merocyanine-dye and Leukemia--Promyelocytic--Acute

If the interplay between caspase proteases and mitochondria decide the fate of the cell during apoptosis, they may constitute useful molecular targets for novel drug design. We have shown that photoactivated merocyanine 540 (pMC540) triggers caspase-mediated apoptosis in HL60 leukemia and M14 melanoma cells. Because pMC540 is a mixture of photoproducts, we set out to purify the biologically active component(s) from this mixture and to investigate their ability to directly activate intracellular caspases and/or trigger mitochondrial events associated with apoptosis. Two photoproducts, namely C1 and C2, purified and characterized by mass spectroscopy and nuclear magnetic resonance (NMR) analysis, effectively induced apoptosis in HL60 and M14 cells. Interestingly, both C1 and C2 induced non-receptor-dependent activation of caspase 8, which was responsible for the downstream activation of caspase 3 and cell death. Both compounds induced the release of cytochrome C from mitochondria of tumor cells and from purified rat liver mitochondria; however, different mechanisms were operative in cytochrome C translocation in response to C1 or C2. C1-induced cytochrome C release was mediated by the mitochondrial permeability transition (MPT) pore and accompanied by a decrease in mitochondrial transmembrane potential (triangle uppsim), whereas cytochrome C release in response to C2 was independent of MPT pore opening. These findings do not exclude the possibility that changes in mitochondrial triangle uppsim are critical for apoptosis in some instances, but support the notion that this may not be a universal step in the apoptotic process. Thus, identification of two novel anticancer agents that directly activate effector components of the apoptotic pathway could have potential implications for the development of newer chemotherapeutic drugs.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Caspases; Cytochrome c Group; Enzyme Activation; Humans; Leukemia, Promyelocytic, Acute; Melanoma; Membrane Potentials; Mitochondria; Mitochondria, Liver; Photochemistry; Pyrimidinones; Rats; Tumor Cells, Cultured

Merocyanine 540 (MC540) is a photosensitizing dye that has been used in several preclinical models and in a phase I clinical trial for the extracorporeal purging of tumor cells from autologous bone marrow grafts. The mechanism of the cytotoxic activity of MC540 is not yet fully understood, and the subcellular targets of MC540-mediated photodynamic damage remain to be identified. The human neutrophil provides an attractive model with which to study the effects of photoactivated MC540 on several well-defined cellular functions. As we report in this paper, simultaneous exposure of neutrophils to MC540 and light inhibited phagocytosis, random migration, chemotaxis, hydrogen peroxide production, and oxygen consumption. By contrast, the ability of neutrophils to kill engulfed bacteria and to produce superoxide radical was not compromised. Intracellular ATP levels and the activities of the cytosolic enzymes superoxide dismutase, catalase, and myeloperoxidase were only slightly reduced. Even in HL-60 leukemia cells, which bind more dye and are more readily killed by MC540-mediated photodynamic therapy than neutrophils, superoxide dismutase, catalase, and myeloperoxidase activities remained at normal or near-normal levels. These results are compatible with the view that plasma membrane components are primary targets of MC540-mediated photodynamic damage.

Topics: Adenosine Triphosphate; Catalase; Chemotaxis, Leukocyte; Humans; Hydrogen Peroxide; Leukemia, Promyelocytic, Acute; Light; Neutrophils; Oxygen Consumption; Peroxidase; Phagocytosis; Photosensitizing Agents; Pyrimidinones; Superoxide Dismutase; Tumor Cells, Cultured

The potential of various photoradiation therapy for the in vitro purging of residual tumor cells from autologous bone marrow (BM) transplants is discussed in this paper. The results with fluorescent dyes, Dihematoporphyrin Ether (DHE) and Merocyanine-540 (MC-540) are detailed. Following photoradiation of cells with white light, both DHE and MC-540 showed high cytocidal activity towards lymphoid and myeloid neoplastic cells, but had significantly less effect on normal granulocyte-macrophage (CFU-GM), erythroid (BFU-E) and mixed colony-forming (CFU-GEMM) progenitor cells. Acute promyelocytic leukemia (HL-60), non-B, non-T, cALLa positive acute lymphoblastic leukemia (Reh), and diffuse histiocytic B-cell lymphoma (SK-DHL-2) cell lines were exposed to different drug concentrations in combination with white light at a constant illumination rate of 50,000 lux. With DHE doses varying from 2.0 to 2.5 ug/ml and MC-540 concentrations of 15 to 20 ug/ml, clonogenic tumor cells could be reduced by more than 4 logs, when treated alone or in mixtures with normal irradiated human marrow cells. However, preferential cytotoxicity towards neoplastic cells was highly dependent on the mode of light activation. MC-540 had no substantial effect on malignant lymphoid (SK-DHL-2) and myeloid (HL-60) cells, and on normal marrow myeloid (CFU-GM) precursors, when the drug incubation was performed in the dark and followed by light exposure of washed cells. Equal doses of MC-540 (15-20 ug/ml) could preferentially eliminate tumor cells under conditions of simultaneous light and drug treatment (30 minutes at 37 degrees C). Using DHE (2.5 ug/ml), 29.3%, 46.8%, and 27.5% of normal marrow CFU-GM, BFU-E, and CFU-GEMM, respectively, were spared, following sequential drug and light exposure of cells, while simultaneous treatment reduced both normal (CFU-GM) and neoplastic cells below the limits of detection. The data from various centers is briefly discussed with special emphasis on clinical trials. Our results provide a useful model for leukemia and lymphoma cells and suggest that these phototherapy experiments can be implemented into clinical trials.

Topics: Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Cell Survival; Colony-Forming Units Assay; Dihematoporphyrin Ether; Hematoporphyrins; Humans; Leukemia, Lymphoid; Leukemia, Promyelocytic, Acute; Lymphoma; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents; Transplantation, Autologous; Tumor Cells, Cultured