Biomedical subjects

Nicholas Mitsiades

Publications and source records attributed to Nicholas Mitsiades.

At least 55 records · Page 3Linked to original sources

PubMed2002

Essential role of caveolae in interleukin-6- and insulin-like growth factor I-triggered Akt-1-mediated survival of multiple myeloma cells.

Caveolae, specialized flask-shaped lipid rafts on the cell surface, are composed of cholesterol, sphingolipids, and structural proteins termed caveolins; functionally, these plasma membrane microdomains have been implicated in signal transduction and transmembrane transport. In the present study, we examined the role of caveolin-1 in multiple myeloma cells. We show for the first time that caveolin-1, which is usually absent in blood cells, is expressed in multiple myeloma cells. Analysis of myeloma cell-derived plasma membrane fractions shows that caveolin-1 is co-localized with interleukin-6 receptor signal transducing chain gp130 and with insulin-like growth factor-I receptor. Cholesterol depletion by beta-cyclodextrin results in the loss of caveola structure in myeloma cells, as shown by transmission electron microscopy, and loss of caveolin-1 function. Interleukin-6 and insulin-like growth factor-I, growth and survival factors in multiple myeloma, induce caveolin-1 phosphorylation, which is abrogated by pre-treatment with beta-cyclodextrin. Importantly, inhibition of caveolin-1 phosphorylation blocks both interleukin-6-induced protein complex formation with caveolin-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. beta-Cyclodextrin also blocks insulin-like growth factor-I-induced tyrosine phosphorylation of insulin-responsive substrate-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. Therefore, cholesterol depletion by beta-cyclodextrin abrogates both interleukin-6- and insulin-like growth factor-I-triggered multiple myeloma cell survival via negative regulation of caveolin-1. Taken together, this study identifies caveolin-1 and other structural membrane components as potential new therapeutic targets in multiple myeloma.

Caveolae↗

PubMed2002

Identification of genes regulated by 2-methoxyestradiol (2ME2) in multiple myeloma cells using oligonucleotide arrays.

Our previous study demonstrated that 2-methoxyestradiol (2ME2), an estrogen derivative, induces apoptosis in multiple myeloma (MM) cells; however, the related transcriptional events are unclear. In the present study, we used oligonucleotide microarrays to identify genes altered during 2ME2-induced apoptosis in MM cells. 2ME2 triggers an early transient induction of genes known to trigger cell death and repression of growth/survival-related genes. Many genes regulating cell defense/repair machinery also were transiently induced. Since 2ME2 also induces apoptosis in MM cells resistant to conventional therapies such as dexamethasone (Dex), we compared the gene profiles of 2ME2-treated and Dex-resistant MM cells. Our results suggest that 2ME2 overcomes Dex resistance by modulating genes that confer chemoresistance in MM cells. Microarray results were confirmed by Northern and Western blot analyses. A comparative analysis of selected genes from freshly isolated MM patient cells and 2ME2-treated MM.1S MM cells further provides an in vivo relevance of our in vitro studies. Collectively, these findings suggest genetic events mediating anti-MM activity of 2ME2, as well as mechanisms whereby 2ME2 overcomes Dex resistance in MM cells. These studies may therefore allow improved therapeutic use of 2ME2, based upon targeting genes that regulate MM cell growth and survival.

2-Methoxyestradiol↗

PubMed2002

Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocin-induced diabetes.

