Anti-malarial drug artesunate ameliorates oxidative lung damage in experimental allergic asthma

Oxidative stress is a critical pathophysiological factor in the development of allergic airway inflammation, resulting in oxidative damage to lipids, proteins, and DNA. Our recent report revealed potent anti-inflammatory effects of the antimalarial drug artesunate in experimental allergic asthma. The present study investigated potential antioxidative effects of artesunate in a murine model of allergic asthma in comparison with dexamethasone, a potent corticosteroid. Mice were sensitized and challenged with ovalbumin and developed airway inflammation and oxidative lung damage. Artesunate markedly suppressed ovalbumin-induced increases in total cell, eosinophil, and neutrophil counts. In contrast, dexamethasone failed to inhibit neutrophil recruitment. Levels of the oxidative damage markers 8-isoprostane, 8-hydroxy-2-deoxyguanosine, and 3-nitrotyrosine were potently repressed by artesunate. However, dexamethasone showed weaker inhibitory effects on 3-nitrotyrosine production. Ovalbumin-induced increases in the expression of the pro-oxidants iNOS and NADPH oxidase (NOX1, 2, 3, and 4) were significantly abated by artesunate. Gene expression of regulatory subunits of NOX, p22phox and p67phox, was also reduced by artesunate. The expression and activities of the antioxidants superoxide dismutase and catalase were substantially reversed with artesunate in ovalbumin-challenged mice. Artesunate significantly enhanced nuclear levels of nuclear factor erythroid-2-related factor 2 (Nrf2) in lung tissues from ovalbumin-challenged mice and in TNF-α-stimulated human bronchial epithelial cells. Our findings implicate a potential therapeutic value for artesunate in the treatment of asthma via the amelioration of oxidative damage in allergic airways, and it may act by suppressing pro-oxidants and restoring the activities and expression of antioxidants via activation of Nrf2. Artesunate may be a potential novel anti-asthma drug capable of controlling both inflammation and oxidative damage in chronic severe asthma.     

Involvement of insulin/phosphoinositide 3-kinase/Akt signal pathway in 17 beta-estradiol-mediated neuroprotection

In the present study, we tested the hypothesis that 17beta-estradiol (betaE2) is a neuroprotectant in the retina, using two experimental approaches: 1) hydrogen peroxide (H(2)O(2))-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both betaE2 and 17alpha-estradiol (alphaE2) significantly protected against H(2)O(2)-induced retinal neuron degeneration; however, progesterone had no effect. betaE2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5-bisphosphate and [(32)gammaATP] were used as substrate. Phospho-Akt levels were also transiently increased by betaE2 treatment. Addition of the estrogen receptor antagonist tamoxifen did not reverse the protective effect of betaE2, whereas the PI3K inhibitor LY294002 inhibited the protective effect of betaE2, suggesting that betaE2 mediates its effect through some PI3K-dependent pathway, independent of the estrogen receptor. Pull-down experiments with glutathione S-transferase fused to the N-Src homology 2 domain of p85, the regulatory subunit of PI3K, indicated that betaE2 and alphaE2, but not progesterone, identified phosphorylated insulin receptor beta-subunit (IRbeta) as a binding partner. Pretreatment with insulin receptor inhibitor, HNMPA, inhibited IRbeta activation of PI3K. Systemic administration of betaE2 significantly protected the structure and function of rat retinas against light-induced photoreceptor cell degeneration and inhibited photoreceptor apoptosis. In addition, systemic administration of betaE2 activated retinal IRbeta, but not the insulin-like growth factor receptor-1, and produced a transient increase in PI3K activity and phosphorylation of Akt in rat retinas. The results show that estrogen has retinal neuroprotective properties in vivo and in vitro and suggest that the insulin receptor/PI3K/Akt signaling pathway is involved in estrogen-mediated retinal neuroprotection.     

Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor

The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated Tyr-Xaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr --> Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.     

Bcl-xL mediates a survival mechanism independent of the phosphoinositide 3-kinase/Akt pathway in prostate cancer cells

