Alteration in erythropoietin-induced cardioprotective signaling by postinfarct ventricular remodeling

Postinfarct remodeling impairs mechanisms of ischemic preconditioning. We examined whether myocardial response to activation of the erythropoietin (EPO) receptor is modified by postinfarct remodeling. Four weeks after induction of myocardial infarction (MI) by coronary ligation in post-MI group (post-MI) or a sham operation in sham group (sham), rat hearts were isolated and subjected to 25-min global ischemia/2-h reperfusion. Infarct size was expressed as a percentage of risk area (i.e., left ventricle) from which scarred infarct was excluded (%I/R). The heart weight was 15% larger in post-MI, but there was no intergroup difference in plasma EPO levels or myocardial EPO receptor levels. EPO infusion (5 U/ml) significantly reduced %I/R from 59.9 +/- 4.1 to 36.2 +/- 4.2 in sham and from 58.1 +/- 5.0 to 35.2 +/- 4.0 in post-MI. This EPO-induced protection was sensitive to a phosphatidylinositol 3-kinase (PI3K) inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), in sham. However, neither LY294002 nor wortmannin inhibited the EPO-induced protection in post-MI. Phosphorylation of Janus kinase 2 by EPO was attenuated and phosphorylation of Akt was not detected in post-MI. A guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, and a mitochondrial ATP-sensitive K(+) channel (mitoK(ATP) channel) blocker, 5-hydroxydecanoate, inhibited EPO-induced protection in both sham and post-MI. Suppressor of cytokine signaling (SOCS)-1 protein level was higher by 50% in post-MI than in sham, although SOCS-3 levels were similar. These findings suggest that postinfarct remodeling disrupts cellular signaling from the EPO receptor to PI3K, presumably by increased SOCS-1. However, in the remodeled myocardium, lack of PI3K/Akt activation by the EPO receptor seems to be compensated by a mechanism upstream of the guanylyl cyclase-mitoK(ATP) channel pathway to achieve EPO-induced protection.     

Ischemic postconditioning mediates cardioprotection via PI3K/GSK-3β/β-catenin signaling pathway in ischemic rat myocardium

Previous studies have shown that PI3K/GSK-3β/β-catenin signaling pathway plays a vital role in ischemic preconditioning. The present study attempts to evaluate whether PI3K/GSK-3β/β-catenin signaling pathway might be responsible for the cardioprotection in ischemic postconditioning. Male Sprague-Dawley rats underwent 30 min of left anterior descending coronary artery occlusion and 2 h of reperfusion. One hundred twenty rats were randomized into six groups: sham, ischemia/reperfusion (I/R), ischemic postconditioning (Post), 15 μg · kg wortmannin (PI3K inhibitor) plus ischemic postconditioning (Wort + Post), wortmannin plus I/R (Wort + I/R), and 0.6 mg · kg SB216763 (GSK-3β inhibitor) plus I/R (SB + I/R). Wortmannin and SB216763 were administered, respectively, 10 and 5 min before reperfusion. Myocardial infarct size; number of apoptotic cardiomyocytes; total Akt, GSK-3β; phosphorylated Akt, GSK-3β; β-catenin in cytosol and nucleus; and Bcl-2 protein were assessed. It was found that Post and SB + I/R reduced infarct size (32.3% [SD, 2.8%], 32.7% [SD, 2.1%], vs. 53.4% [SD, 3.2%], respectively, P < 0.05) and apoptotic index of cardiomyocytes (23.2% [SD, 1.8%], 23.8% [SD, 1.8%], vs. 47.3% [SD, 5.8%], respectively, P < 0.05); compared with I/R, wortmannin abolished the cardioprotection of ischemic postconditioning. Post and SB + I/R increased phosphorylated Akt, phosphorylated GSK3β, β-catenin in cytosol and nucleus, and Bcl-2 expression versus I/R. These results indicate that ischemic postconditioning could induce myocardial protection via PI3K/GSK-3β/β-catenin signaling pathway, activation of which results in accumulation of β-catenin and upregulation of its target genes Bcl-2.     

