A Small-Molecule Inhibitor of Glucose Transporter 1 ......Therapeutic Discovery A Small-Molecule Inhibitor of Glucose Transporter 1 Downregulates Glycolysis, Induces Cell-Cycle Arrest,

Therapeutic Discovery

A Small-Molecule Inhibitor of Glucose Transporter 1Downregulates Glycolysis, Induces Cell-Cycle Arrest,and Inhibits Cancer Cell Growth In Vitro and In Vivo

Yi Liu1,5, Yanyan Cao1,5, Weihe Zhang2, Stephen Bergmeier2, Yanrong Qian2,5, Huzoor Akbar3,Robert Colvin1,5, Juan Ding1,5, Lingying Tong2,5, Shiyong Wu2,5, Jennifer Hines2,5, and Xiaozhuo Chen1,2,3,4,5

AbstractThe functional and therapeutic importance of the Warburg effect is increasingly recognized, and glycolysis

has become a target of anticancer strategies. We recently reported the identification of a group of novel small

compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation–like

mechanism.We hypothesized that the compounds target Glut1 and are efficacious in vivo as anticancer agents.

Here, we report that a novel representative compound WZB117 not only inhibited cell growth in cancer cell

lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 at

10 mg/kg resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin.

Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which

express Glut1 as their sole glucose transporter. Cancer cell treatment withWZB117 led to decreases in levels of

Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATP-

sensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated

retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued

compound-treated cancer cells, suggesting that the reductionof intracellularATPplays an important role in the

anticancer mechanism of the molecule. Senescence induction and the essential role of ATP were reported for

the first time in Glut1 inhibitor–treated cancer cells. Thus, WZB117 is a prototype for further development of

anticancer therapeutics targeting Glut1-mediated glucose transport and glucose metabolism.Mol Cancer Ther;

11(8); 1672–82. �2012 AACR.

IntroductionThe Warburg effect (1–3), or upregulated glycolysis,

recently has been intensively studied and recognized asone of the critical missing pieces of the puzzle for under-standing cancer and formulating more effective antican-cer strategies (4–6). Almost all cancers analyzed upregu-lated glucose transport and aerobic glycolysis regardlessof their oxygen status (7, 8). Furthermore, cancer cellswere found to be addicted to glucose and very sensitive toglucose concentration changes (7, 9). Glucose deprivationis sufficient to induce growth inhibition and cell death incancer cells (10–12). The increased glucose transport incancer cells has been attributed primarily to the upregula-

tion of glucose transporter 1 (Glut1), 1 of themore than 10glucose transporters that are responsible for basal glucosetransport in almost all cell types (13, 14). Glut1 has notbeen targeted until very recently due to the lack of potentand selective inhibitors. First, Glut1 antibodies wereshown to inhibit cancer cell growth (15). Other Glut1inhibitors and glucose transport inhibitors, such as fas-entin (16) and phloretin (17), were also shown to beeffective in reducing cancer cell growth. A group ofinhibitors of glucose transporters has been recently iden-tifiedwith IC50 values lower than 20mmol/L for inhibitingcancer cell growth (18). However, no animal or detailedmechanism studies have been reported with theseinhibitors.

Recently, a small molecule named STF-31 was identi-fied that selectively targets the vonHippel-Lindau (VHL)-deficient kidney cancer cells (19). STF-31 inhibits VHL-deficient cancer cells by inhibiting Glut1. It was furthershown that daily intraperitoneal injection of a solubleanalogue of STF-31 effectively reduced the growth oftumors of VHL-deficient cancer cells grafted on nudemice (19). On the other hand, STF-31 appears to be aninhibitor with a narrow cell target spectrum.

We recently reported the identification of a novel groupof small compounds that inhibit glucose transport and cell

Authors' Affiliations: Departments of 1Biological Science, 2Chemistry andBiochemistry, and 3Biomedical Science, 4Edison Biotechnology Institute,and 5Molecular and Cellular Biology Graduate Program, Ohio University,Athens, Ohio

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Xiaozhuo Chen, Edison Biotechnology Institute,Ohio University, the Ridges, Athens, OH 45701. Phone: 740-593-9699;Fax: 740-593-4795; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0131

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(8) August 20121672

on March 27, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst June 11, 2012; DOI: 10.1158/1535-7163.MCT-12-0131

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growth in several cancer cell lines and these 2 inhibitoryactivities were correlated (20). We further showed thatthese compounds inhibit cancer cell growth using a glu-cose deprivation–like mechanism (20). The compounds’inhibitory activities were greater than most of the otherGlut1-inhibitory agents reported. On the basis of theseactivities and the universal presence of Glut1 in cancercells, we hypothesized that these novel compoundsdownregulate glucose transport and glycolysis by inhi-bitingGlut1 and that they should be effective in inhibitingcancer growth in vivo. To test these hypotheses,WZB117, astructural and functional analogue derived from the com-pounds used in our previous studies (21) with higherpotencies, was used to treat multiple cancer cell lines andtumor-bearing nude mice to determine its in vivo antican-cer efficacy and identify its inhibition target and antican-cer mechanism.

