Journal of Cellular Biochemistry 93:1134–1142 (2004)

Inhibition of Cell Proliferation in Human Breast TumorCells by Antisense Oligonucleotides Against FacilitativeGlucose Transporter 5

K.K. Chan, Judy Y.W. Chan, Karen K.W. Chung, and Kwok-Pui Fung*

Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong, China

Abstract In recent years, successful examples of antisense oligonucleotide (AS) therapy for genetic diseases havestimulated scientists to investigate its application on cancer diseases. AS can be used to down-regulate the mRNA andprotein expression by annealing to specific region of the target mRNA which is responsible for the malignancy. Glucosetransporter 5 (Glut5) is a tissue specific transporter that can be found on breast cancer tissues but not on normal breasttissues. Therefore, it is of clinical interest to investigate whether AS against Glut5 mRNA can tackle breast cancer. In thisstudy, two cell lines, MCF-7 which is estrogen-receptor positive and MDA-MB-231 which is estrogen-receptor negative,were used tomimic breast cancer tissues at early and late stages, respectively. A 15-base sequence around the start codonof Glut5 was used. It was found that AS against Glut5 exerted anti-proliferative effect on both of these two breast tumorcell lines and seemed to exert its effect via the suppression of expression of Glut5 proteins in the cells. AS against Glut5exhibited no effect on humanhepatomaHepG2cellswhich donot possess anyGlut5. The results imply an alternativewayin treating breast tumor as the AS against Glut5, unlike tamoxifen, takes effect on breast tumor cells via suppressing theexpression ofGlut5 that they specifically possess, and regardlesswhether the breast tumors are estrogen dependent or not.J. Cell. Biochem. 93: 1134–1142, 2004. � 2004 Wiley-Liss, Inc.

Key words: antisense; glucose transporter; breast tumor

Thirteen mammalian glucose transporters(Gluts) have been identified so far. They arenamed Glut1-12 and H(þ)-myo-inositol cotran-sporter (HMIT), while Glut6 was found to be apseudogene that cannot be detected in proteinlevel [Wood and Trayhurn, 2003]. There is aclose relationship between the Glut expressionand malignant transformation. Some oncoge-nes can regulate glucose transport in tumorcells. In general, oncogene-transformed cellswill exhibit an increase in glucose transport. Itwas found that src-transformed chicken embryo

fibroblasts and raf-1 transformed 3T3-L1 fibro-blasts had an elevated glucose transport thanthe native one [White et al., 1991; Fingarand Birnbaum, 1994]. This change in glucosetransport can be achieved by increasing theexpression of a particular glucose transporter,decreasing the degradation rate of the trans-porter, or decreasing theKmthrough structuralmodification in Glut [Ahmed and Berridge,1998]. Over-expression of a particular Glut wasalso common in various cancers. For example,Glut1 was found to be over-expressed in breastand colorectal carcinoma [Younes et al., 1995;Haber et al., 1998]. Other cancers such as hepa-toma and ovarian carcinoma also over-expressGlut2 and Glut3, respectively [Grobholz et al.,1993; Kurata et al., 1999]. Besides, high glucoseutilization accompanied with a high prolifera-tion rate could be observed in tumor cells.Due totheir different substrate specificity and affinity,Gluts exhibit a tissue-specific localization.Among the family, Glut5, the most divergentmember, was found to be a fructose transporterwith a Km varied from 6 to 10 mM [Miyamotoet al., 1994]. It can only be located on the brush

� 2004 Wiley-Liss, Inc.

Grant sponsor: Research Grants Council, Hong Kong,China (earmarked grant); Grant number: CUHK 4148/98M; Grant sponsor: Research Grants Council, Hong Kong,China (direct grant); Grant number: 2040955.

