C22:0- and C24:0-dihydroceramides Confer Mixed Cytotoxicity in T-Cell Acute Lymphoblastic Leukemia Cell Lines

Michael W Holliday Jr,Min H Kang,Stephen B Cox,Barry J Maurer,Leah J Siskind

doi:10.1371/journal.pone.0074768

Abstract

We previously reported that fenretinide (4-HPR) was cytotoxic to acute lymphoblastic leukemia (ALL) cell lines in vitro in association with increased levels of de novo synthesized dihydroceramides, the immediate precursors of ceramides. However, the cytotoxic potentials of native dihydroceramides have not been defined. Therefore, we determined the cytotoxic effects of increasing dihydroceramide levels via de novo synthesis in T-cell ALL cell lines and whether such cytotoxicity was dependent on an absolute increase in total dihydroceramide mass versus an increase of certain specific dihydroceramides. A novel method employing supplementation of individual fatty acids, sphinganine, and the dihydroceramide desaturase-1 (DES) inhibitor, GT-11, was used to increase de novo dihydroceramide synthesis and absolute levels of specific dihydroceramides and ceramides. Sphingolipidomic analyses of four T-cell ALL cell lines revealed strong positive correlations between cytotoxicity and levels of C22:0-dihydroceramide (ρ = 0.74–0.81, P ≤ 0.04) and C24:0-dihydroceramide (ρ = 0.84–0.90, P ≤ 0.004), but not between total or other individual dihydroceramides, ceramides, or sphingoid bases or phosphorylated derivatives. Selective increase of C22:0- and C24:0-dihydroceramide increased level and flux of autophagy marker, LC3B-II, and increased DNA fragmentation (TUNEL assay) in the absence of an increase of reactive oxygen species; pan-caspase inhibition blocked DNA fragmentation but not cell death. C22:0-fatty acid supplemented to 4-HPR treated cells further increased C22:0-dihydroceramide levels (P ≤ 0.001) and cytotoxicity (P ≤ 0.001). These data demonstrate that increases of specific dihydroceramides are cytotoxic to T-cell ALL cells by a caspase-independent, mixed cell death mechanism associated with increased autophagy and suggest that dihydroceramides may contribute to 4-HPR-induced cytotoxicity. The targeted increase of specific acyl chain dihydroceramides may constitute a novel anticancer approach.

Highlights

The synthetic retinoid N-(4-hydroxyphenyl)retinamide has demonstrated cytotoxic activity in vitro to cell lines of multiple cancer types, including T-cell acute lymphoblastic leukemia (ALL) [1,2,3,4]
We previously reported that 4-HPR increased the activities of serine palmitoyltransferase and dihydroceramide synthase in a neuroblastoma cell line resulting in an increased ‘ceramides’ fraction and that 4-HPR increased ceramides coincident with cytotoxicity in a dose- and time-dependent manner in acute lymphoblastic leukemia cell lines [2,20]
Our results indicate that increased levels of certain, but not all, native acyl chain dihydroceramides are cytotoxic to T-cell ALL cell lines

Summary

Introduction

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) has demonstrated cytotoxic activity in vitro to cell lines of multiple cancer types, including T-cell acute lymphoblastic leukemia (ALL) [1,2,3,4]. 4-HPR stimulated the de novo sphingolipid pathway leading to a time- and dosedependent increase of dihydroceramides in multiple model systems [9,10,11,12,13,14,15]. We previously reported that 4-HPR increased the activities of serine palmitoyltransferase and dihydroceramide synthase in a neuroblastoma cell line resulting in an increased ‘ceramides’ fraction and that 4-HPR increased ceramides coincident with cytotoxicity in a dose- and time-dependent manner in acute lymphoblastic leukemia cell lines [2,20]. Recent work with more advanced methodologies has demonstrated that 4-HPR increases dihydroceramides due to concurrent inhibition of dihydroceramide desaturase 1 (DES1) [13,14,15]

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