TIGAR Has a Dual Role in Cancer Cell Survival …...NADPH, lower reactive oxygen species (ROS) and...

Therapeutics, Targets, and Chemical Biology

TIGAR Has a Dual Role in Cancer Cell Survival throughRegulating Apoptosis and Autophagy

Jia-Ming Xie1, Bin Li2, Hong-Pei Yu1, Quan-Geng Gao2, Wei Li1, Hao-Rong Wu1, and Zheng-Hong Qin3

AbstractThe p53-induced glycolysis and apoptosis regulator (TIGAR) inhibits glycolysis, resulting in higher intracellular

NADPH, lower reactive oxygen species (ROS) and autophagy activity. In this study, we investigated whetherTIGAR might exert dual impacts on cancer cell survival based on its ability to inhibit both apoptosis andautophagy. In liver or lung cancer cells treated with the anticancer drug epirubicin, TIGAR levels increased in adose- and time-dependent manner. TIGAR silencing enhanced epirubicin-induced elevations in ROS levels andapoptosis rates, in a manner that was blocked by ectopic addition of NADPH or N-acetyl cysteine. These findingswere correlatedwith reduced tumorigenicity and increased chemosensitivity inmouse xenograft tumor assays. Inparallel, TIGAR silencing also enhanced the epirubicin-induced activation of autophagy, in amanner thatwas alsoblocked by ectopic addition of NADPH. Notably, TIGAR silencing also licensed epirubicin-mediated inactivationof the mTOR pathway, suggesting TIGAR also exerted a negative impact on autophagy. However, genetic orpharmacologic inhibition of autophagy increased epirubicin-induced apoptosis in TIGAR-silenced cells. Overall,our results revealed that TIGAR inhibits both apoptosis and autophagy, resulting in a dual impact on tumor cellsurvival in response to tumor chemotherapy. Cancer Res; 74(18); 5127–38. �2014 AACR.

IntroductionTumor-suppressor gene TP53 plays an important role in

the regulation of cellular metabolism, specifically glycolysisand oxidative phosphorylation (OXPHOS) via transcriptionalregulation of its downstream genes TP53-induced glycolysisregulator (TIGAR) and synthesis of cytochrome c oxidase(SCO2; refs. 1 and 2). It has been known that cancer cellsutilize glycolysis, which yields less ATP and can occur inhypoxic tissues that cannot obtain sufficient ATP throughoxidation/phosphorylation. A theory has been proposed toexplain this phenomenon known as "the Warburg effect" (3).The role of TP53 in the regulation of energy metabolismthrough TIGAR and SCO2 provides new insights into the

puzzles of cancer cell metabolism and strategies for cancertherapy (4).

TIGAR functions to lower fructose-2,6-bisphosphate (Fru-2,6-P2) levels in cells, resulting in an inhibition of glycolysis (5).The TIGAR protein shows similarity to the bisphosphatasedomain of PFK-2/FBPase-2 (6-phos-phofructo-2-kinase/fruc-tose-2,6-bisphosphatase), an enzyme that has an essentialfunction in the regulation of glycolysis (5, 6). The concept ofWarburg effect depict that cancer cells preferentially utilize theglycolytic pathway to produce ATP even in the presence ofoxygen, thus the ability of TIGAR to inhibit cell glycolysis seemsto be harmful for cancer cell survival. However, a number ofrecent studies have reported that TIGAR expression wassignificantly elevated in human cancers such as glioblastoma(7), invasive breast cancers (8), and colorectal cancers (unpub-lished observations). The question that urgently needs to beaddressed is why cancer cells needmore TIGAR if their survivalis dependent on glycolysis?

TIGAR also functions to decrease intracellular reactiveoxygen species (ROS) levels through increasing NADPH gen-eration. ROS-adaptive response may play a critical role inprotecting cells against cytotoxic effects of anticancer agents(9, 10) and high intracellular concentrations of glutathione(GSH) have been implicated in resistance to several chemo-therapeutic agents (5). Bensaad and colleagues reported thatTIGAR protects cells from ROS-associated apoptosis (5). Thesemay be the reason why the cancer cells need high levels ofTIGAR for survival. On the other hand, TIGAR-mediated ROSreduction may limit autophagy activity. Considering autop-hagy can function to decrease ROS levels, inhibit apoptosis andsupport energy production in nutrient starvation or metabolicstress conditions (11), the impact of TIGAR-mediated

1Department of General Surgery, The Second Affiliated Hospital of Soo-chowUniversity, Suzhou, China. 2Department ofGeneral Surgery, The FirstHospital of Wu Jiang, Suzhou, China. 3Department of Pharmacology andLaboratory of Nervous Diseases, Jiangsu Key Laboratory of TranslationalResearch and Therapy for Neuro-Psycho-Diseases, Soochow UniversitySchool of Pharmaceutical Science, Suzhou, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

J.-M. Xie and B. Li contributed equally to this article.

Corresponding Authors: Hao-Rong Wu, Department of General Surgery,The Second Affiliated Hospital of Soochow University, 1055 San XiangRoad, Suzhou 215006, China. Phone: 86-512-67783308; Fax: 86-512-67783308; E-mail: [email protected]; and Zheng-Hong Qin,Department of Pharmacology and Laboratory of Aging and Nervous Dis-eases, SoochowUniversity School of Pharmaceutical Sciences, 199RenAiRoad, Suzhou 215123, China. Phone: 86-512-65882071; Fax: 86-512-65882071; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-3517

�2014 American Association for Cancer Research.

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Published OnlineFirst August 1, 2014; DOI: 10.1158/0008-5472.CAN-13-3517

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autophagy inhibition in tumor cell survival remains to beinvestigated. In addition, whether TIGAR plays a role inautophagy regulation independent of ROS also needs to bedefined. A more complete understanding the role of TIGAR inROS and autophagy regulation will pave a new avenue fordeveloping new chemotherapeutic drugs. This study providesthe evidence showing that TIGAR has dual roles in chemo-sensitivity of tumor cells through inhibition of apoptosis andautophagy.

