PI3K/Akt/mTOR信号通路在非小细胞肺癌中作用的研究进展

肺癌是当今世界上严重威胁人类健康与生命的恶性肿瘤，每年有超过100万人死于肺癌。P13K／Akt通路在肿瘤治疗方面已经有了比较广泛的研究，活化受体酪氨酸激酶和它们下游信号递质的研究也已经为肺癌的治疗打开了一个非常有前号的领域。由于P13K／Akt通路在下游多点活化受体酪氨酸激酶，因此被广泛地研究并开发新的抗肿瘤制剂。mTOR已经被确定是P13K／Akt的下游靶点。雷帕霉素及其衍生物具有高度选择性，mTOR的抑制剂对于多种肿瘤的治疗效果已取得了很好的研究结果。在此我们将对肺癌中P13K／Akt／mTOR信号通路的分子基础做一综述，并进一步讨论有关肺癌的多靶点治疗策略。     

促红细胞生成素对乳鼠心肌细胞缺氧／复氧损伤的保护作用

目的:观察促红细胞生成素(eryrthropoietin,EP)对乳鼠心肌细胞缺氧/复氧(hypoxia/reoxygenation,H/R)损伤的影响及其相关机制探讨.方法:分离培养SD乳鼠心肌细胞,建立H/R模型.心肌细胞随机分为4组:①H/R组缺氧2 h,复氧4 h;②EP组在缺氧前1 h予EP(10 U/ml),随即缺氧2 h,复氧4 h;③Wortmannin EP组(W EP) 在EP预处理前30 min予3-磷脂酰肌醇激酶(phosphatidylinositol-3-kinase,PI3-K)特异性阻断剂Wortmannin(100 nmol/L);④正常对照组流式细胞仪检测细胞凋亡率.Western blot法检测心肌细胞EP受体表达和p-AKt/AKt比值.结果:EP可明显降低缺氧/复氧引起的心肌细胞凋亡,并明显增强SD乳鼠心肌细胞p-AKt/AKt比值;而Wortmannin减弱了EP的抗细胞凋亡作用,显著降低了p-AKt/AKt比值.结论:细胞凋亡参与了心肌缺氧/复氧损伤;EP可减少缺氧/复氧引起的SD乳鼠心肌细胞凋亡,其机制可能与EP激活PI-3K/AKt信号通路有关.     

Impaired phosphorylation and insulin-stimulated translocation to the plasma membrane of protein kinase B/Akt in adipocytes from Type II diabetic subjects

Aims/hypothesis. To examine protein kinase B/Akt distribution and phosphorylation in response to insulin in different subcellular fractions of human fat cells from healthy subjects and subjects with Type II (non-insulin-dependent) diabetes mellitus. Methods. We prepared subcellular fractions of plasma membranes (PM), low density microsomes and cytosol and examined gene and protein expression as well as serine and threonine phosphorylation in response to insulin. Results. Protein kinase B/Akt mRNA as well as total protein kinase B/Akt protein in whole-cell lysate and cytosol were similar in both groups. Insulin increased protein kinase B/Akt translocation to the the plasma membrane about twofold [(p < 0.03) in non-diabetic cells but this effect was impaired in diabetic cells (∼ 30 %; p > 0.1)]. In both groups, protein kinase B/Akt threonine phosphorylation considerably increased in low density microsomes and cytosol whereas serine phosphorylation was predominant in the plasma membrane. Phosphatidylinositol-dependent kinase 1, which partially activates and phosphorylates protein kinase B/Akt on the specific threonine site, was predominant in cytosol but it was also recovered in low density microsomes. Serine phosphorylation in response to insulin was considerably reduced (50–70 %; p < 0.05) in diabetic cells but threonine phosphorylation was less reduced (∼ 20 %). Wortmannin inhibited these effects of insulin supporting a role for PI3-kinase activation. Conclusion/interpretation. Insulin stimulates a differential subcellular pattern of phosphorylation of protein kinase B/Akt. Furthermore, insulin-stimulated translocation of protein kinase B/Akt to the plasma membrane, where serine phosphorylation and full activation occurs, is impaired in Type II diabetes. Threonine phosphorylation was much less reduced. This discrepancy may be related to differential activation of phosphatidylinositol 3-kinase in the different subcellular compartments and phosphatidylinositol-dependent kinase 1 having high affinity for phosphatidylinositol phosphate 3. [Diabetologia (2000) 43: 1107–1115] Received: 9 March 2000 and in revised form 22 May 2000     

