磷酰肌醇—3激酶途径在慢性髓细胞白血病细胞增殖和抗凋亡中的作用

目的 探讨磷酰肌醇—3激酶途径在K562和HL60细胞增殖中的作用。方法 用磷酰肌醇—3激酶(PI3K)特异抑制剂Wortmannin抑制PI3K活性，观察K562细胞和HL60细胞增殖能力的变化。结果 显示K562和HL60细胞在培养24小时、48小时、72小时的增殖抑制率分别为41．33％、57．46％、65．85％和32．14％、17．14％、13．14％。生长曲线显示Wortmannin可显著抑制K562细胞的增殖，而对HL60细胞增殖无明显影响。结论 Wortmannin可以通过抑制PI3K通路抑制K562细胞的增殖。     

川芎嗪对血小板中肌醇磷脂和2OK蛋白质磷酸化的抑制作用

在Mg~(2+)存在下.用血小板膜与(γ—~(32)P)ATP保温,以观察川芎嗪对~(32)P掺入磷脂和蛋白质的影响,结果表明:血小板膜中存在磷脂酰肌醇(PI)激酶和磷脂酰肌醇—4—磷酸(PIP)激酶,而胞浆中缺乏或极少;ATP促进肌醇磷脂磷酸化;川芎嗪抑制血小板中PIP和20K蛋白质的磷酸化.半数抑制浓度分别为40μmol·L~(-1)和110μmol·L     

磷酰肌醇-3激酶途径与bcr/abl基因介导的白血病细胞增殖和抗凋亡信号传导

目的探讨磷酰肌醇-3激酶途径在K562、NB4和HL60细胞增殖和凋亡抗性中的不同作用。方法短期培养法直接法G显带检测K562细胞和CML患者骨髓原代细胞的染色体核型,RQ-PCR检测K562细胞和CML患者骨髓原代细胞的bcr/abl基因;用磷酰肌醇-3激酶特异抑制剂渥曼青霉素(WT)抑制磷酰肌醇-3激酶活性,经细胞生长曲线测定、半固体集落形成实验、流式细胞膜联蛋白V标记技术检测细胞凋亡百分比和凋亡指数。观察K562、NB4和HL60细胞增殖能力及凋亡抗性的变化。统计学采用t检验。结果K562细胞G显带检出Ph染色体,RQ-PCR检测K562细胞存在bcr/abl基因;与标准Ph染色体表达的CML患者骨髓原代细胞的bcr/abl基因完全吻合;K562、NB4和HL60细胞在24h、48h、72h的增殖抑制率分别为41.33%、57.46%、65.85%和26.29%、5.51%、2.10%及32.14%、17.14%、13.14%。生长曲线显示WT抑制磷酰肌醇-3激酶途径可显著抑制K562细胞的增殖(P<0.05),而对NB4和HL60细胞增殖无明显影响(P>0.05)。K562、NB4和HL60细胞加和不...     

LY294002对多药耐药急性白血病细胞株HL60/VCR的凋亡及细胞周期的影响研究

目的:研究磷脂酰肌醇-3-激酶(PI3K)抑制剂LY294002对多药耐药急性白血病细胞株HL60/VCR凋亡与细胞周期的影响及可能机制。方法:取耐长春新碱(VCR)的HL60/VCR多药耐药急性白血病细胞株分为未加药的对照组及加入不同浓度的LY294002(其终浓度为1.0、2.5、5.0、10μmol.L-1)组,MTT法检测2种细胞的半数抑制浓度(IC50),流式细胞仪检测细胞凋亡及细胞周期分布,半定量逆转录-聚合酶链反应(RT-PCR)检测抗凋亡蛋白Bcl-2、细胞周期调控分子CyclinD1mRNA表达。结果:与对照组比较,LY294002可明显降低HL60/VCR对VCR的IC50(P<0.05),且与剂量相关;LY294002可显著增加HL60/VCR细胞凋亡率,阻滞细胞于G0/G1期,降低HL60/VCR细胞的Bcl-2、CyclinD1mRNA的表达(P均<0.05)。结论:LY294002可能是通过影响Bcl-2及CyclinD1基因的表达,从而促进HL60/VCR细胞凋亡及抑制细胞增殖。     

