Journal of Surgical Oncology 2011;104:466–471

MDR1 (Multidrug Resistence 1) Can Regulate GCS

(Glucosylceramide Synthase) in Breast Cancer Cells

XIAOFANG ZHANG, PhD,1 XIAOJUAN WU, MD,1 JUAN LI, PhD,2 YANLIN SUN, PhD,1 PENG GAO, PhD,1

CUIJUAN ZHANG, PhD,1 HUI ZHANG, MD,1 AND GENGYIN ZHOU, MD1*

1Department of Pathology, Shandong University School of Medicine, Jinan, Shandong, P.R. China2Department of Gynecology and Obstetrics, Shandong Provincial Hospital, Jinan, P.R.China

Background and Objectives: Besides MDR1/P-glycoprotein (MDR1/P-gp), glucosylceramide synthase (GCS), an enzyme, which transfers

UDP–glucose to ceramide to form glucosylceramide was also related with multidrug resistance (MDR) in breast cancer. Although many

research showed that GCS could affect mdr1 in cancer cells, nobody knows that whether mdr1 can affect GCS in breast cancer. Our study

aims to verify that.

Methods: A plasmid with multidrug resistence 1(mdr1) cDNA was transfected into the sensitive breast cancer cell line MCF-7, while an

RNA interference (RNAi) vector targeted mdr1 was transfected into the MDR cell line MCF-7/ADM. Then RT-PCR, Western blot, MTT, and

flow cytometry were used to assess the expression and function of mdr1 and GCS.

Result: The data displayed that up-regulation of mdr1 could increase the expression of GCS, while the RNAi-expression plasmids could

decrease that. Meantime, the changes of ceramide are opposed to that of GCS and are the same to the alteration of apoptosis rate.

Conclusions: Our results demonstrate that MDR1 could increase cellular apoptosis by regulating the expression of GCS in breast cancer

cells.

J. Surg. Oncol. 2011;104:466–471. � 2011 Wiley-Liss, Inc.

KEY WORDS: multidrug resistance gene-1; glucosylceramide synthase; multidrug resistance; breast cancer

INTRODUCTION

Over-expression of the drug efflux pump P-gp has been regarded

as the major cause of MDR in breast cancer. However, a drug resist-

ance phenotype comprises many, often interacting mechanisms of

resistance [1]. These include increased DNA repair, altered target

sensitivity, decreased apoptotic response, and numerous aberrant sig-

nal transduction pathways. Recently, some research revealed that

accumulation of glucosylceramide (GC) is a characteristic of some

multidrug-resistant cancer cells and tumors derived from patients

who are less responsive to chemotherapy [2,3]. The mechanism of

increase was related to GC synthesis and not to a decrease in degra-

dation. This led to the hypothesis that elevated glucosylceramide

synthase (GCS, EC 2.4.1.80) activity is a novel form of multidrug

resistance (MDR) and that inhibition of GCS is a promising thera-

peutic strategy for combating MDR.

GCS is a pervasive enzyme, which transfers UDP–glucose to

ceramide to form GC [4]. Liu et al. [5–7] reported that down-regula-

tion of GCS in the MDR breast cancer cells MCF-7/AdrR can

restore their sensitivity to various cytotoxic drugs and these exper-

iments confirmed that GCS have effects on MDR in breast cancer.

However, Liscovitch and Ravid [8] verified that MCF-7/AdrR cells

(re-designated NCI/ADR-RES) are derived from OVCAR-8 human

ovarian carcinoma cells, so the result cannot reflect the truth in

breast cancer. But our research in 2009 confirmed that suppression

of GCS could reverse multidrug resistence in breast cancer [9].

MDR1/P-glycoprotein (MDR1/P-gp), a member of ABC trans-

porter family, which confers the ‘‘classical’’ MDR phenotype, acts as

a drug efflux pump, lowering intracellular drug levels to sublethal

concentrations and helps cells to escape from death [10]. As more

has been learned about P-gp, it has been shown to transport GC

across membranes [11]. In addition, P-gp may play a role in regulat-

ing some caspase-dependent apoptotic pathways [12]. Our work in

2009 showed that, GCS can modulate the expression of mdr1 so as

to influence the drug resistance in cancer cell line [13]. Then if mdr1

can also affect GCS is still puzzling. Our work was aim to verify it.

MATERIALS AND METHODS

Cell Lines and Cell Culture

The sensitive breast cancer cell line MCF-7 was obtained from

the American National Cancer Institute. And the cell MCF-7/ADM

was induced by low concentration of adriamycin (10, 20, 40, 100,

and 200 ng/ml) step by step on the background of MCF-7 cell line.

