Hexadecylphosphocholine interferes with the intracellular transport of cholesterol in HepG2 cells

Abstract

We have shown, in a previous publication, that nontoxic concentrations of hexadecylphosphocholine exert an antiproliferative effect on HepG2 cells. Hexadecylphosphocholine also interferes with the biosynthesis of cholesterol and phosphatidylcholine. We have now extended our studies to try to establish the molecular mechanism by which hexadecylphosphocholine disrupts cholesterol homeostasis. Using radiolabelled substrates we determined the effect of hexadecylphosphocholine on cholesterol synthesis, the destiny of cholesterol from low‐density lipoprotein and the transport of cholesterol between the plasma membrane and the endoplasmic reticulum. Protein levels and gene expression of the main proteins involved in cholesterol homeostasis were analysed by western blotting and RT‐PCR, respectively. HepG2 cells exposed to hexadecylphosphocholine showed an increase in cholesterol biosynthesis when acetate, but not mevalonate, was used as a substrate. The activity of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (EC 1.1.1.34) and low‐density lipoprotein receptor, as well as the corresponding mRNA expression, increased after 24 h of treatment with hexadecylphosphocholine. Cholesteryl linoleate in low‐density lipoprotein uptake and further hydrolysis of these esters increased but the cholesterol esterification was reduced after 6 h of treatment with alkylphosphocholine. Cholesterol transport from the plasma membrane to the endoplasmic reticulum was impaired by hexadecylphosphocholine. In conclusion, hexadecylphosphocholine interfered with the transport of cholesterol from the cell surface to the endoplasmic reticulum, leading to a depletion of cholesterol in the endoplasmic reticulum and a deregulation of cholesterol biosynthesis. The accumulation of cholesterol within the cell and the reduction in phosphatidylcholine synthesis produces an alteration in the phosphatidylcholine/cholesterol ratio that may well be responsible for the antiproliferative activity exhibited by hexadecylphosphocholine in HepG2 cells.

Alterations in the homeostasis of phospholipids and cholesterol by antitumor alkylphospholipids

“…Furthermore, the steady-state protein levels and mRNA expression of HMGCR and LDLR increased after incubation with the APC. HMGCR activity was not, however, directly modulated by miltefosine as assayed in vitro both in HepG2 cell lysates and rat-liver microsomes, and exposure of cells to miltefosine did not significantly affect the decay rate of the reductase [58]. On the other hand, incubation with miltefosine did not significantly change the expression of ACAT.…”

“…However, cholesterol esterification from several radioactive exogenous fatty acids was markedly reduced after treatment with miltefosine, even in the presence of the cholesterol analog 25-hydroxycholesterol added to the culture medium; in addition, the reesterification rate of cholesterol from LDL decreased after treatment with the APC [35,58]. This inhibition of intracellular CE synthesis was observable after only one hour of exposure to miltefosine [39].…”

Interaction of Alkylphospholipid Formulations with Breast Cancer Cells in the Context of Anticancer Drug Development

“…In leukemic cells treatment with alkylphospholipids induces the formation of membrane raft aggregates containing Fas/CD95 death receptor and the adaptor molecule Fas-associated death domain-containing protein (FADD), which are critical in the triggering of apoptosis (Gajate et al, 2009). Miltefosine and other alkylphospholipids also alter intracellular cholesterol traffic and metabolism leading to an increased uptake, synthesis and accumulation of cholesterol in the cell (Carrasco et al, 2008;Jimenez-Lopez et al, 2006;Marco et al, 2009). As cholesterol and sphingomyelin content are critical for the integrity and functionality of membrane lipid rafts, the disturbance of the cholesterol/sphingomyelin ratio could alter signaling pathways associated with these membrane domains.…”

Antitumoral Alkylphospholipids Induce Cholesterol Efflux from the Plasma Membrane in HepG2 Cells

“…An important characteristic of APLs is their amphiphilic properties, enabling them to interact with cell membranes and affect cell metabolism at different levels (reviewed by . Within this context, studies carried out in our laboratory have demonstrated that, after long-term treatment, the APL hexadecyl 2-(trimethylazaniumyl)ethyl phosphate (hexadecylphosphocholine; HePC), also known as miltefosine, alters phosphatidylcholine metabolism (Jimé-nez-López et al, 2004) and intracellular cholesterol trafficking and metabolism, all of which lead to an increased uptake, synthesis, and accumulation of cholesterol in the cell (Jimé-nez-López et al, 2006;Carrasco et al, 2008;Marco et al, 2009). Thus, we extended our studies to analyze the effects of a variety of APLs, such as [1-O-octadecyl-2-O-methyl-racglycero-3-phosphocholine] (edelfosine), [(Z)-docos-13-enyl] 2-(trimethylazaniumyl)ethyl phosphate (erucylphosphocholine; ErPC), and 1,1-dimethylpiperidin-1-ium-4-yl) octadecyl phosphate (perifosine) upon intracellular cholesterol homeostasis and found that all of them impede the esterification of plasma-membrane cholesterol via acyl-CoA/cholesterol acyltransferase activity .…”