反义磷脂酰肌醇3-激酶寡脱氧核苷酸抑制白介素-1诱导的NF-κB活化

目的　研究反义磷脂酰肌醇 3 激酶 (PI 3 激酶 )寡脱氧核苷酸 (oligodeoxynucleotides,ODN)对核因子 κB(NF κB)活化的影响。方法　Lipofectin介导反义PI 3 激酶ODN转染HepG2细胞 ,以逆转录PCR法检测PI 3 激酶mRNA表达水平 ,用SandwichELISA法检测NF κB的活化。结果　(1)反义PI 3 激酶ODN抑制PI 3 激酶mRNA表达。 (2 )反义PI 3 激酶ODN呈剂量 (1～ 8μg)和时间 (5～ 2 4h)依赖性地抑制NF κB活化 ,2 μg反义PI 3 激酶ODN与细胞共孵育 8h时抑制效果最好。结论　反义PI 3 激酶ODN通过阻断PI 3 激酶表达抑制NF κB活化 ,预示反义PI 3 激酶ODN可潜在抑制IL 1的炎症活性     

磷脂酰肌醇3-激酶调控白介素-18诱导核因子-кB活化(英文)

目的:研究磷脂酰肌醇(PI)3-激酶在白介素18(IL-18)诱导核因子-кB(NF-кB)活化中的作用。方法:Lipofectin介导反义PI 3-激酶寡核苷酸转染HepG2细胞。用逆转录PCR法检测PI 3-激酶mRNA表达水平,以Sandwich ELISA法检测NF-кB的活化。结果:(1)反义PI 3-激酶寡核苷酸抑制PI 3-激酶mRNA表达。(2)IL-18诱导NF-кB活化。(3)反义PI3-激酶寡核苷酸呈时间(5-24h)和浓度(1-8mg/L)依赖性地抑制IL-18诱导的NF-кB活化。结论:PI 3-激酶调控白介素-18诱导的NF-кB活化。     

抗癌药物磷脂酰肌醇3激酶抑制剂

磷脂酰肌醇3激酶(PI3Ks)是脂激酶家族成员,可通过磷脂酰肌醇的3位磷酸化产生磷脂酰肌醇三磷酸脂(PIP3)来调节细胞的代谢和生长.PI3K途径是人体癌细胞中最常发生突变的途径之一,可引起信号的放大,因此是小分子抑制剂的较好作用靶位,可为癌症的治疗提供机会.近几年涉及PI3K的抑制作用的专利和相关报道急剧增加,本文综述了PI3K抑制剂作为抗癌药物的发现与研究进展,并阐述了具有代表性的7个结构各异的PI3K抑制剂.这些化合物代表了一大批具有多种同分异构特异性且活性较强的酶抑制剂(其IC50在毫微摩尔水平).目前,首类PI3K抑制剂将要进入临床试验阶段,期望这些临床数据将有助于研究人员弄清楚各PI3K异构体对肿瘤的抑制作用及相关的不良反应.     

磷脂酰肌醇3—激酶调控白介素—18诱导核因子—κB活化

目的：研究磷脂酰肌醇（PI）3－激酶在白介素18（IL－18）诱导核因子－κB（NF－κB）活化中的作用。方法：Lipofectin介导反义PI 3－激酶寡核苷酸转染HepG2细胞。用逆转录PCR法检测PI 3－激酶mRNA表达水平,以Sandwich ELISA法检测NF－κB的活化。结果：1）反义PI 3－激酶寡核苷酸抑制PI 3－激酶mRNA表达。（2）IL－18诱导NF－κB活化。（3）反义PI 3－激酶寡核苷酸呈时间（5－24h)和浓度（1－8mg/L)依赖性地抑制IL－18诱导的NF－κB活化。结论：PI 3－激酶调控白介素－18诱导的NF－κB活化。     

通关藤诱导白血病细胞U937,HL60细胞凋亡的实验研究

目的探讨通关藤抑制白血病细胞增殖作用及其机制。方法以不同浓度的通关藤提取物制剂处理白血病细胞U937、HL60,1~5 d,以四甲基偶氮唑盐(MTT)法检测对细胞增殖的影响,以Annexin V/PI双染法检测细胞的凋亡程度,Western blot检测凋亡相关蛋白caspase3,PARP改变,以JC-1染色法检测线粒体跨膜电位(ΔΨm)水平。结果通关藤提取物制剂呈时间和剂量依赖性抑制U937、HL60细胞增殖,50μL/mL时能明显降低线粒体跨膜电位(ΔΨm),活化caspase 3,剪切PARP,诱导细胞凋亡。结论通关藤提取物制剂对U937、HL60白血病细胞有显著的抑制和诱导凋亡作用,能通过降低线粒体跨膜电位途径触发白血病细胞凋亡。     