Diabetic macular edema, resulting from increased microvascular permeability, is the most prevalent cause of vision loss in diabetes. The mechanisms underlying this complication remain poorly understood. In the current study, diabetic vascular permeability (blood-retinal barrier breakdown) is demonstrated to result from a leukocyte-mediated Fas-FasL-dependent apoptosis of the retinal vasculature. Following the onset of streptozotocin-induced diabetes, FasL expression was increased in rat neutrophils (P<0.005) and was accompanied by a simultaneous increase in Fas expression in the retinal vasculature. Static adhesion assays demonstrated that neutrophils from diabetic, but not control, rats induced endothelial cell apoptosis in vitro (P<0.005). The latter was inhibited via an antibody-based FasL blockade (P<0.005). In vivo, the inhibition of FasL potently reduced retinal vascular endothelial cell injury, apoptosis, and blood-retinal barrier breakdown (P<0.0001) but did not diminish leukocyte adhesion to the diabetic retinal vasculature. Taken together, these data are the first to identify leukocyte-mediated Fas-FasL-dependent retinal endothelial cell apoptosis as a major cause of blood-retinal barrier breakdown in early diabetes. These data imply that the targeting of the Fas-FasL pathway may prove beneficial in the treatment of diabetic retinopathy.

Animals↗

PubMed2002

CD40 induces human multiple myeloma cell migration via phosphatidylinositol 3-kinase/AKT/NF-kappa B signaling.

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells in the bone marrow and their egress into peripheral blood with progression to plasma cell leukemia. Our previous study defined a functional role of CD40 activation in MM cell homing and migration. In this study, we examine signaling events mediating CD40-induced MM cell migration. We show that cross-linking CD40, using either soluble CD40L (sCD40L) or anti-CD40 monoclonal antibody (mAb), induces phosphatidylinositol 3-kinase (PI3K) activity and activates its downstream effector AKT in MM.1S cells. CD40 activation also activates the MAP kinase (MEK) pathway, evidenced by phosphorylation of extracellular signal-regulated mitogen-activated protein kinase (ERK), but not c-jun amino-terminal kinase (JNK) or p38, in a dose- and time-dependent manner. Using pharmacologic inhibitors of PI3K and MEK, as well as adenoviruses expressing dominant-negative and constitutively expressed AKT, we demonstrate that PI3K and AKT activities are required for CD40-induced MM cell migration. In contrast, inhibition of ERK/MEK phosphorylation only partially (10%-15%) prevents migration, suggesting only a minor role in regulation of CD40-mediated MM migration. We further demonstrate that CD40 induces nuclear factor (NF)-kappa B activation as a downstream target of PI3K/AKT signaling, and that inhibition of NF-kappa B signaling using specific inhibitors PS1145 and SN50 completely abrogates CD40-induced MM migration. Finally, we demonstrate that urokinase plasminogen activator (uPA), an NF-kappa B target gene, is induced by CD40; and conversely, that uPA induction via CD40 is blocked by PI3K and NF-kappa B inhibitors. Our data therefore indicate that CD40-induced MM cell migration is primarily mediated via activation of PI3K/AKT/NF-kappa B signaling, and further suggest that novel therapies targeting this pathway may inhibit MM cell migration associated with progressive MM.

CD40 Antigens↗

PubMed2002

The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications.

The proteasome inhibitor PS-341 inhibits nuclear factor-kappaB (NF-kappaB) activation, induces apoptosis in cancer cells, including multiple myeloma (MM) cells, and has marked clinical activity as a monotherapy for MM. In this study, we found that subtoxic concentrations of PS-341 potently sensitized MM cell lines and patient cells to DNA-damaging chemotherapeutic agents such as doxorubicin and melphalan, including cells resistant to these drugs and those isolated from a patient who had relapsed after PS-341 monotherapy. Moreover, PS-341 abolished cell adhesion-mediated drug resistance. Using gene expression profiling and proteomic analysis, we demonstrate that PS-341, among its other proapoptotic effects, down-regulates the expression of several effectors involved in the cellular response to genotoxic stress. These data suggest that, in addition to down-regulating the expression of apoptosis inhibitors, PS-341 inhibits genotoxic stress response pathways and thereby restores sensitivity to DNA-damaging chemotherapeutic agents. These studies, therefore, provide the framework for clinical use of this agent in combination with conventional chemotherapy.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2002

Molecular sequelae of proteasome inhibition in human multiple myeloma cells.