糖基化终产物通过PI3k/Akt信号通路诱导肾小管上皮细胞转分化

目的:观察PI3k/Akt信号通路在糖基化终产物(AGEs)诱导的肾小管上皮细胞转分化中的作用。方法:大鼠肾小管上皮细胞随机分为对照组(C组),糖基化终产物组(D组)和PI3K抑制剂组(W组)。D、W组加入AGE-BSA(100mg·L-1);W组在AGE-BSA处理前用PI3K抑制剂预处理1h。加AGE-BSA1,6,12,24,48h时用免疫细胞化学和Westernblot检测α-SMA和p-Akt的表达。结果:D组α-SMA在6h时出现阳性表达,24h达高峰;p-Akt在1h出现阳性表达,12h达高峰,48h开始下降。结论:PI3k/Akt信号转导系统触发了糖基化终产物诱导的大鼠肾小管上皮细胞转分化。     

淀粉样β蛋白片段25~35下调大鼠海马PI3K/Akt/p70S6K信号传导通路(英文)

目的探讨阿尔茨海默病(AD)的淀粉样β蛋白(Aβ)的沉积是否损害神经细胞存活的信号传导通路。方法实验分为生理盐水对照组;Aβ25-35组;Aβ25-35+布洛芬组;Aβ25-35+布洛芬+LY294002组;Aβ25-35+LY294002组。大鼠分别灌胃给予布洛芬7.5或15 mg.kg-1,每日1次,连续3周后,左侧脑室内注射Aβ25-35(10 μL, 1 mmol.L-1),之后继续灌胃给予布洛芬1周。PI3K特异性阻断剂LY294002 (5 μL, 4 mmol.L-1)在注射Aβ25 -35前1 h左侧脑室内注射。注射Aβ25-35后1周,取海马CA1区,Western免疫印迹法观察P53,Bax, FasL, Bcl-2, Akt和p70S6K的蛋白表达水平。应用半胱氨酸天冬氨酸蛋白酶(caspase)3活性测定试剂盒分析caspase 3活性变化,RT-PCR方法观察p70s6k mRNA表达水平。结果脑室内注射Aβ25-35可引起大鼠海马CA1区磷酸化Akt/PKB和磷酸化p70S6K表达明显降低,分别从对照组1.32±0.14和0.769±0.028下降到0.69±0.08和0.479±0.032。同时,海马CA1区促凋亡蛋白P53, Bax和FasL表达及caspase 3活性明显增加,抗凋亡蛋白Bcl-2表达明显降低。预先注射LY294002可使caspase 3活性较单独注射Aβ25-35组进一步增加。给Aβ25-35前后连续给予布洛芬4周可明显对抗Aβ25-35引起的上述变化。LY294002可明显减弱布洛芬上调磷酸化Akt/PKB和磷酸化p70S6K表达的作用。结论 Aβ25-35引起抗凋亡通路PI3K/Akt/p70S6K下调可能参与AD的神经元损伤。布洛芬具有较好的对抗作用,这可能与上调PI3K/Akt/p70S6K通路中的一些蛋白有关。     

Involvement of c-jun N-terminal kinase in G2/M arrest and caspase-mediated apoptosis induced by cardiotoxin III (Naja naja atra) in K562 leukemia cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, may have a potentiality as a structural template for rational drug design in killing cancer cells. Treatment of K562 cells with 0.3 microM of CTX III resulted in G2/M phase cell cycle arrest that was associated with a marked decline in protein levels of G2/M regulatory proteins including cyclin A, cyclin B1, Cdk2 and Cdc25C. In contrast to no effect on the phosphorylation of ERK, p38 MAPK and Akt, an activation of JNK was noted when K562 cells were exposed to CTX III. CTX III-mediated G2/M phase arrest and apoptosis were reduced by treatment with the JNK-specific inhibitor SP600125, but not by ERK and p38MAPK inhibitors. Further investigation showed that the specific JNK inhibitor, SP600125, reduced the activation of caspase-3, caspase-9, and reversed the decline in the expression of cyclin B1. Taken together, our data show for the first time that JNK, but not ERK, p38MAPK or Akt signaling, plays an important role in CTX III-mediated G2/M arrest and apoptosis in K562 cancer cells.     

非小细胞肺癌中磷酸化Akt蛋白表达研究

背景与目的表皮生长因子受体(EGFR)信号传导通路下游分子Akt的激活在肺癌进展的过程中有重要作用,但磷酸化Akt蛋白的表达是否与EGFR表达存在相关性以及是否能提示患者的预后目前尚无定论。本研究旨在评价国人非小细胞肺癌组织中磷酸化Akt蛋白表达的意义。方法应用组织微阵列和免疫组织化学技术检测167例NSCLC手术标本中磷酸化Akt蛋白的表达。结果磷酸化Akt蛋白在NSCLC中的阳性表达率为52.1%(87/167),阳性表达率与性别、发病年龄、吸烟情况、病理类型、分化程度、病理分期及预后之间的相关性无统计学显著意义(P>0.05),并且磷酸化Akt与表皮生长因子受体蛋白表达之间的相关性亦无统计学意义(P=0.122)。结论国人非小细胞肺癌组织中磷酸化Akt阳性表达与患者临床病理特征无明显相关性,并且磷酸化Akt蛋白对患者预后无提示意义。     

Targeting the PI3K/Akt/mTOR pathway: Effective combinations and clinical considerations