Materials and MethodsCell culture

Human lung cancer cell line A549 cells and human hepa-tocellular carcinoma derived HepG2 cells were obtained fromthe American Type Culture Collection and cultured withDulbecco's Modified Eagle Medium (DMEM; GIBCO,11965500) containing 10% fetal bovine serum (FBS; WISTENInc., 086150008), 100 IU/mL penicillin, and 100 IU/mL strep-tomycin in a humidified incubator at 37�C under 5% CO2

atmosphere, and passaged at preconfluent densities by use of0.25% trypsin solution every 2 to 3 days. Cells were stored andused within 3 months after resuscitation of frozen aliquots.

Measurement of cell viabilityThe short-term effects of epirubicin or TIGAR knockdown

on tumor cell growthwas assessedwith 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, M5655)assay. Cells were cultured in 96-well plates (1,000 cells per well)in complete DMEM. Cells were treated with 1.25 to 20 mg/mLepirubicin (Pfizer; 10mgper bottle) or vehicle for 12 to 48 hoursand then subjected to MTT assay. Percentage of growthinhibition was calculated as (ODvehicle � ODtreatment)/ODvehicle

� 100%, where OD was measured at 570 nm with a microplatereader (BieTek). In each experiment, quintuplicate wells wereused for each drug concentration, and assays were repeated inthree independent experiments.

The long-term effects of TIGAR knockdown on tumor cellproliferation was analyzed with colony-formation assay. Cellstransfected with TIGAR siRNA after 12 hours were plated in 6-well plates at a density of 200 cells per well and epirubicin wasadded 24 hours later. Medium was changed every 3 days. After14 days, the colonies were stained with Giemsa for 15 minutesand rinsed with deionized distilled water, photographed usinga Nikon D800 digital camera (36.3 megapixel; Nikon Corp.) andscored using Image J software (W.S. Rasband, Image J, NIH,Bethesda, MD).

Transfection and RNA interferenceTo inhibit TIGAR expression, 2 small interference RNAs

(siRNA) matching region 115 to 133 in exon 3 (GCAG-CAGCTGCTGGTATAT; TIGAR siRNA1) and region 565 to583 in exon 6 (TTAGCAGCCAGTGTCTTAG; TIGAR siRNA2)of the human TIGAR cDNA sequence were synthesized byGenePharma, and a scramble sequence (TTACCGAGACCG-TACGTAT) was synthesized as a negative control. To inhibitautophagy, ATG5 siRNAs (GCAACTCTGGATGGGATTG,ATG5 siRNA1; CATCTGAGCTACCCGGATA, ATG5 siRNA2)were also synthesized.

Amethod called reverse transit transfectionwas adapted fortransient transfection using the Lipofectamine 2000 reagent(Invitrogen, 11668019). HepG2 or A549 cells were added direct-ly to the Lipofectamine 2000-TIGAR siRNAs or ATG5 siRNAscomplexes diluted in Opti-MEM Reduced Serum Medium(GIBCO, 31985070) and transfection occurred while cells wereattaching to the wall of well. For A549 cells, cells were plated ata density of 5 � 105 cells in 6-well plates and, were thentransfected with TIGAR siRNAs using Lipofectamine 2000reagent diluted in Opti-MEM Reduced Serum Medium 24hours later. The final concentration of TIGAR siRNA and ATG5siRNA was 80 and 40 nmol/L. Complete medium was added toeach well 6 hours after transfection. Cells were trypsinized andharvested for Western blot analysis or flow cytometry at theindicated times.

To assess the transfection efficiency, HepG2 cells weretransfected with LV-TIGAR-shRNA (TIGAR#2:50-GATTAG-CAGCCAGTGTCTTAG-30; TIGAR#3: 50-GCTTACATGAGAAG-TCTGTTT-30; Genechem) and 72 hours after transfection, theexpression of green fluorescent protein (GFP) was detectedwith a fluorescence microscopy. The transfection efficiencywas estimated about 90%. For establish stable TIGAR knock-down cells, HepG2 cells were transfected with LV-TIGAR-shRNA (Genechem) and were selected in cell culture mediumcontaining 1 mg/mL poromycin for 1 week. The efficiency ofTIGAR knockdownwas determinedwithWestern blot analysis.Cells were then cultured in culture medium for in vivo xeno-graft experiments.

Flow cytometry detection of apoptosis and ROSCell apoptosis was quantified with double staining of fluo-

rescein isothiocyanate (FITC) conjugated Annexin-V and pro-pidium iodide (PI; Biouniquer, BU-AP0103). Ten thousandscells per sample were acquired with a FACScan flow cytometer(FACScan). Freshly trypsinized cells were pooled,washed twicewith binding buffer, and processed following the manufac-turer's instructions. Cells fluorescence was analyzed with flowcytometry using the Cell Quest Pro software (BeckmanCoulter).

20,70-Dichloro-dihydrofluorescein diacetate (H2-DCFDA,Molecular Probes) was metabolized by nonspecific esterasesto the nonfluorescence product, 20,70-dichloro-dihydrofluores-ceine, which was oxidized to the fluorescent product DCF byROS. ROS levels were determined by incubating the cells withcell culture medium containing 10 mmol/L H2-DCFDA for 30minutes at 37�C. Then, the cells were washed twice in PBS,trypsinized, resuspended in PBS, and measured for ROS con-tent with FACS (FACScan, Becton Dickinson). Assays wereperformed in triplicates and were repeated in three indepen-dent experiments.

Western blot analysisProtein was extracted from cells using cell lysis solution

supplemented with protease inhibitors (Roche, 04693159001)and phosphorylase inhibitors (Roche, 04906845001). Proteinconcentration was determined with a BCA Protein Assay Kit(Pierce, 23227). Equal amounts of protein were fractionatedon Tris-glycine SDS-polyacrylamide gels and subjected to

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electrophoresis and transferred to NC membranes. Mem-branes were blocked with TBS containing 5% (w/v) dry milkwith 0.1% Tween 20, washed with TBS containing 0.1% Tween20 (TBST), and then incubated overnight at 4�C with specificantibodies against TP53 (1:1,000; Cell Signaling Technology,#2524), TIGAR (1:1,000; Abcam, ab37910), LC3 (1:1,000; Abcam,ab62721), p62 (1:1,000; American Research Product, 12-1107),ATG5 (1:1,000; Cell Signaling Technology, #2630), p-mTOR(1:1,000; Cell Signaling Technology, #2971), S6 (1:1,000; CellSignaling Technology, #2217), p-S6 (1:1,000; Cell SignalingTechnology, #4856), 4EBP-1 (1:1,000; Cell Signaling Techno-logy, #9452) and p-4EBP-1 (1:1,000; Cell Signaling Technology,#9451) in nonfat milk containing 0.1% NaN3. After washingin TBST, membranes were incubated with fluorescencesecondary antibodies (1:10,000; Jackson ImmunoResearch,anti-rabbit, 711-035-152, anti-mouse, 715-035-150) at roomtemperature for 1 hour. Immunoreactivity was detected usingOdyssey Infrared Imager (Li-COR Biosciences). The signalintensity of primary antibody binding was quantitatively ana-lyzed with Image J software (W.S. Rasband, Image J, NIH) andwas normalized to a loading control b-actin (1:4,000; Sigma,A5441).

