SHP2和MKP5在P2Y嘌呤受体介导的人前列腺癌细胞侵袭中的调控机制研究

目的　探讨蛋白质酪氨酸磷酸酶(SHP2)及丝裂原活化蛋白激酶磷酸酶5 (MKP5 )在P2Y嘌呤受体激活人不同转移潜能的前列腺癌细胞系MAPKs信号通路中的调节机制及其与细胞侵袭能力的相关性。方法　构建野生型和突变型SHP2及野生型MKP5表达质粒并分别稳定转染入1E8(高转移)和(或)2B4(不转移)细胞。应用免疫沉淀法检测细胞SHP2的酪氨酸磷酸化的程度,免疫印迹法检测ERK1 /2、p38的磷酸化,用Matrigel穿膜实验检测细胞的体外侵袭能力。结果　ATP可刺激细胞的SHP2发生磷酸化,且在1E8中的激活程度高于2B4。野生型SHP2转染可使2B4细胞中ATP对ERK1 /2的激活时效明显延长;而突变型SHP2延迟并降低1E8细胞中ATP对ERK1 /2的激活水平;两者对细胞p38的活性均无明显影响。ATP刺激可使不转移2B4细胞穿膜数明显增多(比对照组增加72 2% ±14 0% ),野生型SHP2转染可使经ATP刺激的细胞穿膜数进一步增加18 7%。ATP刺激可使高转移1E8细胞穿膜数进一步增加(112 8% ±32 0% ),而转染突变型SHP2可部分(40 9% )抑制ATP的促细胞侵袭作用。转染野生型MKP5在明显抑制p38磷酸化的同时,也能部分抑制ATP的促侵袭作用(抑制率在1E8和2B4分别为22 4%和28 7% )。结论　SHP2正性调节P2Y嘌呤受体介导的ERK活化,并可促进前列腺癌细胞的侵袭能力。而MKP5     

MEK-MAPK信号通路在P2Y嘌呤受体活化对人前列腺癌细胞效应中的作用

目的 探讨细胞外信号调节激酶(ERK1／2)及p38通路在P2Y嘌呤受体活化对前列腺癌细胞产生的生物学效应中所起的作用。方法 脂质体法将负显性MAPK激酶1(KA—MEK1)转染高转移性前列腺癌1E8细胞;Western印迹法检测ERK1／2活化水平;应用细胞计数、软琼脂集落形成实验、体外侵袭实验检测ERK1／2及p38通路在P2Y嘌呤受体活化对前列腺癌细胞生长、集落形成、体外侵袭效应中的作用;用流式细胞术检测ATP对凋亡的影响。结果 转染KA—MEK1抑制了细胞的ERK1／2活性。常规培养6d时,KA—MEK1细胞的活细胞数比对照组细胞减少了约71％;以100μmol／L ATP连续刺激6d,KA—MEK1细胞的生长被进一步抑制17.2％;用10μmol／L p38抑制剂SB203580预处理细胞,可以封闭ATP所诱导的额外生长抑制效应。说明持续P2Y受体活化可抑制前列腺癌细胞生长,ERK1／2和p38通路均在其中起作用。细胞凋亡并非ATP生长抑制作用的主要机制。在软琼脂集落形成实验中,KA—MEK1细胞比对照组细胞形成的集落小,而且数目减少了约75％;在抑制了ERK1／2活性后,以ATP预处理并不显著改变细胞的集落形成能力;进一步在抑制了：ERK1／2活性的基础上,以SB203580处理细胞,对细胞集落形成能力也无明显影响。说明在影响癌细胞集落形成方面,主要是ERK1／2通路起作用。在体外侵袭实验中,KA—MEK1细胞的穿膜细胞数比对照组细胞减少了41％;以ATP刺激12h可以促进前列腺癌细胞体外侵袭,其中KA—MEK1细胞的穿膜细胞增加率低于对照组细胞;进一步以SB203580处理细胞,可以抑制ATP诱导的促侵袭效应。说明ERK1／2和p38通路在ATP促侵袭效应中均发挥主要作用。结论P2Y嘌呤受体激活程度的差异可导致不同效应,并涉及不同信号传导通路。持续P2Y嘌呤受体活化抑制前列腺癌细胞生长,其中有ERK1／2及p38通路的参与;短暂P2Y嘌呤受体活化抑制前列腺癌细胞集落形成但促进前列腺癌细胞体外侵袭,在前者ERK1／2起重要的作用,后者有ERK1／2和p38通路的参与。     

