Elevated AKT activity protects the prostate cancer cell line LNCaP from TRAIL-induced apoptosis

We find that the prostate cancer cell lines ALVA-31, PC-3, and DU 145 are highly sensitive to apoptosis induced by TRAIL (tumor-necrosis factor-related apoptosis-inducing ligand), while the cell lines TSU-Pr1 and JCA-1 are moderately sensitive, and the LNCaP cell line is resistant. LNCaP cells lack active lipid phosphatase PTEN, a negative regulator of the phosphatidylinositol (PI) 3-kinase/Akt pathway, and demonstrate a high constitutive Akt activity. Inhibition of PI 3-kinase using wortmannin and LY-294002 suppressed constitutive Akt activity and sensitized LNCaP cells to TRAIL. Treatment of LNCaP cells with TRAIL alone induced cleavage of the caspase 8 and XIAP proteins. However, processing of BID, mitochondrial release of cytochrome c, activation of caspases 7 and 9, and apoptosis did not occur unless TRAIL was combined with either wortmannin, LY-294002, or cycloheximide. Blocking cytochrome c release by Bcl-2 overexpression rendered LNCaP cells resistant to TRAIL plus wortmannin treatment but did not affect caspase 8 or BID processing. This indicates that in these cells mitochondria are required for the propagation rather than the initiation of the apoptotic cascade. Infection of LNCaP cells with an adenovirus expressing a constitutively active Akt reversed the ability of wortmannin to potentiate TRAIL-induced BID cleavage. Thus, the PI 3-kinase-dependent blockage of TRAIL-induced apoptosis in LNCaP cells appears to be mediated by Akt through the inhibition of BID cleavage.     

hPEBP4 resists TRAIL-induced apoptosis of human prostate cancer cells by activating Akt and deactivating ERK1/2 pathways

The treatment options available for prostate cancer are limited because of its resistance to therapeutic agents. Thus, a better understanding of the underlying mechanisms of the resistance of prostate cancer will facilitate the discovery of more efficient treatment protocols. Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is recently identified by us as an anti-apoptotic molecule and a potential candidate target for breast cancer treatment. Here we found the expression levels of hPEBP4 were positively correlated with the severity of clinical prostate cancer. Furthermore, hPEBP4 was not expressed in TRAIL-sensitive DU145 prostate cancer cells, but was highly expressed in TRAIL-resistant LNCaP cells, which show highly activated Akt. Interestingly, hPEBP4 overexpression in TRAIL-sensitive DU145 cells promoted Akt activation but inhibited ERK1/2 activation. The hPEBP4-overexpressing DU145 cells became resistant to TRAIL-induced apoptosis consequently, which could be reversed by PI3K inhibitors. In contrast, silencing of hPEBP4 in TRAIL-resistant LNCaP cells inhibited Akt activation but increased ERK1/2 activation, resulting in their sensitivity to TRAIL-induced apoptosis that was restored by the MEK1 inhibitor. Therefore, hPEBP4 expression in prostate cancer can activate Akt and deactivate ERK1/2 signaling, leading to TRAIL resistance. We also demonstrated that hPEBP4-mediated resistance to TRAIL-induced apoptosis occurred downstream of caspase-8 and at the level of BID cleavage via the regulation of Akt and ERK pathways, and that hPEBP4-regulated ERK deactivation was upstream of Akt activation in prostate cancer cells. Considering that hPEBP4 confers cellular resistance to TRAIL-induced apoptosis and is abundantly expressed in poorly differentiated prostate cancer, silencing of hPEBP4 suggests a promising approach for prostate cancer treatment.     

