Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.     

Phosphatidylinositol 3-Kinase/Akt Plays a Part in Tumor Necrosis Factor-alpha-induced Interleukin-6 Synthesis in Osteoblasts.

We previously showed that tumor necrosis factor-alpha (TNF-alpha) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) is involved in TNF-alpha-stimulated IL-6 synthesis in MC3T3-E1 cells. TNF-alpha induced the phosphorylation of Akt depending upon time. Akt inhibitor, 1L-6-hydroxymethyl- CHIRO-inositol 2-( R)-2- O-methyl-3- O-octadecylcarbonate, significantly suppressed the TNF-alpha-stimulated IL-6 synthesis, but the inhibitory effect was partial. The phosphorylation of Akt induced by TNF-alpha was markedly attenuated by LY294002 and wortmannin, inhibitors of PI3-kinase. Wortmannin and LY294002 significantly reduce the TNF-alpha-induced IL-6 synthesis. On the contrary, the suppressive effects of Akt inhibitor, wortmannin or LY294002 on TNF-alpha-induced phosphorylation of p44/p42 MAP kinase were minor. PD98059, a specific inhibitor of MEK, had little effect on the TNF-alpha-induced phosphorylation of Akt. A combination of Akt inhibitor and PD98059 suppressed the TNF-alpha-induced IL-6 synthesis in an additive manner. These results strongly suggest that PI3-kinase/Akt plays a role in the TNF-alpha-stimulated IL-6 synthesis mainly independent of p44/p42 MAP kinase in osteoblasts.     

TNF-alpha-induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/Akt pathway in human hepatocytes.

Human hepatocytes usually are resistant to TNF-alpha cytotoxicity. In mouse or rat hepatocytes, repression of NF-kappaB activation is sufficient to induce TNF-alpha-mediated apoptosis. However, in both Huh-7 human hepatoma cells and Hc human normal hepatocytes, when infected with an adenovirus expressing a mutated form of IkappaBalpha (Ad5IkappaB), which almost completely blocks NF-kappaB activation, >80% of the cells survived 24 h after TNF-alpha stimulation. Here, we report that TNF-alpha activates other antiapoptotic factors, such as sphingosine kinase (SphK), phosphatidylinositol 3-kinase (PI3K), and Akt kinase. Pretreatment of cells with N,N-dimethylsphingosine (DMS), an inhibitor of SphK, or LY 294002, an inhibitor of PI3K that acts upstream of Akt, increased the number of apoptotic cells induced by TNF-alpha in Ad5IkappaB-infected Huh-7 and Hc cells. TNF-alpha-induced activations of PI3K and Akt were inhibited by DMS. In contrast, exogenous sphingosine 1-phosphate, a product of SphK, was found to activate Akt and partially rescued the cells from TNF-alpha-induced apoptosis. Although Akt has been reported to activate NF-kappaB, DMS and LY 294002 failed to prevent TNF-alpha-induced NF-kappaB activation, suggesting that the antiapoptotic effects of SphK and Akt are independent of NF-kappaB. Furthermore, apoptosis mediated by Fas ligand (FasL) involving Akt activation also was potentiated by DMS pretreatment in Hc cells. Sphingosine 1-phosphate administration partially protected cells from FasL-mediated apoptosis. These results indicate that not only NF-kappaB but also SphK and PI3K/Akt are involved in the signaling pathway(s) for protection of human hepatocytes from the apoptotic action of TNF-alpha and probably FasL.     

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

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

Corneal cell survival in adenovirus type 19 infection requires phosphoinositide 3-kinase/Akt activation

Adenovirus type 19 is a major cause of epidemic keratoconjunctivitis, the only ocular adenoviral infection associated with prolonged corneal inflammation. In this study, we investigated the role of phosphoinositide 3-kinase (PI3K) and Akt and their downstream targets in adenovirus infection, and here we report the novel finding that adenovirus type 19 utilizes the PI3K/Akt pathway to maintain corneal fibroblast viability in acute infection. We demonstrate phosphorylation of GSK-3beta and nuclear translocation of the p65 subunit of NF-kappaB, both downstream targets of the PI3K/Akt pathway, in adenovirus-infected corneal fibroblasts in a PI3K-dependent manner. Inhibition of PI3K had no effect on early viral gene expression, suggesting normal viral internalization, but pretreatment with the PI3K inhibitor LY294002 or overexpression of dominant negative Akt induced early cytopathic effect and caspase-mediated cell death in adenovirus-infected cells. Early cell death could be circumvented despite LY294002 by overexpression of constitutively active Akt. Furthermore, we show an interaction between cSrc and the p85 regulatory subunit of PI3K in infected cells through a phosphorylation-dependent mechanism. The results presented in this paper provide the first direct evidence that PI3K-mediated Akt activation in adenovirus-infected corneal cells may contribute to viral pathogenesis by the prolongation of cell viability.     

