Elsevier

Experimental Cell Research

Volume 292, Issue 2, 15 January 2004, Pages 385-392
Experimental Cell Research

Sphingomyelin synthase as a potential target for D609-induced apoptosis in U937 human monocytic leukemia cells

https://doi.org/10.1016/j.yexcr.2003.10.001Get rights and content

Abstract

Tricyclodecan-9-yl-xanthogenate (D609) is a selective tumor cytotoxic agent. However, the mechanisms of action of D609 against tumor cells have not been well established. Using U937 human monocytic leukemia cells, we examined the ability of D609 to inhibit sphingomyelin synthase (SMS), since inhibition of SMS may contribute to D609-induced tumor cell cytotoxicity via modulating the cellular levels of ceramide and diacylglycerol (DAG). The results showed that D609 is capable of inducing U937 cell death by apoptosis in a dose- and time-dependent manner. The induction of U937 cell apoptosis was associated with an inhibition of SMS activity and a significant increase in the intracellular level of ceramide and decrease in that of sphingomyelin (SM) and DAG, which resulted in an elevation of the ratio between ceramide and DAG favoring the induction of apoptosis. In addition, incubation of U937 cells with C6-ceramide and/or H7 (a selective PKC inhibitor) reduced U937 cell viability; whereas pretreatment of the cells with a PKC activator, PMA or 1-oleoyl-2-acetylglycerol (OAG), attenuated D609-induced U937 cell apoptosis. These results suggest that SMS is a potential target of D609 and inhibition of SMS may contribute to D609-induced tumor cell death via modulation of the cellular levels of ceramide and DAG.

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)

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    Similarly, we reported that TNF-α-mediated transcriptional induction of intracellular adhesion molecule-1 (ICAM-1) through the NF-κB pathway was suppressed in SMS2-deficient MEFs [159], suggesting that SMS2 is involved in the regulation of the inflammatory response induced by a variety of chemokines and cytokines. Treatment with D609 induced apoptotic cell death with an increase in ceramide in U937 cells [160,161] or MDA-MB-435 cells [162], suggesting that SMS promotes anti-apoptotic function by maintaining a lower level of ceramide/SM balance in cancer cells. SMS1 was reported to suppress apoptosis by decreasing ceramide content in numerous stimuli including oxidative stress in Neuto 2A cells [163], photodamage in Jurkat T cells [164,165], and FasL in Jurkat cells [166].

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    Macrophages are the primary target of LPS [28]. Taking into account that SMS inhibition in these cells can be of potential interest in preventing fatal LPS-induced inflammation, here we studied this question using a competitive SMS inhibitor, tricyclodecane-9-yl xanthogenate (D609) [13,23,29–32]. As an influence of D609 on a putative mammalian phosphatidylcholine-specific phospholipase C (PC-PLC) has also been postulated [33–35], we first established that D609 indeed inhibited the activity of SMS.

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    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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1

These authors contributed equally to this work.

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