Actin cytoskeleton dynamics promotes leptin-induced vascular smooth muscle hypertrophy via RhoA/ROCK- and phosphatidylinositol 3-kinase/protein kinase B-dependent pathways

Obesity is associated with increased leptin production that may contribute to cardiovascular pathology through a multiplicity of effects. Leptin has been shown to contribute to vascular remodeling through various mechanisms, including production of vascular smooth muscle (VSMC) hypertrophy; however, the mechanisms underlying the vascular hypertrophic effect of leptin remain unknown. In the present study, we investigated the contributions of the RhoA/Rho kinase (ROCK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways, actin dynamics, and the expression of serum-response factor (SRF) in the hypertrophic effects of leptin on vascular tissue. Strips of rat portal vein (RPV) were cultured with or without leptin at 3.1 nM for 1 to 3 days. Leptin significantly increased RhoA activity by 163 +/- 20%, whereas phosphorylation of downstream factors, including LIM kinase 1 and cofilin-2, was increased by 160 +/- 25 and 290 +/- 25%, respectively. Leptin also significantly phosphorylated Akt by 130 +/- 30%, which was inhibited by the PI3K inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). RhoA/ROCK and PI3K/Akt activation was associated with a significant increase in RPV wet weight (11 +/- 1%), protein synthesis (45 +/- 7%), SRF expression (136 +/- 11%), and polymerization of actin, as reflected by an increase in the F-/G-actin ratio, effects that were significantly attenuated by a leptin receptor (leptin obese receptor) antibody, the ROCK inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) (Y-27632) as well as the PI3K inhibitor LY294002. Our results indicate that the activation of RhoA/ROCK and PI3K/Akt plays a pivotal role in leptin signaling, leading to the development of VSMC hypertrophy through a mechanism involving altered actin dynamics.     

Spectrum of activity and molecular correlates of response to phosphatidylinositol ether lipid analogues, novel lipid-based inhibitors of Akt

The serine/threonine kinase Akt is a promising target in cancer. We previously identified five phosphatidylinositol ether lipid analogues (PIA) that inhibited Akt activation and selectively killed lung and breast cancer cells with high levels of Akt activity. To assess the spectrum of activity in other cell types and to compare PIAs with other inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, we compared growth inhibition by PIAs against the PI3K inhibitors LY294002 and wortmannin and the mTOR inhibitor rapamycin in the NCI60 cell line panel. Although each of these compounds inhibited the growth of all the cell lines, distinct patterns were observed. The PIAs were the least potent but the most cytotoxic. The broad spectrum of activity of PIAs was confirmed in vivo in hollow fiber assays. The response to PIAs was significantly correlated with levels of active but not total Akt in the NCI60, as assessed using COMPARE analysis. However, a number of molecular targets were identified whose expression was more highly correlated with sensitivity to PIAs than active Akt. Expression of these molecular targets did not overlap with those that correlated with sensitivity to LY294002, wortmannin, or rapamycin. A COMPARE analysis of the National Cancer Institute chemical screening database revealed that the patterns of activity of PIAs correlated best with patterns of activity of other lipid-based compounds. These studies show that although PIAs are widely active in cancer cells, which correlates with the presence of its intended target, active Akt, PIAs are biologically distinct from other known inhibitors of the PI3K/Akt/mTOR pathway.     

Akt／GSK-3β信号通路和线粒体渗透性转换孔在吸入性麻醉药预处理对老年大鼠心肌缺血保护的研究

目的Akt／GSK．3β信号通路的激活参与了吸人麻醉药心肌保护预处理。在该研究中观察了老年和成年大鼠在异氟醚心肌保护预处理中的差异,包括Akt／GSK-3β信号通路和线粒体渗透性转换孔在异氟醚预处理中的差异。方法雄性Fisher344大鼠按照各自的年龄,成年（3—5月）、老年（20～24月）,随机分配到阴性对照组（sc）、缺血再灌注组（I／R）、异氟醚组（ISO）中。ISO组,大鼠接受30min1．0MAC的异氟醚预处理。I／R组,大鼠接受30min的心肌缺血。方案A中,大鼠接受2h再灌注,用来检测梗死面积。方案B中,大鼠接受10min再灌注,用Westcrn来检测P-Akt,Akt,P—GSK-3β,GSK．3β的表达;并且用分光光度法分析组织中烟酰胺腺嘌呤二核苷酸（NAD＋）水平,作为反映线粒体渗透性转换孔（mPTP）开放的指标。结果成年组大鼠,异氟醚预处理组（YISO＋I／R）心肌梗死面积为29．9％±1．9％,明显低于缺血再灌注组（YI／R,51．8％±2．1％,P〈0．001）。而老年组大鼠,异氟醚预处理失去心肌保护作用（OISO＋I／R--39．9％4-3．7％VSOI／R=46．9％±2．5％,P〉0．05）。比较成年阴性对照组（YSC组）和成年缺血再灌注组（YI／R组）,异氟醚预处理显著提高了成年异氟醚组（YISO组）和成年异氟醚＋缺血再灌注组（YISO＋I／R组）的Akt和GSK-3B的磷酸化水平（P〈0．05）。但是老年组大鼠,Akt和GSK-39磷酸化水平已经在阴性对照组（osc组）提高（对比YSC组）。异氟醚预处理没有进一步提高老年异氟醚组（OISO组）和老年异氟醚＋缺血再灌注组（OISO＋I／R组）的Akt和GSK-3B的磷酸化水平（P〈0．05）。同样,异氟醚预处理减少了成年组大鼠心肌组织中NAD’的减少,而异氟醚预处理失去了对老年组大鼠的NAD＋流失保护作用。结论老年大鼠失去异氟醚预处理心肌保护作用,失去进一步提高Akt、GSK．3β的磷酸化水平能力和失去关闭mPTP是其机理之一。     

