Inhibition of MG-63 cell proliferation and PDGF-stimulated cellular processes by inhibitors of phosphatidylinositol 3-kinase

Studies on a platelet-derived growth factor (PDGF) responsive osteosarcoma cell line, MG-63, were initiated to determine the effects of phosphatidylinositol (Ptdlns) 3-kinase inhibitors on serum-stimulated cell proliferation and PDGF-stimulated DNA replication, actin rearrangements, or Ptdlns 3-kinase activity. In a dose-dependent manner, the fungal metabolite wortmannin and a quercetin derivative, LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), inhibited serum-stimulated MG-63 cell proliferation. The mitogenic effects of PDGF on MG-63 cells, as determined by incorporation of [³H]-thymidine, were also substantially inhibited in the presence of 0.10 μM wortmannin or 10 μM LY294002. Furthermore, MG-63 cells stimulated by PDGF form distinct actin-rich, finger-like membrane projections which are completely inhibited by either 0.10 μM wortmannin or 10 μM LY294002. At these same concentrations, wortmannin and LY294002 were also effective at reducing levels of phosphatidylinositol 3-phosphate in PDGF-stimulated MG-63 cells. Treatment of these cells with increasing concentrations of wortmannin reduced the level of PDGF stimulated tyrosine phosphorylation of the PDGF receptor but did not significantly affect the amount of the Ptdlns 3-kinase regulatory subunit, p85, associated with the receptor. Additionally, pretreatment of cells with 0.250 μM wortmannin followed by stimulation with PDGF resulted in a slightly reduced level of receptor autokinase activity; however, similar treatment with 50 μM LY294002 did not affect the level of autokinase activity. These results demonstrate the effects of two different Ptdlns 3-kinase inhibitors on serum- and PDGF-stimulated MG-63 cell proliferation and PDGF-stimulated morphological changes and suggest a greater role for Ptdlns 3-kinase in these processes. J. Cell. Biochem. 64:182–195. © 1997 Wiley-Liss, Inc.     

In vitro increase of the fluid-phase endocytosis induced by pulsed radiofrequency electromagnetic fields: importance of the electric field component

Nowadays, due to the wide use of mobile phones, the possible biological effects of electromagnetic fields (EMF) become a public health general concern. Despite intensive research, there are no widely accepted theories about the interactions between EMFs and living cells, and the experimental data are often controversial. We examined the effects of mobile phones EMF (envelope frequency of 217 Hz, carrier frequency of 900 MHz and pulse duration of 580 μs) or its pure, low-frequency pulsed electric field component on fluid-phase endocytosis. In both cases, with exposures exceeding 10 min, an increase of the fluid-phase endocytosis rate was observed (≈1.5-fold), on three different cell types. This increase is an all-or-nothing type of response that is occurring for threshold values comprised between 1.3 and 2.6 W/kg for the delivered EMF powers and between 1.1 and 1.5 V/cm for the electric fields intensities depending upon the cell type. The electric component of these EMFs is shown to be responsible for the observed increase. Variations of frequency or pulse duration of the electric pulses are shown to be without effect. Thus, EMF, via their electrical component, can perturb one of the most fundamental physiological functions of the cells—endocytosis.     

Inhibition of the PI3 kinase/Akt pathway enhances doxorubicin-induced apoptotic cell death in tumor cells in a p53-dependent manner

Constitutive activation of the PI3 kinase/Akt pathway is associated with the neoplastic phenotype of a large number of human tumor cells. As the anti-apoptotic role of the PI3 kinase/Akt pathway has been established, we have examined whether specific blockade of this pathway sensitizes tumor cells to DNA-damaging agent-induced cytotoxicity by enhancing apoptotic cell death. Although a PI3 kinase inhibitor, LY294002, by itself does not induce apoptotic cell death, LY294002 selectively and markedly enhances the apoptosis-inducing efficacy of doxorubicin: such an enhanced cell death is only detected in tumor cells in which the PI3 kinase/Akt pathway is constitutively activated, and it is totally dependent on the functional p53 pathway. These results suggest that the combination of a PI3 kinase/Akt pathway inhibitor and doxorubicin provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the PI3 kinase/Akt pathway is constitutively activated and the p53 pathway is functional.     

PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 activation

The Ras/Raf/extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway is known to cross-talk with other signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, the role of PI3K in ERK-1/2 activation induced by tyrosine kinase receptors was not fully understood. Here, we report that two structurally distinct PI3K inhibitors, wortmannin and LY294002, inhibited insulin-induced activation of ERK1/2 but had no effect on EGF-induced activation of ERK1/2 in hepatocellular carcinoma BEL-7402 and SMMC-7721 cells, breast cancer MCF-7 cells, and prostate cancer LNCaP cells. Although protein kinase C could act as a mediator between PI3K and ERK1/2, protein kinase C inhibitor chelerythrine chloride did not inhibit insulin-induced ERK1/2 activation. Both insulin- and EGF-induced ERK1/2 activation are strictly dependent on Ras activation, however, wortmannin only inhibited insulin-induced, but not EGF-induced Ras activation. These results indicate that PI3K plays different roles in the activation of Ras/ERK1/2 signaling by insulin and EGF, and that insulin-stimulated, but not EGF-stimulated, ERK1/2 and Akt signalings diverge at PI3K.     