All the cells were maintained in RPMI-1640 medium (Gibco, OK)

containing 10% FBS at 378C in a humidified atmosphere containing

5% CO2.

Construction of Recombinant Vector

The RNA interference (RNAi) sequence targeted to MDR1 was

selected according to the previous studies [14].The RNAi sequences

Grant sponsor: National Natural Science Foundation of China; Grantnumber: 30972929; Grant sponsor: Independent Innovation Foundation ofShandong University (IIFSDU).

X. Wu has contributed equally to this work.

*Correspondence to: Gengyin Zhou, MD, Department of Pathology,Shandong University School of Medicine, 44#, Wenhua Xi Road, Jinan,Shandong, 250012 P.R. China. Fax No.: þ86 531 88383168.E-mail: zhougy@sdu.edu.cn

Received 18 November 2010; Accepted 31 March 2011

DOI 10.1002/jso.21958

Published online 2 May 2011 in Wiley Online Library(wileyonlinelibrary.com).

� 2011 Wiley-Liss, Inc.

targeted toMDR1 were 50-GATCTCGTATTGACAGCTATTCGAAT-CAAGAGATTCGAATAGCTGTCAATACTTTTTTGGAAA-30; 50-TCGACTTCCAAAAAAGTATTGACAGCTATTCGAATCTCTTGA-

TTCGAATAGCTGTCAATACGG-30. These oligonucleotides were

annealed after phosphorylation of their 50 terminate, and then subcl-

oned into pSUPER to generate pSUPER-MDR1. And the mdr1

expression vector pSF91m3 was a gift of Professor C. Baum

(Department of Hematology/Oncology, MHH, Germany) [15].

Transfect Ion of Cells

Before transfect ion, cells were seeded in six-well plates at the

density of 1 � 106 cells per well and incubated at 378C in an atmos-

phere with 5% CO2 for 12 hr. For each well, 10 ml (2 mg/ml) of

lipofectamine (Invitrogen, Carlsbad, CA), or 5 ml (1 mg/ml) of vec-

tor was diluted into 250 ml of 1640 culture medium without serum.

After incubated for 10 min at room temperature, the diluted vector

and lipofectamine were mixed together and incubated for 20 min.

Then the mixture was adding to the cells washed three times by

medium without serum. Six hours later, the medium was replaced

with 1 ml of complete 1640 culture medium (The final concentration

of plasmid was 5 mg/ml). As control, 10 ml of lipofectamine and

5 ml (1 mg/ml) of pSUPER were also transfected. The MCF-7 cells

transfected with pSF91m3 were marked as MCF-7/MDR and MCF-

7/ADM treated by pSUPER or pSUPER-MDR1 were marked ADM/

pSUPER or ADM/RNAi, respectively.

Semi-Quantitative Reverse Transcription-PCR Analysis

Each group was cultured for another 48 hr after tranfecton. Total

RNA was isolated using Trizol (Invitrogen) and RT-PCR analysis of

MDR1 mRNA or GCS mRNA expression was performed as

described previously [1,16]. The sequence of the sense and anti-

sense primers for mdr1 mRNA is: 50-ACT GAG CCT GGA GGT

GAA GA-30 and 50-CCA CCA GAG AGC TGA GTT CC-30. Thesequence of the sense and anti-sense primers for GCS mRNA is: 50-CCT TTC CTC TCC CCA CCT TCC TCT-30 and 50-GGT TTC

AGA AGA GAG ACA CCT GGG-30. b-actin was used as internal

control set. The ratio of mdr1/b-actin or GCS/b-actin by scanning

densitometry was named mdr1 index or GCS index, respectively.

Western Blot to Analysis P-gp and GCS Protein

All the cells were cultured for another 48 hr after transfection,

then the confluent cells were lysed in a buffer containing 50 mM

Tris–HCl (pH 8.0), 150 mM NaCl, 0.5% Triton-X100, 2 mM EDTA

(PH8.0), 5 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, and

10 mg/ml aprotinin for 20 min on ice. The complex was centrifuged

at 12,000g for 10 min at 48C. As described before [17], equal

aliquots of protein (50 mg) were resolved using 4–12% gradient

PAGE. The transferred nitrocellulose blot was blocked with 5% fat-

free milk powder in TBS at room temperature for 2 hr. The mem-

brane was immunoblotted with murine monoclonal antibody C219

against human P-gp (0.7 mg/ml, Santa Cruz) or with GCS-1.2

antiserum (diluted 1:1,000) in 5% fat-free milk in TBS-0.1% Tween-

20.As control for equivalent protein loading, the filters were simul-

taneously incubated with rabbit polyclonal antibody against human

b-actin (diluted 1:1,000). Detection was performed using enhanced

chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).