Evaluation of LC3 fluorescent punctaHepG2 cells were seeded onto cover glass (Fisher Scientific,

#032910-9) in 24-well plate andwere transfectedwith scramblesiRNA, TIGAR siRNA1, or TIGAR siRNA2 24 hours later. Cellswere treated with or without the treatment of 5 mg/mLepirubicin for 12 hours. After 72 hours of transfection, cellswere washed with PBS (pH 7.4) and fixed with 4% paraformal-dehyde and then blocked in PBS containing 1% normal bovineserum albumin and 0.1% Triton X-100 for 1 hour at roomtemperature. Cells were then incubated with rabbit polyclonalanti-LC3 antibody (Abcam, ab62721) followed by incubationwith FITC-conjugated anti-rabbit secondary antibodies (1:200;Boster, BA1105). After 1-hour incubation and several rinses,cells were incubated with 0.5 mg/mL 4,6-diamidino-2-pheny-lindole (DAPI) for 10 minutes. Sections were then washed inPBS and cover slipped with fluoromount Aqueous MountingMedium (Sigma, F4680). The slides were analyzed with NikonC1 plus laser scanning confocal microscopy (Nikon D-EclipseC1 plus). Six fields were analyzed for each of the samplesstained with a given antibody, and the number of LC3 puncta/cell was evaluated as the total number of dots divided by thenumber of nuclei in eachmicroscopic field and at least 150 cellswere examined in each treatment. Duplicates of three inde-pendent experiments were analyzed for each group.

Transmission electron microscopyHepG2 cells were seeded into 6-well plates and transfected

with TIGAR siRNA2 or scramble siRNA for 72 hours, epirubicinwas applied 12 hours before the end of transfection. Cells werethen fixed for 2 hours with 2.5% glutaraldehyde in 0.1 Mphosphate buffer (pH 7.4), postfixed in 1% OsO4 dehydratedin graded ethanol and then embedded in epoxy resin. Blockswere cut on an ultramicrotome into ultrathin sections, whichwere poststained with uranyl acetate and lead citrate, andviewed under a Hitachi 7100 electron microscopy.

Redox-state analysisFor GSH/oxidized glutathione (GSSG) measurement, a GSH

and GSSG Assay Kit (Beyotime) was used according to themanufacturer's protocol. NADPH levels of cells were detectedwith enzychromTM NADPþ/NADPH Assay Kit (ECNP-100;BioAssay Systems) according to the manufacturer's protocol.

In vivo tumor growth analysisHepG2 cells and stable TIGAR knockdown HepG2 cells (1�

106) were subcutaneously inoculated into the right oxter of 6-week-old female athymic nude mice (Shanghai SLAC Labora-tory Animal Co. Ltd.). Two weeks after tumor formation, 2mg/kg epirubicin was intraperitoneally administrated every 3days for another 2 weeks, and thenmice were anesthetized andphotographed. After the mice were sacrificed, the tumors wereremoved and photographed and weighted. Tumor proteinswere extracted forWestern blot analysis. All animal procedureswere approved and monitored by the local Animal Care andUse Committee in Soochow University (License NO. Syxk;Su-0062).

Statistical analysisAll data were presented as means � SEM. Data were sub-

jected to one-way ANOVA using the GraphPad Prism softwarestatistical package (GraphPad Software). When a significantgroup effect was found, post hoc comparisons were performedusing the Newman–Keuls t test to examine special groupdifferences. Independent group t testswere used for comparingtwo groups. Significant differences at P < 0.05, 0.01, and 0.001are indicated by �, �� , ��, respectively. All calculations wereperformed using the 14.0 SPSS software package (SPSS Inc.).

ResultsEpirubicin-induced TIGAR expression

Epirubicin, a DNA-damaging anticancer agent inducedgrowth inhibition in both HepG2 cells (Fig. 1A) and A549 cells(Supplementary Fig. S1A) in a concentration- and time-depen-dent manner. To detect TIGAR expression in response toepirubicin treatment, HepG2 cells were seeded onto 6-wellplates for 24 hours and were then treated with differentconcentrations of epirubicin (1.25–20 mg/mL) for 12 hours or5.0 mg/mL epirubicin for different length of time (12–48 hours).An increase in TIGAR protein levels was observed at the lowconcentration of epirubicin (1.25–5 mg/mL), but returned tothat of control levels at 10mg/mL (Fig. 1C). A time-course studyshowed that TIGAR expression was elevated at 12 to 24 hours,but quickly declined and fell down to that of control at 36 hours(Fig. 1E). As TIGAR was known as a TP53-inducible gene, theexpression of TP53 in HepG2 cells treated with differentconcentrations of epirubicin or 5.0 mg/mL epirubicin fordifferent length of time was detected with immunoblotting.The results showed that the elevated expression of TIGARcorrelated with the expression of TP53 as seen in Fig. 1B and D.Similar results were obtained in A549 cells (Supplementary Fig.S1B–E). To further investigate whether the elevation of TIGARwas correlated with the increased expression of TP53, PFTa, aTP53 inhibitor was used. Western blot analysis showed thatexpression of Bax and TIGAR were decreased in a time- and

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dose-dependent manner (Supplementary Fig. S2), which indi-cated that TIGAR induction was dependent on TP53 in HepG2cells.