*Correspondence to: Kwok-Pui Fung, Department ofBiochemistry, The Chinese University of Hong Kong,Shatin, Hong Kong, China. E-mail: kpfung@cuhk.edu.hk

Received 28 July 2003; Accepted 17 November 2003

DOI 10.1002/jcb.20270

border membrane of small intestinal entero-cytes and spermatozoa. Moreover, it is highlyrestricted to neoplastic mammary tissuerather than normal mammary epithelial cells[Zamora-Leon et al., 1996].Antisense molecules are oligonucleotides

whose sequences are complementary to RNAtranscribed from the target gene. These mole-cules block gene expression by interacting withitsRNA transcript [Agrawal andZhao, 1998]. Inprinciple, antisense oligonucleotides (AS) canbe developed and applied against any gene pro-duct. The greatest impact of AS is anticipated inthe area of cancer because effective treatmentsdonot currently exist and because differences ingenetic profile exist between diseased andhealthy individuals [Kronenwett and Haas,1998].For examples,we [Chanetal., 2000;Chenet al., 2002] and other researchers [Noguchiet al., 2000] found that AS against Glut1 couldsuppress the growth of human tumor cells.In this study, we tried to suppress the Glut5

expressionon twobreast tumor cell lines,MCF-7and MDA-MB-231 cells, by using a 15-basephosphorothioated oligonucleotides. In thisclass of oligonucleotides, oneof theoxygenatomsin the phosphate group is replaced with a sulfuratom. The resulting compound is negativelycharged and much more resistant to nucleases[Agrawal and Zhao, 1998]. MCF-7 cells areestrogen-receptor positive and MDA-MB-231cells are estrogen-receptor negative, mimickingbreast cancer at early and late stages, respec-tively. Both MCF-7 and MDA-MB-231 cellsshowed a reduction in both Glut5 mRNA andprotein levels after treatment. It is also accom-panied with a decrease in cell proliferation rate.

METHODS

Cell Culture

Human tumor cell lines MCF-7, MDA-MB-231, and HepG2 were purchased from ATCC(Rockville, MD). They were grown in RPMI-1640 medium (Gibco BRL, Carlsbad, CA) sup-plemented with 10% fetal calf serum andcultured in a humidified atmosphere of 5%CO2 at 378C. Lipofectin was used as a carrierto enhance the uptake of the oligonucleotides.Lipofectin in the same amount (w/w 1:1) as theoligonucleotide was mixed together in serumfree medium. After a 15 h transfection at 378C,the serum-free medium was removed andreplaced with fresh complete medium. The cells

were further incubated at 378C 5% CO2 untilanother assay was carried out.

Design of Oligonucleotide Sequence

The sequence of AS against Glut5 was selec-ted to be complementary to human Glut5 cDNAsequence around the start codon region startedat base 70. For 15 base long AS against Glut5,the sequence was: 50-TTG CTC CAT GCT TGC-30. The sequence of sense control for Glut5 was:50-GCA AGC ATG GAG CAA-30. All the oligo-nucleotideswere phophorothioated at each baseto increase their stability.

Cell Proliferation Assay

2� 104 cells per well were seeded on 96-wellplate. After 24 h incubation time, transfectionwas carried out [Chan et al., 2000]. At theappropriate time, themediumwas removed andwashedwithPBSonce.Volumeof 50ml of a 5mg/ml solution ofMTTwasadded into eachwell andthe plate was incubated at 378C for 2 h. Then,150 ml of DMSO was added and incubated atroom temperature for further 30 min. The opti-cal density of eachwell wasmeasured at 540 nmby an ELISA plate reader (BioRad, Hercules,CA).

Thymidine Incorporation

In a 24-well plate, 6� 104 cells per wellwere seeded. After treating with 2 mM ASagainst Glut5, the medium was discardedand 0.5 mCi [methyl-3H]-thymidine (AmershamBiosciences, Piscataway,NJ) in culturemediumwas added to eachwell. The platewas incubatedat 378C for 3 h. The medium was then removedand washed with PBS. One milliliter of 10%TCA was then added and incubated for 1 h atroom temperature. TCA was discarded andfollowed bywashing twice in PBS. Two hundredmicroliters of 0.1% SDS and 50 ml of 1 N NaOHwere added to lyse the cells. The lysate was thenmeasured for scintillation counting and itsprotein content was evaluated by BCA method.