胰岛素对新生大鼠缺氧/复氧诱导心肌细胞凋亡的影响

目的探讨胰岛素对缺氧/复氧所诱导心肌细胞凋亡的影响及其机制。方法通过给原代培养乳鼠心室肌细胞行缺氧2h/复氧4h,建立缺氧/复氧(anoxia/reoxygenation)心肌细胞损伤模型。于复氧期开始随机给予0.9%生理盐水(VC组)、胰岛素(INS组)、LY294002(LY组)、胰岛素 LY294002(INS LY组)干预。于复氧4h后,利用2,4二硝基苯肼显色法检测乳酸脱氢酶(LDH)活性,硫代巴比妥酸显色法检测丙二醛(MDA)含量,原位末端标记法(TUNEL)和DNA梯带法(DNALadder)标测细胞凋亡,免疫印迹法(Westernblotting)检测磷酸化Akt表达,并比较各组间差异。结果与VC组相比,INS组中LDH活性、MDA含量、凋亡指数(AI)显著降低(P<0.01),磷酸化Akt表达明显增加(P<0.01);但上述指标变化可被LY294002(PI3K抑制剂)所抑制。结论在复氧早期给予胰岛素干预可显著地减少缺氧/复氧所诱导的心肌细胞凋亡,其保护机制与PI3K/Akt所介导的抗细胞凋亡作用有关。     

肠三叶因子介导PI3K/Akt信号通路保护胃黏膜上皮细胞紧密连接

目的：探讨肠三叶因子对胃黏膜上皮细胞紧密连接的保护作用,并研究PI3K/Akt信号通路在其中的作用机制。方法：体外培养GES-1细胞,分别设正常对照组,LPS组,ITF组,LPS＋ITF组,LPS＋ITF＋LY294002组,ITF＋LY294002组。正常对照组：正常培养;LPS组：加入浓度为10mg/L的LPS;ITF组：加入浓度为100μg/L的ITF;LPS＋ITF组：加入浓度为10mg/L的LPS,同时加入100μg/L的ITF;LPS＋ITF＋LY294002组：加入浓度为10mg/L的LPS、100μg/L的ITF,同时加入PI3K/Akt信号通路的抑制剂LY294002（15μM）;ITF＋LY294002组：加入100μg/L的ITF,同时加入15μM的LY294002。培养48h,采用Western blot检测ITF对PI3K/Akt信号通路的作用,采用免疫荧光和Western blot检测细胞紧密连接蛋白Occludin和ZO-1的变化情况。结果：Western blot检测结果说明,与对照组相比,ITF提高了pAkt蛋白的表达水平,而LY294002抑制了ITF激活的pAkt蛋白的表达,说明ITF可以通过激活PI3K/Akt信号通路来调控GES-1细胞的生理活动。免疫荧光和Western blot结果显示LPS导致GES-1细胞的紧密连接遭到破坏,降低紧密连接蛋白Occludin和ZO-1的表达水平,而ITF可以通过激活PI3K/Akt信号通路来保护GES-1细胞的紧密连接的完整性。结论：ITF保护胃黏膜上皮细胞紧密连接的完整性,提高紧密连接蛋白的表达水平,其发挥作用的主要分子机制是通过激活PI3K/Akt信号通路来实现的。     

