In Vitro Antimetastatic Effect of Phosphatidylinositol 3-Kinase Inhibitor ZSTK474 on Prostate Cancer PC3 Cells

In this study, we investigated the mechanisms of protein kinase B (PKB) activation and its role in cumulus cells during in vitro meiotic resumption of porcine oocytes. PKB activity in cumulus cells was significantly decreased by 12 h cultivation of cumulus-oocyte complexes (COCs) in basic medium. However, the addition of phosphodiesterase inhibitors, hypoxanthine or 3-isobutyl-1-methylxanthine, maintained the level of PKB activity in cumulus cells at comparable with that in cumulus cells just after collection from their follicles. When COCs were cultured with phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, PKB activity was significantly decreased, and both caspase 3 activity and the proportion of apoptotic cells were significantly increased as compared with those in cumulus cells just after collection from their follicles. Moreover, the inhibitory effect of hypoxanthine on spontaneous meiotic resumption was overcome by addition of LY294002. On the other hand, markedly high activity of PKB and high intensity of the phosphorylated PKB band were observed in cumulus cells of COCs which were cultured with FSH. The addition of 20 microM LY294002 to FSH-containing medium induced an apoptosis of cumulus cells, whereas little apoptotic-positive signal was detected in COCs cultured with 5 microM LY294002 and FSH. However, the inhibitory effects of LY294002 on progesterone production by cumulus cells and germinal vesicle breakdown in oocytes reached a maximum at 5 microM. Thus, high activity of the PI 3-kinase-PKB pathway in cumulus cells plays an important role in FSH regulation of cell function. Judging from these results, it is estimated that PI 3-kinase in cumulus cells is required for both the suppression of spontaneous meiotic resumption and the induction of gonadotropin-stimulated meiotic resumption.

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Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

Molecular Biology of the Cell ◽

10.1091/mbc.7.3.355 ◽

1996 ◽

Vol 7 (3) ◽

pp. 355-367 ◽

Cited By ~ 99

Author(s):

D J Spiro ◽

W Boll ◽

T Kirchhausen ◽

M Wessling-Resnick

Keyword(s):

Transferrin Receptor ◽

Kinase Inhibitor ◽

Morphological Changes ◽

Endocytic Pathway ◽

Receptor Internalization ◽

Phosphatidylinositol 3 Kinase ◽

Vitro Assay ◽

Phosphatidylinositol 3

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

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Effects of phosphatidylinositol 3-kinase inhibitor on human cervical carcinoma cells in vitro

Frontiers of Medicine in China ◽

10.1007/s11684-009-0067-9 ◽

2009 ◽

Vol 3 (3) ◽

pp. 341-346

Author(s):

Yuan Zhang ◽

Xiaoyan Zhang ◽

Yanhui Li ◽

Xuan Du ◽

Zehua Wang ◽

...

Keyword(s):

Cervical Carcinoma ◽

Kinase Inhibitor ◽

Carcinoma Cells ◽

Phosphatidylinositol 3 Kinase ◽

Human Cervical Carcinoma ◽

Human Cervical Carcinoma Cells ◽

Phosphatidylinositol 3

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The Identification of Raptor as a Substrate for p44/42 MAPK

Endocrinology ◽

10.1210/en.2010-1271 ◽

2011 ◽

Vol 152 (4) ◽

pp. 1264-1273 ◽

Cited By ~ 11

Author(s):

Paul Langlais ◽

Zhengping Yi ◽

Lawrence J. Mandarino

Keyword(s):

Kinase Inhibitor ◽

Mammalian Target Of Rapamycin ◽

Adaptor Protein ◽

Phosphorylation Sites ◽

Phosphatidylinositol 3 Kinase ◽

Mtor Inhibition ◽

Mtor Complex 1 ◽

Mapk Inhibitor ◽

Phosphatidylinositol 3

Abstract The adaptor protein raptor is the functional identifier for mammalian target of rapamycin (mTOR) complex 1 (mTORC1), acting to target mTOR to specific substrates for phosphorylation and regulation. Using HPLC-electrospray ionization tandem mass spectrometry, we confirmed the phosphorylation of raptor at Ser696, Thr706, Ser721, Ser722, Ser855, Ser859, Ser863, Thr865, Ser877, Ser881, Ser883, and Ser884 and identified Tyr692, Ser699, Thr700, Ser704, Ser854, Ser857, Ser882, Ser886, Ser887, and Thr889 as new, previously unidentified raptor phosphorylation sites. Treatment of cells with insulin increased the phosphorylation of raptor at Ser696, Ser855, Ser863, and Thr865 and suppressed the phosphorylation of Ser722. Ser696 phosphorylation was insensitive to mTOR inhibition with rapamycin, whereas treatment of cells with the MAPK inhibitor PD98059 inhibited the insulin-stimulated phosphorylation of raptor at Ser696. In vitro incubation of raptor with p42 MAPK significantly increased raptor phosphorylation (P < 0.01), whereas phosphorylation of a Ser696Ala mutant was decreased (P < 0.05), suggesting MAPK is capable of directly phosphorylating raptor at Ser696. Mutation of Ser696 to alanine interfered with insulin-stimulated phosphorylation of the mTOR downstream substrate p70S6 kinase. Incubation of cells with the MAPK inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor wortmannin decreased the insulin stimulated phosphorylation of raptor, suggesting that the MAPK and phosphatidylinositol 3-kinase pathways may merge at mTORC1.

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