Materials and MethodsCompound inhibitors and other chemicalsCompound WZB117 was synthesized as previously

reported (20, 21). Compound solutions were freshly pre-pared bydissolving the compounds in dimethyl sulfoxide(DMSO) before each experiment. Chemicals oligomycin,cisplatin, paclitaxel, and ATP were from Sigma-Aldrich.

Cell line, cell culture, and experimental controlsHuman non–small cell lung cancer (NSCLC) cell lines

H1299 and A549, human breast ductal carcinoma MCF7,as well as human nontumorigenic NL20 lung andMCF12A breast cells were purchased from AmericanType Culture Collection and were not authenticated.All these cells were maintained in American TypeCulture Collection recommended cell culture media andconditions.Cells were treated with compound WZB117 for 24 or

48 hours. WZB117 (10 mmol/L) was used in the experi-ments unless otherwise noted. Mock-treated and glu-cose deprivation samples served as negative andpositive controls, respectively. In glucose deprivation,Dulbecco’s Modified Eagle’s Media (DMEM) withreduced glucose concentration (2 mmol/L or 8% ofglucose concentration in the regular cell culture medi-um) was prepared by mixing glucose-free DMEM withregular DMEM.

Glucose uptake assay in cancer cells and in humanred blood cellsThe inhibitory activity of compounds on glucose trans-

port was analyzed by measuring the cell uptake of 2-deoxy-D-[3H] glucose as previously described (20, 21).Similar procedurewas used for glucose uptake assay in

human red blood cells (RBC), except that RBCs werewashed and collected by centrifugation at 2,000 � g for5 minutes as they are suspension cells, and the treatedRBCs were solubilized in 0.1% SDS before radioactivitywas measured.

Cell proliferation (MTT) and clonogenic assaysCell proliferation and viability rates were measured

using the MTT Proliferation Assay Kit (Cayman) or clo-nogenic assays (21).

Hypoxia studiesCancer cell study in hypoxia was conducted using the

Anaerobe Gas Generating Pouch System with indicator(BD GasPak EZ). The pouch formed an oxygen-free envi-ronment in which the compound-treated cells were incu-bated for 24 hours. After the hypoxic incubation, thetreated cells were measured for their viability by theMTTassay.

Animal studyMale NU/J nude mice of 6 to 8 weeks of age were

purchased from The Jackson Laboratory and were fedwith the Irradiated Teklad Global 19% protein rodent dietfrom Harlan Laboratories. To determine the in vivo anti-cancer efficacy of compound WZB117 on human tumorxenograft growth, NSCLC A549 cells in exponentialgrowth phase were harvested, washed, precipitated, andresuspended in PBS. Each mouse was injected subcuta-neously with 5 � 106 cancer cells in the flank. Compoundtreatment started 3 days after the cancer cells injection andwhen all tumors became palpable. Tumor cell–injectedmice were randomly divided into 2 groups: control group(n ¼ 10) treated with PBS/DMSO (1:1, v/v) and WZB117treatment group (n¼ 10) treatedwithWZB117 (10mg/kgbodyweight) dissolved in PBS/DMSO solution (1:1, v/v).Mice were given intraperitoneal injection with eitherPBS/DMSO vehicle or compound WZB117 (10 mg/kg)daily for 10 weeks. Tumor sizes were measured every7 days with calipers, and tumor volume (L � W2/2) wascalculated and presented as means � SEM. All of theprocedures involved in animal study were conducted inconformationwith the guidelines of both Ohio University(Athens, OH) and NIH.

Protein target studies I: RBC membrane vesiclepreparation and glucose uptake assay

RBC and RBC-derived vesicles were prepared usingpublished protocols (22) with minor modifications. Theglucose uptake assay using sealed vesicles was similarto that in RBCs, except that the centrifugation was at18,000 � g for 20 minutes to precipitate the vesicles aftereach washing step.

Protein target studies II: docking studiesA molecular model of WZB117 was constructed using

Spartan 10 (Wavefunction Inc.; ref. 23). Following molec-ular mechanics energy minimization with the Merckmolecular force field, the compound structures wereexported toMacromodel (Schr€odinger) and docked to theGlut1 homology modeled PDB structure 1SUK (24) Pro-tein and grid preparations were conducted using theGlide module of FirstDiscovery 2.7 (Schr€odinger) withdefault protocols (25) and centered in the middle of the

A Glut1 Inhibitor Reduces Cancer Growth In Vitro and In Vivo

www.aacrjournals.org Mol Cancer Ther; 11(8) August 2012 1673




transport channel with the bounding box encompassingthe entire channel.WZB117was then docked using Glide,and the best docked structure for the compound wasselected on the basis of the Glide-calculated Emodel value.

