PI3K和Akt蛋白在异丙肾上腺素所致大鼠心肌肥厚中的表达

目的研究异丙肾上腺素(ISO)致大鼠心肌肥厚中PI3K和Akt在心肌组织中的表达,为探讨心肌肥厚的信号转导机制和逆转心肌肥厚提供形态学资料.方法健康成年SD大鼠20只,随机分为实验组、对照组,每组10只.实验组给予异丙肾上腺素处理.1周后处死大鼠,取心肌组织,常规石蜡切片,HE染色,观察心肌组织的病理变化,测量心肌肥厚指标;免疫组织化学染色和免疫荧光染色,检测p-PI3K和p-Akt的表达及分布.结果实验组大鼠心肌肥厚指标与对照组相比均明显升高;免疫组织化学检测显示,实验组心肌组织p-PI3K和p-Akt蛋白表达面积和平均光密度较对照组高.免疫荧光检测实验组心肌组织p-PI3K和p-Akt蛋白表达较对照组高.结论小剂量持续给予 ISO 能建立大鼠心肌肥厚模型;p-PI3K和p-Akt蛋白表达均与心肌肥厚的发生和发展过程相关,PI3K/Akt信号通路激活,可能是导致心肌肥厚的机制之一.     

Nicorandil alleviates apoptosis in diabetic cardiomyopathy through PI3K/Akt pathway

Nicorandil exerts myocardial protection through its antihypoxia and antioxidant effects. Here, we investigated whether it plays an anti‐apoptotic role in diabetic cardiomyopathy. Sprague‐Dawley rats were fed with high‐fat diet; then single intraperitoneal injection of streptozotocin was performed. Rats with fasting blood glucose (FBG) higher than 11.1 mmol/L were selected as models. Eight weeks after the models were built, rats were treated with nicorandil (7.5 mg/kg day and 15 mg/kg day respectively) for 4 weeks. H9c2 cardiomyocytes were treated with nicorandil and then stimulated with high glucose (33.3 mmol/L). TUNEL assay and level of bcl‐2, bax and caspase‐3 were measured. 5‐HD was used to inhibit nicorandil. Also, PI3K inhibitor (Miltefosine) and mTOR inhibitor (rapamycin) were used to inhibit PI3K/Akt pathway. The results revealed that nicorandil (both 7.5 mg/kg day and 15mg/kg day) treatment can increase the level of NO in the serum and eNOS in the heart of diabetic rats compared with the untreated diabetic group. Nicorandil can also improve relieve cardiac dysfunction and reduce the level of apoptosis. In vitro experiments, nicorandil (100 µmol) can attenuate the level of apoptosis stimulated by high glucose significantly in H9C2 cardiomyocyte compared with the untreated group. The effect of nicorandil on apoptosis was blocked by 5‐HD, and it was accompanied with inhibition of the phosphorylation of PI3K, Akt, eNOS, and mTOR. After inhibition of PI3K/Akt pathway, the protective effect of nicorandil is restrained. These results verified that as a NO donor, nicorandil can also inhibit apoptosis in diabetic cardiomyopathy which is mediated by PI3K/Akt pathway.     

Dexamethasone suppresses osteogenesis of osteoblast via the PI3K/Akt signaling pathway in vitro and in vivo

The hypofunction of osteoblasts induced by glucocorticoids (GCs) has been identified as a major contributing factor for GC-induced osteoporosis (GIO). However, the biological mechanism underlying the effect of GC in osteoblasts are not fully elucidated. Recent studies implicated an important role of phosphoinositide 3-kinase (PI3K)/protein kinase B(Akt) signaling pathway in the regulation of bone growth. We propose that the PI3K/Akt signaling may be implicated in the process of GC-induced osteogenic inhibition in osteoblasts. In this study, primary osteoblasts were used in vitro and in rats in vivo to evaluate the biological significance of the PI3K/Akt pathway in GC-induced bone loss. In vivo, dexamethasone (Dex)-treated rats had low bone mineral density and decreased expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), and phosphorylated Akt (p-Akt) in bone tissue. In vitro study shows that Dex over the dose of 10^–8 M remarkably inhibited cellular osteogenesis, as represented by decreased cell viability, lessened ALP activity, and suppressed osteogenic protein expressions including ALP and OCN. Meanwhile, a dramatic downregulation in the PI3K/Akt pathway phosphorylation was also observed in Dex-treated osteoblasts. These changes were marked rescued by treatment with a PI3K agonist 740Y-P. Moreover, downregulation of ALP and OCN expressions by LY294002 can mimic the suppressive effects of Dex. These data together reveal that the suppressed PI3K/Akt pathway is involved in the regulatory action of Dex on osteogenesis.     

