SIRT1 and AMPK Mediate Hypoxia-Induced Resistance of …cancerres.aacrjournals.org/content/canres/74/1/298.full.pdfSIRT1 and AMPK Mediate Hypoxia-Induced Resistance of Non–Small

Therapeutics, Targets, and Chemical Biology

SIRT1 and AMPK Mediate Hypoxia-Induced Resistance ofNon–Small Cell Lung Cancers to Cisplatin and Doxorubicin

Dong Hoon Shin, Yong-Joon Choi, and Jong-Wan Park

AbstractSIRT1 is an NADþ-dependent protein deacetylase induced by metabolic stresses, such as nutrition or oxygen

deprivation. Although SIRT1 contributes to aging and metabolic disorders, its role in cancer progression andtherapeutic responses remains controversial. Because hypoxia occurs widely in solid tumors, where it provokesdrug resistance, we investigated the involvement of SIRT1 in hypoxia-induced chemoresistance. SIRT1 wasdownregulated in a panel of non–small cell lung carcinoma (NSCLC) cells exposed to hypoxia for 48 hours. Themastermetabolic kinase AMP-activated protein kinase (AMPK)was inactivated under the same conditions, likelydue to attenuation of the SIRT1/LKB1-mediated AMPK activation process. Notably, hypoxic inactivation of thisSIRT1–AMPKpathway led to cisplatin and doxorubicin resistance.Mechanistic investigations suggested that thispathway supported the cytotoxic response to cisplatin and doxorubicin by licensing an apoptotic processcontrolled bymitochondria.We confirmed the involvement of this pathway in amouse xenograftmodel of humanNSCLC. Furthermore, we demonstrated that a SIRT1 activator SRT1720 augmented the antitumor effects ofcisplatin, and these effects could be blocked by administration of an AMPK inhibitor compound C. Takentogether, our results offer preclinical proof-of-concept to target the SIRT1–AMPK pathway as a strategy toovercome hypoxia-induced chemoresistance in NSCLC. Cancer Res; 74(1); 298–308. �2013 AACR.

IntroductionSolid tumors almost always contain hypoxic areaswhen they

are radiologically detected. In tumors, oxygen consumption isincreased by proliferating cancer cells and infiltrating immunecells, and also oxygen delivery is impaired due to the abnormalvasculature network and the high interstitial pressure (1, 2).When cancer cells are challenged intermittently or constantlyby hypoxic stress, they set their metabolism to an energy-saving mode and refrain from proliferating. Many clinical andexperimental studies have demonstrated that cancer cellsexposed to hypoxia acquire resistance to anticancer drugs.Basically, drugs are poorly delivered to hypoxic areas withcirculation disturbance, and cancer cells are better able tosurvive after adapting to the hypoxic insult (3).Manymoleculeshave been suggested to be responsible for the hypoxia-induceddrug resistance (4). However, the mechanisms underlyingchemoresistance are quite different according to cancer con-texts and drugs, and complicated even in a cell-line treatedwith single drug.

Sirtuins belong to the class III histone deacetylase (HDAC)family that includes seven isoforms (SIRT1-7) in mammaliancells. They require NADþ as a substrate to deacetylate thelysine residues in histones and non-histone proteins (5, 6).SIRT1 is considered as the prototype of mammalian sirtuinsbecause its structure is most similar to that of the yeast Sir2.SIRT1, which is mainly located in the nucleus, regulates geneexpressions by changing the chromatin structures and bymodulating the activities of transcription factors (7, 8). As itsactivity depends on the ratio of NADþ/NADH, SIRT1 acts as aredox sensor to cope with metabolic imbalance under nutri-tion- or oxygen-deficient conditions (9). As a whole, SIRT1helps cells utilize glucose and survive under harmful condi-tions. In addition, SIRT1 is critical in diverse biologic processes,such as cell division, differentiation, senescence, and tumor-igenesis (10, 11).

Cumulative evidence has supported the involvement ofSIRT1 in cancer progression. SIRT1 exerts its tumor suppres-sive activity by inhibiting oxidative stress, inflammation, pro-liferation, and angiogenesis, by counteracting genotoxic stress,and by inducing apoptosis and autophagy (12–15). In contrast,SIRT1 also provides cancer cells with survival and expansionadvantages, which are achieved through p53 inactivation, Mycactivation, and epithelial-to-mesenchymal transition (16–18).Besides these Janus-faced roles in cancer promotion, SIRT1might determine tumor responses to anticancer drugs.Although SIRT1 has not been intensively explored in terms ofcancer chemotherapy, previous studies have supported thenotion that SIRT1 plays some positive roles in tumor acqui-sition of chemoresistance (19). Whether SIRT1 participates inthe hypoxia-induced chemoresistance is unclear.

Authors' Affiliation: Departments of Pharmacology and Biomedical Sci-ence, Ischemic/Hypoxic Disease Institute, Cancer Research Institute,Seoul National University College of Medicine, Seoul, Korea

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

Corresponding Author: Jong-Wan Park, Department of Pharmacology,Seoul National University College of Medicine, 103 Daehang-ro, Jongno-gu, Seoul 110-799, Korea. Phone: 82-2740-8289; Fax: 82-2745-7996;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-2620

�2013 American Association for Cancer Research.

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During hypoxia, the cellular ratio of NADþ/NADH declinesbecause the dehydrogenation of NADH to NADþ in the mito-chondrion is impaired, which results in decreased functiona-lity of SIRT1 (20). In addition, the increased NADH triggers therepressor C-terminal-Binding Protein-1 (CtBP) to associate withHypermethylated In Cancer 1 (HIC1) on the SIRT1 promoter,reducing the expression of SIRT1 (21). Taken together, SIRT1action is generally regarded to be inhibited under hypoxia.On the basis of the previous concept about the SIRT1-

mediated chemoresistance, theoretically, SIRT1 inhibitionunder hypoxia may sensitize cancer cells to anticancer drugs.However, in reality, chemoresistance is induced under hypoxia.To clarify this dichotomy, the present study was designed andperformed. In particular, we focused on the role of SIRT1 in thehypoxia-induced resistance against cisplatin or doxorubicin innon–small-cell lung carcinoma (NSCLC).

