Phosphatidylinositol ether lipid analogues that inhibit AKT also independently activate the stress kinase, p38alpha, through MKK3/6-independent and -dependent mechanisms.

Previously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified kinases. No kinase was inhibited, but one isoform of p38, p38alpha, was uniformly activated 2-fold. Molecular modeling of p38alpha revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38alpha(-/-) cells, we showed that p38alpha is not required for PIA-induced apoptosis but is required for H(2)O(2)- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38alpha(-/-) cells increased apoptosis. These studies indicate that p38alpha is activated by PIAs through a novel mechanism and show that p38alpha activation contributes to PIA-induced cell death. Independent modulation of Akt and p38alpha could account for the profound cytotoxicity of PIAs.

Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells.

Airway epithelial cells are constantly exposed to environmental insults such as air pollution or tobacco smoke that may contain high levels of reactive nitrogen and reactive oxygen species. Previous work from our laboratory demonstrated that the reactive oxygen species (ROS), hydrogen peroxide (H(2)O(2)), specifically activates neutral sphingomyelinase 2 (nSMase2) to generate ceramide and induce apoptosis in airway epithelial cells. In the current study we examine the biological consequence of exposure of human airway epithelial (HAE) cells to reactive nitrogen species (RNS). Similar to ROS, we hypothesized that RNS may modulate ceramide levels in HAE cells and induce apoptosis. We found that nitric oxide (NO) exposure via the NO donor papa-NONOate, failed to induce apoptosis in HAE cells. However, when papa-NONOate was combined with a superoxide anion donor (DMNQ) to generate peroxynitrite (ONOO(-)), apoptosis was observed. Similarly pure ONOO(-)-induced apoptosis, and ONOO(-)-induced apoptosis was associated with an increase in cellular ceramide levels. Pretreatment with the antioxidant glutathione did not prevent ONOO(-)-induced apoptosis, but did prevent H(2)O(2)-induced apoptosis. Analysis of the ceramide generating enzymes revealed a differential response by the oxidants. We confirmed our findings that H(2)O(2) specifically activated a neutral sphingomyelinase (nSMase2). However, ONOO(-) exposure did not affect neutral sphingomyelinase activity; rather, ONOO(-) specifically activated an acidic sphingomyelinase (aSMase). The specificity of each enzyme was confirmed using siRNA to knockdown both nSMase2 and aSMase. Silencing nSMase2 prevented H(2)O(2)-induced apoptosis, but had no effect on ONOO(-)-induced apoptosis. On the other hand, silencing of aSMase markedly impaired ONOO(-)-induced apoptosis, but did not affect H(2)O(2)-induced apoptosis. These findings support our hypothesis that ROS and RNS modulate ceramide levels to induce apoptosis in HAE cells. However, we found that different oxidants modulate different enzymes of the ceramide generating machinery to induce apoptosis in airway epithelial cells. These findings add to the complexity of how oxidative stress promotes lung cell injury.

Nitric oxide-enhanced caspase-3 and acidic sphingomyelinase interaction: a novel mechanism by which airway epithelial cells escape ceramide-induced apoptosis.

Reactive nitrogen species (RNS) are implicated in the pathophysiology of inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease. The molecular mechanisms and signaling events involved in lung cell injury by RNS are still poorly understood. In the current study, we observe a novel anti-apoptotic response to nitric oxide (NO) exposure (via the NO donors 3-morpholine-syndnonimine (SIN1) or papa-NONOate) of human airway epithelial (HAE) cells. NO exposure via the NO donors increased cellular ceramide levels via ceramide synthase but did not trigger an apoptotic response. Rather, exposure to the NO donors promoted an increase in the protein-protein interaction between acidic sphingomyelinase (aSMase) and caspase-3, with aSMase sequestering caspase-3 and preventing its cleavage. In contrast, when aSMase was silenced in HAE cells or was knocked out in mice, an increase in cleaved caspase-3 was observed. This elevated caspase-3 cleavage was further augmented upon NO exposure (via SIN1 or papa-NONOate) of HAE cells and could be prevented by an inhibitor to ceramide synthase. These results demonstrate a novel mechanism of NO modulation of apoptosis, in which HAE cells exposed to NO via an NO donor induces ceramide generation via ceramide synthase. However, this ceramide induction does not lead to apoptosis unless aSMase is knocked down, allowing the release of caspase-3, its activation and execution of apoptosis.

nSMase2 activation and trafficking are modulated by oxidative stress to induce apoptosis.

