MEK/ERK signaling pathway in apoptosis of SW620 cell line and inhibition effect of resveratrol

Objective:To study the involvement of MAPK MEK/ERK signaling transduction pathway in the apoptosis process of SW620 tumor cell line and the inhibition effect of resveratrol.Methods:SW620 cell lines were divided into 5 groups,namely,control group.PD98059 group,low-dose resveratrol group,mid-dose resveratrol group and high-dose resveratrol group.The inhibition rate of cell proliferation was detected by MTT method.The expression of apoptotic molecules and MEK/ERK signaling pathway related proteins were assayed by realtime PCR and Western blotting.Results:Compared with control group,the proliferation of cells treated with resveratrol was significantly inhibited.In the case of apoptotic molecules,the expression of Bax,Caspase 3 and Caspase 9 was increased significantly while the expression of anti-apoptotic molecule Bcl2 was decreased significantly in resveratrol groups with a dosedependent manner.In the case of molecules in MEK/ERK signaling pathway,the expression of Ras,Raf,MEK and ERKl/2 was decreased significantly in resveratrol groups with a dose-dependent manner.Conclusions:PD98059 and resveratrol can effectively inhibit the proliferation of SW620 through inhibiting the MEK/ERK signaling pathway.

IKKε通过调控异常的MAPK/MEK/ERK信号转导通路参与急性主动脉夹层形成的机制研究

目的分析MAPK/MEK/ERK信号转导通路与急性主动脉夹层形成的关系及IKKε的调控作用,为临床相关治疗和新药研发提供参考。方法清洁级C57BL/6小鼠随机分为5组:对照组、模型组、IKKε-IN-1低剂量、中剂量和高剂量干预组。除对照组外,其余各组小鼠给予2 500 ng/(kg·min)血管紧张素Ⅱ(对照组给予生理盐水)14 d。自第7天起IKKε-IN-1低剂量、中剂量和高剂量干预组小鼠分别给予IKKε-IN-1治疗(1 mg/kg、5 mg/kg和25 mg/kg)。采用老龄小鼠埋泵缓释血管紧张素Ⅱ建立急性主动脉夹层动物模型,并采用IKKε特异性拮抗剂IKKε-IN-1进行干预,分析各组小鼠血浆MAPK/MEK/ERK信号转导通路相关蛋白表达。结果清洁级C57BL/6小鼠皮下植入缓释泵输注血管紧张素Ⅱ7 d后模型组小鼠收缩压为153±11.4 mm Hg(对照组为105±10.4 mm Hg),14 d后收缩压变为176±12.6 mm Hg,而IKKε-IN-1低剂量、中剂量和高剂量干预组小鼠的收缩压明显低于模型组(P<0.05),且呈剂量依赖性(P<0.05);与对照组相比,急性主动脉夹层小鼠Ras、Raf、MEK、ERK1/2、基质金属蛋白酶2(MMP2)及基质金属蛋白酶6(MMP6)蛋白表达明显增强而金属蛋白酶组织抑制因子1(TIMP1)和金属蛋白酶组织抑制因子2(TIMP2)的表达明显减弱(P<0.05),而IKKε-IN-1低剂量、中剂量和高剂量干预组上述蛋白的表达明显恢复(P<0.05),且呈剂量依赖性(P<0.05)。结论激活的MAPK/MEK/ERK信号转导通路及基质金属蛋白酶参与了急性主动脉夹层形成,且此过程与IKKε的调控密切相关。

Effect of Guizhi Fuling Pill combined with GnRH analog on cell proliferation and invasion as well as MEK/ERK pathway in endometriosis lesions

Objective: To study the effect of Guizhi Fuling Pill combined with gonadotropin-releasing hormone analog (GnRH-a) on cell proliferation and invasion as well as MEK/ERK pathway in endometriosis lesions. Methods: Patients who were diagnosed with endometriosis in Bazhong Hospital of Traditional Chinese Medicine between November 2014 and March 2017 were selected as the research subjects and randomly divided into two groups, observation group received preoperative Guizhi Fuling Pill combined with GnRH analog therapy, and control group received preoperative GnRH analog monotherapy. After surgical resection, the endometriosis lesion was collected to determine the mRNA expression of proliferation and invasion-related genes as well as the protein expression of MEK/ERK pathway molecules. Results: Id-1, Sema3A, c-IAP1, OPN and uPA mRNA expression as well as p-MEK, p-EKR1/2, caspase-3 and MMP2 protein expression in endometriosis lesion of observation group were significantly lower than those of control group while Bak, Smac, PAI-1, TIMP1 and TIMP2 mRNA expression as well as caspase-3 protein expression were significantly higher than those of control group. Conclusion: Guizhi Fuling Pill combined with GnRH analog can inhibit the cell proliferation and invasion as well as the MEK/ERK pathway activation in endometriosis lesions.