Materials and MethodsReagents and antibodiesSRT1720, EX527, A769662, cisplatin, and doxorubicin were

purchased from Selleck Chemicals and compound C from EMDMillipore. Antibodies against SIRT1, PGC-1a, cytochrome c,LaminB, Caspase-9, PARP, andb-actinwere obtained fromSantaCruz Biotechnology; antibodies against AMPKa, pAMPKa,mTOR, pmTOR, Caspase-3, and Beclin-1 from Cell SignalingTechnology; antibodies against Bcl-2 family (Bad, Bak, Bid, Bax,Bim, Bcl-x, Mcl-1, Bcl-2, and Bag-1) from Abcam. Anti-HIF-1aantiserum was raised in rabbits, as previously described (22).

Cell culture and hypoxic incubationNSCLC (H1299 and A549), fibrosarcoma (HT1080), gastric

cancer (MKN28), ovarian cancer (SKOV3), breast cancer(MDAMB-231), brain cancer (SHSY5Y and SKNMC), hepatoma(Hep3B and HepG2), renal cancer (786O and RCC4), cervicalcancer (HeLa and SiHa), colon cancer (HT29, HCT116 and CT26),and head and neck cancer (YD10B) cell lines were obtained fromthe American Type Culture Collection; other NSCLC cell lines(HCC15, HCC366, and HCC827) from Korea Cell Line Bank. Thecell lines were passaged for less than 6 months after being auth-enticated by DNA fingerprinting, which was performed using theAmplFLSTR Identifiler PCR Amplification Kit by Korean CellLine Bank. The cells were cultured in minimum essential medi-um, Dulbecco's Modified Eagle Medium, or RPMI-1640, supple-mented with 10% heat-inactivated FBS. Cells were incubated in ahumidified atmosphere at 37�C at 20% O2/5% CO2 for normoxiccondition or at 1% O2/5% CO2 for hypoxic condition.

Cell viability assayAfter cells (2 � 103) were cultured in 96-well plates under

normoxia or hypoxia for 2 days, cell viability was measuredusing MTT dye. Quadruplicate wells were used for each ana-lysis and at least three independent experiments were done.

Immunoblotting and immunoprecipitationCell lysates were prepared in a lysis buffer containing 1%

NP40, 5 mmol/L sodium orthovanadate, and protease inhibi-tors. After being boiled in a denaturing SDS sample buffer,

samples were subjected to SDS–PAGE and then transferredonto polyvinylidene difluoride membranes (Bio-Rad). Mem-branes were incubated overnight at 4�C with a primary anti-body, incubated for 1 hour with a HRP-conjugated secondaryantibody, and visualized using the ECLPlus Kit (Thermo FisherScientific). For immunoprecipitation, cell lysates were incu-bated with 5 mL of anti-LKB1 or anti-AMPK antiserum, orpreimmune serum at 4�C for 2 hours, and immune complexeswere further incubated with protein A/G-Sepharose beads (GEHealthcare) at 4�C for 4 hours. Immunocomplexes were elutedby 10-minute boiling in a denaturing SDS sample buffer andsubjected to immunoblotting with anti-SIRT1, anti-LKB1, oranti-acetyl-lysine antibody.

Analysis of mitochondrial morphologyCells were rinsed with 0.1 mol/L cacodylate buffer and fixed

in 3.5% glutaraldehyde (pH 7.4) for 24 hours at 4�C. After beingwashed in the cacodylate buffer containing 5% sucrose, cellswere post-fixed in 1% osmium tetroxide for 1 hour at 4�C. Cellswere gradually dehydrated in ethanol (50% to 100%) andinfiltrated with Spurr's resin (Electron Microscopy Sciences)over 2 hours. The resin was replaced with fresh Spurr's resinpoured in inverted BEEM embedding capsules (Ted Pella, Inc.)and allowed to be polymerized at 60�C for 36 hours. Beamcapsules were snapped off and attached cells were analyzed byelectron microscopy. Parameters of mitochondrial morphol-ogy (area and perimeter) were quantified using the ImageJprogram (NIH, Bethesda, MD).

Tumor xenograftAll animal procedures were performed in accordance with a

protocol approved by the Seoul National University AnimalCare and Usage Committee (Approval number, SNU-130104).Nude mice (BALB/cAnNCrj-nu/nu) from Charles RiverJapan Inc. (Shin-Yokohama) were injected at a dorsal flanksite with 1 � 106 cells in saline. Tumor volume was measuredwith calipers (volume ¼ a � b2 � 0.52, where a is the widestwidth and b is the perpendicular width) once every 3 days.When tumors reached a volume of 80–100 mm3 (termed day0 in this study), mice were injected intraperitoneally (i.p.)3 times a week with DMSO, cisplatin, SRT1720, or compoundC. On day 21, mice were sacrificed by cervical dislocation. Eachtumor was cut into two parts, which were fixed with 4%formalin or frozen in liquid nitrogen.

Statistical analysisEach result is expressed as the mean and SE or SD, which

was calculated using Microsoft Excel 2010. We used unpaired,two-sided Student t test for all tests and statistical differencewas considered significant when P < 0.05.