The proteasome inhibitor PS-341 inhibits IkappaB degradation, prevents NF-kappaB activation, and induces apoptosis in several types of cancer cells, including chemoresistant multiple myeloma (MM) cells. PS-341 has marked clinical activity even in the setting of relapsed refractory MM. However, PS-341-induced apoptotic cascade(s) are not yet fully defined. By using gene expression profiling, we characterized the molecular sequelae of PS-341 treatment in MM cells and further focused on molecular pathways responsible for the anticancer actions of this promising agent. The transcriptional profile of PS-341-treated cells involved down-regulation of growth/survival signaling pathways, and up-regulation of molecules implicated in proapoptotic cascades (which are both consistent with the proapoptotic effect of proteasome inhibition), as well as up-regulation of heat-shock proteins and ubiquitin/proteasome pathway members (which can correspond to stress responses against proteasome inhibition). Further studies on these pathways showed that PS-341 decreases the levels of several antiapoptotic proteins and triggers a dual apoptotic pathway of mitochondrial cytochrome c release and caspase-9 activation, as well as activation of Jun kinase and a Fas/caspase-8-dependent apoptotic pathway [which is inhibited by a dominant negative (decoy) Fas construct]. Stimulation with IGF-1, as well as overexpression of Bcl-2 or constitutively active Akt in MM cells also modestly attenuates PS-341-induced cell death, whereas inhibitors of the BH3 domain of Bcl-2 family members or the heat-shock protein 90 enhance tumor cell sensitivity to proteasome inhibition. These data provide both insight into the molecular mechanisms of antitumor activity of PS-341 and the rationale for future clinical trials of PS-341, in combination with conventional and novel therapies, to improve patient outcome in MM.

Antineoplastic Agents↗

PubMed2002

Molecular mechanisms mediating antimyeloma activity of proteasome inhibitor PS-341.

We have recently shown that proteasome inhibitor PS-341 induces apoptosis in drug-resistant multiple myeloma (MM) cells, inhibits binding of MM cells in the bone marrow microenvironment, and inhibits cytokines mediating MM cell growth, survival, drug resistance, and migration in vitro. PS-341 also inhibits human MM cell growth and prolongs survival in a SCID mouse model. Importantly, PS-341 has achieved remarkable clinical responses in patients with refractory relapsed MM. We here demonstrate molecular mechanisms whereby PS-341 mediates anti-MM activity by inducing p53 and MDM2 protein expression; inducing the phosphorylation (Ser15) of p53 protein; activating c-Jun NH(2)-terminal kinase (JNK), caspase-8, and caspase-3; and cleaving the DNA protein kinase catalytic subunit, ATM, and MDM2. Inhibition of JNK activity abrogates PS-341-induced MM cell death. These studies identify molecular targets of PS-341 and provide the rationale for the development of second-generation, more targeted therapies.

Antineoplastic Agents↗

PubMed2002

Proteasome inhibitor PS-341 inhibits human myeloma cell growth in vivo and prolongs survival in a murine model.

The proteasome is a ubiquitous and essential intracellular enzyme that degrades many proteins regulating cell cycle, apoptosis, transcription, cell adhesion,angiogenesis, and antigen presentation. We have shown recently that the proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human myeloma cells in vitro. In this study, we examined the efficacy, toxicity, and in vivo mechanism of action of PS-341 using a human plasmacytoma xenograft mouse model. One hundred immunodeficient (beige-nude-xid) mice were used in two independent experiments. The mice were injected s.c. with 3 x 10(7) RPMI-8226 myeloma cells. When tumors became measurable (9.2 days; range, 6-13 days after tumor injection), mice were assigned to treatment groups receiving PS-341 0.05 mg/kg (n = 13), 0.1 mg/kg (n = 15), 0.5 mg/kg (n = 14), or 1.0 mg/kg (n = 14) twice weekly via tail vein, or to control groups (n = 13) receiving the vehicle only. Significant inhibition of tumor growth, even with some complete tumor regression, was observed in PS-341-treated mice. The median overall survival was also significantly prolonged compared with controls (30 and 34 days for high dose-treated mice versus 14 days for controls; P < 0.0001). PS-341 was well tolerated up to 0.5 mg/kg, but some mice treated at 1.0 mg/kg became moribund and lost weight. Analysis of tumors harvested from treated animals showed that PS-341 induced apoptosis and decreased angiogenesis in vivo. These studies therefore demonstrate that PS-341 has significant in vivo antimyeloma activity at doses that are well tolerated in a murine model, confirming our in vitro data and further supporting the early clinical promise of PS-341 to overcome drug resistance and improve patient outcome.

