PI3K pathway inhibitor LY294002 alters Jurkat T cell biobehaviours via ERK1/2-ICBP90 mediation

Little is known about whether there is a relationshipbetweenPI3K/AKT, ERK1/2 and an inverted CCAAT box binding protein (ICBP90) in biological behaviours of tumour cells. The aim of this study was to determine thisusing Jurkat T cells. Compared to PD98059 (an ERK1/2 signaling inhibitor), DAPT (a Notch signaling inhibitor) or adriamycin (a classical anti-tumour drug), the inhibition of Jurkat T cell growth by LY294002 (a PI3K/Akt signaling inhibitor) was more obvious. LY294002 combined with adriamycin appeared to have a synergistic effect. LY294002 strongly blocked Jurkat T cells at each phase of cell cycle with a decrease of DNA content, superior to adriamycin. Consistent with these changes, the expression of phosphorylated ERK1/2 was markedly decreased in the LY294002-treated Jurkat T cells, leading to the reduction of ICBP90 production, followed by moderate attenuation of TGF-β secretion. The results suggest that deactivation of PI3K/Akt signalling can surpress Jurkat T cell growth through inhibiting cell proliferation and blocking the cell cycle. ICBP90 may mediate the PI3K/AKT-ERK1/2 signalling to regulate leukemia cell growth.     

Agmatine Protects Against 6-OHDA-Induced Apoptosis,and ERK and Akt/GSK Disruption in SH-SY5Y Cells

6-Hydroxydopamine (6-OHDA), a metabolite of dopamine is known to induce dopaminergic cell toxicity which makes that a suitable agent inducing an experimental model of Parkinson’s disease (PD). Agmatine has been shown to protect against some cellular and animal PD models. This study was aimed to assess whether agmatine prevents 6-OHDA-induced SH-SY5Y cell death and if yes, then how it affects Akt/glycogen synthesis kinase-3β (GSK-3β) and extracellular signal-regulated kinases (ERK) signals. The cells were treated with different drugs, and their viability was examined via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and morphological observation. Western blot studies were done to assess cleaved caspase-3, Akt/GSK-3β, and ERK proteins. 6-OHDA-induced cell death and caspase-3 cleavage, while agmatine prevented those changes. 6-OHDA also decreased the amount of phosphorylated Akt (pAkt)/Akt while increased GSK-3β activity which was prevented by agmatine. Additionally, this toxin increased pERK/ERK ratio which was averted again by agmatine. The PI3/Akt inhibitor, LY294002, impeded the changes induced by agmatine, while ERK inhibitor (PD98059) did not disturb the effects of agmatine, and by itself, it preserved the cells against 6-OHDA toxicity. This study revealed that agmatine is protective in 6-OHDA model of PD and affects Akt/GSK-3β and ERK pathways.     

低浓度哇巴因对负鼠肾小管上皮细胞增殖的影响

目的:用低血清培养液来模拟肾脏供血不足的营养不良状态,研究低浓度哇巴因对低血清培养下OK细胞(负鼠肾小管上皮细胞)增殖的影响。方法:用低浓度哇巴因(1-30n M)处理0.2%血清培养下OK细胞,MTT实验和Brdu掺入法检测哇巴因对OK细胞增殖的影响;Western blot检测Akt和ERK1/2的磷酸化水平;用LY294002和PD98059分别抑制PI3K/Akt和ERK1/2蛋白激酶活性,观察抑制PI3K/Akt和ERK1/2对哇巴因促进OK细胞增殖的影响。结果:低浓度哇巴因(1-30n M)促进OK细胞的增值,上调OK细胞中Akt和ERK1/2磷酸化水平。用LY294002和PD98059特异抑制Akt和ERK1/2的活化能够抑制哇巴因的促增殖作用。结论:低浓度哇巴因(1-10n M)能够促进OK细胞的增值,PI3K/Akt和ERK1/2信号通路参与哇巴因对OK细胞促增殖作用的调节。     

