Jamie S. Mader

Cationic antimicrobial peptides as novel cytotoxic agents for cancer treatment.

Cancer treatment by conventional chemotherapy is hindered by toxic side effects and the frequent development of multi-drug resistance by cancer cells. Cationic antimicrobial peptides (CAPs) are a promising new class of natural-source drugs that may avoid the shortcomings of conventional chemotherapy because certain CAPs exhibit selective cytotoxicity against a broad spectrum of human cancer cells, including neoplastic cells that have acquired a multi-drug-resistant phenotype. Tumour cell killing by CAPs is usually by a cell membrane-lytic effect, although some CAPs can trigger apoptosis in cancer cells via mitochondrial membrane disruption. Furthermore, certain CAPs are potent inhibitors of blood vessel development (angiogenesis) that is associated with tumour progression. This article reviews the mechanisms by which CAPs exert anticancer activity and discusses the potential application of selected CAPs as therapeutic agents for the treatment of human cancers.

Bovine lactoferricin selectively induces apoptosis in human leukemia and carcinoma cell lines

Bovine lactoferricin (LfcinB) is a cationic, amphipathic peptide that is cytotoxic for human and rodent cancer cells. However, the mechanism by which LfcinB causes the death of cancer cells is not well understood. Here, we show that in vitro treatment with LfcinB rapidly induced apoptosis in several different human leukemia and carcinoma cell lines as determined by DNA fragmentation assays and phosphatidylserine headgroup inversion detected by Annexin V binding to the surface of cancer cells. Importantly, LfcinB treatment did not adversely affect the viability of untransformed human lymphocytes, fibroblasts, or endothelial cells. Studies with different LfcinB-derived peptide fragments revealed that the cytotoxic activity of LfcinB resided within the amino acid sequence FKCRRWQWRM. Treatment of Jurkat T leukemia cells with LfcinB resulted in the production of reactive oxygen species followed by caspase-2-induced dissipation of mitochondrial transmembrane potential and subsequent activation of caspase-9 and caspase-3. Selective inhibitors of caspase-2 (Z-VDVAD-FMK), caspase-9 (Z-LEHD-FMK), and caspase-3 (Z-DEVD-FMK) protected both leukemia and carcinoma cells from LfcinB-induced apoptosis. Conversely, a caspase-8 inhibitor (Z-IETD-FMK) had no effect, which argued against a role for caspase-8 and was consistent with the finding that death receptors were not involved in LfcinB-induced apoptosis. Furthermore, Jurkat T leukemia cells that overexpressed Bcl-2 were less sensitive to LfcinB-induced apoptosis, which was characterized by mitochondrial swelling and the release of cytochrome c from mitochondria into the cytosolic compartment. We conclude that LfcinB kills cancer cells by triggering the mitochondrial pathway of apoptosis at least in part through the generation of reactive oxygen species.

Liposome reconstitution of a minimal protein-mediated membrane fusion machine

Biological membrane fusion is dependent on protein catalysts to mediate localized restructuring of lipid bilayers. A central theme in current models of protein‐mediated membrane fusion involves the sequential refolding of complex homomeric or heteromeric protein fusion machines. The structural features of a new family of fusion‐associated small transmembrane (FAST) proteins appear incompatible with existing models of membrane fusion protein function. While the FAST proteins function to induce efficient cell–cell fusion when expressed in transfected cells, it was unclear whether they function on their own to mediate membrane fusion or are dependent on cellular protein cofactors. Using proteoliposomes containing the purified p14 FAST protein of reptilian reovirus, we now show via liposome–cell and liposome–liposome fusion assays that p14 is both necessary and sufficient for membrane fusion. Stoichiometric and kinetic analyses suggest that the relative efficiency of p14‐mediated membrane fusion rivals that of the more complex cellular and viral fusion proteins, making the FAST proteins the simplest known membrane fusion machines.

Adenosine inhibits activation-induced T cell expression of CD2 and CD28 co-stimulatory molecules: role of interleukin-2 and cyclic AMP signaling pathways.