ResultsHypoxia-induced resistance to cisplatin anddoxorubicinare due to SIRT1 downregulation in NSCLC cells

Six cancer cell lines were treated with cisplatin or doxorubicinunder normoxic (21% O2) or hypoxic (1% O2) conditions for 48hours. The IC50 value for cisplatin or doxorubicin significantly

Roles of SIRT1 and AMPK in Hypoxia-Induced Drug Resistance

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increased under hypoxia in H1299, A549, Hep3B, and HT1080,but did not in HCT116 and MCF7 (Fig. 1A). We checked SIRT1in 21 different cancer cell lines and also hypoxia inducible factor1a (HIF-1a) to verify cellular responses to hypoxia. SIRT1expression in hypoxia was variable in cell lines (SupplementaryFig. S1). Notably,fiveNSCLC lines (H1299, A549, HCC15, HCC366,and HCC827) displayed hypoxic repression of SIRT1 at themRNA and protein levels (Fig. 1B and C). Given that HIFs arecrucial in hypoxia-induced gene regulation, we examined theregulation of SIRT1 expression by HIF-1a or HIF-2a. SIRT1expression under either normoxia or hypoxia was not affected byoverexpression or silencing of HIF-1a and HIF-2a (Supplemen-tary Fig. S2), suggesting that SIRT1 is expressed independently of

HIFs.We next examined the role of SIRT1 in chemoresistance bymanipulating SIRT1 expression. Under either normoxic or hyp-oxic conditions, the IC50 values for cisplatin and doxorubicin inH1299 andA549 cellswere significantly reducedby SIRT1 expres-sion, whereas these were raised by SIRT1 knockdown (Fig. 1Dand Supplementary Fig. S3A). These results indicate that SIRT1overexpression sensitizes NSCLC cells to the anticancer drugswhereas its knockdown confers drug resistance. Given that theeffects of SIRT1were shown in normoxic and hypoxic conditions,SIRT1 may sensitize cancer cells to the drugs regardless of theoxygen tension. Moreover, even in HCT116 cells that did notshow drug resistance and SIRT1 downregulation during hypoxia(Fig. 1B), the IC50 values for both drugs were decreased or

Figure 1. Hypoxia-inducedresistances against cisplatin anddoxorubicin are attributed to SIRT1repression in NSCLC cells. A, cellviabilities with increasingconcentrations of cisplatin anddoxorubicin under normoxic andhypoxic condition for 48 hourswere determined by MTT assay.IC50 values are presented as themeans � SDs (n ¼ 4); �, P < 0.05.B, SIRT1 protein levels in theindicated cancer cells subjected to48-hour hypoxia were determinedby Western blotting. The density ofeach blot was quantified using theImageJ program (NIH), and b-actinwas a loading control. Graycolumns, results from NSCLCcells. C, qRT-PCR was done tocheck the SIRT1 mRNA levels inNSCLCs subjected to 48-hourhypoxia. Each bar represents themean þ SD (n ¼ 3); �, P < 0.05. D,H1299 and A549 cells, which hadbeen transfected with SIRT1plasmid (2mg) or siRNA (80nmol/L),were treated with cisplatin (CP)under normoxic or hypoxicconditions for 48 hours. Cellviabilities (the means � SD, n ¼ 4)were presented as percentages ofthe values in untreated groups, andaverage IC50 values arewrittenwithboxes. � and #, P < 0.05 versusnormoxic control and versushypoxic control, respectively. E,cells within 0.4% top agar werestained with crystal violet andcounted. Each bar represents themean þ SD (n ¼ 4; �, P < 0.05).DOXO, doxorubicin.

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increased by SIRT1 expression or knockdown, respectively (Sup-plementary Fig. S3B). The chemosensitization by SIRT1 may notbe a hypoxia-specific event. To confirm the role of SIRT1 in re-sponse to chemotherapy, we examined the effects of a SIRT1activator, SRT1720, and a SIRT1 inhibitor, EX527, on cisplatin- ordoxorubicin-induced cell death. Hypoxia-induced chemoresis-tance in both cell lines was attenuated by SRT1720, but wasaugmented by EX527 (Supplementary Fig. S4). To test whetherSIRT1 determines the cytotoxic effects of cisplatin and doxoru-bicin in naturally occurring hypoxia, a colony formation assaywas carried out because typically the core part of a colony ishypoxic due to limited oxygen diffusion (23). As was expected,colony numbers substantially decreased in the presence ofcisplatin or doxorubicin. The anticancer effects were potentiatedby SRT1720, but diminished by EX527 (Fig. 1E). All of theseresults suggest that the SIRT1 downregulation during hypoxia

induces tumor resistances against cisplatin and doxorubicinand that SIRT1 restoration sensitizes hypoxic NSCLC cells tothe drugs.

AMPK is inactivated in NSCLC cells during hypoxia,leading to tumor resistance to cisplatin and doxorubicin

SIRT1 and AMP-activated protein kinase (AMPK) act asmetabolic sensors to revise the energy metabolisms accordingto nutritional states. They play this role independently, or do socooperatively, by regulating each other and by sharing com-mon target molecules (24, 25). Therefore, we measured phos-pho-Thr172 AMPKa (an active, catalytic subunit of the AMPKcomplex) levels in NSCLC cells subjected to hypoxia (Supple-mentary Fig. S1). As was shown for SIRT1, phosphorylatedAMPKa (pAMPKa) was diminished under hypoxia in NSCLCcells (Fig. 2A). The pAMPKa level correlated well (r2 ¼ 0.94)

Figure 2. AMPKa mediatesSIRT1-dependent, hypoxia-induced chemoresistance. A,AMPKa and its phosphorylatedform were analyzed by Westernblotting in the cell lysates usedin Fig. 1B and quantified usingImageJ. Gray columns, resultsfrom NSCLCs. B, H1299 and A549cells, which had been transfectedas indicated, were incubated undernormoxic or hypoxic conditionsfor 48 hours. Proteins wereanalyzed using Western blotting.C, transfected cells wereincubated for 48 hours asindicated. Cell lysates wereadded to a plate precoated withinsulin receptor substrate-1, andS789 phosphorylation of thesubstrate was analyzedspectrophotometrically at 450 nm.Each bar represents themean þ SD (n ¼ 4). D, transfectedH1299 cells were incubated for48 hours as indicated, celllysates were subjected toimmunoprecipitation with anti-LKB1, and then to immunoblottingwith the indicated antibodies. E,transfected cells were treated withcisplatin (CP) for 48 hours, and cellviabilities were measured usingMTT. Results (means� SDs, n¼ 4)were expressed as percentages ofthe values of untreated groups, andaverage IC50 values arewrittenwithboxes. � and #, P < 0.05 versusnormoxic control and versushypoxic control, respectively.