All analyses were performed in triplicate in three separate

experiments.

Cytotoxicity Assay

Assays were performed as described previously [10]. Briefly, cells

were seeded in 96-well plates (1,000 cells/well) in 0.1 ml RPMI

1640 medium containing 10% FBS and cultured at 378C for 24 hr

before addition of adriamycin or vinblastine, both of which were

obtained from Sigma–Aldrich, SAINT LOUIS MO. Drugs were

added in FBS-free medium (0.1 ml).After cultured for 72 hr, cells

were stained with 150 ml sterile MTT dye (5 mg/ml; Sigma–Aldrich)

for 4 hr at 378C, subsequently, culture medium was removed and

150 ml of dimethyl sulfoxide (DMSO; Sigma–Aldrich) was added

and thoroughly mixed for 10 min. Absorbance at 490 nm was

recorded using an automatic multi-well spectrophotometer (Bio-Rad-

Coda, Richmond, CA).

Fig. 1. The expression of MDR1 and GCS mRNA and MDR1 andGCS protein after treated for 48 hr. A: The upper figure shows theresult of the RT-PCR products by electrophoresis analysis: The bandsfrom left to right were MCF-7, MCF-7/MDR, MCF-7/ADM, ADM/pSUPER, and ADM/RNAi, respectively. b-actin was analyzed inparallel as a loading control. The lower figure shows the relativemRNA levels of each group (the ratio of MDR1 or GCS to b-actinin MCF-7/ADM was set as 100%) and the white pillars and blackpillars indicate GCS mRNA and MDR1 mRNA, respectively. Dataare mean � SD of three repeat trials, �P < 0.01. B: Protein expres-sion in the cells by Western blot analysis.

MDR1 can Regulate GCS 467

Journal of Surgical Oncology

Apoptosis Analysis

Apoptotic rates were assessed with flow cytometry using the

Annexin V-fluorescein isothiocyanate/propidium iodide (PI) kit

(Bipec Biopharma Corp., MA). Samples were washed with ice-cold

PBS twice and resuspended in binding buffer at a density of

1 � 106 cells/ml. The cells were stained with Annexin V-FITC and

gently votexed. After 15 min of incubation at 4–88C in the dark, PI

was added to the cells for another 5 min incubation at 4–88C in the

dark. The results were analyzed by flow cytometry (FACScan; BD

Biosciences, MD).

Detection of Cellular Ceramide

Cells were seeded into 24-well plates (5,000 cells/well) and there

was a glass slide in every well. After 24 hr, cells were transfected

with or without corresponding plasmid.48 hr later, the glass slides

were fixed with methanol for 40 min. After permeabilization and

block, cells were incubated with anti-ceramide antibody (1:10, MID

15B4, Enzo Life Sciences, PA) overnight at 48C. The slides were

then probed with HRP-labeled polymer conjugated to secondary anti-

body for 30 min.

Statistical Analysis

Statistical analysis was performed by SPSS 13.0 software

package. All data were presented as mean � standard deviation and

one-way ANOVA and Dunnett’s T3 was used to determine the stat-

istical significance. P < 0.05 was considered statistically significant.

RESULTS

Expression of mdr1 mRNA and GCS mRNA in the

Transfected Cells

Semiquantitative RT-PCR was adopted to detect the expression of

mdr1 mRNA and GCS. The size of RT-PCR product for MDR1 and

GCS was 157 bp (3,014–3,170) and 303bp(162–464), respectively

and that of b-actin was 396 bp. As shown in Figure 1A, compared to

MCF-7 cells, the relative expression of MDR1 mRNA in MCF-7 /

MDR was 184-fold of that in MCF-7 cells (P < 0.01) and that of

GCS mRNA was about 1.49-fold, significantly higher than that of

MCF-7 cells (P < 0.05). Compared to MCF-7/ADM cells (the

relative mRNA levels in MCF-7/ADM cells was considered as

100%), the relative expression of MDR1 mRNA in the ADM/RNAi

cells decreased to 38.75 � 9.7% and that of GCS mRNA decreased

to 38.34 � 16%, significantly lower than that of the controls

(P < 0.01).