Cbl-b promotes chemotherapy-induced apoptosis in rat basophilic leukemia cells by suppressing PI3K/Akt activation and enhancing MEK/ERK activation

The ubiquitin ligase Cbl-b is a negative regulator of the PI3K/Akt pathway, the survival pathway implicated in chemotherapy resistance. However, it remains unclear whether Cbl-b can regulate chemosensitivity through modulating Akt activation. In this study, VP-16-induced RBL-2H3 cells apoptosis was accompanied by the activation of Akt and ERK. The PI3K inhibitor LY294002, not the ERK inhibitor PD98059, enhanced the apoptosis. In addition, down-regulation of Cbl-b was also detected. Over expression of Cbl-b significantly enhanced VP-16-induced cell apoptosis with inhibition of Akt activity, while a dominant negative (DN) RING Finger domain mutation completely abolished this enhancement. On the other hand, ERK activity was enhanced by Cbl-b, and the ERK inhibitor PD98059 reversed Cbl-b-enhanced apoptosis. The consistent results were also showed in the process of Ara-c treatment. These observations indicate that Cbl-b promotes RBL-2H3 apoptosis induced by VP-16 or Ara-c, probably through inhibition of Akt and activation of ERK.     

Erythropoietin prevents PC12 cells from 1-methyl-4-phenylpyridinium ion-induced apoptosis via the Akt/GSK-3β/caspase-3 mediated signaling pathway

Apoptosis is a contributing cause of dopaminergic neuron loss in Parkinson disease. Recent work has shown that erythropoietin (EPO) offers protection against apoptosis in a wide variety of tissues. We demonstrate that exposure of PC12 cells to 1-methyl-4-phenylpyridinium ion (MPP⁺) with recombinant human EPO, significantly decreased apoptosis as measured by TUNEL and caspase-3 activity when compared to MPP⁺ treatment alone. EPO induced sustained phosphorylation of Akt and its substrate, GSK-3β, reduced caspase-3 activities in PC12 cells. The anti-apoptotic effect of EPO was abrogated by co-treatment with LY294002, the specific blocker of phosphatidylinositol 3-kinase (PI3K). The effects of EPO on GSK-3β and caspase-3 activities were also blocked by LY294002. LiCl, the inhibitor of GSK-3β, downregulated the caspase-3 activity and blocked the apoptosis induced by MPP⁺. Finally, we determined that EPO transiently activated the ERK signaling pathway, but PD98059, a specific inhibitor of ERK, does not alter the survival effect of EPO in this model system. Thus, these findings indicate that EPO protects against apoptosis in PC12 cells exposed to MPP⁺, through the Akt/GSK-3β/caspase-3 signaling pathway, but the ERK pathway is not involved in the EPO-dependent survival enhancing effect in this model system. The authors Yan Wu and You Shang are equally contributed to this work.     

Inhibition of the PI3K pathway sensitizes fludarabine-induced apoptosis in human leukemic cells through an inactivation of MAPK-dependent pathway

In the present study, we have investigated the effects of PI3K/Akt pathway on the response of human leukemia cells to fludarabine. Inhibition of PI3K/Akt pathway with a selective inhibitor (e.g., LY294002, or wortmannin) in leukemic cells markedly potentiated fludarabine-induced apoptosis. Inhibition of the PI3K/Akt downstream target mTOR by rapamycin also significantly enhanced fludarabine-induced apoptosis. The co-treatment of fludarabine/LY294002 resulted in significant attenuation in the levels of both phospho-Erk1/2 and phospho-Akt, as well as a marked increase in the level of phospho-JNK. The broad spectrum caspase inhibitor BOC-D-fmk markedly blocked fludarabine/LY-induced apoptosis, had no effect on cytochrome c release to the cytosol, and did abrogate caspase and PARP cleavage. This indicates that mitochondrial dysfunction is upstream of the caspase cascade. Moreover, constitutive activation of the MEK/Erk pathway completely blocked apoptosis induced by the combination of fludarabine/LY294002. Additionally, either constitutive activation of Akt or blockage of the JNK pathway significantly diminished apoptosis induced by the combination. Collectively, these findings demonstrate that inactivation of MAPK, Akt, and activation of the JNK pathway contributes to the induction of apoptosis induced by fludarabine/LY. Comparatively, MAPK inactivation plays a crucial role in fludarabine/LY-induced apoptosis. These results also strongly suggest that combining fludarabine with an inhibitor of the PI3K/Akt/mTOR pathway may represent a novel therapeutic strategy for hematological malignancies.     