Regulation of Akt by EGF-R inhibitors,a possible mechanism of EGF-R inhibitor-enhanced TRAIL-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine and a potent inducer of apoptosis. Epidermal growth factor (EGF) signaling is well known to involve in tumor survival and overexpression of EGF receptor (EGF-R) attributes to decreased responsiveness to many available therapies in cancer treatment. We investigated whether EGF-R inhibitors enhance TRAIL-induced apoptosis. We exposed A549 cells to Genistein, PD153035, and PD158780 for 12h and then treated with recombinant TRAIL protein. TRAIL alone induced 25% cell death after a 3-h treatment, but in cells pretreated with EGF-R inhibitors, TRAIL induced cell death to more than 70% after 3h treatment. Genistein enhanced TRAIL-induced apoptosis in a time- and dose-dependent manner. Western blot analyses showed that pretreatment with Genistein down-regulated the protein levels of total Akt and phosphorylated active Akt. Genistein also decreased the protein level of Bcl-XL that is regulated by Akt. These molecules are well characterized to act against induction of apoptotic cell death. Therefore, our data suggest that EGF-R inhibitor may sensitize A549 cells to TRAIL-induced apoptosis by regulating expression of these proteins. EGF-R inhibitors may play an important role in the anti-cancer activity of TRAIL protein, especially in TRAIL-resistant tumors that arise by expressing constitutively active Akt.     

Delta9-tetrahydrocannabinol-induced apoptosis in Jurkat leukemia T cells is regulated by translocation of Bad to mitochondria

Plant-derived cannabinoids, including Delta9-tetrahydrocannabinol (THC), induce apoptosis in leukemic cells, although the precise mechanism remains unclear. In the current study, we investigated the effect of THC on the upstream and downstream events that modulate the extracellular signal-regulated kinase (ERK) module of mitogen-activated protein kinase pathways primarily in human Jurkat leukemia T cells. The data showed that THC down-regulated Raf-1/mitogen-activated protein kinase/ERK kinase (MEK)/ERK/RSK pathway leading to translocation of Bad to mitochondria. THC also decreased the phosphorylation of Akt. However, no significant association of Bad translocation with phosphatidylinositol 3-kinase/Akt and protein kinase A signaling pathways was noted when treated cells were examined in relation to phosphorylation status of Bad by Western blot and localization of Bad to mitochondria by confocal analysis. Furthermore, THC treatment decreased the Bad phosphorylation at Ser(112) but failed to alter the level of phospho-Bad on site Ser(136) that has been reported to be associated with phosphatidylinositol 3-kinase/Akt signal pathway. Jurkat cells expressing a constitutively active MEK construct were found to be resistant to THC-mediated apoptosis and failed to exhibit decreased phospho-Bad on Ser(112) as well as Bad translocation to mitochondria. Finally, use of Bad small interfering RNA reduced the expression of Bad in Jurkat cells leading to increased resistance to THC-mediated apoptosis. Together, these data suggested that Raf-1/MEK/ERK/RSK-mediated Bad translocation played a critical role in THC-induced apoptosis in Jurkat cells.     

Amiloride potentiates TRAIL-induced tumor cell apoptosis by intracellular acidification-dependent Akt inactivation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor gene family, is considered as one of the most promising cancer therapeutic agents due to its ability to selectively induce tumor cell apoptosis. In this study, we investigated whether the Na(+)/H(+) exchanger inhibitor, amiloride, promotes TRAIL-induced apoptotic death both in sensitive and resistant tumor cells, HeLa and LNCaP cells, respectively, and its underlying molecular mechanism. Amiloride enhanced TRAIL-induced apoptosis and activation of caspase-3 and -8 in both cells. This compound increased TRAIL-induced mitochondrial cytochrome c release and poly(ADP-ribose) polymerase cleavage. Moreover, amiloride-induced intracellular acidification, and inhibited the phosphorylated activation of the serine/threonine kinase Akt, which is known to promote cell survival, in both tumor cells. These data suggest that amiloride sensitizes both tumor cells to TRAIL-induced apoptosis by promoting Akt dephosphorylation and caspase-8 activation via the intracellular acidification and that Na(+)/H(+) exchanger inhibitors may play an important role in the anti-cancer activity of TRAIL, especially, in TRAIL-resistant tumors with highly active and expressed Akt.     

Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells

The caspase-8 homologue FLICE-inhibitory protein (FLIP) functions as a caspase-8 dominant negative, blocking apoptosis induced by the oligomerization of the adapter protein FADD/MORT-1. FLIP expression correlates with resistance to apoptosis induced by various members of the tumor necrosis factor family such as TRAIL. Furthermore, forced expression of FLIP renders cells resistant to Fas-mediated apoptosis. Although FLIP expression is regulated primarily by MEK1 activity in activated T cells, the oncogenic signaling pathways that regulate FLIP expression in tumor cells are largely unknown. In this report, we examined the roles of the MAP kinase and phosphatidylinositol (PI) 3-kinase signaling pathways in the regulation of FLIP expression in tumor cells. We observed that the MEK1 inhibitor PD98059 reduced FLIP levels in only 2 of 11 tumor cell lines tested. In contrast, disruption of the PI 3-kinase pathway with the specific inhibitor LY294002 reduced Akt (protein kinase B) phosphorylation and the levels of FLIP protein and mRNA in all cell lines evaluated. The introduction of a dominant negative Akt adenoviral construct also consistently reduced FLIP expression as well as the phosphorylation of the Akt target glycogen synthase kinase-3. In addition, infection of the same cell lines with a constitutively active Akt adenovirus increased FLIP expression and the phosphorylation of GSK-3. These data add FLIP to the growing list of apoptosis inhibitors in which expression or function is regulated by the PI 3-kinase-Akt pathway.     

The short splice form of Casper/c-FLIP is a major cellular inhibitor of TRAIL-induced apoptosis.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor family that selectively induces apoptosis of cancer cells. However, some cancer cells or subpopulations within cancer cell lines are resistant to TRAIL-induced apoptosis. We developed a retroviral cDNA library-based functional cloning approach to unambiguously identify putative inhibitory genes of TRAIL-induced apoptosis. This effort identified the short splice form of Casper/c-FLIP, Casper-S/c-FLIPs, as a major cellular protein that confers resistance to TRAIL-induced apoptosis. Furthermore, we found that Casper deficient embryonic fibroblasts (EFs) were highly sensitive while their wild-type counterparts were completely resistant to TRAIL-induced apoptosis. Retroviral-mediated transduction of Casper-S/c-FLIPs into Casper(-/-) EFs restored resistance to TRAIL. These data suggest that Casper-S/c-FLIPs is a major cellular inhibitor of TRAIL-induced apoptosis.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces rheumatoid arthritis synovial fibroblast proliferation through mitogen-activated protein kinases and phosphatidylinositol 3-kinase/Akt

A hallmark of rheumatoid arthritis (RA) is the pseudo-tumoral expansion of fibroblast-like synoviocytes (FLSs), and the RA FLS has therefore been proposed as a therapeutic target. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been described as a pro-apoptotic factor on RA FLSs and, therefore, suggested as a potential drug. Here we report that exposure to TRAIL-induced apoptosis in a portion (up to 30%) of RA FLSs within the first 24 h. In the cells that survived, TRAIL induced RA FLS proliferation in a dose-dependent manner, with maximal proliferation observed at 0.25 nm. This was blocked by a neutralizing anti-TRAIL antibody. RA FLSs were found to express constitutively TRAIL receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) on the cell surface. TRAIL-R2 appears to be the main mediator of TRAIL-induced stimulation, as RA FLS proliferation induced by an agonistic anti-TRAIL-R2 antibody was comparable with that induced by TRAIL. TRAIL activated the mitogen-activated protein kinases ERK and p38, as well as the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway with kinetics similar to those of TNF-alpha. Moreover, TRAIL-induced RA FLS proliferation was inhibited by the protein kinase inhibitors PD98059, SB203580, and LY294002, confirming the involvement of the ERK, p38, and PI3 kinase/Akt signaling pathways. This dual functionality of TRAIL in stimulating apoptosis and proliferation has important implications for its use in the treatment of RA.     

