Multi-level targeting of the phosphatidylinositol-3-kinase pathway in non-small cell lung cancer cells

Introduction

We assessed expression of p85 and p110α PI3K subunits in non-small cell lung cancer (NSCLC) specimens and the association with mTOR expression, and studied effects of targeting the PI3K/AKT/mTOR pathway in NSCLC cell lines.

Methods

Using Automated Quantitative Analysis we quantified expression of PI3K subunits in two cohorts of 190 and 168 NSCLC specimens and correlated it with mTOR expression. We studied effects of two PI3K inhibitors, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and NVP-BKM120, alone and in combination with rapamycin in 6 NSCLC cell lines. We assessed activity of a dual PI3K/mTOR inhibitor, NVP-BEZ235 alone and with an EGFR inhibitor.

Results

p85 and p110α tend to be co-expressed (p<0.001); p85 expression was higher in adenocarcinomas than squamous cell carcinomas. High p85 expression was associated with advanced stage and poor survival. p110α expression correlated with mTOR (ρ = 0.276). In six NSCLC cell lines, addition of rapamycin to {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or NVP-BKM120 was synergistic. Even very low rapamycin concentrations (1 nM) resulted in sensitization to PI3K inhibitors. NVP-BEZ235 was highly active in NSCLC cell lines with IC₅₀s in the nanomolar range and resultant down-regulation of pAKT and pP70S6K. Adding Erlotinib to NVP-BEZ235 resulted in synergistic growth inhibition.

Conclusions

The association between PI3K expression, advanced stage and survival in NSCLC suggests that it might be a valuable drug target. Concurrent inhibition of PI3K and mTOR is synergistic in vitro, and a dual PI3K/mTOR inhibitor was highly active. Adding EGFR inhibition resulted in further growth inhibition. Targeting the PI3K/AKT/mTOR pathway at multiple levels should be tested in clinical trials for NSCLC.     

Effects of Bone Marrow-Derived Mesenchymal Stem Cells on the Axonal Outgrowth through Activation of PI3K/AKT Signaling in Primary Cortical Neurons Followed Oxygen-Glucose Deprivation Injury

Background

Transplantation with bone marrow-derived mesenchymal stem cells (BMSCs) improves the survival of neurons and axonal outgrowth after stroke remains undetermined. Here, we investigated whether PI3K/AKT signaling pathway is involved in these therapeutic effects of BMSCs.

Methodology/Principal Findings

(1) BMSCs and cortical neurons were derived from Sprague-Dawley rats. The injured neurons were induced by Oxygen–Glucose Deprivation (OGD), and then were respectively co-cultured for 48 hours with BMSCs at different densities (5×10³, 5×10⁵/ml) in transwell co-culture system. The average length of axon and expression of GAP-43 were examined to assess the effect of BMSCs on axonal outgrowth after the damage of neurons induced by OGD. (2) The injured neurons were cultured with a conditioned medium (CM) of BMSCs cultured for 24 hours in neurobasal medium. During the process, we further identified whether PI3K/AKT signaling pathway is involved through the adjunction of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a specific phosphatidylinositide-3-kinase (PI3K) inhibitor). Two hours later, the expression of pAKT (phosphorylated AKT) and AKT were analyzed by Western blotting. The length of axons, the expression of GAP-43 and the survival of neurons were measured at 48 hours.

Results

Both BMSCs and CM from BMSCs inreased the axonal length and GAP-43 expression in OGD-injured cortical neurons. There was no difference between the effects of BMSCs of 5×10⁵/ml and of 5×10³/ml on axonal outgrowth. Expression of pAKT enhanced significantly at 2 hours and the neuron survival increased at 48 hours after the injured neurons cultured with the CM, respectively. These effects of CM were prevented by inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusions/Significance

BMSCs promote axonal outgrowth and the survival of neurons against the damage from OGD in vitro by the paracrine effects through PI3K/AKT signaling pathway.     

Co-Depletion of Cathepsin B and uPAR Induces G0/G1 Arrest in Glioma via FOXO3a Mediated p27Kip1 Upregulation

Background

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined.