Western blot analyses and RNA isolationand real-time PCR

Western blot analyses were conducted using the stan-dard protocol. Antibodies for Glut1 (H-43), eIF2a, andcyclophosphamide–Adriamycin–vincristine–prednisone(CHOP) were from Santa Cruz; PGAM1 antibody wasfrom Novus Biologicals. Antibody for p-eIF2a was fromInvitrogen. All other antibodies were fromCell Signaling.

RNAfrom treatedA549 cellswas isolatedusingRNeasytotal RNA extraction kit (Qiagen), and cDNA was syn-thesized with the Bio-Rad iScript Select cDNA SynthesisKit (Bio-Rad). The produced cDNA was used to specifi-cally quantify the transcript of SLC2A1 (Glut1) using theBio-Rad iCyclerwith theBio-Rad iQSyBrGreen SupermixKit. The RT2-PCR primer sets for human SLC2A1 andb-actinwere from SuperArray. For quantifying transcriptlevels, dCt method was used. b-actin mRNA was used asan internal control for normalizing Glut1 mRNA.

Lactate and ATP measurements and ATP rescuestudy

Extracellular lactate concentrationwasmeasured usingthe Lactate Assay Kit II (BioVision).

Intracellular ATP concentration was measured usingATPlite luminescence ATP detection assay system fromPerkin-Elmer. Briefly, cells were seeded at a density of50,000 cells in eachwell of a 96-well plate. ATP levelsweremeasured after 6, 12, and 24 hours of treatment. Proteinconcentration of cells in each well was determinedfor both lactate and ATP measurements for signalnormalization.

In the cell rescue study, ATP of various concentrationswere added in cell culture medium of cancer cells in 96-well plates with or without 30 mmol/L WZB117. Intracel-lular ATP levels and cell viability were measured by anMTT assay 24 hours after the treatment.

Cell-cycle analysis and detection of apoptoticand necrotic cells

Cell cycle was analyzed as previously described (20).For identification of apoptotic and necrotic cells among

WZB117-treated A549 cells, the treated cells were stainedwith propidium iodide andAnnexin V-FITC according tothe manufacturer’s instructions (BD Pharmingen) andthen subjected to flow cytometric analysis.

Senescence studySenescence was examined by a senescence associate

b-galactosidase (b-gal) assay kit (Cell Signaling) undermicroscope for both b-gal expression and enlarged cellmorphology compared with untreated cells. Cells werephotographed using a microscope (ECLIPSE E600;Nikon).

Statistical analysisSamples were in triplicate or hexad in cell studies. Each

experiment was repeated at least twice with the exceptionof the animal study. Data are reported asmean� SD.Datawere analyzed using one-way ANOVA. P � 0.05 wasconsidered statistically significant.

Results and DiscussionWe previously showed that the human dietary com-

pound a-PGG mimics insulin action by binding andactivating the insulin receptor (IR), resulting in glucoseuptake in target cells (26). We recently reported thata-PGG inhibits cancer cells through IR-mediated apopto-sis in human colon cancer RKO cells (27). In the process ofstructural and function optimization for the anti-diabetesactivity of PGG-derived compounds, numerous glucosetransport inhibitory compounds were synthesized andtested in cancer cells (21, 28). These compounds inhibitedcancer cell growth in a glucose deprivation–like manner(20). This study was done to determine the anticancerefficacy in vivo and identify the anticancer mechanism ofthe inhibitors using the compound WZB117.

Glucose uptake assays showed that WZB117 (Fig. 1A)inhibits glucose transport in cancer cells in a dose-depen-dent manner (Fig. 1B). It also revealed that the inhibitionof glucose transport induced byWZB117 occurred within1minute after the assay started (Fig. 1C, second timepointpartially overlapped with the time point at 0), suggestingthat the inhibitory activity is likely to be via a direct andfast mechanism. Cell viability assay showed thatWZB117inhibited cancer cell proliferation with an IC50 of approx-imately 10 mmol/L (Supplementary Fig. S1A). The inhib-itory activity of WZB117 on cancer cell growth was alsoconfirmed with a clonogenic assay (Fig. 1D), which alsoindicates that the inhibition is irreversible in nature.WZB117 treatment resulted in significantly more cellgrowth inhibition in lung cancer A549 cells than in non-tumorigenic lungNL20 cells (Fig. 1E). Similar resultswerealso observed in breast cancer MCF7 cells and their non-tumorigenic MCF12A cells (Supplementary Fig. S1B).When WZB117 was added to cancer cells grown underhypoxic conditions, more cell growth inhibition wasobserved than under normoxic conditions (Fig. 1F). Theseresults suggested that cancer cells are very sensitive andvulnerable to biologic changes under hypoxic conditions,which further sensitized cancer cells to the glucose trans-port inhibitor WZB117. Synergistic anticancer effectsbetween WZB117 and anticancer drug cisplatin or pacli-taxel were also observed (Supplementary Fig. S1C).

