# Acid Ceramidase Expression Modulates the Sensitivity of A375 Melanoma Cells to Dacarbazine

Carmen Bedia, Josefina Casas, Nathalie Andrieu-Abadie, Gemma Fabriàs,

Thierry Levade

► To cite this version:

Carmen Bedia, Josefina Casas, Nathalie Andrieu-Abadie, Gemma Fabriàs, Thierry Levade. Acid Ceramidase Expression Modulates the Sensitivity of A375 Melanoma Cells to Dacarbazine. Journal of Biological Chemistry, 2011, 286 (32), pp.28200 - 28209. 10.1074/jbc.m110.216382. inserm-04963218

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# Acid Ceramidase Expression Modulates the Sensitivity of A375 Melanoma Cells to Dacarbazine*§

Received for publication, December 28, 2010, and in revised form, June 20, 2011 Published, JBC Papers in Press, June 23, 2011, DOI 10.1074/jbc.M110.216382

Carmen Bedia<sup>†§1</sup>, Josefina Casas<sup>¶</sup>, Nathalie Andrieu-Abadie<sup>†§</sup>, Gemma Fabriàs<sup>¶</sup>, and Thierry Levade<sup>†§||</sup><sup>2</sup>

From †INSERM UMR 1037, CHU Rangueil, BP 84225, Toulouse 31432 Cedex 4, France, the §Centre de Recherche en Cancérologie de Toulouse, Université Toulouse III Paul-Sabatier, Toulouse, France, the ¶Department of Biomedicinal Chemistry, Institut de Química Avançada de Catalunya, Consejo Superior de Investigaciones Científicas, 08034 Barcelona, Spain, and the ‖Laboratoire de Biochimie Métabolique, Institut Fédératif de Biologie, Hôpital Purpan, 31059 Toulouse, France

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 intra-cellular 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.

Melanoma is the most deadly form of skin cancer that originates from melanocytes, the melanin-producing cells located in the basal layer of the epidermis. The incidence of melanoma has been increasing over decades, especially among fair-skinned people (1). Early-stage melanoma can be readily cured by surgical excision of the primary lesion, but advanced melanoma, often a metastatic disease, has a poor median survival, ranging from 2 to 8 months, with only 5% surviving >5 years (2).

Metastatic melanoma is very resistant to many forms of traditional chemotherapy. To date, dacarbazine (DTIC)<fnref n="3" /> is considered to be the reference single agent for the management of advanced melanoma, with objective tumor responses in ~15-25% of patients. However, >95% of the responses are partial, with a 6-year median survival of 2% (3). Temozolomide is an orally available DTIC analog that has shown efficacy in the treatment of a variety of solid tumors, with the advantage of central nervous system penetration. Temozolomide and DTIC have demonstrated equal efficacy in terms of objective response rate and overall survival for patients with advanced metastatic melanoma (4). Since the original approval of DTIC in 1975 for the treatment of metastatic melanoma, considerable effort has been expended in attempts to improve survival. However, treatments based on the combination of these agents with other cytotoxics or immunotherapy have not resulted in response rates of durable remission, enough to increase the median survival of the patients (3).

Sphingolipids are a complex family of bioactive lipids that play important structural and second messenger roles (5). Ceramide is the central molecule to both metabolism and signaling of sphingolipids. It has been implicated in several cellular processes like cell cycle arrest, apoptosis, and cell senescence (6, 7). Therefore, dysregulated ceramide metabolism is linked to numerous human diseases, including cancer (6, 7). Consistent with this, many anticancer agents have been shown to modulate ceramide synthesis by regulating individual components of the *de novo* pathway. For example, daunorubicin stimulates both *de novo* ceramide synthesis and sphingomyelin hydrolysis (8, 9), camptothecin and fludarabine induce *de novo* ceramide production in various human cancer cells (10), and etoposide enhances serine palmitoyltransferase in leukemia cells (11). Other cytotoxic drugs such as cytosine arabinoside are able to activate neutral sphingomyelinase to generate ceramide in leukemic cells (12). In contrast to the actions of ceramide, sphingosine 1-phosphate (S1P), generated from ceramide hydrolysis and further phosphorylation of the resulting sphingosine, is known as a regulator of cell proliferation, inflammation, vasculogenesis, and resistance to apoptotic cell death (13). Hence, the relative balance between ceramide and S1P has been proposed to influence the cell fate in response to cytotoxic stimuli.

