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Published as doi: 10.1096/fj.07-092981.
 (The FASEB Journal. 2008;22:2541-2551.)
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A role for ceramide in driving cancer cell resistance to doxorubicin

Yong-Yu Liu*,1, Jing Yuan Yu{dagger}, Dongmei Yin*, Gauri Anand Patwardhan*, Vineet Gupta*, Yoshio Hirabayashi{ddagger}, Walter M. Holleran§, Armando E. Giuliano{dagger}, S. Michal Jazwinski, Valerie Gouaze-Andersson{dagger}, David P. Consoli{dagger} and Myles C. Cabot{dagger}

* Department of Basic Pharmaceutical Sciences, University of Louisiana at Monroe, Monroe, Louisiana, USA;

{dagger} Department of Experimental Therapeutics, John Wayne Cancer Institute at Saint John’s Health Center, Santa Monica, California, USA;

{ddagger} Neuronal Circuit Mechanisms Research Group, RIKEN Brain Science Institute, Saitama, Japan;

§ Departments of Dermatology and Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USA; and

Departments of Medicine, Tulane University School of Medicine, New Orleans, Louisiana, USA

1Correspondence: Department of Basic Pharmaceutical Sciences, University of Louisiana at Monroe, 700 University Ave., Monroe, LA 71209, USA. E-mail: yliu{at}ulm.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Advanced cancers acquire resistance to chemotherapy, and this results in treatment failure. The cellular mechanisms of chemotherapy resistance are not well understood. Here, for the first time, we show that ceramide contributes to cellular resistance to doxorubicin through up-regulating the gene expression of glucosylceramide synthase (GCS). Ceramide, a cellular lipid messenger, modulates doxorubicin-induced cell death. GCS catalyzes ceramide glycosylation, converting ceramide to glucosylceramide; this process hastens ceramide clearance and limits ceramide-induced apoptosis. In the present study, we evaluated the role of the GCS gene in doxorubicin resistance using several paired wild-type and drug-resistant (doxorubicin-selected) cancer cell lines, including breast, ovary, cervical, and colon. GCS was overexpressed in all drug-resistant counterparts, and suppressing GCS overexpression using antisense oligonucleotide restored doxorubicin sensitivity. Characterizing the effect mechanism showed that doxorubicin exposure increased ceramide levels, enhanced GCS expression, and imparted cellular resistance. Exogenous C6-ceramide and sphingomyelinase treatments mimicked the influence of doxorubicin on GCS, activating the GCS promoter and up-regulating GCS gene expression. Fumonisin B1, an inhibitor of ceramide synthesis, significantly suppressed doxorubicin-up-regulated GCS expression. Promoter truncation, point mutation, gel-shift, and protein-DNA ELISA analysis showed that transcription factor Sp1 was essential for ceramide-induced GCS up-regulation. These data indicate that ceramide-governed GCS gene expression drives cellular resistance to doxorubicin.—Liu, Y.-Y., Yu, J. Y., Yin, D., Patwardhan, G. A., Gupta, V., Hirabayashi, Y., Holleran, W. M., Giuliano, A. E., Jazwinski, S. M., Gouaze-Andersson, V., Consoli, D. P., Cabot, M. C. A role for ceramide in driving cancer cell resistance to doxorubicin.


Key Words: glucosylceramide synthase • Sp1 • transcription • gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CHEMOTHERAPY IS THE CORNERSTONE of treatment for disseminated malignancies, but development of drug resistance remains problematic (1 2 3) . In breast cancer, more than 40% of patients with resectable and 80% of patients with unresectable diseases show a poor response to chemotherapy (1 , 2) . Less consistent with the intent to kill tumor cells is the unfortunate property of anticancer agents to induce drug resistance, resulting in chemotherapy failure. Several reports show that chronic exposure of cancer cells to elevated levels of front-line anticancer drugs, like doxorubicin, vinblastine, and paclitaxel, causes multidrug resistance (MDR) (4 5 6 7) . Increased expression of genes associated with drug action or cell proliferation is the cornerstone for MDR in human cancers. Drug resistance genes such as MDR1 and Bcl-2 are highly expressed in tumors that respond poorly to chemotherapy and in MDR cancer cell lines (8 9 10) . However, mechanisms of chemotherapy-induced gene alterations in drug resistance are poorly understood.

Recent studies show that glucosylceramide synthase (GCS; EC2.41.80) is associated with drug resistance in cancer (11 12 13 14 15) . GCS is a glycosyltransferase in sphingolipid metabolism; it transfers a glucose residue from UDP-glucose to ceramide for synthesis of glucosylceramide (16) . Enforced overexpression of GCS by gene transfection conferred cellular resistance to doxorubicin, daunorubicin, and tumor necrosis factor {alpha} (17 18 19) . GCS expression is up-regulated in MDR cancer cell lines (20) , in chemoresistance leukemia (21) , and in metastatic breast cancer (22) .