Changes of P-gp and GCS Protein Amount in the

Transfected Cells

P-gp and GCS protein expression were analyzed by Western

blot, as shown in Figure 2B. After thansfection for 48 hr, MCF-7/

MDR cells were found to have a great rise of both P-gp and

GCS protein expression. While, compared with the high expression

of P-gp and GCS in MCF-7/ADM cells, the expression of both

were found to have significantly reduction in the cells ADM/

RNAi.

Fig. 2. Cytoroxicity of each group cells by MTT assay. Cells transfected with RNAi plasmids or without any treatment were then givendifferent concentration of adriamycin or vinblastine as indication and incubated for an additional 72 hr. The measure of the cell survival ratewas done as described in the Materials and Methods Section. Data are mean � SD of three repeat trials, ��P < 0.01.

468 Zhang et al.

Journal of Surgical Oncology

Evaluation of Chemosensitivity in Transfected Cells

After transfection, we used adriamycin and vinblastine to assess

the influence of plasmid on cellular response to anti-neoplasm

drugs. The results showed that IC50 (IC50 is the drug concentration

(mmol/L) that results in 50% inhibition of cell growth) for Adriamy-

cin increased from 0.1127 � 0.0294mmol/L to 5.334 �0.2027 mmol/L after being transfected with the vector pSF91m3

(P < 0.01), and that for vinblstine increased from 1.537 �0.4403 mmol/L to 11.31 � 1.063 mmol/L (P < 0.01) (Fig. 2).

In MCF-7/ADM cells, the IC50 for adriamycin was 21.66 �2.792 mmol/L, while that for vinblatine was 49.43 � 3.975 mmol/L.

After transfection with RNAi-expression plasmids, the IC50 for

adriamycin or vinblastine of the cells ADM/RNAi was lowered to

2.800 � 0.4060 mmol/L and 12.79 � 1.325 mmol/L, respectively

(P < 0.01) (Fig. 2).

Alteration of Apoptosis Rate in the Transfected Cells

The apoptosis rate was detected by flow cytometry using the

Annexin V-fluorescein isothiocyanate/PI kit, and the Annexin

V-positive, PI-negative cells were scored as early apoptotic. The

apoptosis rates estimated in the present study only included the early

apoptotic cells, which were marked as LR in Figure 3. After

transfection with pSF91m3, the apoptosis rate decreased from 12.3%

to 8.6%, while after transfection with the RNAi plasmid, the rate of

the early apoptosis increased significantly, from 9.6% to 26.1%.

Changes of Cellular Ceramide After Transfection

The cellular ceramide was detected by immunocytochemistry.

Staining in the cytoplasmic was considered positive (Fig. 4) and

1,000 cells/slide were counted at random. The positive rate

decreased from 25.6% to 1.2% after being transfected with the vector

pSF91m3 (P < 0.01) and that increased from 0.4% to 57.8% after

transfection with the RNAi plasmid (P < 0.01).

DISCUSSION

Breast cancer has become the biggest threaten to women’s health.

MDR to chemotherapeutic agents is a major obstacle to successful

treatment in patients with breast cancer. Over-expression of the

members of the adenosine triphosphate (ATP)-binding cassette

(ABC) membrane transporter family is one of the most important

factors. MDR1/P-glycoprotein (MDR1/P-gp), a member of ABC

transporter family, which confers the ‘‘classical’’ MDR phenotype, is

expressed in almost 50% of human cancers [15].

Fig. 3. Apoptosis rate of each group cells The apoptosis rate was detected by flow cytometry using the Annexin V-fluorescein isothiocya-nate/PI kit, and the Annexin V-positive, PI-negative cells were scored as early apoptotic were marked as LR.

MDR1 can Regulate GCS 469

Journal of Surgical Oncology

Recently, an enzyme, GCS, whose most direct function is now

known to be intracellular glycosylation of ceramide for synthesis of

GC, has become a point of interest [18]. GCs have been found to

involve in many cellular processes such as cell proliferation, onco-

genic transformation, differentiation, and tumor metastasis [19]. In

1996, Lavie et al. [2] first reported that chemotherapy resistant

MCF-7-AdrR breast cancer cells accumulate GC in comparison to

wild-type MCF-7 cells. After that, GC was found to confer to MDR

in many other cancers [3,4,20].So, some people guessed that elevated

GCS activity is a novel form of MDR and that inhibition of GCS is

a promising therapeutic strategy for reversing MDR. Then, Liu et al.

