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.     

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.     

mTORC1 Regulates Flagellin-Induced Inflammatory Response in Macrophages

Bacterial flagellin triggers inflammatory responses. Phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) regulate the production of pro- and anti-inflammatory cytokines that are induced by extrinsic antigens, but the function of mTORC1 in flagellin-induced inflammatory response is unknown. The purpose of this study was to examine the role and the mechanism of PI3K/Akt/mTOR pathway in flagellin-induced cytokine expression in mouse macrophages. We observed that flagellin upregulated TNF-α time- and dose-dependently. Flagellin stimulated rapid (<15 min) PI3K/Akt/mTOR phosphorylation that was mediated by TLR5. Inhibition of PI3K with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin, and of mTORC1 with rapamycin decreased flagellin-induced TNF-α and IL-6 expression and cell proliferation. The activation of NF-κB p65 and STAT3 was regulated by mTORC1 via degradation of IκBα and phosphorylation of STAT3 in response to flagellin, respectively. Thus, the PI3K/Akt/mTORC1 pathway regulates the innate immune response to bacterial flagellin. Rapamycin is potential therapy that can regulate host defense against pathogenic infections.     

Follistatin could promote the proliferation of duck primary myoblasts by activating PI3K/Akt/mTOR signalling

FST (follistatin) is essential for skeletal muscle development, but the intracellular signalling networks that regulate FST-induced effects are not well defined. We sought to investigate whether FST promotes the proliferation of myoblasts through the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signalling. In the present study, we transfected the pEGFP-duFST plasmid and added PI3K and mTOR inhibitors to the medium of duck primary myoblasts. Then, we analysed the cellular phenotypic changes that occurred and analysed the expression of target genes. The results showed that FST promoted myoblast proliferation, induced the mRNA expression of PI3K, Akt, mTOR, 70-kDa ribosomal protein S6K (S6 kinase) and the protein expression of phospho-Akt (Thr³⁰⁸), mTOR, phospho-mTOR (serine 2448), phospho-S6K (Ser⁴¹⁷), inhibited the mRNA expression of FoxO1, MuRF1 (muscle RING finger-1) and the protein expression of phospho-FoxO1 (Ser²⁵⁶). Moreover, we found that the overexpression of FST could alleviate the inhibitory effect of myoblast proliferation caused by the addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor. Additionally, the overexpression of duck FST also relieved the inhibition of myoblast proliferation caused by the addition of rapamycin (an mTOR inhibitor) through PI3K/Akt/mTOR signalling. In light of the present results, we hypothesize that duck FST could promote myoblast proliferation, which is dependent on PI3K/Akt/mTOR signalling.     

Glimepiride Promotes Osteogenic Differentiation in Rat Osteoblasts via the PI3K/Akt/eNOS Pathway in a High Glucose Microenvironment

Our previous studies demonstrated that glimepiride enhanced the proliferation and differentiation of osteoblasts and led to activation of the PI3K/Akt pathway. Recent genetic evidence shows that endothelial nitric oxide synthase (eNOS) plays an important role in bone homeostasis. In this study, we further elucidated the roles of eNOS, PI3K and Akt in bone formation by osteoblasts induced by glimepiride in a high glucose microenvironment. We demonstrated that high glucose (16.5 mM) inhibits the osteogenic differentiation potential and proliferation of rat osteoblasts. Glimepiride activated eNOS expression in rat osteoblasts cultured with two different concentrations of glucose. High glucose-induced osteogenic differentiation was significantly enhanced by glimepiride. Down-regulation of PI3K P85 levels by treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a PI3K inhibitor) led to suppression of P-eNOS and P-AKT expression levels, which in turn resulted in inhibition of RUNX2, OCN and ALP mRNA expression in osteoblasts induced by glimepiride at both glucose concentrations. ALP activity was partially inhibited by 10 µM {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Taken together, our results demonstrate that glimepiride-induced osteogenic differentiation of osteoblasts occurs via eNOS activation and is dependent on the PI3K/Akt signaling pathway in a high glucose microenvironment.     

Hepatocyte Growth Factor Increases Vascular Endothelial Growth Factor-A Production in Human Synovial Fibroblasts through c-Met Receptor Pathway

Background

Angiogenesis is essential for the progression of osteoarthritis (OA). Hepatocyte growth factor (HGF) is an angiogenic mediator, and it shows elevated levels in regions of OA. However, the relationship between HGF and vascular endothelial growth factor (VEGF-A) in OA synovial fibroblasts (OASFs) is mostly unknown.

