Sphingomyelin synthase as a potential target for D609-induced apoptosis in U937 human monocytic leukemia cells
Introduction
Tricyclodecan-9-yl-xanthogenate (D609) is a member of the family of compounds called xanthates [1]. Initially, it was shown that incubation of various transformed and malignant cells with low concentrations (<10 μg/ml) of D609 in an acidic condition (pH 6.8) reverted their morphology, growth pattern, and serum dependence to normal phenotypes [2], [3]. Incubation of these cells with higher concentrations (>10 μg/ml) of D609 alone, or with low concentrations (5 or 10 μg/ml) of D609 plus C10–14 monocarboxylic acids (20–60 μg/ml) at physiological pH (7.4), induced significant cell death [3], [4], [5]. However, incubation with monocarboxylic acids alone had no significant effect on the cell viability. The list of transformed and malignant cell types that are sensitive to D609 toxicity is expanding and now includes bovine papilloma virus type 1 (BPV-1)- and SV40-transformed animal and human fibroblasts, various leukemia/lymphoma cells, and different solid tumor cells with only a few exceptions [3], [4], [5], [6]. Even some drug-resistant tumor cells, such as methotrexate-and Adriamycin-resistant L1210 and S180 cells, are susceptible to D609 cytotoxicity [5]. In contrast, under the same in vitro cell culture conditions, D609 did not show any cytotoxicity against normal human fibroblasts or peripheral blood lymphocytes [3]. In fact, D609 enhanced mitogen-stimulated mouse splenic lymphocyte proliferation and cytokine production (our unpublished results). These observations suggest that unlike other known chemotherapeutic agents that usually inhibit tumor cell growth and induce tumor cell death nonspecifically by inhibiting DNA replication or inducing DNA damage, the antitumor effect of D609 is likely the result of inhibition of a tumor-specific target.
However, the mechanisms of action of D609 against tumor cells remain to be elucidated. Originally, it was suggested that D609 functions as a specific inhibitor of the phosphatidylcholine-specific phospholipase C (PC-PLC), mainly based on in vitro cell-free studies using the bacterial enzyme [7], [8]. PC-PLC utilizes phosphatidylcholine (PC) as substrate and hydrolyzes PC to produce diacylglycerol (DAG) and phosphocholine (PhoCho) [8], [9], [10]]. Many of the biological activities of D609, including its antitumor activity, have been attributed to the inhibition of PC-PLC [7], [8], [9], [11], [12]. However, the mammalian enzyme of PC-PLC has not been characterized. Recently, it was reported that D609 also inhibits SMS, which transfers the PhoCho from PC to ceramide and produces DAG and sphingomyelin (SM) [13], [14], [15]. These observations raise the possibility that SMS may account for some of the cellular effects that had been attributed to PC-PLC [13], [14], because both enzymes utilize PC as substrate and produce DAG as one of their products.
Both PC-PLC and SMS have been implicated in cell transformation [13], [16], [17], [18], [19]. Inhibition of either PC-PLC or SMS, or both, reduces the intracellular level of DAG while inhibition of SMS also increases the level of ceramide [8], [13]. Down-regulation of PKC activity by inhibiting DAG production or direct inhibition of PKC with an inhibitor suppresses tumor cell proliferation and sensitizes tumor cells to irradiation and chemotherapy [20], [21], whereas increased ceramide induces cell cycle arrest, senescence, or apoptosis in a cell-type-dependent manner [22], [23], [24], [25]. Increases in intracellular ceramide occur as a result of exposure of cells to diverse stimuli, including irradiation and various chemotherapeutic agents. These stimuli can induce either the hydrolysis of sphingomyelin or the de novo production of ceramide [22], [23], [24], [25]. Inhibition of ceramide metabolism also results in elevation of intracellular ceramide, which has been actively pursued as a way to enhance tumor responsiveness and to ameliorate tumor drug resistance to cancer therapy [22], [23], [24], [25]. Since inhibition of SMS blocks the conversion of ceramide to SM, one of the main ceramide metabolic pathways, it is conceivable that inhibition of SMS by D609 can increase the intracellular level of ceramide and decrease that of DAG, which may contribute to D609-induced tumor cytotoxicity. This hypothesis was examined by the present study using a well-studied human monocytic leukemia cell line, U937 cells.