GIRK通道在匹罗卡品癫痫模型中的表达改变

目的：研究匹罗卡品诱导癫痫发作后不同时期G蛋白耦联内向整流钾通道（GIRK通道）在海马神经元细胞膜表面表达变化情况，并探索可能的调控机制。 方法：将成年雄性SD大鼠随机分为对照组和匹罗卡品造模组，利用致痫剂匹罗卡品诱导癫痫持续状态（Status epilepticus，SE）后，分别在急性期（24h）和慢性期（30d）取海马组织提取膜蛋白，利用Western Blot方法检测海马膜蛋白GIRK通道亚基蛋白GIRK1、GIRK2的表达改变，并同时检测海马组织PI3K/Akt通路激活情况。建立细胞癫痫模型，观察PI3K抑制剂渥曼青霉素和LY294002对海马神经元内向整流钾通道（Kir）电流的影响。 结果：匹罗卡品诱导大鼠SE发作后急性期细胞膜上GIRK1和GIRK2蛋白的表达较对照组相比显著下调（P<0.05）。海马膜蛋白中GIRK1的表达在大鼠SE发作后慢性期较对照组下降（P<0.05），而GIRK2蛋白表达量没有显著改变（P>0.05）。p-Akt（308）、Akt蛋白表达量的比值在匹罗卡品诱导大鼠SE发作后急性期与对照组相比显著上升（P<0.05），而在SE发作后慢性期无显著差异（P>0.05）。在细胞癫痫模型中，PI3K抑制剂渥曼青霉素和LY294002对痫样放电神经元的Kir电流未发现显著影响。 结论：在大鼠匹罗卡品诱导SE发作后急性期，海马中GIRK通道蛋白表达量下调， PI3K/Akt通路存在明显激活。癫痫急性期GIRK通道表达的调控机制尚需进一步研究。     

氢吗啡酮后处理经PI3K/Akt通路减轻大鼠心肌缺血-再灌注细胞凋亡

目的:探讨PI3K/Akt信号通路在氢吗啡酮后处理减轻大鼠心肌缺血-再灌注细胞凋亡中的作用。方法:健康雄性SD大鼠40只,按照随机数表法随机分为5组,每组8只,假手术组（Sham组）、缺血-再灌注组（I/R组）、缺血-再灌注+氢吗啡酮组（I/R+H组）、缺血-再灌注+PI3K抑制剂组（I/R+W组）、缺血-再灌注+氢吗啡酮+ PI3K抑制剂组（I/R+ H+ W组）。采用左冠状动脉前降支结扎30 min、再灌注120 min的方法建立心肌缺血-再灌注损伤模型。实验结束后,用TTC染色法测心肌梗死面积;用比色法检测血清乳酸脱氢酶（LDH）漏出量;末端标记法（TUNEL）测心肌细胞凋亡;Western blot法检测p-Akt、Bcl-2、Bax蛋白表达。采用SPSS 13.0软件行统计分析。采用单因素方差分析进行组间比较。结果:与Sham组比较,I/R组心肌梗死面积、血清LDH漏出量及心肌细胞凋亡增多,p-Akt表达和Bax表达上调,Bcl-2表达下调（ P<0.05）;与I/R组比较,I/R+H组心肌梗死面积、血清LDH漏出量及心肌细胞凋亡减少,心肌p-Akt及Bcl-2表达上调,Bax表达下调（ P<0.05）;与I/R+H组比较,I/R+H+W组心肌梗死面积、血清LDH漏出量及心肌细胞凋亡增多,p-Akt表达和Bcl-2表达下调,Bax表达上调（ P<0.05）。 结论:氢吗啡酮后处理可减轻心肌缺血-再灌注引起的心肌细胞凋亡,其心肌保护机制可能与激活PI3K/Akt信号通路有关。     

