Altered Sphingolipid Metabolism inN-(4-Hydroxyphenyl)- retinamide-resistant A2780 Human Ovarian Carcinoma Cells

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.

Involvement of c-Fos in fenretinide-induced apoptosis in human ovarian carcinoma cells

“…5,[12][13][14] Other studies have reported that apoptosis in response to HPR primarily occurs by a receptor-independent mechanism, which is accompanied by generation of reactive oxygen species (ROS), 15,16 alteration of mitochondrial membrane potential, 17,18 a decrease in Bcl-2 expression, 19 or an increase in ceramide levels. [20][21][22] The proto-oncogene c-Fos encodes a nuclear DNA binding phosphoprotein that dimerizes with the gene product of c-Jun to form the transcription factor activating protein 1 (AP-1). AP-1 converts extracellular signals into changes in the expression of specific target genes, which harbor the AP-1 DNA binding site(s) in their promoter regions.…”

“…29 We also reported that HPR was able to induce ceramide levels in sensitive but not in resistant cells. 22 Using A2780 and A2780/ HPR cells and a panel of other human ovarian cancer cell lines, we examined the effects of HPR treatment on c-Fos expression to determine the role of the proto-oncogene in HPR-induced apoptosis. Our findings indicated that c-Fos inducibility and HPR-induced apoptosis are closely associated.…”

Fenretinide sensitizes multidrug-resistant human neuroblastoma cells to antibody-independent and ch14.18-mediated NK cell cytotoxicity

“…In 4-HPR-resistant ovarian carcinoma cells, increased ganglioside levels were demonstrated after treatment with 4-HPR compared to 4-HPR sensitive cells [73]. These data suggest that 4-HPR might be able to enhance GD2-directed immunotherapy via an increased GD2 expression on 4-HPR-resistant NB cells mediated by an evasion mechanism from toxic ceramide accumulation.…”

“…4-HPR treatment is known to induce an increase of gangliosides on 4-HPR-resistant ovarian cancer [73] and SH-SY5Y cells [69]. We expanded this attempt to multi-drug or partially resistant NB cells and show that 4-HPR induced an increase of GD2 expression.…”

“…Since the drug-resistant phenotype in cancer cells is often associated with an increase in ceramide glycosylation [72,73,77], we determined whether 4-HPR resistance in CHLA-136 cell line is correlated with an over expression of glucosylceramide synthase (GCS). As shown in Fig.…”