with SIRT1 level in NSCLC cells (Supplementary Fig. S5A). Thisresult encouraged us to check the dependence of AMPKaphosphorylation on SIRT1. AMPKa phosphorylation was reg-ulated SIRT1 dependently under both normoxic and hypoxicconditions (Fig. 2B). In contrast, themTOR phosphorylation atSer2448 was negatively regulated by SIRT1, suggesting that theSIRT1-activated AMPK inhibits mTOR phosphorylation. An invitro assay of AMPK activity also demonstrated that SIRT1activates AMPK (Fig. 2C). Seeking to determine how SIRT1activates AMPK,wewere prompted by a previous report (26) tocheck the possibility that SIRT1 deacetylates and activatesLKB1, which functions to activate AMPK. When SIRT1 wasdownregulated during hypoxia or knocked-down using siRNA,the acetylated form of LKB1 noticeably increased, but was

reduced in hypoxia by SIRT1 expression (Fig. 2D), suggestingthat SIRT1 activates AMPK in NSCLC cells by deacetylatingLKB1. Moreover, AMPKa sensitized NSCLC cells to cisplatin(Fig. 2E) and doxorubicin (Supplementary Fig. S5B).

The SIRT1–AMPKa pathway underlies hypoxia-inducedchemoresistance in NSCLC cells

To confirm that the hypoxic inactivation of the SIRT1–AMPK pathway confers drug resistance to NSCLC cells, NSCLCcells were treated with SIRT1 and AMPK modulators incombination (Fig. 3A). A SIRT1 inhibitor (EX527) induced drugresistances under normoxia, which was reversed by an AMPKactivator (A769662). In contrast, a SIRT1 activator (SRT1720)sensitized cancer cells to cisplatin and doxorubicin under

Figure 3. The SIRT1–AMPKapathway determines cellularsensitivities to cisplatin anddoxorubicin in NSCLC. A, cellswere treated with cisplatin (CP),doxorubicin (DOXO), SRT1720(100nmol/L), EX527 (100nmol/L),A769662 (10 mmol/L), andcompound C (1 mmol/L) undernormoxic or hypoxic conditionsfor 48 hours, and then theirviabilities were measured byMTT. B, transfected cells weretreated with cisplatin (CP) ordoxorubicin (DOXO) for 48 hours,and their viabilities weredetermined by MTT. C, cells,which had been transfected withthe indicated plasmids orsiRNAs, were treated withSRT1720 or EX527 incombinationwith cisplatin (CP) ordoxorubicin (DOXO) and thenincubated under normoxic orhypoxic conditions for 48 hours.Cell viabilities were determinedbyMTT. D, cells were transfectedwith pSIRT1_H363Y and/orpAMPKa, and then treated withcisplatin (CP) or doxorubicin(DOXO) for 48 hours. Each barrepresents themeanþSD (n¼ 4);�, P < 0.05.

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hypoxia, whichwas reversed by anAMPK inhibitor (compoundC). Likewise, another SIRT1 activator, resveratrol augmentedthe anticancer actions of both drugs in H1299 and A549 cellsunder hypoxia, but such effects of resveratrol were abolished bycompound C (Supplementary Fig. S6). To rule out off-targeteffects of small molecules, we checked cell viabilities using theplasmids and siRNAs for SIRT1 and AMPKa (Fig. 3B). Consis-tent with the results shown in Fig. 3A, SIRT1 knockdownsignificantly induced resistance to cisplatin and doxorubicinunder normoxia, and this effect was abolished by AMPKaoverexpression. Under hypoxia, SIRT1 overexpression aug-mented the anticancer effects of cisplatin and doxorubicin,but the SIRT1 effects were attenuated by AMPKa knockdown.Moreover, the EX527-induced chemoresistance in normoxiawas overcome by AMPKa expression, whereas the SRT1720-induced chemosensitization in hypoxia was attenuated byAMPKa knockdown (Fig. 3C). To determinewhether the SIRT1deacetylase function determines tumor responses to drugs, adeacetylase-defective, dominant-negative mutant of SIRT1(H363Y) was expressed (27). SIRT1_H363Y induced cisplatinand doxorubicin resistance under normoxia, which wasreversed by AMPKa expression (Fig. 3D). These results suggestthat the SIRT1–AMPK pathway determines the sensitivities ofNSCLC cells to cisplatin and doxorubicin.

SIRT1–AMPK inactivationunderhypoxiaprotects tumorcells from apoptosis in the presence of cisplatin ordoxorubicinTo understand how the SIRT1–AMPK pathway determines

tumor responses to cisplatin and doxorubicin, various apo-

ptotic markers in cisplatin- or doxorubicin-treated NSCLCcells weremeasured. The number of terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling (TUNEL)-posi-tive cells was reduced by SIRT1 knockdown, but was increasedby AMPKa expression. The TUNEL-positive cell populationwas significantly reduced under hypoxia. This effect of hypoxiawas attenuated by SIRT1 expression, which was reversed byAMPKa knockdown (Fig. 4A and Supplementary Fig. S7A).Likely, caspase-9 in NSCLC cells treated with the drugs wasactivated in a SIRT1- and AMPK-dependent manner (Fig. 4Band Supplementary Fig. S7B). As the release of mitochondrialcytochrome c (Cyt-c) is the initial event of apoptosis, weevaluated the Cyt-c release by costaining Cyt-c (green) withMitotracker (red; Supplementary Fig. S8). Normally, Cyt-ccolocalizes with Mitotracker, which produces yellow fluores-cence due to a green–red overlap. However, the yellow areasunder normoxia were reduced in cancer cells treated with thedrugs, but increased with siSIRT1, which was reversed byAMPKa expression. Under hypoxia, however, Cyt-c still local-ized within mitochondria even in the presence of the drugs,but it was dispersed by SIRT1 expression, which was reversedby AMPKa knockdown. The localization of Cyt-c was alsorechecked in cytoplasmic and mitochondrial fractions byWestern blotting (Supplementary Fig. S9). To confirm theinvolvement of SIRT1 in drug-induced apoptosis, wemeasuredthe cleaved forms of caspase-3 and PARP. Caspase-3 and PARPwere cleaved under normoxia in the presence of cisplatinor doxorubicin. The drug-induced cleavages were attenuatedunder hypoxia, which was reversed by SIRT1 expression (Fig.4C). Moreover, the hypoxia-induced resistance to cisplatin was