Animals↗

PubMed2002

Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications.

Interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1) promote the proliferation of multiple myeloma (MM) cells and protect them against dexamethasone (Dex)-induced apoptosis. We have previously shown that Apo2 ligand/TNF-Related apoptosis inducing ligand (Apo2L/TRAIL) induces apoptosis of MM cells, including cells either sensitive or resistant to Dex and cytotoxic drugs, and overcomes the growth and survival effect of IL-6; conversely, NF-kappaB transcriptional activity attenuates their Apo2L/TRAIL-sensitivity. In the current study, we demonstrate that IGF-1 stimulates sustained activation of NF-kappaB and Akt; induces phosphorylation of the FKHRL-1 Forkhead transcription factor; upregulates a series of intracellular anti-apoptotic proteins including FLIP, survivin, cIAP-2, A1/Bfl-1, and XIAP; and decreases Apo2L/TRAIL-sensitivity of MM cells. In contrast, IL-6 does not cause sustained NF-kappaB activation, induces less pronounced Akt activation and FKHRL-1 phosphorylation than IGF-1, and increases the expression of only survivin. Forced overexpression of constitutively active Akt in MM-1S cells reduced their sensitivity to Apo2L/TRAIL and to doxorubicin (Doxo). In contrast, the Akt inhibitor IL-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate induced cell death of both Dex- and Doxo-sensitive and -resistant cells; opposed the protective effect of constitutive Akt activity against Apo2L/TRAIL; and abrogated the NF-kappaB activation, increase of anti-apoptotic proteins and protection against Apo2L/TRAIL induced by IGF-1. These findings therefore define an important role of the Akt pathway in modulating tumor cell responsiveness to Apo2L/TRAIL, delineate molecular mechanisms for the survival effects of IGF-1, and characterize differential pathophysiologic sequelae of IGF-1 vs IL-6 on MM cells. Importantly, they provide the basis for future clinical trials in MM combining conventional or novel agents with strategies designed to neutralize IGF-1.

Apoptosis↗

PubMed2002

Cytokines modulate telomerase activity in a human multiple myeloma cell line.

Telomerase is a ribonucleoprotein DNA polymerase that elongates the telomeres of chromosomes to compensate for losses that occur with each round of DNA replication and maintain chromosomal stability. Interleukin 6 (IL-6) and insulin-like growth factor 1 (IGF-1) are proliferative and survival factors for human multiple myeloma (MM) cells. To date, however, the effects of IGF-1 and IL-6 on telomerase activity and associated sequelae in MM cells have not been characterized. In this study, we evaluated the effects of IGF-1 and IL-6 on telomerase activity in MM cell lines (MM.1S, U266, and RPMI 8226), as well as patient MM cells. We show that these cytokines up-regulate telomerase activity without alteration of human telomerase reverse transcriptase (hTERT) protein expression. We also demonstrate that increased telomerase activity triggered by these cytokines is mediated by phosphatidylinositol 3'-kinase (PI3k)/Akt/nuclear factor kappaB (NFkappaB) signaling. We confirm involvement of PI3k/Akt/NFkappaB signaling because the PI3k inhibitors wortmannin and LY294002 or the inhibitor of NFkappaB (IkappaB) kinase inhibitor PS-1145 block constitutive and cytokine-induced up-regulation of telomerase activity. Furthermore, we show that dexamethasone (Dex) reduces telomerase activity through the inhibition of hTERT expression before the induction of apoptosis. Importantly, IGF-1 and IL-6 abrogate Dex-induced down-regulation of telomerase activity and apoptosis. The protective effect of those cytokines against Dex-induced down-regulation of telomerase activity is blocked by both wortmannin and PS-1145, whereas the protection against Dex-induced apoptosis is blocked by wortmannin but not PS-1145. Therefore, our results demonstrate that telomerase activity is related not only to transcriptional regulation of hTERT by NFkappaB but also to posttranscriptional regulation because of phosphorylation of hTERT by Akt kinase. These studies therefore demonstrate that telomerase activity is associated with cell growth, survival, and drug resistance in MM cells.