<footnote>* This work was supported in part by INSERM, Université Paul Sabatier, the Vaincre les Maladies Lysosomales Foundation, the Spanish Ministry of Science and Innovation (Grant SAF2008-00706), and the Generalitat de Catalunya (Grant 2009-SGR-1072).</footnote>

<footnote>§ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3, Materials, and Table S1.</footnote>

<footnote n="1">¹ Supported by the Obra Social La Caixa and Agència de Gestió d' Ajuts Universitaris i de Recerca (Grant 2007-BP-A00129), Generalitat de Catalunya. Present address: Dept. of Biomedicinal Chemistry, Institut de Química Avançada de Catalunya, CSIC, 08034 Barcelona, Spain.</footnote>

<footnote n="2">² To whom correspondence should be addressed: INSERM UMR1037, Equipe no. 4, Centre de Recherche en Cancérologie de Toulouse, CHU Rangueil, BP 84225, 31432 Toulouse cedex 4, France. Tel.: 33-5-61-32-20-60 or 6-14-14-72-61; Fax: 33-5-61-32-56-22 or 5-61-32-20-84; E-mail: thierry.levade@inserm.fr.</footnote>

<footnote n="3">³ The abbreviations used are: DTIC, dacarbazine; aCDase, acid ceramidase; AMC, 7-amino-4-methylcoumarin; NAC, N-acetylcysteine; ROS, reactive oxygen species; S1P, sphingosine 1-phosphate; z, benzyloxycarbonyl; fmk, fluoromethyl ketone.</footnote>
Ceramidases are the enzymes that catalyze ceramide hydrolysis to release sphingosine and the corresponding fatty acid. According to their pH optima of activity, acid, neutral, and alkaline ceramidases can be distinguished (14). Among these ceramidases, acid ceramidase (aCDase), encoded by the *ASAH1* gene, appears to play an important role in cancer progression. For instance, aCDase is up-regulated in prostate cancer (15, 16), conferring resistance of cancer cells to radiation (17). Also, pharmacological inhibition of aCDase has been reported to completely prevent tumor growth of human colon cancer cells metastatic to the liver (18) and of prostate cancer xenografts (19). In addition, down-regulation of *ASAH1* using specific siRNA reduced tumor growth in an *in vivo* model of hepatocellular carcinoma (20).

Given that cytotoxic drugs regulate the metabolism of sphingolipids, we have explored the effects of DTIC in human A375 melanoma cells. This work shows for the first time that DTIC promotes aCDase degradation in lysosomes and the subsequent increase of intracellular ceramide levels in A375 cells. We also demonstrate that the modulation of aCDase expression affects the sensitivity of these tumor cells to DTIC treatment.

## EXPERIMENTAL PROCEDURES

**Cell Lines**—The human melanoma cell line A375 was obtained from the American Type Culture Collection. The A375 cell line that stably expresses the tetracycline repressor was obtained by transfection of the pcDNA6/TR vector with Lipofectamine 2000 (Invitrogen) and further selection with blasticidin (3 µg/ml). The cDNA of *ASAH1* was subcloned into pcDNA5/TO, and the sequence was verified. This vector was transfected into pcDNA6/TR A375 cells with Lipofectamine 2000 to obtain a new cell line (A375 *ASAH1*) that overexpresses *ASAH1* under the control of a tetracycline/doxycycline-responsive promoter. The antibiotic selection of this new cell line was performed with blasticidin (3 µg/ml) and hygromycin B (250 µg/ml). Ectopic expression of *ASAH1* was induced with doxycycline at 1 µg/ml for 24 h before treatment with DTIC. Cells were grown in a humidified 5% CO₂ atmosphere at 37 °C in DMEM medium containing Glutamax (Invitrogen).

**Sequences, Vectors, and siRNA Transfections**—The cDNA coding for human *ASAH1* was kindly provided by Dr. J. A. Medin (University of Toronto, Canada). The pcDNA6/TR and pcDNA5/TO plasmids belong to the T-REx™ system from Invitrogen. The pEGFP-LC3 vector was kindly provided by Dr. T. Yoshimori (Osaka University, Japan). siRNA against *ASAH1*, *Atg7*, and emerin (ON-TARGETplus SMART pool) were purchased from Dharmacon Thermo Scientific, and their transfection was performed at 10 nM using the Hi Perfect transfection reagent (Qiagen).

**Other Reagents**—Dacarbazine, doxycycline, N-acetylcysteine, pepstatin A, leupeptin, and monodansylcadaverine were supplied from Sigma, blasticidin and hygromycin B were from Invitrogen, CA074-Me was obtained from Enzo Life Sciences, z-VAD-fmk was from Bachem, Ac-DEVD-AMC from Alexis, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide substrate was purchased from Euromedex.