Epidermal growth factor receptor-dependent,NF-kappaB-independent activation of the phosphatidylinositol 3-kinase/Akt pathway inhibits ultraviolet irradiation-induced caspases-3, -8, and -9 in human keratinocytes

Both phosphatidylinositol 3-kinase (PI3K)/Akt and NF-kappaB pathways function to promote cellular survival following stress. Recent evidence indicates that the anti-apoptotic activity of these two pathways may be functionally dependent. Ultraviolet (UV) irradiation causes oxidative stress, which can lead to apoptotic cell death. Human skin cells (keratinocytes) are commonly exposed to UV irradiation from the sun. We have investigated activation of the PI3K/Akt and NF-kappaB pathways and their roles in protecting human keratinocytes (KCs) from UV irradiation-induced apoptosis. This activation of PI3K preceded increased levels (3-fold) of active/phosphorylated Akt. UV (50 mJ/cm2 from UVB source) irradiation caused rapid recruitment of PI3K to the epidermal growth factor receptor (EGFR). Pretreatment of KCs with EGFR inhibitor PD169540 abolished UV-induced Akt activation/phosphorylation, as did the PI3K inhibitors LY294002 or wortmannin. This inhibition of Akt activation was associated with a 3-4-fold increase of UV-induced apoptosis, as measured by flow cytometry and DNA fragmentation ELISA. In contrast to Akt, UV irradiation did not detectably increase nuclear localization of NF-kappaB, indicating that it was not strongly activated. Consistent with this observation, interference with NF-kappaB activation by adenovirus-mediated overexpression of dominant negative IKK-beta or IkappaB-alpha did not increase UV-induced apoptosis. However, adenovirus-mediated overexpression of constitutively active Akt completely blocked UV-induced apoptosis observed with PI3K inhibition by LY294002, whereas adenovirus mediated overexpression of dominant negative Akt increased UV-induced apoptosis by 2-fold. Inhibition of UV-induced activation of Akt increased release of mitochondrial cytochrome c 3.5-fold, and caused appearance of active forms of caspase-9, caspase-8, and caspase-3. Constitutively active Akt abolished UV-induced cytochrome c release and activation of caspases-9, -8, and -3. These data demonstrate that PI3K/Akt is essential for protecting human KCs against UV-induced apoptosis, whereas NF-kappaB pathway provides little, if any, protective role.     

PAK4 confers cisplatin resistance in gastric cancer cells via PI3K/Akt- and MEK/ERK-dependent pathways

CDDP [cisplatin or cis-diamminedichloroplatinum(II)] and CDDP-based combination chemotherapy have been confirmed effective against gastric cancer. However, CDDP efficiency is limited because of development of drug resistance. In this study, we found that PAK4 (p21-activated kinase 4) expression and activity were elevated in gastric cancer cells with acquired CDDP resistance (AGS/CDDP and MKN-45/CDDP) compared with their parental cells. Inhibition of PAK4 or knockdown of PAK4 expression by specific siRNA (small interfering RNA)-sensitized CDDP-resistant cells to CDDP and overcome CDDP resistance. Combination treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 [the inhibitor of PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B or PKB) pathway] or PD98509 {the inhibitor of MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] pathway} with PF-3758309 (the PAK4 inhibitor) resulted in increased CDDP efficacy compared with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or PD98509 alone. However, after the concomitant treatment of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PD98509, PF-3758309 administration exerted no additional enhancement of CDDP cytotoxicity in CDDP-resistant cells. Inhibition of PAK4 by PF-3758309 could significantly suppress MEK/ERK and PI3K/Akt signalling in CDDP-resistant cells. Furthermore, inhibition of PI3K/Akt pathway while not MEK/ERK pathway could inhibit PAK4 activity in these cells. The in vivo results were similar with those of in vitro. In conclusion, these results indicate that PAK4 confers CDDP resistance via the activation of MEK/ERK and PI3K/Akt pathways. PAK4 and PI3K/Akt pathways can reciprocally activate each other. Therefore, PAK4 may be a potential target for overcoming CDDP resistance in gastric cancer.     