Apoptosis↗

PubMed2002

Apoptotic signaling induced by immunomodulatory thalidomide analogs in human multiple myeloma cells: therapeutic implications.

Thalidomide (Thal) achieves responses even in the setting of refractory multiple myeloma (MM). Although increased angiogenesis in MM bone marrow and the antiangiogenic effect of Thal formed the empiric basis for its use in MM, we have shown that Thal and its immunomodulatory analogs (IMiDs) directly induce apoptosis or growth arrest of MM cells, alter adhesion of MM cells to bone marrow stromal cells, inhibit the production of cytokines (interleukin-6 and vascular endothelial growth factor) in bone marrow, and stimulate natural killer cell anti-MM immunity. In the present study, we demonstrate that the IMiDs trigger activation of caspase-8, enhance MM cell sensitivity to Fas-induced apoptosis, and down-regulate nuclear factor (NF)-kappa B activity as well as expression of cellular inhibitor of apoptosis protein-2 and FLICE inhibitory protein. IMiDs also block the stimulatory effect of insulinlike growth factor-1 on NF-kappa B activity and potentiate the activity of TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), dexamethasone, and proteasome inhibitor (PS-341) therapy. These studies both delineate the mechanism of action of IMiDs against MM cells in vitro and form the basis for clinical trials of these agents, alone and coupled with conventional and other novel therapies, to improve outcome in MM.

Adjuvants, Immunologic↗

PubMed2002

Biologic sequelae of nuclear factor-kappaB blockade in multiple myeloma: therapeutic applications.

The transcription factor nuclear factor-kappaB (NF-kappaB) confers significant survival potential in a variety of tumors. Several established or novel anti-multiple myeloma (anti-MM) agents, such as dexamethasone, thalidomide, and proteasome inhibitors (PS-341), inhibit NF-kappaB activity as part of their diverse actions. However, studies to date have not delineated the effects of specific inhibition of NF-kappaB activity in MM. We therefore investigated the effect of SN50, a cell-permeable specific inhibitor of NF-kappaB nuclear translocation and activity, on MM cells. SN50 induced apoptosis in MM cell lines and patient cells; down-regulated expression of Bcl-2, A1, X-chromosome-linked inhibitor-of-apoptosis protein (XIAP), cellular inhibitor-of-apoptosis protein 1 (cIAP-1), cIAP-2, and survivin; up-regulated Bax; increased mitochondrial cytochrome c release into the cytoplasm; and activated caspase-9 and caspase-3, but not caspase-8. We have previously demonstrated that tumor necrosis factor-alpha (TNF-alpha) is present locally in the bone marrow microenvironment and induces NF-kappaB-dependent up-regulation of adhesion molecules on both MM cells and bone marrow stromal cells, with resultant increased adhesion. In this study, TNF-alpha alone induced NF-kappaB nuclear translocation, cIAP-1 and cIAP-2 up-regulation, and MM cell proliferation; in contrast, SN50 pretreatment sensitized MM cells to TNF-alpha-induced apoptosis and cleavage of caspase-8 and caspase-3, similar to our previous finding of SN50-induced sensitization to apoptosis induced by the TNF-alpha family member TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L. Moreover, SN50 inhibited TNF-alpha-induced expression of another NF-kappaB target gene, intercellular adhesion molecule-1. Although the p38 inhibitor PD169316 did not directly kill MM cells, it potentiated the apoptotic effect of SN50, suggesting an interaction between the p38 and NF-kappaB pathways. Our results therefore demonstrate that NF-kappaB activity in MM cells promotes tumor-cell survival and protects against apoptotic stimuli. These studies provide the framework for targeting NF-kappaB activity in novel biologically based therapies for MM.