Adenosine is an immunosuppressive molecule that is associated with the microenvironment of solid tumors. Mouse T cells activated with anti‐CD3 antibody in the presence of adenosine with or without coformycin (to prevent adenosine breakdown by adenosine deaminase) exhibited decreased tyrosine phosphorylation of some intracellular proteins and were inhibited in their ability to proliferate and synthesize interleukin (IL)‐2. In addition, adenosine interfered with activation‐induced expression of the co‐stimulatory molecules CD2 and CD28. Activation‐induced CD2 and CD28 expression was also diminished when T cells were activated in the presence of anti‐IL‐2 and anti‐CD25 antibodies to neutralize IL‐2 bioactivity. Collectively, these data suggest that CD2 and CD28 up‐regulation following T cell activation is IL‐2‐dependent; and that adenosine inhibits activation‐induced T cell expression of CD2 and CD28 by interfering with IL‐2‐dependent signaling. The inhibitory effect of adenosine on activation‐induced CD2 and CD28 expression could not be attributed to cyclic AMP (cAMP) accumulation resulting from the stimulation of adenylyl cyclase‐coupled adenosine receptors, even though cAMP at concentrations much higher than those generated following adenosine stimulation was inhibitory for both CD2 and CD28 expression. We conclude that adenosine interferes with IL‐2‐dependent T cell expression of co‐stimulatory molecules via a mechanism that does not involve the accumulation of intracellular cAMP. J. Cell. Biochem. 89: 975–991, 2003.

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.

Thy-1 Signaling in the Context of Costimulation Provided by Dendritic Cells Provides Signal 1 for T Cell Proliferation and Cytotoxic Effector Molecule Expression, but Fails to Trigger Delivery of the Lethal Hit

Cross-linking of the GPI-anchored protein Thy-1 results in T cell proliferation and IL-2 synthesis. However, the exact function of Thy-1 in the process of T cell activation remains unknown, as does the effect of costimulation on Thy-1-driven T cell responses. In this study, we have investigated the ability of Thy-1 to substitute for traditional signal 1 in the context of costimulation provided by dendritic cells. Dendritic cells dramatically enhanced T cell proliferation and IL-2 synthesis in response to Thy-1 triggering by anti-Thy-1 mAb. This effect was not dependent on dendritic cell Fcγ receptors, but was a result of B7-mediated costimulation (signal 2). T cells were also activated when microbeads coated with a combination of anti-Thy-1 and anti-CD28 mAbs were used to supply signals 1 and 2, respectively. Thy-1-stimulated T cells adhere to target cells and express perforin, granzyme B, and Fas ligand, but fail to kill target cells due to an inability to reorganize their secretion machinery. Moreover, in contrast to TCR signaling, Thy-1 triggering failed to induce cytotoxicity in redirected lysis assays. We conclude that Thy-1 triggering can partially substitute for signal 1, which, in combination with a strong signal 2, leads to robust T cell proliferation, IL-2 synthesis, and cytotoxic effector molecule expression, but does not induce cytolytic function. The block at the level of cytotoxic effector function that results when T cells are activated in the absence of a classical, Ag-specific signal 1 may constitute a mechanism to ensure the specificity of CTL responses and prevent potentially harmful promiscuous cytotoxicity.

Ryanodine receptor signaling is required for anti‐CD3‐induced T cell proliferation, interleukin‐2 synthesis, and interleukin‐2 receptor signaling

Ryanodine receptors (RyR) are involved in regulating intracellular Ca++ mobilization in T lymphocytes. However, the importance of RyR signaling during T cell activation has not yet been determined. In this study, we have used the RyR‐selective antagonists, ruthenium red and dantrolene, to determine the effect of RyR blockade on T cell receptor‐mediated activation events and cytokine‐dependent T cell proliferation. Both ruthenium red and dantrolene inhibited DNA synthesis and cell division, as well as the synthesis of interleukin (IL)‐2 by T lymphocytes responding to mitogenic anti‐CD3 antibody. Blockade of RyR at initiation of culture or as late as 24 h after T cell receptor stimulation inhibited T cell proliferation, suggesting a requirement for sustained RyR signaling during cell cycle progression. Although flow cytometry revealed that RyR blockade had little effect on activation‐induced expression of the α chain (CD25) of the high affinity IL‐2 receptor, the inhibitory effect of RyR antagonists could not be reversed by the addition of exogenous IL‐2 at initiation of culture. In addition, both ruthenium red and dantrolene had a strong inhibitory effect on IL‐2‐dependent proliferation of CTLL‐2 T cells. These data indicate that RyR are involved in regulating IL‐2 receptor signaling that drives T cell progression through the cell cycle. We conclude that RyR‐associated Ca++ signaling regulates T cell proliferation by promoting both IL‐2 synthesis and IL‐2‐dependent cell cycle progression.