We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H(2)O(2)), induces apoptosis of human airway epithelial (HAE) cells. Under oxidant exposure, a lung sphingomyelinase (SMase) is activated and displays continued ceramide generation and pro-apoptotic signaling, thus leading to the pathological apoptosis that causes lung injury. In a search for a specific SMase that is modulated by oxidative stress, we recently cloned nSMase2 from monkey lung tissue and HAE cells. Here, we show that this nSMase2 is up-regulated by an oxidant (H(2)O(2)) and is inhibited by an antioxidant (glutathione (GSH)). Moreover, nSMase2 subcellular localization is governed by oxidant exposure, which leads to its preferential trafficking to the plasma membrane, where it generates ceramide and induces apoptosis. On the other hand, exposure to GSH results in nSMase2 trafficking to the nucleus, where it neither generates ceramide nor induces apoptosis.

Tumor necrosis factor-{alpha} decreases Akt protein levels in 3T3-L1 adipocytes via the caspase-dependent ubiquitination of Akt.

TNF-alpha is a mediator of insulin resistance in sepsis, obesity, and type 2 diabetes and is known to impair insulin signaling in adipocytes. Akt (protein kinase B) is a crucial signaling mediator for insulin. In the present study we examined the posttranslational mechanisms by which short-term (<6-h) exposure of 3T3-L1 adipocytes to TNF-alpha decreases Akt levels. TNF-alpha treatment both increased the ubiquitination of Akt and decreased its protein level. The decrease in protein was associated with the presence of an (immunoreactive) Akt fragment after TNF-alpha treatment, indicative of Akt cleavage. The broad-spectrum caspase inhibitor t-butoxycarbonyl-Asp(O-Me)-fluoromethyl ketone markedly suppressed these effects of TNF-alpha. The caspase-6 inhibitor Z-Val-Glu(OMe)-Ile-Asp(OMe)-CH(2)F potently suppressed Akt ubiquitination, degradation, and fragment formation, whereas the proteasome inhibitor Z-Leu-Leu-Leu-CHO modestly attenuated the decline in Akt levels. Exposure to TNF-alpha also enhanced the association of Akt with an E3 ligase activity. Adipocytes preexposed to TNF-alpha for 5 h and then stimulated with insulin for 30 min exhibited decreased levels of Akt, phosphorylated Akt, as well as phosphorylated Mdm2, which is a known direct substrate of Akt, and glucose uptake. Caspase inhibition attenuated these inhibitory effects of TNF-alpha. Collectively, our results suggest that TNF-alpha induces the caspase-dependent degradation of Akt via the cleavage and ubiquitination of Akt, which results in its degradation through the 26S proteasome. Furthermore, the caspase- and proteasome-mediated degradation of Akt due to TNF-alpha exposure leads to impaired Akt-dependent insulin signaling in adipocytes. These findings expand the mechanism by which TNF-alpha impairs insulin signaling.

Tobacco components stimulate Akt-dependent proliferation and NFkappaB-dependent survival in lung cancer cells.