P2Y嘌呤受体活化的PI-3K信号通路在前列腺癌细胞生长和侵袭中的作用

目的研究P2Y嘌呤受体激动剂对前列腺癌细胞PI-3K信号通路的激活及对肿瘤细胞生长和侵袭的影响。方法以高转移的人前列腺癌细胞系1E8为研究对象,以Western blot法,用特异性识别磷酸化Akt的抗体检测PI-3K信号通路的活化情况;以细胞计数、流式细胞术、Matrigel侵袭实验等方法检测P2Y受体激活的PI-3K／Akt信号通路在前列腺癌细胞生长和侵袭中的作用。结果P2Y嘌呤受体激动剂以时间和剂量依赖的方式激活了1E8细胞中的PI-3K／Akt信号通路。其持续刺激导致的生长抑制作用主要通过将细胞阻滞在S期实现（刺激组S期细胞分数比对照组提高22．3％）。应用特异性抑制剂LY294002阻断PI-3K通路明显影响1E8细胞的生长,但不能逆转P2Y受体介导的生长抑制作用。P2Y受体瞬时激活对前列腺癌细胞体外侵袭的促进作用主要通过增强细胞的运动能力（刺激组划痕修复率比对照组提高21．2％）,而非提高基质金属蛋白酶（MMP）-2和MMP-9的酶解活性实现,且此促侵袭作用可被LY294002明显抑制。结论P2Y嘌呤受体可以激活人前列腺癌细胞的PI．3K信号通路,并通过这条通路促进肿瘤细胞的运动能力进而促进侵袭;该通路是前列腺癌细胞生长的重要调节通路,但不参与P2Y嘌呤受体对前列腺癌细胞生长抑制的调节。     

液泡型ATP酶C亚基ATP6V0C在人前列腺癌细胞系中的表达及其意义

目的探讨液泡型ATP酶C亚基ATP6V0C在不同转移潜能人前列腺癌细胞系中的表达及其意义。方法采用半定量RT-PCR、荧光实时定量RT-PCR和Western blot法检测ATP6V0C在人不同转移潜能前列腺癌细胞系PC-3M-1E8、PC-3M(高转移潜能)和PC-3M-2B4、PC-3(低转移潜能)中的表达。结果 ATP6V0C在PC-3M-1E8、PC-3M细胞系中的mRNA及蛋白表达量均明显高于在PC-3M-2B4、PC-3细胞系中的表达,其中,ATP6V0C在PC-3M-1E8中的表达最高,差异具有统计学意义(P<0.05)。结论 ATP6V0C在高转移潜能人前列腺癌细胞系中的表达明显高于低转移潜能人前列腺癌细胞系,证明其和肿瘤的转移密切相关,有可能成为判断人前列腺癌侵袭和转移的重要指标及治疗前列腺癌的新靶点。     

H_2S对ATP诱导小胶质细胞活化的影响

目的:研究硫化氢(hydrogen sulfide,H_2S)对三磷酸腺苷(adenosine triphosphate,ATP)诱导大鼠小胶质细胞炎性因子释放的调节,并探讨小胶质细胞条件培养基对神经元样细胞凋亡的影响。方法:选定的大鼠小胶质细胞随机分4组:(1)正常对照组:常规培养;(2)ATP组:细胞接种24 h后用ATP处理;(3)Na HS(sodium hydrosulfide,H_2S供体)+ATP组:ATP处理前用Na HS预孵育30 min,且Na HS始终存在于反应体系中;(4)KN-62(异喹啉衍生物,嘌呤受体拮抗剂)+ATP组:ATP处理前用KN-62预孵育30 min。用ELISA检测各组细胞上清液中TNF-α、IL-6的变化,用Western Blot检测各组丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)P38和JNK表达水平。用人神经母细胞瘤细胞(SH-SY5Y)研究条件培养基对神经元的影响,其方法与上述大鼠小胶质细胞处理相同。用倒置显微镜观察SH-SY5Y细胞形态变化及凋亡情况,用MTT法检测各组细胞活力。结果:当ATP浓度3 mmol/L时,大鼠小胶质细胞释放TNF-α和IL-6水平增加(P0.05),同时上调大鼠小胶质细胞p-P38、p-JNK蛋白表达水平(P0.05)。当ATP浓度5 mmol/L时,小胶质细胞活化后的条件培养基可以使SH-SY5Y细胞形态发生改变,其细胞活力降低(P0.05)。ATP引起的上述变化可以通过Na HS预处理而逆转(P0.05),其作用与P2X7R阻断剂KN-62相类似。结论:H_2S可下调被ATP诱导的p-P38和pJNK MAPK蛋白表达,减少TNF-α和IL-6等炎性因子的释放,从而对神经元产生保护作用。     