[5] found that increased competence to glycosylate ceramide

conferred adriamycin resistance in MCF-7 breast cancer cells by

transfection with GCS cDNA, while using GCS inhibitor 1-phenyl-2-

palmitoylamino-3-morpholino-propanol (PPMP) or transfection of

doxorubicin-resistant MCF-7-AdrR cells with GCS antisense both

restored cell sensitivity to doxorubicin or vinblastine and paclitaxe-

land. Ladisch found that blocking GCS with D, L-threo-phenyl-2-hex-

adecanoylamino-3-pyrrolidino-1-propanol (PPPP), was able to

elevate ceramide levels and enhance vincristine cytotoxicity via pro-

grammed cell death [21]. All the following works demonstrated that

GCS was potentially one MDR-related drug resistance mechanism.

So, it is necessary to know the impact factors of GCS. Some

researches have revealed that both testosterone and increased intra-

cellular ceramide could induce endogenous up-regulation of GCS

[22,23]. As we all know, the expression of P-gp in MCF-7-AdrR is

much higher than that in MCF-7, and the elevated expression of

GCS in the same cell lines informs us that there may be relation

between P-gp and GCS.

However, Liscovitch and Ravid [8] verified that MCF-7/AdrR

cells (re-designated NCI/ADR-RES) are derived from OVCAR-8

human ovarian carcinoma cells; so many results in the cells MCF-7/

AdrR cannot reflex the truth of breast cancer. Then can P-gp modu-

late GCS mediated MDR? Although some studies revealed that P-gp

could transport short-chain fluorescent analogs of sphingomyelin

and GC across membranes and regulate some caspase-dependent

apoptotic pathways so as to regulate GC-based MDR [11,12],

there has been no direct proof to confirm that if P-gp can regulate

GCS.

In order to confirm the following question, we constructed recom-

bined plasmid with RNAi sequences targeted MDR1. Semiquantative

RT-PCR analysis indicated that when the MDR1 mRNA increased,

the GCS mRNA increased too; while the MDR1 mRNA in the cells

reduced, the expression of GCS mRNA also down-regulated. The

changes of MDR1 protein and GCS protein also have consistency by

using Western blot analysis. As far as we all know, GCS can catalyze

ceramide to form GC which lead cells to escape death [19], and our

work demonstrated that when mdr1 gene over-expresses in the cells,

the cellular ceramide becomes lower and the early apoptotic rate is

decreased; while mdr1 gene is suppressed, the cellular ceramide be

higher and the rate of early apoptosis increases significantly. From

the results we can see that mdr1 could affect apoptosis of cells by

regulating GCS.

However, our result is a little different with Shabbits’ [24] in

2002. In that article, the author transfected mdr1 gene into ER-nega-

tive cell line MDA-MB-435 to form MDA435/LCC6MDR1, the data

show that P-gp could not directly influence the expression of GCS,

but affect the translocation of newly formed GlcCer across the Golgi

membrane. However, our study shows that mdr1 could affect the

expression of GCS directly in MCF-7 and MCF-7/ADM cells. As we

all know, MCF-7 is ER-positive. Ruckhaberle et al. [25] found that

the expression of GCS may be related to the ER status in breast

cancer samples. So, we deduced that the ER status might be the

reason of the difference between our data and Shabbits’.

In 2010, Liu et al. [26] reported that GCS up-regulates MDR1

expression in the regulation of cancer drug resistance through cSrc

and b-catenin signaling in the ovarian cell line NCI/ADR-RES. This

study revealed the importance of GCS in the mechanism of cancer

drug resistance. Here in, our work shows that mdr1 also affects GCS

reversely in the breast cancer cell line.

Fig. 4. Expression of cellular ceramide. The cellular ceramide was detected by immunocytochemistry. Staining in the cytoplasmic wasconsidered positive. [Color figure can be viewed in the online issue, available at wileyonlinelibrary.com.]

470 Zhang et al.

Journal of Surgical Oncology

CONCLUSIONS

In conclusion, our study for the first time revealed that mdr1

could regulate GCS in mRNA and protein levels in breast cancer

cells.

ACKNOWLEDGMENTS

We greatly appreciate the gift of GCS antiserum from Dr. R.

Pagano and Dr. D. Marks (Mayo Clinical and Foundation, Rochester,

MN, USA. This work was supported by the National Natural Science

Foundation of China (No. 30972929) and the Independent Innovation

Foundation of Shandong University (IIFSDU).

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