Magnolol induces apoptosis via inhibiting the EGFR/PI3K/Akt signaling pathway in human prostate cancer cells

We observed that treatment of prostate cancer cells for 24 h with magnolol, a phenolic component extracted from the root and stem bark of the oriental herb Magnolia officinalis, induced apoptotic cell death in a dose‐ and time‐dependent manner. A sustained inhibition of the major survival signal, Akt, occurred in magnolol‐treated cells. Treatment of PC‐3 cells with an apoptosis‐inducing concentration of magnolol (60 µM) resulted in a rapid decrease in the level of phosphorylated Akt leading to inhibition of its kinase activity. Magnolol treatment (60 µM) also caused a decrease in Ser(¹³⁶) phosphorylation of Bad (a proapoptotic protein), which is a downstream target of Akt. Protein interaction assay revealed that Bcl‐xL, an anti‐apoptotic protein, was associated with Bad during treatment with magnolol. We also observed that during treatment with magnolol, translocation of Bax to the mitochondrial membrane occurred and the translocation was accompanied by cytochrome c release, and cleavage of procaspase‐8, ‐9, ‐3, and poly(ADP‐ribose) polymerase (PARP). Similar results were observed in human colon cancer HCT116Bax^+/? cell line, but not HCT116Bax^?/? cell line. Interestingly, at similar concentrations (60 µM), magnolol treatment did not affect the viability of normal human prostate epithelial cell (PrEC) line. We also observed that apoptotic cell death by magnolol was associated with significant inhibition of pEGFR, pPI3K, and pAkt. These results suggest that one of the mechanisms of the apoptotic activity of magnolol involves its effect on epidermal growth factor receptor (EGFR)‐mediated signaling transduction pathways. J. Cell. Biochem. 106: 1113–1122, 2009. © 2009 Wiley‐Liss, Inc.     

1,7-Bis(4-hydroxyphenyl)-1,4-heptadien-3-one induces lung cancer cell apoptosis via the PI3K/Akt and ERK1/2 pathways

1,7-Bis(4-hydroxyphenyl)-1,4-heptadien-3-one (EB30) is a diarylheptanoid-like compound isolated from Viscum coloratum. This curcumin analog exhibits significant cytotoxic activity against HeLa, SGC-7901, and MCF-7 cells. However, little is known about the anticancer effects and mechanisms of EB30 in human lung cancer. The current study reports that EB30 significantly reduced the cell viability of A549 and NCI-H292 human lung cancer cells. Further examination revealed that EB30 not only induced cell cycle arrest and promoted the generation of reactive oxygen species (ROS) but also induced cell apoptosis through the intrinsic and extrinsic signaling pathways. Furthermore, EB30 upregulated the expression levels of p-ERK1/2 and p-P90RSK, whereas downregulating the phosphorylation of Akt and P70RSK. Cell viability was further inhibited by the combination of EB30 with LY294002 (a specific PI3K inhibitor) or U0126 (a MEK inhibitor). The current study indicates that EB30 is a potential anticancer agent that induces cell apoptosis via suppression of the PI3K/Akt pathway and activation of the ERK1/2 pathway.     

α‐Lipoic acid inhibits sevoflurane‐induced neuronal apoptosis through PI3K/Akt signalling pathway

Sevoflurane is a widely used anaesthetic agent, including in anaesthesia of children and infants. Recent studies indicated that the general anaesthesia might cause the cell apoptosis in the brain. This issue raises the concerns about the neuronal toxicity induced by the application of anaesthetic agents, especially in the infants and young children. In this study, we used Morris water maze, western blotting and immunohistochemistry to elucidate the role of α‐lipoic acid in the inhibition of neuronal apoptosis. We found that sevoflurane led to the long‐term cognitive impairment in the young rats. This adverse effect may be caused by the neuronal death in the hippocampal region, mediated through PI3K/Akt signalling pathway. We also showed that α‐lipoic acid offset the effect of sevoflurane on the neuronal apoptosis and cognitive dysfunction. This study elucidated the potential clinical role of α‐lipoic acid, providing a promising way in the prevention and treatment of long‐term cognitive impairment induced by sevoflurane general anesthesia. Copyright © 2016 John Wiley & Sons, Ltd.     

