Antiglioma effects of N6‐isopentenyladenosine,an endogenous isoprenoid end product,through the downregulation of epidermal growth factor receptor

Malignant gliomas are highly dependent on the isoprenoid pathway for the synthesis of lipid moieties critical for cell proliferation. The isoprenoid derivative N6‐isopentenyladenosine (iPA) displays pleiotropic biological effects, including a direct anti‐tumor activity in several tumor models. The antiglioma effects of iPA was then explored in U87MG cells both in vitro and grafted in mice and the related molecular mechanism confirmed in primary derived patients' glioma cells. iPA powerfully inhibited tumor cell growth and induced caspase‐dependent apoptosis through a mechanism involving a marked accumulation of the pro‐apoptotic BIM protein and inhibition of EGFR. Indeed, activating AMPK following conversion into its iPAMP active form, iPA stimulated EGFR phosphorylation and ubiquitination along a proteasome‐mediated pathway which was responsible for receptor degradation and its downstream signaling pathways inhibition, including the STAT3, ERK and AKT cascade. The inhibition of AMPK by compound C prevented iPA‐mediated phosphorylation of EGFR, known to precede receptor loss. As expected the block of EGFR degradation, by exposure to the proteasome inhibitor MG132, significantly reduced iPA‐induced cell death. Given the importance of receptor degradation in iPA‐mediated cytotoxicity, we also documented that the EGFR expression levels in a panel of primary glioma cells confers them a high sensitivity to iPA treatment. In conclusion our study provides the first evidence of iPA antiglioma effect. Indeed, as glioma is driven by aberrant signaling of growth factor receptors, particularly the EGFR, iPA, alone or in association with EGFR targeted therapies, might be a promising therapeutic tool to achieve a potent anti‐tumoral effect.     

Ubiquitination and degradation of the Arg tyrosine kinase is regulated by oxidative stress

The c-Abl and Arg nonreceptor tyrosine kinases are activated in the response of cells to oxidative stress. The present studies demonstrate that treatment of cells with 0.1 mM H2O2 is associated with increased tyrosine phosphorylation of Arg and little effect on Arg levels. By contrast, exposure to 1.0 mM H2O2 decreased Arg phosphorylation. Treatment with 1.0 mM H2O2 was also associated with ubiquitination and degradation of Arg. The results show that Arg is stabilized in response to 0.1 mM H2O2 by autophosphorylation of Y-261, consistent with involvement of the Arg kinase function in regulating Arg levels. The results further demonstrate that c-Abl-mediated phosphorylation of Arg on Y-261 similarly confers Arg stabilization. In concert with these results, phosphorylation of Arg on Y-261 blocked H2O2-induced ubiquitination and thereby Arg degradation and inactivation. These findings demonstrate that Arg phosphorylation and degradation are differentially regulated by the degree of oxidative stress, and that Arg stability is conferred by phosphorylation of Y-261.     

PI 3-kinase/Akt and STAT3 are required for the prevention of TGF-β-induced Hep3B cell apoptosis by autocrine motility factor/phosphoglucose isomerase

We established Hep3B cells stably-expressing wild-type and mutated AMF/PGI with differing enzymatic activities in order to investigate how AMF/PGI affects TGF-β-induced apoptosis, and demonstrated that AMF/PGI against TGF-β-induced apoptosis was correlated with its enzymatic activity. AMF/PGI did not alter TGF-β-receptor expression nor affect TGF-β-induced PAI-1 gene promoter or Smad3/4 activity. AMF/PGI induced PI 3-kinase activity, IRS and Akt phosphorylation, which can further regulate BAD phosphorylation. Constitutively-active p110 enhanced AMF/PGI-mediated anti-apoptosis activity, and dominant negative Akt alleviated anti-TGF-β-induced apoptosis. We also demonstrated that STAT3 is a weak anti-apoptotic agent but has an increased anti-apoptotic effect in cooperation with PI 3-kinase/Akt.     

The novel Akt inhibitor API-1 induces c-FLIP degradation and synergizes with TRAIL to augment apoptosis independent of Akt inhibition

API-1 (pyrido[2,3-d]pyrimidines) is a novel small-molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of cellular FLICE-inhibitory protein (c-FLIP) levels and TRAIL-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of death receptor 4 (DR4) or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1 but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Because other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity.     