pH变化对非洲爪蟾卵母细胞ATP受体激活电流的影响

目的：观察pH变化对非洲爪蟾卵母细胞ATP受体激活电流（I_ATP）的影响。方法：采用双极电压钳技术记录卵母细胞膜ATP受体介导的电流。结果：检测的绝大多数细胞对ATP敏感，pH为7.4的ATP引起细胞特征性膜电流，并呈浓度依赖性。该电流包括3个部分：快内向电流（D1），慢内向电流（D2）和慢外向电流（H）。当细胞外pH降低到6.0、5.5时，10-3 mol/L 的ATP引起的IATP幅值明显被抑制，并以对D1电流抑制最明显。胞内应用G蛋白抑制剂阻断P2Y受体，对D1电流影响不大，D2电流明显减小。胞外应用P2X受体拮抗剂，对D1电流有明显的阻断作用。结论：pH值降低对爪蟾卵母细胞IATP的抑制主要通过对P2X受体的影响而实现。     

PKB在类胰蛋白酶诱导基因表达中的作用

目的：探讨蛋白激酶B(PKB)在类胰蛋白酶（tryptase）诱导基因表达中的作用。 方法： 采用RT-PCR和Western blotting 方法，检测tryptase对ECV304细胞PKB（Akt）的表达及其活性和对转录因子AP-1、NF-κB p65亚单位、JNK、p38MAPK、趋化因子IL-8表达的影响。 结果： 在ECV304中1 μg/L tryptase可使PKB蛋白质磷酸化水平增加并促进PKB、转录因子NF-κB P65亚单位、AP-1和趋化因子IL-8的表达，但对JNK、p38MAPK表达影响不大。PI3K特异性抑制剂LY294002可抑制PKB的表达增加，同时可抑制NF-κB P65亚单位和IL-8的表达增加；反义PKB质粒瞬时转染ECV304，可抑制PKB、AP1、NF-κB P65亚单位和IL-8的表达增加；PAR2的抗体可抑制PKB的磷酸化，但不能阻断PKB表达。 结论： 在ECV304细胞tryptase经其膜受体PAR2通过PI3K促进PKB的磷酸化而激活之，通过其下游途径促进趋化因子IL-8、转录因子AP-1、NF-κB P65亚单位和PKB本身的表达增加。     

P2Y13受体在神经系统的研究进展

ATP是维持机体细胞正常代谢所必须的能量载体,也是一种重要的神经递质。"嘌呤受体"是指ATP及其代谢产物ADP、AMP和腺苷所结合的受体。Burnstock等(1978年)将嘌呤受体分为P1和P2受体两类,P1受体又称腺苷受体;P2受体分为P2X(离子通道受体)和P2Y(G蛋白偶联受体)受体。由于分子克隆技术的迅速发展,迄今已从人体组织细胞     

北京鸭红细胞膜G—蛋白与磷酯酶C的偶联及腺苷受体的调节

GDP、GTP和GTPγS激活北京鸭红细胞膜上的磷酯酶C,在10~(-7)～10~(-4)mol/L的范围内,使磷酸肌醇产量增加。其中GTPγS作用最强,分别使IP_2和IP_3增强550％和580％。GTP与GDP的效力相近,使IP_2增加140％和150％;IP_3增加230％和330％。A-嘌呤能受体激动剂6-N-cyclohe-xyladenosine(CHA)对抗ATP通过P-嘌呤能受体激活的磷酯酶C,使其活性下降60％。CHA的这种作用可被其特异拮抗剂8-cyclopentyl-1,3-dimethylxanthine对抗。以上结果表明鸭红细胞是研究受体、G-蛋白及磷酯酶C偶联的良好模型。表明不同嘌呤能受体在调节磷酯酶C中的相反作用。     

