Significance The formation of new blood vessels (neoangiogenesis) accompanies tissue regeneration and healing, but is also crucial for tumor growth, hence understanding how capillaries are stimulated to grow in response to local cues is essential for the much sought-after aim of controlling this process. We have elucidated a Ca 2+ signaling pathway involving NAADP, TPCs, and lysosomal Ca 2+ release activated in vascular endothelial cells by VEGF, the main angiogenic growth factor, and we show that the angiogenic response can be abolished, in cultured cells and in vivo, by inhibiting components of this signaling cascade. The specificity of this pathway in terms of VEGF receptor subtype, intracellular messengers, target channels and Ca 2+ storage organelles, offers new targets for novel antiangiogenic therapeutic strategies.

Toll-like receptors (TLRs) are known to play a key role in the innate immune system particularly in inflammatory response against invading pathogens. Recent reports strongly indicate that they play important roles in cancer cells. Prostate cancer represents one of the most common cancer for which no cure is available once metastatic and androgen refractory. Since TLR3 has been recently suggested as a possible therapeutic target in some cancer cell lines, we studied TLR3 expression and functionality in two human prostate cancer cell lines, LNCaP and PC3. We report that both cell lines express TLR3 and that the TLR3 agonist poly (I:C) activates mitogen-activated protein kinases and induces inhibition of proliferation as well as caspase-dependent apoptosis. By using pharmacological and genetic approaches, we demonstrate the involvement of TLR3 in poly (I:C)-induced effects. We also show that a novel interferon-independent pathway involving protein kinase C (PKC)-alpha activation, upstream of p38 and c-jun N-terminal kinase, is responsible for poly (I:C) pro-apoptotic effects on LNCaP cells. To our knowledge, this is the first report describing a role of PKC-alpha in poly (I:C)-mediated apoptosis. The comprehension of the mechanisms underlying TLR3-mediated apoptosis can contribute tools to develop new agonists useful for the treatment of prostate cancer.

We hypothesized that cytolytic T lymphocytes (CTL) may utilize extracellular ATP (ATPO) during the effector phase of the CTL-target cell interactions and that CTL could be the source of ATPO. It is demonstrated here that incubation of CTL with activating ligands [Con A or monoclonal antibody (mAb) to the T-cell antigen receptor (TCR)] results in the extracellular Ca2+-independent accumulation of the ATP0. The addition of the ATP-degrading enzymes into the mixture of CTL and target cells results in a strong inhibition of the CTL-mediated, TCR-triggered lethal-hit delivery to the target cell. In a parallel control experiment, the employed enzymes did not affect target cell-induced, TCR-triggered exocytosis of granules from CTL. Thus, the removal of ATPO with enzymes does not interfere with the activation of CTL by the target cell but does block lytic events. Cloned helper T lymphocytes also accumulate ATPO after incubation with anti-TCR mAb or Con A, suggesting the possibility that ATPO, which acts in concert with ectoprotein kinases and/or purinergic receptors, may be of general use as a messenger in cellular interactions of T lymphocytes.The molecular mechanisms of the cell-mediated cytotoxicity are still poorly understood. It is generally accepted (1) that the recognition of the target cell (TC) by the T-cell antigen receptor (TCR) is required for the triggering of the biochemical events leading to the "lethal-hit" delivery (2, 3), and a substantial amount of suggestive evidence supports the hypothesis that TC death is due to the exocytosis of the pore-forming proteins from cytolytic T lymphocytes (CTL) (4, 5).Exocytosis of cytolytic granules from CTL, as studied by using the serine esterase-release assay (6-10), was shown to be absolutely dependent on extracellular Ca2+ (10), while the TCR-mediated TC lysis can proceed even in the absence of extracellular Ca2+ (1,11,12). This leads to the conclusion that exocytosis of cytolytic granules may not be the only mechanism of CTL-mediated cytotoxicity (11-13).In search for the alternative mechanism ofcytotoxicity that would be consistent with the observations of the Ca2+-independent lethal-hit delivery, we suggested (14, 15) the possibility of extracellular ATP (ATPO) acting in concert with the cell-surface ATP0-receptors and/or ectoprotein kinases (14-20) as a part of the molecular interactions leading to the CTL-induced TC death. Additionally, we characterized the CTL ecto-ATPase (15) and found that ATPO can kill different tumor TC in both the presence and the absence of Ca2 , whereas CTL are much more resistant to the lysis of ATPO.To further implicate the ATPO in lymphocyte effector functions, it was necessary to explain the source of the ATPO and to demonstrate the effect ofATPO removal on the effector functions of CTL. It is shown here that activated CTL could be the source of the ATPO. Addition of the ATP-degrading enzymes (during the assay) blocks the cytolytic process but does not affect other processes that follow the TC-induced, TCR-mediated trans...