Studies on the mechanism of selective antiproliferative effects of alkyllysophospholipids in mammary epithelial cells

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Samadder, Pranati
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The antiproliferative and cell-selective effects of alkyllysophospholipids (ALPs) have been well established by studies from numerous laboratories. A number of hypotheses have been proposed to explain the mechanisms underlying these effects. Previous studies with MCF-7 cells led to the proposal that ET-18-OCH$\sb3$, the prototypic ALP, perturbs the association of Raf-1 with cell membranes and thereby prevents its sustained activation. This in turn leads to inhibition of downstream kinases such as MAP kinase which leads to inhibition of cell proliferation. If inhibition of Raf-1 activation is responsible for the cytostatic activity of ALPs then the inability of ET-18-OCH$\sb3$ to inhibit the proliferation of some cells could be due to the utilization of Raf-1-independent pathways to activate MAP kinase or differences in the mechanism of activation of Raf-1 in ALP-sensitive and insensitive cells. Before exploring this hypothesis, studies were conducted to validate the suggestion that ALPs inhibit cell proliferation by inhibiting Raf-1 activation. After confirming the role of inhibition of Raf-1 activation in the mechanism of action of ALPs, studies were conducted to explore the mechanisms responsible for the observed elective effects. Serum-free media were developed and MCF-7 cells were adapted for growth in the media. The cells that ere adapted for serum-free growth, MCF-7Ad$\sp+$, became insensitive to growth inhibition by ET-18-OCH$\sb3.$ These cells, along with human normal mammary epithelial (HNME) cells that were also resistant to growth inhibition by ET-18-OCH$\sb3$, were used for our studies. We demonstrated that unlike in MCF-7 cells where inactive Raf is present in the cytosol, in HNME and MCF-7Ad$\sp+$ cells Raf-1 was found in both the membranes and the cytosol in unstimulated quiescent cells. Stimulation of both cells with growth factors resulted in the activation of Raf-1 without further translocation of cytosolic Raf-1. Preincubation of both cells with ET-18-OCH$\sb3$ had little effect on membrane Raf-1 levels and MAP kinase activity and phosphorylation. In contrast, MAP kinase activity in MCF-7 cells was severely inhibited by preincubation with ET-18-OCH$\sb3.$ A progressive increase in membrane Raf-1 levels during the adaptation process which correlated with increasing resistance to ET-18-OCH$\sb3$ was demonstrated. Since ET-18-OCH$\sb3$ inhibits the translocation of Raf-1 to the membrane, the constitutive association of Raf-1 in the membrane by-passes the step that is inhibited by ET-18-OCH$\sb3$ and thereby confers resistance to the growth-inhibitory effect of the compound. Our studies allow us to conclude that Raf-1 is a key intracellular target of choline-containing ALPs in epithelial cells and inhibition of Raf-1 activation by the compounds leads to inhibition of growth. Our results also strongly suggest that differences in the mechanism of activation of Raf-1 may explain why some cells are unaffected by concentrations of ET-18-OCH$\sb3$ that severely inhibit the proliferation of other cells. (Abstract shortened by UMI.)