Suzanne J. Furlong

Curcumin-induced apoptosis in PC3 prostate carcinoma cells is caspase-independent and involves cellular ceramide accumulation and damage to mitochondria.

Curcumin, the principal curcuminoid of tumeric, has potent anticancer activity. To determine the mechanism of curcumin-induced cytotoxicity in prostate cancer cells, we exposed PC3 prostate carcinoma cells to 25 to 100 μ M curcumin for 24 to 72 h. Curcumin treatment of PC3 cells caused time- and dose-dependent induction of apoptosis and depletion of cellular reduced glutathione (GSH). Exogenous GSH and its precursor N-acetyl-cysteine, but not ascorbic acid (AA) or ebselen, decreased curcumin accumulation in PC3 cells and also prevented curcumin-induced DNA fragmentation. The failure of AA and ebselen to protect PC3 cells from curcumin-induced apoptosis argued against the involvement of reactive oxygen species; rather, GSH-mediated inhibition of curcumin-induced cytotoxicity was due to reduced curcumin accumulation in PC3 cells. Curcumin-treated PC3 cells showed apoptosis-inducing cellular ceramide accumulation and activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Caspase-3, caspase-8, and caspase-9 were activated, and cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria following curcumin treatment. Interestingly, curcumin-induced apoptosis was not prevented by p38 MAPK, JNK, or caspase inhibition. We conclude that curcumin-induced cytotoxicity was due to cellular ceramide accumulation and damage to mitochondria that resulted in apoptosis mediated by AIF and other caspase-independent processes.

Mast Cells Down-Regulate CD4+CD25+ T Regulatory Cell Suppressor Function via Histamine H1 Receptor Interaction

Mast cells promote both innate and acquired immune responses, but little is known about the effect of mast cells on T regulatory (Treg) cell function. In this study, we show for the first time that the capacity of murine CD4+CD25+ Treg cells to suppress in vitro proliferation by CD4+CD25− T responder (Tresp) cells in response to anti-CD3/anti-CD28 mAb-coated beads was reduced in the presence of syngeneic bone marrow-derived mast cells (BMMC) activated by FcεR cross-linking. Activated BMMC culture supernatants or exogenous histamine also inhibited Treg cell suppressor function while the histamine H1 receptor-specific antagonist loratadine, but not the H2 receptor-specific antagonist famotidine, restored Treg cell suppressor function in the presence of activated BMMC or activated BMMC culture supernatants. Moreover, treatment of Treg cells with loratadine, but not famotidine, rescued Treg cell suppressor function in the presence of exogenous histamine. In addition, the H1 receptor-specific agonist 2-pyridylethylamine dihydrochloride inhibited Treg cell suppressor function to an extent that was comparable to histamine, whereas the H2 receptor-specific agonist amthamine dihydrobromide was without effect. Both Treg cells and Tresp cells expressed H1 receptors. Exposure to histamine caused Treg cells to express lower levels of CD25 and the Treg cell-specific transcription factor Foxp3. Taken together, these data indicate that BMMC-elaborated histamine inhibited Treg cell suppressor function by signaling through the H1 receptor. We suggest that histamine released as a result of mast cell activation by microbial products might cause a transient decrease in Treg cell suppressor function, thereby enhancing the development of protective immunity.

Bovine lactoferricin induces caspase-independent apoptosis in human B-lymphoma cells and extends the survival of immune-deficient mice bearing B-lymphoma xenografts.