After showing the anticancer activity of WZB117 incultured cancer cells, we went on to address the questionwhether WZB117 inhibits cancer growth in animal tumormodels. The animal study showed that after daily intra-peritoneal injection ofWZB117 at 10 mg/kg bodyweight,the sizes of the compound-treated tumors were onaverage more than 70% smaller than those of themock (PBS/DMSO)-treated tumors (Fig. 2A and B).

Liu et al.

Mol Cancer Ther; 11(8) August 2012 Molecular Cancer Therapeutics1674




Notably, 2 of the 10 compound-treated tumors disap-peared during the treatment and never grew back evenat the end of the study (Fig. 2B). Body weight measure-ment and analysis revealed that the mice treated withWZB117 lost about 1 to 2 grams of bodyweight comparedwith the mock-treated mice (Supplementary Fig. S2A)with most of the weight loss in the fat tissue (Supplemen-taryTable S1). Blood counts andanalysis ofmice at the endof the study showed that lymphocytes and platelets werechanged in the compound-treated mice compared withthe vehicle-treated mice, but the cell counts remained inthe normal ranges (Supplementary Table S2). One of theconcerns for using glucose transport inhibitors was thatthe inhibitor might produce hyperglycemia in the treatedmice. It was found that a single injection of WZB117produced only mild and temporary hyperglycemia thatdisappeared 1 to 2 hours after the compound injectionwithout generating persistent hyperglycemia (Supple-mentary Fig. S2B and unpublished observations). The

relatively high anticancer efficacy and relatively low tox-icity of WZB117 observed in animals may be partiallyexplained by cancer cells’ higher sensitivity and vulner-ability to glucose concentration changes induced byWZB117 than normal cells (Fig. 1E) and by cancer cells’sensitivity to glucose transport inhibition under hypoxiaconditions (Fig. 1F), in which a majority of cancer cellswere growing in animals.

We previously found that our small-molecule inhibi-tors of glucose transport inhibited glucose transport inall the cancer cell lines tested internally. With additionaldata (Fig. 1C), we speculated that the target of theseinhibitors is Glut1, as Glut1 is responsible for basalglucose transport in almost all cell types (13), and Glut1was upregulated in many cancer cells tested (8, 20) Totest this hypothesis, RBCs were chosen as a cell modelfor determining whether Glu1 is the target of compoundWZB117 because RBCs express Glut1 as their sole glu-cose transporter (29) and are an established model for

Figure 1. Small-molecule WZB117and its inhibitory actions on glucoseuptake and cancer cell growth.Glucose transport and cellproliferation of WZB117-treatedcancer cells was measured byglucose uptake andMTT cell viabilityassays, respectively. A, structure ofWZB117. WZB117 is a structuralanalogue ofWZB115 (21)with amorepotent anticancer activity and amolecular weight of 368.31 Da. B,WZB117 inhibits glucose transport inA549 cancer cells in a dose-dependent manner. C, WZB117rapidly and completely inhibitsglucose transport in cancer cells.WZB117 (30 mmol/L) was used totreat A549 cells. Glucose uptake inthe treated cells was measured at 0,1, 5, 30, 60, and 120minutes after theaddition of 2-deoxy-D-[3H] glucose.D, WZB117 treatment led toirreversible cell growth inhibition in 3cancer cell lines as determined byclonogenic assays. E, WZB117inhibits cell proliferation in the humanlung cancer cell line A549significantly more than it does inNL20 nontumorigenic lung cells 48hours after treatment. ��, P� 0.001.F, WZB117 treatment under hypoxiccondition further reduced cancercells' proliferation rate. A549 cellswere treated with or without 10 mMWZB117 and were then immediatelytransferred and maintained in ahypoxic pouch. The viability of thetreated cells was measured 24 hoursafter treatment. A549 cells in normalor low-glucose cell culture mediatreated under normoxia or hypoxiaconditions served as controls.��, P � 0.01.