Figure 4. The SIRT1–AMPKapathway mediates apoptosis inNSCLC. Transfected H1299 andA549 cells were treated withcisplatin (CP) for 48 hours.Apoptosis was analyzed bymeasuring the indicated markers:A, TUNEL staining; B, caspase-9cleavage; C, caspase-3 and PARPcleavages. D, the transfected cellswere treated with cisplatin (CP)and/or Z-LEHD-FMK (100 mmol/L)for 48 hours. Cell viabilities weremeasured by MTT assay and eachbar represents the mean þ SD(n ¼ 4; �, P < 0.05).






attenuated by SIRT1 expression, which was reversed by acaspase inhibitor (Fig. 4D). These results indicate that thehypoxic inactivation of the SIRT1–AMPK pathway renderscancer cells resistant to caspase-mediated apoptosis inducedby anticancer drugs.

Hypoxic inactivation of the SIRT1–AMPK pathwayreduces apoptosis by inhibiting mitochondrial activity

Mitochondria play a central role in apoptotic cell deathunder genotoxic stress induced by anticancer agents (28, 29).

As both SIRT1 and AMPK promote mitochondrial bio-genesis (25), we hypothesized that they determine tumorsensitivities to cisplatin and doxorubicin by ensuring mito-chondrial biogenesis. To examine mitochondrial structure,transmission electron microscopy was used to examineNSCLC cells in normoxic and hypoxic conditions (Fig. 5A, left).Mitochondrial parameters like area, number, and perimeterwere measured using the ImageJ program (Fig. 5A, right).Mitochondria decreased in number and size during hypoxia.These changes were prevented by SIRT1 restoration, which

Figure 5. The hypoxic inhibition ofthe SIRT1–AMPKa pathwaydecreases mitochondrialbiogenesis in NSCLC. A, cells,which had been transfected asindicated in the box, wereincubated under normoxic orhypoxic conditions for 48 hours.Mitochondrial number andmorphology were analyzed on thebasis of electron microscopicimages (scale bar, 1 mm; left). Area(mm2) and perimeter (mm) ofmitochondria were measuredusing ImageJ. Bars, mean þ SD(n¼ 4); �, P < 0.05 (right) versus "a"level; #, P < 0.05 between twogroups. B, ATP levels wereanalyzed using the EnzyLight Kitunder the same conditions to A.Results are presented as therelative values to "a" level. Bars,means þ SD (n ¼ 4); �, P < 0.05.

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was attenuated by AMPKa knockdown. In addition, the mito-chondrial parameters were reduced under normoxia by SIRT1knockdown, which was reversed by AMPKa expression. Toevaluate mitochondrial function, ATP levels were measured inNSCLC cells (Fig. 5B). The normoxic ATP levels were reducedby SIRT1 knockdown, which was recovered by AMPKa expres-sion. ATP levels decreased during hypoxia were recovered bySIRT1 expression, which was attenuated by AMPKa knock-down. These results suggest that the inactivation of the SIRT1–AMPK pathway leads to mitochondrial suppression underhypoxia. These findings strongly indicate that the hypoxicinactivation of the SIRT1–AMPK pathway blocks mitochon-driogenic apoptosis through mitochondrial suppression andsubsequently desensitizes cancer cells to anticancer drugs.

The anticancer effect of cisplatin is augmented in H1299tumors by activation of the SIRT1–AMPK pathway

We examined whether the anticancer effect of cisplatin ismodulated through the SIRT1–AMPK pathway in H1299 xeno-grafted tumors. Cisplatin reduced tumor volume andweight by23% and 50%, respectively, 21 days after the first drug injection.Cisplatin in combinationwith SRT1720 retarded tumor growthmore efficiently, but the combination effect was abolished byan additional treatment with compound C (Fig. 6A and B).Body weights of mice on day 21 were not significantly differentamong the six experimental groups (Fig. 6C). Representativephotographs of excised tumors are shown in SupplementaryFig. S10. To evaluate AMPK activity and apoptosis in xeno-grafts, Western blotting of tumor tissue homogenates was

Figure 6. The activation of theSIRT1–AMPKa pathwayovercomes the tumor resistance tocisplatin in NSCLC tumorxenografts. H1299 cells wereimplanted in the flank of nudemice.After tumor volume reached about100 mm3, tumor-bearing micewere treated 3 times a week withcisplatin (3 mg/kg, i.p.), SRT1720(0.2 mg/kg, i.v.), and/or compoundC (1 mg/kg, i.p.). A, tumor volumesweremeasured once per 3 days for21 days. The results of tumorvolumes are expressed as themeans� SEM (n¼ 10); �, P < 0.05.B, tumors were removed frommiceand weighed (means þ SEM,n ¼ 10). C, body weights(means � SEM, n ¼ 10) of micewere not significantly differentamong all groups. D, theexpressions of pAMPKa (T172),PGC-1a, cleaved caspase-9,and cleaved caspase-3 weredetected in four xenografts pereach group by Western blotting,and the blot intensities wereanalyzed using ImageJ. Eachbar represents the mean þ SEM(n ¼ 4); �, P < 0.05.






carried out. AMPKa in grafted tumors was activated bySRT1720 injection but was inactivated by compound C injec-tion. The combined treatment with SRT1720 and cisplatinincreased the levels of cleaved caspase-9 and caspase-3. How-ever, the addition of compound C attenuated the combinationeffect (Supplementary Figs. S11A and S11B). The tumor levelsof activated AMPKa and caspases were quantified using theImageJ program and compared among the groups (Fig. 6D).Apoptotic death of cancer cells in H1299 xenografts wasexamined using TUNEL staining. SRT1720 sensitized tumorsto cisplatin but compound C desensitized tumors (Supple-mentary Fig. S11C). The in vivo results supported the notionthat the SIRT1–AMPK pathway can be a target for sensitizingNSCLC cells to cisplatin.

