Abstract
In the present work, we studied the effects of fenretinide (N-(4-hydroxyphenyl)retinamide (HPR)), a hydroxyphenyl derivative of all-trans-retinoic acid, on sphingolipid metabolism and expression in human ovarian carcinoma A2780 cells. A2780 cells, which are sensitive to a pharmacologically achievable HPR concentration, become 10-fold more resistant after exposure to increasing HPR concentrations. Our results showed that HPR was able to induce a dose- and time-dependent increase in cellular ceramide levels in sensitive but not in resistant cells. This form of resistance in A2780 cells was not accompanied by the overexpression of multidrug resistance-specific proteins MDR1 P-glycoprotein and multidrug resistance-associated protein, whose mRNA levels did not differ in sensitive and resistant A2780 cells. HPR-resistant cells were characterized by an overall altered sphingolipid metabolism. The overall content in glycosphingolipids was similar in both cell types, but the expression of specific glycosphingolipids was different. Specifically, our findings indicated that glucosylceramide levels were similar in sensitive and resistant cells, but resistant cells were characterized by a 6-fold lower expression of lactosylceramide levels and by a 6-fold higher expression of ganglioside levels than sensitive cells. The main gangliosides from resistant A2780 cells were identified as GM3 and GM2. The possible metabolic mechanisms leading to this difference were investigated. Interestingly, the mRNA levels of glucosylceramide and lactosylceramide synthases were similar in sensitive and resistant cells, whereas GM3 synthase mRNA level and GM3 synthase activity were remarkably higher in resistant cells.
Highlights
Sphingolipid metabolism plays a pivotal role in the mechanism of apoptosis induced in tumor cells
We studied the effects of fenretinide (N-(4-hydroxyphenyl)retinamide (HPR)), a hydroxyphenyl derivative of all-trans-retinoic acid, on sphingolipid metabolism and expression in human ovarian carcinoma A2780 cells
A2780 and A2780/HPR cells were incubated in the presence of [1-3H]sphingosine for 2 h, followed by a 24-h chase. [1-3H]sphingosine is efficiently incorporated by cultured cells and converted into ceramide, which is further utilized for the synthesis of more complex sphingolipids
Summary
Chemicals—Commercial chemicals were the purest available, common solvents were distilled before use, and water was doubly distilled in a glass apparatus. 25 l of 0.1% Triton-X 100 (v/v) in chloroform/methanol (2:1) and 25 l of 0.2% sodium cholate (w/v) in chloroform/methanol (2:1) were mixed with 2–100 pmol of [1-3H(sphingosine)]C16Cer (corresponding to 4.5 nCi) from a stock solution in chloroform/methanol (2:1) and dried under N2 To this mixture, 7.5 l of water was added, and the tubes were sonicated for 3 min, heated for 5 s at 80 °C, and put on ice. Ceramidase activities were assayed by adding 12.5 l of 400 mM sodium acetate buffer, pH 4.5 (for the acidic enzyme), 12.5 l of 400 mM Tris-HCl, pH 7.4 (for the neutral enzyme), or 12.5 l of 400 mM glycine-NaOH, pH 9.0 (for the alkaline enzyme), 5 l 50 mM MgCl2, and 10 l of cell homogenate (containing 200 g of protein) in a total reaction volume of 50 l. The protein content was determined according to Lowry [61], using bovine serum albumin as the reference standard
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