体外缺血缺氧条件下大鼠骨髓间充质干细胞凋亡发生的机制研究

目的：研究体外大鼠骨髓间充质干细胞（Bone marrow-derived mesenchymal stem cells, BMSCs）在缺血缺氧条件下发生凋亡的作用机制。方法：采取大鼠骨髓,以密度梯度离心分离出单个核细胞（MNCs）,于体外培养并由牛垂体提取物（PEX）诱导扩增传代培养出骨髓间充质干细胞（MSCs）。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,骨髓间充质干细胞（BMSCs）在缺血缺氧条件下培养,通过Annexin V／PI双染细胞凋亡检测比较不同组别细胞的凋亡率和蛋白印迹法（western blot）来观察细胞中蛋白的变化。结果：①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞培养成功。②对照组（无缺血缺氧）与缺血缺氧组比较,缺血缺氧组的凋亡率显著性增加,而通过磷酸化Akt的表达量显著性增加提示PI3K（Phosphoinositide-3kinase）／Akt（ProteinkinaseB,PKB）信号通路被激活（P〈0．05）;同时缺血缺氧组与缺血缺氧＋PI3K／Akt抑制剂（LY294002）组比较,缺血缺氧＋PI3K／Akt抑制剂（LY294002）组的凋亡率显著降低,而通过磷酸化Akt的表达量显著减少提示PI3K/Akt信号通路被抑制（P〈0．05）。结论：PI3K／Akt信号通路对体外缺血缺氧条件下培养的骨髓间充质干细胞凋亡发生有关键性作用。     

体外缺血缺氧条件下大鼠骨髓间充质干细胞凋亡发生的机制研究

目的:研究体外大鼠骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells,BMSCs)在缺血缺氧条件下发生凋亡的作用机制。方法:采取大鼠骨髓,以密度梯度离心分离出单个核细胞(MNCs),于体外培养并由牛垂体提取物(PEX)诱导扩增传代培养出骨髓间充质干细胞(MSCs)。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,骨髓间充质干细胞(BMSCs)在缺血缺氧条件下培养,通过Annexin V/PI双染细胞凋亡检测比较不同组别细胞的凋亡率和蛋白印迹法(western blot)来观察细胞中蛋白的变化。结果:①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞培养成功。②对照组(无缺血缺氧)与缺血缺氧组比较,缺血缺氧组的凋亡率显著性增加,而通过磷酸化Akt的表达量显著性增加提示PI3K(Phosphoinosi-tide-3kinase)/Akt(ProteinkinaseB,PKB)信号通路被激活(P<0.05);同时缺血缺氧组与缺血缺氧+PI3K/Akt抑制剂(LY294002)组比较,缺血缺氧+PI3K/Akt抑制剂(LY294002)组的凋亡率显著降低,而通过磷酸化Akt的表达量显著减少提示PI3K/Akt信号通路被抑制(P<0.05)。结论:PI3K/Akt信号通路对体外缺血缺氧条件下培养的骨髓间充质干细胞凋亡发生有关键性作用。     

Distinctive regulation and function of PI 3K/Akt and MAPKs in doxorubicin-induced apoptosis of human lung adenocarcinoma cells

Regulation and function of PI 3K/Akt and mitogen-activated protein kinases (MAPKs) in doxorubicin-induced cell death were investigated in human lung adenocarcinoma cells. Doxorubicin induced dose-dependent apoptosis of human lung adenocarcinoma NCI-H522 cells. Prior to cell death, both Akt and the MAPK family members (MAPKs: ERK1/2, JNK, and p38) were activated in response to the drug treatment. The kinetics of the inductions for Akt and MAPKs are, however, distinct. The activation of Akt was rapid and transient, activated within 30 min of drug addition, then declined after 3 h, whereas the activations of three MAPKs occurred later, 4 h after addition of the drug and sustained until cell death occurred. Inhibition of PI 3K/Akt activation had no effect on MAPKs' activation, suggesting that the two pathways are independently activated in response to the drug treatment. Inhibition of PI 3K/Akt and p38 accelerated and enhanced doxorubicin-induced cell death. On the contrary, inhibition of ERK1/2 or JNK had no apparent effect on the cell death. Taken together, these results suggest that PI 3K/Akt and MAPKs signaling pathways are all activated, but with distinct mechanisms, in response to doxorubicin treatment. Activation of PI 3K/Akt and p38 modulates apoptotic signal pathways and inhibits doxorubicin-induced cell death. These responses of tumor cells to cancer drug treatment may contribute to their drug resistance. Understanding of the mechanism and function of the responses will be beneficial for the development of novel therapeutic approaches for improvement of drug efficacy and circumvention of drug resistance.     