Based on these observations, we treated HepG2 and A549cells with 5 mg/mL epirubicin for 12 hours in rest of experi-ments, as this treatment protocol induced aminor inhibition ofcell viability but a significant increase in TIGAR protein levels.

Downregulation of TIGAR-enhanced epirubicin-inducedapoptosis

To investigate the effects of TIGAR on the cell growth ofHepG2 and A549 cells, 2 different TIGAR siRNA molecules(TIGAR siRNA1, TIGAR siRNA2) were designed to silenceTIGAR expression. HepG2 cells were transfectedwith scramblesiRNA, TIGAR siRNA1 or TIGAR siRNA2 at the concentration of80 nmol/L for 72 hours. The transfection efficiency was �90%as detected with a fluorescence microscopy (SupplementaryFig. S3A)Western blot analysis of TIGAR protein levels showed89% and 91% of silencing efficiency with TIGAR siRNA1 andTIGAR siRNA2, respectively (Fig. 2A). A 65.6% (TIGAR siRAN1)and 71.2% (TIGAR siRNA2) of silencing efficiency was alsodetected in A549 cells (Supplementary Fig. S3B). The short-term effect of TIGAR knockdown on cell growth wasexamined with an MTT assay in both A549 and HepG2 cells.Knockdown of TIGAR had no significant effect on the cell

proliferation under normal condition. However, TIGAR knock-down significantly enhanced the inhibitory effects of epirubi-cin on growth of HepG2 cells, suggesting a protective effect ofTIGAR on cancer cell survival (Fig. 2B). Similar effects wereobtained in A549 cells with combination of TIGAR knockdownand epirubicin treatment (Supplementary Fig. S3C). To eval-uate the long-term effect of TIGAR knockdown on cell prolif-eration, colony-formation assay was used. The formation ofclones of HepG2 cells after TIGAR knockdownwas only slightlyreduced under normal condition. The epirubicin-inducedinhibition of the colony formation was greatly enhanced byknockdown of TIGAR (Fig. 2C and D).

To further investigate whether downregulation of TIGARenhances the chemosensitivity of HepG2 and A549 cells, weanalyzed apoptosis of HepG2 and A549 cells in response toepirubicin after knockdown of TIGAR. TIGAR interferenceenhanced the apoptotic index when HepG2 cells treated with5.0 mg/mL epirubicin compared with those treated with epir-ubicin alone (Fig. 2E and F). Similar effects of TIGAR knock-down on epirubicin-induced apoptosis were observed in A549cells (Supplementary Fig. S3D). Furthermore, this studyshowed that knockdown of TIGAR promoted apoptosis partlythrough the activation of the conventional caspase-dependentapoptotic pathway with a significant enhancement in theactivation of caspase 3 (Supplementary Fig. S4A). The

Figure 1. Epirubicin inhibited cell growth and induced TIGAR expression. A, the dose response and the time course of the effect of epirubicin on cell viability.HepG2 cells were treated with different concentrations of epirubicin (1.25–20 mg/mL) for 12, 24, 36, and 48 hours. Cell viability was detected with MTTassay. B and D, the effects of epirubicin on protein levels of TP53. HepG2 cells were treated with different concentrations of epirubicin (0, 1.25, 2.5, 5, 10, and20 mg/mL) for 12 hours or 5 mg/mL epirubicin for different lengths of time (12, 24, 36, and 48 hours). The protein levels of TP53 were detected withWestern blotting. b-Actin proteinwas used as a loading control. Quantitative analysis was performedwith Image J. C and E, the effects of epirubicin on proteinlevels of TIGAR. The HepG2 cells were treated as described above. Values are means � SD from three independent experiments. �, P < 0.05;��, P < 0.01; ��, P < 0.001; ns, P > 0.05 versus control group.

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apoptosis was inhibited with the pan-caspase inhibitorZ-VAD-FMK (Supplementary Fig. S4B and S4C). These resultssuggest that TIGAR knockdown enhances the chemosensitiv-ity of tumor cells to epirubicin.

Inhibition of TIGAR expression reduced tumorigenicityand enhanced chemosensitivity to epirubicin in vivoTo evaluate the effects of TIGAR on tumor growth and

chemosensitivity of hepatocellular carcinoma to epirubicin,a xenograft nude mouse model was used. HepG2 cells withstable TIGAR knockdown (Fig. 3A and B) were inoculated intothe right oxter of 6-week-old female athymic nudemice. Amildinhibition of tumor growth in vivo was observed in stableTIGAR knockdown cells as compared with vector-transfected

cells. Epirubicin treatment (2 mg/kg) reduced tumor size andthe effect was substantially enhanced in the stable TIGARknockdown group (Fig. 3C–E). The reduced expression ofTIGAR was confirmed with GFP-fluorescence (Fig. 3F) andWestern blot analysis (Fig. 3G). These in vivo results revealedthat inhibition of TIGAR expression can reduce tumorigenicityand enhance the chemosensitivity of hepatocellular carcinomato epirubicin.

TIGAR knockdown enhanced epirubicin-inducedelevation of ROS levels

TIGAR is reported to inhibit glycolysis and decrease intra-cellular ROS levels, thus protects cells from ROS-associatedapoptosis. To detect whether this increased chemosensitivity

Figure 2. Downregulation ofTIGAR inhibited cell survival andenhanced epirubicin-inducedapoptosis. A, knockdownefficiency of TIGAR in HepG2 cells.Transient transfection of siRNAswas applied to knockdown TIGARexpression for 72 hours andepirubicin was added 12 hoursbefore the end of the experiment.The protein levels of TIGAR weredetected with Western blotting.b-Actin protein was used as aloading control. Quantitativeanalysis was performedwith ImageJ. B, short-term effects ofepirubicin on cell growth afterTIGAR knockdown. HepG2 cellswere treated as described aboveand cell growth was assessed withMTT assay. C, long-term effects ofepirubicin on cell growth afterTIGAR knockdown. HepG2 cellswere treated as described aboveand cell growth was assessed bycolony-formation assay. D,quantitative analysis of clonenumbers of HepG2 cells afterTIGAR knockdown with or withoutepirubicin treatment. E, the effectsof epirubicin on apoptosis afterTIGAR knockdown. HepG2 cellswere treated as described aboveand apoptosis was detected withflow cytometry detection. F,quantitative analysis of percentageof apoptotic HepG2 cells afterTIGAR knockdown with or withouttreatment of epirubicin. Values aremeans � SD from threeindependent experiments.�, P < 0.05; ��, P < 0.01;��, P < 0.001; ns, P > 0.05versus corresponding group.