Section snippets
Reagents
D609 was synthesized and purified as described by Rao [1], and its purity was >97%. Cell-permeable and biologically active short-chain ceramide (C6-ceramide) was obtained from the Synthetic Lipid Core at the Department of Biochemistry and Molecular Biology, Medical University of South Carolina. Phorbol 12-myristate 13-acetate (PMA), 3-[4,5-yl] 1 2,5 bromide (MTT), 1-(5-isoquinolinysulfonyl)-2-methylpiperazine (H7), and 1-oleoyl-2-acetylglycerol (OAG) were obtained from Sigma (St. Louis, MO).
Results and discussion
As shown in Fig. 1, U937 cells incubated with various concentrations of D609 exhibited a dose- and time-dependent decrease in viability. The EC50 value of D609 against U937 cells after 48 h incubation was 125 μM (or 33 μg/ml). To determine if D609 kills tumor cells by induction of apoptosis, we measured the changes in the mitochondrial transmembrane potential (MTP), externalization of plasma membrane phosphatidylserine (PS), and DNA fragmentation in U937 cells after treatment with D609. A
Acknowledgements
This work was supported in part by the research grants from the Charlotte Geyer Foundation and Department of Defense through the Hollings Cancer Center (to Dr. Daohong Zhou) and the NIH grant HL43707 (to Dr. Yusuf A. Hannun).
References (32)
- et al.
Reversion of bovine papillomavirus-induced transformation and immortalization by a xanthate compound
Exp. Cell Res.
(1985) - et al.
Selective killing of tumor cells by xanthates
Cancer Lett.
(1987) - et al.
Induction of apoptosis and potentiation of TNF- and Fas-mediated apoptosis in U937 cells by the xanthogenate compound D609
Exp. Cell Res.
(1997) - et al.
Antitumoral activity of a xanthate compound: I. Cytotoxicity studies with neoplastic cell lines in vitro
Cancer Lett.
(1989) - et al.
Involvement of SAPK/JNK pathway in X-ray-induced rapid cell death of human T-cell leukemia cell line MOLT-4
Cancer Lett.
(2000) - et al.
TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced “acidic” sphingomyelin breakdown
Cell
(1992) - et al.
Interruption of growth signal transduction by an antiviral and antitumoral xanthate compound
Exp. Cell Res.
(1988) Interruption of TPA-induced signals by an antiviral and antitumoral xanthate compound: inhibition of a phospholipase C-type reaction
Biochem. Biophys. Res. Commun.
(1989)- et al.
Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does sphingomyelin synthase account for the putative phosphatidylcholine-specific phospholipase C?
J. Biol. Chem.
(1998) - et al.
Differential effects of sphingomyelin hydrolysis and resynthesis on the activation of NF-kappa B in normal and SV40-transformed human fibroblasts
J. Biol. Chem.
(2000)
Basic fibroblast growth factor-induced proliferation of primary astrocytes. Evidence for the involvement of sphingomyelin biosynthesis
J. Biol. Chem.
Reversion of Ras- and phosphatidylcholine-hydrolyzing phospholipase C-mediated transformation of NIH 3T3 cells by a dominant interfering mutant of protein kinase C lambda is accompanied by the loss of constitutive nuclear mitogen-activated protein kinase/extracellular signal-regulated kinase activity
J. Biol. Chem.
Ablation of Go alpha-subunit results in a transformed phenotype and constitutively active phosphatidylcholine-specific phospholipase C
J. Biol. Chem.
Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill
J. Immunol. Methods
Analysis of apoptotic cells by flow and laser scanning cytometry
Methods Enzymol.
Activity of protein kinase C modulates the apoptosis induced by polychlorinated biphenyls in human leukemic HL-60 cells
Toxicol. Lett.
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2019, Experimental NeurologyCitation Excerpt :However, SMSs are showed as the key enzymes for the biosynthesis of SM. Inhibition or knockdown of SMS1/2 reduces SM levels (Meng et al., 2004; Li et al., 2007; Van der Luit et al., 2007; Ding et al., 2008). Consistent with this result, we indeed observe reduced levels of SM in SMS1-knockdown hippocampus and D609-treated cultured cells.
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These authors contributed equally to this work.