RT-PCR Analysis

Four micromolar of AS against Glut5 wasused to treat the tumor cells. TRIzol Reagent(Gibco BRL) was used to extract the total RNAafter transfection and recovery. The RNA pelletwas dried briefly and dissolved in DEPC-water.Three to four micrograms of the total RNA wasused to synthesize the first strand cDNA byusing the SuperScript Preamplification System(Gibco BRL). The forward primer sequence of

Treatment of Breast Cancer by AS Against Glut5 1135

Glut5 was: 50-TAG GGC AAG CTT CTG AAGTGT ACC CGG AAA AGG-30 while the reverseprimer sequence was: 50-TAG GGC GCG GCCGCG AAA AGT GAT CAG GTT CAT-30. Theseprimers flanking a 700base pairs productwouldamplify the Glut5 mRNA. For glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the for-ward primer sequence was: 50-ACC ACA GTCCAT GCC ATC AC-30 while the reverse primersequencewas: 50-TCCACCACCCTGTTGCTGTA-30. The amplified products were then elec-trophoresed on a 1% agarose gel and stainedwith ethidium bromide.

Western Hybridization

Four micromolar of AS against Glut5 wasused to treat the tumor cells. Protein lysate wasextracted at appropriate time point. The proteincontent of each sample was estimated by BCAprotein assay. Twenty to twenty-fivemicrogramsamples were used to run SDS–PAGE (4.5%stacking gel and 8% running gel). The proteinswere then transferred to a polyvinylidenefluoride (PVDF) microporous membrane bysemi-dry transfer system (BioRad). The mem-brane was blocked in 10% non-fat milk (in TBS-T) before incubating with monoclonal rabbitantibody produced against human Glut5. Afteradding anti-rabbit IgG antibody conjugatedwith horseradish peroxidase (Santa Cruz Bio-technology, Santa Cruz, CA), ECL detectionreagents (Amersham) were used and the emit-ting signal was detected by exposure to auto-radiography film (Kodak, Rochester, NY). Thebands were quantified by using densitometer(Molecular Dynamics, Amersham Biosciences).

Fructose Uptake Assay

Twomicromolar ofASagainstGlut5wasusedin transfection. At appropriate time, the med-ium was removed and the cells were washedwith PBS. Fructose (0.1 mM) and 0.5 mCi 14C-fructose for fructose uptake assay (Amersham)in PBS was added to each well for 15 min at378C. The PBS was discarded and washedwith 10 mM fructose in normal saline. Afterthat, 400 ml of 0.1% SDS was added. Volume of10 ml was used for protein content determina-tion and the rest was for scintillation counting.

Statistical Analysis

Data were expressed as the mean value�SDin different experiments. Student’s t-test wasperformed in the experiments. P< 0.05 indi-

cated that the difference between the tested andcontrol groups was statistically significant.

RESULTS

RT-PCR Analysis

The total RNA were extracted from both celllines after treated with AS against Glut5 andthe Glut5 mRNA level was measured by usingRT-PCR. As observed in Figure 1A, the level ofGlut5 mRNA in MCF-7 cells was suppressed by77.5% at 5 h after transfection when comparedwith the level in serum-free control. At 48 hpost-transfection, the effect of the AS againstGlut5 was still valid by which the suppressionwas found to be 46.5%. For MDA-MB-231 cells,suppressions of Glut5 mRNA were also observ-ed at 5 and 24 h after the transfection, with thesuppression of 88.8 and 80.7%, comparing withserum-free controls, respectively (Fig. 1B).When comparing with the level of Glut5 mRNAin antisense treated tumor cells, the degree ofsuppression is highest at 24 h after transfectioninMCF-7andMDA-MB-231 tumor cells (Fig. 2).

Western Analysis

The probable change in Glut5 protein expres-sion in MCF-7 and MDA-MB-231 cells aftertransfection ofASagainstGlut5weremeasuredby Western hybridization. The results showedthat after recovery from transfection for 1 day,the amount of Glut5 proteins in AS treated cellswere lower than the sense (S) control (Fig. 3).Combined with the results in RT-PCR, thereduction of Glut5 mRNA by AS of Glut5 hadsubsequently led to suppression of translationof Glut5 mRNA to Glut5 protein. This suppres-sion was shown in both MCF-7 and MDA-MB-231 cells. After 24 h of transfection, in MCF-7cells, the Glut5 protein amounts were about 4%less than the sense control while about 20%reduction was detected in MDA-MB-231 cells.The suppression of translation could also beobserved at 48 h after transfection. In MCF-7cells, the suppression of Glut5 protein by AS ofGlut5 was found to be about 36% comparingwith sense control. On the other hand, theGlut5protein level was about 18% less in the anti-sense treated MDA-MB-231cells when com-pared with sense control.

