PI3K/AKT/mTOR信号通路在肾上腺皮质癌组织的表达特点

目的分析PI3K/AKT/mTOR信号通路在肾上腺皮质癌组织中的表达特点及其意义。方法选取2008年1月至2012年12月经手术治疗且具有完整临床、病理资料的肾上腺皮质癌标本15例,以正常肾上腺组织(肾癌患者施行手术时所取下)12例作为对照。研究这些组织中该信号通路关键蛋白p-AKT、p-mTOR、p-S6以及血管内皮生长因子(VEGF)的表达情况。结果与对照组比较,p-AKT(12/15,80%)、p-mTOR(11/15,73.3%)、p-S6(13/15,86.7%)以及VEGF(12/15,80%)在肾上腺皮质癌中明显高表达。结论 PI3K/AKT/mTOR信号通路关键蛋白p-AKT、p-mTOR、p-S6以及VEGF参与肾上腺皮质癌的发生发展。     

Evidence of the immunomodulatory role of dual PI3K/mTOR inhibitors in transplantation: an experimental study in mice

The PI3K/mTOR signaling cascade is fundamental in T‐cell activation and fate decisions. We showed the distinct regulation of PI3K/mTOR in regulatory and effector T‐cells and proposed the potential therapeutic benefit of targeting this pathway to control the balance between effector and regulatory T‐cell activities. Substantial adverse effects in long‐term clinical usage of rapamycin suggest the use of alternative treatments in restraining effector T‐cell function in transplant patients. We hypothesize that dual PI3K/mTOR inhibitors may represent an immunosuppressant alternative. Here we show that dual PI3K/mTOR PI‐103 and PKI‐587 inhibitors interfered IL‐2‐dependent responses in T‐cells. However, in contrast to the inhibitory effects in non‐Treg T‐cell proliferation and effector functions, dual inhibitors increased the differentiation, preferential expansion, and suppressor activity of iTregs. Rapamycin, PI‐103, and PKI‐587 targeted different signaling events and induced different metabolic patterns in primary T‐cells. Similar to rapamycin, in vivo administration of PI‐103 and PKI‐587 controlled effectively the immunological response against allogeneic skin graft. These results characterize specific regulatory mechanisms of dual PI3K/mTOR inhibitors in T‐cells and support their potential as a novel therapeutic option in transplantation.     

NVP‐BEZ235, a dual pan class I PI3 kinase and mTOR inhibitor,promotes osteogenic differentiation in human mesenchymal stromal cells

Osteoblasts are bone‐forming cells derived from mesenchymal stromal cells (MSCs) that reside within the bone marrow. In response to a variety of factors, MSCs proliferate and differentiate into mature, functional osteoblasts. Several studies have shown previously that suppression of the PI3K and mTOR signaling pathways in these cells strongly promotes osteogenic differentiation, which suggests that inhibitors of these pathways may be useful as anabolic bone agents. In this study we examined the effect of BEZ235, a newly developed dual PI3K and mTOR inhibitor currently in phase I–II clinical trials for advanced solid tumors, on osteogenic differentiation and function using primary MSC cultures. Under osteoinductive conditions, BEZ235 strongly promotes osteogenic differentiation, as evidenced by an increase in mineralized matrix production, an upregulation of genes involved in osteogenesis, including bone morphogenetic proteins (BMP2, ‐4, and ‐6) and transforming growth factor β1 (TGF‐β1) superfamily members (TGFB1, TGFB2, and INHBE), and increased activation of SMAD signaling molecules. In addition, BEZ235 enhances de novo bone formation in calvarial organotypic cultures. Using pharmacologic inhibitors to delineate mechanism, our studies reveal that suppression of mTOR and, to a much lesser extent PI3K p110α, mediates the osteogenic effects of BEZ235. As confirmation, shRNA‐mediated knockdown of mTOR enhances osteogenic differentiation and function in SAOS‐2 osteoblast‐like cells. Taken together, our findings suggest that BEZ235 may be useful in treating PI3K/mTOR‐dependent tumors associated with bone loss, such as the hematologic malignancy multiple myeloma. © 2010 American Society for Bone and Mineral Research.     