TGF-beta1 represses activation and resultant death of microglia via inhibition of phosphatidylinositol 3-kinase activity

Overactivation of microglial cells may cause severe brain tissue damage in various neurodegenerative diseases. Therefore, the overactivation of microglia should be repressed by any means. The present study investigated the potential mechanism and signaling pathway for the repressive effect of TGF-beta1, a major anti-inflammatory cytokine, on overactivation and resultant death of microglial cells. A bacterial endotoxin LPS stimulated expression of inducible NO synthase (iNOS) and caused death in cultured microglial cells. TGF-beta1 markedly blocked these LPS effects. However, the LPS-evoked death of microglial cells was not solely attributed to excess production of NO. Because phosphatidylinositol 3-kinase (PI3K) was previously shown to play a crucial role in iNOS expression and cell survival signals, we further studied whether PI3K signaling was associated with the suppressive effect of TGF-beta1. Like TGF-beta1, the PI3K inhibitor LY294002 blocked iNOS expression and death in cultured microglial cells. Both TGF-beta1 and LY294002 decreased the activation of caspases 3 and 11 and the mRNA expression of various kinds of inflammatory molecules caused by LPS. TGF-beta1 was further found to decrease LPS-induced activation of PI3K and Akt. TGF-beta1 and LY294002 suppressed LPS-induced p38 mitogen-activated kinase and c-Jun N-terminal kinase activity. In contrast, TGF-beta1 and LY294002 enhanced LPS-induced NF-kappaB activity. Our data indicate that TGF-beta1 protect normal or damaged brain tissue by repressing overactivation of microglial cells via inhibition of PI3K and its downstream signaling molecules.     

Contribution of caveolin-1 alpha and Akt to TNF-alpha-induced cell death

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor-alpha (TNF-alpha)-resistant lines from L929 cells. Using this approach, we discovered that caveolin-1 alpha is required for TNF-alpha-induced cell death in L929 cells. The need for caveolin-1 alpha in TNF-alpha-induced cell death was confirmed by the restoration of sensitivity to TNF-alpha after ectopic reconstitution of caveolin-1 alpha/beta expression. This caveolin-1 alpha-mutated line was also resistant to H(2)O(2) and staurosporine, but not to lonidamine. HepG2 cells are known to lack endogenous caveolins. HepG2 cells stably transfected with caveolin-1 alpha/beta were found to be much more sensitive to TNF-alpha than either parental cells transfected with caveolin-1 beta or parental cells transfected with an empty vector. In contrast to its extensively documented antiapoptotic effect, the elevated activity of Akt appears to be important in sensitizing caveolin-1-expressing cells to TNF-alpha, since pretreatment of cells with the phosphatidylinositide 3-kinase (PI3K) inhibitor LY-294002 or wortmannin completely blocked PI3K activation and markedly improved the survival of TNF-alpha-treated L929 cells. The survival rates of caveolin-1 alpha-normal and caveolin-1 alpha-deficient L929 cells were comparable after treatment with PI3K inhibitor and TNF-alpha. Similar results were obtained with HepG2 cells that stably expressed caveolin-1 alpha/beta or -beta and parental cells transfected with an empty vector. In summary, our results indicate that caveolin-1 alpha preferentially sensitizes L929 cells to TNF-alpha through the activation of a PI3K/Akt signaling pathway.     

Inhibition of the PI3 kinase/Akt pathway enhances doxorubicin-induced apoptotic cell death in tumor cells in a p53-dependent manner

Constitutive activation of the PI3 kinase/Akt pathway is associated with the neoplastic phenotype of a large number of human tumor cells. As the anti-apoptotic role of the PI3 kinase/Akt pathway has been established, we have examined whether specific blockade of this pathway sensitizes tumor cells to DNA-damaging agent-induced cytotoxicity by enhancing apoptotic cell death. Although a PI3 kinase inhibitor, LY294002, by itself does not induce apoptotic cell death, LY294002 selectively and markedly enhances the apoptosis-inducing efficacy of doxorubicin: such an enhanced cell death is only detected in tumor cells in which the PI3 kinase/Akt pathway is constitutively activated, and it is totally dependent on the functional p53 pathway. These results suggest that the combination of a PI3 kinase/Akt pathway inhibitor and doxorubicin provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the PI3 kinase/Akt pathway is constitutively activated and the p53 pathway is functional.     