induced by TIGAR knockdown was associated with increasedROS levels, we detected the ROS levels in the cells transfectedwith TIGAR siRNAs and treated with epirubicin. The resultsshowed that ROS levels were slightly increased in HepG2 cellsafter TIGAR knockdown. The treatment with epirubicin afterTIGAR knockdown produced greater increase in ROS levelscompared with control cells and cells treated with TIGARscramble siRNA (Fig. 4A and B). To confirm that the enhance-ment of epirubicin-induced apoptosis after TIGAR knockdownwas attributable to the increased cellular ROS levels, NADPH

was supplemented to HepG2 cells. Cells were transfected withTIGAR siRNA2 and were treated with 10 mmol/L NADPH1 hour before epirubicin treatment. Addition of NADPH sig-nificantly reduced ROS levels in cells transfected with TIGARsiRNA2 and treated with epirubicin (Fig. 4A and B). Similarresults were seenwith the ROS scavenger NAC (SupplementaryFig. S5A and B). GSH/GSSG and NADPH levels after TIGARknockdown with or without treatment of epirubicin were alsodetected and results showed a significant decrease in GSH/GSSG and NADPH levels after TIGAR knockdown HepG2 cells

Figure 3. Inhibition of TIGARexpression enhancedchemosensitivity to epirubicin invivo. A, transfection efficiency oflentivirus inHepG2cells.Cellsweretransfected with LV-TIGAR-shRNAat a MOI value of 10. GFP-greenfluorescence was detected with afluorescence microscopy after 72hours. B, Western blot analysis ofTIGAR protein expression afterHepG2 cells transfected withLV-shTIGAR and selected with1 mg/mL poromycin for 1 week.GAPDH was used as a loadingcontrol. C, HepG2 cells and stableTIGAR knockdown HepG2 cells(1 � 106) were subcutaneouslyinoculated into the right oxter of6-week-old female athymic nudemice. Two weeks after tumorformation, 2 mg/kg epirubicin wasintraperitoneally administratedevery 3 days for another 2 weeks.Mice were anesthetized andphotographed. D, the visualcomparison of tumor size from 6mice in each group. E, tumorweights from 6 mice in each groupwere measured. Values aremeans � SD. �, P < 0.05 versuscontrol group; ns, P > 0.05;#, P < 0.05 versus correspondinggroup. F, GFP-greenfluorescence was detected withfluorescence microscope inxenograft tumor tissues (frozensection, 10-mm thickness),DAPI-blue fluorescence field wasphotographed at the same vision.G, Western blot analysis of TIGARprotein expression in xenografttumor tissues.GAPDHwasusedasa loading control.

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treated with 5 mg/mL epirubicin (Fig. 4C and D). These effectswere partly blockedwith supplementation of 10mmol/LNAC1hour before epirubicin treatment (Supplementary Fig. S5C andD). The colony-formation assay also showed a protective effectof NAC on tumor cell growth (Supplementary Fig. S5E). Apo-ptosis of HepG2 cells after TIGAR knockdown with or withouttreatment of 5 mg/mL epirubicin was significantly reducedafter applying of NADPH (Fig. 4E); however, compared withcontrol cells, the apoptotic rate of HepG2 cells treated with 5.0mg/mL epirubicin after TIGAR knockdown with addition ofNADPH was still higher than that of epirubicin alone, suggest-ing that enhancement of chemosensitivity by knockdown ofTIGAR is partially mediated by elevation of ROS levels.

TIGAR knockdown enhanced epirubicin-inducedautophagy activationTo investigate the effects of epirubicin on autophagy,

HepG2 cells were seeded onto 6-well plates for 24 hours andwere then treated with different concentrations of epirubicin(1.25–20 mg/mL) for 12 hours or 5 mg/mL epirubicin fordifferent length of time (12–48 hours). The data showed thatautophagy was activated if treated with higher concentration(10–20 mg/mL) of epirubicin for longer time (36–48 hours)with increases in LC3- II/LC3-I ratio and decreases in p62levels (Fig. 5A–D). It was reported that TIGAR can inhibitautophagy through downregulating ROS levels. To determine

whether autophagy was activated by knockdown of TIGAR,HepG2 cells were left untreated or treated with 5 mg/mLepirubicin with or without TIGAR knockdown and the LC3levels were detected with Western blot analysis. The resultsshowed that knockdown of TIGAR alone did not significantlyincrease the LC3-I/LC3-II ratio. Treatment of HepG2 cellswith epirubicin (5 mg/mL) did not significantly induce acti-vation of autophagy at the time examined. However, knock-down of TIGAR resulted in significant activation of autophagyafter treatment of epirubicin (Fig. 5E). Meanwhile, the greaterincrease in the LC3 fluorescence intensity and the number ofLC3 patches were seen in HepG2 cells after treatment withepirubicin and TIGAR knockdown (Fig. 5F). The greaterdecrease in p62 was also found to in response to epirubicintreatment after TIGAR knockdown (Fig. 5G). To furtherconfirm the enhancement of autophagy activity by knock-down of TIGAR, HepG2 cells were transfected with scramblesiRNA or TIGAR siRNA2 with or without treatment of epir-ubicin and electron microscopy was used to detect theformation of autophagosomes. Results showed that comparedwith control, the formation of auatophagosomes was detectedin HepG2 cells after TIGAR knockdown or treated with 5.0mg/mL epirubicin; however, the increased number of autop-hagosomes, secondary lysosomes, and vacuolated mitochon-dria were seen in HepG2 cells treated with combined epir-ubicin and TIGAR knockdown (Fig. 6). These results suggest

