merocyanine-dye and Neuroblastoma

Although a requirement of zinc (Zn) for normal brain development is well documented, the extent to which Zn can modulate neuronal proliferation and apoptosis is not clear. Thus, we investigated the role of Zn in the regulation of these two critical events. A low Zn availability leads to decreased cell viability in human neuroblastoma IMR-32 cells and primary cultures of rat cortical neurons. This occurs in part as a consequence of decreased cell proliferation and increased apoptotic cell death. In IMR-32 cells, Zn deficiency led to the inhibition of cell proliferation through the arrest of the cell cycle at the G0/G1 phase. Zn deficiency induced apoptosis in both proliferating and quiescent neuronal cells via the intrinsic apoptotic pathway. Reductions in cellular Zn triggered a translocation of the pro-apoptotic protein Bad to the mitochondria, cytochrome c release, and caspase-3 activation. Apoptosis is the resultant of the inhibition of the prosurvival extracellular-signal-regulated kinase, the inhibition of nuclear factor-kappa B, and associated decreased expression of antiapoptotic proteins, and to a direct activation of caspase-3. A deficit of Zn during critical developmental periods can have persistent effects on brain function secondary to a deregulation of neuronal proliferation and apoptosis.

Topics: Analysis of Variance; Animals; Antioxidants; Apoptosis; Caspase 3; Cell Cycle; Cell Proliferation; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytochromes c; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Embryo, Mammalian; Female; Gene Expression Regulation; Humans; In Situ Nick-End Labeling; Mitogen-Activated Protein Kinases; Neuroblastoma; Neurons; Photosensitizing Agents; Pregnancy; Proto-Oncogene Proteins c-bcl-2; Pyrimidinones; Rats; Rats, Sprague-Dawley; Serine; Signal Transduction; Thioctic Acid; Time Factors; Zinc

Paediatric solid tumours exhibit steep dose-response curves to alkylating agents and are therefore considered candidates for high-dose chemotherapy and autologous stem cell support. There is growing evidence that autologous stem cell grafts from patients with solid tumours are frequently contaminated with live tumour cells. The objective of this study was to perform, in a preclinical purging model, an initial assessment of the safety and efficacy of a two-step purging procedure that combined Merocyanine 540-mediated photodynamic therapy (MC540-PDT) with a brief exposure to the alkyl-lysophospholipid, Edelfosine. Human and murine bone marrow cells and Neuro-2a murine neuroblastoma, SK-N-SH human neuroblastoma, SK-ES-1 and U-2 OS human osteosarcoma, G-401 and SK-NEP-1 human Wilms' tumour, and A-204 human rhabdomyosarcoma cells were exposed to a fixed dose of MC540-PDT followed by a brief incubation with graded concentrations of Edelfosine. Survival was subsequently assessed by in vitro clonal assay or, in the case of CD34-positive haematopoietic stem cells, by an immunohistochemical method. Combination purging with MC540-PDT and Edelfosine depleted all tumour cells by >4 log while preserving at least 15% of murine granulocyte/macrophage progenitors (CFU-GM), 34% of human CFU-GM, and 31% of human CD34-positive cells. The data suggest that combination purging with MC540-PDT and Edelfosine may be useful for the ex vivo purging of autologous stem cell grafts from patients with paediatric solid tumours.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Survival; Dose-Response Relationship, Drug; Hematopoietic Stem Cell Transplantation; Humans; Mice; Neuroblastoma; Osteosarcoma; Phospholipid Ethers; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Rhabdomyosarcoma; Tumor Cells, Cultured; Wilms Tumor

The effect of merocyanine 540 (Mc 540) mediated photoirradiation on both neoplastic and normal hemopoietic progenitor cells was studied. Bone marrow (BM) cells from children with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) at initial diagnosis, ALL in remission, neuroblastoma and normal children as well as cells of Reh-6 and HL-60 cell lines were incubated with Mc 540 in the presence of human albumin (HA) and exposed to different argon laser 514 nm doses. Cell survival was estimated using Trypan Blue supravital stain following a 24-h incubation and leukemic cell lines were studied in continuous cell cultures of 4 weeks duration. Our results showed that HA protects normal BM cells from Mc 540 mediated phototoxicity. A 99.9999% inhibition of Reh-6 and HL-60 was noted at irradiation doses where the corresponding mean survival of normal BM cells was 77.4 +/- 12 and 70.3 +/- 10%, respectively. BM leukemic cells from children with ALL and AML were also very sensitive to Mc 540 photoirradiation in contrast to neuroblastoma cells where only a three-fold reduction was observed. Finally, the survival of normal BM progenitors was 38% for colony forming unit erythroid CFU-E, 37% for burst forming unit erythroid BFU-E, 55% for CFU-GM and 29% for CFU-GEMM. In conclusion it seems that Mc 540 mediated photoirradiation in neoplastic cells exerts selective cytotoxicity and can be used in ex vivo purging of malignant cells in BM.