Celecoxib promotes c-FLIP degradation through Akt-independent inhibition of GSK3

Celecoxib is a COX-2 inhibitor that reduces the risk of colon cancer. However, the basis for its cancer chemopreventive activity is not fully understood. In this study, we defined a mechanism of celecoxib action based on degradation of cellular FLICE-inhibitory protein (c-FLIP), a major regulator of the death receptor pathway of apoptosis. c-FLIP protein levels are regulated by ubiquitination and proteasome-mediated degradation. We found that celecoxib controlled c-FLIP ubiquitination through Akt-independent inhibition of glycogen synthase kinase-3 (GSK3), itself a candidate therapeutic target of interest in colon cancer. Celecoxib increased the levels of phosphorylated GSK3, including the α and β forms, even in cell lines, where phosphorylated Akt levels were not increased. Phosphoinositide 3-kinase inhibitors abrogated Akt phosphorylation as expected but had no effect on celecoxib-induced GSK3 phosphorylation. In contrast, protein kinase C (PKC) inhibitors abolished celecoxib-induced GSK3 phosphorylation, implying that celecoxib influenced GSK3 phosphorylation through a mechanism that relied upon PKC and not Akt. GSK3 blockade either by siRNA or kinase inhibitors was sufficient to attenuate c-FLIP levels. Combining celecoxib with GSK3 inhibition enhanced attenuation of c-FLIP and increased apoptosis. Proteasome inhibitor MG132 reversed the effects of GSK3 inhibition and increased c-FLIP ubiquitination, confirming that c-FLIP attenuation was mediated by proteasomal turnover as expected. Our findings reveal a novel mechanism through which the regulatory effects of c-FLIP on death receptor signaling are controlled by GSK3, which celecoxib acts at an upstream level to control independently of Akt.     

Proteasome Inhibitors Induce a p38 Mitogen-activated Protein Kinase (MAPK)-dependent Anti-apoptotic Program Involving MAPK Phosphatase-1 and Akt in Models of Breast Cancer

Proteasome inhibitors represent a novel class of anti-tumor agents that have clinical efficacy against hematologic malignancies, but single-agent activity against solid tumors such as breast cancer has been disappointing, perhaps due to activation of anti-apoptotic survival signals. To evaluate a possible role for the p38 mitogen-activated protein kinase (MAPK), A1N4-myc human mammary epithelial, and BT-474 and MDA-MB-231 breast carcinoma cells, were studied. Exposure of these lines to pharmacologic p38 blockade enhanced proteasome inhibitor-mediated apoptosis, as did overexpression of dominant negative (DN)-p38-α and -β-MAPK isoforms. Inhibition of p38 resulted in suppression of induction of anti-apoptotic MAPK phosphatase (MKP)-1, in association with enhanced activation of the pro-apoptotic c-Jun-N-terminal kinase (JNK). Moreover, infection of cells treated with a proteasome inhibitor/p38 inhibitor combination with Adenovirus (Ad) inducing over-expression of MKP-1 suppressed apoptosis compared with controls. Further targets of p38 MAPK were also studied, and proteasome inhibition activated phosphorylation of MAPK-activated protein kinase-2, heat shock protein (HSP)-27, and the AKT8 virus oncogene cellular homolog (Akt). Inhibition of p38 MAPK resulted in decreased phospho-HSP-27 and phospho-Akt, while down-regulation of HSP-27 with a small interfering RNA decreased phosphorylation of Akt, directly linking activation of p38 to Akt. Finally, inhibition of Akt with phosphatidylinositol-3-kinase inhibitors increased apoptosis, as did over-expression of DN-Akt. These studies support the hypothesis that proteasome inhibitors activate an anti-apoptotic survival program through p38 MAPK that involves MKP-1 and Akt. Further, they suggest that strategies targeting MKP-1 and Akt could enhance the anti-tumor efficacy of proteasome inhibitors against breast cancer.     