Ceramide generated by cells in response to diverse stress factors retards cell proliferation and programs cell death (16 , 23) . In recent years, several reports have shown that ceramide regulates gene expression (24 25 26 27 28 29) . For example, ceramide up-regulates the expression of acid sphingomyelinase (28) , p21WAF1/CIP1 (25 , 26) , and cyclooxygenase-2 (COX-2) (27) , and it down-regulates the expression of c-myc (25) and glutathione S-transferase (30) . Earlier work from other groups showed that ceramide increases GCS enzyme activity and GCS mRNA levels (24 , 29) . Herein, we show that doxorubicin induces GCS overexpression through endogenous formation of ceramide. Because high GCS expression reduces intracellular ceramide residence time, we propose that this is a factor contributing to doxorubicin resistance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
The human breast adenocarcinoma cell line MCF-7 and its drug-resistant counterpart MCF-7-AdrR (NCI/ADR-Res) (6) were kindly provided by Dr. Kenneth Cowan (University of Nebraska Medical Center Eppley Cancer Center, Omaha, NE, USA) and Dr. Merrill Goldsmith (National Cancer Institute, Bethesda, MD, USA). The ovarian cancer cell line A2780-AD, which is resistant to doxorubicin (4) , was kindly provided by Dr. Thomas C. Hamilton (Fox Chase Cancer Center, Philadelphia, PA, USA). The human cervical carcinoma cell line KB-3–1 and its doxorubicin-selected KB-A1 subline (31) were from Dr. Michael M. Gottesman (National Cancer Institute). The human colon cancer cell line SW620, the doxorubicin-selected SW620Ad1000 (32) , and the doxorubicin-selected human breast cancer MCF-7-P500 (32) cell lines were kindly provided by Drs. Susan Bates and Antonio Fojo (National Cancer Institute). Cells were maintained in RPMI 1640 medium containing 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 584 mg/liter L-glutamine. A2780-AD cells were cultured in medium containing 100 nM doxorubicin in addition to the above components.

Cytotoxicity assay
These assays were performed as previously described (11 , 17 , 33) . Cells (4x103 to 104/well) were plated in 96-well plates with 10% FBS RPMI 1640 medium. After 24 h growth, cells were shifted to 5% FBS RPMI 1640 medium containing increasing concentrations of anticancer agents as indicated and cultured for another 72 h. Cell viability was determined using the Promega 96 Aqueous cell proliferation assay kit or the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA).

To observe the influence of antisense oligonucleotide against GCS on drug response, cells were pretreated (33) with OligofectAMINE alone (vehicle) or OligofectAMINE plus ODN-7 (200 nM) for 4 h, and then cultured another 72 h in 5% FBS medium containing increased concentrations of anticancer agents.

RNA extraction and mRNA analysis
Cellular RNA was purified using a total RNA isolation RNeasy Mini kit (Qiagen, Valencia, CA, USA). Equal amounts of RNA (100 ng) were used for reverse transcriptase-polymerase chain reaction (RT-PCR), as previously described (11 , 33) . Under upstream primer (5'-CTGGAAACATTCTTTGAATTGGAT-3') and downstream primer (5'-TCTCATTAAACAAGACATTCCTGTC-3') conditions, a 441-base pair (bp) fragment in the region of GCS gene (498–938) was produced using a high-fidelity single-tube RT-PCR system, ProSTAR HF (Stratagene, La Jolla, CA, USA).

For quantitative RT-PCR, the cDNA was synthesized using the SuperScript First-Strand synthesis system and random hexamer reverse transcription primers (Invitrogen, Carlsbad, CA, USA). Under upstream primer (5'-GACCTGGCCTTGGAGGGAAT-3') and downstream primer (5'-GAGACACCTGGGAGCTTGCT-3') conditions, a 149-bp fragment in the region of GCS gene (303–451) was produced using a QuantiFast SYBR Green PCR kit (Qiagen) with a MyiQ Real-Time PCR Detection System (Bio-RAD Laboratories, Hercules, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 200 bp; up primer 5'-ATGGGGAAGGTGAAGGTCGG-3'; down primer 5'-TCCACCACCCTGTTGCTGTA-3') was used as an endogenous control for normalization. Quantitation was carried out using human GCS DNA standard curves generated by serial dilution of pcDNA 3.1-GCS plasmid (11) .

Western blot
Western blots were performed using a modified procedure (11 , 19) . Confluent cells (1x107 cells/100 mm dish) were washed twice with PBS containing 1.0 mM PMSF and detached with trypsin-EDTA solution. Cells pelleted by centrifugation were solubilized in 1.0 ml cold TNT buffer (20 mM Tris-HCl, pH 7.4; 200 mM NaCl; 1.0% Triton X-100; 1.0 mM PMSF; and 1.0% aprotinin) for 60 min with shaking. Insoluble debris was excluded by centrifugation at 12,000 g for 45 min at 4°C. The detergent-soluble fraction was loaded in equal aliquots (50 µg protein/lane) and resolved using 4–20% gradient SDS-PAGE. The transferred blot was blocked (3% fat-free milk powder in 10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.05% Tween-20), and immunoblotted with GCS antiserum (1:1000) in binding solution (0.5% BSA in 10 mM Tris-HCl, pH 8.0; 150 mM NaCl) at 4°C for 18 h. The detection method was enzyme-linked chemiluminescence plus (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). GCS antiserum (designated fraction GCS-6.2 antiserum) was provided by Dr. R. Pagano and Dr. D. Marks (Mayo Clinical and Foundation, Rochester, MN, USA) (34) .

