Acid ceramidase expression modulates the sensitivity of A375 melanoma cells to dacarbazine

Abstract

Dacarbazine (DTIC) is the treatment of choice for metastatic melanoma, but its response in patients remains very poor. Ceramide has been shown to be a death effector and to play an important role in regulating cancer cell growth upon chemotherapy. Among ceramidases, the enzymes that catabolize ceramide, acid ceramidase (aCDase) has been implicated in cancer progression. Here we show that DTIC elicits a time- and dose-dependent decrease of aCDase activity and an increase of intracellular ceramide levels in human A375 melanoma cells. The loss of enzyme activity occurred as a consequence of reactive oxygen species-dependent activation of cathepsin B-mediated degradation of aCDase. These events preceded autophagic features and loss of cell viability. Down-regulation of acid but not neutral or alkaline ceramidase 2 resulted in elevated levels of ceramide and sensitization to the toxic effects of DTIC. Conversely, inducible overexpression of acid but not neutral ceramidase reduced ceramide levels and conferred resistance to DTIC. In conclusion, we report that increased levels of ceramide, due to enhanced degradation of aCDase, are in part responsible for the cell death effects of DTIC. These results suggest that down-regulation of aCDase alone or in combination with DTIC may represent a useful tool in the treatment of metastatic melanoma.

FIGURE 1.

Dacarbazine-induced changes in intracellular ceramide concentrations. A375 melanoma cell lines were incubated for 48 h in the presence or absence of DTIC (100 μg/ml). Cellular lipids were extracted, and sphingolipid concentrations were determined by LC/MS. Data represent the levels in picomoles/10⁶ cells of individual ceramide species (A); total ceramide species (B) and sphingosine (So) and sphingosine 1-phosphate (S1P) (C). Data are the means ± S.E. of five independent determinations.

FIGURE 2.

Dacarbazine-induced loss of acid ceramidase activity. A, aCDase activity in A375 melanoma cells treated with DTIC (100 μg/ml) for the indicated times (mean ± S.E. of three independent experiments). B, time course of cell viability loss in A375 cells treated with 100 μg/ml DTIC (mean ± S.E. of two independent experiments performed in sextuplicate). C, dose dependence of decrease in aCDase activity (filled squares) in A375 cells incubated for 48 h with the indicated concentrations of DTIC (data are from a representative experiment performed in duplicate). In the same cell lysates, the activity of acid β-galactosidase (open squares) was determined. D, Western blot analysis of aCDase and sphingosine kinase 1 in A375 cells treated with DTIC (100 μg/ml) for 48 h.

FIGURE 3.

Effect of protease inhibitors on the loss of aCDase induced by dacarbazine. A, effect of lysosomal protease inhibitors on aCDase activity in A375 cells treated for 48 h with or without the indicated concentrations of DTIC and preincubated for 2 h in the absence or presence of leupeptin (50 μm) or pepstatin A (100 μm). B, effect of the cathepsin B inhibitor CA074-Me on the loss of aCDase activity. Cells were preincubated for 2 h with CA074-Me (5 μm) and then treated for 24 h with or without DTIC (25 μg/ml). C, effect of the broad caspase inhibitor z-VAD-fmk on aCDase activity. Cells were preincubated with z-VAD-fmk (20 μm) or DMSO for 2 h, and then treated for 48 h with or without DTIC (25 μg/ml). Data are the means ± S.E. of three independent experiments.

FIGURE 4.

Effect of NAC on acid ceramidase activity (A), cathepsin B activity (B), and cell viability (C) in dacarbazine-treated melanoma cells. A and C, A375 cells were preincubated for 2 h in the presence or absence of NAC (10 mm) and then treated for 48 h with the indicated concentration of DTIC. Data are means ± S.E. of three independent experiments performed in triplicate. B, cathepsin B activity was determined in cells preincubated or not for 2 h with 10 mm NAC and then treated for the indicated times with DTIC (10 μg/ml). Enzyme activity is expressed in percentage of that measured in cells preincubated with or without NAC for only 2 h, but not treated by DTIC (mean ± S.E. of three independent experiments).

FIGURE 5.

Temporal relationship between accumulation of C16 ceramide and autophagy. A, fluorescence microscopy pictures of A375 cells. Cells stably expressing the pEGFP-LC3 vector were treated for the indicated times with DTIC (100 μg/ml). Cells were stained with monodansylcadaverine (MDC, 50 μm) for 30 min and fixed with paraformaldehyde before observation. B, levels of ceramide species in A375 cells treated for the indicated times with DTIC (100 μg/ml). Basal concentrations of individual ceramide species were 60, 14, 17, 72, and 91 pmol/10⁶ cells for C16:0, C18:0, C22:0, C24:0, and C24:1, respectively. C, aCDase activity was determined in cells after transfection of siRNA against Atg7 or emerin for 24 h, and then treated for 48 h with or without 100 μg/ml DTIC (data are from a representative experiment performed in duplicate). Inset, Western blot analysis of Atg7 in cells transfected with siRNA against Atg7 and emerin.

FIGURE 6.

Effects of aCDase down-regulation in A375 melanoma cells. A, A375 cells were transfected with siRNA against aCDase and emerin for 72 h, then quantitative RT-PCR and Western blot analysis were performed (data are means ± S.E. of three independent experiments). B, aCDase activity of cells transfected with siRNA against aCDase or emerin for 24 h, then treated with or without DTIC for 48 h (data are representative of two independent experiments performed in duplicate). C, A375 cells were transfected with siRNA against ASAH1 or a control siRNA, and 24 h later, cells were treated or not with DTIC (100 μg/ml) for 48 h. Lipids were extracted, and levels of total ceramides, sphingosine (So), and S1P were quantified by LC/MS. Data are means ± S.E. of three independent experiments performed in duplicate. D, cell viability of A375 cells transfected with aCDase or emerin siRNA for 24 h, then treated with the indicated concentrations of DTIC for 48 h (mean ± S.E. of three independent experiments performed in triplicate).

FIGURE 7.

Effects of aCDase overexpression in A375 melanoma cells. A, A375 ASAH1 cells were treated or not with doxycycline (DOX, 1 μg/ml) for 24 h. Then, aCDase activity measurement and Western blot analysis were performed (data are representative of more than five experiments). B, A375 ASAH1 cells were induced or not with doxycycline for 24 h, then treated or not with DTIC (10 μg/ml) for 48 h. Lipids were extracted, and levels of total ceramides, sphingosine (So), and S1P were quantified by LC/MS. Data are means ± S.E. of three independent experiments performed in duplicate. C, aCDase activity was measured in A375 cells preincubated for 24 h with or without doxycycline and then treated for 48 h with the indicated concentrations of DTIC (mean ± S.E. of two independent experiments performed in duplicate). D, cell viability was measured in 24-h induced and non-induced cells treated for 48 h with the indicated concentrations of DTIC. Results are means ± S.E. of three independent experiments performed in triplicate.