PI3K/AKT/mTOR信号通路在肝癌细胞自体吞噬中的作用研究

目的探讨在肝癌细胞中PI3K／AKT／mTOR信号通路在氨基酸缺乏诱导的自体吞噬中的作用。方法观察HCCLM3、MHCC97L及SMMC-7721肝癌细胞在氨基酸缺乏情况下及与P13K抑制剂3-MA、Wortmannin及LY294002，mTOR抑制剂雷帕霉素协同作用下细胞活力变化。GFP—LC3荧光法观察细胞内自噬体的形成。Western印迹检测LC3-Ⅱ蛋白的表达。结果经无氨基酸培养液EBSS处理后48h，HCCLM3、MHCC97L及SMMC-7721细胞存活率分别为（78．9±3．8）％、（57．2±13．1）％及（3．4±3．0）％（P〈0．01）。与HCCLM3、MHCC97L相比，细胞中的自噬体在SMMC-7721细胞中明显增多，分别为（13．1±3．5）％、（20．0±1．1）％及（48．9±4．5）％（P〈0．01）。Western印迹显示在EBSS处理后早期即有LC3—Ⅱ蛋白的明显表达。在EBSS基础上雷帕霉素20μmol／L处理24h，3种细胞株活力明显下降。3-MA、Wortmannin及LY294002处理也加速EBSS诱导的细胞死亡。结论高转移潜能肝癌细胞比较耐受自噬样细胞死亡。P13K／AKT／mTOR信号通路对自噬样细胞死亡可能起重要调节作用。     

连翘苷调节相关信号通路对地塞米松诱导的成骨细胞自噬和凋亡的影响

目的 探讨连翘苷(phillyrin, PHN)调节磷脂酰肌醇3-激酶(PI3K)/丝氨酸苏氨酸激酶(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对地塞米松(dexamethasone, DEX)诱导的成骨细胞自噬和凋亡的影响。方法 将MC3T3-E1细胞分为对照组(NOR组)、DEX组(10μmol/L DEX处理MC3T3-E1细胞)、L-PHN组(5μmol/L PHN处理MC3T3-E1细胞)、M-PHN组(10μmol/L PHN处理MC3T3-E1细胞)、H-PHN组(20μmol/L PHN处理MC3T3-E1细胞)、ZSTK474组(用20μmol/L PHN和2μmol/L的PI3K/AKT/mTOR信号通路抑制剂ZSTK474处理MC3T3-E1细胞);MTT法检测细胞毒性和细胞活力;流式细胞术检测MC3T3-E1细胞凋亡;透射电子显微镜(TEM)观察自噬小体;Western blot法检测MC3T3-E1细胞中自噬、凋亡和PI3K/AKT/mTOR通路相关蛋白表达;ALP活性及ALP染色检测MC3T3-E1细胞分化能力。结果 0～80μmol/L PHN对...     

LY294002, a PI3K pathway inhibitor,prevents leptin‐induced adverse effects on spermatozoa in Sprague‐Dawley rats