Ceramide analysis
Analysis was performed as previously described (17 , 35) . Cells were seeded in 6-well plates (6x104 cells/well) in 10% FBS RPMI 1640 medium. After 20 h culture, cells were treated with doxorubicin in 5% FBS RPMI 1640 medium for the indicated periods. Cellular lipids were radiolabeled by adding [3H]palmitic acid (2.5 µCi/ml) to the culture medium for 24 h. Tritium-labeled ceramide was isolated by lipid extraction and thin-layer chromatography (TLC). Ceramides (ceramide, dihydroceramide) were resolved using a solvent system containing chloroform/acetic acid (90:10, v/v). Commercial ceramide standards (Avanti Polar Lipids, Alabaster, AL, USA) were cochromatographed. After development, lipids were visualized with iodine vapor staining and identified by migration. The ceramide area was scraped into 0.5 ml water. EcoLume counting fluid (4.5 ml) was added, the samples were mixed, and radioactivity was quantitated by liquid scintillation spectrometry. [9,10-3H]Palmitic acid (56.5 Ci/mmol) was purchased from DuPont/NEN (Boston, MA, USA). Silica Gel-G TLC plates were from Analtech (Newark, DE, USA).

Cellular ceramide glycosylation assay
Cellular ceramide glycosylation assay was performed as previously described with slight modification (36 , 37) . Cells (1x106 cells/35 mm dish) were grown 12 h in 10% RPMI 1640 medium and switched to 1% bovine serum albumin (fatty acid free) medium containing 5 µM NBD C6-ceramide complexed to BSA (Invitrogen). After 2 h incubation at 37°C, cells were then rinsed twice with ice-cold phosphate-buffered saline (pH 7.4), and 1.3 ml of ice-cold methanol containing 2% acetic acid was added. The cells were scraped free, transferred to glass vials, and lipids were extracted by the addition of chloroform (1.3 ml) followed by water (1.3 ml). The resulting organic lower phase was evaporated and resuspended in 100 µl of chloroform/methanol (1:1, v/v), and aliquots were applied to partisil high-performance TLC plates with fluorescent indicator (Whatman, Florham Park, NJ, USA). The samples were resolved using chloroform/methanol/3.5 N ammonium hydroxide (85:15:1, v/v/v) and visualized with a AlphaImager HP imaging system (Alpha Innotech, San Leandro, CA, USA) for analysis glucosylceramide and ceramide. TLC areas, aligned with bands of lipid standards on the fluorescent chromatograms, were scraped into 96-well plates with 200 µl of methanol. The samples were quantitated using by Synergy HT multidetection microplate reader (BioTek, Winnooski, VT, USA) at excitation of 466 and emission of 539 nm. For quantitation, calibration curves were established after TLC separation of NBD C6-ceramide (Invitrogen) and NBD C6-glucosylceramide (N-hexanol-NBD-glucosylceramide; Matreya, Pleasant Gap, PA, USA).

GCS promoter plasmids and promoter activity assay
GCS promoter (38) was truncated based on element sites. The PCR fragments were inserted in the pGL3.1 basic vector to drive the luciferase reporter gene. Point mutation of the Sp1 elements in the GCS promoter was performed as previously described (39) . Briefly, the point mutations were generated using the QuikChangeTM mutagenesis kit (Stratagene). The AAAT motif replaced the GGGC in Sp1 binding site (–556 to –561) (38) , and the mutant sequences of pGCS-Luc1 M2 were confirmed by direct sequencing at a DNA sequencing core facility (Louisiana State University, Baton Rouge, LA, USA).

MCF-7 or MCF-7-AdrR cells (1x106 cells/well) were seeded in 6-well plates and grown in 10% FBS RPMI 1640 medium overnight. After rinsing with serum-free medium, the luciferase-reporter plasmid of the GCS promoter (4 µg DNA) was introduced into cells by incubation with Lipofectamine 2000 (Invitrogen) in serum-free medium for 4 h. After 48 h growth in 10% FBS medium, the transiently transfected cells were harvested in lysis buffer. The luciferase assay system (Promega, Madison, WI, USA) was used to assess luciferase activity. Briefly, the soluble fraction (25 µl) was incubated with luciferase substrate at room temperature (20–25°C) for 1 min, and the light intensity catalyzed by luciferase was measured using a scintillation counter or Synergy HT multidetection microplate reader. Luciferase activity was normalized to β-galactosidase and protein concentration in each sample. pGL3.1-basic and pGL3.1-control vectors were used as a transfection control. For induction, MCF-7 cells were grown in 5% FBS RPMI 1640 medium containing the indicated concentrations of reagents. C6-ceramide (N-hexanoyl-D-erythro-sphingosine) and C6-dihydroceramide (N-hexanoyl-D-erythro-sphinganine) were purchased from Matreya, Inc. Sphingomyelinase (human placenta, 100 U/mg protein) and doxorubicin hydrochloride were purchased from Sigma (St. Louis, MO, USA), and fumonisin B1 was from Biomol (Plymouth Meeting, PA, USA). Natural ceramide (egg) was purchased from Avanti Polar Lipids; Sp1 siRNA (h) silencing human Sp1 transcription factor was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein concentrations in cell lysates were measured by BCA protein assay (Pierce, Rockford, IL, USA).