Cell Proliferation Assay

The effect of theASagainstGlut5 on theproli-feration rate of both cell lines was investigated

1136 Chan et al.

by using MTT assay. For the 15-base Glut5 AS,anti-proliferative effect was observed in bothMCF-7 and MDA-MB-231 cells (Fig. 4). Theanti-proliferative effect exhibited a concentra-tion-dependent manner. The non-specific effectwas relative low as indicated by a higher

percentage of survival in sense (S) treatment.In addition, the anti-proliferative effect of theAS was similar in both cell lines. Similarpercentage was observed at the same concen-tration of AS.

To verify the specificity of anti-Glut5 oligo-nucleotide on cells that expressed Glut5 only,the AS against Glut5 was applied to HepG2cells. HepG2 cell line is a hepatocarcinoma cellline that expresses Glut1, Glut2, and Glut3 butno Glut5 expression was detected [Yamamotoet al., 1990]. Therefore, HepG2 cell line is a goodindicator for the specificity of the effect of ASagainst Glut5 in the present study. From theresults on Figure 4C, the anti-proliferative ef-fect of the AS against Glut5 on MDA-MB-231cells was significantly higher than that onHepG2 cells. The difference was found to besignificant (P< 0.05) and the level of antisense

Fig. 1. The suppression of Glut5 mRNA in (A) MCF-7 and (B)MDA-MB-231 cells after treated with antisense oligonucleotide(AS) against Glut5. Total RNA was extracted from cells treatedwith serum-freemedium (SF), sense sequence (S), and AS againstGlut5, respectively, at the time point indicated after transfection.The RNA was then reverse transcribed into cDNA which wouldcarried out PCR by specific primers. The amounts of samples

added were normalized by amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The PCR products werethen run in 1% agarose gel and stained with ethidium bromide.The intensity of the bands were analyzed by densitometer. Theexperiments were repeated twice. Only one of the results wasshown here. Not more than 10% in SDwere obtained in the twoexperiments.

Fig. 2. Comparison of the Glut5 mRNA in MCF-7 and MDA-MB-231 cells treated with AS against Glut5 at different timepoints. A, MCF-7 cells; B, MDA-MB-231 cells.

Treatment of Breast Cancer by AS Against Glut5 1137

effect on HepG2 cells was similar with the non-specific effect caused by the sense oligonucleo-tides.This result indicated thatASagainstGlut5was more effective on tumor cells with Glut5expression, i.e., MCF-7 andMDA-MB-231 cells.

Thymidine Incorporation

To confirm that the growth of the tumor cellswas inhibited, the amount of DNA synthesisduring S phase can be detected by monitoringthe incorporation of radioactive DNA precur-sors [methyl-3H]-thymidine. From the resultson Figure 5, the counts measured in AS againstGlut5 treated MCF-7 and MDA-MB-231 cellswere lower than both serum-free and sensecontrols. This anti-proliferative effect could alsobe observed until 48 h after transfection whilethe differences of thymidine incorporated on bothcell lines were enhanced than 24 h recovery.

Fructose Uptake Measurement

Carbon-14 labeled fructose was used to followthe change in cellular uptake. For MCF-7 cells,the fructose uptake was reduced by the ASagainst Glut5 either at 24 or 48 h after trans-fection (Fig. 6A). The amount of fructose uptakewas significantly lowerwhen comparedwith thesense control. For MDA-MB-231 cells, similarresults were observed.

DISCUSSION

AS is a short stretch of synthetic, chemicallymodified nucleic acids. High specificity of anti-sense mechanism can even distinguish sequen-ces difference between one base [Mercatanteand Kole, 2000]. Moreover, the suppressiveeffect can occur at the level of both mRNA andprotein. These characteristics make AS be apotential agent on many therapies [Giovineet al., 1998; Miayake et al., 2000].