LY294002对Jeko-1细胞的增殖抑制及作用机制研究

本研究旨在探讨磷脂酰肌醇3-激酶/丝氨酸苏氨酸激酶（PI3K/Akt）特异性抑制剂LY294002对人套细胞淋巴瘤Jeko-1细胞生长、细胞凋亡的影响及其作用机制.四甲基偶氮唑盐（MTT）方法检测细胞增殖率;流式细胞术检测细胞凋亡水平;Western blot检测凋亡相关蛋白Cyclin D1、Bcl-2、procaspase-3及PI3 K/Akt信号通路相关蛋白p-Akt、p-mTOR、p-P70S6K的变化.结果表明,LY294002能抑制Jeko-1细胞的增殖.Jeko-1细胞经LY294002 0、5、10和20 μmol/L作用24 h后,凋亡率分别为（3.25±1.27）％、（11.34±2.35）％、（22.81±2.74）％和（43.61±3.48）％,差异有统计学意义（P＜0.01）;Westem blot检测发现,凋亡相关蛋白Cyclin D1、Bcl-2、procaspase-3表达下降,PI3K/Akt信号通路相关蛋白p-Akt、p-mTOR、p-P70S6K磷酸化水平降低.结论：LY294002可明显抑制Jeko-1细胞增殖,其机制可能通过下调PI3K/Akt信号通路中的p-Akt、p-mTOR、p-P70S6K磷酸化水平,抑制PI3K/Akt信号通路,促进细胞凋亡.     

Effects induced by inhibitors of the phosphatidylinositol 3‐kinase/Akt and nitric oxide synthase/guanylyl cyclase pathways on the isometric contraction in rat aorta: a comparative study

This work was aimed to determine whether isometric contraction in Wistar rat aorta is related to the phosphatidylinositol 3‐kinase (PI3K)/Akt‐dependent activation of endothelial nitric oxide synthase (eNOS). Basically, we hypothesized that additional increases in active tone occur after the pharmacological inhibition of a transduction pathway involved in NO synthesis or action. In intact aortic rings contracted with phenylephrine or high K⁺, the cumulative administration of the PI3K inhibitor, LY294002, elicited significant decreases – but not supplementary increases – in tone. In endothelium‐intact tissues, on the other hand, the Akt1/2 kinase inhibitor did not alter phenylephrine‐ and K⁺‐induced isometric contractions. The PI3K inhibitor wortmannin (1 × 10^?7 m ) produced a significant supplementary contraction only in endothelium‐intact aortic rings precontracted with phenylephrine. Higher concentrations of this inhibitor produced relaxations of phenylephrine and high K⁺‐constricted endothelium‐intact and endothelium‐denuded aortic rings. LY294002 and wortmannin did not cause any potentiating effect on phenylephrine‐ and angiotensin II‐induced concentration‐dependent contractile responses in endothelium‐intact tissues. In intact aortic rings contracted with phenylephrine or high K⁺, the addition of the NOS inhibitor, L‐NAME, or the guanylyl cyclase inhibitor, ODQ, further augmented tone in a concentration‐dependent manner, and these supplementary contractions were significantly reduced by endothelium removal. Taken together, our data suggest that the PI3K/Akt pathway is not counteracting aortic isometric contractions by activation of the eNOS. It appears, on the other hand, that the smooth muscle PI3K can stimulate contraction without activation of the protein kinase Akt in response to GPCR agonists and high K⁺.     

1alpha,25-dihydroxyvitamin D(3)-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex

1alpha,25-dihydroxyvitamin D(3) (D(3)) promotes the maturation of myeloid cells and surface expressions of CD14 and CD11b, markers of cell differentiation in response to D(3). To examine how these responses are regulated, THP-1 cells were grown in serum-free medium and incubated with D(3). This was associated with rapid and transient increases in phosphatidylinositol 3-kinase (PI 3-kinase) activity. Furthermore, induction of CD14 expression in response to D(3) was abrogated by (a) the PI 3-kinase inhibitors LY294002 and wortmannin; (b) antisense oligonucleotides to mRNA for the p110 catalytic subunit of PI 3-kinase; and (c) a dominant negative mutant of PI 3-kinase. In THP-1 cells, induction of CD11b expression by D(3) was also abrogated by LY294002 and wortmannin. Similarly, LY294002 and wortmannin inhibited D(3)-induced expression of both CD14 and CD11b in peripheral blood monocytes. In contrast to CD14 and CD11b, hormone-induced expression of the Cdk inhibitor p21 in THP-1 cells was unaffected by either wortmannin or LY294002. These findings suggest that PI 3-kinase selectively regulates D(3)-induced monocyte differentiation, independent of any effects on p21.     

