Wortmannin抑制PI3-K途径对K562及NB4细胞增殖的影响

目的 :为探讨Wortmannin抑制磷酰肌醇 - 3激酶 (PI- 3K)途径对K5 62 ,NB4细胞增殖的影响 ,探寻慢性髓细胞性白血病 (CML)的治疗新途径 .方法 :用磷酰肌醇 - 3激酶 (PI - 3K)特异抑制剂Wort mannin抑制PI - 3K活性 ,观察慢性髓细胞性白血病细胞系K5 62细胞和急性早幼粒细胞性白血病细胞系NB4细胞在 2 4,48,72h增殖能力的变化 .t检验统计分析 .结果 :K5 62和NB4细胞在 2 4,48,72h的增殖抑制率分别为 3 4 67% ,5 7 46% ,65 85 %和 2 6 2 9% ,5 5 1% ,2 10 % .集落形成实验以GM -CSF为主要生长刺激物的培养体系在 3 7℃ ,5 %CO2 孵箱培养 14d后细胞系的集落数和集落形成率分别为K5 62 :80 75±10 2 4和 16 15 % ,K5 62 +WT :3 8 0 0± 12 75和 7 60 % ,NB4:2 9 5 0± 5 97和 5 90 % ,NB4+WT :3 0 5 0± 5 74和 6 10 % .集落形成抑制率为 :5 2 94%和 3 3 9% .结论 :Wortmannin可显著抑制K5 62细胞的增殖和集落形成 ,而对NB4细胞无明显影响 (P均 <0 0 5 ) .Wortmannin可以通过抑制PI - 3K通路抑制K5 62细胞的增殖 ,而对NB4细胞增殖无明显影响     

Rab5在胰腺癌细胞BxPC3中对Notch1活性的调节

目的 本研究拟阐明内吞调节蛋白Rab5在Notch活化中的作用.方法 用RNA干扰的方法抑制BxPC3细胞中Rab5蛋白的表达,用Western blot测定Notch1活性型Notch ICD的表达.用Wortmannin和LY294002抑制PI3激酶的活性,观察Notch活性的变化.结果 抑制Rab5蛋白的表达,或者抑制PI3激酶的活性后,Notch1的活性型Notch ICD的表达量均显著下降,同时BxPC3细胞的生长受到抑制.结论 在胰腺癌细胞BxPC3中内吞调节蛋白质Bab5和PI3激酶在Notch活化中起着关键的作用,提示Notch的活化依赖于内吞.     

Macrophage migration inhibitory factor induces phosphorylation of Mdm2 mediated by phosphatidylinositol 3-kinase/Akt kinase: Role of this pathway in decidual cell survival

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has an anti-apoptotic effect through several downstream targets, which includes activation of the transformed mouse 3T3 cell double-minute 2 (Mdm2) protein, its translocation to the nucleus and degradation of the tumor suppressor p53. We show that Mif, the Macrophage Migration Inhibitory Factor, an important cytokine at the maternal fetal interface in several species, triggers phosphorylation of Mdm2 protein in a PI3K/Akt-dependent manner, thereby preventing apoptosis in cultured mouse decidual cells. Inhibition of Akt and PI3K suppresses the pathway. Mif treatment also changes the nuclear translocation of p53 and interferes with the apoptotic fate of these cells when challenged with reactive oxygen species. In conclusion, an important mechanism has been found underlying decidual cell survival through Akt signaling pathway activated by Mif, suggesting a role for this cytokine in decidual homeostasis and in the integrity of the maternal-fetal barrier that is essential for successful gestation.     

Tamoxifen enhances erlotinib-induced cytotoxicity through down-regulating AKT-mediated thymidine phosphorylase expression in human non-small-cell lung cancer cells

Tamoxifen is a triphenylethylene nonsteroidal estrogen receptor (ER) antagonist used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Thymidine phosphorylase (TP) is an enzyme of the pyrimidine salvage pathway which is upregulated in cancers. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels through AKT inactivation. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, combination treatment with PI3K inhibitors (LY294002 or wortmannin) and tamoxifen further decreased the TP expression and cell viability of NSCLC cells. Knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available small molecular inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical treatment of NSCLC. Compared to a single agent alone, tamoxifen combined with erlotinib resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-AKT and phospho-ERK1/2, and reduced TP protein levels. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and erlotinib for the treatment of NSCLC.     