Methodology/Principal Findings

Cathespin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27^Kip1 and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by αVβ3/PI3K/AKT/FOXO pathway as observed by the decreased αVβ3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27^Kip1 and FOXO3a when treated with {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 (10 µM) and increased luciferase expression with the siRNA and {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 treatments when the FOXO binding promoter region of p27^Kip1 was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly.

Conclusion/Significance

Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27^Kip1 accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.     

Ursolic Acid Attenuates Diabetic Mesangial Cell Injury through the Up-Regulation of Autophagy via miRNA-21/PTEN/Akt/mTOR Suppression

ObjectiveTo investigate the effect of ursolic acid on autophagy mediated through the miRNA-21-targeted phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in rat mesangial cells cultured under high glucose (HG) conditions.MethodsRat glomerular mesangial cells were cultured under normal glucose, HG, HG with the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or HG with ursolic acid conditions. Cell proliferation and hypertrophy were assayed using an MTT assay and the ratio of total protein to cell number, respectively. The miRNA-21 expression was detected using RT-qPCR. The expression of phosphatase and tensin homolog (PTEN)/AKT/mTOR signaling signatures, autophagy-associated protein and collagen I was detected by western blotting and RT-qPCR. Autophagosomes were observed using electron microscopy.ResultsCompared with mesangial cells cultured under normal glucose conditions, the cells exposed to HG showed up-regulated miRNA-21 expression, down-regulated PTEN protein and mRNA expression, up-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and down-regulated LC3II expression. Ursolic acid and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 inhibited HG-induced mesangial cell hypertrophy and proliferation, down-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and up-regulated LC3II expression. However, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 did not affect the expression of miRNA-21 and PTEN. Ursolic acid down-regulated miRNA-21 expression and up-regulated PTEN protein and mRNA expression.ConclusionsUrsolic acid inhibits the glucose-induced up-regulation of mesangial cell miRNA-21 expression, up-regulates PTEN expression, inhibits the activation of PI3K/Akt/mTOR signaling pathway, and enhances autophagy to reduce the accumulation of the extracellular matrix and ameliorate cell hypertrophy and proliferation.     

Resveratrol Reduces Prostate Cancer Growth and Metastasis by Inhibiting the Akt/MicroRNA-21 Pathway

The consumption of foods containing resveratrol produces significant health benefits. Resveratrol inhibits cancer by reducing cell proliferation and metastasis and by inducing apoptosis. These actions could be explained by its ability to inhibit (ERK-1/2), Akt and suppressing the levels of estrogen and insulin growth factor -1 (IGF-1) receptor. How these processes are manifested into the antitumor actions of resveratrol is not clear. Using microarray studies, we show that resveratrol reduced the expression of various prostate-tumor associated microRNAs (miRs) including miR-21 in androgen-receptor negative and highly aggressive human prostate cancer cells, PC-3M-MM2. This effect of resveratrol was associated with reduced cell viability, migration and invasiveness. Additionally, resveratrol increased the expression of tumor suppressors, PDCD4 and maspin, which are negatively regulated by miR-21. Short interfering (si) RNA against PDCD4 attenuated resveratrol’s effect on prostate cancer cells, and similar effects were observed following over expression of miR-21 with pre-miR-21 oligonucleotides. PC-3M-MM2 cells also exhibited high levels of phospho-Akt (pAkt), which were reduced by both resveratrol and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a PI3-kinase inhibitor). MiR-21 expression in these cells appeared to be dependent on Akt, as {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 reduced the levels of miR-21 along with a concurrent increase in PDCD4 expression. These in vitro findings were further corroborated in a severe combined immunodeficient (SCID) mouse xenograft model of prostate cancer. Oral administration of resveratrol not only inhibited the tumor growth but also decreased the incidence and number of metastatic lung lesions. These tumor- and metastatic-suppressive effects of resveratrol were associated with reduced miR-21 and pAkt, and elevated PDCD4 levels. Similar anti-tumor effects of resveratrol were observed in DU145 and LNCaP prostate cancer cells which were associated with suppression of Akt and PDCD4, but independent of miR-21.These data suggest that resveratrol’s anti-tumor actions in prostate cancer could be explained, in part, through inhibition of Akt/miR-21 signaling pathway.     