Enzyme substrates included Rbm14-12 (aCDase substrate) produced in our laboratories (21), z-Arg-Arg-AMC, a cathepsin B

B fluorogenic substrate from Calbiochem, and 4-methylumbelliferyl-β-D-galactopyranoside (β-galactosidase substrate) obtained from Sigma.

**LC/MS Analysis of Sphingolipids**—Sphingolipid extracts, fortified with internal standards (N-dodecanoylsphingosine, N-dodecanoylglucosylsphingosine, D-erythro-dihydrospingosine, N-dodecanoylsphingosylphosphorylcholine, and C17-D-erythro-dihydrospingosine-1-phosphate, 0.2 nmol each), were prepared as described (22) and analyzed. The LC/MS analysis consisted of a Waters Aquity UPLC system connected to a Waters LCT Premier orthogonal accelerated time of flight mass spectrometer (Waters), operated in positive electrospray ionization mode. Full scan spectra from 50 to 1500 Da were acquired, and individual spectra were summed to produce data points each of 0.2 s. Mass accuracy and reproducibility were maintained by using an independent reference spray via the LockSpray interference. The analytical column was a 100 × 2.1-mm inner diameter, 1.7-µm C8 Acquity UPLC bridged ethylene hybrid (Waters). The two mobile phases were phase A: MeOH/H₂O/HCOOH (74:25:1, v/v) and phase B: MeOH/HCOOH (99:1, v/v); both contained 5 mM ammonium formate. The column was held at 30 °C. Quantification was carried out using the extracted ion chromatogram of each compound, using 50 mDa windows. The linear dynamic range was determined by injecting standard mixtures. Positive identification of compounds was based on the accurate mass measurement with an error of <5 ppm and its LC retention time, compared with that of a standard (±2%).

For lysosphingolipid quantification, analysis of the extracts was performed by LC/MS/MS with a system consisting of a Waters Alliance 2690 LC pump equipped with an autosampler and connected to a Quattro LC triple-quadrupole mass spectrometer from Micromass (Manchester, UK). Separation was achieved on a Purospher STAR-RP-18 column (125 × 2 mm, 5 µm) (Merck, Darmstadt). The two mobile phases were phase A and phase B as described above. A gradient was programmed: 0.0 min, 50% B; 2 min, 50% B; 7 min, 100% B; 17 min, 100% B; 19 min, 50% B; and 26 min, 50% B. The flow rate was 0.3 ml min⁻¹. MS/MS detection was performed with an electrospray interface operating in the positive ion mode acquiring the following selected reaction monitoring transitions: C17 D-erythro-dihydrospingosine-1-phosphate, 368–252, collision energy 18 eV; and S1P, 380–264 Da, collision energy 16 eV.

**Acid Ceramidase, Lysosomal β-Galactosidase, and Cathepsin B Activities**—To measure aCDase activity, cultured cells were collected and washed twice with PBS. Cell pellets were resuspended in 100 µl of a cold 0.2 M sucrose solution and then sonicated. Cell lysates were centrifuged at 15,000 × g for 3 min. The supernatant was collected and used for protein quantification (23) to work with equal amounts of protein. The enzymatic assay was carried out in 96-well plates (21). Each well contained a mixture of 74.5 µl of 25 mM sodium acetate buffer, pH 4.5, 0.5 µl of a 4 mM Rbm14-12 substrate solution in ethanol, and a fixed amount of protein (20 µg) in a volume of 25 µl of a 0.2 M sucrose solution. The plate was incubated at 37 °C for 3 h. Then, the enzymatic reaction was stopped by adding 50 µl of methanol and 100 µl of a 2.5 mg/ml NaIO₄ fresh solution in 100 mM glycine/NaOH buffer, pH 10.6, in each well. The plate was pro-

<figure><img src="image_2.png" /></figure>
## Acid Ceramidase, Melanoma, and Dacarbazine

tected from light for 2 h, and then the released fluorescence was quantified using a microplate fluorescence reader ($\lambda_{ex}$, 360 nm; $\lambda_{em}$, 446 nm). The amount of umbelliferone released was calculated from the fluorescence intensity by using calibration curves with umbelliferone in the range from 0 to 3000 pmol.