Among various molecular strategies by which prostate cancer cells evade apoptosis, phosphoinositide 3-kinase (PI3K)/Akt signaling represents a dominant survival pathway. However, different prostate cancer cell lines such as LNCaP and PC-3 display differential sensitivity to the apoptotic effect of PI3K inhibition in serum-free media, reflecting the heterogeneous nature of prostate cancer in apoptosis regulation. Whereas both cell lines are equally susceptible to LY294002-mediated Akt dephosphorylation, only LNCaP cells default to apoptosis, as evidenced by DNA fragmentation and cytochrome c release. In PC-3 cells, Akt deactivation does not lead to cytochrome c release, suggesting that the intermediary signaling pathway is short-circuited by an antiapoptotic factor. This study presents evidence that Bcl-xL overexpression provides a distinct survival mechanism that protects PC-3 cells from apoptotic signals emanating from PI3K inhibition. First, the Bcl-xL/BAD ratio in PC-3 cells is at least an order of magnitude greater than that of LNCaP cells. Second, ectopic expression of Bcl-xL protects LNCaP cells against LY294002-induced apoptosis. Third, antisense down-regulation of Bcl-xL sensitizes PC-3 cells to the apoptotic effect of LY294002. The physiological relevance of this Bcl-xL-mediated survival mechanism is further underscored by the protective effect of serum on LY294002-induced cell death in LNCaP cells, which is correlated with a multifold increase in Bcl-xL expression. In contrast to Bcl-xL, Bcl-2 expression levels are similar in both cells lines, and do not respond to serum stimulation, suggesting that Bcl-2 may not play a physiological role in antagonizing apoptosis signals pertinent to BAD activation in prostate cancer cells.     

Involvement of the phosphoinositide 3-kinase/Akt signaling pathway in the resistance to therapeutic treatments of human leukemias

A major factor undermining successful cancer treatment is the occurrence of resistance to conventional treatments such as chemotherapy and ionizing radiation. Evidence accumulated over the recent years has indicated the phosphoinositide 3-kinase/Akt signal transduction pathway as one of the major factors implicated in cancer resistance to conventional therapies. Indeed, the phosphoinositide 3-kinase/Akt axis regulates the expression and/or function of many anti-apoptotic proteins which strongly contributes to cancer cell survival. As a result, small molecules designed to specifically target key components of this signaling network are now being developed for clinical use as single therapeutic agents and/or in combination with other forms of therapy to overcome resistance. Initially, the phosphoinositide 3-kinase/Akt signal transduction pathway has been mainly investigated in solid tumors. Recently, however, this network has also been recognized as an important therapeutic target in human leukemias. Specific inhibition of this signalling pathway may be a valid approach to treat these diseases and increase the efficacy of standard types of therapy.     

Hyaluronan oligosaccharides inhibit anchorage-independent growth of tumor cells by suppressing the phosphoinositide 3-kinase/Akt cell survival pathway

Hyaluronan oligosaccharides (molecular weight: approximately 2.5 x 10(3)) inhibit growth of several types of tumors in vivo. In vitro, the oligomers inhibit anchorage-independent growth of several tumor cell types. In accordance with this finding, the oligomers also induce apoptosis and stimulate caspase-3 activity under anchorage-independent conditions. Since inhibitors of phosphoinositide 3-kinase (PI 3-kinase) mimic the action of hyaluronan oligomers and since the PI 3-kinase/Akt (protein kinase B) cell survival pathway has previously been implicated in anchorage-independent growth of tumor cells, we examined the effect of oligomers on PI 3-kinase and its downstream activities in TA3/St murine mammary carcinoma and HCT 116 human colon carcinoma cells. We observed that 50-150 microg/ml hyaluronan oligomers inhibit PI 3-kinase activity and phosphorylation of Akt to approximately the same extent as optimal doses of wortmannin and LY294002, known inhibitors of PI 3-kinase. Similar inhibition of downstream events, i.e. phosphorylation of BAD and FKHR, was also observed. These effects were not observed on treatment with similar concentrations of chitin oligomers, chondroitin sulfate, or hyaluronan polymer. High molecular weight (approximately 2 x 10(6)) and low molecular weight (approximately 8 x 10(4)) preparations of hyaluronan polymer were equally ineffective. The effects of hyaluronan oligomers on these parameters were similar in magnitude to the effect of treatment with activity-blocking antibody against CD44. We interpret these results to indicate that the oligomers competitively block binding of endogenous hyaluronan polymer to CD44, consequently giving rise to attenuated signaling. Finally, we observed that hyaluronan oligomers, but not chitin oligomers, chondroitin sulfate, or hyaluronan polymer, stimulate expression of PTEN, a phosphatase that degrades the major signaling product of PI 3-kinase action, phosphoinositide 3,4,5-trisphosphate. We conclude that perturbation of hyaluronan-CD44 binding leads to suppression of the PI 3-kinase/Akt cell survival pathway and consequently to inhibition of anchorage-independent growth in culture and tumor growth in vivo.     

