IFN-gamma inhibition of TRAIL-induced IAP-2 upregulation, a possible mechanism of IFN-gamma-enhanced TRAIL-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine molecule of TNF family and a potent inducer of apoptosis. The anticancer activities of TNF family members are often modulated by interferon (IFN)-gamma. Thus, we investigated whether IFN-gamma enhances TRAIL-induced apoptosis. We exposed HeLa cells to IFN-gamma for 12 h and then treated with recombinant TRAIL protein. No apoptosis was induced in cells pretreated with IFN-gamma, and TRAIL induced 25% cell death after 3 h treatment. In HeLa cells pretreated with IFN-gamma, TRAIL induced cell death to more than 70% at 3 h, indicating that IFN-gamma pretreatment sensitized HeLa cells to TRAIL-induced apoptosis. We investigated molecules that might be regulated by IFN-gamma pretreatment that would affect TRAIL-induced apoptosis. Western blotting analyses demonstrated that TRAIL treatment increased the level of IAP-2 protein and IFN-gamma pretreatment inhibited the upregulation of IAP-2 protein by TRAIL protein. Our data indicate that TRAIL can signal to activate both apoptosis induction and antiapoptotic mechanism, at least, through IAP-2 simultaneously. IFN-gamma or TRAIL treatment alone did not change expression of other pro- or antiapoptotic proteins such as DR4, DR5, FADD, Bax, IAP-1, XIAP, Bcl-2, and Bcl-XL. Our findings suggest that IFN-gamma may sensitize HeLa cells to TRAIL-induced apoptosis by preventing TRAIL-induced IAP-2 upregulation, and IFN-gamma may play a role in anticancer therapy of TRAIL protein through such mechanism.     

Reconstitution of galectin-3 alters glutathione content and potentiates TRAIL-induced cytotoxicity by dephosphorylation of Akt

We investigated the role of galectin-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic death in human breast carcinoma BT549 cells. We observed that parental galectin-3 null BT549 cells (BT549(par)) as well as control vector transfected (BT549(neo)) cells were resistant to TRAIL, while galectin-3 cDNA-transfected BT549 cells (BT549(gal-3)) were sensitive to TRAIL. Data from flow cytometry and immunoblotting analyses reveal that reconstitution of galectin-3 promoted cell death and PARP cleavage as well as caspase (-8, -9, and -3) activation during TRAIL treatment. However, unlike TRAIL treatment, galectin-3 transfectants were resistant to UV-B-induced PARP cleavage. Data from cDNA array analysis show that galectin-3 did not significantly enhance or reduce any apoptosis-related gene expression. Moreover, although galectin-3 restored pre-mRNA splicing activity and resulted in elevation of FLIPs protein, experiments with FLIPs cDNA-transfected cells show that overexpression of FLIPs did not sensitize cells to TRAIL. Interestingly, BT549(gal-3) cells demonstrated a approximately 2-fold increase in total glutathione content as well as a approximately 5-fold increase in GSSG content in comparison to BT549(par) and BT549(neo) cells, suggesting that galectin-3 overexpression may alter intraceullular oxidation/reduction reactions affecting the metabolism of glutathione and other thiols. In addition, galectin-3 overexpression inactivated Akt by dephosphorylation. Finally, overexpression of constitutively activated Akt protected BT549(gal-3) cells from TRAIL-induced cytotoxicity. Taken together, our data suggest that galectin-3-enhanced TRAIL-induced cytotoxicity is mediated through dephosphorylation of Akt, possibly through a redox-dependent process.     

Genistein enhances TRAIL-induced apoptosis through inhibition of p38 MAPK signaling in human hepatocellular carcinoma Hep3B cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some carcinoma cancer cells. However, it was found that treatment with TRAIL in combination with nontoxic concentrations of genistein sensitized TRAIL-resistant human hepatocellular carcinoma Hep3B cells to TRAIL-mediated apoptosis. Combined treatment with genistein and TRAIL-induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis were correlated with the induction of caspase activity that resulted in the cleavage of poly(ADP-ribose) polymerase (PARP). Both cell viability and the cleavage of PARP induced by combined treatment were significantly inhibited by caspase-3, -8 and -9 inhibitors, which demonstrates the important roles of caspases in the observed cytotoxic effects. Genistein treatment also triggered the inhibition of p38-β mitogen-activated protein kinase (MAPK) activation. Pretreatment with SB203580 resulted in significantly increased sub-G1 population and loss of mitochondrial membrane potential (MMP) in TRAIL-induced apoptosis. By contrast, overexpression of p38 MAPK protected apoptosis by co-treatment with genistein and TRAIL, suggesting that the p38 MAPK act as key regulators of apoptosis in response to treatment with a combination of genistein and TRAIL in human hepatocellular carcinoma Hep3B cells.     

Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression.     

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.     

p53-Independent up-regulation of a TRAIL receptor DR5 by proteasome inhibitors: a mechanism for proteasome inhibitor-enhanced TRAIL-induced apoptosis

Gliomas are the most common brain tumors in adults and account for more than half of all brain tumors. Despite intensive clinical investigations, average survival for the patients harboring the malignancy has not been significantly improved. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), shown to have potent and cancer-selective killing activity, has drawn considerable attention as a promising anti-cancer therapy. In an attempt to develop TRAIL as an anti-cancer therapy for gliomas, tumor suppressor activity of TRAIL was assessed using human glioma cell lines such as U373MG, U343MG, U87MG and LN18. U343MG, U87MG and LN18 cells were susceptible to TRAIL; however, U373MG cells were completely refractory to TRAIL. Resistance to the applied therapies is a key issue in cancer treatment; thus, various combination treatments were evaluated using U373MG cells to identify a better regimen. Unlike Doxorubicin, Etoposide, Actinomycin D and Wortmannin, a proteasome inhibitor MG132 significantly enhanced TRAIL-induced apoptosis. Similarly, other proteasome inhibitors, including Lactacystin, Proteasome inhibitor I and Velcade (Bortezomib), also enhanced apoptotic activity of TRAIL. Among these proteasome inhibitors, Velcade, the only approved drug, was as effective as MG132 in enhancing TRAIL-induced apoptosis. Both Velcade and MG132 increased the protein levels of DR5, a TRAIL receptor known to be up-regulated by p53, in U373MG cells where p53 is mutated. Our data indicate that proteasome inhibitors up-regulate DR5 in a p53-independent manner and a combination therapy comprising TRAIL and Velcade become a better treatment regimen for gliomas.     

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.     

TRAIL-induced apoptosis in gliomas is enhanced by Akt-inhibition and is independent of JNK activation

Patients with malignant gliomas have a poor prognosis and new treatment paradigms are needed against this disease. TRAIL/Apo2L selectively induces apoptosis in malignant cells sparing normal cells and is hence of interest as a potential therapeutic agent against gliomas. To determine the factors that modulate sensitivity to TRAIL, we examined the differences in TRAIL-activated signaling pathways in glioma cells with variable sensitivities to the agent. Apoptosis in response to TRAIL was unrelated to DR5 expression or endogenous p53 status in a panel of 8 glioma cell lines. TRAIL activated the extrinsic (cleavage of caspase-8, caspase-3 and PARP) and mitochondrial apoptotic pathways and reduced FLIP levels. It also induced caspase-dependent JNK activation, which did not influence TRAIL-induced apoptosis. Because the pro-survival PI3K/Akt pathway is highly relevant to gliomas, we assessed whether Akt could protect against TRAIL-induced apoptosis. Pretreatment with SH-6, a novel Akt inhibitor, enhanced TRAIL-induced apoptosis, suggesting a protective role for Akt. Conversely, TRAIL induced caspase-dependent cleavage of Akt neutralizing its anti-apoptotic effects. These results demonstrate that TRAIL-induced apoptosis in gliomas involves both activation of death pathways and downregulation of survival pathways. Additional studies are warranted to determine the therapeutic potential of TRAIL against gliomas.Supported in part by the NIH grant PO1 CA55261     

Direct interaction of Smac with NADE promotes TRAIL-induced apoptosis

Second mitochondria-derived activator of caspase (Smac) has been implicated in the activation of apoptosis in response to cell stress. We screened for Smac/DIABLO-binding protein for further understanding of Smac-mediated apoptosis. We identified NADE, previously known as p75NTR-associated cell death executor, as a Smac-binding protein. Smac-NADE interaction was mapped to the N-terminal region of Samc and the C-terminal region of NADE. Co-expression of NADE and Smac promotes TRAIL-induced apoptosis in MCF-7 cells. Interestingly, the co-presence of Smac and NADE inhibits XIAP-mediated Smac ubiquitination. In conclusion, our results provide the first evidence that the interaction between Smac and NADE regulates apoptosis through the inhibition of Smac ubiquitination.     

