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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.

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Conflict of interest statement

Competing Interests: SBC is employed by Research and Testing Laboratory, LLC. A patent application by the Texas Tech University System (TTUS) is pending, on which BJM is an Inventor. As TTUS has a revenue-sharing policy for faculty-inventors, should this patent be issued and commercialized, BJM may share in patent-derived revenues. Patent application: Num. 20120121691 A1, dated May 17, 2012, entitled, " Method for Increasing the Production of a Specific ACYL-Chain Dihydroceramide(s) for Improving the Effectiveness of Cancer Treatments." Assignee: Texas Tech University System. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1

Figure 1. Schematic of the de novo …

Figure 1. Schematic of the de novo ceramide pathway.

Rate-limiting enzyme, serine palmitoyltransferase (SPT), condenses…

Figure 1. Schematic of the de novo ceramide pathway. Rate-limiting enzyme, serine palmitoyltransferase (SPT), condenses serine and palmitoyl-CoA to 3-ketosphinganine, which is subsequently reduced to sphinganine. Dihydroceramide synthases 1-6 (CerS 1-6), each utilizing a preferred subset of fatty acid-derived acyl-CoAs, add a fatty acyl chain (green) to sphinganine to produce dihydroceramides. Dihydroceramide desaturase (DES1) converts dihydroceramides to ceramides by introduction of a 4,5-trans double bond into the sphinganine backbone of dihydroceramide. 4-HPR stimulates both SPT and CerS in certain cancer cell lines. Both 4-HPR and GT-11, a synthetic ceramide derivative, inhibit DES1. Asterisks (*) indicate variable carbon length and saturation.
Figure 2

Figure 2. Effects of sphinganine and GT-11…

Figure 2. Effects of sphinganine and GT-11 on dihydroceramides, ceramides and cytotoxicity.

A - C …
Figure 2. Effects of sphinganine and GT-11 on dihydroceramides, ceramides and cytotoxicity. A-C) CCRF-CEM cells were treated with sphinganine (4 µM, S) ± GT-11 (0.5 µM, G) for six hours and sphingolipids analyzed. A) Total dihydroceramides (DHCer) and ceramides (Cer) were normalized to drug/fatty acid vehicle-treated cells (control) and plotted as fold change (Y-axis). Error bar, propagated standard deviation. Individual dihydroceramides (B) and ceramides (C) were normalized to control and plotted as fold change (Z-axis). Dihydroceramides and ceramides are identified by acyl chain (x:y), where x is the number of carbons and y is the number of double bonds in the acyl chain (X-axis). Significant (A, P ≤ 0.001; B and C, P ≤ 0.05) fold changes between treatment and vehicle control levels are indicated by asterisks (*). D) Cytotoxicity of sphinganine and GT-11. Indicated cell lines were treated with sphinganine (0-4 µM) and/or GT-11 (0.5 µM). The cytotoxicity of GT-11-alone is represented by the sphinganine (0 µM) data point. Cytotoxicity assayed by DIMSCAN cytotoxicity assay at +48 hours. Data were normalized to vehicle-treated control and plotted as Survival Fraction (Y-axis). Error bar, SEM. Sphinganine + GT-11 resulted in significantly increased (P ≤ 0.001) cytotoxicity relative to sphinganine-only for all concentrations and across all cell lines.
Figure 3

Figure 3. Effects of specific fatty acids…

Figure 3. Effects of specific fatty acids on sphinganine ± GT-11-induced dihydroceramides and cytotoxicity.

A …
Figure 3. Effects of specific fatty acids on sphinganine ± GT-11-induced dihydroceramides and cytotoxicity. A, B) Effects on dihydroceramides. CCRF-CEM cells were treated with (A) sphinganine (1 µM), or (B) sphinganine (1 µM) + GT-11 (0.5 µM), and supplemented with the indicated fatty acids (5 µM) for six hours, followed by sphingolipid assay. To evaluate the effects resulting from addition of each fatty acid, data for (A) and (B) were normalized either to cells that received sphinganine-only with no fatty acid supplementation (A), or to sphinganine + GT-11 without fatty acid (B), and plotted as fold change (Z-axis). Fatty acids are identified by x:y, where x is the number of carbons and y is the number of double bonds in the fatty acid chain (Y-axis). Significant (P ≤ 0.05) differences from sphinganine-only are indicated by asterisks (*). C) Effects on cytotoxicity. CCRF-CEM cells were treated with sphinganine-only (0-4 µM), or sphinganine (0-4 µM) + GT-11 (0.5 µM), supplemented with the indicated fatty acids (5 µM). Cytotoxicity assayed by DIMSCAN cytotoxicity assay at +48 hours. Data were normalized to control and plotted as survival fraction (Y-axis). Error bar, SEM. Data, grouped by sphinganine dose, were analyzed by one-way ANOVA.
Figure 4

Figure 4. Mechanisms of C22:0-dihydroceramide induced cell…

Figure 4. Mechanisms of C22:0-dihydroceramide induced cell death.