FGFRL1 affects chemoresistance of small‐cell lung cancer by modulating the PI3K/Akt pathway via ENO1

Fibroblast growth factor receptor‐like 1 (FGFRL1), a member of the FGFR family, has been demonstrated to play important roles in various cancers. However, the role of FGFRL1 in small‐cell lung cancer (SCLC) remains unclear. Our study aimed to investigate the role of FGFRL1 in chemoresistance of SCLC and elucidate the possible molecular mechanism. We found that FGFRL1 levels are significantly up‐regulated in multidrug‐resistant SCLC cells (H69AR and H446DDP) compared with the sensitive parental cells (H69 and H446). In addition, clinical samples showed that FGFRL1 was overexpressed in SCLC tissues, and high FGFRL1 expression was associated with the clinical stage, chemotherapy response and survival time of SCLC patients. Knockdown of FGFRL1 in chemoresistant SCLC cells increased chemosensitivity by increasing cell apoptosis and cell cycle arrest, whereas overexpression of FGFRL1 in chemosensitive SCLC cells produced the opposite results. Mechanistic investigations showed that FGFRL1 interacts with ENO1, and FGFRL1 was found to regulate the expression of ENO1 and its downstream signalling pathway (the PI3K/Akt pathway) in SCLC cells. In brief, our study demonstrated that FGFRL1 modulates chemoresistance of SCLC by regulating the ENO1‐PI3K/Akt pathway. FGFRL1 may be a predictor and a potential therapeutic target for chemoresistance in SCLC.     

Over-expression of microRNA-494 up-regulates hypoxia-inducible factor-1 alpha expression via PI3K/Akt pathway and protects against hypoxia-induced apoptosis

Background

Hypoxia-inducible factor-1 alpha (HIF-1α) is one of the key regulators of hypoxia/ischemia. MicroRNA-494 (miR-494) had cardioprotective effects against ischemia/reperfusion (I/R)-induced injury, but its functional relationship with HIF-1α was unknown. This study was undertaken to determine if miR-494 was involved in the induction of HIF-1α.

Results

Quantitative RT-PCR showed that miR-494 was up-regulated to peak after 4 hours of hypoxia in human liver cell line L02. To investigate the role of miR-494, cells were transfected with miR-494 mimic or miR-negative control, followed by incubation under normoxia or hypoxia. Our results indicated that overexpression of miR-494 significantly induced the expression of p-Akt, HIF-1α and HO-1 determined by qRT-PCR and western blot under normoxia and hypoxia, compared to negative control (p < 0.05). While {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 treatment markedly abolished miR-494-inducing Akt activation, HIF-1α and HO-1 increase under both normoxic and hypoxic conditions (p < 0.05). Moreover, apoptosis detection using Annexin V indicated that overexpression of miR-494 significantly decreased hypoxia-induced apoptosis in L02 cells, compared to control (p < 0.05). MiR-494 overexpression also decreased caspase-3/7 activity by 1.27-fold under hypoxia in L02 cells.

Conclusions

Overexpression of miR-494 upregulated HIF-1α expression through activating PI3K/Akt pathway under both normoxia and hypoxia, and had protective effects against hypoxia-induced apoptosis in L02 cells. Thus, these findings suggested that miR-494 might be a target of therapy for hepatic hypoxia/ischemia injury.     