Topics: Bone Marrow; Bone Marrow Cells; Bone Marrow Purging; Cell Line; Cell Survival; Child; Dose-Response Relationship, Radiation; Hematopoietic Stem Cells; HL-60 Cells; Humans; Lasers; Leukemia, Myeloid, Acute; Neuroblastoma; Photolysis; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Pyrimidinones; Radiation-Sensitizing Agents

Mechanism of merocyanine 540 (MC540) mediated photosensitization in glioblastoma (U-87MG) and neuroblastoma (Neuro 2a) cells was investigated. Photoinduced lipid peroxidation was measured in the presence of mechanistic probes-deuterium oxide (D2O), sodium azide, superoxide dismutase (SOD), mannitol and sodium benzoate. In both the types of cells, the photoinduced lipid peroxidation was enhanced in D2O whereas it showed inhibition in the presence of sodium azide. SOD also inhibited the lipid peroxidation while sodium benzoate and mannitol had no effect. These results suggest that photosensitization of U-87MG and Neuro 2a cells by MC 540 involves both type I (free radical mediated) and type II (singlet oxygen mediated) mechanisms.

Topics: Animals; Drug Screening Assays, Antitumor; Glioblastoma; Humans; Lipid Peroxidation; Mice; Neuroblastoma; Photosensitizing Agents; Pyrimidinones; Tumor Cells, Cultured

In order to evaluate the selective killing of merocyanine 540 (MC 540) mediated photoirradiation in neoplastic cells, bone narrow cells from children with leukaemia or neuroblastoma and normal children as well as peripheral blood cells and Reh-6 and HL-60 cell lines were studied. Cell suspensions were incubated with MC 540 and exposed to various argon laser 514 nm doses. Cell survival was estimated with trypan blue supravital stain following a 24 h incubation and has been followed in continuous cell cultures of 4 weeks duration. Our results showed that the inhibition of survival of neoplastic haemopoietic cells by laser in the presence of MC 540 is proportional to the MC 540 and photoirradiation doses. A 99.9999% inhibition of Reh-6 and HL-60 was noted at irradiation doses where the corresponding mean survival of normal bone narrow cells was (33.6 +/- 15.5)% and (50.6 +/- 10.7)% respectively. Peripheral blood mononuclear cells were not sensitive to MC 540 mediated photoirradiation. The inhibition of survival of bone marrow metastatic neuroblastoma cells was (69.9 +/- 4.1)%. In conclusion, it seems that MC 540 mediated photoirradiation in neoplastic cells exerts selective cytotoxicity and can be used in ex vivo purging of malignant cells in the bone marrow.

Topics: Bone Marrow; Cell Line; Cell Survival; Child; Dose-Response Relationship, Radiation; Hematopoietic Stem Cells; HL-60 Cells; Humans; Kinetics; Leukemia; Light; Neuroblastoma; Photosensitizing Agents; Pyrimidinones; Tumor Cells, Cultured

Binding and photodynamic action of merocyanine 540 (MC540) has been studied in glioma (U-87MG) and neuroblastoma (Neuro 2A) cells as a function of dye concentration, incubation time of cells with MC540 and growth phase of cells. In the plateau phase, U-87MG cells accumulated more MC540 as compared to exponentially growing cells, whereas in Neuro 2A cells the opposite effect was observed. Exponentially growing U-87MG cells were more photosensitive than plateau phase cells. However, the photosensitivity of Neuro 2A cells was not dependent on the growth phase. Thus, MC540 mediated photosensitization may be useful for photodynamic therapy of brain tumours.