TRAIL apoptosis is enhanced by quercetin through Akt dephosphorylation

TNF-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapy that preferentially induces apoptosis in cancer cells. However, many neoplasms are resistant to TRAIL by mechanisms that are poorly understood. Here we demonstrated that human prostate cancer cells, but not normal prostate cells, are dramatically sensitized to TRAIL-induced apoptosis and caspase activation by quercetin. Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells. We have shown that quercetin can potentiate TRAIL-induced apoptotic death. Human prostate adenocarcinoma DU-145 and LNCaP cells were treated with various concentrations of TRAIL (10-200 ng/ml) and/or quercetin (10-200 microM) for 4 h. Quercetin, which caused no cytotoxicity by itself, promoted TRAIL-induced apoptosis. The TRAIL-mediated activation of caspase, and PARP (poly(ADP-ribose) polymerase) cleavage were both enhanced by quercetin. Western blot analysis showed that combined treatment with TRAIL and quercetin did not change the levels of TRAIL receptors (death receptors DR4 and DR5, and DcR2 (decoy receptor 2)) or anti-apoptotic proteins (FLICE-inhibitory protein (FLIP), inhibitor of apoptosis (IAP), and Bcl-2). However, quercetin promoted the dephosphorylation of Akt. Quercetin-induced potent inhibition of Akt phosphorylation. Taken together, the present studies suggest that quercetin enhances TRAIL-induced cytotoxicity by activating caspases and inhibiting phosphorylation of Akt.     

Quercetin sensitizes pancreatic cancer cells to TRAIL-induced apoptosis through JNK-mediated cFLIP turnover

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that can selectively kill cancer cells. Nonetheless, many cancers are resistant to TRAIL, and the molecular mechanisms of TRAIL resistance in cancer, particularly pancreatic cancer, are still unclear. In this study, we tested the hypothesis that quercetin, a flavonoid, induces apoptosis in TRAIL-resistant pancreatic cancer cells. Although quercetin alone had no significant cytotoxic effect, when combined with TRAIL, it promoted TRAIL-induced apoptosis that required mitochondrial outer membrane permeabilization. A BH3-only protein BID knockdown dramatically attenuated TRAIL/quercetin-induced apoptosis. The expression levels of cellular FLICE-like inhibitory protein (cFLIP) decreased in a dose-dependent manner in the presence of quercetin, and overexpression of cFLIP was able to robustly rescue pancreatic cancer cells from TRAIL/quercetin-induced apoptosis. Additionally, quercetin activated c-Jun N-terminal kinase (JNK) in a dose-dependent manner, which in turn induced the proteasomal degradation of cFLIP, and JNK activation also sensitized pancreatic cancer cells to TRAIL-induced apoptosis. Thus, our results suggest that quercetin induces TRAIL-induced apoptosis via JNK activation-mediated cFLIP turnover.     

肿瘤细胞抗TRAIL凋亡诱导的分子机制

肿瘤坏死因子相关的凋亡诱导配体(tumornecrosisfactor-relatedapoptosis-inducingligand,TRAIL)是肿瘤坏死因子(tumornecrosisfactor,TNF)超家族的成员之一,它能选择性诱导肿瘤细胞凋亡,对大多数正常细胞无杀伤作用。研究表明,某些恶性肿瘤抵抗TRAIL诱导的凋亡,且TRAIL重复作用使一些TRAIL敏感的细胞产生获得性抗性,这是TRAIL应用于肿瘤治疗的重大障碍。现对与TRAIL凋亡诱导通路直接相关的抗TRAIL机制及由Akt等途径介导的抗性分子机制进行综述。     

Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells

Vascular endothelial growth factor (VEGF) utilizes a phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling pathway to protect endothelial cells from apoptotic death. Here we show that PI 3-kinase/Akt signaling promotes endothelial cell survival by inhibiting p38 mitogen-activated protein kinase (MAPK)-dependent apoptosis. Blockade of the PI 3-kinase or Akt pathways in conjunction with serum withdrawal stimulates p38-dependent apoptosis. Blockade of PI 3-kinase/Akt also led to enhanced VEGF activation of p38 and apoptosis. In this context, the pro-apoptotic effect of VEGF is attenuated by the p38 MAPK inhibitor SB203580. VEGF stimulation of endothelial cells or infection with an adenovirus expressing constitutively active Akt causes MEKK3 phosphorylation, which is associated with decreased MEKK3 kinase activity and down-regulation of MKK3/6 and p38 MAPK activation. Conversely, activation-deficient Akt decreases VEGF-stimulated MEKK3 phosphorylation and increases MKK/p38 activation. Activation of MKK3/6 is not dependent on Rac activation since dominant negative Rac does not decrease p38 activation triggered by inhibition of PI 3-kinase. Thus, cross-talk between the Akt and p38 MAPK pathways may regulate the level of cytoprotection versus apoptosis and is a new mechanism to explain the cytoprotective actions of Akt.     

The small heat shock protein alpha B-crystallin is a novel inhibitor of TRAIL-induced apoptosis that suppresses the activation of caspase-3

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor alpha family of cytokines that preferentially induces apoptosis in transformed cells, making it a promising cancer therapy. However, many neoplasms are resistant to TRAIL-induced apoptosis by mechanisms that are poorly understood. We demonstrate that the expression of the small heat shock protein alpha B-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) correlates with TRAIL resistance in a panel of human cancer cell lines. Stable expression of wild-type alpha B-crystallin, but not a pseudophosphorylation mutant impaired in its assembly and chaperone function, protects cancer cells from TRAIL-induced caspase-3 activation and apoptosis in vitro. Furthermore, selective inhibition of alpha B-crystallin expression by RNA interference sensitizes cancer cells to TRAIL. In addition, wild-type alpha B-crystallin promotes xenograft tumor growth and inhibits TRAIL-induced apoptosis in vivo in nude mice, whereas a pseudophosphorylation alpha B-crystallin mutant impaired in its anti-apoptotic function inhibits xenograft tumor growth. Collectively, these findings indicate that alpha B-crystallin is a novel regulator of TRAIL-induced apoptosis and tumor growth. Moreover, these results demonstrate that targeted inhibition of alpha B-crystallin promotes TRAIL-induced apoptosis, thereby suggesting a novel strategy to overcome TRAIL resistance in cancer.     

Procaspase-8失调与肿瘤细胞对TRAIL的耐受

肿瘤坏死因子相关凋亡诱导配体(TRAIL)可激活胱天蛋白酶（caspase）家族蛋白系列级联反应,最终诱导细胞凋亡. TRAIL选择性地诱导肿瘤细胞凋亡而不损伤正常细胞,使其成为治疗癌症的潜在药物靶点. 目前已知,细胞型FADD样白介素-1-β转换酶抑制蛋白(c FLIP)和凋亡抑制蛋白(IAPs)是肿瘤细胞对TRAIL耐受的主要原因.胱天蛋白酶原-8（procaspase-8）是TRAIL凋亡信号途径中的凋亡起始蛋白. 然而近年发现,在某些肿瘤细胞中procaspase-8功能失调常会阻碍凋亡信号传导,使肿瘤细胞对TRAIL诱导的凋亡产生耐受. 本文就其机制进行概述.     