Blocking of PI3K/AKT induces apoptosis by its effect on NF-κB activity in gastric carcinoma cell line SGC7901

NF-κB plays an important role in many aspects of tumorigenesis and tumor progression by its antiapoptosis effect. Hence, NF-κB has been regarded as a therapeutic target in cancer, because inhibition of NF-κB not only induces enhancing apoptosis but also causes increasing sensitivity to radiation or chemotherapy in several tumor cells. The activation of NF-κB is presumed to be associated with PI3K/Akt signal pathway in gastric carcinoma, but the underlying molecular mechanism remains unclear. Our work demonstrates that blocking PI3K/Akt by LY294002 inhibits the NF-κB activity with significantly increased apoptosis in gastric cancer cell. Furthermore, when the cells were pretreated with IKK siRNA and/or IκB siRNA then exposed to LY294002, the results suggest that the regulatory significantly increased apoptosis in gastric cancer cell. Furthermore, when the cells were pretreated, effect of PI3K/AKT on NF-κB activity is associated with the influence of PI3K/AKT on IKK/IκB. The apoptosis induced by blocking PI3K/AKT might be ascribed to inhibition of NF-κB activity through IKK/IκB at least in part.     

Inhibition of phosphatidylinositol 3-kinase-Akt signaling blocks growth,promotes apoptosis,and enhances sensitivity of small cell lung cancer cells to chemotherapy

A promising therapeutic alternative to inhibition of growth factor receptors is the inhibition of downstream signal transduction pathways. Such an approach may be especially important in tumors that can use signals from multiple growth factor receptors for growth and survival. Both stem cell factor (SCF) and insulin-like growth factor (IGF)-I, components of prominent small cell lung cancer (SCLC) autocrine loops, as well as FCS, can potently activate phosphatidylinositol 3-kinase (PI3K)-Akt signaling, albeit with different kinetics. SCF-induced PI3K-Akt activation occurs rapidly but fades within 60 min; IGF-I and FCS-induced activation persists for at least 6 h. SCF and IGF-I-mediated growth was potently inhibited by LY294002 in proportion to its ability to inhibit phosphatidylinositol 3-kinase (PI3K)-Akt signaling. A panel of six SCLC cell lines grown in 10% FCS was also very sensitive to LY294002, with average IC50 and LD50 of 5 and 25 microM, respectively. These drug concentrations suppressed the growth of the MRC-5 pulmonary fibroblast cell line and primary bronchial epithelial cells but did not induce significant cell death. Because LY294002 can also inhibit PI3K-related enzymes, we confirmed the role of the PI3K-Akt pathway in SCLC using doxycycline-regulated expression of a dominant-negative (kinase dead) and a constitutively active (CA; myristolated) Akt allele. Expression of dominant-negative Akt, which could only be achieved at relatively low levels, completely inhibited growth in the absence of exogenous growth factors and inhibited SCF-mediated growth but had no effect on IGF-I-mediated growth at the expression levels attained. Expression of CA Akt markedly augmented growth in the absence of exogenous growth factors but had minimal effect on growth in the presence of saturating concentrations SCF or IGF-I. Because PI3K-Akt signaling is known to promote survival under apoptotic stresses, we determined the effect of this pathway on SCLC sensitivity to etoposide. LY294002 potentiated the effect of low concentrations of etoposide in inhibiting growth and inducing apoptosis. The effect of low concentrations of LY294002 could largely be reversed by expression of CA Akt, suggesting that it was mediated by inhibition of Akt signaling. Expression of CA Akt by itself also induced resistance to etoposide-mediated apoptosis. Taken together, these data demonstrate that PI3K-Akt signaling promotes SCLC growth, survival, and chemotherapy resistance. Therefore, selective inhibitors of PI3K or Akt could potentially be useful as novel therapeutic agents in the treatment of SCLC.     

Constitutively activated Akt-1 is vital for the survival of human monocyte-differentiated macrophages. Role of Mcl-1, independent of nuclear factor (NF)-kappaB, Bad, or caspase activation.

