Noncytotoxic alkyl-lysophospholipid treatment increases sensitivity of leukemic K562 cells to lysis by natural killer (NK) cells

Abstract

Alkyl-lysophospholipids (ALP) are a group of anti-cancer compounds that have previously been shown to have the unique feature of being selectively toxic to neoplastic tissues. Because alkyl-lysophospholipids target the cell membrane as their site of action, our aim was to analyse the immunological effects of a nonlethal ALP treatment on leukemic K562 cells. In this in vitro study we used ET-18-OCH3, one of the most potent ALP derivatives, at different concentrations ranging from 25 up to 100 pg/ml. By measurement of cell viability and of apoptosis, we determined a concentration of 25 pg/ml ET-18-OCH3 and an incubation period of 2 hr as nonlethal for K562 cells; higher concentrations markedly reduced cell viability and led to induction of apoptosis. Similar to the effects induced by nonlethal heat shock, a nontoxic ET-I 8-OCH3 treatment led to a significant increase in the sensitivity of (Mollinedo et al., 1994), are discussed. The biological activities that could be associated with ET-18-OCH3 include activation of macrophages, inhibition of angiogenesis in a human endothelial cell line and downregulation of the expression of cell-adhesion molecules (Candal et al., 1994). It has been reported that macrophages acquire cytotoxic activity against tumor cells (Andreesen et al., 1978) induced by ALP treatment. All these findings together indicate that different target sites could be involved in the activity of ALP and that beside cytocidal effects, the immunological relevance of an ALP treatment has to be considered as well. In the present report we correlate an increased sensitivity of K562 cells to lysis by non-MHC restricted, interleukin-2 (IL-2) stimulated NK effector cells with the effects of nonlethal ET-18-OCH3 treatment. MATERIAL AND METHODS Cells and cell cultureThe human chronic myelogenous leukemia cell line K562 (ATCC, Rockville, MD, CCL 243) was grown in RPMI 1640 (GIBCO, Eggenstein, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS) (GIBCO), 6 mM L-glutamine (GIBCO) and antibiotics (penicillin 100 I.U./ml and streptomycin 100 pg/ml, GIBCO). Cells were incubated at 37°C in a humidified atmosphere at 5% COz and 95% air. All studies were carried out in log-phase growth. ET-18-OCH3 and the treatment of cells with ET-18-OCH3ET-18-OCH3 (1-octadecyl-2-methyl-rac-glycero-3-phosphocholine) was dissolved at 500 pg/ml as a stock solution in RPMI 1640 medium containing 10% FCS by heating at 50°C for 30 min and sonication. The solution was sterilized by filtration through a 0.2 p, m filter and stored at -20°C. ET-18-OCH3 was added to the cell cultures (1 x lo6 cells/ml) at different concentrations ranging from 25 up to 100 pg/ml. After treatment for 2 hr the cells were washed 3 times with phosphate buffered saline (PBS) and then incubated with fresh culture medium in the absence of ET-18-OCH3 for 2 hr and for 6 hr. These time points were chosen in accordance to the cytotoxicity assay. Measurement of cell viability and apoptosis by flow cytornetiyCell viability was evaluated by propidium iodide (P...

Potential immunological functions of salivary Hsp70 in mucosal and periodontal defense mechanisms

“…The first of these involves the appearance of Hsp70 on the surface of certain tumor cells or virally infected cells, leading to phagocytosis and lysis of these cells [10,55,56,67,74,79]. New findings indicate that surface expression of Hsp70 on tumor cells is accompanied by Hsp70-release involving lipid rafts and exosomes and leading to an activation of natural killer (NK) cells also in the environment [3,6,33].…”

“…Because of its chaperoning ability, uncomplexed Hsp70 binds other peptides, and as a complex induces receptor-mediated uptake into antigen-presenting cells (i.e. macrophages, dendritic cells) to cross-present this complex as an antigen (coupled with MHC-I or MHC-II molecules) to cytotoxic T cells and NK cells [8,10,51,74,75,79]. Although bacterial Hsp70 associates mainly with bacterial antigens, human Hsp70 associates peptides such as self peptides, tumor peptides, or viral peptides because these are derived from proteins synthesized in human cells [75].…”

“…Although bacterial Hsp70 associates mainly with bacterial antigens, human Hsp70 associates peptides such as self peptides, tumor peptides, or viral peptides because these are derived from proteins synthesized in human cells [75]. This means that the antigenpresenting function of Hsp70 is important not only against bacteria, but also as an initiator of immune defense against tumor cells and viral infections [8,10,51,71,74,77,79].…”

“…Langerhans and dendritic cell [23]) and there is also a considerable flux of neutrophils through the gingival sulcus into saliva even in health. Regarding these, an important potential function of salivary Hsp70 could be the modulation of antigen sampling, since uncomplexed Hsp70 binds other peptides, and as a complex induces receptor-mediated uptake into antigen-presenting cells [8,10,51,74,75,79].…”

Heat shock protein 70 and glycoprotein 96 are differentially expressed on the surface of malignant and nonmalignant breast cells

“…Hsp70 correlates with MHC-independent natural killer (NK) cell cytotoxicity of tumor cells, which can be blocked using specific antibodies to Hsp70 (Botzler et al 1996a(Botzler et al , 1996bMulthoff 1997;Roigas et al 1998). In contrast to Hsp70, the role of surface gp96 in NK cytotoxicity has not been evaluated, but studies showing that secretion of gp96 mediates NK cell expansion suggest similar function (Strbo et al 2002).…”