脑内P2X7受体研究进展

三磷酸腺苷（ATP）是细胞供能和储能的物质,在研究中发现它还是中枢神经系统的一种重要神经递质。1970年Burnstock G证实ATP是非胆碱能和非肾上腺素能神经释放的神经递质,正式提出了神经元细胞膜表面存在嘌呤受体。1985年他将ATP结合的受体命名为嘌呤能受体,并将其分为P1（腺苷）、P2（腺嘌呤核苷酸）两大类受体。     

Differential activation and regulation of mitogen-activated protein kinases through the antigen receptor and CD40 in human B cells.

In human B cells, antigen receptor ligation and CD40 ligation are known to activate the extracellular-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) pathways, which in turn regulate many important B cell functions. We previously reported that antigen receptor ligation activated the ERK pathway whereas CD40 ligation activated the JNK/stress-activated protein kinase (SAPK) pathway. Here, we demonstrate that another SAPK, p38/Hog1, is activated by both antigen receptor ligation or CD40 ligation in a human B-lymphoblastoid cell line and tonsillar B cells. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, partially inhibited ERK2 and p38 activation triggered through the B cell receptor whereas activation of JNK1 and p38 through CD40 was not affected. PD98059, a specific inhibitor of mitogen-activated extracellular response kinase kinase (MEK), significantly inhibited ERK2 activation and partially inhibited p38 activation triggered by anti-IgM antibody treatment, but did not affect CD40-dependent signaling events. In addition, anti-IgM antibody-induced signaling pathways were shown to be PKC-dependent in contrast to the CD40-induced signaling pathways. Thus, the B cell receptor and CD40 recruit the ERK, JNK and p38 pathways by using different upstream effectors.     

The Proinflammatory Cytokine, IL-6, and its Interference with bFGF Signaling and PSMA in Prostate Cancer Cells

The aim of the present work was to study the expression of the proinflammatory cytokine, interleukin-6 (IL-6), mediated by bFGF signaling and its possible crosstalk with prostate-specific membrane antigen (PSMA) in LNCaP and PC3-PSMA prostate cancer cell lines. PC3 cells stably transfected with PSMA gene were used for restoring PSMA expression. LNCaP and PC3-PSMA cells were exposed to 10 ng/mL of basic fibroblast growth factor (bFGF). IL-6 production was measured by ELISA assay, and levels of PSMA expression were assessed by flow cytometry. AKT, ERK1/2, and p38 phosphorylation were detected by Western blot. bFGF enhances IL-6 production in LNCaP and PC3-PSMA prostate cancer cells. The effect of bFGF on stimulating IL-6 secretion was greater in LNCaP than in PC3-PSMA cells. In the presence of bFGF, PSMA expression was activated after 4 days of treatment in LNCaP and PC3-PSMA cells. This activation was not maintained after long term of treatment in both metastatic cell lines. Solely MAPKs pathways (ERK1/2 and p38) were activated after bFGF stimulation in both metastatic cell lines, whereas AKT did not show any activation. The interference of the proinflammatory cytokine, IL-6, with bFGF signaling and PSMA, should be of high clinical relevance in the treatment of metastatic prostate cancer. In developing novel therapeutic modalities targeting IL-6, significant attention should be given to PSMA and its inactivation to fight against prostate cancer.     