Apoptosis↗

PubMed2002

Intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human multiple myeloma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2 ligand) effectively kills multiple myeloma (MM) cells in vitro irrespective of refractoriness to dexamethasone and chemotherapy. Because clinical trials with this anticancer agent are expected shortly, we investigated the signaling pathway of TRAIL-induced apoptosis in MM. We detected rapid cleavage of caspases-8, -9, -3, and -6, as well as the caspase substrates poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor-45 (DFF45), but not caspase-10, upon TRAIL treatment in sensitive MM cells, pointing to caspase-8 as the apical caspase of TRAIL signaling in MM cells. These phenomena were not observed or were significantly delayed in TRAIL-resistant MM cells, suggesting that resistance may arise from inhibition at the level of caspase-8 activation. Higher levels of expression for various apoptosis inhibitors, including FLICE-inhibitory protein (FLIP), and lower procaspase-8 levels were present in TRAIL-resistant cells and sensitivity was restored by the protein synthesis inhibitor cycloheximide (CHX) and the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), which both lowered FLIP and cellular inhibitor of apoptosis protein-2 (cIAP-2) protein levels. Forced expression of procaspase-8 or FLIP antisense oligonucleotides also sensitized TRAIL-resistant cells to TRAIL. Moreover, the cell permeable nuclear factor (NF)-kappaB inhibitor SN50, which sensitizes TRAIL-resistant cells to TRAIL, also inhibited cIAP2 protein expression. Finally, CHX, BIM, and SN50 facilitated the cleavage and activation of procaspase-8 in TRAIL-resistant cells, confirming that inhibition of TRAIL-induced apoptosis occurs at this level and that these agents sensitize MM cells by relieving this block. Our data set a framework for the clinical use of approaches that sensitize MM cells to TRAIL by agents that inhibit FLIP and cIAP-2 expression or augment caspase-8 activity.

Antineoplastic Agents↗

PubMed2002

NF-kappa B as a therapeutic target in multiple myeloma.

We have shown that thalidomide (Thal) and its immunomodulatory derivatives (IMiDs), proteasome inhibitor PS-341, and As(2)O(3) act directly on multiple myeloma (MM) cells and in the bone marrow (BM) milieu to overcome drug resistance. Although Thal/IMiDs, PS-341, and As(2)O(3) inhibit nuclear factor (NF)-kappaB activation, they also have multiple and varied other actions. In this study, we therefore specifically address the role of NF-kappaB blockade in mediating anti-MM activity. To characterize the effect of specific NF-kappaB blockade on MM cell growth and survival in vitro, we used an IkappaB kinase (IKK) inhibitor (PS-1145). Our studies demonstrate that PS-1145 and PS-341 block TNFalpha-induced NF-kappaB activation in a dose- and time-dependent fashion in MM cells through inhibition of IkappaBalpha phosphorylation and degradation of IkappaBalpha, respectively. Dexamethasone (Dex), which up-regulates IkappaBalpha protein, enhances blockade of NF-kappaB activation by PS-1145. Moreover, PS-1145 blocks the protective effect of IL-6 against Dex-induced apotosis. TNFalpha-induced intracellular adhesion molecule (ICAM)-1 expression on both RPMI8226 and MM.1S cells is also inhibited by PS-1145. Moreover, PS-1145 inhibits both IL-6 secretion from BMSCs triggered by MM cell adhesion and proliferation of MM cells adherent to BMSCs. However, in contrast to PS-341, PS-1145 only partially (20-50%) inhibits MM cell proliferation, suggesting that NF-kappaB blockade cannot account for all of the anti-MM activity of PS-341. Importantly, however, TNFalpha induces MM cell toxicity in the presence of PS-1145. These studies demonstrate that specific targeting of NF-kappaB can overcome the growth and survival advantage conferred both by tumor cell binding to BMSCs and cytokine secretion in the BM milieu. Furthermore, they provide the framework for clinical evaluation of novel MM therapies based upon targeting NF-kappaB.