In our study, we have tried to investigate theeffect of AS against Glut5 on breast tumor cells,basing on the findings that alteration of glucosetransporter-mediated glucose transport wouldaffect the growth of tumor cells [Fung et al.,1986; Noguchi et al., 2000; Chen et al., 2002].Glut5, a breast tumor specific glucose transpor-ter, was chosen to be the target because thenormal breast tissues do not express Glut5[Zamora-Leon et al., 1996]. A previous researchfound that suppression of Glut1 mRNA couldsuppress the growth of a gastric cancer cell line[Noguchi et al., 2000]. We [Chan et al., 2000;Chen et al., 2002] also found that AS againstGlut1couldsuppress thegrowthofhumanhepa-toma HepG2 cell line. It was speculated thatupset of the Glut5 mediated glucose transportwould also inhibit the growth of breast tumorcells.

Fig. 3. The suppression of Glut5 in protein levels inMCF-7 andMDA-MB-231 cell lines after treatment of AS against Glut5.At 24 h (1) and 48 h (2) after transfection, cells with differenttreatments were trypsinized and the proteins were extractedas described in ‘‘Methods.’’ The protein samples were thenelectrophoresed in SDS–PAGE. The Glut5 proteins were blottedagainst anti-rabbit Glut5 antibodies and developed by ECLmethod.

The intensities of the bands were analyzed by densitometer.A, MCF-7 cells; B, MDA-MB-231cells; SF, serum-free medium;S, sense oligonucleotide; AS, antisense oligonucleotide. Theexperiments were repeated twice. Only one of the results wasshown here. Not more than 10% in SDwere obtained in the twoexperiments.

1138 Chan et al.

By RT-PCR, the amount of mRNA in ASagainstGlut5 treatedMCF-7andMDA-MB-231cells is less than the serum-free and sensecontrol. From our results, the suppression ofGlut5 mRNA could be observed as soon as 5 hafter transfection.The reduction ofmRNAcouldonly be observed in antisense treatment (Figs. 1and 2). This illustrated that only AS againstGlut5 bound to target RNA specifically but notthe sense oligonucleotides. The suppression ofGlut5 mRNA lasted for 48 h after transfection.The level of mRNA recovered back to normallevel as the serum-free and sense controls for24 h further, i.e., at 96 h after transfection (datanot shown). Moreover, the trend of changes inmRNA levelwas similar in both tumor cell lines.The suppression was most pronounced at 24 hafter the transfection. The level of Glut5mRNAthen increased gradually. This may be due to

the beginning of degradation of the AS insidethe cells. Single dose of AS against Glut5 couldlast for about 48 h.

The suppression of mRNA of Glut5 by ASagainst Glut5 should inevitably lead to declinein the corresponding protein expression.Through the changes in protein level, the phe-notypes or physiological conditions of cells willbe affected. The amount of Glut5 proteins wasless in AS against Glut5 treated cells than theamount in sense control (Fig. 3). The suppres-sion of Glut5 proteins could be observed at 24and 48 h after the transfection. In MCF-7 cells,the degree of suppression of Glut5 protein wasless than the suppression in MDA-MB-231cells at 24 h after transfection. However, asthe recovery period kept going, this subtleinhibition of protein synthesis in MCF-7 cellswas changed to bemore pronounced at 48h. The

Fig. 4. The effect of AS against Glut5 on the growth of MCF-7and MDA-MB-231 cells. Different concentrations of oligonu-cleotide as indicated was added to 2� 104 cells/well in 96-wellplate. Equal amount of lipofectin (w/w)was used to complexwiththe oligonucleotide in serum-free medium. After 15 h transfec-tion, the cells were recovered in fresh complete medium forfurther 48 h. The percentage of cell confluence relative to serum-free treated controlwasmeasured byMTT assay. Sense sequence

(S) was also added to differentiate any non-specific effectobserved in antisense treatment (AS). Each bar represented themean value of five replicates �SD. HepG2 cells were treated asthe same conditions as MDA-MB-231 cells. S-HepG2: HepG2cells treated with Glut5 sense oligonucleotides; AS-HepG2:HepG2 cells treated with Glut5 antisense oligonucleotides; AS-MDA: MDA-MB-231 cells treated with Glut5 antisense oligo-nucleoides. *P<0.05.