Methodology/Principal Findings

Here we found that stimulation of OASFs with HGF induced concentration- and time-dependent increases in VEGF-A expression. Pretreatment with PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002), Akt inhibitor, or mTORC1 inhibitor (rapamycin) blocked the HGF-induced VEGF-A production. Treatment of cells with HGF also increased PI3K, Akt, and mTORC1 phosphorylation. Furthermore, HGF increased the stability and activity of HIF-1 protein. Moreover, the use of pharmacological inhibitors or genetic inhibition revealed that c-Met, PI3K, Akt, and mTORC1 signaling pathways were potentially required for HGF-induced HIF-1α activation.

Conclusions/Significance

Taken together, our results provide evidence that HGF enhances VEGF-A expression in OASFs by an HIF-1α-dependent mechanism involving the activation of c-Met/PI3K/Akt and mTORC1 pathways.     

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.     

Odorant Stimulation Promotes Survival of Rodent Olfactory Receptor Neurons via PI3K/Akt Activation and Bcl-2 Expression

Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorant-induced Bcl-2 expression, as well as the effects on odorant-induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.     

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.     

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.     

Dexmedetomidine Reduces Isoflurane-Induced Neuroapoptosis Partly by Preserving PI3K/Akt Pathway in the Hippocampus of Neonatal Rats

Prolonged exposure to volatile anesthetics, such as isoflurane and sevoflurane, causes neurodegeneration in the developing animal brains. Recent studies showed that dexmedetomidine, a selective α2-adrenergic agonist, reduced isoflurane-induced cognitive impairment and neuroapoptosis. However, the mechanisms for the effect are not completely clear. Thus, we investigated whether exposure to isoflurane or sevoflurane at an equivalent dose for anesthesia during brain development causes different degrees of neuroapoptosis and whether this neuroapoptosis is reduced by dexmedetomidine via effects on PI3K/Akt pathway that can regulate cell survival. Seven-day-old (P7) neonatal Sprague-Dawley rats were randomly exposed to 0.75% isoflurane, 1.2% sevoflurane or air for 6 h. Activated caspase-3 was detected by immunohistochemistry and Western blotting. Phospho-Akt, phospho-Bad, Akt, Bad and Bcl-xL proteins were detected by Western blotting in the hippocampus at the end of exposure. Also, P7 rats were pretreated with various concentrations of dexmedetomidine alone or together with PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, and then exposed to 0.75% isoflurane. Terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 were used to detect neuronal apoptosis in their hippocampus. Isoflurane, not sevoflurane at the equivalent dose, induced significant neuroapoptosis, decreased the levels of phospho-Akt and phospho-Bad proteins, increased the expression of Bad protein and reduced the ratio of Bcl-xL/Bad in the hippocampus. Dexmedetomidine pretreatment dose-dependently inhibited isoflurane-induced neuroapoptosis and restored protein expression of phospho-Akt and Bad as well as the Bcl-xL/Bad ratio induced by isoflurane. Pretreatment with single dose of 75 µg/kg dexmedetomidine provided a protective effect similar to that with three doses of 25 µg/kg dexmedetomidine. Moreover, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, partly inhibited neuroprotection of dexmedetomidine. Our results suggest that dexmedetomidine pretreatment provides neuroprotection against isoflurane-induced neuroapoptosis in the hippocampus of neonatal rats by preserving PI3K/Akt pathway activity.     

Heat shock protein 90β inhibits apoptosis of intestinal epithelial cells induced by hypoxia through stabilizing phosphorylated Akt

Intestinal epithelial cell (IEC) apoptosis induced by hypoxia compromise intestinal epithelium barrier function. Both Akt and Hsp90 have cytoprotective function. However, the specific role of Akt and Hsp90β in IEC apoptosis induced by hypoxia has not been explored. We confirmed that hypoxia-induced apoptosis was reduced by Hsp90β overexpression but enhanced by decreasing Hsp90β expression. Hsp90β overexpression enhanced BAD phosphorylation and thus reduced mitochondrial release of cytochrome C. Reducing Hsp90β expression had opposite effects. The protective effect of Hsp90β against apoptosis was negated by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, an Akt inhibitor. Further study showed that Akt phosphorylation was enhanced by Hsp90β, which was not due to the activation of upstream PI3K and PDK1 but because of stabilization of pAkt via direct interaction between Hsp90β and pAkt. These results demonstrate that Hsp90β may play a significant role in protecting IECs from hypoxia-induced apoptosis via stabilizing pAkt to phosphorylate BAD and reduce cytochrome C release. [BMB Reports 2013;46(1): 47-52]     