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and have been frequently used in studying glucosetransport (30, 31). The glucose uptake assays revealedthat WZB117 indeed inhibited the glucose transport inRBCs (Fig. 3A). The WZB117 treatment did not induceother changes to RBCs such as hemolysis (unpublishedobservation). To eliminate other possibilities, the glu-cose uptake assays were repeated in RBC-derived vesi-cles, in which all the intracellular proteins and enzymeswere removed and only membrane-bound and mem-brane-associated proteins remained (32). The right side-out vesicles (ROV) exhibit the same membrane orien-tation as RBC whereas inside-out vesicles (IOV) showopposite membrane orientation as the membrane ofRBCs. However, as Glut1 is a glucose uniporter andcan transport glucose in both directions across the cellmembrane, Glut1 located on either IOV or ROV shouldbe able to transport glucose down the glucose gradient.As expected, WZB117 indeed inhibited glucose trans-

port in both IOV and ROV (Fig. 3B and C), stronglysupporting the hypothesis that Glut1 is the target ofWZB117.

To find additional evidence for direct WZB117-Glut1interactions, ligand docking studies were conductedusing the Glide module of FirstDiscovery 2.7 (Schr€odin-ger). Glide conducts flexible ligand docking to a rigidreceptor using a grid-based docking method and scoringfunction (23). The docking study revealed that the bindingofWZB117 to Glut1 involved 3 hydrogen bonds, one eachwith Asn34, Arg126, and Trp412 (Fig. 3D). These aminoacid residues are located in the central channel region ofGlut1.

After showing that Glut1 was very likely to be thetarget of WZB117, we went on to determine the sequenceof molecular events of WZB117 treatment on glycoly-sis. Real-time quantitative reverse transcription PCR(RT2-PCR) and Western blot analysis of Glut1 revealedthat similar to the glucose deprivation control, the level ofGlut1mRNAwas upregulated 24 hours after the treatment(Fig. 4A),whereasGlut1 protein levelwas decreased by theWZB117 treatment as early as 12 hours (Fig. 4B). Theseapparently inconsistent results could be explained thus:Inhibition of glucose transport by WZB117 decreased glu-cose supply to cancer cells, resulting in an urgent require-ment for increasing glucose import and the upregulationof Glut1 mRNA level. However, because of the limitedsupply of glucose required for the processing of glycosy-lated membrane-bound proteins including Glut1, Glut1protein levels were not increased. The possibility of theinvolvement of other mechanisms such as AMP-activatedprotein kinase (AMPK)/mTOR signaling–mediated arrestof protein synthesis cannot be ruled out.

The addition ofWZB117 to cancer cells led to reductionof extracellular lactate levels (Fig. 4C) and intracellularATP (Fig. 4D) as early as 6 to 12 hours after the WZB117treatment with a further decline at 24 hours. Importantly,addition of extracellular ATP to the cell culture media atthe time of compound addition significantly increasedintracellular ATP levels (Supplementary Fig. S3A) andrescued the compound-treated cancer cells 24 hours afterthe treatment (Fig. 4E). If the addition of extracellular ATPwas delayed for 12 hours or longer, the ATP started losingits rescue ability (Supplementary Fig. S3B). These resultssuggest that the reduced ATP level is largely responsiblefor the cancer cell inhibitory activity of the compound, atleast for the first 24 hours of the compound treatment. ThesameATPadditionwas ineffective in rescuing cancer cellstreated by paclitaxel (Fig. 4E), a drug that inhibits cancercells using a mechanism not directly involving ATP.Although ATP has been known to cross cell plasmamembrane (33, 34), the mechanism by which ATP enterscells is not presently known. This is the first time thatextracellular ATP is shown to be important in rescuingcancer cells with deprived glucose transport and glucosemetabolism. Extracellular ATP may contribute to onco-genesis and cancer metabolism significantly more thanpreviously thought.

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Figure2. SmallmoleculeWZB117 inhibits cancer growth in tumor-bearingnudemice. A, daily intraperitoneal injection ofWZB117 at 10mg/kg bodyweight for 10weeks resulted inmore than 70% reduction in tumor volumeof human A549 lung cancer grafted on nude mice. PBS/DMSO (1:1, v/v)were injected in themock-treated controlmice.N¼10 for each treatmentgroup. �, P < 0.05. B, photographs of untreated or WZB117-treatedtumor-bearing nudemice with representative tumors. Photographs weretaken 8 weeks after the compound treatment. The middle imagesrepresent tumors close to the average tumor sizes of the groups. Thetumor on the mouse of the WZB117-treated group (bottom right)disappeared during the study.

Liu et al.