Inhibition of PI3K-Akt Signaling Blocks Exercise-Mediated Enhancement of Adult Neurogenesis and Synaptic Plasticity in the Dentate Gyrus

Background

Physical exercise has been shown to increase adult neurogenesis in the dentate gyrus and enhances synaptic plasticity. The antiapoptotic kinase, Akt has also been shown to be phosphorylated following voluntary exercise; however, it remains unknown whether the PI3K-Akt signaling pathway is involved in exercise-induced neurogenesis and the associated facilitation of synaptic plasticity in the dentate gyrus.

Methodology/Principal Findings

To gain insight into the potential role of this signaling pathway in exercise-induced neurogenesis and LTP in the dentate gyrus rats were infused with the PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or vehicle control solution (icv) via osmotic minipumps and exercised in a running wheel for 10 days. Newborn cells in the dentate gyrus were date-labelled with BrdU on the last 3 days of exercise. Then, they were either returned to the home cage for 2 weeks to assess exercise-induced LTP and neurogenesis in the dentate gyrus, or were killed on the last day of exercise to assess proliferation and activation of the PI3K-Akt cascade using western blotting.

Conclusions/Significance

Exercise increases cell proliferation and promotes survival of adult-born neurons in the dentate gyrus. Immediately after exercise, we found that Akt and three downstream targets, BAD, GSK3β and FOXO1 were activated. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 blocked exercise-induced phosphorylation of Akt and downstream target proteins. This had no effect on exercise-induced cell proliferation, but it abolished most of the beneficial effect of exercise on the survival of newly generated dentate gyrus neurons and prevented exercise-induced increase in dentate gyrus LTP. These results suggest that activation of the PI3 kinase-Akt signaling pathway plays a significant role via an antiapoptotic function in promoting survival of newly formed granule cells generated during exercise and the associated increase in synaptic plasticity in the dentate gyrus.     

Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

Background

Traumatic brain injury (TBI) induces a complex sequence of apopototic cascades that contribute to secondary tissue damage. The aim of this study was to investigate the effects of salidroside, a phenolic glycoside with potent anti-apoptotic properties, on behavioral and histological outcomes, brain edema, and apoptosis following experimental TBI and the possible involvement of the phosphoinositide 3-kinase/protein kinase B (PI3K)/Akt signaling pathway.

Methodology/Principal Findings

Mice subjected to controlled cortical impact injury received intraperitoneal salidroside (20, or 50 mg/kg) or vehicle injection 10 min after injury. Behavioral studies, histology analysis and brain water content assessment were performed. Levels of PI3K/Akt signaling-related molecules, apoptosis-related proteins, cytochrome C (CytoC), and Smac/DIABLO were also analyzed. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, was administered to examine the mechanism of protection. The protective effect of salidroside was also investigated in primary cultured neurons subjected to stretch injury. Treatment with 20 mg/kg salidroside_significantly improved functional recovery and reduced brain tissue damage up to post-injury day 28. Salidroside_also significantly reduced neuronal death, apoptosis, and brain edema at day 1. These changes were associated with significant decreases in cleaved caspase-3, CytoC, and Smac/DIABLO at days 1 and 3. Salidroside increased phosphorylation of Akt on Ser473 and the mitochondrial Bcl-2/Bax ratio at day 1, and enhanced phosphorylation of Akt on Thr308 at day 3. This beneficial effect was abolished by pre-injection of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Moreover, delayed administration of salidroside at 3 or 6 h post-injury reduced neuronal damage at day 1. Salidroside treatment also decreased neuronal vulnerability to stretch-induced injury in vitro.

Conclusions/Significance

Post-injury salidroside improved long-term behavioral and histological outcomes and reduced brain edema and apoptosis following TBI, at least partially via the PI3K/Akt signaling pathway.     

