Quaternary ammonium analogs of ether lipids inhibit the activation of protein kinase C and the growth of human leukemia cell lines.

PURPOSE: ET-18-OCH3 (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is a representative of the first generation of antitumor ether lipids and is a model in the development of new compounds including a series of quaternary ammonium analogs (QAA). In the present study, we evaluated the QAA as inhibitors of cell growth and studied their mechanism of action. METHODS: We compared the effects of the QAA on the proliferation of human leukemia cell lines which are sensitive (HL-60) or resistant to ET-18-OCH3 (HL-60R and K562). In addition we used cell fractionation and enzymatic assays to determine the effects of QAA on protein kinase C (PKC) translocation in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA). RESULTS: The QAA and ET-18-OCH3 were approximately equally effective inhibitors of HL-60 cell growth. However, the QAA were more effective inhibitors of K562 and HL-60R cell proliferation. The HL-60R cells, which were selected for resistance to ET-18-OCH3, were also resistant to BM 41.440 which is structurally similar. In serum-free medium, the phosphorus-containing compounds (ET-18-OCH3, BM 41.440 and He-PC) were much more effective inhibitors (8-20-fold) of the K562 cell line while the activities of the QAA were only moderately increased (1.2-2.3-fold). When serum albumin was added to the serum-free medium, the K562 cells became resistant to ET-18-OCH3, suggesting that albumin is responsible for the differential sensitivity. The QAA compounds, which inhibit PKC activity in vitro, inhibited cell proliferation. However, a QAA which did not inhibit PKC did not inhibit cell proliferation. The phorbol ester TPA stimulates PKC translocation and causes HL-60 cell differentiation to adherent macrophage-type cells. The QAA inhibited TPA-induced cell differentiation and PKC translocation indicating that they also inhibit PKC in intact cells. CONCLUSIONS: The cellular effects of the QAA appear to be due to inhibition of PKC. In addition, these data indicate that albumin, which is important as a mediator of the uptake of ET-18-OCH3, has only a small effect on the uptake of QAA. Together these data indicate that the QAA are potential anticancer agents, showing a significant ability to inhibit growth of leukemia cell lines that are resistant to ET-18-OCH3.

Protein kinase CbetaII activation by 1-beta-D-arabinofuranosylcytosine is antagonistic to stimulation of apoptosis and Bcl-2alpha down-regulation.

1-beta-D-Arabinofuranosylcytosine (ara-C) stimulates the formation of both diglyceride and ceramide in the acute myelogenous leukemia cell line HL-60 (Strum, J. C., Small, G. W., Pauig, S. B., and Daniel, L. W. (1994) J. Biol. Chem 269, 15493-15497). ara-C also causes apoptosis in HL-60 cells which can be mimicked by exogenous ceramide. However, the signaling role for ara-C-induced diacylglycerol (DAG) is not defined. We found that Bcl-2 levels were increased by treatment of HL-60 cells with exogenous DAG or 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, exogenous ceramide treatment caused a decrease in cellular Bcl-2 levels. Thus, ara-C stimulates the synthesis of two second messengers with opposing effects on Bcl-2. Since the effects of ara-C-induced DAG could be due to protein kinase C (PKC) activation, we determined the effects of ara-C on PKC isozymes. ara-C caused an increase in membrane-bound PKCbetaII (but not PKCalpha or PKCdelta). ara-C or TPA-induced translocation of PKCbetaII was inhibited by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), and ara-C-induced apoptosis was stimulated by pretreatment of the cells with ET-18-OCH3. ET-18-OCH3 also inhibited stimulation of Bcl-2 by TPA and enhanced the decrease in Bcl-2 observed in ara-C-treated cells. These data indicate that ara-C-induced apoptosis is limited by ara-C-stimulated PKCbetaII through effects on Bcl-2. To further determine the role of PKC, we used antisense oligonucleotides directed toward PKCbetaII. The antisense, but not the sense, oligonucleotide inhibited PKCbetaII activation and enhanced ara-C-induced apoptosis. These data demonstrate that the stimulation of apoptosis by ara-C is self-limiting and can be enhanced by inhibition of PKC.

Second generation hybrid polar compounds are potent inducers of transformed cell differentiation.

Hybrid polar compounds, of which hexamethylenebisacetamide (HMBA) is the prototype, are potent inducers of differentiation of murine erythroleukemia (MEL) cells and a wide variety of other transformed cells. HMBA has been shown to induce differentiation of neoplastic cells in patients, but is not an adequate therapeutic agent because of dose-limiting toxicity. We report on a group of three potent second generation hybrid polar compounds, diethyl bis-(pentamethylene-N,N-dimethylcarboxamide) malonate (EMBA), suberoylanilide hydroxamic acid (SAHA), and m-carboxycinnamic acid bis-hydroxamide (CBHA) with optimal concentrations for inducing MEL cells of 0.4 mM, 2 microM, and 4 microM, respectively, compared to 5 mM for HMBA. All three agents induce accumulation of underphosphorylated pRB; increased levels of p2l protein, a prolongation of the initial G1 phase of the cell cycle; and accumulation of hemoglobin. However, based upon their effective concentrations, the cross-resistance or sensitivity of an HMBA-resistant MEL cell variant, and differences in c-myb expression during induction, these differentiation-inducing hybrid polar compounds can be grouped into two subsets, HMBA/EMBA and SAHA/CBHA. This classification may prove of value in selecting and planning prospective preclinical and clinical studies toward the treatment of cancer by differentiation therapy.