The acid β-galactosidase activity was determined on the same cell lysates used for aCDase activity assay. This assay is based on a published fluorometric method (24) with some modifications to adapt it to the 96-well plate format. Briefly, each well contained 20 µl of a 0.5 M sodium acetate buffer, pH 4.5, 20 µl of a 20 mM EDTA solution, 40 µl of a 3 mM solution of 4-methylumbelliferyl-β-D-galactopyranoside (final concentration of 1.2 mM), 10 µl of lysate, and 10 µl of ultrapure water. After incubation at 37 °C for 30 min, the reaction was stopped by the addition of 100 µl of a 100 mM glycine/NaOH buffer, pH 10.6. The fluorescence released was quantified using a microplate fluorescence reader ($\lambda_{ex}$, 360 nm; $\lambda_{em}$, 446 nm) as described above.

The cathepsin B activity was measured using the fluorogenic substrate z-Arg-Arg-AMC. The assay was carried out in 96-well plates. The incubation mixture of each well consisted of 95 µl of 0.4 M sodium acetate buffer, pH 5.5, containing 30 µg of protein extracts prepared as described above, and 5 µl of a 1 mM ethanolic solution of the substrate (final concentration, 50 µM). After a 1-h incubation at 37 °C, the reaction was stopped by adding 100 µl of a 100 mM glycine/NaOH buffer, pH 10.6. The amount of the released product was measured by fluorometry as described for the above assays.

*Viability Assay*—Cell viability was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, according to the manufacturer's instructions.

*Morphological Analyses*—A375 cells stably transfected with pEGFP-LC3 vector were treated at different times with DTIC, and then stained with monodansylcadaverine (50 µM) for 30 min and fixed with paraformaldehyde. Cells were examined under a fluorescence Leica DMR microscope equipped with a digital camera.

*Western Blot*—Cells were washed with PBS and harvested. Cell pellets were resuspended in 0.1 ml of ice-cold PBS containing 10% glycerol, 1% Triton X-100, 1 mM NaVO₄, 10 mM NaF, and a protease inhibitor mixture (Roche Applied Science), and further lysed by brief sonication. Samples were centrifuged at 10,000 × g for 10 min. Supernatants were collected, and protein content was determined by the Bradford method (Bio-Rad). Proteins (30–50 µg of cell lysates) were separated on a 12.5% polyacrylamide gel. After transfer onto a nitrocellulose membrane (Millipore), immunoblotting was performed using a rabbit polyclonal anti-acid ceramidase antibody (a kind gift from Dr. K. Sandhoff, Bonn, Germany), rabbit anti-SphK1 (Sigma), rabbit anti-β-actin, or rabbit anti-Atg7 (Cell Signaling). Density of the bands was quantified with ImageJ (National Institutes of Health).

*Quantitative RT-PCR*—Non-confluent melanoma cells were harvested, and RNA was extracted (RNeasy kit, Qiagen) according to the manufacturer's protocol and treated with DNase (Qiagen). RNA quality was assessed by automated gel electrophoresis (Experion, Bio-Rad). RNA (2 µg) was reverse-transcribed (SuperScript II, Invitrogen) and used as a template

for quantitative PCR. The reactions were done in duplicate on the StepOne instrument (Applied Biosystems) using SYBR Green PCR kit and primer assay (QuantiTect, Qiagen). Results were quantified using the system software. mRNA of TATA box binding protein, transferrin receptor, and β-actin were analyzed for normalization.

*Flow Cytometry*—Cells were washed and harvested by trypsinization. The analysis of LC3-EGFP fluorescence was assessed using a FACScan (BD Biosciences) cytometer as described previously (25). The fluorescence intensity observed on the FL1-H histogram inversely correlated with autophagic flux.

*Statistical Analysis*—Means ± S.E. were calculated in the different assays. Statistical significance between groups was assessed using the Student's t test. Differences were considered significant at *, *p* < 0.05; **, *p* < 0.01; and ***, *p* < 0.001.

## RESULTS

*Dacarbazine Treatment of Melanoma Cells Increases Ceramide Levels*—We first investigated the effects of DTIC treatment on the cellular levels of sphingolipids. To do this, A375 melanoma cells were treated with DTIC at 100 µg/ml for 48 h, the concentration corresponding to its IC₅₀ *in vitro* (see supplementary Table S1). Mass spectrometry analysis of the lipidic extracts revealed an increase of ceramide levels, especially those of 16- and 18-carbon chain length (~1.6- and 1.4-fold, respectively) (Fig. 1, A and B). This is particularly relevant in the case of C16:0 ceramide, because it is one of the major ceramide species in mammalian cells.<fnref n="4" /> Also, we found that sphingosine and S1P levels were reduced by 50 and 75%, respectively (Fig. 1C). Whereas glucosylceramide species were slightly increased, ~1.3-fold, sphingomyelin levels remained unaltered (data not shown). These observations on the sphingolipid profile focused our attention to ceramidases, because these degrading enzymes are responsible for the catabolism of ceramides to sphingosine.