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.     

Hypoxic Conditioned Medium from Rat Cerebral Cortical Cells Enhances the Proliferation and Differentiation of Neural Stem Cells Mainly through PI3-K/Akt Pathways

Purpose

To investigate the effects of hypoxic conditioned media from rat cerebral cortical cells on the proliferation and differentiation of neural stem cells (NSCs) in vitro, and to study the roles of PI3-K/Akt and JNK signal transduction pathways in these processes.

Methods

Cerebral cortical cells from neonatal Sprague–Dawley rat were cultured under hypoxic and normoxic conditions; the supernatant was collected and named ‘hypoxic conditioned medium’ (HCM) and ‘normoxic conditioned medium’ (NCM), respectively. We detected the protein levels (by ELISA) of VEGF and BDNF in the conditioned media and mRNA levels (by RT-PCR) in cerebral cortical cells. The proliferation (number and size of neurospheres) and differentiation (proportion of neurons and astrocytes over total cells) of NSCs was assessed. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and SP600125, inhibitors of PI3-K/Akt and JNK, respectively, were applied, and the phosphorylation levels of PI3-K, Akt and JNK were measured by western blot.

Results

The protein levels and mRNA expressions of VEGF and BDNF in 4% HCM and 1% HCM were both higher than that of those in NCM. The efficiency and speed of NSCs proliferation was enhanced in 4% HCM compared with 1% HCM. The highest percentage of neurons and lowest percentage of astrocytes was found in 4% HCM. However, the enhancement of NSCs proliferation and differentiation into neurons accelerated by 4% HCM was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and SP600125, with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 having a stronger inhibitory effect. The increased phosphorylation levels of PI3-K, Akt and JNK in 4% HCM were blocked by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and SP600125.

Conclusions

4%HCM could promote NSCs proliferation and differentiation into high percentage of neurons, these processes may be mainly through PI3-K/Akt pathways.     

Phosphatidylinositol 3-Kinase and Rab5 GTPase Inversely Regulate the Smad Anchor for Receptor Activation (SARA) Protein Independently of Transforming Growth Factor-β1

SARA has been shown to be a regulator of epithelial cell phenotype, with reduced expression during TGF-β1-mediated epithelial-to-mesenchymal transition. Examination of the pathways that might play a role in regulating SARA expression identified phosphatidylinositol 3-kinase (PI3K) pathway inhibition as sufficient to reduce SARA expression. The mechanism of PI3K inhibition-mediated SARA down-regulation differs from that induced by TGF-β1 in that, unlike TGF-β1, PI3K-dependent depletion of SARA was apparent within 6 h and did not occur at the mRNA or promoter level but was blocked by inhibition of proteasome-mediated degradation. This effect was independent of Akt activity because neither reducing nor enhancing Akt activity modulated the expression of SARA. Therefore, this is likely a direct effect of p85α action, and co-immunoprecipitation of SARA and p85α confirmed that these proteins interact. Both SARA and PI3K have been shown to be associated with endosomes, and either {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or p85α knockdown enlarged SARA-containing endocytic vesicles. Inhibition of clathrin-mediated endocytosis blocked SARA down-regulation, and a localization-deficient mutant SARA was protected against down-regulation. As inhibiting PI3K can activate the endosomal fusion-regulatory small GTPase Rab5, we expressed GTPase-deficient Rab5 and observed endosomal enlargement and reduced SARA protein expression, similar to that seen with PI3K inhibition. Importantly, either interference with PI3K via {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or p85α knockdown, or constitutive activity of the Rab5 pathway, enhanced the expression of smooth muscle α-actin. Together, these data suggest that although TGF-β1 can induce epithelial-to-mesenchymal transition through reduction in SARA expression, SARA is also basally regulated by its interaction with PI3K.     

Macrophage-Activating Lipopeptide-2 Requires Mal and PI3K for Efficient Induction of Heme Oxygenase-1

Aims

This study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes.