Ghrelin stimulates angiogenesis via GHSR1a-dependent MEK/ERK and PI3K/Akt signal pathways in rat cardiac microvascular endothelial cells

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHSR), is thought to exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function such as cell proliferation, migration, survival and angiogenesis. However, the effect of ghrelin on angiogenesis and the corresponding mechanisms have not yet been extensively studied in cardiac microvascular endothelial cells (CMECs) isolated from left ventricular myocardium of adult Sprague-Dawley (SD) rats. In our study, we found that ghrelin and GHSR are constitutively expressed in CMECs. Ghrelin significantly increases CMECs proliferation, migration, and in vitro angiogenesis. The ghrelin-induced angiogenic process was accompanied by phosphorylation of ERK and Akt. MEK inhibitor PD98059 abolished ghrelin-induced phosphorylation of ERK, but had no effect on Akt phosphorylation. PI3K inhibitor LY294002 abolished ghrelin-induced phosphorylation of Akt, but had no effect on ERK phosphorylation. Ghrelin-induced angiogenesis was partially blocked by treatment with PD98059 or LY294002. In addition, this angiogenic effect was almost completely inhibited by PD98059+LY294002. Pretreatment with GHSR1a blocker [D-Lys3]-GHRP-6 abolished ghrelin-induced phosphorylation of ERK, Akt and in vitro angiogenesis. In conclusion, this is the first demonstration that ghrelin stimulates CMECs angiogenesis through GHSR1a-mediated MEK/ERK and PI3K/Akt signal pathways, indicating that two pathways are required for full angiogenic activity of ghrelin. This study suggests that ghrelin may play an important role in myocardial angiogenesis.     

M1 muscarinic receptors block caspase activation by phosphoinositide 3-kinase- and MAPK/ERK-independent pathways

When PC12 cells are deprived of trophic support they undergo apoptosis. We have previously shown that survival of trophic factor-deprived PC12M1 cells can be promoted by activation of the G protein-coupled muscarinic receptors. The mechanism whereby muscarinic receptors inhibit apoptosis is poorly understood. In the present study we investigated this mechanism by examining the effect of muscarinic receptor activation on the serum deprivation-induced activity of key players in apoptosis, the caspases, in PC12M1 cells. The results showed that m1 muscarinic activation inhibits caspase activity induced by serum deprivation. This effect appeared to be caused by the prevention of activation of caspases such as caspase-2 and caspase-3, and not by the inhibition of existing activity. Muscarinic receptor activation also stimulated the mitogen-activated protein kinase/extracellular signaling-regulated kinase (MAPK/ERK) and phosphoinositide (PI) 3-kinase signaling pathways. The PI 3-kinase pathway inhibitors wortmannin and LY294002, as well as the MAPK/ERK pathway PD98059 inhibitor, did not however suppress the inhibitory effect of the muscarinic receptors on caspase activity. The results therefore suggested that the muscarinic survival effect is mediated by a pathway that leads to caspase inhibition by MAPK/ERK- and PI 3-kinase-independent signaling cascades.     