*Dacarbazine Elicits Acid Ceramidase Degradation*—We centered our efforts on the study of aCDase, because several studies have connected this enzyme to cancer promotion and resistance to therapy, particularly of prostate cancer (15–17). Likewise, a bioinformatic approach has found aCDase to be among the most important marker genes for melanoma (26). These precedents and the above results of mass spectrometry led us to explore a possible relationship between this enzyme and the sensitivity of A375 melanoma cells to DTIC.

Interestingly, time-course experiments revealed a time-dependent decrease of aCDase activity after treatment with DTIC. After 48 h of treatment, cells lost ~80% of aCDase basal levels (Fig. 2A). Cell viability was also reduced with time after treatment, being significantly affected from 16 h onwards, when aCDase activity was reduced by 50% (Fig. 2B). Also, the degradation of aCDase activity showed to be dose-dependent with a loss of >50% already at a 25 µg/ml concentration (Fig. 2C). However, under the same conditions, lysosomal β-galac-

<footnote n="4">⁴ A significant increase in total ceramide levels was observed after quantification using the DAG kinase method (984 ± 161 pmol/mg of protein in DTIC-treated cells versus 500 ± 144 pmol/mg of protein in control cells; *n* = 7; *p* < 0.001).</footnote>
Acid Ceramidase, Melanoma, and Dacarbazine

<figure><img src="image_3.png" /></figure>

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.

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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.

tosidase activity was not decreased, meaning that the integrity of lysosomes was not compromised. Western blot experiments revealed a lower aCDase protein content in cells treated with DTIC,<fnref n="5" /> whereas levels of sphingosine kinase 1 remained unchanged (Fig. 2D). However, levels of *ASAH1* transcripts after treatment remained unchanged (data not shown), thus pointing to a post-transcriptional degradation of aCDase. Of note, aCDase activity was not decreased in A375 cells treated with cytotoxic concentrations of cisplatin (80 µM, data not shown), meaning that aCDase degradation is not a common response to cytotoxicities.

To investigate the mechanism responsible for aCDase degradation, A375 cells were preincubated with protease inhibitors,

including the cysteine and serine protease inhibitor leupeptin and the aspartyl protease inhibitor pepstatin A. As a result, we observed that only leupeptin was able to protect cells from aCDase degradation elicited by low doses of DTIC (Fig. 3A). Cathepsin B is a lysosomal cysteine protease that has been shown to be implicated in aCDase degradation after treatment with desipramine (27). Thus, this enzyme was tested as a possible cause for aCDase degradation. Preincubation of A375 melanoma cells with the cathepsin B inhibitor CA074-Me resulted in a near total protection of aCDase activity from DTIC-induced degradation (Fig. 3B). Next, we wanted to explore whether aCDase degradation was the consequence of caspase activation, as the activity of effector caspases measured using the fluorogenic substrate Ac-DEVD-AMC increased at 48 h after DTIC treatment (data not shown). To check this,

<figure><img src="image_5.png" /></figure>

<footnote n="5">⁵ Any molecular mass species smaller than 13 kDa were not observed, possibly due to the lack of recognition of such fragments by the antibody used.</footnote>
Acid Ceramidase, Melanoma, and Dacarbazine

<figure><img src="image_6.png" /></figure>

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.

A375 melanoma cells were preincubated with z-VAD-fmk, a broad caspase inhibitor, and then treated with DTIC. We observed that aCDase activity was not protected from degradation, meaning that caspases were not involved in this activity loss (Fig. 3C).

Dacarbazine-induced Generation of ROS Occurs Prior to Acid Ceramidase Degradation—Cellular metabolism of DTIC is believed to lead to the production of ROS in the cytoplasm giving H₂O₂, that diffuses to the lysosomes causing cell damage (28). Also, it has been reported that temozolomide is able to induce autophagy in cells (29), although the relationship between production of ROS, lysosomal damage, and autophagy has not been clarified yet for DTIC.

To investigate the role of ROS in aCDase degradation, possibly through cathepsin B activation, we preincubated A375 cells with N-acetylcysteine (NAC), a precursor of glutathione, a major intracellular antioxidant. As shown in Fig. 4A, NAC protected A375 cells from aCDase degradation, triggered by low doses of DTIC (from 5 to 25 µg/ml). As a consequence, after DTIC treatment at 10 µg/ml, the increase of ceramide levels was only 20% to that of cells not preincubated with NAC (data not shown). Regarding cathepsin B activity, a considerable increase of its activity was observed after DTIC treatment, which peaked at 24 h, and was completely abrogated in the presence of NAC (Fig. 4B). These findings suggest that the production of ROS after DTIC treatment leads to aCDase degradation, probably through cathepsin B activation. Cell death after treatment with low doses of DTIC was also attenuated in the presence of NAC (Fig. 4C).