Isosilybin B causes androgen receptor degradation in human prostate carcinoma cells via PI3K-Akt-Mdm2-mediated pathway

The identification and development of novel nontoxic phytochemicals that target androgen and androgen receptor (AR) signaling remains a priority for prostate cancer (PCA) control. In the present study, we assessed the antiandrogenic efficacy of isosilybin B employing human PCA LNCaP (mutated AR), 22Rv1 (mutated AR) and LAPC4 (wild-type AR) cells. Isosilybin B (10-90 microM) treatment decreased the AR and prostate specific antigen (PSA) levels in LNCaP, 22Rv1 and LAPC4 cells, but not in non-neoplastic human prostate epithelial PWR-1E cells. Isosilybin B treatment also inhibited synthetic androgen R1881-induced nuclear localization of AR, PSA expression and cell growth, and caused G(1) arrest. In mechanistic studies identifying AR degradation, isosilybin B caused increased phosphorylation of Akt (Ser-473 and Thr-308) and Mdm2 (Ser-166), which was linked with AR degradation as pretreatment with PI3K inhibitor (LY294002)-restored AR level. Further, overexpression of kinase-dead Akt largely reversed isosilybin B mediated-AR degradation suggesting a critical role of Akt in AR degradation. Antibody pull-down results also indicated that isosilybin B treatment enhances the formation of complex between Akt, Mdm2 and AR, which promotes phosphorylation-dependent AR ubiquitination and its degradation by proteasome. Together, present findings identify a novel mechanism for isosilybin B-mediated anticancer effects in human PCA cells.     

Potent antitumor agent proteasome inhibitors: a novel trigger for Bcl2 phosphorylation to induce apoptosis.

The sustained cytotoxicity conferred by proteasome inhibitors against a broad spectrum of human cancer cells is mediated by a delicate mechanism of programmed cell death. Similar to microtubule disarraying agents, the cell death induced by these potent antitumor agents precedes blocking in cell cycle transition at G2-M phase. The microtubule damaging antineoplastic drugs can kill tumor cells by inducing phosphorylation of antiapoptotic proteins such as Bcl2, Bcl-xL or MCL-1. The simultaneous apoptosis with Bcl2 phosphorylation was evident in cancer cells challenged with the proteasome inhibitor, MG132. Our studies suggest that the proteasome inhibitor MG132 induced tumor cell killing is mediated through Bcl2 phosphorylation.     

Hsp90-Akt phosphorylates ASK1 and inhibits ASK1-mediated apoptosis

Hsp90 client protein Akt has been shown to inhibit cell apoptosis in part by inhibiting proapoptotic kinase ASK1 (apoptosis signal-regulating kinase 1) activity. In the present study, we show that Hsp90 inhibits hydrogen peroxide (H(2)O(2))-induced ASK1-p38 activation in endothelial cells (EC). The inhibitory effect of Hsp90 on ASK1-p38 activities is diminished when the Akt phosphorylation site on ASK1 (pSer83) is absent or when Akt is genetically deleted in cells, suggesting that Hsp90 and Akt function together to inhibit ASK1-p38 signaling. Thus, inhibition of Hsp90 by 17-allyamino-17-demethoxygeldanamycin (17-AAG) or phosphatidylinositol 3-kinase (PI3K) LY294002 induced and synergized ASK1 activation and ASK1-mediated EC apoptosis. Furthermore, we show that in resting EC Hsp90, Akt and ASK1 form a ternary complex in which both Akt and ASK1 bind to the middle domain of Hsp90, suggesting that Hsp90 may hold Akt and ASK1 in close proximity. The N-terminal domain of ASK1 containing the Akt phosphorylation site (pSer83) associates with Akt in resting state. However, Akt is released from the N-terminal domain concomitant with binding to the C-terminal domain of ASK1 in response to ASK1 activator H(2)O(2), inhibitor of Hsp90 17-AAG and Akt inhibitor LY294002, leading to a more stable Hsp90-Akt-ASK1 complex. We conclude that Hsp90-Akt forms a complex with ASK1 and protect EC from stress-induced apoptosis.     

The phosphatidylinositide 3'-kinase/Akt survival pathway is a target for the anticancer and radiosensitizing agent PKC412, an inhibitor of protein kinase C.

Activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt survival pathway protects against apoptotic stress stimuli. Therefore, compounds that down-regulate this pathway are of clinical interest for single and combined anticancer treatment modalities. Here we demonstrate that the cytotoxic effect of the protein kinase C (PKC)-inhibitor N-benzoylated staurosporine (PKC412) is mediated via the PI3K/Akt pathway. Dose-dependent down-regulation of the proliferative activity, activation of the apoptotic machinery, and cell killing by PKC412 (0-1 microM) in Rat1a-fibroblasts and H-ras-oncogene-transformed fibroblasts correlated with a decrease of Akt phosphorylation and a reduced phosphorylation of the endogenous Akt-substrate GSK3-alpha. Expression of the dominant-active myristoylated form of Akt abrogated this cytotoxic effect of PKC412. Experiments with Apaf-1-deficient cells revealed that PKC412-induced cytotoxicity depends on an intact apoptosome but that the decrease of Akt phosphorylation is not attributable to apoptosis execution. Comparative experiments indicate that PKC412 and the parent-compound staurosporine down-regulate this survival pathway upstream or at the level of Akt but by a different mechanism than the PI3K-inhibitor LY294002. Furthermore, inhibition of this pathway by PKC412 is relevant for sensitization to ionizing radiation. These results demonstrate the specific role of this signaling pathway for the PKC412-mediated down-regulation of an apoptotic threshold and its cytotoxicity.     

The involvement of PI 3-K/Akt-dependent up-regulation of Mcl-1 in the prevention of apoptosis of Hep3B cells by interleukin-6

Interleukin-6 (IL-6) is a pleitrophic cytokine that not only regulates growth and differentiation of many cell types, but also induces production of acute phase proteins (AAP) in hepatocytes. Our previous works have demonstrated that both PI 3-K/Akt and STAT3 pathways were concomitantly activated and cooperatively mediated the anti-apoptotic effect of IL-6. This investigation reports that IL-6 protected cells against apoptosis induced by a variety of agents including, TGF-beta, UV and retinoic acid (RA) in Hep3B cells, suggesting that IL-6 is a fundamental determinant of hepatic cell survival. Mcl-1, but not other Bcl-2 family members, was rapidly up-regulated by IL-6, with a peak (approximately 3-4-fold) appearing at 4 h. Transient transfection of cells with a mcl-1 antisense vector, resulting in a 50-60% reduction of the anti-apoptotic effect of IL-6, indicating that Mcl-1 is a downstream effector of IL-6. Which signaling pathway transduced by IL-6 responsible for the Mcl-1 up-regulation was further investigated. In Hep3B cells, the JAK/STAT3, ERK, and PI 3-K/Akt pathways were activated by IL-6 stimulation. Blocking JAK/STAT3 activation with a dominant-negative mutant STAT3F or a JAK inhibitor AG490 could not influence IL-6-mediated Mcl-1 up-regulation. Similarly, PD98059 treatment, a MEK specific inhibitor, also failed to inhibit Mcl-1 expression. However, the IL-6-induced Mcl-1 up-regulation was effectively attenuated in the presence of PI 3-K inhibitors, LY294002 and wortmannin. Expression of dominant-negative Akt, but not Etk, could abrogate the IL-6-induced increase of Mcl-1. In conclusion, our results suggest that the anti-apoptotic effect of IL-6 is mediated, at least in part, by Mcl-1 expression and that is mainly through the PI 3-K/ Akt-dependent pathway.     

p34 (SEI-1) inhibits ROS-induced cell death through suppression of ASK1

Apoptosis signal-regulating kinase 1 (ASK1) is a key factor for controlling several cellular events including the cell cycle, senescence and apoptosis, in response to reactive oxygen species (ROS). However, the mechanisms that regulate ASK1 protein levels remain largely unexplored. In this study, we demonstrate that p34 (SEI-1) , a positive cell cycle regulator with an oncogenic potential, inhibits ROS-induced cell death by suppressing ASK1. We first found that p34 (SEI-1) -expressing cells have enhanced resistance to hydrogen peroxide (H 2O 2). Moreover, ectopic expression of p34 (SEI-1) clearly inhibited H 2O 2-induced phosphorylation of ASK1 in the colon cancer cell lines- HCT116 and SW620-in association with a decrease in ASK1 protein levels. Interestingly, p34 (SEI-1) induced ubiquitination of ASK1, however, no direct interaction was found between p34 (SEI-1) and ASK1. These results suggest that p34 (SEI-1) inhibits ROS-induced cell death through by indirectly inducing ubiquitination of ASK1.     