Acute Effects of Ethanol on Pharmacologically Isolated Kainate Receptors in Cerebellar Granule Neurons: Comparison with NMDA and AMPA Receptors

Abstract: Comparisons of acute ethanol's effects on individual members of the three major families of ionotropic glutamate receptors (kainate, AMPA, and NMDA) have been performed only with recombinant receptors. However, no study has compared the acute effects of ethanol on individual members of each one of these receptor families in the same neuron. We accomplished this task by using cultured cerebellar granule neurons and LY303070 (GYKI-53784), a noncompetitive and selective AMPA receptor antagonist. Ethanol concentrations of 25, 50, 75, and 100 m M decreased the amplitude of pharmacologically isolated kainate-activated currents by 3 ± 1, 9 ± 2, 14 ± 2, and 22 ± 3% (n = 8), respectively. The magnitude of the ethanol-induced inhibition of nonselective kainate-activated currents, i.e., in the absence of LY303070, and currents activated by submaximal AMPA concentrations was not significantly different from that obtained with isolated kainate currents. However, the magnitude of the ethanol-induced inhibition of NMDA receptor-activated currents was about twofold greater than that of kainate and/or AMPA receptors.     

Class III Phosphoinositide 3‐Kinase/VPS34 and Dynamin are Critical for Apical Endocytic Recycling

Recycling is a limiting step for receptor‐mediated endocytosis. We first report three in vitro or in vivo evidences that class III PI3K/VPS34 is the key PI3K isoform regulating apical recycling. A substractive approach, comparing in Opossum Kidney (OK) cells a pan‐class I/II/III PI3K inhibitor (LY294002) with a class I/II PI3K inhibitor (ZSTK474), suggested that class III PI3K/VPS34 inhibition induced selective apical endosome swelling and sequestration of the endocytic receptor, megalin/LRP‐2, causing surface down‐regulation. GFP‐(FYVE)x2 overexpression to sequester PI(3)P caused undistinguishable apical endosome swelling. In mouse kidney proximal tubular cells, conditional Vps34 inactivation also led to vacuolation and intracellular megalin redistribution. We next report that removal of LY294002 from LY294002‐treated OK cells induced a spectacular burst of recycling tubules and restoration of megalin surface pool. Acute triggering of recycling tubules revealed recruitment of dynamin‐GFP and dependence of dynamin‐GTPase, guidance directionality by microtubules, and suggested that a microfilamentous net constrained endosomal swelling. We conclude that (i) besides its role in endosome fusion, PI3K‐III is essential for endosome fission/recycling; and (ii) besides its role in endocytic entry, dynamin also supports tubulation of recycling endosomes. The unleashing of recycling upon acute reversal of PI3K inhibition may help study its dynamics and associated machineries.     

胰高血糖素样肽-1抑制高糖诱导的新生大鼠心室肌细胞氧化应激效应

本文旨在观察胰高血糖素样肽-1(glucagon like peptide-1,GLP-1)对高糖所致新生大鼠心室肌细胞氧化应激的影响,并探讨PI3K-Akt通路在其中所起的作用。将酶消化法分离的经α-肌动蛋白免疫荧光法鉴定的原代培养72~96h的新生大鼠心室肌细胞分为5组:正常对照组、高糖组、高糖+GLP-1组、高糖+GLP-1+LY294002(PI3K-Akt通路的抑制剂)组、高渗对照组。采用硫代巴比妥酸显色法测定细胞上清液中丙二醛(malonaldehyde,MDA)含量,用黄嘌呤氧化酶法测定细胞上清液中超氧化物歧化酶(superoxide dismutase,SOD)的活性,运用PCR凝胶电泳检测NADPH P47phox亚基mRNA的变化,运用荧光显微镜及流式细胞术检测心室肌细胞内的ROS含量,运用流式细胞术及DNAladder法检测心室肌细胞的凋亡,Western blot检测各组心肌细胞Akt磷酸化水平。结果显示:(1)与正常对照组相比,高糖组细胞生长状态较差,搏动频率减慢甚至消失,细胞凋亡率显著增加,DNA ladder呈现凋亡独有的梯状图谱,胞浆MDA水平增高,上清液中SOD活性下降,心肌...     

Effects of astragalus polysaccharide on apoptosis of myocardial microvascular endothelial cells in rats undergoing hypoxia/reoxygenation by mediation of the PI3K/Akt/eNOS signaling pathway

The study explores the effect of astragalus polysaccharide (APS) mediating P13K/Akt/eNOS signaling pathway on apoptosis of myocardial microvascular endothelial cells (MMECs) in hypoxia/reoxygenation (H/R). MMECs were classified into blank, H/R, H/R + 25 mg/L APS, H/R + 50 mg/L APS, H/R + 100 mg/L APS, H/R + LY, and HR + 100 mg/L APS + LY groups. Cell viability was detected using MTT assay and apoptotic cell morphological changes by Hoechst staining. NO content, cell cycle and apoptosis, PI3K/Akt/eNOS signaling pathway proteins were detected using nitrate reductase assay, flow cytometry and Western blotting. An increased cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins, and a decreased apoptosis rate was observed in the H/R + 50 mg/L APS and H/R + 100 mg/L APS groups compared with the H/R and H/R + 25 mg/L APS groups. Compared with the H/R + 50 mg/L APS group, the apoptosis rate decreased, whereas the cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins increased in the H/R + 100 mg/L APS group. The H/R + LY and HR + 100 mg/L APS + LY groups followed opposite trends. In comparison to the HR + 100 mg/L APS group, the apoptosis rate in the H/R + LY and HR + 100 mg/L APS + LY groups increased, and the cell survival rate, NO content and expression of PI3K/Akt/eNOS signaling pathway associated proteins decreased. Collectively, APS improves the damage caused by H/P by mediating PI3K/Akt/eNOS signaling pathway.