PLX8394,a RAF inhibitor,inhibits enterovirus 71 replication by blocking RAF/MEK/ERK signaling

Enterovirus 71(EV71)poses a serious threat to human health,with scattered outbreaks worldwide.There are several vaccines against a few EV71 strains but no efficient drug for the treatment of EV71 infection.Therefore,it is urgent and of significance to develop anti-EV71 drugs.Here,we found that PLX8394,a RAF inhibitor,possesses high antiviral activity against EV71 in vitro,being superior to the traditional clinical drug ribavirin.Moreover,PLX8394 exhibits broad-spectrum antiviral activity against enteroviruses.Notably,in a suckling mouse model,PLX8394 provided a 70%protection rate for EV71-infected mice,reduced the viral load in liver and heart tissues,and relieved the inflammatory response.A mechanistic study showed that PLX8394 inhibited EV71 by suppressing the RAF/MEK/ERK signaling pathway.Thus,PLX8394 lays a foundation for the development of new drugs against EV71.

MAPK/ERK regulation of P53 in human epidermoid carcinoma cell line A431

Objective:To observe the impact of activation and inhibition of mitogen activated protein kinases(MAPK)/extracellular signalregulated protein kinase(ERK)signaling pathway on the proliferation and apoptosis of cutaneous squamous cell carcinoma(SCC).cells and investigate the interaction mechanism between MAPK/ERK signaling pathway and tumor suppressor gene P53 in SCC.Methods:Human A431 cells were cultured and divided into MAPK/ERK inhibition groups with low-,medium-and highconcentration of inhibitors(PD98059+DMSO),MAPK/ERK activation groups with low-,medium-and high-concentration of stimuli(IGF+PBS)and blank control group(DMSO).The cell proliferation in vitro was detected by MTT assay,with the cell apoptosis detected by flow cytometry(FCM)and the protein expression of P-ERK and P53 detected by western blot in each group.Results:The A431 cell proliferation was inhibited by different concentrations of PD98059 with a clear concentration-effect and time-effect relationship(p<.05);and the cell proliferation was promoted by the different concentrations of IGF with a clear concentration-effect and time-effect relationship(p<.05).The FCM results showed a significant increase in the apoptosis rate of A431 cells which were treated with PD98059,with a clear concentration-effect relationship(p<.05);while the apoptosis rate was decreased significantly after A431 cells were treated with IGF,also with a concentration-effect relationship(p<.05).The western blot results showed that the expression of P-ERK protein was decreased but the expression of P53 was increased after A431 cells were treated with PD98059.With the concentration of PD98059 going up,the decrease in P-ERK and the increase in P53 were more significant(p<.05);while the expression of P-ERK protein was increased but the expression of P53 was decreased after A431 cells were treated with IGF.With the concentration of IGF going up,the increase in P-ERK and the decrease in P53 were more significant(p<.05).According to Pearson correlation analysis,the expression of P53 was negatively correlated to that of P-ERK(p<.05).Conclusions:After MAPK/ERK signaling pathway was activated by IGF in A431 cells,the expression of pro-apoptotic factor P53 was decreased with the ability of cell proliferation enhanced and the ability of apoptosis reduced.However,after the inhibition of MAPK/ERK signaling pathway,the expression of pro-apoptotic factor P53 was increased with the ability of cell proliferation reduced and the ability of apoptosis increased.