TNF-alpha induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myocytes

The activation of phosphatidylinositol (PI) 3-kinase and Akt/protein kinase B (PKB) by tumor necrosis factor (TNF)-alpha and their roles on stimulation of protein synthesis were investigated in cultured neonatal rat cardiac myocytes. Treatment of cells with TNF-alpha resulted in enlargement of cell surface area and stimulation of protein synthesis without affecting myocyte viability. TNF-alpha induced marked activation of PI3-kinase and Akt/PKB, and the activation of PI3-kinase and Akt/PKB was rapid (maximal at 10 and 15 min, respectively) and concentration dependent. Akt/PKB activation by TNF-alpha was inhibited by a PI3-kinase-specific inhibitor LY-294002 and adenovirus-mediated expression of a dominant negative mutant of PI3-kinase, indicating that TNF-alpha activates Akt/PKB through PI3-kinase activation. Furthermore, TNF-alpha-induced protein synthesis was inhibited by pretreatment with LY-294002 and expression of a dominant negative mutant of PI3-kinase or Akt/PKB. These results indicate that activation of the PI3-kinase-Akt/PKB pathway plays an essential role in protein synthesis induced by TNF-alpha in cardiac myocytes.     

Reversal of boswellic acid analog BA145 induced caspase dependent apoptosis by PI3K inhibitor LY294002 and MEK inhibitor PD98059

PI3K/Akt and ERK pathways are important for growth and proliferation of many types of cancers. Therefore, PI3K inhibitor LY294002 (LY) and MEK1/2 inhibitor PD98059 (PD) are used to sensitize many types of cancer cell lines to chemotherapeutic agents, where AKT and ERK pathways are over activated. However, in this study, we show for the first time that PD could protect the leukemia cells independent of ERK pathway inhibition, besides, we also report a detailed mechanism for antiapoptotic effect of LY in HL-60 cells against the cytotoxicity induced by a boswellic acid analog BA145. Apoptosis induced by BA145 is accompanied by downregulation of PI3K/Akt and ERK pathways in human myelogenous leukemia HL-60 cells, having activating N-Ras mutation. Both LY and PD protected the cells against mitochondrial stress caused by BA145, and reduced the release of cytochrome c and consequent activation of caspase-9. LY and PD also diminished the activation of caspase-8 without affecting the death receptors. Besides, LY and PD also reversed the caspase dependent DNA damage induced by BA145. Further studies revealed that LY and PD significantly reversed the inhibitory effect of BA145 on cell cycle regulatory proteins by upregulating hyperphosphorylated retinoblastoma, pRB (S795) and downregulating p21 and cyclin E. More importantly, all these events were reversed by caspase inhibition by Z-VAD-fmk, suggesting that both LY and PD act at the level of caspases to diminish the apoptosis induced by BA145. These results indicate that inhibitors of PI3K/Akt and ERK pathways can play dual role and act against chemotherapeutic agents.     

Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways

The aim of this study was to investigate the effects of apelin on proliferation and apoptosis of mouse osteoblastic MC3T3-E1 cells. APJ was expressed in MC3T3-E1 cells. Apelin did not affect Runx2 expression, alkaline phosphatase (ALP) activity, osteocalcin and type I collagen secretion, suggesting that it has no effect on osteoblastic differentiation of MC3T3-E1 cells. However, apelin stimulated MC3T3-E1 cell proliferation and inhibited cell apoptosis induced by serum deprivation. Our study also shows that apelin decreased cytochrome c release and caspase-3, capase-8 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Apelin activated c-Jun N-terminal kinase (JNK) and Akt (phosphatidylinositol 3-kinase downstream effector), and the JNK inhibitor SP600125, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) inhibited its effects on proliferation and serum deprivation-induced apoptosis. Furthermore, apelin protected against apoptosis induced by the glucocorticoid dexamethasone or TNF-alpha. Apelin stimulates proliferation and suppresses serum deprivation-induced apoptosis of MC3T3-E1 cells and these actions are mediated via JNK and PI3-K/Akt signaling pathways.     