Thermosensitization and induction of apoptosis or cell-cycle arrest via the MAPK cascade by parthenolide, an NF-κB inhibitor, in human prostate cancer androgen-independent cell lines

Parthenolide (PTL), a nuclear factor-κB (NF-κB) inhibitor, has a significant thermo-enhancement effect. Modification of thermosensitivity by treatment with PTL prior to hyperthermia was investigated in the human prostate cancer androgen-independent cell lines PC3 and DU145. In addition, we analyzed the mechanisms related to induction of apoptosis or G?/M cell-cycle arrest via the effects of ERK1/2, p38 and SAPK/JNK signaling on mitogen-activated protein kinase (MAPK). Lethal damage caused by mild hyperthermia at 41.0?C or 42.0?C in both cell lines resulted in a low level of thermosensitivity, while sequential combination with PTL showed significant thermosensitization. Step-up hyperthermia (SUH) (42?C for 30 min, 43.0?C or 43.5?C for various periods) reduced the thermosensitivity of the cells to second heating. However, PTL given as pre-treatment prior to SUH prevented SUH-induced thermal tolerance and resulted in significant thermosensitization. Induction of apoptosis by the combination of PTL and hyperthermia at 44.0?C was determined by the ratio of sub-G1 division cells using flow cytometry, which was increased significantly in comparison with single treatment, and was more effective in PC3 than DU145 cells. The behavior of ERK1/2, p38, and SAPK/JNK signaling in the MAPK cascade by treatment with PTL and hyperthermia were examined by Western blotting. As for PC3 cells, ras-downstream p-ERK1/2 was activated and p-p38 slightly activated by combined treatment with PTL and hyperthermia in comparison with each alone. As for DU145 cells, ERK1/2 was not changed, while p38 and SAPK/JNK were slightly activated by combination treatment. These results were related to increases in the induction of apoptosis, G?/M cell cycle arrest, and lethal damage of cells via the MAPK cascade. Together, our findings demonstrate that PTL is an effective thermosensitizing agent for multidisciplinary therapy for human prostate cancer.     

Cooperativity between extracellular adenosine 5'-triphosphate and activation of N-methyl-D-aspartate receptors in long-term potentiation induction in hippocampal CA1 neurons

The mechanism of ATP-induced long-term potentiation (LTP) was studied pharmacologically using guinea-pig hippocampal slices. LTP, induced in CA1 neurons by 10 min application of 10 microM ATP, was blocked by co-application of the N-methyl-D-aspartate (NMDA) receptor antagonist, D,L-2-amino-5-phosphonovalerate (5 or 50 microM). In ATP-induced LTP, the delivery of test synaptic inputs (once every 20 s) to CA1 neurons could be replaced by co-application of NMDA (100 nM) during ATP perfusion. These results suggest that, in CA1 neurons, a co-operative effect between extracellular ATP and activation of NMDA receptors is required to trigger the process involved in ATP-induced LTP. In addition, ATP-induced LTP was blocked by co-application of an ecto-protein kinase inhibitor, K-252b (40 or 200 nM), whereas a P2X purinoceptor antagonist, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid 4-sodium (50 microM), or a P2Y purinoceptor antagonist, basilen blue (10 microM), had no effect.The results of the present study, therefore, indicate that the mechanisms of ATP-induced LTP involve the modulation of NMDA receptors/Ca(2+) channels and the phosphorylation of extracellular domains of synaptic membrane proteins, one of which could be the NMDA receptor/Ca(2+) channel.     

Roles of NF-κB and MAPK signaling pathways in morphological and cytoskeletal responses of microglia to double-stranded RNA

Following virus infection of the central nervous system, microglia become activated and undergo morphological as well as functional transformations, thereby initiating effective antiviral actions. Herein, we have examined the contribution of nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways to cell shape determination and cytoskeletal organization in microglia upon stimulation with double-stranded RNA (dsRNA), a conserved molecular pattern of virus infection. Under non-proliferative condition, microglial MG6-1 cells displayed a distinctive morphology with spinescent processes and small somata. Following dsRNA stimulation, the process-bearing microglial cells exhibited swift and drastic changes in cell morphology, filamentous actin (F-actin) structure, and intracellular signaling. In the dsRNA-stimulated microglial cells, the activation of c-Jun N-terminal kinase (JNK) pathway was involved in morphological alteration into an ameboid state. We also found that p38 signaling pathway negatively regulates the formation of cytoplasmic vacuoles in microglial cells. Furthermore, the dsRNA-induced accumulation of F-actin was partly mediated by NF-κB, JNK, and p38 pathways. These results indicate that NF-κB and MAPK signaling pathways mediate morphological and cytoskeletal changes during dsRNA-induced microglial activation.