Autophagy occurred as early as 6 hours after the com-pound treatment (Supplementary Fig. S3C), suggestingthat autophagy and extracellular ATPmight work togeth-er to rescue compound-treated cancer cells by providingneeded biomaterial and energy, respectively.Oligomycin, a specific mitochondrial inhibitor, did

not reduce cell proliferation rate at a concentration of50 nmol/L (data not shown). However, when WZB117was used to treat A549 cells together with 50 nmol/Loligomycin, the presence of oligomycin further reducedthe proliferation rate of A549 cells compared with thesamples treated with WZB117 alone (Fig. 4F). This resultindicates that at 50 nmol/L, an effective mitochondria-inhibitory dose (35), oligomycin alone did not change cellproliferation rate but sensitized cancer cells to the inhib-itory activity ofWZB117 (Fig. 4F).Mitochondria inhibitionis known to upregulate glycolysis (35). These data suggestthat when a Glut1/glycolysis inhibitor, WZB117 in thiscase, was added to oligomycin-treated cells, these cellswere unable to compensate for oligomycin-induced inhi-bition of mitochondria with upregulating glycolysis. The

double inhibitions further reduced the proliferation rateof the treated cancer cells. This result also shows thatWZB117 ismore effective in inhibiting cell proliferation incells that have some degree of mitochondrial dysfunctionand dependence on glycolysis such as cancer cells. Thisfinding can also partially explain whyWZB117 was moreeffective in inhibiting cancer cells than their noncancerouscell counterparts (Fig. 1E and Supplementary Fig. S1B).

Other key glycolytic enzymes, first rate-limiting hexo-kinase II (36), and cancer cell–specific PKM2 (37), werereduced at 6 and/or 12 hours but upregulated at 24 hours,whereas PGAM1 (38), an enzyme catalyzes an alternativestep for pyruvate synthesis, was not affected by WZB117treatment (Fig. 4G).

After identifying and characterizing some of the bio-logic and biochemical changes in cancer cells related toglucose transport and glucose metabolism, other cellgrowth, survival, and cell death processeswere examinedin WZB117-treated cancer cells to identify molecular par-ticipants and consequences of the treatment.Western blotanalyses showed that key cell growth signaling proteins

Figure 3. Protein target studies:WZB117 inhibits glucose transport inhuman RBCs and RBC-derivedvesicles by binding and inhibitingGlut1 as shown by docking studies.Glut1 antibody- and cytochalasin B–treated RBCs were used as controlsin glucose uptake assays of RBCs(A), IOV (B), and ROV (C).Experiments were repeated 3 times.�, P < 0.05; ��, P < 0.01; and��, P < 0.001. D, docked structureand interactions of WZB117 bindingto Glut1. The image on top showsthat WZB11 binds to the centralchannel region of Glut1. The imageon the bottom shows the detailedinteractions (formation of 3 hydrogenbonds) between WZB117 and aminoacid residues of Glut1.

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such asAkt (39),mTOR (40), andAMPK(41)were affectedby the compound treatment inways similar to the changesfound in glucose deprivation controls (Fig. 5A). Phos-phorylated Akt and mTOR were found to decrease 6 and12 hours after the compound treatment. Notably, the

upregulation of phosphorylation of the energy (ATP)sensor AMPK coincided with the start of the decline inATP levels (Fig. 4D) and with the time when extracellularATP started to lose its rescue ability (Supplementary Fig.S3B). All these changes suggest that the treated cancer

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Figure 4. Glycolysis studies: WZB117 treatment resulted in changes in levels of glycolytic proteins and metabolites and addition of ATP rescuedWZB117-treated A549 cells. A549 cells were treated with WZB117 for various times and then mRNA, proteins, and metabolites of the cells were measured.Glucose deprivation samples served as controls. �,P� 0.05; ��,P� 0.01; ��,P� 0.001.Western blot analyseswere conducted 3 times. Intensities of proteinbands were first normalized with their respective b-actin controls and then further normalized by arbitrarily setting the relative intensity of the mock-treated sample of that time point as 1 in histograms. A, RT-PCR analysis of Glut1 mRNA level. B, Glut1 protein levels of WZB117-treated A549 cells analyzedby Western blotting. C, extracellular lactate levels secreted by A549 cells treated with or without WZB117. Lactate concentration of mock-treated sampleswas assigned as 100%. D, intracellular ATP levels of cancer cells treated with or without WZB117. Mock-treated and glucose deprivation samplesserved as negative andpositive controls, respectively, and theATPconcentration ofmock-treated sampleswas assigned as 100%.E, addition of extracellularATP rescuedWZB117-treated but not paclitaxel-treated A549 cells. Cells were treatedwith various concentrations of ATP in the presence of either 30 mmol/LWZB117 or 1 mmol/L paclitaxel for 24 hours, and the cell viability was measured. F, synergistic anticancer effect between WZB117 and a mitochondriainhibitor oligomycin. A549 cells were treated with 1 mmol/LWZB117 in the absence or presence of 50 nmol/L oligomycin. G, glycolytic enzyme changes overtime in WZB117-treated A549 cells. This experiment was repeated 3 times.