Circ_0053943 complexed with IGF2BP3 drives uveal melanoma progression via regulating N6-methyladenosine modification of Epidermal growth factor receptor

Numerous studies have characterized the critical role of circular RNAs(circRNAs)as regulatory factors in the progression of multiple cancers.However,the biological functions of circRNAs and their underlying molecular mechanisms in the progression of uveal melanoma(UM)remain enigmatic.In this study,we identified a novel circRNA,circ_0053943,through re-analysis of UM microarray data and quantitative RT-PCR.Circ_0053943 was found to be upregulated in UM and to promote the proliferation and metastatic ability of UM cells in both in vitro and in vivo settings.Mechanistically,circ_0053943 was observed to bind to the KH1 and KH2 domains of insulin-like growth factor 2 mRNA-binding protein 3(IGF2BP3),thereby enhancing the function of IGF2BP3 by stabilizing its target mRNA.RNA sequencing assays identified epidermal growth factor receptor(EGFR)as a target gene of circ_0053943 and IGF2BP3 at the transcriptional level.Rescue assays demonstrated that circ_0053943 exerts its biological function by stabilizing EGFR mRNA and regulating the downstream mitogen-activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK)signaling pathway.Collectively,circ_0053943 may promote UM progression by stabilizing EGFR mRNA and activating the MAPK/ERK signaling pathway through the formation of a circ_0053943/IGF2BP3/EGFR RNA-protein ternary complex,thus providing a potential biomarker and therapeutic target for UM.

Synergistic inhibition of MEK and reciprocal feedback networks for targeted intervention in malignancy

The RAS-RAF-MEK-ERK signaling pathway(MAPK signaling pathway) plays a significant role in multiple pathological behaviors and is most frequently dysregulated in more than 30% of human cancers.As key elements in this pathway, MEK1/2 play crucial roles in tumorigenesis and the inhibition of apoptosis, which makes their inhibition an attractive antitumor strategy.Dozens of potent non-ATP-competitive allosteric MEK1/2 inhibitors have been developed that have produced substantial improvement in clinical outcomes over the past decade.However, the efficacy of these agents is limited, and response rates are variable in a wide range of tumors that harbor RAS and RAF mutations due to the development of resistance, which is derived mainly from the persistence of MAPK signaling and increased activation of the mutual feedback networks.Both intrinsic and acquired resistance to MEK inhibitors necessitates the synergistic targeting of both pathways to restore the therapeutic effects of a single agent.In this review, the significant role of the MAPK pathway in carcinogenesis and its therapeutic potential are comprehensively examined with a focus on MEK inhibitors.Then, the activation of feedback networks accompanying MEK inhibition is briefly reviewed.Combination strategies that involve the simultaneous inhibition of the original and resistance pathways are highlighted and elaborately described on the basis of the latest research progress.Finally, the obstacles to the development of MEK-related combination systems are discussed in order to lay the groundwork for their clinical application as frontline treatments for individual patients with MAPK-hyperactivated malignancies.

Hypoxia-inducible factor-1α–mediated upregulation of CD99 promotes the proliferation of placental mesenchymal stem cells by regulating ERK1/2

BACKGROUND As human placenta-derived mesenchymal stem cells(hP-MSCs)exist in a physiologically hypoxic microenvironment,various studies have focused on the influence of hypoxia.However,the underlying mechanisms remain to be further explored.AIM The aim was to reveal the possible mechanisms by which hypoxia enhances the proliferation of hP-MSCs.METHODS A hypoxic cell incubator(2.5%O2)was used to mimic a hypoxic microenvironment.Cell counting kit-8 and 5-ethynyl-20-deoxyuridine incorporation assays were used to assay the proliferation of hP-MSCs.The cell cycle was profiled by flow cytometry.Transcriptome profiling of hP-MSCs under hypoxia was performed by RNA sequencing.CD99 mRNA expression was assayed by reverse transcription-polymerase chain reaction.Small interfering RNA-mediated hypoxia-inducible factor 1α(HIF-1α)or CD99 knockdown of hP-MSCs,luciferase reporter assays,and the ERK1/2 signaling inhibitor PD98059 were used in the mechanistic analysis.Protein expression was assayed by western blotting;immunofluorescence assays were conducted to evaluate changes in expression levels.RESULTS Hypoxia enhanced hP-MSC proliferation,increased the expression of cyclin E1,cyclin-dependent kinase 2,and cyclin A2,and decreased the expression of p21.Under hypoxia,CD99 expression was increased by HIF-1α.CD99-specific small interfering RNA or the ERK1/2 signaling inhibitor PD98059 abrogated the hypoxia-induced increase in cell proliferation.CONCLUSION Hypoxia promoted hP-MSCs proliferation in a manner dependent on CD99 regulation of the MAPK/ERK signaling pathway in vitro.