Recent data from mice deficient for phosphatase and tensin homologue deleted from chromosome 10 or src homology 2 domain-containing 5' inositol phosphatase, phosphatases that negatively regulate the phosphatidylinositol 3-kinase (PI3K) pathway, revealed an increased number of macrophages in these animals, suggesting an essential role for the PI3K pathway for macro-phage survival. Here, we focused on the role of the PI3K-regulated serine/threonine kinase Akt-1 in modulating macrophage survival. Akt-1 was constitutively activated in human macrophages and addition of the PI3K inhibitor, LY294002, suppressed the activation of Akt-1 and induced cell death. Furthermore, suppression of Akt-1 by inhibition of PI3K or a dominant negative (DN) Akt-1 resulted in loss of mitochondrial transmembrane potential, activation of caspases-9 and -3, and DNA fragmentation. The effects of PI3K inhibition were reversed by the ectopic expression of constitutively activated Akt-1 or Bcl-x(L). Inhibition of PI3K/Akt-1 pathway either by LY294002 or DN Akt-1 had no effect on the constitutive or inducible activation of nuclear factor (NF)-kappaB in human macrophages. However, after inhibition of the PI3K/Akt-1 pathway, a marked decrease in the expression of the antiapoptotic molecule Mcl-1, but not other Bcl-2 family members was observed, and Mcl-1 rescued macrophages from LY294002-induced cell death. Further, inhibition of Mcl-1 by antisense oligonucleotides, also resulted in macrophage apoptosis. Thus, our findings demonstrate that the constitutive activation of Akt-1 regulates macrophage survival through Mcl-1, which is independent of caspases, NF-kappaB, or Bad.     

热休克蛋白27介导异氟醚预处理延迟相对缺血/再灌注损伤兔心肌的保护作用研究

目的 探讨热休克蛋白27(HSP27)在异氟醚预处理延迟相对缺血/再灌注(I/R)兔心肌保护中的作用机制.方法 将30只新西兰大白兔随机分成假手术组(C组)、I/R组和体积分数为2%的异氟醚预处理组(S组)3组,每组10只.C组吸入纯氧2 h,24 h后仅行左冠状动脉(冠脉)套线而不阻断血流160 min;I/R组吸入纯氧2 h,24 h后结扎左冠脉前降支阻断血流40 min,再灌注120 min;S组吸入2%的异氟醚和纯氧2 h,24 h后处理同I/R组.再灌注后抽血测定各组丙二醛(MDA)含量;用伊文思蓝和氯化三苯四唑(TTC)染色法测定心肌梗死面积;用蛋白质免疫印迹法(Western blotting)测定各组HSP27和核转录因子-κB(NF-κB)的蛋白表达.结果 异氟醚预处理延迟相可降低I/R损伤心肌梗死面积[(19.7±2.8)%比(37.8±1.75)%],上调HSP27表达[(84.5±4.3)灰度值比(53.1±3.8)灰度值],下调NF-κB表达[(58.6±4.2)灰度值比(119.3±5.6)灰度值],降低MDA含量[(5.24±0.45)kU/L比(9.42±0.83)kU/L],差异均有统计学意义(P均＜0.05).结论 HSP27介导了异氟醚预处理延迟相对I/R心肌的保护作用.     

Inhibition of the phosphatidylinositol 3-kinase/Akt pathway sensitizes MDA-MB468 human breast cancer cells to cerulenin-induced apoptosis

Fatty acid synthase is overexpressed in cancer especially in tumors with a poor prognosis. The specific fatty acid synthase inhibitor cerulenin can induce apoptosis in cancer cells. Likewise, phosphatidylinositol 3-kinase (PI3K)/Akt kinase activities are elevated in primary tumors and cancer cell lines. Here, we tested whether inhibition of PI3K/Akt pathway would sensitize cancer cells to cerulenin-induced apoptosis. We show that LY294002, an inhibitor of PI3K, sensitized MDA-MB468 breast cancer cells to cerulenin-induced apoptosis. In MDA-MB468 cells, cerulenin- and LY294002-mediated apoptosis was associated with caspase-3 activation and the release of cytochrome c from mitochondria to cytosol. In addition, we observed additional species of Bak in mitochondria, suggesting a possible Bak activation. Treatment of cells with cerulenin and LY294002 down-regulated the protein levels of X chromosome-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis 1 (cIAP-1), and Akt, whereas the levels of mitogen-activated protein/extracellular signal-regulated kinase kinase and other antiapoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xl) did not change. Interestingly, the nonspecific caspase inhibitor, z-VAD-FMK, inhibited the down-regulation of Akt, XIAP, and cIAP-1 in cerulenin- and LY294002-treated cells. In conclusion, these studies show that inhibition of PI3K can sensitize cerulenin-induced apoptosis in MBA-MB468 breast cancer cells via activation of caspases, down-regulation of antiapoptotic proteins, such as XIAP, cIAP-1 and Akt, and possibly, activation of Bak in mitochondria.     