<figure><img src="image_7.png" /></figure>

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).

Acid Ceramidase Degradation Is Involved in Dacarbazine-induced Autophagy of Melanoma Cells—Next, DTIC-induced autophagy of A375 melanoma cells was investigated. It was assessed by flow cytometry of A375 cells stably transfected with
Acid Ceramidase, Melanoma, and Dacarbazine

<figure><img src="image_8.png" /><figcaption>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.</figcaption></figure>

pEGFP-LC3. This method is based on the fact that the EGFP molecule is sensitive to low pH, making its fluorescence disappear when LC3-EGFP is delivered into lysosomes, i.e. when autophagolysosomes are formed (25). Using this method we observed that, although A375 cells lost 10% of fluorescence intensity under DTIC treatment, preincubation with NAC totally prevented the fluorescence loss (data not shown). Thus, protection by NAC from DTIC-induced autophagy is mediated by ROS formation. However, it was still unclear whether the DTIC-induced loss of aCDase activity was involved in the autophagy and the ensuing cell death observed.

To further explore these mechanisms, the onset of autophagy was assessed by EGFP-LC3 dot formation and monodansylcadaverine staining (30). When A375 melanoma cells were treated with DTIC (100 µg/ml), we observed that the punctuate structures, corresponding to autophagosomes and autophagolysosomes, appeared between 8 and 16 h (Fig. 5A). Remarkably, occurrence of the autophagic features was concomitant to the decrease of aCDase activity after DTIC treatment (Fig. 2A). Also, C16:0 and C18:0 ceramide levels,

assessed by mass spectrometry, started to increase between 8 and 16 h after treatment (Fig. 5B). These observations suggest that there is a temporal relationship between aCDase degradation, ceramide accumulation and the onset of autophagy.

To investigate whether aCDase degradation was a consequence of the autophagic process triggered by DTIC, A375 melanoma cells were transfected with an siRNA against Atg7, to inhibit autophagy (31). Despite efficient Atg7 silencing, aCDase degradation was still taking place, meaning that aCDase degradation likely occurred upstream of autophagy induced by DTIC (Fig. 5C).

**Silencing of Acid Ceramidase Sensitizes While Overexpression Renders A375 Melanoma Cells More Resistant to Dacarbazine**—
To further investigate the implication of aCDase on cell survival after DTIC treatment, aCDase expression was modulated. For *ASAH1* down-regulation, melanoma A375 cells were transfected with specific siRNA against *ASAH1*. After treatment with siRNA at a concentration of 10 nM, *ASAH1* expression, assessed by quantitative RT-PCR, was reduced to 15% (Fig. 6A).

<figure><img src="image_9.png" /></figure>
Acid Ceramidase, Melanoma, and Dacarbazine

<figure><img src="image_10.png" /></figure>

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).

aCDase activity was also dramatically reduced (Fig. 6B). Under these conditions, we found a small (10%), but not statistically significant increase in total ceramides. However, when we explored the values of the molecular ceramide species, an increase was observed for C16 and C18 species (22 and 26% increase, data not shown).<fnref n="6" /> These little changes are probably due to compensation by other ceramide-metabolizing enzymes and/or residual aCDase activity. Nevertheless, under treatment with DTIC, the combination of ASAH1 silencing and aCDase degradation made aCDase activity almost undetectable (Fig. 6B). This was translated into a 20% increase of ceramide levels (especially those of C16, C18, C22, and C24:1 ceramides with increases between 30 and 40%) and decreased levels of sphingosine and S1P (~40 and 60%, respectively), compared with those of DTIC-treated cells transfected with control siRNA (see Fig. 6C). Under conditions of down-regulation of ASAH1, cell viability was reduced by ~20%. Combination of ASAH1 down-regulation and DTIC treatment at doses ranging from 5 to 200 µg/ml resulted in a significantly increased sensitivity of A375 cells to DTIC (Fig. 6D). Viability of A375 cells was reduced by 45 to 65% of that of cells transfected with control siRNA.