Retrospective studies have shown that patients with tobacco-related cancers who continue to smoke after their diagnoses have lower response rates and shorter median survival compared with patients who stop smoking. To provide insight into the biologic basis for these clinical observations, we tested whether two tobacco components, nicotine or the tobacco-specific carcinogen, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), could activate the Akt pathway and increase lung cancer cell proliferation and survival. Nicotine or NNK, rapidly and potently, activated Akt in non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) cells. Nicotinic activation of Akt increased phosphorylation of multiple downstream substrates of Akt in a time-dependent manner, including GSK-3, FKHR, tuberin, mTOR and S6K1. Since nicotine or NNK bind to cell surface nicotinic acetylcholine receptors (nAchR), we used RT-PCR to assess expression of nine alpha and three beta nAchR subunits in five NSCLC cell lines and two types of primary lung epithelial cells. NSCLC cells express multiple nAchR subunits in a cell line-specific manner. Agonists of alpha3/alpha4 or alpha7 subunits activated Akt in a time-dependent manner, suggesting that tobacco components utilize these subunits to activate Akt. Cellular outcomes after nicotine or NNK administration were also assessed. Nicotine or NNK increased proliferation of NSCLC cells in an Akt-dependent manner that was closely linked with changes in cyclin D1 expression. Despite similar induction of proliferation, only nicotine decreased apoptosis caused by serum deprivation and/or chemotherapy. Protection conferred by nicotine was NFkappaB-dependent. Collectively, these results identify tobacco component-induced, Akt-dependent proliferation and NFkappaB-dependent survival as cellular processes that could underlie the detrimental effects of smoking in cancer patients.

Preferential inhibition of Akt and killing of Akt-dependent cancer cells by rationally designed phosphatidylinositol ether lipid analogues.

Activation of the PI3k/Akt pathway controls key cellular processes and contributes to tumorigenesis in vivo, but investigation of the PI3k/Akt pathway has been limited by the lack of specific inhibitors directed against Akt. To develop Akt inhibitors, we used molecular modeling of the pleckstrin homology (PH) domain of Akt to guide synthesis of structurally modified phosphatidylinositol ether lipid analogues (PIAs). Here, we characterize the biochemical and cellular effects of PIAs. Of 24 compounds tested, five PIAs with modifications at two sites on the inositol ring inhibited Akt with IC(50)s < 5 micro M. Molecular modeling identified putative interactions of PIAs with the phosphoinositide-binding site in the PH domain of Akt, and growth factor-induced translocation of Akt to the plasma membrane was inhibited by PIA administration. Inhibition of Akt occurred rapidly and was maintained for hours. PIAs decreased phosphorylation of many downstream targets of Akt without affecting upstream kinases, such as PI3k or phosphoinositide-dependent kinase-1, or members of other kinase pathways such as extracellular signal-regulated kinase. Importantly, PIAs increased apoptosis 20-30-fold in cancer cell lines with high levels of endogenous Akt activity but only 4-5-fold in cancer cell lines with low levels of Akt activity. These studies identify PIAs as effective Akt inhibitors, and provide proof of principle for targeting the PH domain of Akt.

Sphingosine-1-phosphate inhibition of apoptosis requires mitogen-activated protein kinase phosphatase-1 in mouse fibroblast C3H10T 1/2 cells.

The roles of extracellular regulated kinase (ERK) activation and mitogen-activated protein kinase phosphatase-1 (MKP-1) were examined in sphingosine-1-phosphate (S1P)-mediated inhibition of apoptosis in C3H10T 1/2 fibroblast cells. Apoptosis induced by the ceramide analog, C8-ceramine, was inhibited by S1P (ceramine/S1P). Stress activated protein kinase or c-Jun N-terminal kinase (SAPK/JNK) activation was significantly higher after ceramine and ceramine/S1P treatments. Ceramine/S1P treatment also significantly increased ERK activation and MKP-1 protein levels. ERK activation was required for the inhibition of apoptosis by S1P as shown using the mitogen-activated protein kinase kinase inhibitor, PD98059. Transfection with a dominant negative mutant construct of the MKP-1 gene prevented S1P inhibition of apoptosis and resulted in sustained SAPK/JNK activity. The MKP-1 mutant did not affect ERK activity, indicating that MKP-1 preferentially down-regulates SAPK/JNK in C3H10T 1/2 cells. Finally, the S1P activation of ERK and inhibition of apoptosis were reduced by pertussis toxin treatment, suggesting that G-protein-coupled receptors, such as the endothelial differentiation gene (EDG) receptor, play a role. Thus, both ERK activation and MKP-1, which down-regulates SAPK/JNK, are required for S1P-mediated inhibition of apoptosis.