Although current treatments based on the use of B-cell-specific anti-CD20 monoclonal antibodies and aggressive combinatorial chemotherapy have improved the survival of patients suffering from B-cell non-Hodgkins lymphoma (NHL), some individuals fail to respond to treatment and relapses remain common. New and more effective treatments for B-cell NHL are therefore required. Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that is cytotoxic for several human tumor cell lines but does not harm healthy cells. Here we show that in vitro treatment with LfcinB caused Raji and Ramos human B-lymphoma cells to die by apoptosis, as indicated by DNA fragmentation, chromatin condensation, and nuclear disintegration. LfcinB killed B-lymphoma cells more efficiently at low serum concentrations and was inhibited in the presence of exogenous bovine serum albumin, suggesting partial neutralization of cationic LfcinB by anionic serum components. LfcinB-induced apoptosis in B-lymphoma cells was caspase-independent since caspase-3 activation was not detected by Western blotting and the general caspase inhibitor z-VAD-fmk did not prevent LfcinB-induced DNA fragmentation. Importantly, immune-deficient SCID/beige mice that were inoculated intravenously with Ramos B-lymphoma cells in order to model B-cell NHL exhibited extended survival following systemic administration of LfcinB, indicating that LfcinB warrants further investigation as a novel therapeutic agent for the possible treatment of B-cell NHL.

Signaling through TLR7 enhances the immunosuppressive activity of murine CD4+CD25+ T regulatory cells

Although signaling through certain TLRs is known to modulate the function of T lymphocytes, the effect of TLR7 stimulation on CD4+CD25+ Treg cell activity has not yet been elucidated. In this study, we show that mouse CD4+CD25+ Treg cells express TLR7 mRNA and protein. We therefore used the TLR7 agonists imiquimod, gardiquimod, and single‐stranded poly(U) to show that TLR7 stimulation enhanced the ability of murine Treg cells to suppress anti‐CD3/anti‐CD28 mAb‐coated bead‐stimulated proliferation of syngeneic CD4+CD25– Tresp cells. In contrast, imiquimod failed to enhance the suppressor function of Treg cells from mice deficient in the MyD88 adaptor protein involved in TLR7 and other TLR signal transduction. Imiquimod increased murine Treg cell‐mediated suppression of Tresp cell proliferation induced by anti‐TCRβ mAb in the presence of syngeneic BMDCs, and Treg cells from gardiquimod‐treated mice exhibited enhanced in vitro suppressor function. Moreover, levels of Tresp cell‐secreted IL‐2 and IFN‐γ were reduced further in the presence of Treg cells plus imiquimod in comparison with Treg cells alone. In addition, imiquimod treatment increased CD25 expression by Treg cells and caused exogenous IL‐2 to enhance Treg cell suppressor function. Furthermore, combined treatment with imiquimod and IL‐2 increased Foxp3 expression by Treg cells. Collectively, these findings suggest that TLR7 signaling enhanced the suppressor function of Treg cells by sensitizing Treg cells to IL‐2‐induced activation. We speculate that TLR7‐stimulated enhancement of Treg cell suppressor function may modulate host T cell responses against ssRNA viruses.

Bevacizumab diminishes experimental autoimmune encephalomyelitis by inhibiting spinal cord angiogenesis and reducing peripheral T-cell responses.

Abstract Angiogenesis in the animal model of multiple sclerosis experimental autoimmune encephalomyelitis (EAE) is regulated by vascular endothelial growth factor (VEGF) and angiopoietin-2. We determined whether VEGF blockade with the anti-VEGF monoclonal antibody bevacizumab could inhibit angiogenesis and affect peripheral pathogenic immune responses in EAE. Mice treated with bevacizumab from the time of onset of clinical signs showed reduced clinical and pathologic scores. Bevacizumab suppressed angiogenesis and reduced angiopoietin-2 expression at Day 21 but had no effect on VEGF upregulation at Day 14. Messenger RNA levels for the angiogenesis-related protein CD105 were increased at Day 14. Bevacizumab reduced vascular permeability in the spinal cord at Day 14 and Day 21. In peripheral lymph nodes, it induced retention of CD4-positive T cells and inhibited T-cell proliferation. It also reduced mononuclear cell infiltration into spinal cord and the relative proportion of T cells. Isolated lymphoid cells showed reduced secretion of the T-helper 17 (Th-17) cell cytokine interleukin 17 and the Th-1 cytokine interferon-&ggr;. When bevacizumab was added to naive T cells or to antigen-stimulated T cells from mice with untreated EAE in vitro, it had no effect on proliferation or the secretion of interleukin 17 or interferon-&ggr;. These data indicate that bevacizumab ameliorates vascular and T-cell responses during EAE, but its effects on T cells may be indirect, possibly by suppressing angiogenesis.