As a reciprocal approach, ASAH1 was overexpressed in A375 melanoma cells. The TRex-inducible expression system (Invitrogen) was used to establish the A375 ASAH1 cell

line, which after addition of doxycycline at 1 µg/ml, ectopically expressed aCDase. Western blot analysis and aCDase activity assays indeed attested the effectiveness of this system (Fig. 7A). Ceramide levels in A375 ASAH1 cells were considerably decreased (by 40–50%), especially those of C16, C22, and C24:1 ceramides, in DTIC-treated cells after induction as compared with non-induced cells (Fig. 7B). This was accompanied by a significant (50%) increase in sphingosine levels, but no changes in the S1P content were detected in DTIC-treated A375 ASAH1-induced cells compared with the non-induced (Fig. 7B).

When induced A375 ASAH1 cells were treated with DTIC at different doses, almost no loss of aCDase activity was detected (Fig. 7C). Indeed, whereas induced cells conserved >80% of aCDase activity, non-induced cells exhibited <20% of the initial activity.

Next, cell viability after DTIC treatment in induced and non-induced A375 ASAH1 cells was determined. Cells that overexpressed aCDase were more resistant to DTIC treatment, most significantly at doses below 50 µg/ml (Fig. 7D). When the effects of another cytotoxic drug, cisplatin, were explored, no difference was observed between induced and non-induced cells (see supplemental Fig. S1), meaning that protection of A375 melanoma cells by overexpression of aCDase may be restricted to dacarbazine.

The viability results obtained both by down-regulation and overexpression experiments suggest that aCDase activity modulates A375 cell sensitivity to DTIC likely through changes in

<footnote n="6">⁶ The changes in ceramide levels were accompanied by a significant decrease in sphingosine content (Fig. 6C) and a significant increase in glucosylceramide level (data not shown).</footnote>
A

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B

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C

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D

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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.

the intracellular levels of ceramide. In support to this idea, another human melanoma cell line, UACC257, proved to be very resistant to DTIC (see **supplemental Table S1**). This cell line exhibited a 10-fold higher aCDase activity than that of A375 (**supplemental Fig. S2**), suggesting a direct correlation between aCDase activity and resistance to DTIC. Such a correlation was observed in other human melanoma cell lines between ASAH1 mRNA levels, acid ceramidase content, and enzyme activity and sensitivity to DTIC (**supplemental Fig. S2**).

DISCUSSION

The present work identifies aCDase as a novel target for DTIC. We demonstrate for the first time that DTIC, the most active agent for treating metastatic melanoma, triggers a ROS-dependent, cathepsin-mediated proteolytic degradation of aCDase in a time- and dose-dependent manner. This is followed by ceramide accumulation and ensuing cell death with

autophagic features. Genetic manipulation of aCDase expression showed that the expression/activity of this lipid hydrolase is able to modulate the response of melanoma cells to DTIC.

Our results show that aCDase degradation is probably carried out by cathepsin B, because its specific inhibition with CA074-Me protects cells from aCDase activity loss. This finding is reminiscent of the involvement of cathepsins B and L in the aCDase degradation induced by chemical treatments, such as desipramine, chloroquine, or chlorpromazine (27).

It is known that temozolomide and DTIC are DNA-alkylating agents. Recently, it has been proposed that they can also be metabolized in the cytosol to 5-(3-monomethyl-1-triazeno)imidazole-4-carboxamide, to further produce ROS that diffuse to lysosomes (28). Blocking these species by using NAC in DTIC-treated A375 cells conferred protection from increase in cathepsin B activity, aCDase degradation, ceramide accumula-

<figure><img src="image_15.png" /></figure>
tion, cell death, and autophagy. These results strongly suggest that the production of ROS after DTIC treatment is a causal event in aCDase degradation, probably through cathepsin B activation. Furthermore, ROS would be also implicated in cell death and autophagy. This is in accordance with a previous work demonstrating that ROS participate in TNFα-induced stimulation of autophagy (32).

Autophagy encompasses processes by which cytoplasmic materials reach lysosomes for degradation. Under physiological conditions, autophagy plays a role in cell maintenance and adaptation under numerous stimuli (33, 34). Cellular autophagic activity is normally low under basal conditions, but some cancer cells, due to their localization, often in hypoxic or nutrient-deprived environments, exhibit high rates of autophagy. For instance, A375 melanoma cells display elevated basal levels of autophagy, which made it difficult to measure little changes in autophagy induced by DTIC treatment. Thus, we could not use Western blot analysis for reliable LC3-II detection and quantification. Instead of this technique, we could assess autophagy by flow cytometry of cells stably expressing LC3-EGFP. Besides its self-defense role, autophagy can also promote a form of cell death, called programmed cell death II, presumably by excessive activation of autophagy to a point from which recovery of the cell is unattainable (35). In cancer therapy, activation of autophagy in apoptosis-resistant tumors could provide a way to induce cell death and prevent tumor growth. In this sense, cytotoxic drugs like arsenic trioxide, imatinib, and rapamycin among others can induce autophagic death of cancer cells (35).