血管生成素-1对HCT-8细胞的作用及其可能机制的研究

目的观察血管生成素-1（Angiopoietin-1,Ang-1）蛋白对无血清DMEM培养基培养的结肠癌细胞（HCT-8）存活率的影响,探讨其与PI3-’kinase/Akt通路的关系。方法将不同浓度（0、0.05、0.2、0.4、0.82、.0 mg/L）的Ang-1蛋白作用于HCT-8细胞,用MTT法检测细胞增殖,根据实验结果选定Ang-1蛋白的后续实验浓度,加入Ang-1及LY294002,应用Western blotting蛋白免疫印记法分析相关蛋白（Tie-2、PI3K、Akt）的变化。结果 Ang-1（0.05、0.2、0.4、0.82、.0 mg/L）＋DMEM组与无血清DMEM培养基组比较,Tie-2、PI3K、Akt三种蛋白在HCT-8细胞中的表达均增强（P〈0.01,P〈0.01,P〈0.01）,DMEM＋Ang-1＋LY294002组三种蛋白的表达均减弱（P〉0.05,P〈0.01,P〈0.01）。结论较低浓度（0.05 mg/L）的血管生成素-1蛋白在结肠癌细胞中即有抗凋亡作用,并且随着剂量的增加抗凋亡作用平稳,其诱导凋亡的机理可能与Tie-2/PI3-’kinase/Akt调节的通路有关,应用该途径的抑制剂LY294002可抑制结肠癌细胞的生长,实现抗肿瘤作用。     

Inhibition of PI3K signaling triggered apoptotic potential of curcumin which is hindered by Bcl-2 through activation of autophagy in MCF-7 cells

Curcumin is a natural anti-cancer agent derived from turmeric (Curcuma longa). Curcumin triggers intrinsic apoptotic cell death by activating mitochondrial permeabilization due to the altered expression of pro- and anti-apoptotic Bcl-2 family members. Phosphoinositol-3-kinase (PI3K) and Akt, key molecular players in the survival mechanism, have been shown to be associated with the Bcl-2 signaling cascade; therefore, evaluating the therapeutic efficiency of drugs that target both survival and the apoptosis mechanism has gained importance in cancer therapy. We found that Bcl-2 overexpression is a limiting factor for curcumin-induced apoptosis in highly metastatic MCF-7 breast cancer cells. Forced overexpression of Bcl-2 also blocked curcumin-induced autophagy in MCF-7 cells, through its inhibitory interactions with Beclin-1. Pre-treatment of PI3K inhibitor LY294002 enhanced curcumin-induced cell death, apoptosis, and autophagy via modulating the expression of Bcl-2 family members and autophagosome formation in MCF-7 breast cancer cells. Atg7 silencing further increased apoptotic potential of curcumin in the presence or absence of LY294002 in wt and Bcl-2+ MCF-7 cells. The findings of this study support the hypothesis that blocking the PI3K/Akt pathway may further increased curcumin-induced apoptosis and overcome forced Bcl-2 expression level mediated autophagic responses against curcumin treatment in MCF-7 cells.     

Inhibition of phosphatidylinositol 3-kinase sensitizes ovarian cancer cells to carboplatin and allows adjunct chemotherapy treatment

Signal transduction pathways associated with cancer progression and chemotherapeutic resistance are being investigated as molecular targets for chemotherapy. The phosphatidylinositol 3-kinase (PI3K) pathway has been found to be frequently amplified and have increased activity in ovarian cancer. The current study investigates the efficacy of an antagonist of PI3K, LY294002, in inhibiting ovarian cancer cell growth and survival both in vitro and in vivo. The hypothesis tested is that inhibition of PI3K signaling makes ovarian cancer cells susceptible to the effects of platinum-based chemotherapy. Observations show that LY294002 is an effective inhibitor of ovarian cancer cell growth and survival in vitro. Inhibition of PI3K/Akt signaling increased the sensitivity of ovarian cell cultures to the cytotoxic effects of carboplatin. The combined treatment of LY294002 and carboplatin was needed to optimally promote cellular apoptosis and decrease ovarian cancer cell survival in vitro. To extend these observations, a model involving in vivo i.p. growth of human ovarian tumors in a nude mouse was used. LY294002 in combination with carboplatin was more effective in inhibiting ovarian cancer cell xenograft growth than either agent alone. The results of this study suggest that the combined treatment of carboplatin and LY294002 can effectively decrease ovarian tumor progression and support the use of a PI3K inhibitor (e.g., LY294002) as an adjunct platinum-based drug therapy for treatment of ovarian cancer.