Antineoplastic Agents↗

PubMed2002

Nonsteroidal anti-inflammatory drugs prevent early diabetic retinopathy via TNF-alpha suppression.

Leukocyte adhesion to the diabetic retinal vasculature results in blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell injury and death. Intercellular adhesion molecule-1 (ICAM-1) and the leukocyte integrin CD18 are required for these processes. Diabetes was induced in Long Evans rats, resulting in a two- to threefold increase in retinal leukocyte adhesion. Following one week of diabetes, neutrophil CD11a, CD11b, and CD18 expression was increased significantly, as were retinal ICAM-1 levels. Animals were treated with aspirin, a cyclooxygenase 2 (COX-2) inhibitor (meloxicam), or a soluble tumor necrosis factor alpha (TNF-alpha) receptor/Fc construct (TNFR-Fc, etanercept). High-dose aspirin, etanercept, and high-dose meloxicam each reduced leukocyte adhesion and suppressed blood-retinal barrier breakdown. High-dose aspirin also reduced the expression of CD11a, CD11b, and CD18, whereas meloxicam and etanercept did not. High-dose aspirin, etanercept, and high-dose meloxicam each reduced retinal ICAM-1 expression. Aspirin and meloxicam both lowered retinal TNF-alpha levels. Notably, aspirin, meloxicam, and etanercept did not change retinal vascular endothelial growth factor levels. High-dose aspirin, etanercept and high-dose meloxicam, each suppressed the retinal expression of eNOS and the DNA-binding capacity of retinal nuclear factor-kappaB. High-dose aspirin also suppressed Erk kinase activity, which is involved in CD18 up-regulation. Taken together, these data identify COX-2 and TNF-alpha as operative in the early signature pathologies of diabetic retinopathy, a newly recognized inflammatory disease.

Animals↗

PubMed2002

Regulation of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in thyroid carcinoma cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand selectively kills neoplastic cells, including thyroid carcinoma cells (Mitsiades et al: Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res 2000, 60:4122-41299). We investigated the mechanisms regulating Apo2L/TRAIL-induced apoptosis in thyroid carcinoma cells, as well as the impact of insulin-like growth factor (IGF)-1, interferon-gamma, and TNF-alpha. We found that the emergence of resistance to Apo2L/TRAIL, after prolonged incubation with this cytokine, was associated with increased levels of FLICE inhibitory protein (FLIP), and was overcome by cycloheximide and bisindolylmaleimide, that specifically down-regulated FLIP expression, as well as by transfection of a FLIP anti-sense oligonucleotide. IGF-1 activated Akt; up-regulated the caspase inhibitors FLIP, cIAP-2, XIAP, and survivin; and attenuated Apo2L/TRAIL-induced apoptosis. This effect was inhibited by the IGF-1 receptor neutralizing antibody aIR3, the PI-3K inhibitor wortmannin, and the heat shock protein-90 chaperone inhibitor geldanamycin. Transfection of constitutively active Akt protected from TRAIL. Conversely, interferon-gamma and TNF-alpha had a sensitizing effect. We conclude that FLIP may negatively regulate Apo2L/TRAIL-induced apoptosis in thyroid carcinomas. Microenvironmental paracrine survival factors, such as IGF-1, up-regulate caspase inhibitors, including FLIP, and protect from Apo2L/TRAIL in a PI-3K/Akt-dependent manner. T helper-1 cytokines and compounds that selectively abrogate the IGF-1 signaling pathway may be helpful adjunct agents in Apo2L/TRAIL-based anti-cancer therapeutic regimens.

