A549 cells(P<0.01).The expression of CX3CR1 at mRNA level in the A549 cells was increased after treatment with HS-5-CM.MAPK/ERK inhibitor U0126 inhibited the activation of MAPK/ERK signaling pathway(P<0.01), and re-duced the migration ability(P<0.01)and the expression of CX3CR1(P<0.05)in the A549 cells.CONCLUSION:HS-5-CM significantly promotes the A549 cell viability and migration ability.Activation of MAPK/ERK signaling pathway and the expression of CX3CR1 may play a important role in this process.

Inhibition of Yiqi Jiedu formula on proliferation of nasopharyngeal carcinoma cells by MAPK/ERK signaling pathway / 中国中药杂志

To study the effect of aqueous extracts of Yiqi Jiedu formula (YQ) on the proliferation of CNE2 cells in human nasopharyngeal carcinoma, and investigate its mechanism to provide a new theoretical basis for the clinical application of YQ. CNE2 cells were treated with different concentrations (0.125, 0.25, 0.5, 0.25 g·L⁻¹) of YQ, positive control medicine (cisplatin 4.0 mg·L⁻¹), inhibitor PD98059 (50 μmol·L⁻¹), activator isoproterenol hydrochloride (20 μmol·L⁻¹), activator isoproterenol hydrochloride (ISO)+YQ 0.5 g·L⁻¹. Then cell labeling by using real-time analyzer (RTCA) and CCK 8 method were used to detect cell proliferation activity, and the half inhibitory concentration (IC₅₀) was calculated. The cell cycle distribution was detected by fluorescence double dye flow cytometry PI staining, and Western blot method was used to detect the expression levels of related protein and MAPK/ERK signaling pathway. The results of RTCA and CCK-8 test showed that as compared with the control group, YQ group could effectively inhibit the proliferation of CNE2 cells (<0.01), with a dose and time dependence, and 48 h IC₅₀ value was 0.5 g·L⁻¹. The results of cell cycle showed that after 48 h of water extract treatment, the cell cycle was significantly changed, the proportion of G₀/G₁ was reduced, the ratio of G₂/M increased, and the cell cycle was in G₂/M period (<0.01). Western blot results showed that after 48 h treatment with different concentrations of aqueous extract, cell cycle-related proteins cyclinD1, cyclinD3 and CDK2 expression levels were down-regulated; MAPK/ERK signaling pathway related protein p-c-Raf, p-MEK, p-ERK1/2 expression level significantly lower as compared with the control group (<0.05). After adding activator and inhibitor in MAPK/ERK signaling pathway on this basis, the results showed that after adding activator ISO, cell proliferation was significantly higher than that in the Control group; the cycle related proteins cyclinD1, cyclinD3, and CDK2 expression levels were increased; at the same time, key protein p-c-Raf, p-MEK, p-ERK1/2 expression levels in the signal pathways were relatively increased. While after the addition of inhibitor PD98059, the cell proliferation was significantly lower than that in the Control group, and the expression level of corresponding protein was decreased, which was significantly different from the Control group (<0.05). So YQ could block cell cycle and inhibit the proliferation of CNE2 cells mainly by reducing the expression of MAPK/ERK signaling pathway key protein p-c-Raf, p-MEK and p-ERK1/2.

Molecular Mechanism of Inducing GLC-82 Cells Apoptosis by Ethanol Extract from Wedelia prostrate (Hook.et Arn.) Hemsl / 中国生物工程杂志

Objective:To study the antitumor mechanism of W40,a monomer purified from Wedelia prostrate (Hook.et Arn.) Hemsl.Methods:The effects of W40 on the cell proliferative of GLC-82 cells were detected by MTT assay and colony formation assay.The migratory abilities of GLC-82 cells were observed by wound healing assay.Cell apoptosis was evaluated by Annexin V-FITC/PI staining analysis.The levels of apoptosis-relative proteins and cell proliferation-related proteins,such as Caspase-3,PARP,Stat3 and ERK,were detected by Western blotting.Results:MTF assay showed that W40 had a significant cytotoxic effect on non-small cell lung cancer GLC-82 cells.Colony formation assays showed that W40 significantly inhibited GLC-82 cells proliferation.The migration of GLC-82 cells was inhibited by W40 in a dose-dependent manner.Flow cytometry showed that the apoptotic rate increased gradually in a concentration-dependent manner.W40 down-regulated Stat3 as decreasing p-Stat3 and downstream proteins of Bcl-2 and Mcl-1.At the same time,W40 up-regulated the expression of pro-apoptotic protein Bax,and increased the cleavaged Caspase-9,Caspase-3 and PARP.W40 also down-regulated BRAF / MAPK / ERK signal pathway as decreasing p-BRAF,p-MEK and p-ERK.Conclusions:W40 induced apoptosis by inhibiting BRAF / MAPK / ERK and Stat3 signaling pathways.