This study examined the effects of PI3K and AMPK signalling pathway inhibitors on leptin‐induced adverse effects on rat spermatozoa. Sprague‐Dawley rats, aged 14–16 weeks, were randomised into control, leptin‐, leptin + dorsomorphin (AMPK inhibitor)‐, and leptin+LY294002 (PI3K inhibitor)‐treated groups with six rats per group. Leptin was given once daily for 14 days via the intraperitoneal (i.p.) route at a dose of 60 ug kg^?1 body weight. Rats in the leptin and inhibitor‐treated groups received concurrently either dorsomorphin (5 mg kg^?1 day^?1) or LY294002 (1.2 mg kg^?1 day^?1) i.p. for 14 days. Controls received 0.1 ml of normal saline. Upon completion, sperm count, sperm morphology, seminiferous tubular epithelial height (STEH), seminiferous tubular diameter (STD), 8‐hydroxy‐2‐deoxyguanosine (8‐OHdG) and phospho‐Akt/total Akt ratio were estimated. Data were analysed using ANOVA. Sperm count, STEH and STD were significantly lower, while the percentage of spermatozoa with abnormal morphology and the level of 8‐OHdG were significantly higher in rats treated with leptin and leptin + dorsomorphin when compared to those in controls and LY294002‐treated rats. Testicular phospho‐Akt/total Akt ratio was significantly higher in leptin and leptin + LY294002‐treated rats. In conclusion, LY294002 prevents leptin‐induced changes in rat sperm parameters, suggesting the potential role of the PI3K signalling pathway in the adverse effects of leptin on sperm parameters.     

Orally bioavailable GSK-3alpha/beta dual inhibitor increases markers of cellular differentiation in vitro and bone mass in vivo.

GSK-3, a component of the canonical Wnt signaling pathway, is implicated in regulation of bone mass. The effect of a small molecule GSK-3 inhibitor was evaluated in pre-osteoblasts and in osteopenic rats. GSK-3 inhibitor induced osteoblast differentiation in vitro and increased markers of bone formation in vitro and in vivo with concomitant increased bone mass and strength in rats. INTRODUCTION: Inactivation of glycogen synthase kinase -3 (GSK-3) leads to stabilization, accumulation, and translocation of beta-catenin into the nucleus to activate downstream Wnt target genes. To examine whether GSK-3 directly regulates bone formation and mass we evaluated the effect of 603281-31-8, a small molecule GSK-3 alpha/beta dual inhibitor in preosteoblastic cells and in osteopenic rats. MATERIALS AND METHODS: Murine mesenchymal C3H10T1/2 cells were treated with GSK-3 inhibitor (603281-31-8) and assayed for beta-catenin levels, activity of Wnt-responsive promoter, expression of mRNA for bone formation, and adipogenic markers and alkaline phosphatase activity. In vivo, 6-month-old rats were ovariectomized (OVX), allowed to lose bone for 1 month, and treated with GSK-3 inhibitor at 3 mg/kg/day orally for 60 days. At the end of treatment, BMD was measured by DXA, bone formation rate by histomorphometry, vertebral strength (failure in compression), and the expression levels of osteoblast-related genes by real-time PCR. RESULTS: Treatment of C3H10T1/2 cells with the GSK-3 inhibitor increased the levels of beta-catenin accompanied by activation of Wnt-responsive TBE6-luciferase reporter gene. This was associated with an increased expression of mRNA for bone sialoprotein (1.4-fold), collagen alpha1 (I) (approximately 2-fold), osteocalcin (1.2-fold), collagen alpha1(V) (1.5-fold), alkaline phosphatase (approximately 160-fold), and runx2 (1.6-fold), markers of the osteoblast phenotype and bone formation activity. Alkaline phosphatase mRNA expression paralleled alkaline phosphatase activity. The mRNA levels of collagens alpha1 (I), alpha1 (V), biglycan, osteonectin, and runx-2 increased on treatment with the GSK-3 inhibitor in rat femur compared with the OVX control. DXA analyses revealed significant increases in BMC and BMD in cancellous and cortical bone of OVX rats treated with GSK-3 inhibitor. This was associated with increased strength (peak load, energy, and stiffness) assessed by lumbar vertebra load to failure in compression. Histomorphometric analyses showed that 603281-31-8 robustly increased bone formation but did not exclude a small effect on osteoclasts (resorption). CONCLUSIONS: An orally active, small molecule GSK-3 inhibitor induced osteoblast differentiation and increased markers of bone formation in vitro, and increased markers of bone formation, bone mass, and strength in vivo, consistent with a role for the canonical Wnt pathway in osteogenesis.     

