The resistance of many human cancers to immunebased therapies, including adoptive immunotherapy and the administration of therapeutic cancer vaccines, has been attributed to tumor-associated immune suppression, due in part to immunosuppressive molecules located within the tumor microenvironment. Adenosine is a purine nucleoside found within the interstitial fluid of solid tumors at concentrations that are able to inhibit cell-mediated immune responses to tumor cells. It is well established that extracellular adenosine inhibits T lymphocyte activation and effector function, including T cell adhesion to tumor cells and cytotoxic activity, by signaling primarily through A 2a and A 3 adenosine receptors on the surface of T cells. Importantly, A 2a adenosine receptordeficient mice exhibit enhanced anti-tumor immune responses by CD8 + T cells, as well as a dramatic reduction in the growth of experimental tumors in comparison to wild-type controls. A 2a adenosine receptor signaling has also been implicated in adenosine-mediated inhibition of cytokine production and cytotoxic activity by activated natural killer (NK) cells, although the process of NK cell granule exocytosis is apparently suppressed via a distinct and as yet uncharacterized adenosine receptor. In this report, we review the evidence that adenosine is a potent inhibitor of cellular immune responses and may therefore be a major barrier to the successful immunotherapy of human carcinomas. The signaling pathways through which adenosine exerts its inhibitory effects on cell-mediated immune responses are also discussed. The accumulated evidence suggests that the effectiveness of immune-based therapies for solid tumors may be enhanced by selective antagonism of the adenosine receptor subtypes through which adenosine inhibits the antitumor activity of T lymphocytes and NK cells. Contents

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.

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 microM 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.

Bovine lactoferricin (LfcinB) is a cationic peptide that selectively induces caspase-dependent apoptosis in human leukemia and carcinoma cell lines. Ceramide is a second messenger in apoptosis signaling that has been shown to increase the cytotoxicity of various anti-cancer drugs. In this study, we determined whether manipulation of intracellular ceramide levels enhanced LfcinB-induced apoptosis of estrogen-nonresponsive MDA-MB-435 breast carcinoma cells. LfcinB caused DNA fragmentation and morphological changes consistent with apoptosis in MDA-MB-435 breast cancer cell cultures, but did not affect the viability of untransformed mammary epithelial cells. MDA-MB-435 breast cancer cells also exhibited DNA fragmentation and morphological changes consistent with apoptosis following exposure to the cell-permeable ceramide analog C 6. An additive increase in DNA fragmentation was observed when both LfcinB and C 6 ceramide were added to MDA-MB-435 breast cancer cell cultures. A greater than additive increase in DNA fragmentation was seen when LfcinB was used in combination with tamoxifen, which prevents the metabolism of endogenous ceramide to glucosylceramide by glucosylceramide synthase, as well as blocking estrogen receptor signaling. However, a selective inhibitor of glucosyl-ceramide synthase,1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, failed to further increase DNA fragmentation by LfcinB, suggesting that tamoxifen enhanced LfcinB-induced apoptosis in breast cancer cells via a mechanism that did not involve glucosylceramide synthase inhibition. We conclude that combination therapy with LfcinB and tamoxifen warrants further investigation for possible use in the treatment of breast cancer.

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-γ. 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-γ. 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.