Figure 4. TIGAR knockdown enhanced epirubicin-induced elevation of ROS levels. A, ROS levels after TIGAR knockdown and epirubicin treatmentin the presence and absence of NADPH. TheHepG2 cells were transfectedwith scramble siRNA or TIGAR siRNA2 for 60 hours and thenwere left untreated ortreated with 5 mg/mL epirubicin for 12 hours in the presence or absence of NADPH. The ROS levels and cell apoptosis were measured with flow cytometryafter treatment of epirubicin for 12 hours. B, quantitative analysis of ROS levels after TIGAR knockdown and epirubicin treatment in the presence andabsence of NADPH. C, GSH/GSSG ratio in HepG2 cells after TIGAR knockdown with or without treatment of 5 mg/mL epirubicin. D, NADPH levels in HepG2cells after TIGAR knockdown with or without treatment of 5 mg/mL epirubicin. E, cell apoptosis after TIGAR knockdown and epirubicin treatmentin thepresenceandabsenceofNADPH. TheHepG2cellswere treated asdescribed above andapoptosiswasdetectedwith flowcytometry. Values aremeans� SD from three independent experiments. �, P < 0.05; ��, P < 0.01; ��, P < 0.001; ns, P > 0.05 versus corresponding group.






an enhancement in the autophagy activation by TIGARknockdown in response to epirubicin treatment.

To further investigate whether this enhanced autophagyactivity after TIGAR knockdown was caused by the elevation ofROS levels, HepG2 cells transfected with TIGAR siRNA2 weretreatedwith NADPH2 hours before the treatment of 5.0 mg/mLepirubicin. Results showed that the increment of epirubicin-induced autophagy activity by TIGAR knockdownwas partiallyinhibited with supplementation of NADPH (Fig. 5H). Thissuggests that in addition to ROS, other mechanisms may beinvolved in TIGAR-mediated autophagy regulation (11). AsmTOR is a major regulator of autophagy, the inhibition ofwhich has been shown to induce activation of autophagy inresponse to nutrient starvation (12), we then detected thephosphorylation of mTOR and its downstream protein S6 and4EBP1 in response to epirubicin after TIGAR knockdown.Results showed that silencing TIGAR expression alone had nosignificant effect on phosphorylation of mTOR under normalcondition. Treatment of HepG2 cells with epirubicin caused a

robust increase in the levels of phosphorylated mTOR but notits downstream S6 and 4EBP1. However, knockdown of TIGARalmost completely blocked the phosphorylation of mTORinduced by epirubicin (Fig. 7A). Moreover, knockdown ofTIGAR reduced phosphorylated S6 and 4EBPI below the con-trol levels after combined epirubicin treatment (Fig. 7B and C).These results indicate that TIGARmay have a direct regulatoryrole in the mTOR signaling pathway.

Inhibition of autophagy enhanced TIGAR knockdown–induced increases in apoptosis

To investigate whether the activation of autophagy inducedby TIGAR knockdown affects chemosensitivity of epirubicin, 3-MA, an autophagy inhibitor that has been reported to inhibitthe activity of PI3 kinase and block the formation of autopha-gosomes, was applied 12 hours before the end of the treatment.After applying 3-MA, the rate of apoptosis of HepG2 cellstreated with 5 mg/mL epirubicin and TIGAR knockdown wassignificantly increased (Fig. 7D and E). Colony-formation assay

Figure 5. TIGARknockdownenhancedepirubicin-induced autophagy activation. A, the dose-responseof epirubicin on protein levels of p62. HepG2cellsweretreatedwith different concentrations of epirubicin for 12 hours. Cells were lysed and the protein levels of p62were detectedwithWestern blotting. b-Actinwasused as a loading control. Quantitative analysis was performed with Image J. B, the dose–response of epirubicin on protein levels of LC3. HepG2cells were treated as described above. C, the time course of epirubicin on protein levels of p62. HepG2 cells were treated with 5 mg/mL of epirubicin for 12, 24,36, and 48 h. Cells were lysed and the protein levels of p62 were detected with Western blotting. b-Actin was used as a loading control. Quantitativeanalysis was performedwith Image J. D, the time course of epirubicin on protein levels of LC3; HepG2 cells were treated as described above. E, the effects ofTIGAR knockdown on LC3 in the presence and absence of epirubicin. HepG2 cells were transfected with scramble siRNA or TIGAR siRNA1 andTIGAR siRNA2 for 60 hours and then were left untreated or treated with 5 mg/mL epirubicin for 12 hours. The protein levels of LC3were detectedwithWesternblotting. b-Actin was used as a loading control. Quantitative analysis was performed with Image J. F, distribution of LC3 puncta in TIGAR knockdown HepG2cells. HepG2 cells were planted onto 24-well plate and were treated as described above and then fixed with 4% paraformaldehyde and processed forimmunofluorescence. Cells were analyzed with a confocal microscopy. LC3 was stained green and DAPI was stained blue. Scale bar, 5 mm. Thenumber of LC3 dots/cell was quantitatively analyzed. G, effects of TIGAR knockdown on p62 in the presence and absence of epirubicin. HepG2 cells weretreated as described above. H, the effects of NADPH on autophagy induced by TIGAR knockdown. HepG2 cells were transfected with TIGAR siRNA2for 60 hours and then were treated with 5 mg/mL epirubicin for 12 hours; NADPH was added 2 hours before the treatment with epirubicin. The levels of LC3were detected by Western blot analysis. b-Actin was used as a loading control. Values are means � SD from three independent experiments. �, P < 0.05;��, P < 0.01; ��, P < 0.001; ns, P > 0.05 versus control group.

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also showed a decrease in clones after autophagy was inhibitedby 3-MA. It has reported that 3-MA has dual role inmodulationof autophagy via different temporal patterns of inhibition onclass I and class III PI3K (13). To further investigate the effectsof autophagy induced by TIGAR knockdown on cell apoptosisand chemosensitivity, HepG2 cells were cotransfected withATG5 siRNA andTIGAR siRNA for 48 hours. The cotransfectionefficiency was 62.33% for Atg5 and was 72.05% for TIGAR (Fig.7F). HepG2 cells cotransfected with ATG5 siRNA and TIGARsiRNA were then treated with 5 mg/mL epirubicin for 12 hoursand the cell viability was assessed with MTT assay. The resultsshowed that inhibition of autophagy by knockdown of ATG5 inTIGAR-silenced HepG2 cells, cell survival was greatly reducedafter the treatment with epirubicin (Fig. 7G). These resultssuggested that enhanced autophagy activity induced by TIGARknockdown reduces the cytotoxicity of epirubicin.