Akt and XIAP regulate the sensitivity of human uterine cancer cells to cisplatin,doxorubicin and taxol

We have investigated the interrelationship between two anti-apoptotic factors, XIAP and Akt, and their role in chemoresistance of uterine cancer cells. We used one cervical cancer cell line (HeLa) and two endometrial cancer cell lines (KLE and Ishikawa) as a model. The three drugs decreased Akt and XIAP content and induced apoptosis in P-Akt-negative HeLa cells. In P-Akt1/3-positive Ishikawa cells apoptosis induction correlated with XIAP decrease. P-Akt1/2/3-positive KLE cells showed maximum chemoresistance as XIAP and Akt levels/phosphorylation remained stable in response to the three drugs, and only cisplatin could significantly induce apoptosis. We found that XIAP and Akt were functionally linked in uterine cancer cells, as downregulation of XIAP with RNAi decreased P-Akt levels, and inhibition of PI3-K/Akt activity using LY294002 decreased XIAP content. Overexpression of constitutively active Akt isoforms in HeLa cells induced isoform-specific sensitivity to doxorubicin and taxol but not cisplatin. XIAP RNAi increased the cell-specific sensitivity to cisplatin and doxorubicin but not taxol. Finally, we found P-Akt immunoreactivity in epithelial cells from multiple human endometrial carcinoma tumors, suggesting that Akt may also regulate chemosensitivity in uterine cancers in vivo. Altogether these results highlight an intertwined role for specific Akt isoforms and XIAP in chemoresistance of uterine cancer cells.     

NF-kappaB activation but not PI3K/Akt is required for dexamethasone dependent protection against TNF-alpha cytotoxicity in L929 cells

Tumor necrosis factor alpha (TNF-alpha) is one of the best-described cell death promoters. In murine L929 fibroblasts, dexamethasone inhibits TNF-alpha-induced cytotoxicity. Since phosphatidyl inositol 3 kinase (PI3K) and nuclear factor kappa B (NF-kappaB) proteins regulate several survival pathways, we evaluated their participation in dexamethasone protection against TNF-alpha cell death. We interfered with these pathways by overexpressing a negative dominant mutant of PI3K or a non-degradable mutant of inhibitor of NF-kappaB alpha (IkappaBalpha) (the cytoplasmic inhibitor of NF-kappaB) in L929 cells. The mutant IkappaB, but not the mutant PI3K, abrogated dexamethasone-mediated protection. The loss of dexamethasone protection was associated with a diminished accumulation in XIAP and c-IAP proteins.     

The phosphoinositol-3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial 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.     

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.     

Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival

The serine/threonine kinase Akt (also known as protein kinase B) is activated in response to various stimuli by a mechanism involving phosphoinositide 3-kinase (PI3-K). Akt provides a survival signal that protects cells from apoptosis induced by growth factor withdrawal, but its function in other forms of stress is less clear. Here we investigated the role of PI3-K/Akt during the cellular response to oxidant injury. H(2)O(2) treatment elevated Akt activity in multiple cell types in a time- (5-30 min) and dose (400 microM-2 mm)-dependent manner. Expression of a dominant negative mutant of p85 (regulatory component of PI3-K) and treatment with inhibitors of PI3-K (wortmannin and LY294002) prevented H(2)O(2)-induced Akt activation. Akt activation by H(2)O(2) also depended on epidermal growth factor receptor (EGFR) signaling; H(2)O(2) treatment led to EGFR phosphorylation, and inhibition of EGFR activation prevented Akt activation by H(2)O(2). As H(2)O(2) causes apoptosis of HeLa cells, we investigated whether alterations of PI3-K/Akt signaling would affect this response. Wortmannin and LY294002 treatment significantly enhanced H(2)O(2)-induced apoptosis, whereas expression of exogenous myristoylated Akt (an activated form) inhibited cell death. Constitutive expression of v-Akt likewise enhanced survival of H(2)O(2)-treated NIH3T3 cells. These results suggest that H(2)O(2) activates Akt via an EGFR/PI3-K-dependent pathway and that elevated Akt activity confers protection against oxidative stress-induced apoptosis.     

PI3K inhibition potentiates Bcl‐2‐dependent apoptosis in renal carcinoma cells

Inhibitors of PI3‐K/Akt are currently being assessed clinically in patients with advanced RCC. Identification of therapeutic strategies that might enhance the efficacy of PI3‐K/Akt inhibitors is therefore of great interest. As PI3‐K inhibition would be expected to have many pro‐apoptotic effects, we hypothesized that there may be unique synergy between PI3‐K inhibitors and BH3‐mimetics. Towards this end, we assessed the combination of the PI3K inhibitor LY 294002 and the Bcl‐2 family inhibitor ABT‐737 in RCC cell lines. We found that the combinatorial treatment with these agents led to a significant increase in PARP cleavage and cell death in all RCC cell lines. The synergized cell death was correlated with decreased levels of Mcl‐1 and XIAP, and increased levels in Bim, and appears critically dependent upon the activation of caspase 3 and 8. The enhanced lethality observed with the combination also appears dependent upon the regulation of XIAP, Mcl‐1 and Bim levels. Our results suggest that the combination of PI3‐K inhibitors with BH3‐mimetics may be a viable therapeutic strategy in RCC.