DiscussionLung cancer is themost common cancer worldwide in terms

of incidence and mortality, and NSCLC constitutes approxi-mately 80% of all primary lung cancers (30, 31). Chemotherapyfor NSCLC is based on the combined treatment of cisplatin andanother anticancer drug, including gemcitabine, irinotecan,taxanes, and vinorelbine (32). Although these combinationregimens have produced better therapeutic results than cis-platin alone, the best regimens can only give an overallresponse rate of less than 50% (33). Strategies to furtherimprove the survival of patients with NSCLC involve discoveryor development of more effective drugs, or the sensitization ofNSCLC cells to preexisting drugs. For the latter strategy, themechanisms underlying chemoresistance need to be clearlyunderstood. Yet, this goal is difficult to achieve becauseresistance is multifactorial, involving pharmacokinetic resis-tance, cellular resistance, and the microenvironment. Thepresent study focused on the mechanism underlying hypox-ia-induced chemoresistance, and implicated hypoxic repres-sion of SIRT1 in the resistance of NSCLC cells to cisplatin anddoxorubicin.

In the present study, we first investigated whether SIRT1participates in the hypoxia-induced chemoresistance inNSCLC cells. As SIRT1 was downregulated during hypoxia, wetested the possibility that the hypoxia-induced chemoresis-tance could be attributed to SIRT1 repression. SIRT1 wasrequired for the cytotoxic actions of cisplatin and doxorubicin,which was shown in the experiments controlling SIRT1 expres-sion and activity. SIRT1 activated AMPK by deacetylating andactivating the AMPK activator LKB1. The involvement of theSIRT1–AMPK pathway in the tumor-killing actions of cisplatinand doxorubicin was also demonstrated by controlling theexpressions and activities of AMPKa and SIRT1. Furthermore,the SIRT1–AMPK pathway promoted mitochondrial biogene-sis and by doing so ensured mitochondriogenic apoptosis inthe presence of cisplatin or doxorubicin. Under hypoxia,however, the drug-mediated apoptosis was attenuated due tomitochondrial suppression induced by the SIRT1–AMPK inac-tivation. The graphical summary of this mechanism is pre-sented in Fig. 7.

Although the relation between SIRT1 and cancer has beenextensively investigated in the past decade, the roles of SIRT1

in tumorigenesis and tumor development are still controver-sial. The oncogenic role of SIRT1 has been supported by clinicaland pharmacologic reports showing that SIRT1 is highlyexpressed in some human cancers and that tumor growth canbe retarded by SIRT1 inhibition (34). For example, Li andcolleagues found that SIRT1 was highly expressed in stem cellsof chronic myelogenous leukemia (CML; ref. 35). They alsodemonstrated that SIRT1 inhibition could suppress the growthof CML both in vitro and in vivo due to p53 activation (35). Incontrast, many studies using mice with genetically modifiedSIRT1 levels have consistently demonstrated that SIRT1 servesas a tumor suppressor (36). Because it targets a variety of keyfactors determining cell fate, SIRT1 could act as a tumorsuppressor or promoter depending on cell context. In thepresent study, we treated H1299 tumor-bearing mice withSRT1720 (SIRT1 activator) for 3 weeks, but observed nosignificant change in tumor growth (Fig. 6A). This resultsuggests that SIRT1 does not play a critical role in H1299tumor growth. Nonetheless, SIRT1 could be a promising targetfor cancer therapy because it participates in the apoptoticprocess induced by anticancer drugs.

SIRT1 regulation under hypoxia is dependent on the cellcontext. As shown in Fig. 1B, the hypoxic repression of SIRT1was not observed in about 50% of tested cell lines. In particular,RCC4, MKN28, and HeLa cells induce SIRT1 expression duringhypoxia. Indeed, the hypoxic regulation of SIRT1 expressionremains as a conflicting phenomenon. Chen and colleagues

Figure 7. A proposed mechanism underlying hypoxia-inducedresistances to cisplatin and doxorubicin in NSCLC cells.

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demonstrated that SIRT1 in Hep3B and HT1080 cell-linesis induced under hypoxia at the transcriptional level by bothHIF-1a and HIF-2a (37). In contrast, Zhang and colleaguesshowed that the SIRT1 transcription in fibroblasts is notice-ably repressed under hypoxia in a CtBP-dependent fashion(21). Presently, SIRT1 was constantly downregulated in thetested NSCLC cell lines. To rule out the possible involvementof HIF-1a/2a in SIRT1 regulation, we overexpressed orknocked-down both HIFs in NSCLC cells, but found no differ-ences in SIRT1 levels under either normoxia or hypoxia. Moredefined experiments should be done to elucidate why SIRT1under hypoxic conditions is differentially regulated among celllines.AMPK is activated in response to poor energy states, such