Phenethyl isothiocyanate exhibits antileukemic activity in vitro and in vivo by inactivation of Akt and activation of JNK pathways

Effects of phenethyl isothiocyanate (PEITC) have been investigated in human leukemia cells (U937, Jurkat, and HL-60) as well as in primary human acute myeloid leukemia (AML) cells in relation to apoptosis and cell signaling events. Exposure of cells to PEITC resulted in pronounced increase in the activation of caspase-3, -8, -9, cleavage/degradation of PARP, and apoptosis in dose- and time-dependent manners. These events were accompanied by the caspase-independent downregulation of Mcl-1, inactivation of Akt, as well as activation of Jun N-terminal kinase (JNK). Inhibition of PI3K/Akt by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 significantly enhanced PEITC-induced apoptosis. Conversely, enforced activation of Akt by a constitutively active Akt construct markedly abrogated PEITC-mediated JNK activation, Mcl-1 downregulation, caspase activation, and apoptosis, and also interruption of the JNK pathway by pharmacological or genetically (e.g., siRNA) attenuated PEITC-induced apoptosis. Finally, administration of PEITC markedly inhibited tumor growth and induced apoptosis in U937 xenograft model in association with inactivation of Akt, activation of JNK, as well as downregulation of Mcl-1. Taken together, these findings represent a novel mechanism by which agents targeting Akt/JNK/Mcl-1 pathway potentiate PEITC lethality in transformed and primary human leukemia cells and inhibitory activity of tumor growth of U937 xenograft model.     

The protective effect of hispidin against hydrogen peroxide-induced apoptosis in H9c2 cardiomyoblast cells through Akt/GSK-3β and ERK1/2 signaling pathway

Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong anti-oxidant, anti-cancer, anti-diabetic, and anti-dementia properties. However, the cardioprotective efficacy of hispidin has not yet been investigated. In the present study, we investigated the protective effect of hispidin against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells and neonatal rat ventricular myocytes. While the treatment of H9c2 cardiomyoblast cells with hydrogen peroxide caused a loss of cell viability and an increase in the number of apoptotic cells, hispidin significantly protected the cells against hydrogen peroxide-induced cell death without any cytotoxicity as determined by XTT assay, LDH release assay, Hoechst 33342 assay, and Western blotting of apoptosis proteins such as caspase-3, Bax, and Bcl-2. Our data also shows that hispidin significantly scavenged intracellular ROS, and markedly enhanced the expression of antioxidant enzymes such as heme oxygenase-1 and catalase, which was accompanied by the concomitant activation of Akt/GSK-3β and ERK1/2 phosphorylation in H9c2 cardiomyoblast cells. The effects of hispidin on Akt and ERK phosphorylation were abrogated by LY294002 (a PI3K/Akt inhibitor) and U0126 (an ERK1/2 inhibitor). The effect of hispidin on GSK-3b activities was also blocked by LY294002. Furthermore, inhibiting the Akt/GSK-3β and ERK1/2 pathway by these inhibitors significantly reversed the hispidin-induced Bax and Bcl-2 expression, apoptosis induction, and ROS production. These findings indicate that hispidin protects against apoptosis in H9c2 cardiomyoblast cells exposed to hydrogen peroxide through reducing intracellular ROS production, regulating apoptosis-related proteins, and the activation of the Akt/GSK-3β and ERK1/2 signaling pathways.     

Protein kinase B/Akt regulates coxsackievirus B3 replication through a mechanism which is not caspase dependent

The role of signaling pathways including the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) during viral infection has gained much recent attention. Our laboratory reported on an important regulatory role for extracellular signal-regulated kinases (ERK1/2), subfamily members of the MAPKs, during coxsackievirus B3 (CVB3) infection. However, the role of the PI3K pathway in CVB3 infection has not been well characterized. CVB3 is the most common known viral infectant of heart muscle that directly injures and kills infected cardiac myocytes during the myocarditic process. In the present study, we investigated the role of protein kinase B (PKB) (also known as Akt), a general downstream mediator of survival signals through the PI3K cascade, in regulating CVB3 replication and virus-induced apoptosis in a well-established HeLa cell model. We have demonstrated that CVB3 infection leads to phosphorylation of PKB/Akt on both Ser-473 and Thr-308 residues through a PI3K-dependent mechanism. Transfection of HeLa cells with a dominant negative mutant of Akt1 or pretreatment of wild-type HeLa cells with the specific PI3K inhibitor LY294002 significantly suppresses viral RNA expression, as reflected in diminished viral capsid protein expression and viral release. Dominant negative Akt1 and LY294002 also increase apoptosis in infected cells, which can be reversed by addition of the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk). Interestingly, blocking of apoptosis by zVAD.fmk does not reverse the viral RNA translation blockade, indicating that the inhibitory effect of dominant negative Akt1 on viral protein expression is not caspase dependent. In addition, we showed that the attachment of virus to its receptor-coreceptor complex is not sufficient for PKB/Akt activation and that postentry viral replication is required for Akt phosphorylation. Taken together, these data illustrate a new and imperative role for Akt in CVB3 infection in HeLa cells and show that the PI3K/Akt signaling is beneficial to CVB3 replication.     

Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways

Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.     

Inhibition of Src-like kinases reveals Akt-dependent and -independent pathways in insulin-like growth factor I-mediated oligodendrocyte progenitor survival

Insulin-like growth factor I (IGF-I) has been previously shown to promote survival of oligodendrocyte progenitors; however, the underlying mechanisms are not fully understood. Our aim was to investigate the involvement of phosphatidylinositol 3-kinase (PI3K), MEK1, and Src family tyrosine kinases in IGF-I-mediated oligodendrocyte progenitor survival. In agreement with previous studies, IGF-I promoted cell survival. We show that IGF-I prevented apoptosis induced by growth factor deprivation in a PI3K-dependent and MEK/ERK-independent manner. In addition, IGF-I activated Akt while inhibiting caspase-3 activation, and these effects were reversed by the PI3K inhibitors LY 294002 and wortmannin, but not by the MEK1 inhibitor PD 98059. Interestingly, PP2, a specific Src-like kinase inhibitor, blocked the tyrosine phosphorylation of Src, Fyn, and Lyn and IGF-I-stimulated Akt activation, yet had no significant effects on caspase-3 activation or progenitor survival. To further determine whether Akt is required for IGF-I-mediated survival, oligodendrocyte progenitors were transduced with defective Akt mutants or treated with an Akt inhibitor. Although the Akt mutants and inhibitor decreased Akt activity and reduced basal cell survival, IGF-I could partially rescue oligodendrocyte progenitors by decreasing caspase-3 activation. These results suggest that 1) PI3K is essential for IGF-I-promoted cell survival, 2) downstream activation of Akt-dependent and -independent pathways is involved, and 3) Src-like tyrosine kinases participate in IGF-I-induced Akt activation. Therefore, an unidentified effector(s) of PI3K appears to be involved in conferring complete IGF-I-mediated protection of oligodendrocyte progenitors.     

Involvement of activation of PI3K/Akt pathway in the protective effects of puerarin against MPP+-induced human neuroblastoma SH-SY5Y cell death

In an attempt to clarify the protective effect of puerarin on toxin-insulted dopaminergic neuronal death, this present study was carried out by using a typical Parkinson's disease (PD) model - 1-methyl-4-phenylpyridinium iodide (MPP(+))-induced dopaminergic SH-SY5Y cellular model. Data are presented, which showed that puerarin up-regulated Akt phosphorylation in both of MPP(+)-treated and non-MPP(+)-treated cells. The presence of PI3K inhibitor LY294002 completely blocked puerarin-induced activation of Akt phosphorylation. Moreover, puerarin decreased MPP(+)-induced cell death, which was blocked by phosphoinositide 3-kinase (PI3K) inhibitor LY294002. We further demonstrated that puerarin protected against MPP(+)-induced p53 nuclear accumulation, Puma (p53-upregulated mediator of apoptosis) and Bax expression and caspase-3-dependent programmed cell death (PCD). This protection was blocked by applying a PI3K/Akt inhibitor. Additionally, it was Pifithrin-α, but not Pifithrin-μ, which blocked MPP(+)-induced Puma and Bax expression, caspase-3 activation and cell death. Collectively, these data suggest that the activation of PI3K/Akt pathway is involved in the protective effect of puerarin against MPP(+)-induced neuroblastoma SH-SY5Y cell death through inhibiting nuclear p53 accumulation and subsequently caspase-3-dependent PCD. Puerarin might be a potential therapeutic agent for PD.     