In our A375 cell model, DTIC-induced aCDase degradation was accompanied by accumulation of ceramides (mainly C16 and C18 species) and decreased sphingosine and S1P levels. Besides its pro-apoptotic properties, ceramide has also been described as an inducer of autophagy (36, 37). Consistent with this, our results show a temporal relationship between the degradation of aCDase, the increase of ceramide levels, and the onset of autophagic features after DTIC treatment, suggesting that, in our model, the accumulation of ceramide resulting from aCDase degradation could be involved in the induction of autophagy.<fnref n="7" />

Regarding a possible induction of apoptosis after DTIC treatment, we started to detect cleavage of caspase 3 after 48 h of treatment (data not shown), meaning that apoptosis is not an early event in DTIC-treated A375 melanoma cells. This is in agreement with a previous report on temozolomide-treated malignant glioma cells (29). One possibility is that autophagy contributes to the induction of apoptosis as a result of a cross-talk between these two types of programmed cell death (31). This is exemplified in the work of Djavaheri-Mergny et al. (38),

where activation of autophagy by TNF-α is required for induction of apoptosis and cell death.

Our findings also show that the modulation of *ASAH1* expression can regulate the sensitivity of A375 melanoma cells to DTIC. *ASAH1* down-regulation sensitized cells to the treatment, probably through increased levels of ceramide. Sensitization of cells by *ASAH1* down-regulation (20) or by enzymatic inhibition (19) has already been reported in other cancer models. Interestingly, down-regulation of other ceramidases such as neutral (*ASAH2*) or alkaline ceramidase 2 (*ASAH3L*) did not recapitulate the sensitizing effects of *ASAH1* silencing (only *ASAH3L* silencing slightly increased the sensitivity of cells to DTIC; see supplemental Fig. S3).

Conversely, we report that aCDase overexpression can partially protect A375 cells from cell death at low doses of DTIC. A possible explanation for this limited effect is that, under high doses of DTIC, other mechanisms not related to ceramide metabolism are triggered (e.g. DNA alkylation). These results are in accordance with a previous study showing that overexpression of aCDase in prostate cells confers resistance to several cytotoxic drugs (16). The fact, that the protection from cell death observed after overexpressing aCDase only takes place under DTIC but not cisplatin treatment, suggests that there is a specific mechanism of DTIC involving aCDase (as a matter of fact, cisplatin did not induce a decrease of aCDase activity). Another proof of aCDase involvement in the resistance to DTIC is the direct correlation observed between *ASAH1* mRNA levels, aCDase activity, and the sensitivity to DTIC in several human melanoma cell lines.

Of note, inducible *ASAH2* overexpression was not associated with significant changes in cell viability after treatment with DTIC (supplemental Fig. S3B) as well as other cytotoxics, like cisplatin or etoposide (data not shown). Together with the absence of effects of *ASAH2* down-regulation, this suggests that neutral ceramidase is not involved in the response of A375 to DTIC, probably due to the different localization of the accumulated ceramide.

In conclusion, the present data strongly indicate that metabolism of ceramide by aCDase action is involved in the sensitivity of melanoma cells to DTIC. Taken together, this work further strengthens the importance of aCDase as a key factor in melanoma progression (26, 39). It also suggests that aCDase down-regulation, in combination with DTIC, may be a promising strategy for improved treatment of metastatic melanoma.

Acknowledgments—We thank Dr. J. A. Medin for *ASAH1* cDNA, Dr. T. Yoshimori for the pEGFP-LC3 vector, Dr. K. Sandhoff for the aCDase antibody, Dr. P. Codogno for helpful comments and discussion, Dr. S. C. Sorli for help and advice in molecular biology aspects, and E. Dalmau for LC/MS technical assistance.

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<footnote n="7">⁷ The decreased levels of sphingosine and S1P observed under DTIC treatment may suggest that the increased ceramide levels are not the only cause of the cell death observed. The same conclusion could apply for the decreased cell viability upon *ASAH1* silencing. However, in cells where DTIC failed to elevate ceramide, their sphingosine level remained high, but that of S1P still decreased. Thus, the resistance of the aCDase-overexpressing cells is likely not due to an effect on S1P but rather to a decrease of ceramide content.</footnote>
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