Differentiation of HL-60 cells distinguishes between cytostatic and cytotoxic effects of the alkylphospholipid ET-18-OCH3.

The synthetic dialkylphospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) inhibits growth of the acute myelogenous leukemia cell line HL-60. Incubation of HL-60 cells with demethyl-sulfoxide causes the cells to differentiate to a granulocyte-like phenotype and become quiescent. Incubation of the DMSO-treated cells with ET-18-OCH3 results in a reduction in cell numbers due to cytotoxicity. In contrast, treatment of undifferentiated HL-60 cells with lower concentrations of ET-18-OCH3 leads to growth inhibition. These data indicate that the model of differentiated HL-60 cells currently used for the study of resistance to growth inhibition is inappropriate. HL-60 cells can be used to measure growth inhibition and at higher doses cytotoxicity. However, the differentiated, nonproliferative, cells can only be used to measure direct cytotoxicity. Therefore, the results of studies directly comparing the effects of ET-18-OCH3 in proliferative HL-60 cells and quiescent DMSO-treated HL-60 cells should be reevaluated. An evaluation of the effects of low concentrations of ET-18-OCH3 (0.5-1.5 microM) in proliferative HL-60 cells indicated that ET-18-OCH3 was an effective cytostatic agent at nontoxic concentrations. In summary, studies on the mechanism of action of ET-18-OCH3, or related ether lipids, should carefully investigate differences in the effects at cytostatic versus cytotoxic concentrations.

ET-18-OCH3 inhibits nuclear factor-kappa B activation by 12-O-tetradecanoylphorbol-13-acetate but not by tumor necrosis factor-alpha or interleukin 1 alpha.

1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) is a synthetic diether phospholipid that is competitive with phosphatidylserine binding to the regulatory domain of protein kinase C (PKC). Our previous studies indicate that the selective inhibition of tumor cell growth by ET-18-OCH3 may be due to altered signal transduction mechanisms, including the inhibition of PKC. To further define the mechanism of action of ET-18-OCH3, we have used it to study the role of PKC in regulation of the transcription factor NF-kappa B, which is activated by diverse stimuli. In the 293.27.2 human kidney cell line, as in hematopoietic cells of all lineages, NF-kappa B is stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 alpha (IL-1 alpha). The response to either TNF-alpha or IL-1 alpha is synergistically enhanced by TPA. However, the regulatory mechanisms and signal transduction systems responsible for NF-kappa B activation in response to these different stimuli have not been determined in detail. We have used ET-18-OCH3 and auranofin, which inhibit PKC by different mechanisms, to assess the role of PKC in NF-kappa B activation. ET-18-OCH3 markedly inhibits TPA-induced NF-kappa B activation, as measured by HIV long terminal repeat-directed expression of beta-galactosidase. The IC50 for inhibition by ET-18-OCH3 is approximately 2 microM, a noncytotoxic concentration. Inhibition of TPA-induced NF-kappa B activation was dependent upon preincubation with ET-18-OCH3, and the drug was active at approximately 2 mol% of total cellular phospholipid. ET-18-OCH3 did not inhibit NF-kappa B activation by either TNF-alpha or IL-1 alpha, indicating that there are multiple distinct signal transduction pathways leading to activation of NF-kappa B. We have confirmed these results using auranofin, an antirheumatic drug that is a specific PKC inhibitor interacting with the catalytic domain. Like ET-18-OCH3, auranofin blocked NF-kappa B activation by TPA but not by TNF-alpha or IL-1 alpha. Also like the ether lipid, auranofin only partially blocked the synergy exhibited by TPA and TNF-alpha. To confirm the role of NF-kappa B in this response, we measured NF-kappa B by electrophoretic mobility shift assay. Both ET-18-OCH3 and auranofin inhibited cellular induction of the active NF-kappa B complex in response to TPA but not in response to TNF-alpha.(ABSTRACT TRUNCATED AT 400 WORDS)

Flow cytometric detection of hydrogen peroxide production induced by doxorubicin in cancer cells.

2',7'-Dichlorofluorescin diacetate (DCFH-DA) has been previously used to study the oxidative burst of neutrophils induced by different stimuli. The method is based on the fact that DCFH-DA diffuses through the cell membrane and it is hydrolyzed by intracellular esterases to DCFH, which remains trapped within the cells. DCFH, a nonfluorescent compound, is able to react with free radical products, particularly with hydrogen peroxide, and to generate the fluorescent 2',7'-dichlorofluorescein (DCF). By flow cytometric detection of DCF fluorescence, an indirect measure of reactive oxygen species production in single cells may be obtained. Using a modified procedure to load cells of the human colon adenocarcinoma cell line LoVo with DCFH-DA, a significant fluorescence increase above the basal fluorescence level has been detected after treatment with doxorubicin doses as low as 0.4 microM. This increase is not detectable when the cells are preloaded with catalase, using a scraping method, and it is not due to doxorubicin own fluorescence. These experiments prove that the increase of DCF fluorescence intensity observed during doxorubicin treatment is not due to technical artifacts but it is attributable to free radicals produced in the cells by the drug.