Activation of the PI3K/mTOR Pathway Is Involved in Cystic Proliferation of Cholangiocytes of the PCK Rat

The polycystic kidney (PCK) rat is an animal model of Caroli’s disease as well as autosomal recessive polycystic kidney disease (ARPKD). The signaling pathways involving the mammalian target of rapamycin (mTOR) are aberrantly activated in ARPKD. This study investigated the effects of inhibitors for the cell signaling pathways including mTOR on cholangiocyte proliferation of the PCK rat. Cultured PCK cholangiocytes were treated with rapamycin and everolimus [inhibitors of mTOR complex 1 (mTOC1)], {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 [an inhibitor of phosphatidylinositol 3-kinase (PI3K)] and NVP-BEZ235 (an inhibitor of PI3K and mTORC1/2), and the cell proliferative activity was determined in relation to autophagy and apoptosis. The expression of phosphorylated (p)-mTOR, p-Akt, and PI3K was increased in PCK cholangiocytes compared to normal cholangiocytes. All inhibitors significantly inhibited the cell proliferative activity of PCK cholangiocytes, where NVP-BEZ235 had the most prominent effect. NVP-BEZ235, but not rapamycin and everolimus, further inhibited biliary cyst formation in the three-dimensional cell culture system. Rapamycin and everolimus induced apoptosis in PCK cholangiocytes, whereas NVP-BEZ235 inhibited cholangiocyte apoptosis. Notably, the autophagic response was significantly induced following the treatment with NVP-BEZ235, but not rapamycin and everolimus. Inhibition of autophagy using siRNA against protein-light chain3 and 3-methyladenine significantly increased the cell proliferative activity of PCK cholangiocytes treated with NVP-BEZ235. In vivo, treatment of the PCK rat with NVP-BEZ235 attenuated cystic dilatation of the intrahepatic bile ducts, whereas renal cyst development was unaffected. These results suggest that the aberrant activation of the PI3K/mTOR pathway is involved in cystic proliferation of cholangiocytes of the PCK rat, and inhibition of the pathway can reduce cholangiocyte proliferation via the mechanism involving apoptosis and/or autophagy.     

Varicella-Zoster Virus ORF12 Protein Activates the Phosphatidylinositol 3-Kinase/Akt Pathway To Regulate Cell Cycle Progression

Varicella-zoster virus (VZV) activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and alters cell cycle progression, but the viral protein(s) responsible for these activities is unknown. We previously reported that the VZV open reading frame 12 (ORF12) protein triggers phosphorylation of ERK. Here, we demonstrate that the VZV ORF12 protein also activates the PI3K/Akt pathway to regulate cell cycle progression. Transfection of cells with a plasmid expressing the ORF12 protein induced phosphorylation of Akt, which was dependent on PI3K. Infection of cells with wild-type VZV triggered phosphorylation of Akt, while infection with an ORF12 deletion mutant induced less phosphorylated Akt. The activation of Akt by ORF12 protein was associated with its binding to the p85 subunit of PI3K. Infection of cells with wild-type VZV resulted in increased levels of cyclin B1, cyclin D3, and phosphorylated glycogen synthase kinase 3β (GSK-3β), while infection with the ORF12 deletion mutant induced lower levels of these proteins. Wild-type VZV infection reduced the G₁ phase cell population and increased the M phase cell population, while infection with the ORF12 deletion mutant had a reduced effect on the G₁ and M phase populations. Inhibition of Akt activity with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 reduced the G₁ and M phase differences observed in cells infected with wild-type and ORF12 mutant viruses. In conclusion, we have found that the VZV ORF12 protein activates the PI3K/Akt pathway to regulate cell cycle progression. Since VZV replicates in both dividing (e.g., keratinocytes) and nondividing (neurons) cells, the ability of the VZV ORF12 protein to regulate the cell cycle is likely important for VZV replication in various cell types in the body.     