DADS抑制JAK1/STAT3信号通路诱导人白血病HL-60细胞分化

目的探讨JAKs/STATs信号转导通路在二烯丙基二硫(DADS)诱导人白血病HL60细胞分化中的变化及其调控机制。方法将HL60细胞与DADS或JAKs/STATs信号通路的激酶抑制剂AG490在体外共同培养,观察细胞形态变化,检测药物作用前后细胞NBT还原能力及细胞表面分化抗原CD11b的改变;用Westernblot检测JAKs,STATs各家族成员在DADS诱导HL60细胞分化中的改变;并用免疫细胞化学法检测核转录基因STATs,cmyc,cfos,cjun的表达变化。结果DADS和AG490均可诱导HL60细胞向成熟粒系分化,且DADS在1.25mg·L-1时诱导分化作用达峰值;Westernblot检测JAK1,STAT3的酪氨酸激酶发生了磷酸化改变;免疫细胞化学示STAT3与cmyc基因蛋白核内表达下降,cjun,cfos基因蛋白核内表达上升。结论JAK1,STAT3酪氨酸激酶的磷酸化抑制参与了DADS诱导HL60细胞分化的调控,其机制可能通过调控与HL60细胞增殖分化相关的基因表达,抑制细胞DNA合成,从而抑制细胞增殖,诱导分化。DADS的作用相当于JAK1/STAT3信号通路的阻断剂。     

Effect of desipramine on inositol phosphate formation and inositol phospholipids in rat brain and human platelets.

To examine the mechanism of action of antidepressant drugs, we studied the effect of desipramine (DMI) in vitro on agonist-stimulated inositol phosphate formation and inositol phospholipids in rat brain and human platelets. We observed that DMI inhibited thrombin-stimulated 3H-inositol bisphosphate (IP2) and 3H-inositol trisphosphate (IP3) but not 3H-inositol monophosphate (IP1) formation in human platelets. DMI also inhibited norepinephrine (NE) and serotonin (5-HT) stimulated 3H-IP1 formation in rat cerebral cortex. DMI increased levels of all three 3H-inositol phospholipids, 3H-phosphatidyl inositol (PI), 3H-PI-4-phosphate (PIP), and 3H-PI 4,5-bisphosphate (PIP2), in both platelets and rat cortex. The decreased formation of inositol phosphates and increased levels of [3H]-PI, [3H]-PIP, and [3H]-PIP2 by DMI appears to be due to the inhibition of the enzyme phospholipase C rather than its effects on receptors. It is thus possible that interaction of tricyclic antidepressant drugs with the PI-signaling system may be related to their mechanism of action.     

Effects of a water-soluble antitumor ether phosphonoinositide, D-myo-inositol 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (C4-PI), on inositol lipid metabolism in breast epithelial cancer cell lines

We have demonstrated previously that D-myo-inositol 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (C4-PI), an isosteric phosphonate analog of phosphatidylinositol developed to inhibit inositol lipid metabolism, was unable to inhibit phosphatidylinositol (PI) 3-kinase activity. We now report the effects of the compound on other aspects of inositol metabolism. We demonstrated that C4-PI inhibits the activity of purified recombinant PI-phospholipase C-beta (PLC-beta) at all concentrations tested; it enhanced the activity of PI-PLC-gamma and PI-PLC-delta at low concentrations (10 microM), while severely inhibiting their activities at higher concentrations. In the breast cancer cell lines MCF-7 (estrogen receptor positive) and MDA-MB-468 (estrogen receptor negative), C4-PI had no effect on the uptake of D-myo-inositol but severely inhibited its incorporation into PI. In spite of the drastic decrease in PI synthesis, C4-PI did not affect the levels of inositol incorporated into phosphatidylinositol 4,5-bisphosphate (PIP2) in the cells. In vitro assays showed that C4-PI inhibited PI synthase activity (inhibition of 35% at 50 microM) but had little effect on PI 4-kinase activity (inhibition of 13% at 150 microM). C4-PI inhibited the proliferation of MCF-7 and MDA-MB-468 cell lines with IC(50) values of 12 and 18 microM. Taken together, the results suggest that the accumulation of [3H]inositol in PIP2 in cells incubated with C4-PI may be due to the inhibition of PIP2 hydrolysis in the cells with no effect on its synthesis. The role of these C4-PI-induced effects in the mechanism of growth inhibition by C4-PI remains to be established.     