Methods

Human monocytic THP-1 cells were cultured for transient transfection with plasmids and stimulation with MALP-2 for indicative time intervals. After incubation with MALP-2, cells were collected and disrupted, before being tested for promoter activity using luciferase assay. For analysis of proteins, immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system, and the band intensity was measured by densitometryic analysis. For the detection of co-immunoprecipitation, SDS-PAGE was performed and the membranes were probed using respective antibodies. To investigate the cellular localization of NF-E2-related factor 2 (Nrf2), cells underwent immunofluorescence staining and confocal microscopy, and were analyzed using electrophoretic mobility shift assay.

Results

MALP-2-induced HO-1 expression and promoter activity were abrogated by transfection with dominant negative (DN) plasmids of TLR2 and TLR6, or their neutralizing antibodies. However, inhibition of MyD88 or transfection with the DN-MyD88 was insufficient to attenuate HO-1 expression. In contrast, mutation or silencing of MyD88 adapter-like (Mal) by DN-Mal or siRNA almost completely blocked HO-1 induction. Btk, c-Src and PI3K were also involved in MALP-2-induced HO-1 expression, as revealed by specific inhibitors LFM-A13, PP1 and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, or by transfection with siRNA of c-Src. MALP-2-induced activation of PI3K was attenuated by transfection with DN mutant of Mal, and by pretreatment with LFM-A13 or PP1. Furthermore, MALP-2 stimulated the translocation of Nrf2 from the cytosol to the nucleus and Nrf2 binding to the ARE site in the HO-1 promoter, which could also be inhibited by pretreatment with a PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusions

These results indicated that MALP-2 required TLR2/6, Btk, Mal and c-Src to activate PI3K, which in turn initiated the activation of Nrf2 for efficient HO-1 induction.     

TRAIL-R4 promotes tumor growth and resistance to apoptosis in cervical carcinoma HeLa cells through AKT

Background

TRAIL/Apo2L is a pro-apoptotic ligand of the TNF family that engages the apoptotic machinery through two pro-apoptotic receptors, TRAIL-R1 and TRAIL-R2. This cell death program is tightly controlled by two antagonistic receptors, TRAIL-R3 and TRAIL-R4, both devoid of a functional death domain, an intracellular region of the receptor, required for the recruitment and the activation of initiator caspases. Upon TRAIL-binding, TRAIL-R4 forms a heteromeric complex with the agonistic receptor TRAIL-R2 leading to reduced caspase-8 activation and apoptosis.

Methodology/Principal Findings

We provide evidence that TRAIL-R4 can also exhibit, in a ligand independent manner, signaling properties in the cervical carcinoma cell line HeLa, through Akt. Ectopic expression of TRAIL-R4 in HeLa cells induced morphological changes, with cell rounding, loss of adherence and markedly enhanced cell proliferation in vitro and tumor growth in vivo. Disruption of the PI3K/Akt pathway using the pharmacological inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, siRNA targeting the p85 regulatory subunit of phosphatidylinositol-3 kinase, or by PTEN over-expression, partially restored TRAIL-mediated apoptosis in these cells. Moreover, the Akt inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, restituted normal cell proliferation index in HeLa cells expressing TRAIL-R4.

Conclusions/Significance

Altogether, these results indicate that, besides its ability to directly inhibit TRAIL-induced cell death at the membrane, TRAIL-R4 can also trigger the activation of signaling pathways leading to cell survival and proliferation in HeLa cells. Our findings raise the possibility that TRAIL-R4 may contribute to cervical carcinogenesis.     