Tobacco-specific Carcinogen 4-(Methylnitrosoamino)-1-(3-pyridyl)-1-butanone(NNK) Activating ERK1/2 MAP Kinases and Stimulating Proliferation of Human Mammary Epithelial Cells

Cigarette smoking is correlated with the development of various cancers. 4- （Methylnitresoamino） -1- （3-pyridyl） - 1-butanone（NNK） is one of the major tobacco-specific carcinogens in the cigarette smoke, which increases the risk of breast cancer. In the present study, it was demonstrated that NNK rapidly activated ERK1 and ERK2 MAP kinases in human normal mammary epithelial cells. It was found that there are two different routes for the activation of ERK1/2 with NNK. One is from nicotinic receptor nAchR to MEK1/2, and the other is from tyrosine kinase containing receptor to MEK1/2. The tobacco-specific carcinogen NNK shows a strong proliferative effect on normal human mammary epithelial cells and cancer mammary epithelial cells.

Apolipoprotein A1 suppresses the hypoxia-induced angiogenesis of human retinal endothelial cells by targeting PlGF

AIM:To investigate the anti-angiogenic effect of apolipoprotein A1(apoA1)on primary human retinal vascular endothelial cells(HRECs)and explore the possible mechanism.METHODS:The primary HRECs were transfected with apoA1-GFP recombinant lentiviral and were compared with cells undergoing transfection with empty lentiviral vectors.Hypoxia chambers were used to simulate the anoxic environment of cells under pathological condition.The concentrations of secreted vascular endothelial growth factor(VEGF)and placental growth factor(PlGF)were measured by enzyme-linked immunosorbent assay(ELISA).Cell migration ability was detected by wound healing assay.The sprouting of HRECs was determined by tube formation assay.The protein levels of extracellular signal regulated kinase 1/2(ERK1/2)and phosphor ylated ERK1/2(p-ERK1/2)were measured by Western blot.RESULTS:Overexpressed apoA1 in hypoxia-induced HRECs significantly suppressed PlGF(0.67±0.10 folds,P=0.007).Overexpressed apoA1 also attenuated hypoxiainduced cell migration(0.32±0.11 folds,P<0.0001),tube formation(0.66±0.01 folds,P<0.0001)and the phosphorylation levels of ERK(0.6±0.11 folds,P=0.025).Pretreatment of mitogen-activated protein kinase kinase(MEK)inhibitor(U0126)further reduced the PlGF and angiogenesis in hypoxia-induced HRECs.CONCLUSION:ApoA 1 inhibits the angiogenesis at least in part by inactivating ERK1/2 in hypoxia-induced HRECs.Moreover,apoA1 suppresses the PlGF expression,which selectively associated with pathological angiogenesis.

Apoptosis in glioma-bearing rats after neural stem cell transplantation

Abnormal activation of the Ras/Raf/Mek/Erk signaling cascade plays an important role in glioma. Inhibition of this aberrant activity could effectively hinder glioma cell proliferation and promote cell apoptosis. To investigate the mechanism of gJioblastoma treatment by neural stem ceiJ trans- plantation with respect to the Ras/Raf/Mek/Erk pathway, C6 glioma cells were prepared in sus- pension and then infused into the rat brain to establish a glioblastoma model. Neural stem cells isolated from fetal rats were then injected into the brain of this glioblastoma model. Results showed that Raf-1, Erk and Bcl-2 protein expression significantly increased, while Caspase-3 protein expression decreased. After transplantation of neural stem cells, Raf-1, Erk and Bcl-2 protein expression significantly decreased, while Caspase-3 protein expression significantly in-creased. Our findings indicate that transplantation of neural stem cells may promote apoptosis of glioma cells by inhibiting Ras/Raf/Mek/Erk signaling, and thus may represent a novel treatment approach for glioblastoma.