Role of mitogen-activated protein kinases in Thy-1-induced T-lymphocyte activation.

Thy-1 (CD90) crosslinking by monoclonal antibodies (mAb) in the context of costimulation causes the activation of mouse T-lymphocytes; however, the associated signal transduction processes have not been studied in detail. In this study we investigated the role of mitogen-activated protein kinases (MAPKs) in Thy-1-mediated T-lymphocyte activation using mAb-coated polystyrene microspheres to crosslink Thy-1 and costimulatory CD28 on murine T-lymphocytes. Concurrent Thy-1 and CD28 crosslinking induced DNA synthesis by T-lymphocytes, as well as interleukin (IL)-2 and IL-2 receptor (IL-2R) alpha chain (CD25) expression. Increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and c-Jun N-terminal protein kinase (JNK) was also observed. Pharmacologic inhibition of ERK1/2 or JNK activation inhibited Thy-1-induced DNA synthesis and IL-2 production by T-lymphocytes. p38 MAPK inhibition also decreased DNA synthesis in Thy-1-stimulated T-lymphocytes; however, IL-2 production was increased in these cells. Inhibition of JNK, but not ERK1/2 or p38 MAPK, caused a marked reduction in Thy-1-induced CD25 expression. In addition, inhibition of p38 MAPK or JNK, but not ERK1/2, impaired the growth of IL-2-dependent CTLL-2 T-lymphocytes but did not substantially affect CD25 expression. Finally, exogenous IL-2 reversed the inhibitory effect of ERK1/2 or JNK inhibition on Thy-1-stimulated DNA synthesis by T-lymphocytes but did not substantially reverse JNK inhibition of CD25 expression. Collectively, these results suggest that during Thy-1-induced T-lymphocyte activation, ERK1/2 and JNK promoted IL-2 production whereas p38 MAPK negatively regulated IL-2 expression. JNK signalling was also required for CD25 expression. IL-2R signalling involved both p38 MAPK and JNK in CTLL-2 cells, whereas p38 MAPK was most important for IL-2R signalling in primary T-lymphocytes. MAPKs are therefore essential signalling intermediates for the Thy-1-driven proliferation of mouse T-lymphocytes.

Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4(+) T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 (α chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca(2+) release to inhibit IκB phosphorylation, which is required for nuclear translocation of the transcription factor NFκB. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4(+)CD25(+) regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

The Ca2+ channel blocker flunarizine induces caspase-10-dependent apoptosis in Jurkat T-leukemia cells

Flunarizine is a Ca2+ channel blocker that can be either cytoprotective or cytotoxic, depending on the cell type that is being examined. We show here that flunarizine was cytotoxic for Jurkat T-leukemia cells, as well as for other hematological maligancies, but not for breast or colon carcinoma cells. Treatment of Jurkat cells with flunarizine resulted in caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and laddering of DNA fragments, all of which are hallmarks of apoptosis. Flunarizine-induced DNA fragmentation was inhibited by the caspase-3 inhibitor z-DEVD-fmk, the caspase-8/caspase-10 inhibitor z-IETD-fmk, and the caspase-10 inhibitor z-AEVD-fmk, but was not reduced in caspase-8-deficient Jurkat cells, indicating the involvement of caspase-10 upstream of caspase-3 activation. Interestingly, FADD recruitment to a death receptor was not involved since flunarizine caused DNA fragmentation in FADD-deficient Jurkat cells. Flunarizine treatment of Jurkat cells also resulted in reactive oxygen species production, dissipation of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, and caspase-9 activation, although none of these events were necessary for apoptosis induction. Collectively, these findings indicate that flunarizine triggers apoptosis in Jurkat cells via FADD-independent activation of caspase-10. Flunarizine warrants further investigation as a potential anti-cancer agent for the treatment of hematological malignancies.