A ) Reactive oxygen species levels in…
Figure 4. Mechanisms of C22:0-dihydroceramide induced cell death. A) Reactive oxygen species levels in sphinganine and/or GT-11 treated CCRF-CEM cells supplemented with or without C22:0-fatty acid. CCRF-CEM cells were treated with sphinganine (1 µM) and/or GT-11 (0.5 µM), both with and without C22:0-FA (5 µM). 4-HPR (10 µM) and H2O2 (120 mM, not shown) were employed as positive controls. Cells were stained with 2’, 7’-dichlorofluorescein diacetate and fluorescence analyzed after 6 hours by flow cytometry. Data were normalized to control. Error bars, SEM. B & C) Effect of pan-caspase inhibition on TUNEL positivity. CCRF-CEM cells were pre-treated with pan-caspase inhibitor, Boc-D-FMK (80 µM), or DMSO (final concentration = 0.33%, Boc-D-FMK vehicle control), for one hour prior to treatment with ABT-737 (1 µM, positive control), or C22:0-FA plus sphinganine (1 or 2 µM, S) + GT-11 (0.5 µM, G). C22:1-FA plus sphinganine (1 µM) + GT-11 (0.5 µM, G) and Boc-D-FMK alone included as controls. Cells analyzed by TUNEL assay at +24 hrs. Shown are histograms representative of three separate experiments. Histograms are of indicated treatments analyzed by PI counter-stain of TUNEL samples. D) Effect of pan-caspase inhibition on cytotoxicity. CCRF-CEM cells were pre-treated with pan-caspase inhibitor, Boc-D-FMK (80 µM, Boc), for one hour prior to treatment with ABT-737 (1 µM), C22:0-FA plus sphinganine (2 µM, S) + GT-11 (0.5 µM, G). Cytotoxicity assessed at +12 and +24 hrs by DIMSCAN cytotoxicity assay and represented as Survival Fraction. Asterisks (*) represent significant (P ≤ 0.05) effects of Boc treatment.
Figure 5

Figure 5. Caspase cleavage and LC3B-I/II turnover.

Figure 5. Caspase cleavage and LC3B-I/II turnover.

A ) CCRF-CEM cells treated with drug/fatty acid…
Figure 5. Caspase cleavage and LC3B-I/II turnover. A) CCRF-CEM cells treated with drug/fatty acid vehicle (C), sphinganine (1 µM, S), or sphinganine (1 µM) + GT-11 (0.5 µM) (SG), were supplemented with the indicated fatty acids (5 µM). After 12 hours, total proteins were extracted and procaspase-3 (35 kb), activated caspase-3 (17/19 kb), and LCB3-I/II (14/16 kb), were detected by immunoblotting. β-Actin served as loading control. Treatment with pan-Bcl-2 inhibitor, ABT-737 (1 µM, A), and LC3B-transfected, HEK-293 cell lysate, were used as positive controls. C22:1-fatty acid was used as a negative control for C22:0-fatty acid. Lanes rearranged to ease interpretation. Data representative of three separate experiments are shown. B) Assessment of LC3B-II flux. CCRF-CEM cells were pretreated with or without protease inhibitors (Pepstatin-A and E64d) and treated as described. After 12 hours, total proteins were extracted and LC3B-I/II analyzed by immunoblotting. Data representative of three separate experiments are shown, except for C22:1-fatty acid.
Figure 6

Figure 6. Effects of specific fatty acids…

Figure 6. Effects of specific fatty acids on sphinganine + GT-11-induced dihydroceramide accumulation and cytotoxicity.

Figure 6. Effects of specific fatty acids on sphinganine + GT-11-induced dihydroceramide accumulation and cytotoxicity. A) Effects of fatty acids on dihydroceramide levels. MOLT-4, COG-LL-317h, and COG-LL-332h, cell lines were treated with sphinganine (1 µM) + GT-11 (0.5 µM) supplemented with the indicated fatty acid (5 µM) for six hours and sphingolipids analyzed. Data are normalized to cells that received sphinganine + GT-11 without fatty acid supplementation and dihydroceramide levels plotted as fold change (Z-axis). Significant (P ≤ 0.05) fold change differences are indicated by asterisks (*). B) Effects of fatty acids on cytotoxicity. Cell lines were treated with sphinganine (0-4 µM) ± GT-11 (0.5 µM) and supplemented with C18:0-, C22:0- or C22:1-fatty acids (5 µM). Cytotoxicity assessed at +48 hours by DIMSCAN cytotoxicity assay. Data were normalized to controls and plotted as Survival Fraction (Y-axis). Error bar, SEM.
Figure 7

Figure 7. Effects of specific fatty acids…

Figure 7. Effects of specific fatty acids on 4-HPR-induced dihydroceramide levels and cytotoxicity.

A )…
Figure 7. Effects of specific fatty acids on 4-HPR-induced dihydroceramide levels and cytotoxicity. A) Effects on dihydroceramide levels. COG-LL-317h and COG-LL-332h cells were treated with 4-HPR (1 µM) with or without C18:0- or C22:0-fatty acids (5 µM) for six hours and sphingolipids analyzed. Data were normalized to cells that received 4-HPR without fatty acid supplementation and plotted as fold change (Z-axis). Significant (P ≤ 0.001) differences from 4-HPR without fatty acid indicated by asterisks (*). B) Effects on cytotoxicity. CCRF-CEM, MOLT-4, COG-LL-317h, and COG-LL-332h cell lines were treated with 4-HPR (0-9 µM) ± C18:0- or C22:0-fatty acids (5 µM) and cytotoxicity assessed at +48 hours by DIMSCAN cytotoxicity assay. Data were normalized to controls and represented as Survival Fraction (Y-axis). Error bar, SEM. Significant (P ≤ 0.001) differences in cytotoxicity from 4-HPR without fatty acid are indicated by asterisks (*).
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References

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