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...     

Wortmannin抑制PI3K途径对bcr/abl基因介导的白血病细胞增殖和凋亡抗性影响的研究

目的探讨磷酰肌醇-3激酶(PI3K)途径在K562、NB4和HL60细胞增殖和凋亡抗性中的不同作用。方法用磷酰肌醇-3激酶(PI3K)特异抑制剂Wortmannin(WT)抑制PI3K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(Annexin-V-Flous)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。结果K562、NB4和HL60细胞在24、48、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PI3K途径可显著抑制K562细胞的增殖,对NB4和HL60细胞增殖无明显影响。K562、NB4和HL60细胞加和不加WT,培养14d后的集落形成率分别为16.15%和7.60%,5.90%和6.10%,6.60%和6.40%。集落形成抑制率为52.94%、3.39%和3.03%。K562、NB4和HL60细胞加WT、AraC、WT+AraC作用24h后的凋亡细胞百分比分别为(17.27±1.94)%、(23.25±14.12)%、(17.60±1.27)%;(17.00±3.36)%、(20.55±8.57)%、(22.07±5.62)%;(27.60±4.36)%、(17.56±9.28)%、(20.50±7.97)%。凋亡指数分别为(6.88±2.66)、(7.79±2.75)、(3.03±0.56);(8.91±3.86)、(9.35±4.19)、(3.79±0.93);(13.39±4.49)、(7.61±6.35)、(5.27±2.69)。结论WT可以通过抑制PI3K通     

Wortmannin对表皮生长因子诱导的血管平滑肌细胞增殖及迁移的影响

目的:研究PI3K抑制剂Wortmannin对表皮生长因子(EGF)诱导的大鼠血管平滑肌细胞(VSMCs)增殖、迁移及磷酸化Akt蛋白表达的影响,旨在探讨EGF诱导的VSMCs增殖和迁移的信号通路。方法:以低血清培养的VSMCs作为对照,采用细胞计数、划痕损伤实验和WesternBlotting技术,观察Wortmannin对EGF诱导的VSMCs增殖、迁移及磷酸化Akt蛋白表达的影响。结果:干预后24h,EGF组VSMCs增殖、迁移较对照组明显增强,同时磷酸化Akt蛋白的表达平行增高。而Wortmannin则明显抑制了VSMCs的增殖、迁移和磷酸化Akt蛋白的表达。结论:PI3K/Akt信号通路参与了EGF诱导的VSMCs增殖和迁移。     

夏枯草抑制人淋巴瘤细胞增殖的机制

目的：研究夏枯草对淋巴瘤细胞生长的影响及其可能的机制。方法：参考临床常用剂量,分别采用50g·L^-1夏枯草（夏枯草组）、50g·L^-1夏枯草＋1μmol·L^-1wortmannin（wortmannin组）处理Raji细胞,以加入同体积生理盐水的naji细胞作对照,应用MTT法检测细胞增殖率,Western Blotting检测Akt磷酸化水平和Caspase-3的表达。结果：夏枯草组细胞增殖率明显低于对照组（P〈0．05）;Akt磷酸化水平在夏枯草组中表达明显降低（P〈0．05）,但wortmannin可抑制其磷酸化,夏枯草组的Caspase-3显著增高（P〈0．05）。结论：夏枯草可以明显抑制Raji细胞的生长,且这种抑制作用可能是通过抑制Akt信号转导通路,继而激活细胞凋亡来实现的。     

Activation of telomerase by ionizing radiation: differential response to the inhibition of DNA double-strand break repair by abrogation of poly (ADP-ribosyl)ation, by LY294002, or by Wortmannin

PURPOSE: Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)-activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide. METHODS AND MATERIALS: COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10 Gy. Phosphorylation status of PKB/Akt and of PKCalpha/beta(II) was assessed by western blotting. RESULTS: No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable. CONCLUSIONS: Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than alpha/beta(II) cannot be ruled out.