N-(4-Hydroxyphenyl)retinamide increases dihydroceramide and synergizes with dimethylsphingosine to enhance cancer cell killing

Abstract

Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is cytotoxic in many cancer cell types. Studies have shown that elevation of ceramide species plays a role in 4-HPR cytotoxicity. To determine 4-HPR activity in a multidrug-resistant cancer cell line as well as to study ceramide metabolism, MCF-7/AdrR cells (redesignated NCI/ADR-RES) were treated with 4-HPR and sphingolipids were analyzed. TLC analysis of cells radiolabeled with [ 3 H]palmitic acid showed that 4-HPR elicited a doseresponsive increase in radioactivity migrating in the ceramide region of the chromatogram and a decrease in cell viability. Results from liquid chromatography/ electrospray tandem mass spectrometry revealed large elevations in dihydroceramides (N-acylsphinganines), but not desaturated ceramides, and large increases in complex dihydrosphingolipids (dihydrosphingomyelins, monohexosyldihydroceramides), sphinganine, and sphinganine 1-phosphate. To test the hypothesis that elevation of sphinganine participates in the cytotoxicity of 4-HPR, cells were treated with the sphingosine kinase inhibitor D-erythro-N,N -dimethylsphingosine (DMS), with and without 4-HPR. After 24 h, the 4-HPR/DMS combination caused a 9-fold increase in sphinganine that was sustained through +48 hours, decreased sphinganine 1-phosphate, and increased cytotoxicity. Increased dihydrosphingolipids and sphinganine were also found in HL-60 leukemia cells and HT-29 colon cancer cells treated with 4-HPR. The 4-HPR/DMS combination elicited increased apoptosis in all three cell lines. We propose that a mechanism of 4-HPR -induced cytotoxicity involves increases in dihydrosphingolipids, and that the synergy between 4-HPR and DMS is associated with large increases in cellular sphinganine. These studies suggest that enhanced clinical efficacy of 4-HPR may be realized through regimens containing agents that modulate sphingoid base metabolism. [Mol Cancer Ther 2008;7(9):2967 -76]

Sphingosine Kinase Mediates Resistance to the Synthetic Retinoid N-(4-Hydroxyphenyl)retinamide in Human Ovarian Cancer Cells

“…Ceramide production in HPR-treated tumor cells is somehow peculiar, because it has been suggested that the most relevant mechanism for HPR-induced ceramide generation might be the de novo biosynthesis (HPR is indeed a potent activator of serine palmitoyltransferase and dihydroceramide synthases, the key enzymes in the early steps of ceramide biosynthesis), whereas for many other antitumor drugs sphingomyelin hydrolysis due to the activation of sphingomyelinases is the main source of pro-apoptotic ceramide. Moreover, it has been demonstrated that dihydroceramide, rather than ceramide, accumulates in HPR-treated tumor cells, due to the inhibitory effect of this retinoid on dihydroceramide desaturase (22,24,37). Indeed we recently showed by electrospray ionization-MS analysis that dihydroceramide and not ceramide is increased upon treatment of A2780 cells with HPR (36).…”

“…Indeed we recently showed by electrospray ionization-MS analysis that dihydroceramide and not ceramide is increased upon treatment of A2780 cells with HPR (36). In addition, recent observations suggest that HPR cytotoxicity might at least in part due to the elevation of cellular sphinganine levels (24). Independently from the mechanism underlying the production of cytotoxic (dihydro)ceramide upon chemotherapeutic treatments, it has been suggested that the acquired resistance to chemotherapeutic drugs in tumors and tumor cell lines can be ascribed to alterations in sphingolipid metabolism leading to a reduced potential for dihydroceramide formation or an increased clearance of dihydroceramide.…”

“…Sphinganine production was also enhanced by HPR treatment, and a possible contribution of sphinganine to HPR-induced toxicity has been recently suggested (24). Obviously, increased SK activity associated with HPR resistance would be effective in scavenging sphinganine as well.…”

“…HPR-induced production of ceramide mainly occurs via de novo synthesis, because HPR activates both serine palmitoyltransferase and dihydroceramide synthase (20,21) that catalyze the first steps in sphingolipid biosynthesis (22). Moreover, it has been recently shown that HPR concomitantly inhibits dihydroceramide desaturase (22,23), suggesting that dihydroceramide rather than (or in addition to) ceramide might mediate HPRinduced toxicity (24) possibly involving other mechanisms of death in addition to apoptosis. In this work, we investigate whether the resistance to HPR of human ovarian cancer cells could be associated with the activation of the SK/S1P leading to an altered dihydroceramide/S1P ratio that could prevent or overcome ceramide-mediated HPR-induced cell death.…”

Sphingosine kinase 1 promotes tumor progression and confers malignancy phenotypes of colon cancer by regulating the focal adhesion kinase pathway and adhesion molecules

“…SphK1 was significantly elevated in azoxymethane-induced murine colon cancer tissues, SphK1 knockout mice subjected to azoxymethane had significantly less aberrant formation of crypt foci and cancer development (7). Moreover, SphK1 inhibitor or shRNA enhanced the cytotoxicity and chemosensitivity of colon cancer cells (8,9). Studies also showed that the expression of SphK1 was required for the proliferation of small intestinal tumor cell and SphK1 was able to promote intestinal adenoma progression (10).…”