Systemic Lupus Erythematosus: the involvement of PI3K/Akt/mTOR pathway in cellular cycle and in early apoptosis

ObjectivePI3K/Akt/mTOR signaling pathway plays a critical role in many cellular functions. Until now there are no data regarding this signaling pathway and p27kip1 status in peripheral blood mononuclear cells (PBMCs) of patients with Systemic Lupus Erythematosus (SLE). Therefore, we proposed to analyze in SLE PBMCs the activity of Akt, the expression level of their substrate p27kip1 and the relationship with cell cycle progression as well as the contribution of mTOR to the increased apoptosis of SLE PBMCs.MethodsAkt and mTOR activities were evaluated by measuring their phosphorylation level, using immunoblotting. The expression level of p27kip1 in PBMCs and lymphocytes was analyzed by immunoblotting and FACS. Quantification of apoptosis and the cell cycle distribution were performed using FACS.ResultsThe phosphorylation level of Akt at Thr308 was statistically significant higher while the expression level of p27kip1 was two times lower in SLE than in HC PBMCs. The percentage of lymphocytes accumulated in S and G2/M cell cycle phases are significantly increased in SLE than in HC. Statistical analysis demonstrated that p27kip1 expression level correlated with the percentages of lymphocytes in cell cycle phases. Moreover, in SLE lymphocytes, p27kip1 expression level was indirectly correlated with apoptosis. The phosphorylation level of mTOR was similar in SLE and HC PBMCs, probably due to the therapy. In SLE lymphocytes, where an increased apoptosis was found, mTOR activity was inverse correlated with apoptosis.ConclusionsIncreased activity of Akt, observed in SLE lymphocytes, explains the reduced expression of its substrate p27kip1. This defect seems to be involved in SLE lymphocytes passage by G1/S cell cycle checkpoint. Therefore, SLE lymphocytes accumulate in S and G2/M cell cycle phases towards apoptosis or proliferation. The inverse correlation of mTOR activity and apoptosis rather suggested an anti-apoptotic role of this kinase.     

PI3K/PTEN/AKT/mTOR信号通道在胃癌有关组织中的表达及临床意义

目的 研究PI3K/PTEN/AKT/mTOR信号通道在胃癌组织中的表达及临床意义.方法 采用免疫组化SP法分别检测33例胃癌及30例正常胃组织中mTOR、PTEN的表达情况,并分析二者与性别、年龄、淋巴结转移、浸润深度和分化程度的关系.结果 mTOR蛋白阳性反应产物主要分布于胞质,PTEN的蛋白阳性反应产物主要定位于胞核.将mTOR表达阴性,PTEN表达阳性为一组,与mTOR表达阳性而PTEN表达阴性为一组进行比较,发现对于不同的大体类型,组织学分型,二组间的差异有统计学意义(P<0.05),对于不同的淋巴结转移,临床病理分期,二组间的差异有统计学意义(P<0.01).结论 在PI3K/PTEN/AKT/mTOR信号通道中,mTOR与PTEN的联合表达可作为判断胃癌分期、判断预后的指标和化学治疗的靶点.     