as nutrient deficiency, severe exercise, and other cellularstresses, due to increased levels of AMP, a product of ATPbreakdown. AMP binds to the g-subunit of the AMPKcomplex, which allosterically promotes the LKB1-mediatedphosphorylation and activation of the catalytic a-subunit(26). AMPK restores energy homeostasis by facilitating thecatabolic processes of glucose and fatty acid. Many recentstudies have supported the existence of bidirectional cross-talk between AMPK and SIRT1. In terms of the AMPKregulation of SIRT1, Canto and colleagues demonstratedthat AMPK stimulates SIRT1 activity in myoblasts byincreasing the ratio of NADþ/NADH (38). In contrast, Lanand colleagues demonstrated that SIRT1 activates AMPKsignaling in human embryonic kidney cells by deacetylatingLKB1 at Lys48 (39). Briefly, after being deacetylated bySIRT1, LKB1 is translocated from the nucleus to the cyto-plasm, where it forms an active complex with STE20-relatedadaptor protein (STRAD) and mouse embryo scaffold pro-tein (MO25), and then activates the a-subunit of AMPK. Ofthe two regulatory routes, the SIRT1–AMPK pathway mightbe involved in NSCLC cell responses to cisplatin and doxo-rubicin because the SIRT1 action on drug resistance couldbe reversed by inhibiting AMPK. Moreover, the acetylated(inactive) LKB1 level increased during hypoxia due to SIRT1suppression. However, we did not check whether the AMPK-to-SIRT1 pathway determines anticancer effects.Themetabolic sensors SIRT1 and AMPK have been reported

to promote mitochondrial biogenesis through the peroxisomeproliferator-activated receptor gamma coactivator 1a (PGC-1a) signaling pathway. PGC-1a binds and activates nuclearrespiratory factor 1 and 2, mitochondrial transcription factorA, and estrogen-related receptor gamma transcription factorsthat express the nuclear genes essential for mitochondrialreplication and respiratory function (40). Under energy-defi-cient conditions, the intracellular levels of AMP and NADþ

increase, leading to activation of both AMPK and SIRT1. Then,

PGC-1a is phosphorylated and deacetylated cooperatively bythe two enzymes, and activates the genes required for mito-chondrial biogenesis in the nucleus (38). Hypoxiamay drive cellmetabolism toward the adaptation to energy deprivationbecause the energy level also drops during hypoxia. However,mitochondrial biogenesis should be blocked during hypoxiabecause the mitochondria become useless for ATP generationdue to lack of oxygen and instead produce free electron-driventoxic metabolites like reactive oxygen species. Fig. 7 depicts aproposed mechanism underlying hypoxia-induced mitochon-drial suppression.

On the basis of the present results, we can suggest a newpotential strategy for overcoming the hypoxia-induced che-moresistance in NSCLC. The combination of a SIRT1 acti-vator or/and an AMPK activator with conventional antican-cer drugs like cisplatin and doxorubicin. Considering thatdrug-induced apoptosis is triggered in hypoxia by activatingthe SIRT1–AMPK pathway, this strategy may be useful foraugmenting the anticancer effects of molecularly targeteddrugs. In this case, however, the outcome of the SIRT1–AMPK activation might depend on the target signaling of thedrugs because SIRT1 and AMPK can cross-talk with multiplesignaling pathways that confer a survival benefit on the cells.The advantages and disadvantages of SIRT1–AMPK activa-tion in cancer therapy should be carefully evaluated beforeits clinical application.

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

Authors' ContributionsConception and design: D.H. Shin, J.-W. ParkDevelopment of methodology: D.H. ShinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D.H. Shin, Y.-J. ChoiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D.H. ShinWriting, review and/or revision of the manuscript: D.H. Shin, J.-W. ParkAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D.H. ShinStudy supervision: J.-W. Park

AcknowledgmentsThe authors thank professors Yang-Sook Chun and Chung-Hyun Cho of the

Seoul National University College of Medicine for their kind discussions andcomments.

Grant SupportThis work was supported by a grant (2010-0029948) funded by the National

Research Foundation of Korea.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 11, 2013; revised October 21, 2013; accepted November 7,2013; published OnlineFirst November 15, 2013.

References1. H€ockel M, Vaupel P. Tumor hypoxia: definitions and current clinical,

biologic, and molecular aspects. J Natl Cancer Inst 2001;93:266–76.2. Tr�edan O, Galmarini CM, Patel K, Tannock IF. Drug resistance and

the solid tumor microenvironment. J Natl Cancer Inst 2007;99:1441–54.

3. Cosse JP, Michiels C. Tumour hypoxia affects the responsiveness ofcancer cells to chemotherapy and promotes cancer progression.Anticancer Agents Med Chem 2008;8:790–7.

4. Teicher BA. Hypoxia and drug resistance. Cancer Metastasis Rev1994;13:139–68.






5. Qiu X, Brown KV, Moran Y, Chen D. Sirtuin regulation in calorierestriction. Biochim Biophys Acta 2010;1804:1576–83.

6. Cen Y, Y Youn D, A Sauve A. Advances in characterization of humansirtuin isoforms: chemistries, targets and therapeutic applications.Curr Med Chem 2011;18:1919–35.

7. Oberdoerffer P,MichanS,McVayM,MostoslavskyR, Vann J, Park SK,et al. SIRT1 redistribution on chromatin promotes genomic stability butalters gene expression during aging. Cell 2008;135:907–18.

8. Michan S, Sinclair D. Sirtuins in mammals: insights into their biologicalfunction. Biochem J 2007;404:1–13.

9. Webster BR, Lu Z, Sack MN, Scott I. The role of sirtuins in modulatingredox stressors. Free Radic Biol Med 2012;52:281–90.

10. Saunders L, Verdin E. Sirtuins: critical regulators at the crossroadsbetween cancer and aging. Oncogene. 2007;26:5489–504.

11. Haigis MC, Guarente LP. Mammalian sirtuins—emerging rolesin physiology, aging, and calorie restriction. Genes Dev 2006;20:2913–21.

12. Kume S, Haneda M, Kanasaki K, Sugimoto T, Araki SI, Isono M, et al.Silent information regulator 2 (SIRT1) attenuates oxidative stress-induced mesangial cell apoptosis via p53 deacetylation. Free RadicBiol Med 2006;40:2175–82.