Topics: Fluorescent Dyes; Glioma; Neuroblastoma; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Staining and Labeling; Tumor Cells, Cultured

The neuroblastoma cell lines SK-N-LO, SK-PN-DW and IMR 5 were stained with Merocyanine 540 and exposed to white light. 99% of clonogenic tumour cells were destroyed while 60% of bone marrow progenitor cells survived this treatment.

Topics: Bone Marrow; Bone Marrow Purging; Cell Survival; Hematopoietic Stem Cells; Humans; Light; Neuroblastoma; Photochemotherapy; Photosensitizing Agents; Pyrimidinones; Tumor Cells, Cultured

6-Hydroxydopamine(6-OHDA) and Merocyanine-540(MC-540) have been used clinically for purging of neuroblastoma cells prior to autologous bone marrow transplantation. Both substances were found to be more toxic against neuroblastoma cells than against hematopoietic stem cells. The more pronounced cytotoxic effects of 6-OHDA against neuroblastoma cells were not caused by its selective uptake; the rapid autooxidation at physiological pH leads to the formation of H2O2 already in the incubation medium. Cytotoxic effects were not detected in short-time test systems (4 hour chromium-51 release assay) but only after longer incubation periods. In contrast, MC-540 proved to be toxic almost equally in short- and long-time test systems. 4-Hydroxynonenal(4-HNE) that may be formed in the plasma membrane subsequently to photoactivation of MC-540 was only slightly more toxic to neuroblastoma cells than to hematopoietic cells. Although the use of 6-OHDA and MC-540 in bone marrow purging has some limitations, the sensitivity of neuroblastoma cells against reactive oxygen compounds may be exploited more generally for therapy of this tumor.

Topics: Aldehydes; Antineoplastic Agents; Cell Survival; Hematopoietic Stem Cells; Humans; Hydrogen Peroxide; Hydroxydopamines; Neuroblastoma; Oxidopamine; Pyrimidinones; Tumor Cells, Cultured

Topics: Animals; Bone Marrow; Bone Marrow Transplantation; Hematopoietic Stem Cells; Humans; Leukemia; Leukemia, Experimental; Mice; Neoplastic Stem Cells; Neuroblastoma; Photochemistry; Preoperative Care; Pyrimidinones; Radiation-Sensitizing Agents; Tumor Cells, Cultured; Tumor Stem Cell Assay

The purpose of this study was to determine if photosensitization mediated by the fluorescent dye, merocyanine 540, could be used to preferentially kill murine neuroblastoma cells in simulated autologous remission marrow grafts. Simultaneous exposure of Neuro 2a or NB41A3 neuroblastoma cells to merocyanine 540 and white light reduced the concentration of in vitro-clonogenic tumor cells 50,000-fold. By contrast, the same treatment had little effect on the graft's ability to rescue lethally irradiated syngeneic hosts. Lethally irradiated C57BL/6J X A/J F1 mice transplanted with photosensitized mixtures of neuroblastoma cells and normal marrow cells (1:100 or 1:10) survived without developing neuroblastomas. It is conceivable that merocyanine 540-mediated photosensitization will prove useful for the extracorporeal purging of residual neuroblastoma cells from human autologous remission marrow grafts.

Topics: Animals; Bone Marrow; Bone Marrow Transplantation; Cell Line; Cell Survival; Female; Hematopoietic Stem Cells; Mice; Mice, Inbred C57BL; Neuroblastoma; Photochemotherapy; Pyrimidinones; Radiation-Sensitizing Agents; Transplantation, Autologous; Trypsin

Cells from three different human neuroblastoma cell lines and normal human bone marrow cells were exposed to the lipophilic fluorescent dye, merocyanine 540 (MC 540), and white light. In vitro clonogenic tumor cells were inactivated up to 25,000 times more rapidly than multipotent hematopoietic progenitor cells (CFU-GEMM). It is conceivable that this pronounced difference in sensitivity to MC 540-mediated photolysis can be exploited for the selective killing of residual neuroblastoma cells in autologous remission marrow grafts.

Topics: Bone Marrow; Bone Marrow Transplantation; Cell Line; Cell Survival; Hematopoietic Stem Cells; Humans; Neoplastic Stem Cells; Neuroblastoma; Photolysis; Pyrimidinones; Transplantation, Autologous