DiscussionAlteration of energy metabolism is one of the hallmarks of

cancer cells and has recently inspired particular interest ininvestigating regulation of metabolic pathways in cancer che-motherapy (14). The increased dependency of cancer cells onthe glycolytic pathway for ATP generation provides a biochem-ical basis for the design of therapeutic strategies to preferen-

tially kill cancer cells by pharmacologic inhibition of glycolysis.Thus development of novel glycolytic inhibitors as a new classof anticancer agents is likely to have broad therapeutic appli-cations (15). Several glycolysis inhibitors are now in preclinicaland clinical development, such as lactate dehydrogenase Ainhibitor FX11 (16) or hexokinase inhibitor 2-deoxy-glucose(15, 17) and lonidamine (18). The function of TIGAR as a kind ofglycolytic inhibitor seems to be unfavorable to cancer cellsurvival from the view of glycolysis. However, recent studiesshowed that TIGAR was overexpressed in several types ofcancer cells, and its expression was favorable to tumor cellsurvival (5). The role and mechanisms by which TIGAR affectcancer cell survival need to be further studied.

To determine the response and role of TIGAR in cancer cellswith relation to chemotherapy, this study investigated theregulation of chemosensitivity by TIGAR in HepG2 andA549 cells. Consistent with the previous findings (5, 19, 20),TIGAR expression in A549 and HepG2 cells was elevatedfollowing treatment with epirubicin in face of mild or transientstress. TIGAR knockdown mildly decreased the cell viability,partially because of the increased rate of apoptosis. Further-more, the epirubicin-induced decline in cell viability and theincrease in apoptosis were greatly enhanced when TIGAR wasknocked down. These studies suggest a protective effect of

Figure 6. TIGAR knockdown increased epirubicin-induced autophagosome formation. Electronmicrographs showed the ultrastructure of control HepG2 cellsor HepG2 cells transfected with scramble siRNA or TIGAR siRNA2 with or without treatment of 5.0 mg/mL epirubicin. A and B, HepG2 control cells or cellstransfected with scramble siRNA seemed to be normal ultrastructure with healthy looking mitochondria, lysosomes, and nuclei. C, HepG2 cells transfectedwith TIGAR siRNA2 showed the autophagosomes and secondary lysosomes. Mitochondria vacuolation was found. D, HepG2 cells treated with 5.0 mg/mLalso appeared secondary lysosomes and mitochondria vacuolation, as well as autophagosomes formation. E, HepG2 cells transfected with TIGAR siRNA2combined with treatment of 5.0 mg/mL epirubicin increased the number of vacuoles and the autophagosomes in the cytoplasm. The increases in secondarylysosomes and mitochondrial vacuolation, condensation of chromatin were also observed. Scale bars: 1 mm. Arrows, autophagosomes; #, lysosomes;�, mitochondria.






TIGAR on cancer cell survival under basal and stress condi-tions. The in vivo studies further showed a reduction intumorigenicity and an increase in chemosensitivity to epiru-bicin after TIGAR knockdown, suggesting TIGAR may be atherapeutic target for cancer therapy.

Spitz and colleagues reported that cancer cells producedgreater amount of superoxide and hydrogen peroxide thannormal cells (21, 22). To reduce the hydroperoxide toxifica-tion, the pentose pathway was upregulated to produce moreNADPH. TIGAR can inhibit glycolysis; however, this effect

Figure 7. TIGAR knockdown enhanced epirubicin-induced autophagy by inhibiting the mTOR pathway and inhibition of autophagy enhanced TIGARknockdown–induced increases in apoptosis. A, effects of TIGAR knockdown on phosphorylation ofmTOR in the presence and absence of epirubicin. HepG2cells were treated as described above; the protein levels of phosphorylated mTOR were detected with Western blot analysis. b-Actin was used asa loading control. Quantitative analysis was performed with Image J. B and C, phosphorylation of mTOR downstream protein S6 and 4EBP1 after TIGARknockdown on HepG2 cells in the presence and absence of epirubicin; b-actin was used as a loading control. D and E, the effects of 3-MA on TIGARknockdown–induced increases in apoptosis. Sixty hours following transfection of scramble siRNA or TIGAR siRNA2, HepG2 cells were left untreated ortreated with 5 mg/mL epirubicin for 12 hours in the presence and absence of 10 mmol/L 3-MA. The apoptotic rate was measured with flow cytometryof Annexin V-FITC andPI double stained cells. F, efficiency of co-knockdownof Atg5 and TIGAR.HepG2cellswere cotransfectedwith Atg5 siRNAandTIGARsiRNA for 48 hours, and the protein levels of Atg5 and TIGAR were determined with Western blot analysis. b-Actin protein was used as a loadingcontrol. Quantitative analysis was performedwith Image J. G, effects of Atg5 knockdown on TIGAR knockdown–induced increases in apoptosis. HepG2 cellswere cotransfected with Atg5 siRNA and TIGAR siRNA for 48 hours. Cell viability was detected with MTT assay after TIGAR and Atg5 knockdownwith or without the treatment of epirubicin. Values are means � SD from three independent experiments. �, P < 0.05; ��, P < 0.01; ��, P < 0.001; ns, P > 0.05versus corresponding group.

Xie et al.





may be masked by other regulatory mechanisms in tumorcells. In agreement with Spitz and colleagues (21, 22), weobserved the lactate production was increased in coloncancer tissues compared with corresponding normal tissues.We also observed a high expression of G6PD in colon cancertissues alone with TIGAR over expression (unpublishedobservations); thus, TIGAR can increase the generation ofNADPH by promoting the flow of the pentose phosphatepathway (PPP). To investigate whether TIGAR knockdown–induced enhancement in chemosensitivity is caused by theincreases in ROS, total cellular ROS levels, GSH/GSSG ratio,and NADPH levels were detected. The increases in ROS levelsand decreases in GSH/GSSG ratio or NADPH levels werefound, which confirmed the role of TIGAR in suppressingROS production. To further confirm that the enhancedapoptosis to epirubicin after TIGAR knockdown was medi-ated by elevation of cellular ROS, NADPH and NAC wereapplied. The results demonstrated that cell apoptosis wasapparently reduced if the NADPH or NAC was added to cells.However, the apoptotic rate of cancer cells treated withNADPH after TIGAR knockdown was still higher than thatof the control cells, indicating other mechanisms are alsoinvolved in TIGAR-regulated chemosensitivity. Caspase-3,an important death protease (23) was found to be activatedin cells transfected with TIGAR siRNA or treated withepirubicin, this effect was also amplified with combinationof TIGAR knockdown and epirubicin. Cell apoptosis inducedby TIGAR knockdown, epirubicin, or combination of bothwas partially rescued after supplementation of Z-VAD-FMK,a pan-caspase inhibitor. It suggests that the promotion ofchemosensitivity by TIGAR knockdown is more or lessbecause of the activation of the caspase-dependent apopto-tic pathway.Recent reports showed that TIGAR expression was corre-