Ras regulates neuronal polarity via the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway

The establishment of a polarized morphology is an essential event in the differentiation of neurons into a single axon and dendrites. We previously showed that glycogen synthase kinase-3beta (GSK-3beta) is critical for specifying axon/dendrite fate by the regulation of the phosphorylation of collapsin response mediator protein-2 (CRMP-2). Here, we found that the overexpression of the small GTPase Ras induced the formation of multiple axons in cultured hippocampal neurons, whereas the ectopic expression of the dominant negative form of Ras inhibited the formation of axons. Inhibition of phosphatidylinositol-3-kinase (PI3-kinase) or extracellular signal-related kinase (ERK) kinase (MEK) suppressed the Ras-induced formation of multiple axons. The expression of the constitutively active form of PI3-kinase or Akt (also called protein kinase B) induced the formation of multiple axons. The overexpression of Ras prevented the phosphorylation of CRMP-2 by GSK-3beta. Taken together, these results suggest that Ras plays critical roles in establishing neuronal polarity upstream of the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway and mitogen-activated protein kinase cascade.     

Down-regulation of the tumor suppressor gene retinoic acid receptor beta2 through the phosphoinositide 3-kinase/Akt signaling pathway

The retinoic acid receptor beta2 (RARbeta2) is a potent, retinoid-inducible tumor suppressor gene, which is a critical molecular relay for retinoid actions in cells. Its down-regulation, or loss of expression, leads to resistance of cancer cells to retinoid treatment. Up to now, no primary mechanism underlying the repression of the RARbeta2 gene expression, hence affecting cellular retinoid sensitivity, has been identified. Here, we demonstrate that the phosphoinositide 3-kinase/Akt signaling pathway affects cellular retinoid sensitivity, by regulating corepressor recruitment to the RARbeta2 promoter. Through direct phosphorylation of the corepressor silencing mediator for retinoic and thyroid hormone receptors (SMRT), Akt stabilized RAR/SMRT interaction, leading to an increased tethering of SMRT to the RARbeta2 promoter, decreased histone acetylation, down-regulation of the RARbeta2 expression, and impaired cellular differentiation in response to retinoid. The phosphoinositide 3-kinase/Akt signaling pathway, an important modulator of cellular survival, has thus a direct impact on cellular retinoid sensitivity, and its deregulation may be the triggering event in retinoid resistance of cancer cells.     

Clusterin activates survival through the phosphatidylinositol 3-kinase/Akt pathway

Clusterin is, in its major form, a secreted heterodimeric disulfide-linked glycoprotein (75-80 kDa). It was first linked to cell death in the rat ventral prostate after androgen deprivation. Recent studies have demonstrated that overexpression of clusterin in prostatic cells protects them against tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. However the details of this survival mechanism remain undefined. Here, we investigate how clusterin prevents cells from undergoing TNFalpha-induced apoptosis. We established a double-stable prostatic cell line for inducible clusterin by using the Tet-On gene expression system. We demonstrated that 50% of the cells overexpressing clusterin escaped from TNFalpha- and actinomycin D-induced cell death. Moreover we demonstrated that the incubation of MLL cells with conditioned medium containing the secreted clusterin or the supplementation of purified clusterin in the extracellular medium decreased the TNFalpha-induced apoptosis significantly. This extracellular action implicates megalin, the putative membrane receptor for clusterin to mediate survival. Indeed clusterin overexpression up-regulated the expression of megalin and induced its phosphorylation in a dose-dependent manner. We interestingly showed that clusterin overexpression is associated with the up-regulation of the phosphorylation of Akt. Activated Akt induced the phosphorylation of Bad and caused a decrease of cytochrome c release. These results enable us to pinpoint one mechanism by which secreted clusterin favors survival in androgen-independent prostate cancer cells, implicating its receptor megalin and Akt survival pathway.     

Phosphatidylinositol 3-kinase/Akt stimulates androgen pathway through GSK3beta inhibition and nuclear beta-catenin accumulation

PI3K/Akt plays a critical role in prostate cancer cell growth and survival. Recent studies have shown that the effect of PI3K/Akt in prostate cells is mediated through androgen signaling. The PI3K inhibitor, LY294002, and a tumor suppressor, PTEN, negatively regulate the PI3K/Akt pathway and repress AR activity. However, the molecular mechanisms whereby PI3K/Akt and PTEN regulate the androgen pathway are currently unclear. Here, we demonstrate that blocking the PI3K/Akt pathway reduces the expression of an endogenous AR target gene. Moreover, we show that the repression of AR activity by LY294002 is mediated through phosphorylation and inactivation of GSK3beta, a downstream substrate of PI3K/Akt, which results in the nuclear accumulation of beta-catenin. Given the recent evidence that beta-catenin acts as a coactivator of AR, our findings suggest a novel mechanism by which PI3K/Akt modulates androgen signaling. In a PTEN-null prostate cancer cell line, we show that PTEN expression reduces beta-catenin-mediated augmentation of AR transactivation. Using the mutants of beta-catenin, we further demonstrate that the repressive effect of PTEN is mediated by a GSK3beta-regulated degradation of beta-catenin. Our results delineate a novel link among the PI3K, wnt, and androgen pathways and provide fresh insights into the mechanisms of prostate tumor development and progression.     