Enhancing osteogenic bioactivities of coaxial electrospinning nanoscaffolds through incorporating iron oxide nanoparticles and icaritin for bone regeneration

Bone tissue engineering provides a promising strategy for the treatment of bone defects.Nonetheless,the clinical utilization of biomaterial-based scaffolds is constrained by their inadequate mechanical strength and absence of osteo-inductive properties.Here,we proposed to endow nano-scaffold(NS)constructed by coaxial electrospinning technique with enhanced osteogenic bioactivities and mechanical properties by incorporating biocompatible magnetic iron oxide nanoparticles(IONPs)and icaritin(ICA).Four types of nano-scaffolds(NS,ICA@NS,NS-IONPs and ICA@NS-IONPs)were prepared.The incorporation of ICA and IONPs minimally impact their surface morphological and chemical properties.IONPs enhanced the mechanical properties of NS scaffolds,including hardness,tensile strength,and elastic modulus.In vitro assessments demonstrated that ICA@NS-IONPs exhibited enhanced osteogenic bioactivities towards mouse calvarial pre-osteoblast cell line MC3T3-E1 as evidenced by detecting the alkaline phosphatase(ALP)activity level,expressions of osteogenesis-related genes and proteins as well as mineralized nodule formation.Mechanistic investigations revealed that MEK/ERK(MAP kinase-ERK kinase(MEK)/extracellularsignal-regulated kinase(ERK))signaling pathway could offer a plausible explanation for the osteogenic differentiation of MC3T3-E1 cells induced by ICA@NS-IONPs.Furthermore,the implantation of nano-scaffolds in rat skull defects exhibited a substantial improvement in in vivo bone regeneration.Therefore,IONPs and ICA incorporated coaxial electrospinning nano-scaffolds present a novel strategy for the optimization of scaffolds for bone tissue engineering.

黑色素瘤小分子靶向药物研究进展

黑色素瘤(melanoma)是一种恶性的黑色素细胞肿瘤。我国黑色素瘤的发病率逐年攀升,而中晚期黑色素瘤尚无有效的治疗方法,因此研发新的治疗药物显得尤为迫切。本文对当前黑色素瘤靶向药物进行了总结,重点讨论了MAPK/ERK信号通路的抑制剂(如BRAF抑制剂和MEK抑制剂)。

孕哺期氯化镧暴露对子代大鼠学习记忆及细胞外信号调节激酶通路的影响

目的研究孕哺期氯化镧暴露对子代大鼠大脑皮质细胞外信号调节激酶(extracellular-signal-regulated kinases,ERK)、MAPK/ERK(MEK)及磷酸化水平和Raf-1表达的影响。方法将32只健康成年雌性Wistar大鼠随机分成对照和低、中、高剂量氯化镧组,与雄性成年Wistar大鼠按1∶1比例同笼交配。自受孕起对照组饮用蒸馏水,低、中、高剂量染镧组分别饮用含1.25、2.5、5.0 g/L LaCl3的蒸馏水溶液,并哺育其子代大鼠,至子代大鼠断乳后结束染毒。采用Morris水迷宫测试子代大鼠空间辨别性学习记忆能力,采用透射电镜法研究子代大鼠大脑皮质神经突触超微结构的改变,采用Western blot法测定子代大鼠大脑皮质Raf-1、MEK、ERK蛋白水平和MEK、ERK磷酸化水平。结果各染镧组仔鼠学习记忆能力与对照组相比明显下降,且随着染镧剂量的增加下降越来越明显。与对照组相比,各染镧组仔鼠大脑皮质神经突触超微结构出现异常改变,如突触活性带变短、突触后致密物变薄,其中5.0 g/L LaCl3组神经突触超微结构异常改变最为明显。各染镧组Raf-1蛋白水平低于对照组(P<0.05),且随着染镧剂量的增加而下降。各染镧组p-MEK和p-ERK蛋白水平低于对照组(P<0.05),且随着染镧剂量的增加而降低,其中5.0 g/L LaCl3组p-MEK和p-ERK蛋白水平与对照组、1.25和2.5 g/L LaCl3组比较均下降(P<0.05)。而各染镧组MEK和ERK蛋白水平与对照组比较无明显变化(P>0.05)。结论孕哺期氯化镧暴露导致子代大鼠大脑皮质中Raf-1表达、MEK和ERK磷酸化水平降低,这可能是氯化镧对神经系统毒性损伤的机制之一。