Antipsychotic agent thioridazine sensitizes renal carcinoma Caki cells to TRAIL-induced apoptosis through reactive oxygen species-mediated inhibition of Akt signaling and downregulation of Mcl-1 and c-FLIP(L)

Thioridazine has been known as an antipsychotic agent, but it also has anticancer activity. However, the effect of thioridazine on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitization has not yet been studied. Here, we investigated the ability of thioridazine to sensitize TRAIL-mediated apoptosis. Combined treatment with thioridazine and TRAIL markedly induced apoptosis in various human carcinoma cells, including renal carcinoma (Caki, ACHN, and A498), breast carcinoma (MDA-MB231), and glioma (U251MG) cells, but not in normal mouse kidney cells (TMCK-1) and human normal mesangial cells. We found that thioridazine downregulated c-FLIP(L) and Mcl-1 expression at the post-translational level via an increase in proteasome activity. The overexpression of c-FLIP(L) and Mcl-1 overcame thioridazine plus TRAIL-induced apoptosis. We further observed that thioridazine inhibited the Akt signaling pathway. In contrast, although other phosphatidylinositol-3-kinase/Akt inhibitors ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin) sensitized TRAIL-mediated apoptosis, c-FLIP(L) and Mcl-1 expressions were not altered. Furthermore, thioridazine increased the production of reactive oxygen species (ROS) in Caki cells, and ROS scavengers (N-acetylcysteine, glutathione ethyl ester, and trolox) inhibited thioridazine plus TRAIL-induced apoptosis, as well as Akt inhibition and the downregulation of c-FLIP(L) and Mcl-1. Collectively, our study demonstrates that thioridazine enhances TRAIL-mediated apoptosis via the ROS-mediated inhibition of Akt signaling and the downregulation of c-FLIP(L) and Mcl-1 at the post-translational level.     

Caveolin-1 negatively regulates TRAIL-induced apoptosis in human hepatocarcinoma cells

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) offers promising therapeutic potential based on its ability to induce apoptosis in various cancer cell lines without obvious adverse effect to normal cells. However, the mechanism of the differential sensitivity towards TRAIL-induced apoptosis remains unclear. Here, we demonstrate that caveolin-1 directly regulated TRAIL-induced apoptosis in HepG2 cells. ShRNA-mediated caveolin knockdown sensitized TRAIL-induced apoptosis and disruption of caveolae structure by the cholesterol-extracting reagent, methyl-β-cyclodextrin (MCD), enhanced TRAIL-induced apoptosis. Over-expression of caveolin-1 partially blocked TRAIL-induced apoptosis. The engagement of TRAIL with its receptor DR4 reduced the localization of DR4 in caveolae and resulted in its internalization. Blockade of caveolae-mediated internalization of DR4 by filipin III effectively enhanced TRAIL-induced apoptosis. Collectively, our results reveal a new mechanism by which caveolin-1 negatively regulates TRAIL-induced apoptosis in human hepatocarcinoma cells.     

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.     

Hypoxia inhibits TRAIL-induced tumor cell apoptosis: Involvement of lysosomal cathepsins

Tumor hypoxia interferes with the efficacy of chemotherapy, radiotherapy, and tumor necrosis factor-α. TRAIL (tumor necrosis factor-related apoptosis inducing ligand) is a potent apoptosis inducer that limits tumor growth without damaging normal cells and tissues in vivo. We present evidence for a central role of lysosomal cathepsins in hypoxia and/or TRAIL-induced cell death in oral squamous cell carcinoma (OSCC) cells. Hypoxia or TRAIL-induced activation of cathepsins (B, D and L), caspases (-3 and -9), Bid cleavage, release of Bax and cytochrome c, and DNA fragmentation were blocked independently by zVAD-fmk, CA074Me or pepstatin A, consistent with the involvement of lysosomal cathepsin B and D in cell death. Lysosome stability and mitochondrial membrane potential were reduced in hypoxia and TRAIL-induced apoptosis. However, TRAIL treatment under hypoxic condition resulted in diminished apoptosis rates compared to treatment under normoxia. This inhibitory effect of hypoxia on TRAIL-induced apoptosis may be based on preventing Bax activation and thus protecting mitochondria stability. Our data show that TRAIL or hypoxia independently triggered activation of cathepsin B and D leading to apoptosis through Bid and Bax, and suggest that hypoxic tissue regions provide a selective environment for highly apoptosis-resistant clonal cells. Molecular therapy approaches based on cathepsin inhibitors need to address this novel tumor-preventing function of cathepsins in OSCC.     

