Phorbol ester selectively stimulates the phospholipase D-mediated hydrolysis of phosphatidylethanolamine in multidrug-resistant MCF-7 human breast carcinoma cells

Abstract

The phospholipase D (PLD)-mediated synthesis of phosphatidylethanol (PtdEtOH) and the hydrolysis of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) were examined in drug-sensitive and multidrug-resistant lines of MCF-7 human breast carcinoma cells. In drug-sensitive (MCF-7/WT) cells, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) failed to enhance either the synthesis of PtdEtOH or the hydrolysis of either phospholipid. In the drug-resistant (MCF-7/MDR) cells, 100 nM PMA greatly enhanced both the synthesis of PtdEtOH (approximately 21-fold) and the hydrolysis of PtdEtn (approximately 29-fold), but had no effect on the hydrolysis of PtdCho. The PLD activators sphingosine and H2O2 were found to elicit only a slight (1.28-1.4-fold) stimulatory effect on PtdCho hydrolysis in both the MCF-7/WT and MCF-7/MDR cell types, and had only a small effect on PtdEtn hydrolysis in the MCF-7/WT cells as well. However, these agents significantly (approximately 2.6-3.5-fold) stimulated PtdEtn hydrolysis in the MCF-7/MDR cells. These data indicate that MCF-7/MDR cells contain a PtdEtn-specific PLD activity which can be selectively stimulated by PMA, sphingosine and H2O2.

Tamoxifen stimulates phospholipase D activity by an estrogen receptor‐independent mechanism

“…The fact that drug-sensitive breast carcinoma cells do not contain appreciable levels of functional PLD ( [28], and this work) suggests that this enzyme may not be required for the aggressive growth of these cells. Consequently, in these drugsensitive cells PLD can not mediate the growth inhibitory effect of TAM.…”

“…In contrast, MCF-7/MDR cells contain a PLD system which preferentially hydrolyzes PtdEtn in the presence of PMA and other activators [28]. Similarly, treatment of these cells with 5-50 pM concentrations of TAM in a 10% serum-containing medium caused preferential stimulation of [~4qPtdEtn hydrolysis during either a 20 min (Fig.…”

“…MCF-7 cells were maintained as described earlier [28]. NIH 3T3 clone 7 fibroblasts were obtained from Dr. Douglas R. Lowy (National Cancer Institute, Bethesda, MD, USA) and were cultured continuously in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, penicillin (50 U/ml), streptomycin (50/tg/ml) and glutamine (2 mM).…”

“…3) and then treated with TAM in the presence of 2 mM ethanolamine or 20 mM choline, as appropriate; these latter compounds served to prevent phosphorylation of newly formed [~4C]ethanolamine and [~4C]choline [29,31]. Suspended and attached MCF-7/ MDR [28] and NIH 3T3 cells [31,32] were similarly responsive to PMA, sphingosine and H202 with respect to the activation of PLD, but suspended cells contained 5-10 times less background levels of 14C-labeled precursors. This made evaluation of the effects of lower concentrations of TAM on phospholipid hydrolysis possible.…”

Two phosphatidylethanol classes separated by thin layer chromatography are produced by phospholipase D in rat brain hippocampal slices

“…However, several studies using exogenous substrates [8,9] or labeled endogenous phospholipids [10 14] have shown that other phospholipids or lysophospholipids [15] are also hydrolyzed by PLD in many types of intact cells or subcellular fractions. Substrate specificity has been related to distinct molecular forms of the enzyme localized in different subcellular fractions [16][17][18] or to different sensitivity to regulatory mechanisms [19,20]. A specific feature of PLD is the transphosphatidylation reaction, in which the enzyme transfers the phosphatidyl moiety of phospholipids to a short-chain primary alcohol and forms a metabolically stable phosphatidylalcohol, which is easily separated from the rest of naturally occurring phospholipids by thin layer chromatography (TLC).…”