Propofol Inhibiting the Expression of VEGF through MAPK/ERK Signal Pathways in Oesophageal Carcinoma Cells EC9706 / 医学研究杂志

Objective To investigate the effect of propofol on the expression of VEGF in Oesophageal carcinoma cells EC9706 and its related molecular mechanism.Methods The expression of VEGF in human Oesophageal carcinoma cells EC9706 and normal esophageal epithelium cells HEEC were compared by immunoblotting.EC9706 was treated with different concentrations of propofol (0,2,6,10μg/L).After incubation,the EC9706 cell lines were detected with MTT,flow cytometry,cell invasion and cell scratch tests.The expression of VEGF,and the phosphorylation of p38 (MAPK) and p44/42 (ERK1/2) were detected by qRT-PCR and immunoblotting in each group.Results The expression of VEGF in EC9706 cells was significantly higher than that in HEEC cells.After propofol intervention,the proliferation,migration and invasion of propofol groups were significantly lower than that of Ctrl group,while the apoptotic rate of propofol groups were significantly higher than that of Ctrl group.The expression of VEGF mRNA and protein in propofol groups were significantly lower than that in Ctrl group.The expression and phosphorylation of p38 (MAPK) and p44 / 42 (ERK1 / 2) were inhibited by propofol.Both of these effects were dose-dependent.Conclusion Propofol inhibits the proliferation,invasion and migration of OC cells EC9706 and promotes apoptosis.Its potential mechanism may work by inhibiting the MAPK/ERK signaling pathway,thereby inhibiting VEGF expression.

Induction of Integrin Signaling by Steroid Sulfatase in Human Cervical Cancer Cells

Steroid sulfatase (STS) is an enzyme responsible for the hydrolysis of aryl and alkyl sulfates. STS plays a pivotal role in the regulation of estrogens and androgens that promote the growth of hormone-dependent tumors, such as those of breast or prostate cancer. However, the molecular function of STS in tumor growth is still not clear. To elucidate the role of STS in cancer cell proliferation, we investigated whether STS is able to regulate the integrin signaling pathway. We found that overexpression of STS in HeLa cells increases the protein and mRNA levels of integrin β1 and fibronectin, a ligand of integrin α5β1. Dehydroepiandrosterone (DHEA), one of the main metabolites of STS, also increases mRNA and protein expression of integrin β1 and fibronectin. Further, STS expression and DHEA treatment enhanced phosphorylation of focal adhesion kinase (FAK) at the Tyr 925 residue. Moreover, increased phosphorylation of ERK at Thr 202 and Tyr 204 residues by STS indicates that STS activates the MAPK/ERK pathway. In conclusion, these results suggest that STS expression and DHEA treatment may enhance MAPK/ERK signaling through up-regulation of integrin β1 and activation of FAK.

Mortalin promotes ovarian cancer cell growth through MAPK-ERK signal pathway / 解剖学报

Objective By constructing mortalin stably expressing ovarian cancer cell lines in A 2780 and A2780/cis, we demonstrate the role of mortalin in the ovarian cancer cell growth .Methods CCK-8 assay was used to measure cell viability in the overexpression mortalin group compared with the control group .The flow cytometry analysis was used to understand the effect of upregulated mortalin on the ovarian cancer cell cycle .Western blotting was used to determine the expression and phosphorylation level of MAPK /ERK and JNK/SAPK signal pathways .Results The results showed that increased expression of mortalin could accelerate ovarian cancer cell proliferation and promote G 1 transition, leading to a faster restoration of normal distribution of cell cycle .We found that mortalin overexpression significantly activated p-Raf and p-ERK1/2, but not p-JNK.Conclusion The results demonstrate that mortalin effect on the ovarian cancer cell proliferation contributes to active the MAPK-ERK signaling pathway .