pkh-26 and Leukemia

Modified citrus pectin (MCP) is known for its anti-cancer effects and its ability to be absorbed and circulated in the human body. In this report we tested the ability of MCP to induce the activation of human blood lymphocyte subsets like T, B and NK-cells.. MCP treated human blood samples were incubated with specific antibody combinations and analyzed in a flow cytometer using a 3-color protocol. To test functionality of the activated NK-cells, isolated normal lymphocytes were treated with increasing concentrations of MCP. Log-phase PKH26-labeled K562 leukemic cells were added to the lymphocytes and incubated for 4 h. The mixture was stained with FITC-labeled active form of caspase 3 antibody and analyzed by a 2-color flow cytometry protocol. The percentage of K562 cells positive for PKH26 and FITC were calculated as the dead cells induced by NK-cells. Monosaccharide analysis of the MCP was performed by high-performance anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD).. MCP activated T-cytotoxic cells and B-cell in a dose-dependent manner, and induced significant dose-dependent activation of NK-cells. MCP-activated NK-cells demonstrated functionality in inducing cancer cell death. MCP consisted of oligogalacturonic acids with some containing 4,5-unsaturated non-reducing ends.. MCP has immunostimulatory properties in human blood samples, including the activation of functional NK cells against K562 leukemic cells in culture. Unsaturated oligogalacturonic acids appear to be the immunostimulatory carbohydrates in MCP.

Topics: Antibodies; Antineoplastic Agents, Phytogenic; B-Lymphocytes; Caspase 3; Cell Death; Citrus; Dose-Response Relationship, Drug; Humans; K562 Cells; Killer Cells, Natural; Leukemia; Leukocytes; Lymphocyte Activation; Oligosaccharides; Organic Chemicals; Pectins; Phytotherapy; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer

The application of the fluorescent cell membrane probes PKH2 and PKH 26 GL in the measurement of leukaemic cell growth was examined on four cell lines K562, NALM-6, ACV (a pre-B cell line) and HL-60 using flow cytometry. As the amount of probe per cell reduces at each cell division, the fluorescence can be used to measure cell proliferation. By measuring the mean fluorescence intensity of the cells at the beginning of culture and at various time points, and by combining this information with a viable cell count, it was possible to determine: (1) the number of viable cells; (2) their rate of proliferation; (3) their number of cell divisions; and (4) the maintenance of cells in a viable state over a period of time. It was demonstrated that these parameters could be reliably established using the red fluorescent probe PKH26 GL. In contrast, the green fluorescent probe PKH2 GL showed dye transfer resulting in an underestimation of the number of cell divisions and an overestimation of the maintenance of cells in a viable state. The potential advantages of the use of PKH26 GL over conventional assays for the measurement of leukaemic cell growth parameters are discussed.

Topics: Cell Division; Cell Survival; Flow Cytometry; Fluorescent Dyes; Humans; Leukemia; Organic Chemicals; Staining and Labeling; Tumor Cells, Cultured