Identification of X-linked inhibitor of apoptosis-associated factor-1 as an interferon-stimulated gene that augments TRAIL Apo2L-induced apoptosis

In the course of gene array studies aimed at identifying IFN-stimulated genes associated with interferon beta (IFN-beta)-induced apoptosis, we identified X-linked inhibitor of apoptosis-associated factor-1 (XAF1) as a novel IFN-stimulated gene. XAF1 mRNA was up-regulated by IFN-alpha and IFN-beta in all cells examined. However, IFNs induced high levels of XAF1 protein predominantly in cell lines sensitive to the proapoptotic effects of IFN-beta. In apoptosis-resistant cells including WM164 melanoma, WM35 melanoma, U937 pro-monocytic leukemia, and HT1080 fibrosarcoma cells, XAF1 mRNA was strongly up-regulated but XAF1 protein was up-regulated only weakly or not at all. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a critical mediator of IFN-beta-induced apoptosis, but most melanoma cell lines were resistant to recombinant TRAIL protein. For example, A375 melanoma cells were defective in TRAIL induction by IFN-beta and were resistant to TRAIL-induced apoptosis. However, IFN-beta pretreatment sensitized them to subsequent recombinant TRAIL-induced apoptosis. A375 cells expressing XAF1 constitutively were more sensitive to TRAIL-induced apoptosis compared with empty vector-transfected cells. The degree of sensitization by XAF1 was similar to that provided by IFN pretreatment and was correlated with the level of XAF1 expressed. Furthermore, the overexpression of the zinc-finger portion of XAF1 blocked IFN-dependent sensitization of A375 melanoma cells to the proapoptotic effects of TRAIL. These results suggested that IFN-dependent induction of XAF1 strongly influenced cellular sensitivity to the proapoptotic actions of TRAIL.     

Serum bioactive lysophospholipids prevent TRAIL-induced apoptosis via PI3K/Akt-dependent cFLIP expression and Bad phosphorylation

Serum contains a variety of biomolecules, which play an important role in cell proliferation and survival. We sought to identify the serum factor responsible for mitigating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis and to investigate its molecular mechanism. TRAIL induced effective apoptosis without serum, whereas bovine serum decreased apoptosis by suppressing cytochrome c release and caspase activation. Indeed, albumin-bound lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) inhibited TRAIL-induced apoptosis by suppressing caspase activation and cytochrome c release. LPA increased phosphatidylinositol 3-kinase (PI3K)-dependent Akt activation, cellular FLICE-inhibitory protein (cFLIP) expression, and Bad phosphorylation, resulting in inhibition of caspase-8 activation and Bad translocation to mitochondria. The antiapoptotic effect of LPA was abrogated by PI3K inhibitor, transfection with dominant-negative Akt, and specific downregulation of cFLIP expression using siRNA and further increased by siRNA-mediated suppression of Bad expression. Moreover, sera from ovarian cancer patients showed more protective effect against TRAIL-induced apoptosis than those from healthy donors, and this protection was suppressed by PI3K inhibitor. Our results indicate that albumin-bound LPA and S1P prevent TRAIL-induced apoptosis by upregulation of cFLIP expression and in part by Bad phosphorylation, through the activation of PI3K/Akt pathway.     

Up-regulation of IAPs by PI-3K: a cell survival signal-mediated anti-apoptotic mechanism

Under normal cell physiology, a balance between cell survival and apoptosis is crucial for homeostasis. Many studies have demonstrated that apoptosis is modulated by cell survival stimuli. Active Akt, a common mediator of cell survival signals, has been shown to inhibit apoptosis by attenuating activity of pro-apoptotic factors Bad and caspase-9. However, the anti-apoptotic mechanisms mediated by various cell survival signals are poorly understood. Human prostate cancer LNCaP cells, known to contain constitutively activated Akt as a result of a frame-shift mutation in PTEN, an inhibitor of PI-3K/Akt pathway, were observed to be completely resistant to TRAIL-induced apoptosis. In agreement with the known action of Akt, blockade of the PI-3K/Akt pathway rendered LNCaP cells highly susceptible to TRAIL. Importantly, active PI-3K/Akt prevented processing/activation of caspase-3, a phenomenon associated with the function of inhibitor of apoptosis proteins (IAPs). In fact, inhibition of PI-3K activity using Wortmannin significantly decreased the protein levels of IAPs, concomitantly promoting processing/activation of caspase-3 and TRAIL-induced apoptosis. My data indicate that in addition to blocking Bad and caspase-9 through Akt, PI-3K also inhibits caspase-3 through up-regulating IAPs, thereby attenuates apoptosis.     