Treatment of Breast Cancer by AS Against Glut5 1139

Fig. 5. The anti-proliferative effect of AS againstGlut5 onMCF-7 andMDA-MB-231 cells. After 24 h (1) or 48 h (2) recovery fromtransfection, [methyl-3H]-thymidine in 0.5 mCi was added tocells for further incubation for 3 h. The amount of thymidineuptake was measured by scintillation counting. Five microliters

of the cell lysate was used for measuring of the protein amounts.Data were expressed as the mean value of triplicate experiments�SD (A) MCF-7; (B) MDA-MB-231. The significance of thedifferences were analyzed by Student’s t-test; *P<0.001,**P< 0.02.

Fig. 6. The effect of AS against Glut5 on the fructose uptake ofMCF-7 and MDA-MB-231 cells. The amount of fructose uptakewas measured in antisense treated cells and the sense-sequencetreated cells after (1) 24 hand (2) 48h after transfection.A,MCF-7

cells; B, MDA-MB-231 cells. Data were expressed as the meanvalue of four replicates �SD. The significance of the differenceswere analyzed by Student’s t-test. S, sense oligonucleotide; AS,antisense oligonucleotide. *P<0.001; ***P<0.03.

1140 Chan et al.

inhibitory effect transited from mRNA to pro-tein level seemed to be slower in MCF-7 cellsthan that in MDA-MB-231cells.After applying AS against Glut5, both breast

tumor cell lines showed a reduction in thegrowth rate. The MTT assay indicated a dose-dependent response to the AS against Glut5(Fig. 4). By [methyl-3H]-thymidine incorpora-tion assay, the amounts of DNA synthesis inbothMCF-7 andMDA-MB-231 cells were foundto be significantly suppressed in AS againstGlut5 treatment than that in sense oligonucleo-tide treatment after 24 and 48 h (Fig. 5). More-over, the antisense effect was highly specific onboth breast tumor cells with Glut5 expressiononly (Fig. 5). For those tumor cells withoutGlut5, such as HepG2 cells, only non-specificeffect can be obtained. The high specificity ofusing thisASagainstGlut5may reduceany sideeffects on other tissues when it is applied clini-cally, because most of them (including normalbreast cells) do not possess Glut5. This is one ofthemajor reasons whywe focus onGlut5 ratherthan other glucose transporters.Besides the anti-proliferative effect, some of

the physiological changes have been investi-gated. As expected, the fructose uptake in anti-sense treated tumor cells was found to bereduced to a greater extent in AS against Glut5treated breast tumor cells. It is consistent withthe results showing an inhibition on Glut5translation. The decrease in fructose uptakecould be attained during the period of time inwhich the expression of Glut5 mRNA and thenproteins were suppressed (Fig. 6). Moreover, itseems that there is no up-regulation of otherglucose transporters because there is no com-pensatory increase in glucose uptake afterfructose uptake is suppressed (data not shown).In conclusion, AS against Glut5 can exert

anti-proliferative effect on Glut5-containingbreast tumor cells but not for tumor cells with-out Glut5 expression such as HepG2 cells. Inaddition, AS against Glut5 can take effect onboth MCF-7 cells which are estrogen-receptorpositive and MDA-MB-231 cells which areestrogen-receptor negative. It is unlike tamox-ifen, a common drug for treating breast cancer,which isnot effective on estrogen-receptornega-tive cells, e.g., MDA-MB-231 cells used in thepresent study. As a result, estrogen-receptornegative cells in the breast cancer patient con-tinue to grow and the breast cancer at the laterstage of the patient cannot be treated by tamo-

xifen. The feasibility test of AS against Glut 5treatment in vivo in nude mice bearing MCF-7or MDA-MB-231 cells are in progress.

REFERENCES

Agrawal S, Zhao Q. 1998. Antisense therapeutics. CurrOpin Chem Biol 2:519–528.

Ahmed N, Berridge MV. 1998. Transforming oncogenesregulate glucose transport by increasing transporteraffinity for glucose: Contrasting effects of oncogenesand heat stress in a murinemarrow-derived cell line. LifeSci 63:1887–1903.

Chan JY, Chu AC, Fung KP. 2000. Inhibition ofP-glycoprotein expression and reversal of drug resistanceof human hepatoma HepG2 cells by multidrug resistancegene (mdr1) antisense RNA. Life Sci 67:2117–2124.