Down-regulation of FoxO-dependent c-FLIP expression mediates TRAIL-induced apoptosis in activated hepatic stellate cells

Activated hepatic stellate cells which contribute to liver fibrosis have represented an important target for antifibrotic therapy. In this study, we found that TRAIL inhibited PI3K/Akt-dependent FoxO phosphorylation and relocated FoxO proteins into the nucleus from the cytosol in activated human hepatic stellate LX-2 cells. The accumulated FoxO proteins in the nucleus led to down-regulation of c-FLIP_L/S expression, resulting in the activation of apoptosis-related signaling molecules including the activation of caspase-8, -3, and Bid, as well as mitochondrial cytochrome c release. These results were supported by showing that siRNA-mediated knockdown of FoxO led to restoration of c-FLIP_L/S expression and resistance to TRAIL-induced apoptosis after treatment of LX-2 cells with TRAIL. Furthermore, c-FLIP_L/S-transfected LX-2 cells showed the decreased sensitivity to TRAIL-induced apoptosis. Collectively, our data suggest that sequential activation of FoxO proteins under conditions of suppressed PI3K/Akt signaling by TRAIL can down-regulate c-FLIP_L/S, consequently promoting TRAIL-induced apoptosis in LX-2 cells. Therefore, the present study suggests TRAIL may be an effective strategy for antifibrotic therapy in liver fibrosis.     

TRAIL-induced cleavage and inactivation of SPAK sensitizes cells to apoptosis

Ste20-related proline-alanine-rich kinase (SPAK) has been linked to various cellular processes, including proliferation, differentiation, and ion transport regulation. Recently, we showed that SPAK mediates signaling by the TNF receptor, RELT. The presence of a caspase cleavage site in SPAK prompted us to study its involvement in apoptotic signaling induced by another TNF member, TRAIL. We show that TRAIL stimulated caspase 3-like proteases that cleaved SPAK at two distinct sites. Cleavage had little effect on the activity of SPAK but removed its substrate-binding domain. In addition, TRAIL reduced the activity of SPAK in HeLa cells in a caspase-independent manner. Thus, TRAIL inhibited SPAK by two mechanisms: activation of caspases, which removed its substrate-binding domain, and caspase-independent down-regulation of SPAK activity. Furthermore, reducing the amount of SPAK by siRNA increased the sensitivity of HeLa cells to TRAIL-induced apoptosis. Thus, TRAIL down-regulation of SPAK is an important event that enhances its apoptotic effects.     

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.     

Wnt-expressing rat embryonic fibroblasts suppress Apo2L/TRAIL-induced apoptosis of human leukemia cells

Wnt signaling enhances cell proliferation and the maintenance of hematopoietic cells. In contrast, cytotoxic ligand Apo2L/TRAIL induces the apoptosis of various transformed cells. We observed that co-culture of human pre-B leukemia cells KM3 and REH with Wnt1- or Wnt3a-producing rat embryonic fibroblasts efficiently suppressed Apo2L/TRAIL-induced apoptosis of the lymphoid cells. This suppression occurs at the early stages of the Apo2L/TRAIL apoptotic cascade and, interestingly, the activation of the Wnt pathway alone in human leukemia cells is not sufficient for their full anti-apoptotic protection. We hypothesize that a stimulus emanating specifically from Wnt1- or Wnt3a-expressing rat fibroblasts is responsible for the observed resistance to Apo2L/TRAIL. This anti-apoptotic signaling was significantly hampered by the inhibition of the MEK1/ERK1/2 or NFκB pathways in KM3 and REH cells. Our results imply that paracrine Wnt-related signals could be important for the survival of pre-B cell-derived malignancies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. L. Doubravská and Š. Šímová are joint first authors. V. Kořínek and L. Anděra are senior co-authors.     

HER2 signaling downregulation by trastuzumab and suppression of the PI3K/Akt pathway: an unexpected effect on TRAIL-induced apoptosis

We investigated whether HER2 downregulation by trastuzumab modulates the responsiveness of breast cancer cells to TNF-related apoptosis-inducing ligand (TRAIL). Interestingly, in contrast to increased response to TRAIL in SKBr3 cells, trastuzumab decreased the susceptibility of BT474 cells to TRAIL. This decrease was also observed after exogenous inhibition of PI3-K/Akt kinase, but not MAPK/ERK kinase (MEK)/mitogen-activated protein kinase (MAPK). In BT474 cells, but not SKBr3 cells, inhibition of the HER2/phosphatidylinositol 3' kinase (PI3K)/Akt pathway resulted in downregulation of the pro-apoptotic receptors TRAIL-receptor 1 (TRAIL-R1) and TRAIL-R2. TRAIL-induced caspase-8 activation, Bid processing, drop of DeltaPsi(m), and poly ADP-ribose polymerase (PARP) cleavage but not in caspase-9 activation, and these events were inhibited in HER2/PI3K/Akt-suppressed BT474 cells, which on the other hand exhibited downregulation of Bcl-xL and increased response to mitomycin C. We show that HER2/PI3K/Akt pathway may play a specific pro-apoptotic role in certain cell type by inducing TRAIL-R1 and -R2 expression and thereby enhancing responsiveness to TRAIL.     

Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling

PI3k-Akt and p53 pathways are known to play anti- and pro-apoptotic roles in cell death, respectively. Whether these pathways are recruited in influenza virus infection in highly productive monkey (CV-1) and canine (MDCK) kidney cells was studied here. Phosphorylation of Akt (Akt-pho) was found to occur only early after infection (5–9 h.p.i). Nuclear accumulation and phosphorylation of p53 (p53-pho), and expression of its natural target p21/waf showed low constitutive levels at this period, whereas all three parameters were markedly elevated at the late apoptotic stage (17–20 h.p.i.). Up-regulation of Akt-pho and p53-pho was not induced by UV-inactivated virus suggesting that it required virus replication. Also, mRNAs of p53 and its natural antagonist mdm2 were not increased throughout infection indicating that p53-pho was up-regulated by posttranslational mechanisms. However, p53 activation did not seem to play a leading role in influenza-induced cell death: (i) infection of CV1 and MDCK cells with recombinant NS1-deficient virus provoked accelerated apoptotic death characterized by the lack of p53 activation; (ii) mixed apoptosis-necrosis death developed in influenza-infected human bronchial H1299 cells carrying a tetracycline-regulated p53 gene did not depend on p53 gene activation by tetracycline. Virus-induced apoptosis and signaling of Akt and p53 developed in IFN-deficient VERO cells with similar kinetics as in IFN-competent CV1-infected cells indicating that these processes were endocrine IFN-independent. Apoptosis in influenza-infected CV-1 and MDCK cells was Akt-dependent and was accelerated by Ly294002, a specific inhibitor of PI3k-Akt signaling, and down-regulated by the viral protein NS1, an inducer of host Akt. The obtained data suggest that influenza virus (i) initiates anti-apoptotic PI3k-Akt signaling at early and middle phases of infection to protect cells from fast apoptotic death and (ii) provokes both p53-dependent and alternative p53-independent apoptotic and/or necrotic (in some host systems) cell death at the late stage of infection. These data have been partially presented at The 3rd Orthomyxovirus Research Conference (sponsored by ESWI and NIH). Abstr. p. 23 entitled: “Influenza virus-specific up-regulation of Akt and Mdm2 in infected cells” by Zhirnov O.P., and Klenk H.D., July 28–21, 2005. Queen’s College, Cambridge, United Kingdom; and at The Annual Meeting of Virology in Munich, March 15–18 (2006)—“Influenza virus-specific up-regulation of Akt, Mdm2, and p53 in infected cells” by O. P. Zhirnov and H. D. Klenk; Book of abstracts, p. 339     

Proteasome inhibitor MG132 enhances TRAIL-induced apoptosis and inhibits invasion of human osteosarcoma OS732 cells

MG132 as a proteasome inhibitor could induce apoptosis in various cancer cells. This study aimed to discuss the effect of proteasome inhibitor MG132 on the TRAIL-induced apoptosis of human osteosarcoma OS732 cells. MG132 and TRAIL were applied on OS732 cells respectively or jointly. Cell survival rates, changes of cellular shape, cell apoptosis and cell invasion were analyzed, respectively, by 3-(4,5)-dimethylthiahiazo(-z-y1)-2,5-di-phenytetrazoliumromide (MTT) assay, inverted phase contrast microscope, flow cytometry, and transwell invasion chamber methods. The protein levels of DR5, caspase-3, caspase-8, p27^kip1 and MMP-9 were measured by Western blot analysis. The results indicated that combination of MG132 and TRAIL had the effect of up-regulating expression of DR5, caspase-3, caspase-8 and p27^kip1, down-regulating expression of MMP-9 and inducing apoptosis as well as suppressing the ability of invasion of OS732 cells. The survival rate of combined application of 10 μM MG132 and 100 ng/ml TRAIL on OS732 cells was significantly lower than that of the individual application (p < 0.01). Changes of cellular shape and apoptotic rates also indicated the apoptosis-inducing effect of combined application was much stronger than that of individual application. Cell cycle analysis showed combination of MG132 and TRAIL mostly caused OS732 cells arrested at G2–M-phase. The invasion ability of OS732 cells was restrained significantly in the combined group compared with the individual group and control group.