lated with the ex vivo sensitivity of chronic lymphocyticleukemia cells to fludarabine (24). Lui and colleagues demon-strated that TIGAR/NADPH cascade was involved in antitu-mor effects of a new c-Met tyrosine kinase inhibitor, suggestingthat inhibition of TIGAR may have an antitumor potentiality(25). ECyd, an RNA-directed nucleoside anti-metabolite, elicitsits antitumor effect via TIGAR downregulation (26). Wankaand colleagues demonstrated that TIGAR was elevated shortlyafter irradiation, and silencing TIGAR expression resulted incell viability inhibition and the damnification of DNA repair inglioblastoma cell lines, with the possibility to enhance radio-sensitization (20). In consistent with these findings, thisstudy suggests that TIGAR may be a valuable target forchemotherapy.Bensaad and colleagues demonstrated that TIGAR can

modulate ROS in response to nutrient starvation or meta-bolic stress, and functions to inhibit autophagy (11). Ling Yeand colleagues, reported that knockdown of TIGAR inducedapoptosis and autophagy in HepG2 hepatocellular carcino-ma cells (27). How does TIGAR influence autophagy activityand its consequence on cell viability, however, is not clearlydefined. Autophagy is an evolutionarily conserved mecha-nism for degradation of intracellular substances, responsiblefor the recycle of metabolic substances and the maintenance

of intracellular stability (28). Whether autophagy kills orprotects cancer cells is complex (29). In most chemother-apeutic strategies, elevation of autophagy activity in varyingdegrees after treatment could be observed (30). This can beconsidered as a protective strategy for tumor cells to avoidbeing entirely killed by drugs. The pro-survival ability ofautophagy renders tumor cells resistant to anticancer drugs,which greatly compromises curative efficacy of chemother-apy. A complex interplay between apoptosis and autophagyhas also been reported (31). In many cases, autophagy caninhibit apoptosis by removing damaged mitochondria, pre-venting the release of apoptogenic factors such as cyto-chrome c from the mitochondria and the activation of theapoptotic cascade in cancer cells (32, 33). In agreement withother investigators (11, 27), knockdown of TIGAR alone onlyslightly increased autophagy activity, but significantlyenhanced epirubicin-induced autophagy activation. Toexplore the effects of activated autophagy on cell viabilityafter TIGAR knockdown, 3-MA or ATG5 siRNA was implied.Inhibition of autophagy with 3-MA or ATG5 knockdownfurther increased the epirubicin-induced apoptosis whenTIGAR was knocked down, which suggests a protective effectof autophagy on cancer cell survival.

It has been proposed that TIGAR affects autophagy as aresult of reduction in ROS. In this study, NADPH partiallyblocked TIGAR knockdown–induced enhancement ofautophagy activity, suggesting additional mechanisms maybe involved. This study found that knockdown of TIGARalone had no significant effect on mTOR but robustlydecreased the epirubicin-induced the phosphorylation ofmTOR and its downstream protein S6 and 4EBP1. Thisfinding suggests that TIGAR may influence autophagy activ-ity through regulating ROS levels and a direct inhibitoryeffect on the mTOR pathway.

In summery, TIGAR was induced by epirubicin. Knock-down of TIGAR expression by siRNA enhanced the antitu-mor effects of epirubicin with increased cellular ROS levels,active caspase-3 and the rate of apoptosis. The effects can bepartially blocked by supplementing NADPH or NAC. Knock-down of TIGAR slightly increased activation of autophagybut significantly enhanced epirubicin-induced autophagyactivation. The TIGAR knockdown–induced autophagy acti-vation involved in elevation of ROS and inhibition of mTOR.Inhibition of autophagy activity enhanced epirubicin-induced apoptosis. Therefore, TIGAR exerts dual effects onsurvival of tumor cells, with one favorable effect mediated byreducing ROS and an unfavorable effect because of reducingautophagy activity. However, based on current information,the predominant role of TIGAR on cancer cells is prosurvi-val. This study also suggests that if TIGAR is targeted forchemotherapy, inhibiting autophagy would provide addi-tional benefits.

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

Authors' ContributionsConception and design: J.-M. Xie, B. Li, H.-P. Yu, Z.-H. QinDevelopment of methodology: J.-M. Xie, B. Li, H.-P. Yu, Z.-H. Qin






Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.-M. Xie, B. Li, H.-P. Yu, Z.-H. QinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.-M. Xie, B. Li, H.-P. Yu, Z.-H. QinWriting, review, and/or revision of themanuscript: J.-M. Xie, B. Li, H.-P. Yu,Z.-H. QinAdministrative, technical, or material support (i.e., reporting or orga-nizingdata, constructingdatabases): J.-M. Xie, B. Li, H.-P. Yu, Q.-G. Gao,W. Li,H.-R. Wu, Z.-H. QinStudy supervision: Q.-G. Gao, W. Li, H.-R. Wu

Grant SupportThis work was supported by the National Natural Science Foundation of

China (No. 30930035) and the Priority Academic Program development ofJiangsu Higher Education Institutes.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 12, 2013; revised May 14, 2014; accepted July 9, 2014;published OnlineFirst August 1, 2014.

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2014;74:5127-5138. Published OnlineFirst August 1, 2014.Cancer Res Jia-Ming Xie, Bin Li, Hong-Pei Yu, et al. Apoptosis and AutophagyTIGAR Has a Dual Role in Cancer Cell Survival through Regulating

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