Electrophoretic-mobility shift assay (EMSA) and transcription factor ELISA
MCF-7 cells (5x106 cells/dish) were grown in 100 mm dishes with 10% FBS RPMI 1640 medium for 12 h and switched to 5% FBS RPMI 1640 medium containing doxorubicin (0.5 µM) or C6-ceramide (5.0 µM) for 24 h. Nuclear extracts were prepared with a nuclear extraction kit (Panomics, Fremont, CA, USA). Panomics EMSA kit was used to identify Sp1 transcription factor. Nuclear extracts (5.0 µg protein) were incubated for 30 min at room temperature in a 10 µl reaction containing the appropriate buffer, poly(dI-dC) and Sp1 probe (biotin-labeled, 10 ng). For competition experiments, 33-fold excess unlabeled competitor (unlabeled Sp1 probe, 330 ng) was added to the reaction mixture before incubation. The reaction products were resolved on a 6% nondenaturing polyacrylamide gel in 0.5x Tris-borate-EDTA at 4°C. After blocking, the membrane was incubated with streptavidin-alkaline phosphate conjugate (1:1000 dilution) and then with ECF substrate (GE Healthcare Bio-Sciences). Fluorescence of bound Sp1 oligonucleotide was detected using a Storm Gel and Blot Imaging System (GE Healthcare Bio-Sciences). The biotin-labeled and unlabeled Sp1 probes were purchased from Panomics; the sequence is 5'-ATTCGATCGGGGCGGGGCGAG.

The transcription factor ELISA kit for Sp1 from Panomics was used to assess Sp1 protein-DNA binding activity. Forty micrograms extracted nuclear proteins was incubated with biotin-labeled Sp1 probe in binding buffer at 28°C for 1 h. The bound Sp1 protein was recognized by Sp1 antibody (1:200 dilution) and then second antibody conjugated with horseradish peroxidase. The absorbance of tetramethylbenezidine products was measured at 450 nm.

Statistics
All data represent the mean ± SD. Experiments were repeated 2 or 3 times. Student’s t test was used to compare mean values, using a Microsoft Excel program (Microsoft, Redmond, WA, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GCS is overexpressed in doxorubicin-resistant cancer cells
Enhanced glucosylceramide accumulation and increased GCS activity have been found in drug-resistant variants of human breast cancer (35) , colon cancer (40) , and leukemia (21) . We previously reported that GCS is overexpressed in doxorubicin-resistant breast cancer and colon cancer cells (20) . With the goal of assessing molecular mechanisms that underlie this phenomenon, we have conducted an in-depth characterization of GCS expression in drug-resistant cancer cell lines established through doxorubicin selection pressure. In comparing doxorubicin-selected MDR cancer cell lines with their wild-type counterparts (Fig. 1 A), we found that GCS mRNA levels increased by 4-fold in MCF-7-AdrR, 3-fold in breast cancer MCF-7-P500, 3-fold in ovarian cancer A2780-AD, 7-fold in cervical carcinoma KB-A1, and 4-fold in colon cancer SW620Ad1000 cells. Results from Western blots demonstrate that GCS protein levels are enhanced in all drug-resistant counterparts (Fig. 1B ) compared with each wild-type. Assessing the cellular glycosylation of C6-ceramide showed that GCS activity (ratios of glucosylceramide to ceramide, Fig. 1C ) was from 1.6-fold to 2-fold higher in drug-resistant cell lines compared with corresponding wild-type cells. We further found that GCS promoter activity, measured by transient transfection with pGCS-Luc1, was 15-fold higher (2818 vs. 191) in MCF-7-AdrR, 4-fold higher (799 vs. 191) in MCF-7 P500, 3-fold higher (1767 vs. 547) in A2780-AD, 3-fold higher (500 vs. 178) in KB-A1, and 2-fold higher (847 vs. 428) in SW620Ad1000 cells compared with wild-type parental cells (Fig. 1D ). These data are consistent with our previous report (20) and demonstrated that high GCS expression, due to activated GCS promoter, is a general characteristic in doxorubicin-selected cancer cells.