miR-155靶向调节PIK3R1对类风湿关节炎大鼠模型PI3K/Akt/mTOR信号通路的影响

目的 探讨miR-155靶向调节PIK3R1对类风湿关节炎（rheumatoid arthritis,RA）大鼠PI3K/Akt/mTOR信号通路的影响。方法 SD大鼠随机分为对照组、模型组、miR-155 agomir组、miR-155 antagomir组、miR-155阴性对照组,诱导RA模型,分组处理后,观察关节症状,检测关节炎指数及后足趾容积;HE染色观察大鼠关节组织病理形态;使用试剂盒检测大鼠关节组织炎性因子IL-6、IL-17及IL-18水平;免疫印迹检测大鼠关节组织PI3K/Akt/mTOR信号通路蛋白表达;qRT-PCR实验检测大鼠关节组织miR-155及PIK3R1 mRNA水平;双荧光素酶报告基因实验检测miR-155对PIK3R1的靶向调控作用。结果 与对照组比较,模型组大鼠关节炎症状明显,关节炎指数、后足趾容积、关节组织炎性因子IL-6、IL-17及IL-18水平、关节组织p-PI3K/PI3K、p-Akt/Akt及p-mTOR/mTOR水平、关节组织miR-155水平明显升高（P＜0.05）,PIK3R1 mRNA水平明显降低（P＜0.05）。与模型组、miR-155阴性对照组比较,miR-155 antagomir组大鼠上述指标得到明显改善（P＜0.05）;miR-155 agomir组与miR-155 antagomir组趋势相反（P＜0.05）。结论 miR-155可靶向下调PIK3R1的表达,激活PI3K/Akt/mTOR信号,加重RA大鼠关节炎症损伤,下调miR-155表达,可抑制PI3K/Akt/mTOR信号激活及炎症反应发生发展,改善关节炎症状。     

黄芪多糖通过PI3K/AKT/mTOR促进激素性骨质疏松症大鼠成骨细胞增殖

目的 探讨黄芪多糖在体内外通过PI3K/AKT/mTOR信号通路对糖皮质激素诱导的骨质疏松症（glucocorticoid-induced osteoporosis,GIOP）的保护作用。方法 从分化的骨髓间充质干细胞（bone marrow mesenchymal stem cells,BM-MSCs）中培养成骨细胞,分为PBS组、模型组、LY294002组、黄芪多糖组和LY294002+黄芪多糖干预组。通过CCK-8法和碱性磷酸酶（alkaline phosphatase,ALP）染色检测细胞增殖和分化。MDC染色观察自噬体的形成。Western blot检测Beclin-1、p62等信号通路及自噬相关因子的蛋白表达。大鼠分为对照组、模型组、LY294002组、黄芪多糖组和LY294002+黄芪多糖组。比较各组大鼠的骨密度、骨组织形态学参数、组织中通路和自噬相关因子的表达。结果 黄芪多糖促进成骨细胞的增殖和分化能力（P＜0.05）。与模型组相比,黄芪多糖组PI3K/AKT/mTOR通路相关磷酸化蛋白的表达、成骨细胞的增殖分化能力、自噬体及自噬相关因子的表达均升高,但在LY294002组中发现了相反的结果（P＜0.05）。在体内实验中,与模型组相比,GIOP大鼠通过黄芪多糖干预改善了骨密度和骨形态参数,并提高了软骨组织中自噬相关因子的表达,而LY294002干预则表现出相反的结果（P＜0.05）。LY294002部分逆转了黄芪多糖对GIOP中成骨分化和骨形态参数的影响。结论 黄芪多糖通过PI3K/AKT/mTOR通路对GIOP发挥保护作用,可能与诱导自噬和促进成骨细胞增殖有关。     

Caspase-3/Treg and PI3K/AKT/mTOR pathway is involved in Liver Ischemia Reperfusion Injury (IRI) protection by everolimus

Ischemia-reperfusion injury (IRI) of the liver is a severe complication that can follow hemorrhagic shock and liver surgery. Regulatory T cells (Treg) show the potential of improving outcomes of IRI. Everolimus is an mTOR inhibitor used in liver transplantation and the treatment of tumor patients through PI3K/AKT/mTOR pathway. The present study was designed to investigate the efficacy and mechanism of everolimus on Treg for the treatment of IRI. Hepatocytes were exposed to H₂O₂ and hypoxic conditions to investigate the effects of everolimus on reactive oxygen species ROS-induced and H/R-induced injury in vitro. The effects of everolimus on liver IRI were investigated in a warm ischemia liver model in vivo. Our results indicate that everolimus markedly protected liver IRI in vivo and vitro. Furthermore, everolimus increased the levels of phospho- (p-)AKT, p-mTOR but not p-GSK following IRI. Taken together, our data showed that everolimus protected against IRI via regulation of caspase-3/Treg and the PI3K/AKT/mTOR signalling pathway, which provides new insight into the treatment of liver IRI.