The phosphoinositide 3-kinase/Akt pathway is activated by daunorubicin in human acute myeloid leukemia cell lines.

Daunorubicin induces apoptosis in myeloid leukemia cells by activation of neutral sphingomyelinase and ceramide generation occurring 4-10 min after daunorubicin addition. We show here that daunorubicin is able to increase the phosphoinositide 3-kinase activity and enhance intracellular phosphoinositide 3-kinase lipid products prior to ceramide generation. Daunorubicin activates Akt, a downstream phosphoinositide 3-kinase effector. Interestingly, the phosphoinositide 3-kinase inhibitors wortmannin and LY294002 accelerate daunorubicin-induced apoptosis in U937 cells. The phosphoinositide 3-kinase/Akt pathway has been involved in cell survival following serum deprivation, tumor necrosis factor alpha, anti-Fas and UV radiations. Our results suggest that anti-tumor agents such as daunorubicin may also activate anti-apoptotic signals that could contribute to drug resistance.     

Duvelisib attenuates bleomycin-induced pulmonary fibrosis via inhibiting the PI3K/Akt/mTOR signalling pathway

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that seriously threatens the health of patients. The pathogenesis of IPF is still unclear, and there is a lack of effective therapeutic drugs. Myofibroblasts are the main effector cells of IPF, leading to excessive deposition of extracellular matrix (ECM) and promoting the progression of fibrosis. Inhibiting the excessive activation and relieving autophagy blockage of myofibroblasts is the key to treat IPF. PI3K/Akt/mTOR pathway plays a key regulatory role in promoting fibroblast activation and autophagy inhibition in lung fibrosis. Duvelisib is a PI3K inhibitor that can simultaneously inhibit the activities of PI3K-δ and PI3K-γ, and is mainly used for the treatment of relapsed/refractory chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma tumour (SLL). In this study, we aimed to examine the effects of Duvelisib on pulmonary fibrosis. We used a mouse model of bleomycin-induced pulmonary fibrosis to evaluate the effects of Duvelisib on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of Duvelisib in lung fibroblasts in vitro. The in vivo experiments showed that Duvelisib significantly alleviated bleomycin-induced collagen deposition and improved pulmonary function. In vitro and in vivo pharmacological experiments showed that Duvelisib dose-dependently suppressed lung fibroblast activation and improved autophagy inhibition by inhibiting the phosphorylation of PI3K, Akt and mTOR. Our results indicate that Duvelisib can alleviate the severity of pulmonary fibrosis and provide potential drugs for the treatment of pulmonary fibrosis.     

Suppression of apoptosis by UVB irradiation: survival signaling via PI3-kinase/Akt pathway

UVB irradiation induces apoptosis in several cell types. However, we report here that UVB irradiation prevents induction of apoptosis in cells detached from the extracellular matrix under serum-free conditions. NIH3T3 cells cultured in bovine serum albumin-coated dishes (detached from the extracellular matrix) underwent apoptosis under serum-free conditions, which was inhibited by UVB (<0.1 J/cm(2)) irradiation, keeping suspension conditions, as determined by chromatin condensation and the appearance of a subG1 DNA fraction. Furthermore, UVB irradiation decreased caspase-3/7, -8/6, and -9 activation and eliminated loss of mitochondrial inner transmembrane potential, suggesting suppression upstream of the caspase cascade. Treatment with PI3-kinase inhibitors, wortmannin, and LY294002 partly eliminated the UV-mediated inhibition of cell death and recovered the inhibited caspase-3/7 activity. Phosphorylation of Akt was observed from 15 min after UVB irradiation. These results suggested that UVB irradiation transduced a survival signal via PI3 kinase activation and phosphorylation of Akt, and induced some apoptosis inhibition factors upstream of the caspase cascade.     