Figure 1
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  		Figure 1. Expression of glucosylceramide synthase in wild-type and doxorubicin-resistant human cancer cell lines. A) GCS mRNA levels by quantitative real-time PCR. Cells were seeded in 10% FBS RPMI 1640 medium and grown for 24 h. Isolated total RNA (100 ng) was analyzed by quantitative real-time PCR, high-fidelity RT-PCR, and 1% agarose gel electrophoresis. Housekeeping gene, β-actin, was used as end point control. B) Western blot. For Western blots, detergent-soluble cellular protein (50 µg) was subjected to 4–20% SDS-PAGE. Proteins were transferred to nitrocellulose, and immunoblots were incubated with GCS-6.2 antiserum and detected using ECL plus. C) Cellular GCS assay. Cells were incubated with 5 µM NBD C6-ceramide, and the NBD C6-glucosylceramide was assessed (see Materials and Methods). D) GCS promoter assay. GCS promoter plasmid (pGCS-Luc1, 4 µg DNA/well) was introduced into cells by incubation with Lipofectamine 2000 in serum-free medium for 5 h. After 48 h growth in 10% FBS medium, the transfected cells were harvested to measure luciferase activity. Drug-resistant cells transfected with pGL3-basic plasmid were used as control to calculate the increase in luciferase activity. *P < 0.001 compared with drug-sensitive cells.

To validate whether GCS overexpression is associated with drug resistance, we assessed cellular response to doxorubicin before and after GCS gene suppression. Our previous report showed that ODN-7, a phosphorothioate deoxyribonucleotide, could specifically inhibit human GCS expression (33) . We pretreated cells with ODN-7 (200 nM) and assessed cell viability after 72 h doxorubicin treatment. We found that ODN-7 pretreatment increased doxorubicin sensitivity 36-fold in MCF-7-AdrR, 35-fold in MCF-7 P500, 4-fold in A2780-AD, 76-fold in KB-A1, and 62-fold in SW620-Ad1000 cells. However, ODN-7 pretreatment did not influence doxorubicin EC50 in A2780 and SW620 wild-type cell lines but increased doxorubicin sensitivity 4-fold in MCF-7 and KB-3–1 wild-type cells (Fig. 2 ; Table 1 ).


Figure 2
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  		Figure 2. Influence of GCS suppression on cellular response to doxorubicin. Cells (4000–8000/well) were plated in 96-well plates in RPMI 1640 medium containing 10% FBS. After overnight culture, cells were pretreated with OligofectAMINE alone (right panel) or with OligofectAMINE plus ODN-7 (200 nM; left panel) for 4 h, after which cells were cultured in 5% FBS medium containing increasing concentrations of doxorubicin for 72 h. Cell viability was determined using the Promega 96 Aqueous cell proliferation assay kit. A) Breast cancer cells. B) Ovary carcinoma cells. C) Cervical carcinoma cells. D) Colon carcinoma cells (SW620Ad, SW620Ad1000).


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  		Table 1. Influence of GCS down-regulation on cell response to doxorubicin treatment

Doxorubicin induces GCS expression through ceramide
Exogenous C2-ceramide or endogenous ceramide generated by GCS inhibition elevates GCS activity in MDCK cells (22) . Ceramide produced by sphingomyelinase or short-chain ceramide treatment increases GCS mRNA in human hepatoma Huh7 and mouse melanoma B16 cells (29) . These findings suggest that ceramide might increase GCS expression. To determine if doxorubicin could precipitate ceramide-induced GCS expression, we first assessed ceramide levels in breast cancer cells grown with doxorubicin. The steady-state level of ceramide in doxorubicin-selected MCF-7-AdrR cells was significantly higher than in wild-type MCF-7 cells (Fig. 3 ). Exposure to doxorubicin (0.5 µM, the EC50 for doxorubicin in MCF-7 cells) increased ceramide levels by more than 2-fold in wild-type MCF-7 cells but not in MCF-7-AdrR (Fig. 3) .


Figure 3
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  		Figure 3. Cellular ceramide levels in MCF-7 and MCF-7-AdrR cells before and after doxorubicin exposure. Cells (1x106/well) were plated in 6-well plates and cultured in 5% FBS RPMI 1640 medium containing 0.5 µM doxorubicin for the indicated times. [3H]Palmitic acid (2.5 µCi/ml culture medium) was added to the plates 24 h before the end of the experiment. Ceramide was resolved by TLC in a solvent system containing chloroform/acetic acid (90:10, v/v). *P < 0.01, **P < 0.001 compared with MCF-7 cells, respectively.

To assess the influence of doxorubicin on GCS expression and determine whether ceramide would produce a similar response, we exposed MCF-7 cells to doxorubicin (0.5 µM) and C6-ceramide (2.5 µM, 48 h). Quantitative PCR analysis showed that doxorubicin and C6-ceramide increased GCS mRNA levels by 2.5- and 8-fold, respectively (Fig. 4 A). Ceramide-induced GCS expression was dose-dependent; a significant influence was apparent at a concentration of 2.5 µM (Fig. 4B ). Ceramide also activated the GCS promoter (pGCS-Luc1) in transiently transfected MCF-7 cells (Fig. 4C ), as shown by the dose-dependent increase in luciferase activity. Ceramide did not influence luciferase expression in MCF-7 cells transfected with a truncated form of the GCS promoter, pGCS-Luc3. Significantly, up-regulated GCS expression by exposure to C6-ceramide (5 µM, 48 h), conferred MCF-7 cell resistance to ceramide and doxorubicin; the EC50 values for C6-ceramide and doxorubicin were increased by 2-fold (21.67 vs. 10.88 µM; 0.87 vs. 0.37 µM), respectively (Fig. 4D ). These results suggest that ceramide generated in cells in response to doxorubicin stress up-regulates GCS expression.