Antineoplastic Agents↗

PubMed2002

Constitutive nuclear factor-kappaB activity is crucial for human retinoblastoma cell viability.

Retinoblastoma (Rb) is the most common intraocular malignancy of childhood. Although systemic and intrathecal chemotherapy with local and cranial radiotherapy have improved overall survival, the prognosis for patients with central nervous system involvement is still poor. We investigated the role of the transcription factor nuclear factor (NF)-kappaB, which promotes cell survival in several other models, in the pathophysiology of Rb. The human Rb cell lines Y79 and WERI-Rb1 were treated with the cell permeable peptide SN50, that specifically inhibits the transcriptional activity of NF-kappaB by blocking its translocation into the nucleus. We found that NF-kappaB inhibition up-regulated Bax; down-regulated the anti-apoptotic proteins Bcl-2, A1, and cIAP-2; and induced loss of the mitochondrial transmembrane potential and caspase-independent, calpain-dependent apoptosis in Rb cells. Inhibition of the p38 kinase sensitized cells to SN50-induced cell death, whereas insulin-like growth factor-1 activated NF-kappaB and attenuated the proapoptotic effect of SN50. Finally, NF-kappaB inhibition sensitized Rb cells to doxorubicin. In conclusion, inhibition of NF-kappaB activity in Rb cells leads to loss of mitochondrial transmembrane potential and caspase-independent, calpain-dependent apoptosis. Therapeutic strategies targeting NF-kappaB could be beneficial in the clinical management of Rb, either alone or in combination with conventional chemotherapy.

Antineoplastic Agents↗

PubMed2002

Retinal vascular endothelial growth factor induces intercellular adhesion molecule-1 and endothelial nitric oxide synthase expression and initiates early diabetic retinal leukocyte adhesion in vivo.

Leukocyte adhesion to the diabetic retinal vasculature results in early blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell injury and death. Previous work has shown that intercellular adhesion molecule-1 (ICAM-1) and CD18 are required for these processes. However the relevant in vivo stimuli for ICAM-1 and CD18 expression in diabetes remain unknown. The current study investigated the causal role of endogenous vascular endothelial growth factor (VEGF) and nitric oxide in initiating these events. Diabetes was induced in Long-Evans rats with streptozotocin, resulting in a two- to threefold increase in retinal leukocyte adhesion. Confirmed diabetic animals were treated with a highly specific VEGF-neutralizing Flt-Fc construct (VEGF TrapA(40)). Retinal ICAM-1 mRNA levels in VEGF TrapA(40)-treated diabetic animals were reduced by 83.5% compared to diabetic controls (n = 5, P < 0.0001). VEGF TrapA(40) also potently suppressed diabetic leukocyte adhesion in retinal arterioles (47%, n = 11, P < 0.0001), venules (36%, n = 11, P < 0.0005), and capillaries (36%, n = 11, P < 0.001). The expression of endothelial nitric oxide synthase (eNOS), a downstream mediator of VEGF activity, was increased in diabetic retina, and was potently suppressed with VEGF TrapA(40) treatment (n = 8, P < 0.005). Further, VEGF TrapA(40) reduced the diabetes-related nitric oxide increases in the retinae of diabetic animals. The inhibition of eNOS with N-omega-nitro-L-arginine methyl ester also potently reduced retinal leukocyte adhesion. Although neutrophil CD11a, CD11b, and CD18 levels were increased in 1-week diabetic animals, VEGF TrapA(40) did not alter the expression of these integrin adhesion molecules. Taken together, these data demonstrate that VEGF induces retinal ICAM-1 and eNOS expression and initiates early diabetic retinal leukocyte adhesion in vivo. The inhibition of VEGF bioactivity may prove useful in the treatment of the early diabetic retinopathy.

Animals↗