Molecular mechanism of diclofenac-induced apoptosis of promyelocytic leukemia: dependency on reactive oxygen species, Akt, Bid, cytochrome and caspase pathway

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in a variety of cells, but the mechanism of this effect has not been fully elucidated. We report that diclofenac, a NSAID, induces growth inhibition and apoptosis of HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS), Akt, caspase-8, and Bid. ROS generation occurs in an early stage of diclofenac-induced apoptosis preceding cytochrome c release, caspase activation, and DNA fragmentation. N-Acetyl-L-cysteine, an antioxidant, suppresses ROS generation, Akt inactivation, caspase-8 activation, and DNA fragmentation. Cyclic AMP, an inducer of Akt phosphorylation, suppresses Akt inactivation, Bid cleavage, and DNA fragmentation. LY294002, a PI3 kinase inhibitor, enhances Akt inactivation and DNA fragmentation. Ac-IETD-CHO, a caspase-8 inhibitor, suppresses Bid cleavage and DNA fragmentation. z-VAD-fmk, a universal caspase inhibitor, but not cyclosporin A (CsA), an inhibitor of mitochondrial membrane permeability transition, suppresses DNA fragmentation. These results suggest the sequential mechanism of diclofenac-induced apoptosis of HL-60 cells: ROS generation suppresses Akt activity, thereby activating caspase-8, which stimulates Bid cleavage and induces cytochrome c release and the activation of caspase-9 and-3 in a CsA-insensitive mechanism. Furthermore, we found that 2-methoxyestradiol (2-ME), a superoxide dismutase inhibitor, significantly enhances diclofenac-induced apoptosis; that is, diclofenac combined with 2-ME may have therapeutic potential in the treatment of human leukemia.     

Activation of ERK1/2 and PI3K/Akt by IGF-1 on GAP-43 Expression in DRG Neurons with Excitotoxicity Induced by Glutamate In Vitro

Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor and plays an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons. Whether IGF-1 influences growth-associated protein 43 (GAP-43) expression and activates the extracellular signal-regulated protein kinase (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways in DRG neurons with excitotoxicity induced by glutamate (Glu) remains unknown. In this study, embryonic 15-day-old rat DRG explants were cultured for 48 h and then exposed to IGF-1, Glu, Glu + IGF-1, Glu + IGF-1 + PD98059, Glu + IGF-1 + LY294002, Glu + IGF-1 + PD98059 + LY294002 for additional 12 h. The DRG explants were continuously exposed to growth media as control. The levels of GAP-43 mRNA were detected by real time-PCR analysis. The protein levels of GAP-43, phosphorylated ERK1/2, phosphorylated Akt, total ERK1/2, and total Akt were detected by Western blot assay. GAP-43 expression in situ was determined by immunofluorescent labeling. Apoptotic cell death was monitored by Hoechst 33342 staining. IGF-1 alone increased GAP-43 and its mRNA levels in the absence of Glu. The decreased GAP-43 and its mRNA levels caused by Glu could be partially reversed by the presence of IGF-1. IGF-1 rescued neuronal cell death caused by Glu. Neither the ERK1/2 inhibitor PD98059 nor the PI3K inhibitor LY294002 blocked the effect of IGF-1, but both inhibitors together were effective. To validate the impact of GAP-43 expression by IGF-1, GAP-43 induction was blocked by administration of dexamethasone (DEX). IGF-1 partially rescued the decrease of GAP-43 and its mRNA levels induced by DEX. DEX induced an increase of cell apoptosis. IGF-1 may play an important role in neuroprotective effects on DRG neurons through regulating GAP-43 expression with excitotoxicity induced by Glu and the process was involved in both ERK1/2 and PI3K/Akt signaling pathways.