The Cytoplasmic Tail of FPC Antagonizes the Full-Length Protein in the Regulation of mTOR Pathway

FPC (fibrocystin or polyductin) is a single transmembrane receptor-like protein, responsible for the human autosomal recessive polycystic kidney disease (ARPKD). It was recently proposed that FPC undergoes a Notch-like cleavage and subsequently the cleaved carboxy(C)-terminal fragment translocates to the nucleus. To study the functions of the isolated C-tail, we expressed the intracellular domain of human FPC (hICD) in renal epithelial cells. By 3-dimensional (3D) tubulogenesis assay, we found that in contrast to tubule-like structures formed from control cells, hICD-expressing cells exclusively formed cyst-like structures. By western blotting, we showed that the Akt/mTOR pathway, indicated by increased phosphorylation of Akt at serine 473 and S6 kinase 1 at threonine 389, was constitutively activated in hICD-expressing cells, similar to that in FPC knockdown cells and ARPKD kidneys. Moreover, application of mTOR inhibitor rapamycin reduced the size of the cyst-like structures formed by hICD-expressing cells. Application of either {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or wortmannin inhibited the activation of both S6K1 and Akt. Expression of full-length FPC inhibited the activation of S6 and S6 kinase whereas co-expression of hICD with full-length FPC antagonized the inhibitory effect of full-length FPC on mTOR. Taken together, we propose that FPC modulates the PI3K/Akt/mTOR pathway and the cleaved C-tail regulates the function of the full-length protein.     

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.     

Activation of the Akt Survival Pathway Contributes to TRAIL Resistance in Cancer Cells

The mechanism of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance in cancer cells is not fully understood. Here, we show that the Akt survival pathway plays an important role in TRAIL resistance in human cancer cells. Specifically, we found that TRAIL treatment activates the Akt survival pathway and that inhibition of this pathway by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or knockdown of Akt sensitizes resistant cancer cells to TRAIL. Since Akt is negatively regulated by the tumor suppressor PTEN, we examined the TRAIL sensitivity in PTEN knockdown mouse prostate epithelial cells and found that PTEN^−/− cells are more resistant than PTEN^+/+ cells while the sensitivity of PTEN^+/− cells fell in between. Further, we showed that overexpression of a mutant PTEN confers TRAIL resistance in PTEN^+/+ cells, supporting a role of PTEN in TRAIL sensitivity. In TRAIL resistant breast T47D cells, overexpression of the mutant PTEN further increased their resistance to TRAIL. Taken together, our data indicate that inactivation of functional PTEN and the consequent activation of the Akt pathway prevents TRAIL-induced apoptosis, leading to TRAIL resistance. Therefore, our results suggest that TRAIL resistance can be overcome by targeting PTEN or the Akt survival pathway in cancer cells.     

Nitrogen-containing bisphosphonates inhibit RANKL- and M-CSF-induced osteoclast formation through the inhibition of ERK1/2 and Akt activation

Background

Bisphosphonates are an important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Recent studies have shown that nitrogen-containing bisphosphonates induced apoptosis in rabbit osteoclasts and prevented prenylated small GTPase. However, whether bisphosphonates inhibit osteoclast formation has not been determined. In the present study, we investigated the inhibitory effect of minodronate and alendronate on the osteoclast formation and clarified the mechanism involved in a mouse macrophage-like cell lines C7 and RAW264.7.

Results

It was found that minodronate and alendronate inhibited the osteoclast formation of C7 cells induced by receptor activator of NF-κB ligand and macrophage colony stimulating factor, which are inhibited by the suppression of geranylgeranyl pyrophosphate (GGPP) biosynthesis. It was also found that minodronate and alendronate inhibited the osteoclast formation of RAW264.7 cells induced by receptor activator of NF-κB ligand. Furthermore, minodronate and alendornate decreased phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; similarly, U0126, a mitogen protein kinase kinase 1/2 (MEK1/2) inhibitor, and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited osteoclast formation.

Conclusions

This indicates that minodronate and alendronate inhibit GGPP biosynthesis in the mevalonate pathway and then signal transduction in the MEK/ERK and PI3K/Akt pathways, thereby inhibiting osteoclast formation. These results suggest a novel effect of bisphosphonates that could be effective in the treatment of bone metabolic diseases, such as osteoporosis.