Figure 4
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  		Figure 4. The influence of doxorubicin and ceramide on GCS gene expression. A) RT-PCR analysis of GCS. MCF-7 cells were treated with doxorubicin (Dox, 0.5 µM) or C6-ceramide (Cer, 2.5 µM) in 5% FBS RPMI 1640 medium for 48 h. Total RNA (100 ng) was used to analyze GCS mRNA by real-time PCR. MCF-7-AdrR cells served as a GCS positive control. B) Influence of ceramide concentration on GCS mRNA levels. MCF-7 cells were grown for 48 h in 5% FBS RPMI 1640 medium containing increasing concentrations of C6-ceramide. Extracted total RNA (100 ng/reaction) was used for quantitative PCR analysis. C) Influence of ceramide concentration on GCS promoter activity. After 24 h transfection with pGCS-Luc1 and pGCS-Luc3 plasmids (4 µg DNA/well), MCF-7 cells were grown for 24 h in 5% FBS RPMI 1640 medium containing increasing concentrations of C6-ceramide. Cell lysates (25 µg protein/reaction) were used to measure luciferase activity. D) Influence of ceramide exposure on cellular response to doxorubicin. After exposure to vehicle control or to C6-ceramide (5 µM, 48 h), MCF-7 cells, in 96-well plates (6000 cells/well), were treated with increasing concentrations of doxorubicin or C6-ceramide for 72 h, and cell viability was measured using CellTiter-Glo luminescent assay (Promega). *P < 0.01 compared with cells exposed to vehicle control.

In addition to doxorubicin, we also assessed the influence of another ceramide generator, sphingomyelinase; an inactive ceramide analog (C6-dihydroceramide); and a ceramide synthesis inhibitor (fumonisin B1) on GCS promoter activity and GCS expression. C6-ceramide, sphingomyelinase, and doxorubicin activated the GCS promoter (Fig. 5 A) and significantly increased GCS mRNA and protein levels (Fig. 5B, C ), whereas C6-dihydroceramide or natural long-chain ceramide did not significantly influence GCS expression. Fumonisin B1 pretreatment blocked activation of the GCS promoter and GCS expression in cells exposed to doxorubicin. These results indicate that doxorubicin induces GCS expression in cells via de novo ceramide synthesis but that sphingomyelin-derived ceramide is also active in this respect.


Figure 5
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  		Figure 5. The influence of various agents on GCS promoter activity and GCS gene expression. A) GCS promoter activity analysis. After 24 h transfection with GCS promoter plasmid (pGCS-Luc1, 4 µg DNA/well), MCF-7 cells were maintained in 5% FBS RPMI 1640 medium containing indicated agents for an additional 48 h before luciferase determination. *P < 0.001 compared with vehicle control. B) GCS mRNA levels. MCF-7 cells were cultured in 5% FBS RPMI 1640 medium containing indicated agents for 48 h. The isolated total RNA (100 ng) was then analyzed by quantitative PCR analysis. C) GCS expression. MCF-7 cells were cultured in 5% FBS RPMI 1640 medium containing indicated agents for 48 h. Isolated total RNA (100 ng) was analyzed by high-fidelity RT-PCR and 1% agarose gel electrophoresis. For Western blot, the detergent-soluble fraction (50 µg protein/lane) was resolved using 4–20% gradient SDS-PAGE. The transferred nitrocellulose membranes were immunoblotted with GCS antiserum (1:1000) and detected by ECL. Dox, 0.5 µM doxorubicin; C6-Cer, 5.0 µM C6-ceramide; C6-diH-Cer, 5.0 µM C6-dihydroceramide; SMase, 0.5 U/ml sphingomyelinase; FB1, 25 µM fumonisin B1; N-Cer, 5 µM natural ceramide.

GCS gene transcription and Sp1
GCS gene expression is naturally driven by the GCS promoter (38) . The intact GCS promoter consists of 631 nucleotides (–734 to –103), 80% of which are guanine and cytosine (38) . BLAST analysis has shown potential binding sites for NF{kappa}-B/C-Rel, AhR, Sp1, GATA-1, Ap-2, and CAP in the promoter region. To determine which factors influence GCS expression, we introduced a series of truncated GCS promoter vectors into cells and tested luciferase activity. As shown schematically in Fig. 6 A (left panel), the luciferase reporter is driven by intact or truncated GCS promoters in pGCS-Luc1, pGCS-Luc2, and pGCS-Luc3 vectors. The pGCS-Luc2 vector has no elements for NF{kappa}-B/C-Rel, AhR, GATA-1, and Ap-2, and the pGCS-Luc3 has no element for the Sp1 transcription factor. We found that the luciferase activity of MCF-7 cells transfected with pGCS-Luc3 was ~10% of that of cells transfected with pGCS-Luc1 or pGCS-Luc2 (Fig. 5A , right panel). C6-ceramide increased luciferase activity by 3.8-fold (721 vs. 191) in cells containing pGCS-Luc1 and by 3-fold (687 vs. 231) in cells containing pGCS-Luc2 compared with untreated controls. However, C6-ceramide did not increase luciferase activity in cells transfected with either pGCS-Luc3 or the pGL3-basic vector. Consistently, the point mutation in the Sp1 element (Fig. 6B , left panel) dramatically decreased GCS promoter activity in MCF-7 cells transfected with pGCS-Luc1 M2 plasmid and eliminated the inductive effects of C6-ceramide and doxorubicin on GCS promoter. Sp1 siRNA, which knocks down Sp1 gene expression, substantially reduced GCS promoter activation and GCS mRNA levels but also diminished the up-regulative effects of C6-ceramide and doxorubicin on GCS (Fig. 6B ).