The PI3K/Akt/mTOR pathway is a prototypic survival pathway that is constitutively activated in many types of cancer. Mechanisms for pathway activation include loss of tumor suppressor PTEN function, amplification or mutation of PI3K, amplification or mutation of Akt, activation of growth factor receptors, and exposure to carcinogens. Once activated, signaling through Akt can be propagated to a diverse array of substrates, including mTOR, a key regulator of protein translation. This pathway is an attractive therapeutic target in cancer because it serves as a convergence point for many growth stimuli, and through its downstream substrates, controls cellular processes that contribute to the initiation and maintenance of cancer. Moreover, activation of the Akt/mTOR pathway confers resistance to many types of cancer therapy, and is a poor prognostic factor for many types of cancers. This review will provide an update on the clinical progress of various agents that target the pathway, such as the Akt inhibitors perifosine and PX-866 and mTOR inhibitors (rapamycin, CCI-779, RAD-001) and discuss strategies to combine these pathway inhibitors with conventional chemotherapy, radiotherapy, as well as newer targeted agents. We will also discuss how the complex regulation of the PI3K/Akt/mTOR pathway poses practical issues concerning the design of clinical trials, potential toxicities and criteria for patient selection.     

淀粉样β蛋白片段25～35下调大鼠海马PI3K/Akt/p70S6K信号传导通路

目的 探讨阿尔茨海默病（AD）的淀粉样β蛋白(Aβ)的沉积是否损害神经细胞存活的信号传导通路。方法 实验分为生理盐水对照组；Aβ_25-35组；Aβ_25-35 +布洛芬组；Aβ_25-35+布洛芬+LY294002组；Aβ_25-35+LY294002组。大鼠分别灌胃给予布洛芬7.5或15 mg·kg^-1，每日1次，连续3周后，左侧脑室内注射Aβ_25-35（10 μL, 1 mmol·L^-1，之后继续灌胃给予布洛芬1周。PI3K特异性阻断剂LY294002（5 μL, 4 mmol·L^-1在注射Aβ_25-35前1 h左侧脑室内注射。注射Aβ_25-35后1周，取海马CA1区，Western免疫印迹法观察P53, Bax, FasL, Bcl-2, Akt和p70S6K的蛋白表达水平。应用半胱氨酸天冬氨酸蛋白酶(caspase)3活性测定试剂盒分析caspase 3活性变化，RT-PCR方法观察p70s6k mRNA表达水平。结果 脑室内注射Aβ_25-35可引起大鼠海马CA1区磷酸化Akt/PKB和磷酸化p70S6K表达明显降低，分别从对照组1.32±0.14和0.769±0.028下降到0.69±0.08和0.479±0.032。同时,海马CA1区促凋亡蛋白P53, Bax和FasL表达及caspase 3活性明显增加，抗凋亡蛋白Bcl-2表达明显降低。预先注射LY294002可使caspase 3活性较单独注射Aβ_25-35组进一步增加。给Aβ_25-35前后连续给予布洛芬4周可明显对抗Aβ_25-35引起的上述变化。LY294002可明显减弱布洛芬上调磷酸化Akt/PKB和磷酸化p70S6K表达的作用。结论 Aβ_25-35引起抗凋亡通路PI3K/Akt/p70S6K下调可能参与AD的神经元损伤。布洛芬具有较好的对抗作用，这可能与上调PI3K/Akt/p70S6K通路中的一些蛋白有关。     

Thyroid hormone activates Akt and prevents serum starvation-induced cell death in neonatal rat cardiomyocytes

Thyroid hormone is known to cause hypertrophy, tachycardia, vasorelaxation, and enhanced contractile function. The exact mechanisms responsible for these effects are unknown but classical regulation of gene expression through binding to nuclear receptors has been widely implicated. Data have also accumulated suggesting that TH can exert effects through non-classical mechanisms involving activation of signal transduction pathways. Whether thyroid hormone can activate signal transduction pathways in the heart is unknown. In this study, we treated neonatal rat cardiomyocytes with T3 and determined the expression and phosphorylation of signaling molecules. T3 caused specific activation of Akt/PKB signaling after 24 h of treatment. Since Akt is known to protect against cell death, cells were serum-starved in the presence or absence of T3 to determine whether T3 could protect against serum starvation-induced cell death. Indeed, myocytes treated with T3 displayed enhanced sarcomeric structure after 4 days of serum starvation. T3 increased cell viability as measured by MTT assays, prevented DNA laddering, and reduced TUNEL positive cells, which was associated with increased phosphorylated Akt and glycogen synthase kinase 3beta (GSK-3beta). The protective effect of T3 on cell viability, DNA laddering and TUNEL positive cells were blocked by LY294002, a phosphoinositide-3 kinase (PI3K) inhibitor that blocks Akt signaling. Overall these data suggest that T3 can activate Akt in cardiomyocytes which protects myocytes against cell death.