Modifications of phospholipid metabolism induced by chlorpromazine, desmethylimipramine and propranolol in C6 glioma cells

The effects of chlorpromazine (CPZ), desmethylimipramine (DMI) and propranolol (PRO) on phospholipid metabolism in C6 glioma cells were studied by following the incorporation of 32Pi, [U-14C]glycerol, [2-3H]glycerol and [1-14C]oleate into lipids. The drugs produced a dose-dependent increase in the incorporation of 32Pi and [U-14C]glycerol, but not of [1-14C] oleate, into total phospholipids, that reached a plateau at 200 microM CPZ and 500 microM DMI and PRO. The three drugs shifted the incorporation of precursors from neutral [phosphatidylcholine (PC) and phosphatidylethanolamine (PE)] to acidic phospholipids [phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylglycerol, phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2)] in a dose-dependent, qualitatively similar manner. The incorporation of [2-3H]glycerol into diacylglycerol was also depressed markedly by CPZ. Addition of 1 mM 1,2-dioleoylglycerol, 1-oleoyl-2-acetylglycerol or oleate only partially reversed the decrease in PC labeling caused by CPZ. 12-O-Tetradecanoylphorbol-13-acetate counteracted this effect of CPZ completely but greatly increased PC labeling even in the absence of the drug. Polyphosphoinositides rapidly incorporated 32Pi at early times reaching a plateau in about 40 min. The labeling rate of PI was not parallel to that of PIP or PIP2 and continued to increase even after the polyphosphoinositides had reached a plateau. CPZ increased PI labeling much more than that of PIP and PIP2. These data suggest that cationic amphiphilic drugs may act by inhibiting CTP:phosphocholine cytidylyltransferase, thus decreasing incorporation of precursors into PC and PE; inhibiting PA phosphohydrolase with increased formation of phosphatidyl-CMP, the intermediate for the synthesis of acidic phospholipids; and stimulating the inositol exchange reaction, forming a pool of PI that is not available for PIP and PIP2 synthesis.     

Glucocorticoid inhibition of antigen-induced inositol phosphate formation: possible involvement of phosphatases.

The suppressive effect of glucocorticoids (GC) upon antigen-induced phosphatidylinositol phospholipase C (PI-PLC) activity and inositol phosphate formation by rat basophilic leukemia cells (RBL-2H3) has been characterized. Addition of antigen for a period of 1-30 min enhanced production of [3H]inositol monophosphate (IP1), inositol 1,4-biphosphate (IP2) and inositol 1,4,5-triphosphate (IP3) by about 5-10 fold. Pretreatment with hydrocortisone (HC) and dexamethasone (DEX) reduced formation of the various inositol phosphates (IPs) and degradation of phosphatidylinositol-4-5-biphosphate (PIP2) by an average of 50% Antigen-stimulated phosphorylation of an 18 kDA and other proteins was inhibited by about 60% following pretreatment with the GC. This inhibition was in turn prevented by cycloheximide. Moreover, DEX doubled cellular acid phosphatase activity. The results suggest that the inhibitory effect of GC is possibly mediated, among other things, by protein phosphatase activity.     

蛋白激酶C和磷脂酰肌醇4,5二磷酸之间相互调节作用的研究进展

蛋白激酶C(protein kinase C,PKC)是一个多基因家族,包含多种同工酶,分布广泛且功能复杂,在许多信号转导通路发挥重要作用。磷脂酰肌醇(4,5)二磷酸(PIP2)是分布在细胞膜中的磷脂类信号分子,在细胞中的分布和含量处于动态变化中。PIP2的水解后生成DAG和IP3。DAG可以直接激活PKC,而IP3通过调节细胞内钙离子的浓度从而改变钙依赖型PKCs的活性。同时,PKC通过激活PI4K或PIP5K可以调节细胞膜PIP2水平。PKCs使离子通道蛋白发生磷酸化,改变通道蛋白与PIP2的亲和力,从而影响PIP2对离子通道的调节。该文对PKCs和PIP2在细胞信号转导过程中相互调节的相关研究进展进行综述。     

Changes in membrane potential and phosphoinositides during alpha 1-adrenoceptor stimulation in smooth muscle cells of guinea-pig taenia caeci