PI3K-Akt信号传导通路对糖代谢的调控作用

磷脂酰肌醇3-激酶(PI3Ks)作为酪氨酸激酶和G蛋白偶联受体的主要下游分子,通过催化产生第二信使3,4,5-三磷酸磷脂酰肌醇(PIP3)并激活Akt、糖原合酶激酶-3(GSK-3)、Forkhead转录因子FoxO1、mTOR(mammalian target of rapamycin)等下游分子,将多种生长因子及细胞因子的信号传递到细胞内,从而对细胞增殖、分化、凋亡和葡萄糖转运等多种生物过程起重要的调节作用.PTEN(phosphatase and tensin homologue)是PI3K信号通路的重要负调节因子.本文将对PI3K-Akt信号通路在糖代谢中的作用予以简要综述.     

Erianin induces triple-negative breast cancer cells apoptosis by activating PI3K/Akt pathway

Background: Triple-negative breast cancer (TNBC) is a refractory subtype of breast cancer, 25–30% of which have dysregulation in the PI3K/AKT pathway. The present study investigated the anticancer effect of erianin on TNBC cell line and its underlying mechanism.Methods: After treatment with erianin, MTT assay was employed to determine the MDA-MB-231 and EFM-192A cell proliferation, the nucleus morphological changes were observed by DAPI staining. The cell cycle and apoptotic proportion were detected by flow cytometry. Western blot was performed to determine the cell cycle and apoptosis-related protein expression and PI3K pathways. Finally, the antiproliferative activity of erianin was further confirmed by adding or not adding PI3K agonists SC79.Results: Erianin inhibited the proliferation of MDA-MB-231 and EFM-192A cells in a dose-dependent manner, the IC₅₀ were 70.96 and 78.58 nM, respectively. Erianin could cause cell cycle arrest at the G₂/M phase, and the expressions of p21 and p27 were up-regulated, while the expressions of CDK1 and Cyclin B1 were down-regulated. Erianin also induced apoptosis via the mitochondrial pathway, with the up-regulation of the expression of Cyto C, PARP, Bax, active form of Caspase-3, and Caspase-9. Furthermore, p-PI3K and p-Akt expression were down-regulated by erianin. After co-incubation with SC79, the cell inhibition rate of erianin was decreased, which further confirmed that the attenuated PI3K/Akt pathway was relevant to the pro-apoptotic effect of erianin.Conclusions: Erianin can inhibit the proliferation of TNBC cells and induce cell cycle arrest and apoptosis, which may ascribe to the abolish the activation of the PI3K/Akt pathway.     

Rhabdastrellic acid-A inhibited PI3K/Akt pathway and induced apoptosis in human leukemia HL-60 cells

Increasing evidence suggests that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer treatment. Our investigation indicates that Rhabdastrellic acid-A, an isomalabaricane triterpenoid isolated from the sponge, Rhabdastrella globostellata, inhibits proliferation of HL-60 cells with an IC(50) value of 0.68mug/ml, and induces apoptosis. Rhabdastrellic acid-A also induces cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and caspase-3. Pretreatment of HL-60 cells with the caspase-3 specific inhibitor, DEVD-CHO, prevents Rhabdastrellic acid-A-induced DNA fragmentation and PARP cleavage. Activated PI3K and Akt significantly decreases after treatment with Rhabdastrellic acid-A in HL-60 cells. Expression levels of protein bcl-2, bax remain unchanged in response to Rhabdastrellic acid-A treatment in HL-60 cells. These results suggest that Rhabdastrellic acid-A inhibits PI3K/Akt pathway and induces caspase-3 dependent-apoptosis in HL-60 human leukemia cells.     

PI3K Acts in synergy with loss of PKC to elicit apoptosis via the UPR

It is known that Ras mutations, together with loss of PKC, are apoptotic in various types of mammalian cells. The mechanism of how aberrant Ras transmits this apoptotic signaling remains unclear. Using three V12‐Ha‐ras loop mutants that preferentially bind to and activate one of Ras effectors, we tested the role of Ras downstream pathways in the induction of apoptosis in rat lung epithelia, human lung or prostate cancer cells. After PKC inhibition, the activation of PI3K/Akt renders the susceptibility of cells to apoptosis. We also demonstrate that the amount of ROS is moderately increased in the cells ectopically expressing V12C40 and dramatically elevated by suppression of PKC, which leads to apoptosis through the activation of UPR. Thus, our study suggests that after PKC abrogation, PI3K functions downstream of Ras to perturb the state of cellular redox and signals to ER stress‐regulated apoptotic machinery. J. Cell. Biochem. 107: 76–85, 2009. © 2009 Wiley‐Liss, Inc.