Liu et al.





cells responded to changes in glycolysis and energy statusby downregulating phosphorylation levels of enzymesinvolved in cell growth signaling pathway and energyhomeostasis. AMPK is very likely to act as the key linkbetween theATP reduction and the subsequent cancer cellinhibition.An endoplasmic reticulum stress study showed that the

protein level of GRP78/BiP, an endoplasmic reticulumstressmarker (42, 43), steadily increased from6 to48hoursafter WZB117 treatment. The onset of endoplasmic retic-ulum stress, as indicated by the start of the GRP78/Bipincrease at 6 to 12 hours, coincidedwith the initial indirectsignof glycosylationdysfunction in thedecline of the levelof glycosylated protein Glut1 (Fig. 4B). Increased eIF2aphosphorylation was accompanied by an elevation ofGRP78/BiP, but CHOP expression was not increased toa detectable level (Fig. 5B). These results indicate thatWZB117 induces endoplasmic reticulum stress, whichlikely leads to PKR-like endoplasmic reticulum kinase(PERK) activation and eIF2a phosphorylation (44). How-ever, the stress level is not severe enough to induce CHOPexpression and significant apoptosis as indicated by thevery low level of PARP cleavage (Fig. 5C) and very smallchanges in the numbers of apoptotic cells in the treatedcells (Fig. 6A, right). The role of endoplasmic reticulum

stress played in the inhibition of WZB117-treated cancercells is presently unclear.

WZB117 treatment led to approximately 30% and 50%reductions in cell proliferation rate 24 and 48 hours aftercompound treatment, respectively (Fig. 6A, left). Flowcytometric study,whichused 10,000 cells regardless of thetreatments, showed thatWZB117 treatment resulted in 8%increase in necrosis at 48 hours [Fig. 6A, top right quad-rant of (iv)]with only about 2% apoptosis [Fig. 6A, bottomright quadrant of (iv)]. Flow cytometric analysis revealedthat WZB117 treatment led to cell-cycle arrest. WZB117treatment resulted in approximately 23% and 4% morecells inG0–G1 andG2–Mphases, respectively and approx-imately 30% less S-phase cells (Fig. 6B). This number, 30%,also matched with the MTT assay result at 24 hours [Fig.6A, (ii) of left], indicating that at 24 hours, almost all thereduction in cancer cell proliferation was due to the cell-cycle arrest.

G1 arrest is known to be regulated by phosphorylatedretinoblastoma (pRb), whose activity is regulated by itsphosphorylation. The phosphorylation of Rb is regulatedby cyclin-dependent kinase (CDK)2/cyclin E2 complex(45, 46). As expected, levels of CDK2 and cyclin E2 as wellas pRb were decreased at the same time point (Fig. 6C).These changes were likely to be responsible for, at least in

Figure 5. WZB117 treatment led tochanges in protein factors involved incell growth/survival signaling,endoplasmic reticulum stress, andapoptosis in A549 cells. Glucosedeprivation samples served ascontrols for WZB117 treatment ofA549 cells in these Western blotanalyses. Western blot analyseswere conducted 3 times. Intensitiesof protein bands were firstnormalized with their respectiveb-actin controls and then furthernormalized by arbitrarily setting therelative intensity of the mock-treatedsample of that time point as 1 inhistograms. �, P � 0.05. WZB117treatment resulted in changes in A.Cell growth signaling proteins Akt,AMPK, and mTOR, endoplasmicreticulum stress markers (B), andminimal cleavage (C) in apoptosismarker PARP.

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part, the cell-cycle arrest. p16 is known to be involved inpRb and cell-cycle regulations. However, p16 gene ishomozygously deleted in A549 cells and therefore playsno role in pRb and cell-cycle regulation of A549 cells (47).Additional but presently unknown mechanism may beinvolved in the regulation of pRb and cell-cycle arrest inA549 cells treated with WZB117.

Cell staining and observation revealed enlarged cellmorphology and significant expression of b-gal, a widelyused marker of cancer cell senescence (48), in A549 cells24 hours afterWZB117 treatment (Fig. 6D). Combining theb-gal expression and enlarged cell morphology withirreversible cell inhibition (Fig. 1D) and changes in phos-phorylated Rb, common and important features of senes-

cence (49, 50), itwas concluded thatWZB117-treatedA549cells became senescent concomitant to or following cell-cycle arrest. This is the first time that senescence wasreported in cancer cells treated by a Glut1 inhibitor. Onepossible explanation for WZB117-treated cancer cellsundergoing senescence and necrosis, rather than apopto-sis, is that apoptosis is an ATP-utilizing process whereasnecrosis and senescence are not. The compound treatmentmight deplete intracellular ATP so much that the cancercellswereunable to carry out apoptosis, forcing the cells toundergo senescence and necrosis (51).