Inhibition of Protein Synthesis Alters Protein Degradation through Activation of Protein Kinase B (AKT)

The homeostasis of protein metabolism is maintained and regulated by the rates of protein biosynthesis and degradation in living systems. Alterations of protein degradation may regulate protein biosynthesis through a feedback mechanism. Whether a change in protein biosynthesis modulates protein degradation has not been reported. In this study, we found that inhibition of protein biosynthesis induced phosphorylation/activation of AKT and led to phosphorylation of AKT target substrates, including FoxO1, GSK3α/β, p70S6K, AS160, and the E3 ubiquitin ligase MDM2. Phosphorylation of ribosomal protein S6 was also modulated by inhibition of protein biosynthesis. The AKT phosphorylation/activation was mediated mainly through the PI3K pathway because it was blocked by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. The activated AKT phosphorylated MDM2 at Ser¹⁶⁶ and promoted degradation of the tumor suppressor p53. These findings suggest that inhibition of protein biosynthesis can alter degradation of some proteins through activation of AKT. This study reveals a novel regulation of protein degradation and calls for caution in blocking protein biosynthesis to study the half-life of proteins.     

Sensitization of TRAIL-resistant LNCaP cells by resveratrol (3, 4', 5 tri-hydroxystilbene): molecular mechanisms and therapeutic potential

Background

We have previously shown that prostate cancer LNCaP cells are resistant to TRAIL, and downregulation of PI-3K/Akt pathway by molecular and pharmacological means sensitizes cells to undergo apoptosis by TRAIL and curcumin. The purpose of this study was to examine the molecular mechanisms by which resveratrol sensitized TRAIL-resistant LNCaP cells.

Results

Resveratrol inhibited growth and induced apoptosis in androgen-dependent LNCaP cells, but had no effect on normal human prostate epithelial cells. Resveratrol upregulated the expression of Bax, Bak, PUMA, Noxa, Bim, TRAIL-R1/DR4 and TRAIL-R2/DR5, and downregulated the expression of Bcl-2, Bcl-X_L, survivin and XIAP. Treatment of LNCaP cells with resveratrol resulted in generation of reactive oxygen species, translocation of Bax and p53 to mitochondria, subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, AIF, Smac/DIABLO and Omi/HtrA2), activation of caspase-3 and caspase-9 and induction of apoptosis. The ability of resveratrol to sensitize TRAIL-resistant LNCaP cells was inhibited by dominant negative FADD, caspase-8 siRNA or N-acetyl cysteine. Smac siRNA inhibited resveratrol-induced apoptosis, whereas Smac N7 peptide induced apoptosis and enhanced the effectiveness of resveratrol.

Conclusion

Resveratrol either alone or in combination with TRAIL or Smac can be used for the prevention and/or treatment of human prostate cancer.     

Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts: ACTIONS ON CYTOSKELETAL ORGANIZATION,SURVIVAL, AND RESORPTION*

Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (β), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15–20 min to 65–75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kβ and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.     

CEACAM6 Promotes Gastric Cancer Invasion and Metastasis by Inducing Epithelial-Mesenchymal Transition via PI3K/AKT Signaling Pathway

Overexpressed CEACAM6 in tumor tissues plays important roles in invasion, metastasis and anoikis resistance in a variety of human cancers. We recently reported that CEACAM6 expression is upregulated in Gastric cancer (GC) tissues and promoted GC metastasis. Here, we report that CEACAM6 promotes peritoneal metastases in vivo and is negatively correlated with E-cadherin expression in GC tissues. Overexpressed CEACAM6 induced epithelial-mesenchymal transition (EMT) in GC, as measured by increases in the EMT markers N-cadherin, Vimentin and Slug while E-cadherin expression was decreased in CEACAM6-overexpressing GC cells; opposing results were observed in CEACAM6-silenced cells. Furthermore, E-cadherin expression was negatively correlated with depth of tumor invasion, lymph node metastasis and TNM stage in GC tissues. Additionally, CEACAM6 elevated matrix metalloproteinase-9 (MMP-9) activity in GC, and anti-MMP-9 antibody could reverse the increasing invasion and migration induced by CEACAM6. CEACAM6 also increased the levels of phosphorylated AKT, which is involved in the progression of a variety of human tumors. We further observed that {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, could reverse CEACAM6-induced EMT via mesenchymal-epithelial transition. These findings suggest that CEACAM6 enhances invasion and metastasis in GC by promoting EMT via the PI3K/AKT signaling pathway.