Differential Inhibition of T Lymphocyte Proliferation and Cytokine Synthesis by [6]-Gingerol, [8]-Gingerol, and [10]-Gingerol.

[6]‐Gingerol, [8]‐gingerol, and [10]‐gingerol are pungent components of fresh ginger, extracts of which inhibit various components of the inflammatory response. Because little is known regarding the effect of gingerols with different unbranched alkyl side chain lengths on the activation and effector function of T lymphocytes, we compared the effects of [6]‐gingerol, [8]‐gingerol, and [10]‐gingerol on murine T lymphocyte proliferation, expression of CD25 and CD69 activation markers, cytokine synthesis, and interleukin (IL)‐2 receptor signaling. All three gingerols inhibited DNA synthesis by T lymphocytes, as well as interferon‐γ synthesis. In contrast, only [8]‐gingerol and [10]‐gingerol inhibited CD25 and CD69 expression, and IL‐2 synthesis. None of the gingerols affected IL‐4 synthesis. Exogenous IL‐2 enhanced T lymphocyte proliferation in the presence of [6]‐gingerol but did not significantly increase T lymphocyte proliferation in the presence of [8]‐gingerol or [10]‐gingerol. In line with this finding, [8]‐gingerol and [10]‐gingerol impaired IL‐2‐induced proliferation of CTLL‐2 cells, but constitutive CD25 expression was unaffected, indicating inhibition of IL‐2 receptor signaling. In general, [10]‐gingerol and [8]‐gingerol were more potent inhibitors of T lymphocytes than [6]‐gingerol. Suppression of T lymphocyte responses by gingerols suggests that these phytochemicals may be beneficial in chronic inflammatory conditions associated with excessive or inappropriate T lymphocyte activation. Copyright

Murine experimental autoimmune encephalomyelitis is diminished by treatment with the angiogenesis inhibitors B20-4.1.1 and angiostatin (K1-3).

Angiogenesis is the formation of new blood vessels form pre-existing vasculature whose contribution to inflammatory conditions of the Central Nervous System is being studied in order to generate novel therapeutic targets. This study is the first to investigate the impact of two particular angiogenesis inhibitors on murine Experimental Autoimmune Encephalomyelitis (EAE), an inflammatory disease that mimics aspects of the human disease Multiple Sclerosis. The inhibitors were chosen to reduce angiogenesis by complimentary means. Extrinsic factors were targeted with B20-4.1.1 through its ability to bind to murine Vascular Endothelial Growth Factor (VEGF). Vascular processes connected to angiogenesis were targeted directly with K(1-3), the first three kringle domains of angiostatin. Mice treated with B20-4.1.1 and K(1-3) from onset of signs had reduced clinical scores 18–21 days after EAE induction. Both agents suppressed spinal cord angiogenesis without effect on local VEGF expression. B20-4.1.1 reduced spinal cord vascular permeability while K(1-3) had no effect. T cell infiltration into the spinal cord at day 21 was unaffected by either treatment. B20-4.1.1 reduced peripheral T cell proliferation while K(1-3) had no effect. Lymphoid cells from treated mice produced reduced levels of the T helper-17 (Th-17) cell cytokine interleukin (IL)-17 with no effect on the Th-1 cytokine interferon (IFN)-γ or Th-2 cytokine IL-4. However, when both drugs were added in vitro to naive T cells or to antigen stimulated T cells from mice with untreated EAE they had no effect on proliferation or levels of IL-17 or IFN-γ. We conclude that these angiogenesis inhibitors mitigate EAE by both suppressing spinal cord angiogenesis and reducing peripheral T cell activation.