Immune evasion by murine melanoma mediated through CC chemokine receptor-10.

Human melanoma cells frequently express CC chemokine receptor (CCR)10, a receptor whose ligand (CCL27) is constitutively produced by keratinocytes. Compared with B16 murine melanoma, cells rendered more immunogenic via overexpression of luciferase, B16 cells that overexpressed both luciferase and CCR10 resisted host immune responses and readily formed tumors. In vitro, exposure of tumor cells to CCL27 led to rapid activation of Akt, resistance to cell death induced by melanoma antigen-specific cytotoxic T cells, and phosphatidylinositol-3-kinase (PI3K)-dependent protection from apoptosis induced by Fas cross-linking. In vivo, cutaneous injection of neutralizing antibodies to endogenous CCL27 blocked growth of CCR10-expressing melanoma cells. We propose that CCR10 engagement by locally produced CCL27 allows melanoma cells to escape host immune antitumor killing mechanisms (possibly through activation of PI3K/Akt), thereby providing a means for tumor progression.

1,25-dihydroxyvitamin D inhibits vitamin E succinate-induced apoptosis in C3H10T1/2 cells but not Harvey ras-transfected cells.

In this study, the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on regulation of apoptosis was compared in control C3H10T1/2 mouse fibroblast cells and those transfected with the Harvey ras oncogene. A known apoptotic stimulator, vitamin E succinate (VES), reduced cell number in a time- and dose-dependent manner in both cell types. In an assay for viable cells, there were significantly more C3H10T1/2 cells cotreated with VES and 1,25(OH)2D3 (-5.0 +/- 10.5% of vehicle-treated controls) compared to VES alone treated cells (-60.8 +/- 5.6%). In contrast, 1,25(OH)2D3 did not change the percentage of viable cells following treatment by VES in ras-transfected cells [-67.3 +/- 7.5%, VES alone compared to 57.3 +/-v 15.7% with VES and 1,25(OH)2D3 ]. Further studies confirmed that 1,25(OH)2D3 inhibited VES-mediated apoptosis (1.27 +/- 0.34-fold over vehicle control) compared to VES treatment alone (2.29 +/- 0.56-fold increase) in C3H10T1/2 cells, but not in ras-transfected cells [3.07 +/- 0.43-fold increase, VES treatment alone; 3.64 +/- 0.42-fold increase, VES and 1,25(OH)2D3]. Both C3H10T1/2 and ras-transfected cells treated with VES had increased concentrations of cellular VES with very little change in a-tocopherol, indicating that the cells took up VES intact. In addition, both cell lines contained similar levels of nuclear vitamin D receptor (VDR); however, the ras-transfected cells had reduced VDRE transcriptional activity. In conclusion, VES exerts its effect intact and 1,25(OH)2D3 preferentially protects C3H10T1/2 cells, whereas ras-transformed cells were not protected from VES-mediated apoptosis.

Activation of the PI3K/Akt pathway and chemotherapeutic resistance.

The resistance of many types of cancer to conventional chemotherapies is a major factor undermining successful cancer treatment. In this review, the role of a signal transduction pathway comprised of the lipid kinase, phosphatidylinositol 3-kinase (PI3K), and the serine/threonine kinase, Akt (or PKB), in chemotherapeutic resistance will be explored. Activation of this pathway plays a pivotal role in essential cellular functions such as survival, proliferation, migration and differentiation that underlie the biology of human cancer. Akt activation also contributes to tumorigenesis and tumor metastasis, and as shown most recently, resistance to chemotherapy. Modulating Akt activity is now a commonly observed endpoint of chemotherapy administration or administration of chemopreventive agents. Studies performed in vitro and in vivo combining small molecule inhibitors of the PI3K/Akt pathway with standard chemotherapy have been successful in attenuating chemotherapeutic resistance. As a result, small molecules designed to specifically target Akt and other components of the pathway are now being developed for clinical use as single agents and in combination with chemotherapy to overcome therapeutic resistance. Specifically inhibiting Akt activity may be a valid approach to treat cancer and increase the efficacy of chemotherapy.