Inflammatory cytokines induce phosphorylation and ubiquitination of prostate suppressor protein NKX3.1

Inflammation of the prostate is a risk factor for the development of prostate cancer. In the aging prostate, regions of inflammatory atrophy are foci for prostate epithelial cell transformation. Expression of the suppressor protein NKX3.1 is reduced in regions of inflammatory atrophy and in preinvasive prostate cancer. Inflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin-1beta accelerate NKX3.1 protein loss by inducing rapid ubiquitination and proteasomal degradation. The effect of TNF-alpha is mediated via the COOH-terminal domain of NKX3.1 where phosphorylation of serine 196 is critical for cytokine-induced degradation. Mutation of serine 196 to alanine abrogates phosphorylation at that site and the effect of TNF-alpha on NKX3.1 ubiquitination and protein loss. This is in contrast to control of steady-state NKX3.1 turnover, which is mediated by serine 185. Mutation of serine 185 to alanine increases NKX3.1 protein stability by inhibiting ubiquitination and doubling the protein half-life. A third COOH-terminal serine at position 195 has a modulating effect on both steady-state protein turnover and on ubiquitination induced by TNF-alpha. Thus, cellular levels of the NKX3.1 tumor suppressor are affected by inflammatory cytokines that target COOH-terminal serine residues to activate ubiquitination and protein degradation. Our data suggest that strategies to inhibit inflammation or to inhibit effector kinases may be useful approaches to prostate cancer prevention.     

Downregulation of epidermal growth factor receptor signaling by singlet oxygen through activation of caspase-3 and protein phosphatases

Downregulation of survival signaling pathways contributes to the cytotoxicity of reactive oxygen species (ROS) and may underlie certain therapies for hyperproliferative diseases. We have investigated the role of singlet oxygen, an ROS formed by photosensitization, in the regulation of survival signaling via the epidermal growth factor receptor (EGFR). Exposure of human keratinocytes to singlet oxygen resulted in rapid loss of EGFR, which was not blocked by either inhibition of receptor internalization or by interrupting the major proteolytic pathways (proteasome, lysosome or calpain). However, pretreatment with a caspase-3 inhibitor, DEVD-FMK, inhibited EGFR degradation. Caspase-3 cleavage was detected as early as 5 min after singlet oxygen treatment, and recombinant active caspase-3 completely cleaved EGFR in a keratinocyte membrane fraction. The singlet oxygen-induced loss of EGFR was accompanied by dephosphorylation of EGFR as well as of Akt and extracellular signal-regulated kinase 1/2 (ERK)1/2. Singlet oxygen-induced protein dephosphorylation was not dependent on activation of caspase-3. In contrast, inhibition of protein phosphatases (PPs) with okadaic acid completely blocked dephosphorylation of EGFR, ERK1/2 and Akt as well as degradation of EGFR. These results indicate that the oxidative stress produced by singlet oxygen rapidly disrupts EGFR-mediated signaling by decreasing both the protein level and its phosphorylation. These responses depended on intertwined activation of caspase-3 and PPs.     

The role of homeostatic regulation between tumor suppressor DAB2IP and oncogenic Skp2 in prostate cancer growth

Altered DAB2IP gene expression often detected in prostate cancer (PCa) is due to epigenetic silencing. In this study, we unveil a new mechanism leading to the loss of DAB2IP protein; an oncogenic S-phase kinase-associated protein-2 (Skp2) as E3 ubiquitin ligase plays a key regulator in DAB2IP degradation. In order to unveil the role of Skp2 in the turnover of DAB2IP protein, both prostate cell lines and prostate cancer specimens with a variety of molecular and cell biologic techniques were employed. We demonstrated that DAB2IP is regulated by Skp2-mediated proteasome degradation in the prostate cell lines. Further analyses identified the N-terminal DAB2IP containing the ubiquitination site. Immunohistochemical study exhibited an inverse correlation between DAB2IP and Skp2 protein expression in the prostate cancer tissue microarray. In contrast, DAB2IP can suppress Skp2 protein expression is mediated through Akt signaling. The reciprocal regulation between DAB2IP and Skp2 can impact on the growth of PCa cells. This reciprocal regulation between DAB2IP and Skp2 protein represents a unique homeostatic balance between tumor suppressor and oncoprotein in normal prostate epithelia, which is apparently altered in cancer cells. The outcome of this study has identified new potential targets for developing new therapeutic strategy for PCa.