13. Yoshizaki T,Milne JC, Imamura T, SchenkS,SonodaN,Babendure JL,et al. SIRT1 exerts anti-inflammatory effects and improves insulinsensitivity in adipocytes. Mol Cell Biol 2009;29:1363–74.

14. Kabra N, Li Z, Chen L, Li B, Zhang X,Wang C, et al. SirT1 is an inhibitorof proliferation and tumor formation in colon cancer. J Biol Chem2009;284:18210–7.

15. Potente M, Ghaeni L, Baldessari D, Mostoslavsky R, Rossig L,Dequiedt F, et al. SIRT1 controls endothelial angiogenic functionsduring vascular growth. Genes Dev 2007;21:2644–58.

16. Kim JE, Chen J, Lou Z. DBC1 is a negative regulator of SIRT1. Nature2008;451:583–6.

17. Yuan J, Minter-Dykhouse K, Lou Z. A c-Myc–SIRT1 feedback loopregulates cell growth and transformation. J Cell Biol 2009;185:203–11.

18. Byles V, Zhu L, Lovaas J, Chmilewski L, Wang J, Faller D, et al. SIRT1induces EMT by cooperating with EMT transcription factors andenhances prostate cancer cell migration and metastasis. Oncogene2012;31:4619–29.

19. Song NY, Surh YJ. Janus-faced role of SIRT1 in tumorigenesis. Ann NY Acad Sci 2012;1271:10–9.

20. Jang SY, Kang HT, Hwang ES. Nicotinamide-induced mitophagyevent mediated by high NADþ/NADH ratio and SIRT1 protein activa-tion. J Biol Chem 2012;287:19304–14.

21. Zhang Q, Wang SY, Fleuriel C, Leprince D, Rocheleau JV, Piston DW,et al. Metabolic regulation of SIRT1 transcription via a HIC1: CtBPcorepressor complex. Proc Natl Acad Sci U S A 2007;104:829–33.

22. Yeo EJ, Chun YS, Cho YS, Kim J, Lee JC, Kim MS, et al. YC-1: apotential anticancer drug targeting hypoxia-inducible factor 1. J NatlCancer Inst 2003;95:516–25.

23. Shin HW, Cho CH, Kim TY, Park JW. Sunitinib deregulates tumoradaptation to hypoxia by inhibiting HIF-1a synthesis in HT-29

colon cancer cells. Biochem Biophys Res Commun 2010;398:205–11.

24. Cant�oC, JiangLQ,DeshmukhAS,MatakiC,CosteA, LagougeM,et al.Interdependence of AMPK and SIRT1 for metabolic adaptation tofasting and exercise in skeletal muscle. Cell Metab 2010;11:213–9.

25. Hou X, Xu S, Maitland-Toolan KA, Sato K, Jiang B, Ido Y, et al. SIRT1regulates hepatocyte lipid metabolism through activating AMP-acti-vated protein kinase. J Biol Chem 2008;283:20015–26.

26. Ruderman NB, Xu XJ, Nelson L, Cacicedo JM, Saha AK, Lan F, et al.AMPK and SIRT1: a long-standing partnership? Am J Physiol Endo-crinol Metab 2010;298:E751–E60.

27. Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, et al.Stress-dependent regulation of FOXO transcription factors by theSIRT1 deacetylase. Science 2004;303:2011–5.

28. Kadenbach B, Barth J, Akg€un R, Freund R, Linder D, Possekel S, et al.Regulation of mitochondrial energy generation in health and disease.Biochim Biophys Acta 1995;1271:103–9.

29. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309–11.

30. Parkin DM. Global cancer statistics in the year 2000. Lancet Oncol2001;2:533–43.

31. Belani CP. Chemotherapy regimens in advanced non small-cell lungcancer: recent randomized trials.Clin LungCancer 2000;2Suppl 1:S7–S10.

32. Arriagada R, Bergman B, Dunant A, Le Chevalier T, Pignon JP,Vansteenkiste J, et al. Cisplatin-based adjuvant chemotherapy inpatients with completely resected non-small-cell lung cancer. N EnglJ Med 2004;350:351–60.

33. Bunn P, Kelly K. New chemotherapeutic agents prolong survival andimprove quality of life in non-small cell lung cancer: a review of theliterature and future directions. Clin Cancer Res 1998;4:1087–100.

34. Roth M, Chen WY. Sorting out functions of sirtuins in cancer. Onco-gene 2013;120:1–12.

35. Li L, Wang L, Li L, Wang Z, Ho Y, McDonald T, Holyoake TL, Chen W,Bhatia R. Activation of p53 by SIRT1 inhibition enhances elimination ofCML leukemia stem cells in combination with imatinib. Cancer Cell2012;21:266–81.

36. Herranz D, SerranoM. SIRT1: recent lessons frommouse models. NatRev Cancer 2010;10:819–23.

37. Chen R, Dioum EM, Hogg RT, Gerard RD, Garcia JA. Hypoxiaincreases sirtuin 1 expression in a hypoxia-inducible factor-dependentmanner. J Biol Chem 2011;286:13869–78.

38. Cant�o C, Auwerx J. PGC-1alpha, SIRT1 and AMPK, an energysensing network that controls energy expenditure. Curr Opin Lipidol2009;20:98–105.36.

39. Lan F, Cacicedo JM, Ruderman N, Ido Y. SIRT1 modulation of theacetylation status, cytosolic localization, and activity of LKB1 possiblerole in AMP-activated protein kinase activation. J Biol Chem 2008;283:27628–35.

40. FinckBN,Kelly DP. PGC-1 coactivators: inducible regulators of energymetabolism in health and disease. J Clin Invest 2006;116:615–22.

Shin et al.





2014;74:298-308. Published OnlineFirst November 15, 2013.Cancer Res Dong Hoon Shin, Yong-Joon Choi and Jong-Wan Park Small Cell Lung Cancers to Cisplatin and Doxorubicin

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