Figure 6
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  		Figure 6. GCS promoter structure and transcription factors. A) GCS promoter structure (schematic) and promoter activity. MCF-7 cells (4x105/well) were plated in 6-well plates and grown overnight. Reporter plasmids (4 µg DNA/well) were introduced into cells by incubation with Lipofectamine 2000 in serum-free medium. After 24 h transfection, cells were grown in 5% FBS RPMI 1640 medium containing 5.0 µM C6-ceramide for 24 h. The soluble fraction (25 µg protein/reaction) was used to measure luciferase activity. *P < 0.001 compared with vehicle control. B) The influence of Sp1 on GCS promoter activation and GCS gene expression. For luciferase assay (left panel), reporter plasmids (pGCS-Luc1 wild-type, pGCS-Luc1 M2 mutant; 4 µg DNA/well) were introduced into cells by incubation with Lipofectamine 2000 in serum-free medium; in siRNA group, pGCS-Luc1 plasmid was introduced into cells 24 h after of Sp1 siRNA transfection (100 nM). Cells were grown in 5% FBS RPMI 1640 medium containing 5.0 µM C6-ceramide and 0.5 µM doxorubicin for 24 h. For the mRNA analysis (right panel), MCF-7 cells were transfected with or without Sp1 siRNA (100 nM), following the exposure of doxorubicin (0.5 µM) and C6-ceramide (5.0 µM) for 48 h. *P < 0.001, **P < 0.001 compared with MCF-7 cells transfected with pGCS-Luc1 plasmid grown in medium. C) EMSA and ELISA of Sp1. MCF-7 cells were cultured in 5% FBS RPMI 1640 medium containing C6-ceramide (2.5 µM) or doxorubicin (0.5 µM) and Sp1 siRNA (100 nM) for 24 h. Nuclear extract (5.0 µg protein/lane; 40 µg/well) from each sample was used to detect Sp1 protein-DNA binding activity. Complex-1 and complex-2 are DNA-protein complexes of Sp1 (left panel). Recombinant Sp1 (0.64 µg/well) was used as standard (right panel); *P < 0.05 compared with cells treated with vehicle.

We further examined whether doxorubicin or ceramide would increase Sp1 levels in MCF-7 cells. In gel-shift assays of Sp1 (Fig. 6C ), protein-DNA complex-1 increased 3-fold and 2-fold in cells treated with C6-ceramide and doxorubicin, respectively. Excess unlabeled Sp1 probe and Sp1 antibody competitively blocked the increase of complex-1 under these conditions. In transcription factor ELISA analysis for Sp1, we found that doxorubicin and ceramide significantly increased the levels of bound Sp1. The Sp1 siRNA reduced Sp1 activities, even in cells treated with C6-ceramide (Fig. 6C ). These data suggest that the Sp1 element in the GCS promoter is essential for GCS expression and is responsive to ceramide.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Herein, we present the first evidence demonstrating that doxorubicin up-regulates GCS gene expression to cause drug resistance, as shown both in doxorubicin-selected cell lines and in cells after short-term doxorubicin exposure. We found that doxorubicin activated the GCS promoter and induced GCS gene expression. Previously, Kohno et al. (41) have demonstrated that doxorubicin amplifies the MDR1 gene, resulting in drug resistance.

Development of resistance to doxorubicin is accompanied by myriad genetic alterations that affect membrane transporters, tumor suppressors, and regulators of cell cycle and cell death (42 43 44) . Distinct from other genes involved in drug resistance, the GCS gene encodes an enzyme for lipid metabolism. GCS catalyzes conversion of ceramide to glucosylceramide, which is the backbone for more than 300 glycolipids, including globoside Gb5 and gangliosides GM3 and GD3 (12 , 14) . Enforced overexpression of GCS by gene transfection enhances GCS enzyme activity and confers cancer cell resistance to doxorubicin and daunorubicin (17 , 18) . We previously showed that increased GCS activity can deactivate ceramide-induced apoptosis (11 , 19 , 33) . In the present work, we show that GCS overexpression is a characteristic of doxorubicin resistance, clearly evident from the data in Fig. 1 and previous work (20) . Suppressing GCS gene expression by antisense gene transfection and RNA interference (11 , 45 , 46) or by antisense oligonucleotides (33) may therefore be an effective means to temper a cancer cell’s resistance to cytotoxic chemotherapy.