Taken together, data reported here show that after theexposure to WZB117, cancer cells experienced an imme-diate reduction in glucose transport. Consequently, some

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Figure 6. WZB117 treatment led tocell-cycle arrest, changes in cell-cycle protein, necrosis, andsenescence in A549 cells.WZB117was used to treat A549 cells andglucose deprivation–treatedsamples served as controls.�, P � 0.05; ��, P � 0.01; and��, P � 0.001. A, determination ofrelative proliferation rates and cellcomposition of A549 cells treatedby WZB117 over time. Mock- and/or glucose deprivation–treatedcells served as controls. Left,relative proliferation ratesmeasured by theMTT assay. Right,flow cytometric analysis of cellcomposition of A549 cells treatedwith WZB117 for 24 or 48 hours.Top right quadrant of each graphindicates percentage of necroticcells. Points (i–iv) in the leftcorrespond to the same labeledflow cytometric graphs, whichwere analyzed at the same times.FITC, fluorescein isothiocyanate.B, WZB117 treatment led to cell-cycle arrest as determined by flowcytometric analysis. C, levelchanges of cell-cycle regulatoryproteins. Western blot analyseswere conducted 3 times.Intensities of protein bands werefirst normalized with theirrespective b-tubulin controls andthen further normalized byarbitrarily setting the relativeintensity of the mock-treatedsample of that time point as 1 inhistograms. D, b-Gal staining ofA549 cells treatedwithWZB117 for24 hours. Mock-treated andstained cells served as controls.Magnification, �100.

Liu et al.





key glycolytic enzymes andmetabolites (ATP and lactate)were decreased in the first fewhours. These led to changesin key enzymes, particularly AMPK in ATP sensing andenergy homeostasis. All these changes culminated in cell-cycle arrest, accompanied by senescence and upregula-tion of some glycolytic enzymes, which is likely to be aresponse to senescence (52). Prolonged inhibition of glu-cose transport and reduction of glycolysis induced necro-sis (19, 53), further inhibiting cancer cell growth.Cell-cycleG1 arrest, mediated by downregulation of cyclin E2 andphosphorylation of Rb, and subsequent senescence andnecrosis were the major mechanisms underlying theinhibitory action of WZB117 on cancer cell growth.Reduced ATP levels appear to play an essential role in

the WZB117-induced cancer cell inhibition in the first 24hours of the compound treatment. Figure 7 graphicallydepicts the hypothetical mechanism and time sequence ofmolecular and cellular events described above. Both ATPreduction and senescencewere described for the first timeas potential anticancer mechanisms of Glut1 inhibitors.

All these data indicate that WZB117, a novel Glut1inhibitor, is effective both in vitro and in vivo in inhibitingcancer cell growth and can serve as a prototypicalcompound for the further development of Glut1 andglucose transport inhibitors as a new group of anticancertherapeutics.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Liu, Y. Cao, W. Zhang, S. Bergmeier, J. Hines,X. ChenDevelopment of methodology: Y. Liu, W. Zhang, S. Bergmeier, R. Colvin,J. Hines, X. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):Y. Liu, S. Bergmeier, Y.Qian,H.Akbar, R. Colvin,J. Ding, L. Tong, S. Wu, J. HinesAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis):Y. Liu, Y. Cao,W. Zhang, Y.Qian, R. Colvin,J. Ding, L. Tong, S. Wu, J. Hines, X. ChenWriting, review, and/or revision of the manuscript: Y. Liu, Y. Cao,Y. Qian, H. Akbar, R. Colvin, J. Hines, X. ChenAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases: Y. Liu, S. Bergmeier, X. ChenStudy supervision: X. ChenDesigned and synthesized all the compounds: W. Zhang

AcknowledgmentsThe authors thank Dr. Yan Liu for critical review of the manuscript.

Grant SupportThis work was partially supported by an NSF PFI Grant IIP-0227907 to

the Edison Biotechnology Institute of Ohio University and an Ohio Uni-versity medical school RSAC award to X. Chen.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 13, 2012; revised May 24, 2012; accepted May 28,2012; published OnlineFirst June 11, 2012.

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2012;11:1672-1682. Published OnlineFirst June 11, 2012.Mol Cancer Ther Yi Liu, Yanyan Cao, Weihe Zhang, et al.

In Vivo and In VitroGrowth Glycolysis, Induces Cell-Cycle Arrest, and Inhibits Cancer Cell A Small-Molecule Inhibitor of Glucose Transporter 1 Downregulates

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