Alpha-lipoic acid preconditioning reduces ischemia-reperfusion injury of the rat liver via the PI3-kinase/Akt pathway

In liver resection and transplantation ischemia-reperfusion injury (IRI) is one of the main causes of organ dys- or nonfunction. The aim of the present study was to determine whether alpha-lipoic acid (LA) is able to attenuate IRI. Rat livers were perfused with Krebs-Henseleit buffer with or without LA (+/-wortmannin), followed by ischemia (1 h, 37 degrees C) and reperfusion (90 min). Efflux of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) and hepatic ATP content were determined enzymatically. Activation of NF-kappaB and activating protein 1 (AP-1) was examined by EMSA, and protein phosphorylation was examined by Western blot. Caspase-3-like activity served as an indicator for apoptotic processes. Animals treated intravenously with 500 micromol LA were subjected to 90 min of partial no-flow ischemia followed by reperfusion for up to 7 days. Preconditioning with LA significantly reduced LDH and PNP efflux during reperfusion in isolated perfused rat livers. ATP content was significantly increased in LA-treated livers. Postischemic activation of NF-kappaB and AP-1 was significantly reduced in LA-pretreated organs. Preconditioning with LA significantly enhanced Akt phosphorylation. It showed neither effect on endothelial nitric oxide synthase nor on Bad phosphorylation. Importantly, simultaneous administration of wortmannin, an inhibitor of the phosphatidylinositol (PI)3-kinase/Akt pathway, blocked the protective effect of LA on IRI, demonstrating a causal relationship between Akt activation and hepatoprotection by LA. Interestingly, despite activation of Akt, LA did not reduce postischemic apoptotic cell death. The efficacy of LA treatment in vivo was shown by reduced GST plasma levels and improved liver histology of animals pretreated with LA. This study shows for the first time that the PI3-kinase/Akt pathway plays a central protective role in IRI of the rat liver and that LA administration attenuates IRI via this pathway.     

The phosphoinositide 3-kinase/AKT1 pathway involvement in drug and all-trans-retinoic acid resistance of leukemia cells

Disruption of the apoptotic pathways may account for resistance to chemotherapy and treatment failures in human neoplastic disease. To further evaluate this issue, we isolated a HL-60 cell clone highly resistant to several drugs inducing apoptosis and to the differentiating chemical all-trans-retinoic acid (ATRA). The resistant clone displayed an activated phosphoinositide 3-kinase (PI3K)/AKT1 pathway, with levels of phosphatidylinositol (3,4,5) trisphosphate higher than the parental cells and increased levels of both Thr 308 and Ser 473 phosphorylated AKT1. In vitro AKT1 activity was elevated in resistant cells, whereas treatment of the resistant cell clone with two inhibitors of PI3K, wortmannin or Ly294002, strongly reduced phosphatidylinositol (3,4,5) trisphosphate levels and AKT1 activity. The inhibitors reversed resistance to drugs. Resistant cells overexpressing either dominant negative PI3K or dominant negative AKT1 became sensitive to drugs and ATRA. Conversely, if parental HL-60 cells were forced to overexpress an activated AKT1, they became resistant to apoptotic inducers and ATRA. There was a tight relationship between the activation of the PI3K/AKT1 axis and the expression of c-IAP1 and c-IAP2 proteins. Activation of the PI3K/AKT1 axis in resistant cells was dependent on enhanced tyrosine phosphorylation of the p85 regulatory subunit of PI3K, conceivably due to an autocrine insulin-like growth factor-I production. Our findings suggest that an up-regulation of the PI3K/AKT1 pathway might be one of the survival mechanisms responsible for the onset of resistance to chemotherapeutic and differentiating therapy in patients with acute leukemia.     

Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase.

Angiotensin II (Ang II) exerts contractile and trophic effects in glomerular mesangial cells (MCs). One potential downstream target of Ang II is the protein kinase Akt/protein kinase B (PKB). We investigated the effect of Ang II on Akt/PKB activity in MCs. Ang II causes rapid activation of Akt/PKB (5-10 min) but delayed activation of phosphoinositide 3-kinase (PI3-K) (30 min). Activation of Akt/PKB by Ang II was not abrogated by the PI3-K inhibitors or by the introduction of a dominant negative PI3-K, indicating that in MCs, PI3-K is not an upstream mediator of Akt/PKB activation by Ang II. Incubation of MCs with phospholipase A2 inhibitors also blocked Akt/PKB activation by Ang II. AA mimicked the effect of Ang II. Inhibitors of cyclooxygenase-, lipoxyogenase-, and cytochrome P450-dependent metabolism did not influence AA-induced Akt/PKB activation. However, the antioxidants N-acetylcysteine and diphenylene iodonium inhibited both AA- and Ang II-induced Akt/PKB activation. Dominant negative mutant of Akt/PKB or antioxidants, but not the dominant negative form of PI3-K, inhibited Ang II-induced protein synthesis and cell hypertrophy. These data provide the first evidence that Ang II induces protein synthesis and hypertrophy in MCs through AA/redox-dependent pathway and Akt/PKB activation independent of PI3-K.