The effect of a submaximal concentration of adrenaline (3-5 microM) was studied in taenia caeci smooth muscle cells. Membrane potential hyperpolarization was observed in intact muscle preparations and this response could be separated into two phases, depending on the state of a membrane-bound calcium compartment. The effect of alpha 1-adrenergic stimulation was also measured by [3H]inositol incorporation into phospholipid and inositol phosphate fractions of taenia cell suspensions both in the absence and presence of 2.5 mM extracellular calcium. In the absence of extracellular calcium the inositol phospholipids increased within 15 s after stimulation, followed by enhanced inositol phosphates. With calcium present there was a biphasic increase in the phosphatidylinositol 4,5-bisphosphate (PIP2) fraction with a simultaneous release of inositol phosphates. Lithium ions affected the incorporation of label into the lipids but not into the inositol phosphate fractions. These findings suggest that, in taenia caeci cells, alpha 1-adrenergic-induced membrane hyperpolarization resulting in muscle relaxation is associated with changes in the PIP2 content.     

Chlorpromazine increases the turnover of metabolically active phosphoinositides and elevates the steady-state level of phosphatidylinositol-4-phosphate in human platelets.

Non-permeabilizing concentrations (< 40 microM) of chlorpromazine (CPZ) increase the radioactivity of phosphatidylinositol-4-phosphate (PIP) in platelets pre-labelled with [32P]Pi, but the biochemical mechanisms underlying this increase are poorly understood. Incubation of [32P]Pi-labelled, gel-filtered platelets with 25 microM CPZ for 10 min increased: (1) the mass of PIP from 315 to 476 nmol/10(11) platelets but not the total inositol phospholipid mass, (2) the specific phosphodiester radioactivities in phosphatidylinositol (PI), PIP and phosphatidylinositol-4,5-bisphosphate (PIP2) by 34, 63 and 37%, respectively, and (3) the specific phosphomonoester radioactivities in PIP and PIP2 by 53 and 10%, respectively. In control platelets (no CPZ) the specific radioactivity of the phosphodiester was the same in PI, PIP and PIP2, and the specific radioactivity in the phosphomonoester in PIP and PIP2 was 55% of that of the gamma-phosphoryl in ATP, measured as metabolically active, actin-bound ADP. These results suggest that 55% of each of PI, PIP and PIP2 constitutes a metabolic pool which is labelled by 32P in the platelets, while the remainder is in a metabolically inactive pool and not labelled. CPZ has two major effects: (1) CPZ interferes with the kinase and phosphohydrolase reactions that maintain the steady-state level of PIP in the metabolic phosphoinositide pool, resulting in a 92% increase in the PIP level of this pool, and (2) CPZ causes synthesis (45% in 10 min) of new phosphodiester in the metabolically active phosphoinositides by tentative stimulation of the turnover of the phosphoinositide cycle, de novo phosphoinositide synthesis and/or diacylglycerol formation through phospholipases C and D. The marked alteration by CPZ of phosphoinositide metabolism may be part of the mechanism by which this drug effects its psychotropic action.     

SHIP2: an emerging target for the treatment of type 2 diabetes mellitus

With the rapid increase in the number of patients developing type 2 diabetes mellitus and the lack of optimal therapies, much focus has been placed on the insulin-signaling pathway in the discovery of novel drug targets. Phosphatidyl Inositol 3-Kinase (PI3K) is central to mediating insulin-s metabolic effects. PI3K catalyzes the generation of phosphatidyl inositol (3,4,5) triphosphate (PIP(3)). Inhibition of PI3K activity results in a blockade of insulin signaling including glucose uptake and glyocogen synthesis. Thus, PIP(3) is a critical mediator of insulin action. A family of phosphatidyl inositol phosphatases have been identified that counter-regulate PI3K activity by hydrolyzing PIP(3) to phosphatidyl inositol bisphosphate at either the 3' or 5' position of the inositol ring. Mice lacking one of these enzymes, Src-Homology Inositol Phosphatase-2 (SHIP2), demonstrate increased insulin sensitivity, suggesting that pharmacological inhibition of SHIP2 could alleviate insulin resistance. Recent studies demonstrate elevated SHIP2 expression is associated with insulin resistance in human patients. Comparing the studies on SHIP2 and other phosphatases suggests how inhibition of SHIP2 leads to increased insulin sensitivity without deleterious effects. This review focuses on the emergence of SHIP2 as a target in the insulin-signaling pathway for the treatment of type 2 diabetes.