Chen CP, Li XX, Zhang LR, Min JM, Chan JY, Fung KP,Wang SQ, Zhang LH. 2002. Synthesis of antisenseoligonucleotide-peptide conjugate targeting to GLUT-1inHepG-2 andMCF-7Cells. BioconjugChem13:525–529.

Fingar DC, Birnbaum MJ. 1994. A role for Raf-1 inthe divergent signaling pathways mediating insulin-stimulated glucose transport. J Biol Chem 269:10127–10132.

Fung KP, Choy YM, Chan TW, Lam WP, Lee CY. 1986.Glucose regulates its own transport in Ehrlich ascitestumour cells. Biochem Biophys Res Commun 134:1231–1237.

Giovine M, Gasparini A, Scarfi S, Damonte G, Sturla L,Millo E, Tonetti M, Benatti U. 1998. Synthesis andcharacterization of a specific peptide nucleic acid thatinhibits expression of inducible NO synthase. FEBS Lett426:33–36.

Grobholz R, Hacker HJ, Thorens B, Bannasch P. 1993.Reduction in the expression of glucose transporterprotein GLUT 2 in preneoplastic and neoplastic hepaticlesions and re-expression of GLUT 1 in late stages ofhepatocarcinogenesis. Cancer Res 53:4204–4211.

Haber RS, Rathan A, Weiser KR, Pritsker A, Itzkowitz SH,Bodian C, Slater G, Weiss A, Burstein DE. 1998. GLUT1glucose transporter expression in colorectal carcinoma: Amarker for poor prognosis. Cancer 83:34–40.

Kronenwett R, Haas R. 1998. Antisense strategies for thetreatment of hematological malignancies and solidtumors. Ann Hematol 77:1–12.

Kurata T, Oguri T, Isobe T, Ishioka S, Yamakido M.1999. Differential expression of facilitative glucosetransporter (GLUT) genes in primary lung cancers andtheir liver metastases. Jpn J Cancer Res 90:1238–1243.

Mercatante D, Kole R. 2000. Modification of alternativesplicing pathways as a potential approach to chemother-apy. Pharmacol Ther 85:237–243.

Miayake H, Tolcher A, Gleave ME. 2000. Chemosensitiza-tioon and delayed androgen-independent recurrence ofprostate cancer with the use of antisense Bcl-2 oligonu-cleotides. J Natl Cancer Inst 92:34–41.

Miyamoto K, Tatsumi S, Morimoto A, Minami H, Yama-moto H, Sone K, Taketani Y, Nakabou Y, Oka T, TakedaE. 1994. Characterization of the rabbit intestinal fructosetransporter (GLUT5). Biochem J 303:877–883.

Noguchi Y, Saito A,Miyagi Y, Yamanaka S,Marat D, Doi C,Yoshikawa T, Tsuburaya A, Ito T, Satoh S. 2000.

Treatment of Breast Cancer by AS Against Glut5 1141

Suppression of facilitative glucose transporter 1 mRNAcan suppress tumor growth. Cancer Lett 154:175–182.

White MK, Rall TB,Weber MJ. 1991. Differential regulationof glucose transporter isoforms by the src oncogene inchicken embryo fibroblasts. Mol Cell Biol 11:4448–4454.

Wood IS, Trayhurn P. 2003. Glucose transporters (GLUTand SGLT): Expanded families of sugar transportproteins. Br J Nutr 89:3–9.

YamamotoT,SeinoY,FukumotoH,KohG,YanoH,InagakiN,Yamada Y, Inoue K, Manabe T, Imura H. 1990. Over-ex-

pression of facilitative glucose transporter genes in humancancer. BiochemBiophys ResCommun170:223–230.

YounesM, BrownRW,ModyDR, Fernandez L, Laucirica R.1995. GLUT1 expression in human breast carcinoma:Correlation with known prognostic markers. AnticancerRes 15:2895–2898.

Zamora-Leon SP, Golde DW, Concha II, Rivas CI, Delgado-Lopez F, Baselga J, Nualart F, Vera JC. 1996. Expressionof the fructose transporter GLUT5 in human breastcancer. Proc Natl Acad Sci 93:1847–1852.

1142 Chan et al.