Down-regulation of cFLIP following reovirus infection sensitizes human ovarian cancer cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) shows promise as a chemotherapeutic agent. However, many human cancer cells are resistant to killing by TRAIL. We have previously demonstrated that reovirus infection increases the susceptibility of human lung (H157) and breast (ZR75-1) cancer cell lines to TRAIL-induced apoptosis. We now show that reovirus also increases the susceptibility of human ovarian cancer cell lines (OVCAR3, PA-1 and SKOV-3) to TRAIL-induced apoptosis. Reovirus-induced increases in susceptibility of OVCAR3 cells to TRAIL require virus uncoating and involve increased activation of caspases 3 and 8. Reovirus infection results in the down-regulation of cFLIP (cellular FLICE inhibitory protein) in OVCAR3 cells. Down-regulation of cFLIP following treatment of OVCAR3 cells with antisense cFLIP oligonucleotides or PI3 kinase inhibition also increases the susceptibility of OVCAR3 cells to TRAIL-induced apoptosis. Finally, over-expression of cFLIP blocks reovirus-induced sensitization of OVCAR3 cells to TRAIL-induced apoptosis. The combination of reovirus and TRAIL thus represents a promising new therapeutic approach for the treatment of ovarian cancer.     

Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis

TRAIL can induce apoptosis in melanoma cells and thus may offer new hope for melanoma therapy. However, many melanoma cells are resistant to TRAIL. To examine molecular mechanisms in cell resistance, we analyzed TRAIL-induced DISC in TRAIL-sensitive melanoma cells and showed that apoptosis-initiating caspase-8 and caspase-10 were recruited to the DISC where they became activated through autocatalytical cleavage, leading to apoptosis through cleavage of downstream substrates such as caspase-3 and DFF45. In TRAIL-resistant melanoma cells, however, c-FLIP proteins were recruited to the DISC, resulting in the inhibition of caspase-8 and caspase-10 cleavage in the DISC. Both calmodulin-dependent protein kinase II (CaMKII) protein and enzymatic activity were upregulated in resistant cells and CaMKII inhibitor KN-93 downregulated expression of c-FLIP proteins, thus sensitizing resistant cells to TRAIL-induced apoptosis. Transfection of CaMKII cDNA in sensitive melanoma cells resulted in cell resistance to TRAIL, where transfection of CaMKII dominant-negative cDNA in resistant cells restored TRAIL sensitivity in cells. These results indicate that the CaMKII-mediated pathway for c-FLIP upregulation protects melanoma cells from TRAIL-induced apoptosis and targeting this pathway may provide novel therapeutic strategies in treatment of melanomas.     

Diverse antiapoptotic signaling pathways activated by vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase in prostate cancer cells converge on BAD

It has been demonstrated that vasoactive intestinal polypeptide, epidermal growth factor, and chronic activation of phosphatidylinositol 3-kinase can protect prostate cancer cells from apoptosis; however, the signaling pathways that they use and molecules that they target are unknown. We report that vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase activate independent signaling pathways that phosphorylate the proapoptotic protein BAD. Vasoactive intestinal polypeptide operated via protein kinase A, epidermal growth factor required Ras activity, and effects of phosphatidylinositol 3-kinase were predominantly mediated by Akt. BAD phosphorylation was critical for the antiapoptotic effects of each signaling pathway. None of these survival signals was able to rescue cells that express BAD with mutations in phosphorylation sites, whereas knockdown of BAD expression with small hairpin RNA rendered cells insensitive to apoptosis. Taken together, these results identify BAD as a convergence point of several antiapoptotic signaling pathways in prostate cells.