Among resistance-associated genes, including MDR1, MRP, Bcl-2, and mutant p53, MDR1 overexpression has been extensively examined in doxorubicin-selected cancer cells (4 5 6 , 41) . MDR1 gene expression can be mediated by transcription factors Sp1, YB-1, HSP, NF-IL6, p53, and EGR1 (47 48 49) . However, how cellular messengers of doxorubicin selection elicit gene overexpression for drug resistance remains unclear. A recent report (50) showed that doxorubicin up-regulates GCS in drug-resistant HL-60/ADR leukemia cells. Our study is consistent with the findings of Uchida et al. (50) and further demonstrates that ceramide, as a cellular messenger, contributes to GCS overexpression in doxorubicin exposure.

In addition to DNA intercalation and topoisomerase II interaction (51) , doxorubicin, at low concentration, mainly initiates signal transduction pathways, including those of protein kinase C and ceramide (52 53 54) . With regard to ceramide, activation of ceramide signaling by doxorubicin has been shown to induce apoptosis in many cell models (11 , 54 55 56 57 58) . As a second messenger, ceramide indeed affects myriad cellular processes (16) , including gene expression (59) . Recent studies (60 61 62 63) show that ceramide induces cyclooxygenase (COX)-2, nociceptin/orphanin FQ, acid sphingomyelinase (aSMase), and intercellular adhesion molecule-1gene expression. Ceramide activates NF-{kappa}B, inducing expression of nociceptin/orphani FQ and COX-2 (61 , 63) . Deigner et al. (62) observed that ceramide activates Sp1 and AP-2, inducing aSMase expression. However, with doxorubicin exposure, NF-{kappa}B activation is independent of ceramide (64) . The GCS promoter contains several motif elements for transcription, including NF-{kappa}B/C-Rel, AhR, Sp1, GATA-1, and AP-2 (38) . In the present study, results from truncation and point mutation of promoter, ceramide synthesis inhibition, and gel-shift assays clearly indicate that doxorubicin induces GCS expression by ceramide through Sp1-activated transcription.

Doxorubicin is one of the most active anticancer compounds used widely in the treatment of solid tumors. Cytotoxicity is the chief result of drug-intercalated DNA damage and ceramide-induced apoptosis (51) . Although it is well known that doxorubicin induces drug resistance (3 , 5 , 41) , the signal transduction pathways and transcription processes for doxorubicin-mediated gene expression are poorly understood. Our study shows that doxorubicin-enhanced ceramide induces GCS gene expression, and enhanced capacity for ceramide glycosylation endows cells stressed by doxorubicin-selection to evolve a resistant phenotype. As shown schematically in Fig. 7 , doxorubicin increases ceramide levels by either activation of sphingomyelinase or activation of enzymes of de novo ceramide synthesis, resulting in downstream signaling of apoptosis. However, as the natural lipid substrate of GCS, ceramide up-regulates GCS expression, which occurs through Sp1, as we have shown. We postulate this positive feedback cycle is antiapoptotic and drives cellular resistance to ceramide-generating types of chemotherapy drugs. Further studies to characterize the signal transduction pathways that switch ceramide-induced apoptosis to ceramide-mediated drug resistance via GCS gene expression may provide an approach to prevent anthracycline resistance.


Figure 7
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  		Figure 7. Up-regulation of GCS expression by doxorubicin. Doxorubicin can enhance ceramide formation by activation of either sphingomyelinase or the enzymes of de novo ceramide synthesis (serine palmitoyltransferase, ceramide synthase). In addition to induction of apoptosis, ceramide increases Sp1 transcription factor levels, activates the GCS promoter, and up-regulates GCS gene expression. GCS catalyzes ceramide glycosylation, converting apoptotic ceramide to glucosylceramide. Dox, doxorubicin; SMase, sphingomyelinase; GCS, glucosylceramide synthase.


   ACKNOWLEDGMENTS
 
This work was supported by a Public Health Service/U.S. National Institutes of Health (NIH) grant (P20 RR16456) from the National Center for Research Resources to Y.Y.L and S.M.J.; Department of Defense Breast Cancer Research Program (DAMD17–01-1–0536) to Y.Y.L.; the Public Health Service/NIH (GM77391) and the Susan G. Komen Breast Cancer Foundation to M.C.C.; Associates for Breast and Prostate Cancer Studies (Los Angeles, CA, USA); Fashion Footwear Charitable Foundation (New York, NY, USA) Shoes on Sale/QVC; Strauss Foundation Trust (Sandra Krause); and the Leslie and Susan Gonda (Goldschmied) Foundation. The authors appreciate the advice and help of Dr. V. Hsia and R. Pinnoji, Basic Pharmaceutical Sciences, University of Louisiana at Monroe, in real-time PCR analysis.

Received for publication July 19, 2007. Accepted for publication January 10, 2008.


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